Page 1 !"#$$% &'( ( )* + , - - - - . / - / 0 / - . - - - ( , - 0 / - / 1 ( - - 2 ...

!"#$$%&'( ( )* + , - - - - . / - / 0 / - . - - - ( , - 0/- / 1 ( - - 2 - / - - -

% 3 4 - / # 1 56 / 1 1 & %

! " #

$%

! " # $ !% ! & $ ' ' ($ ' # % % ) % * % ' $ ' + " % & ' !# $, ( $ - . ! "- ( % . % % % % $ $$ - - - - // $$$ 0 & 1

2& )*3/ +) * !4 !& 5!6%55787& % ) 9 : ! * & /- ;8@IRU$/$%RWK/$DQGĮ-LA are metabolized by the same microsomal enzyme system, by alternating desaturation and elongation to make two cascades of metabolic products up to 22 or more carbons long (Figure 5) which occur in tissue structural lipids in animals and in man [Donadio 1991; Holman, 1998]. Nomenclature: PUFA are commonly classified according to a shorthand nomenclature which designates the chain length, number of double bonds and position of the double bond nearest to the terminal methyl group >:LOOLV@7KLVWHUPLQDOFDUERQDWRPLVRIWHQUHIHUUHGWRDVWKHRPHJDȦ DVLWRFFXUVDWWKH opposite end of the molecule to carbon-1 which bears the carboxyl function. The use of the ³RPHJD´QRPHQFODWXUHZas proposed by Holman [1964]. Alternatively, the n-x notation is used LQVWHDGRIȦ-[WRGHVFULEHWKHSRVLWLRQQHDUHVWWRWKHPHWK\OJURXS>7LQRFR@Ȧ-3 fatty acids are represented by alpha-linolenic acid ( ALA, C18:3 cisD9,12,15 ZKLOHȦ-6 fatty acids by linoleic acid (LA, C18:2 cisD9,12). 7KHJHQHUDOIRUPXODVIRUȦ-DQGȦ-6 fatty acids are, 1CH3-2CH¬2-3CH=CH-R

1CH3-2CH¬2-3CH2-4CH¬2-5CH2-6CH=CH-R

ȦRUQFDUERQ ȦRUQFDUERQ

27

respecWLYHO\:KHUHµ5¶UHSUHVHQWVUHVWRIWKHFDUERQFKDLQDQGWKHQXPEHURIFRQMXJDWHGGRXEOH bonds may vary. Distribution: LA and ALA as well as their long chain derivatives (e.g. EPA and DHA), are important components of animal and plant cell membranes. LA is found in triglycerides, cholesterol esters and in very small amounts in phospholipids. EPA is found in cholesterol esters, triglycerides and phospholipids. DHA is found mostly in phospholipids and is one of the most DEXQGDQWFRPSRQHQWVRIWKHEUDLQ¶VVWructural lipids [Teitelbaum and Walker, 2001]. Both these essential fatty acid families have distinctive nutritional and metabolic effects and each has a direct precursor relationship with specific class of eicosanoids. Thus, all eicosanoids formed in the human body are generated from PUFA that must be derived from the diet [Willis, 1987]. Sources: The omega fatty acids are found in fish and marine foods, green leafy vegetables and in certain plant seeds and walnut. (a)

Gifts from the sea: omega-3 fatty acids (EPA and DHA)

7KHORQJFKDLQȦ-38)$HLFRVDSHQWDHQRLFDFLG(3$& Ȧ-3) and docosahexaenoic acid '+$& Ȧ-3) (Figure 6) are predominantly found in certain fish and sea mammals (tuna, trout, herring, salmon, sardine etc.). Infact, the origin of EPA/DHA in aquatic systems are the plants of the sea, microalgae and seaweed. They produce high levels of EPA and DHA which are acquired by the marine fish/mammals through the food chain. Fish oils are concentrated source of EPA and DHA [Simopoulos et al., 1986]. Eggs can also provide small amount of DHA (/HXQJ@'LHWDU\Ȧ-3 fatty acids DUHSUHIHUHQWLDOO\LQFRUSRUDWHGLQWRWKHPXFRVDOSKRVSKROLSLGV DWWKHH[SHQVHRIȦ-6 fatty acids resulting in immunomodulatory effects including alteration of lymphocyte proliferation, neutrophil function, antigen presentation and induction of apoptosis in addition to changes in LQWHVWLQDOVWUXFWXUHDQGIXQFWLRQLQFOXGLQJFKDQJHVLQFHOOXODUJO\FRSURWHLQVDQGWLJKWMXQFWLRQVȦ3 fatty acids have been shown to influence the intestinal BBM uptake of glucose, galactose and cholesterol [Thompson et al., 1988; 1989]. %HQHILFLDOKHDOWKHIIHFWVRIȦ-3 PUFA from plant sources (ALA) in health and diseases ³6HHGVRIKRSH´IRUYHJHWDULDQV 7KHKHDOWKEHQHILWVRIGLHWDU\FRQVXPSWLRQRIIRRGULFKLQPDULQHȦ-3 PUFA are well established. :KHWKHUSODQWȦ-3 PUFAs DUHDVEHQHILFLDODVWKHȦ-3 PUFA from fish/marine food has not been LQYHVWLJDWHGH[WHQVLYHO\Į-/LQROHQLFDFLG$/$ LVDPDMRUȦ-3 FA in plants (Harris, 2005). It is a precursor of EPA but not DHA and must be converted to EPA for its bioeffector role [Mantzioris et al., 1994]. ALA is metabolized to EPA in humans to a significant extent [Valsta et al., 1996]. ALA from vegetable sources including grains and oils, offer an alternative source for those who are unable to regularly consume fish for religious or other reasons [Harper and Jacobson, 2001]. Although plants, sea microalgae and seaweeds are primary source of EPA and DHA, but most are not concentrated sources. They do not contribute significantly to EPA intakes in the western world but are important sources where people use large quantities of seaweeds on a daily basis (e.g. Japan and other parts of Asia). Thus vegetarians can rely to some extent on eggs/microalgae supplements for the supply of essential fatty acids. ALA from vegetable oils and EPA and DHA from fish oil have several parallel beneficial health effects [Ide et al., 2000; Kim and Choi, 2001]. Recent epidemiologic studies have shown that dietary ALA is associated with a lower risk of CVD in men and women [Djousse et al., 2001]. ALA has been associated with a lower rate of fatal and non-fatal coronary events [Nannicini et al., 2006]. Hypotensive effect of flaxseed oil have been documented lately thus constituting another mechanism for cardioprotective effect of ALA [Paschos et al., 2007]. The consumption of ALA 34

rich FXO offers protective effects against cardiovascular disorders compared to LA-rich sunflower oil via their ability to decrease the tendency of platelet to aggregate [Allman, 1995] and inhibition oISURJUHVVLRQRIDUWHULDODWKHURVFOHURVLV>/RUJHULODQG6DOHQ0R]DIIDULDQ@Ȧ-3 ALA KDVEHHQVKRZQWRKDYHJUHDWHUK\SROLSLGHPLFHIIHFWWKDQȦ-6 LA [Kim and Choi, 2001]. Role of ALA in flaxseed oil in prevention of cancer has also been demonstrated [Kelley et al., 1991; Williams et al., 2007]. Utility of flax and flaxseed oil in lupus nephritis and renal failure have also been accounted [Kelley et al., 1991; Basch et al., 2007]. Dietary supplementation with flaxseed oil affects the biochemistry of fatty acid metabolism and thus the balance of proinflammatory mediators and atherogenic lipids, holding a great promise for modulating inflammatory diseases (Chilton et al., 2008). Flaxseed oil has been shown to increase life span of irradiated mice suggesting its prophylactic potential against radiation-induced degenerative changes in liver (Bhatia et al., 2007). 9. KIDNEY ANATOMY AND PHYSIOLOGY: The kidney is a vital organ that plays an essential role in health and disease. The main function of the kidney is to maintain total body fluid volume, its composition and pH within physiologic range. This is achieved collectively by the presence of several millions of architectural and functional XQLWV RI WKH NLGQH\ NQRZQ DV ³QHSKURQ´ $ QHSKURQ FRQVLVWV RI glomerulus with an extended tubular structure. A rat kidney contains 30,000-35,000 nephrons whereas a human kidney is made up of about 1,30,000 nephrons. All these nephrons contribute to maintain renal functions by selective reabsorption of various ions and solutes. 9.1 The structure and function of kidney: The structure of the mammalian kidney apparently looks very homogenous, however, can be viewed as a composite of several tissue organs, geometrically, functionally and metabolically [Schmidt and Guder, 1976] (Figure 7). Thus each nephron consists of group of organs arranged in series coursing through four concentric tissue planes, the cortex, outer and inner zones of the outer medulla and the inner medulla (papilla) [Schmidt and Guder, 1976] (Figure 8). Each tissue plane DOVRKDVLQGLYLGXDO³RUJDQ´FKDUDFWHULVWLFVZLWKUHVSHFWWRWKHLULRQLFFRQWHQWVDQGPHWDEROLFUDWHV [Guder, 1973; Schmidt and Guder, 1976]. The luminal brush border membrane (BBM) of renal proximal tubule is the major site for reabsorption of various solutes including amino acids, sugars and other nutrients, certain ions and minerals such as Na+ and inorganic phosphate (Pi). 35

Reabsorption of most ions and solutes from the tubular lumen is coupled by an active transport with sodium (Na+) via a carrier located on apical side and is driven by an electrochemical gradient of Na+ generated by Na+/K+-ATPase located on basolateral side [Massary and Fleisch, 1980; Bonjour and Caverzasio, 1984]. Thus the transport of Na+ is considered to be a major work function of the kidney upon which all other transports are dependent [Ullrich et al., 1974; 1977]. The energy for the sodium transport is mainly provided by the hydrolysis of ATP at antiluminal membrane site involving Na+/K+ATPase [Balagura-Baruch et al., 1973; Evan et al., 1983]. Since the production of ATP is usually coupled to oxidative metabolism occurring in mitochondria, Na+ transport appears to be linked with the oxidative metabolism or oxygen tension (pO2) of the renal tubular cells. A direct linear relationship between O2 uptake/utilization and Na+ reabsorption has been found [Thurau, 1961; Torelli et al., 1986]. There appears to be a reverse cortico-medullary gradient for tissue oxygen tension (pO2) i.e., pO2 in inner medulla is far lower than in cortical tissue [Aukland and Krog, 1960; 1961; Aukland, 1962; Aperia and Liebow, 1964]. The proximal tubule in cortex has been demonstrated to be the major site for ions and solute reabsorption including water reabsorption which occurs at luminal or brush border membrane (BBM) site of the proximal tubules [Evan et al., 1983]. Thus ions and solutes such as sodium, phosphate, sugars and amino acids etc. are reabsorbed at the BBM of the proximal tubule as secondary active transport which is energized by the transcellular transport of sodium ions (primary active transport) from luminal membrane (in wardly) out of the epithelial cell by basolateral membrane [Hammerman and Schwab, 1984; Kempson and Dousa, 1986]. According to several studies, fatty acids, glutamine, lactate, citrate and glucose in particular are the major substrates which support the transport work of the kidney [Pitts and Macleod, 1975; Pitts, 1975; Mandel and Balaban, 1981]. It is well established that various nephron subsegments located in different tissue zones of the kidney have different functions in solute and fluid transport, as well as in substrate metabolism. For example, the renal cortex is characterized mostly by aerobic oxidative metabolism [Lee et al., 1962] while the renal medulla is the site of anaerobic metabolism and glycolysis. Moreover, the renal cortex is also capable of producing glucose [Guder, 1973; Burch et al., 1978; Maleque, 1980]. The oxidation of glucose in kidney may occur by several different metabolic pathways depending on the location and type of a particular nephron segment in the kidney: (i) the tricarboxylic acid (TCA) cycle, in which glucose undergoes glycolysis to pyruvate, which distributed differentially in the kidney. The renal medulla is the major region for 36

the production of lactate from glucose by glycolytic enzymes [Hems and Gaja, 1972; Guder, 1973] while the oxidative conversion of glucose to CO2 was shown in renal cortex [Lee et al., 1962; Cohen, 1979]. Nephron, which consists of various subsegments, shows distinct structural and functional differences (Figure 8). Thus nephron heterogeneity also adds to the variation in the kidney functions. Both inter- and intra-nephronal heterogeneity exists in the mammalian kidney that depends on the origin and location of the nephrons in the cortical region of the kidney [Lise et al., 1987; Francois and Danielle, 1985]. The nephron which originates in the glomerulus located in VXSHUILFLDOFRUWH[LVNQRZQDV³VXSHUILFLDOQHSKURQ´ZKLOHWKHQHSKURQZKLFKRULJLQDWHVIURPGHHS FRUWLFDOUHJLRQLVFDOOHGDV³GHHS´RU³MX[WDPHGXOODU\QHSKURQ´. These populations of nephrons have been found to be distinct structurally and functionally [Francois and Danielle, 1985]. In internephronal heterogeneity, proximal convoluted tubules of superficial nephrons always touch the surface of the kidney. In intra-nephron or axial heterogeneity, the proximal tubules have been divided into three distinct morphological subsegments namely S1, S2, and S3. The early proximal convoluted tubules (PCT) both in superficial and juxtamedullary nephrons is defined as S1segment and can be identified by its attachment with glomeruli on one side. S2 is defined as the late superficial proximal convoluted tubule, early superficial proximal straight tubule and late juxtamedullary proximal convoluted tubule. S3 is located principally in the outer stripe of outer medulla and terminal superficial proximal straight tubule and entire juxtamedullary proximal straight tubule. S3 is identified by its medullary location and by its connection with thin limbs on distal part. All S3-subsegments (pars recta), as they descend from cortex into the outer stripe of the outer medulla, change from S2 to S3 cell type. Thus the outer stripe of the outer medulla contains proximal tubular cells but only the S3 type [Woodhall et al., 1978]. These segments can be characterized by their biomarker enzymes [Yusufi et al., 1994]. 9.2 Acute Renal Failure: 9.2 Acute Renal Failure: Acute Renal Failure (ARF) is a process rather than a state. Often it denotes a reversible insufficiency of the glomerular and tubular excretory functions which may be triggered by renal or extra-renal mechanisms. ARF denotes a dramatic clinical situation in which both the kidneys stop their function within a short period of time or immediately depending on the severity of ARF.

38

It is best characterized by increase of nitrogenous waste products, such as serum creatinine and blood urea nitrogen and appearance of proteinic and enzymic components in the urine. ARF can be grossly divided into three phases; pathogenic phase, manifestation phase and recovery phase. In the first phase, a progressive disintegration and necrosis especially of tubular epithelial cells has been observed, leading to the functional loss of the kidney which is manifested by the reduction of inulin clearance [Steinhausen and Parekh, 1984]. In the second phase, long lasting effects are observed that severely affect the clearance of both creatinine and inulin and which can continue for several days after recovery begins, depending on the degree of renal damage. In the recovery phase, there is an increase in concentrating ability of the kidney with eventual normalization of kidney functions. A number of environmental variables and heavy metals like chromium, mercury, cadmium, lead, uranium [Bank et al., 1967; Cronin, 1986; Banday et al., 2008b] chemicals [Yagil, 1990; Weinberg et al., 1990] including drugs like aminoglycosides, cephalosporin, cisplatin etc. [Porter and Bennett, 1981; Harris et al., 1990; Basnakian et al., 2002; Banday et al., 2008a] affect the structure and function of the kidney leading to ARF. Generally, ARF caused by drugs and chemicals is much more severe and irreversible and the recovery sometimes is not possible [Steinhausen and Parekh, 1984]. The pathophysiologic mechanism of ARF has been investigated extensively in the last few decades. Four major possible causes of ARF have been generalized which include renal vasoconstriction, glomerular permeability, tubular obstruction and tubular leakage [Mason, 1986]. For many causes of ARF, ROS have been invoked as a pathway for renal injury. The kidney has been studied as an organ that can generate ROS and is vulnerable to the damaging effects of ROS. That ROS contribute to the pathogenesis of ARF is supported by the presence of oxidative stress in models of ARF, the amelioration of renal injury in these models by antioxidant maneuvers, and the worsening of renal injury in such models by manipulations that exacerbate sustained oxidative stress. Oxygen radicals are important mediators of renal damage in ARF causing lipid peroxidation of cell and organelle membranes, disrupting the structural integrity and capacity for cell transport and energy production (Greene and Paller, 1991). Several preventive measures have also been utilized but a definite answer for the pathogenesis and control of acute renal failure, however, remains the topic of future studies.

39

10. INTESTINAL ANATOMY AND PHYSIOLOGY: The digestion and absorption of food components are major functions of the intestinal mucosa. It is the most metabolically active tissue in the body. Many complex compounds while passing through the small intestine are degraded into simple compounds which cross the intestinal epithelium before reaching the various body organs. Thus intestinal epithelium not only regulates diverse absorptive and secretory processes but also process the substances that traverse it. 10.1 The structure and function of intestine: The small intestine extends from the pyloric orifice where it is continuous with the stomach to the ileocecal junction where it continues to the large intestine. It is divided into three distinct structural and functional parts: (i)

Duodenum

(ii)

Jejunum

(iii)

Ileum

To perform the entitled functions, the absorptive surface of small intestine is greatly amplified by transverse folds of mucous membrane (Figure 10). The small intestine is consisting of four layers: (i)

serosa (outermost)

(ii)

muscularis mucosa

(iii)

submucosa

(iv)

mucosa (innermost)

The absorptive cells of the small intestine are highly polarized tall columnar cells with a general architecture and structure that is similar to a number of other epithelial cell types whose major function is absorption [Grosser et al., 1992]. They are distinguished by the presence of an apical striated border which forms the absorbing surface in contact with luminal contents. The plasma membrane of these enterocytes consists mainly of two structurally and functionally different. The mucosa of the small intestine has transverse folds known as villi, which are finger like projections 0.5-1.5 mm in length. The villi are lined with a single layer of epithelial cells and contain a network of capillaries and lymphatic vessels (Lacteals). The free edges of the cells of the 40

microvilli contains fine filaments known as terminal web. The products of digestion may go through the microvillar membrane traversing the terminal web into cytoplasm. Brush Border membrane (BBM) lining the enterocytes (intestinal cells) constitutes one of the most important cellular membranes owing to its role in terminal digestion and absorption functions which are carried out by certain hydrolytic enzymes e.g. disaccharidases, dipeptidases, ROLJRSHSWLGDVHVȖ-glutamyl transferase, maltase, lactase, sucrase and alkaline phosphatase [Kenny and Booth, 1978]. These enzymes are differentially distributed in the thickness of BBM e.g. sucrase being superficially located [Brasitus, 1979] whereas alkaline phosphatase and ATPase are more deeply embedded within the membrane [Sigrist-Nelson et al., 1977]. The absorption of digestive food and various ions occurs by passive as well as active transport [Hopfer et al., 1973; Schultz, 1977]. The transport of water, sugars and amino acids is a Na+ dependent secondary active transport energized by an electrochemical gradient due to differences in Na+ concentrations across the BBM [Hopfer et al., 1973; Schultz, 1977; Hopfer, 1978; Ramaswamy et al., 1991]. The role of proton motive force as a source of energy (in terms of ATP) has also been demonstrated [Rajendran et al., 1987; Vorum et al., 1988; Ganapathy et al., 1991]. This is carried out by sodium pump (Na+/K+ATPase); which is a highly conserved integral membrane protein. The activities of certain enzymes belonging to glycolysis, TCA cycle, HMP shunt pathway, have been reported under various experimental conditions [Budhoski et al., 1982; Farooq et al., 2004; 2006]. The presence of hexokinase, glucokinase, 6-phosphofructokinase, glucose-6-phosphatase, fructose-1, 6-bisphosphatase, oxoglutarate dehydrogenase, citrate synthase, PEP carboxykinase, NADP-malic enzyme and glutamine indicate that gluconeogenesis is also operational in the small intestine

[Ockermann, 1965; Budhoski et al., 1982]. The function

of small intestine appears to be metabolic as opposed to be absorptive. Thus the studies involving metabolic activities may in some way reflect the absorption properties of the intestinal mucosa. 10.2 Gastrointestinal pathophysiology: Small intestinal mucosa is one of the largest areas of contact of the human organism with the environment [Schumann et al., 1999]. It is constantly challenged by diet derived oxidants, mutagens and carcinogens as well as by endogenously generated ROS [Tak Yee, 1999].

42

Figure 10: Anatomy of small intestine.

Figure 11: Structure of intestinal villi

43

The small intestine is thus primary site for great exposure to hazardous and life threatening environmental toxicants such as heavy metals (uranium) and drugs (aminoglycoside antibiotics and antineoplastic agents). Recent studies have clearly demonstrated that the enzymes present in the small intestine are not only altered by dietary stress such as starvation and fasting [Farooq et al., 2004; 2006] but also by antineoplastic and antibiotics drugs such as cisplatin and gentamicin [Arivarasu and Mahmood, 2007; Farooq et al., 2007]. Most prominent pathophysiological mechanism of gastrointestinal toxicity include direct effects on cell membrane, inhibition or stimulation of mucosal proliferation, nerve damage, activation of emetic pathways, disruption of intracellular signal transduction, generation of reactive oxygen substances, activation or inhibition of metabolic enzymes, intracellular toxicity etc. Reactive oxygen metabolites/species are the major causative factors for the mucosal lesions through oxidative stress. The major toxic effects of ROS include direct cytotoxicity towards epithelial cells, net fluid secretion into the lumen and alteration in functions of intestinal microvasculature that lead to increased permeability and membrane damage [Grisham et al., 1990]. 11. STRUCTURE AND FUNCTION OF LIVER: The liver weighing roughly 1.2-1.6 kg performs many of the functions necessary for staying healthy. The liver is the first organ that comes into contact with enterally absorbed nutrients and xenobiotics via portal blood (Kahl, 1999). Two large vessels carry blood to the liver: the hepatic artery comes from the heart and carries blood rich in oxygen, whereas the portal vein brings to the liver, blood rich in nutrients from the small intestine. These vessels are divided into smaller and smaller vessels, ending in capillaries, which further divide into thousands of lobules. Each lobule is composed of hepatocytes, and as blood passes through, they are able to monitor, add or remove substances from it (Sherwood, 1997). Among the most important functions of the liver are: 1)

Removing and excreting body wastes and hormones as well as drugs and other foreign

substances. Enzymes in the liver alter some toxins so they can be more easily excreted in the urine. 2)

Synthesizing plasma proteins, including those necessary for blood clotting. Most of the 12

clotting factors are plasma proteins produced by the liver. 3)

Producing immune factors and removing bacteria, helping the body fight infection. 44

12. Scope of the Thesis: The preventive and therapeutic value of food is a hot topic today. From ancient times, the physicians and scholars in Asia have understood that food has both preventive and therapeutic value and is an integral part of human health. The concept of chemo-prevention of certain diseases using naturally occurring substances that can be included in the diet consumed by human populations is gaining attention. A number of environmental contaminants, chemical and drugs e.g. cisplatin (CP), gentamicin (GM), uranyl nitrate (UN) dramatically alter the structure and function of various tissues and produce multiple adverse effects in the liver, kidney, heart and intestine [Fatima et al., 2004; Barbier et al., 2005; Priyamvada et al., 2008, 2010; Khan et al., 2009 a, b]. Various metabolites and donors of nitric oxide (NO) have been used as useful chemicals for the treatment of various diseases [Nickerson, 1970; Chow and Hong, 2002]. Nitrate and nitrite are used as food preservatives and colouring agents in meat, fish products and cheese [Lundberg et al., 2004]. In addition, potassium nitrate has been used as a diuretic agent and nitrite as a vasodilator, circulatory depressant or an antidote for cyanide poisoning [Bruijns, 1982; Chow and Hong, 2002]. Other precursors or sources of nitrite include N-containing foods, nitrogen containing drugs/chemicals (e.g. nitroglycerin - an anti-microbial agent and coronary dilator, sodium nitroprusside Na2[Fe(CN)6NO] - an anti-hypertensive agent and nitric oxide (NO) [Chow and Hong, 2002]. However, excessive amounts of NO donors/metabolites have been shown to exert adverse effects since nitrate, nitrite and NO can be recycled back and forth. Nitrates are not toxic per se; ingestion of large amounts of nitrates may cause severe gastroenteritis. Methemoglobinemia, anemia and nephritis caused by prolonged exposure to small amounts of nitrates are likely resulting from its reduction to nitrite by bacteria [Dollahite and Rowe, 1974]. A high dietary intake of nitrite has been implicated as a risk factor for human cancer, and formation of nitroso-compounds in the stomach during inflammation is correlated with nitrate in food and water [Ames, 1983; Mirvish, 1995]. A number of biochemical changes, functional impairments and histipathological lesions have been observed in nitrite-treated rats [Abuharfeil et al., 2001]. The major health concerns with nitrite are the risk of methemoglobinaemia and its potential carcinogenic, mutagenic and cytotoxic effects [Stuethr and Nathan, 1989; Ignarro, 1989; Moncada et al., 1991]. On the other hand, excessive and unregulated NO synthesis has been implicated as causal or contributing to 46

pathophysiological conditions including many lethal and debilitating human disorders: arthritis, atherosclerosis, cancer, diabetes, numerous degenerative neuronal diseases, stroke, and myocardial infarction to name a few [Culotta and Koshland, 1992, Gross and Wolin, 1995]. It has now been documented that reactions of H2O2 with nitrite and of superoxide with NO have a common intermediate-peroxynitrite (ONOO-) [Carmichael et al., 1993], which is responsible for nitration of proteins, DNA strand breaks and mutations, and with interference of protein functions [Sampson et al., 1998]. The quest for protective dietary nutrients for the control and management of various chronic diseases has spanned generations of scientific research. Of late, renewed interest and vigor has EHHQVKRZHUHGRQWKHUROHRIRPHJDIDWW\DFLGV)LVKRLOHQULFKHGLQȦ-3 PUFA (EPA and DHA) provide one such dietary source of biologically active components that has been shown to be copreventative and co-therapeutic in a wide variety of ailments [Doughman et al., 2007]. A number of investigations have already demonstrated that diet supplemented with fish oil (FO) HQULFKHGLQȦ-3 PUFA has profound beneficial health effects against various pathologies including cancers, cardiovascular diseases, diabetes, depression, arthritis, asthma and inflammatory and immune disorders of kidney and intestine. RecenWO\ Ȧ-3 PUFA from some plants/seeds e.g. IOD[VHHGRLO);2 VKRZHGPDQ\VLPLODUKHDOWKEHQHILWVDVGHPRQVWUDWHGE\Ȧ-3 PUFA from FO. Dietary fatty acids are known to be incorporated in cellular membranes especially plasma membranes affecting membrane fluidity and the organization of various membrane enzymes and transport proteins [Kogteva and Bezuglov, 1998] influencing the functional aspects of various RUJDQVHVSHFLDOO\RINLGQH\DQGLQWHVWLQHOHDGLQJWR³SRVLWLYH´HQGSRLQWVUHODWLQJWRKHDOWKDQG disease [Seo, 2005]. 5HFHQWO\ZHKDYHVKRZQWKDW)2HQULFKHGLQȦ-3 sucessfully prevented gentamicin and uranyl nitrate induced nephrotoxicty [Priyamvada et al., 2008, 2010]. In the present studies we K\SRWKHVL]HGWKDWGLHWDU\)2DQG);2HQULFKHGLQȦ-3 PUFA, would be able to prevent/reduce NO donors/metabolites induced nephrotoxic and other adverse effects in rat kidney, intestine and liver. To address this hypothesis the present work was undertaken to study detailed biochemical events/cellular response/mechanism of NO donors/metabolites induced nephrotoxic and other adverse effects in rat kidney, intestine and liver. NO donors used in this study were, L-arginine 47

which is an essential amino acid substrate for nitric oxide synthase (NOS) and pharmacological donors like sodium nitroprusside (SNP). SNT was used as a metabolite of NO and also as a donor VLQFH LW LV FRQYHUWHG WR 12 LQ YLYR 7KH HIIHFWV RI GLHWDU\ Ȧ-3 PUFA was also determined to observe any protection provided by them against NO donors/metabolites nephrotoxicity and other adverse effects. The specific objectives of the planned research included: 9 In the first part: Experiments were conducted to study the effects of dietary fish oil (FO) and flaxseed oil (FXO) ERWK HQULFKHG LQ Ȧ-3 on sodium nitrite (SNT) induced adverse effects on various biochemical parameters in serum/urine and on the enzymes of carbohydrate metabolism, brush border membrane (BBM), lysosomes and oxidative stress in renal cortex, medulla, small intestine and liver of rats. The effect of PUFA on the renal transport of Pi was also determined. 9 In the second part: Experiments were conducted to study the effects of dietary fish oil (FO) and flaxseed oil (FXO) ERWK HQULFKHG LQ Ȧ-3 on sodium nitroprusside (SNP) induced adverse effects on various biochemical parameters in serum/urine and on the enzymes of carbohydrate metabolism, brush border membrane (BBM), lysosomes and oxidative stress in renal cortex, medulla, small intestine and liver of rats. The effect of PUFA on the renal transport of Pi was also determined. 9 In the third part: Experiments were conducted to study the effects of dietary fish oil (FO) and flaxseed oil (FXO) ERWK HQULFKHG LQ Ȧ-3 on L-argine (L-ARG) induced adverse effects on various biochemical parameters in serum/urine and on the enzymes of carbohydrate metabolism, brush border membrane (BBM), lysosomes and oxidative stress in renal cortex, medulla, small intestine and liver of rats. The effect of PUFA on the renal transport of Pi was also determined. The results of the present studies showed that NO donors/metabolites treatment caused specific alterations in various serum/urine biochemical parameters and in the enzyme activities of various systems. The results further indicate that dietary FO and FXO, largely prevented/ameliorated NO donors/metabolites-induced alterations in various parameters. These studies would be helpful in further understanding the pathogenesis of NO donors/metabolites-induced toxic insult and its SRVVLEOHSUHYHQWLRQRUSURWHFWLRQE\GLHWDU\Ȧ-3 PUFA from fish oil and flaxseed oil (FXO being 48

D SRWHQWLDO DOWHUQDWLYH VRXUFH RI Ȧ-3 PUFA for vegetarians). Putting forth a good basis for the clinical use of omega-3 fatty acids both as dietary components and as future drugs/adjuvants which are relatively safe and inexpensive, confirming the dicta-³1XWULWLRQ LV WKH IRXQGDWLRQ IRU DOO KHDOWK´

49

A: MATERIALS Animals: Adult male Albino rats (Wistar strain) were purchased from Central Animal House Facility, Jamia Hamdard University, New Delhi, India. Substrates for carbohydrate metabolism and lysosomal enzymes: Sodium pyruvate for lactate dehydrogenase, oxaloacetate for malate dehydrogenase, L-malic acid for malic enzyme, Dglucose-6-phosphate for glucose-6-phosphate dehydrogenase and glucose-6-phosphatase, fructose-1,6-diphosphate for fructose-1,6-bis phosphatase and p-nitrophenyl phosphate for acid phosphatase were purchased from Sisco Research Laboratory (SRL, Mumbai, India). Substrates for BBM marker enzymes of kidney and intestine: p-nitrophenyl phosphate for alkaline phosphatase, g-glutamyl-p-nitroanilide for g-glutamyl transferase and L-leucine-p-nitroaniline for leucine amino peptidase were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Sucrose for sucrase was purchased from SRL (Mumbai, India). Substrates for enzymes involved in free radical scavenging: Pyrogallol for superoxide dismutase and hydrogen peroxide for catalase were purchased from SRL (Mumbai, India). Glutathione reductase required in assay of glutathione peroxidase was purchased from Sigma Chemical Co. (St Louis, MO, USA). Radioactive Chemicals: Radioactive phosphate (32Pi) was purchased from Bhabha Atomic Research Center, India. Animal Diet: The standard rat pellet diet was obtained from Aashirwaad Industries, Chandigarh, India. Oil: Fish oil: Menhaden, Sigma Chemical Co. (St. Louis, MO, USA). Flaxseed oil: Omega Nutrition, Canada Inc., Vancouver. 50

Miscellaneous: All chemicals used were of the finest quality commercially available and their sources are indicated against them. Glass double distilled water was used in all experiments.

51

B: METHODS 1. Animal Protocol: Male Wistar rats weighing 150-200 gm were used in all studies. Animals were acclimatized for a week prior to the experiment on standard rat pellet diet with free access to water. The animal experiments were conducted according to the guidelines of Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of India. Body weights of rats were recorded at the start and completion of the procedure. DietA nutritionally adequate laboratory pellet diet was obtained from Aashirwaad Industries, Chandigarh (India). Pellets were crushed finely and mixed with (a) 15% Fish oil (b) 15% Flaxseed oil and stored in airtight containers. Vitamin E as DL-Į-tocopherol (270 mg/kg chow) was added to each of the modified rat chows in order to meet the increased metabolic requirement for antioxidants on a diet high in polyunsaturated fatty acids. The oil diets were prepared in small batches (for 3d consumption) and supplied everyday to ensure freshness, the left over diet from each cage was discarded. With the exception of the dietary oils, all components of the diet were identical. General Experimental designAfter acclimatization (7 days) the animals were divided into different dietary groups fed on (a) VWDQGDUGUDWGLHWFRQWUROUDWV DQGE Ȧ-3 PUFA supplemented diet (FO rats and FXO rats). (i)

,QILUVWVHULHVRIH[SHULPHQWVHIIHFWRIGLHWDU\FRQVXPSWLRQRIȦ-3 fatty acid enriched fish

oil/flaxseed oil with sodium nitrite treatment. (ii)

,QVHFRQGVHULHVRIH[SHULPHQWVHIIHFWRIGLHWDU\FRQVXPSWLRQRIȦ-3 fatty acid enriched

fish oil/flaxseed oil with sodium nitroprusside treatment. (iii)

,QWKLUGVHULHVRIH[SHULPHQWVHIIHFWRIGLHWDU\FRQVXPSWLRQRIȦ-3 fatty acid enriched fish

oil/flaxseed oil with L-arginine treatment.

52

Body weights of rats were recorded before and after inducing specific experimental conditions. The rats were anesthetized with anesthetic ether and sacrificed. Blood and urine samples were collected. Kidney and small intestine were harvested and processed for the preparation of homogenates and BBMV, utilized for further analyses as described below. Blood collection: Before sacrifice of the rats blood was withdrawn from the left jugular vein with the help of a disposable syringe. Serum was obtained by centrifugation at 2000 x g for 10 min. Urine collection: Three or four rats of each group were placed in standard metabolic cages and the urine was collected for 3 hrs at initiation of the experimental conditions and prior to the day of sacrifice. Urine volumes were recorded and the urine samples were frozen and stored at -80ºC for subsequent analyses. 2. Analysis of Serum and Urine Parameters: The serum samples were deproteinated with 3% TCA in a ratio of 1:3. After incubation for 10 min at room temperature, the samples were centrifuged at 4000 rpm (Remi Centrifuge, India) for 10 min. The protein free supernatant was used to quantitate serum creatinine (Scr) and inorganic phosphate (Pi) and the precipitate was saved to quantitate total phospholipids. Cholesterol was determined directly in the serum samples. (i) Quantitiative determination of creatinine: Creatinine was determined by the method of Levinson and MacFate (1969). To 0.5 ml of the deproteinated serum supernatant, 50 ml of 10% NaOH and 200 µl of saturated picric acid were added and incubated for exactly 20 min at room temperature. A calibration curve was simultaneously prepared using known concentrations of creatinine solution ranging between 2.0-5.0 mg. The sample and the standards were read at 520 nm in Cintra 5, GBC, Scientific Equipment. Pty. Australia. Creatinine was determined in urine samples after a dilution of 1:10 with distilled water following the same procedure. (ii) Determination of inorganic phosphate: The inorganic phosphate (Pi) was measured in protein free (TCA precipitated) serum supernatant by the method of Tausky and Shorr (1953). The serum supernatant (0.5 ml) was diluted to 3 ml with 1.3 ml of glass distilled water and 1.2 ml of FeSO4 reagent (5 gm FeSO4 was dissolved in 10 ml, of 10% w/v ammonium molybdate in 10N H2SO4 and diluted to 100 ml with glass distilled water) was added. A calibration curve was prepared simultaneously with test samples using known concentrations of 53

KH2PO4

(0.018 mmoles-0.28

mmoles). The blue colour obtained was read at 820 nm after 20 min incubation at room temperature in Cintra 5, GBC, Scientific Equipment Pty. Australia. Pi was determined in urine samples after a dilution of 1:10 with distilled water following the same procedure. (iii) Quantitative determination of phospholipids: Phospholipids are determined in the serum TCA-precipitates by the method of Bartlett (1959) as modified by Marinetti (1962). The precipitates were digested with 1 ml of 70% perchloric acid on an electric digestion unit. On cooling to room temperature 3 ml of glass distilled water was added. The phosphate (inorganic) released was estimated by adding 2 ml of FeSO4 reagent by the method of Tausky and Shorr (1953) as described above in Pi estimation. The phospholipid values were obtained after multiplying the phospholipid phosphorus by a factor of 25. (iv) Quantitative determination of cholesterol: Cholesterol was determined by the method of Zlatkis et al., (1953). To 3 ml acetic acid, 30 ml serum sample was added. To this, 2 ml of FeCl3 reagent (prepared by diluting 1 ml of 10% FeCl3 w/v in glacial acetic acid to 100 ml with concentrated H2SO4) was added carefully from the side to allow the formation of a brown ring. The samples were shaken thoroughly, cooled and colour density was read in Cintra 5, GBC, Scientific Equipment Pty. Australia, at 560 nm against a reagent blank. A calibration curve was simultaneously prepared using known concentration (0.02 - 0.2 mg) of cholesterol. (v) Determination of blood urea nitrogen and glucose: Levels of urea and glucose were assayed by standard colorimetric procedures, urea by reaction with diacetyl monoxime and glucose by reaction with o-toluidine using kits from Span diagnostics (Surat, India). Glucose was also assayed in the urine samples by reaction with o-toluidine using kit from Span diagnostics (Surat, India). (vi) Determination of urinary protein: The urine collected was filtered, precipitated by 13% (final concentration) TCA and was then centrifuged at 3000 rpm for 10 min. The protein content was assayed by the method of Lowry et al., (1951). 3. Preparation of tissue homogenates: (i) Kidney homogenate (cortex and medulla) for the determination of metabolic enzymes: After the completion of treatment schedule (SNT, SNP or ARG), the kidneys were carefully separated from the treated and control animals, and homogenized in 0.1 M Tris-HCl buffer pH 7.5 by a glassteflon homogenizer (Thomas PA, USA) by passing 5 pulses; at 4ºC to make a 15% w/v 54

homogenate. The homogenate was then subjected to high speed Ultra-Turrex Kunkel homogenizer (Type T-25, Janke & Kunkel GMBH & Co. KG. Staufen) for 3 pulses of 30 s each with an interval of 30 s between each stroke. Homogenate was centrifuged at 2000 rpm at 4ºC for 10 min in Beckman J2-M1 (Beckman instruments. Inc Palo Alto, C.A. USA) high-speed refrigerated centrifuge to remove the cell debris. The supernatant was saved in aliquots and stored at -20ºC for analyses of metabolic enzymes (Figure 13). (ii) Intestinal mucosal homogenate for the determination of metabolic enzymes: After the completion of treatment schedule (SNP, SNT or ARG) the small intestines from the treated and control animals were removed, washed with 1 mM Tris-HCl, 0.9% NaCl, pH 7.5 and slit open in the middle. The mucosa was gently scraped with a glass slide and was used in the preparation of homogenate. Mucosal scrapings were homogenized in 0.1 M Tris-HCl buffer, pH 7.5, in a glassteflon homogenizer at 4ºC to make a 10% w/v homogenate. The homogenate was then subjected to high speed Ultra-Turrex Kunkel homogenizer for 3 pulses of 30 s each with an interval of 30 s between each stroke. Homogenate was centrifuged at 2000 rpm at 4ºC for 10 min in Beckman J2M1 high-speed refrigerated centrifuge to remove the cell debris. The supernatant was saved in aliquots and stored at -20ºC for analyses of metabolic enzymes (Figure 13). (iii) Liver homogenate for the determination of metabolic enzymes: After the completion of treatment schedule (SNT, SNP or ARG), liver was carefully separated from the treated and control animals, and homogenized in 0.1 M Tris-HCl buffer pH 7.5 by a glass-teflon homogenizer (Thomas PA, USA) by passing 5 pulses; at 4ºC to make a 10% w/v homogenate. The homogenate was then subjected to high speed Ultra-Turrex Kunkel homogenizer (Type T-25, Janke & Kunkel GMBH & Co. KG. Staufen) for 3 pulses of 30 s each with an interval of 30 s between each stroke. Homogenate was centrifuged at 2000 rpm at 4ºC for 10 min in Beckman J2-M1 (Beckman instruments. Inc Palo Alto, C.A. USA) high-speed refrigerated centrifuge to remove the cell debris. The supernatant was saved in aliquots and stored at -20oC for analyses of metabolic enzymes. (iv) Homogenate for enzymatic and non-enzymatic antioxidant parameters: A 10% homogenate of cortex, medulla, intestinal mucosa and liver was prepared in 0.1 M Tris-HCl buffer, pH 7.5, in a glass-teflon homogenizer with 5 complete strokes. The homogenate was then subjected to high speed Ultra-Turrex Kunkel homogenizer for 3 pulses of 30 s each with an interval of 30 s between each stroke. One part of the homogenate was saved at -20ºC for estimation of total±SH and lipid 55

peroxidation and other was centrifuged at 5000 rpm for 15 min at 4ºC and supernatant was stored at -20ºC for analyses of free-radical scavenging enzymes. 4. Preparation of brush border membranes: (i) Cortical Brush Border Membrane (BBM) Preparation: After the completion of treatment schedule (SNT, SNP or ARG), the kidneys from treated and control animals were removed, decapsulated and kept in ice-cold buffered saline (154 mM NaCl, 5 mM Tris-HEPES, pH 7.4). Each kidney was cut horizontally into two halves and the whole cortex (WC) was carefully separated from medullary and papillary portions. The BBM vesicles (BBMV) were prepared by the method of Schimtz et al., (1973) using MgCl2 for precipitation of membranes other than BBM as described by Yusufi and Dousa (1987]) and as outlined in schematic diagram (Figure 13). In each experiment, tissues from three to six animals (control and experimental) were pooled to obtain a sufficient amount of starting material. All the steps involved were strictly carried out at 0-4ºC unless otherwise specified. a. The cortical tissue for the preparation of BBMV was homogenized in a buffered solution containing 50 mM mannitol, 5 mM Tris base/HEPES, pH 7.5 (5ml/gm tissue) with four complete passes by glass-teflon homogenizer. b. The homogenate (10% w/v) was diluted with the above solution (20ml/gm tissue) followed by high speed homogenization (Ultra-Turrex Kunkel homogenizer) with three pulses of 30 s each with 30 s interval between each pulse. Aliquots of cortical homogenates were saved and quickly frozen for further analyses. c. 1 M MgCl2 was added to the homogenate (to a final concentration of 10 mM) and kept on ice for 20 min with intermittent shaking. d. The homogenate was then centrifuged at 5000 rpm (2000 x g) for 10 min in a Beckman J2M1 refrigerated centrifuge using JA-17 rotor. e. The pellet was discarded and the supernatant was recentrifuged at 17,000 rpm (35,000 x g) for 30 min. f. The pellet thus obtained was resuspended in a solution containing 300 mM mannitol, 5 mM Tris base/ HEPES, pH 7.5 with four passes by a loose fitting Dounce homogenizer (Wheaton IL, Thomas PA, USA) and centrifuged at 17,000 rpm (35,000 x g) for 30 min in 15 ml corex glass tube using JA-20 rotor. 56

g. The supernatant was discarded and the white outer portion of the fluffy pellet was resuspended carefully in a small volume of buffered 300 mM mannitol, leaving the dark brown centre of the pellet undisturbed (mitochondrial contamination). The suspension thus obtained was homogenized by hand held Douncer or passed through a gauge needle number 21. This BBM suspension was quickly frozen in small aliquots and used for enzyme analysis while aliquots of freshly prepared membranes were used for transport studies. (ii) Intestinal Brush Border Membrane (BBM) Preparation: After the completion of treatment schedule (SNT, SNP or ARG), the small intestines from treated and control animals were removed, washed with 1 mM Tris-HCl, 0.9% NaCl, pH 7.5 and slit open in the middle. The mucosa was gently scraped with a glass slide and was used in the preparation of intestinal BBM. The intestinal BBM was prepared as described by Kessler et al., (1978) using CaCl2 for precipitation of membranes other than BBM with a few modifications, as outlined in schematic diagram (Figure 13). All procedures were carried out at 0-4oC unless otherwise specified. a. The mucosal scrapings for the preparation of BBMV were homogenized in a buffered solution containing 50 mM mannitol, 2 mM Tris-HCl, pH 7.5 with four complete passes by a glass-teflon homogenizer. b. The mucosal homogenate was diluted with the above Tris-mannitol buffer (15 ml/gm tissue) and further homogenized using Ultra Turrex Kunkel homogenizer at high speed with three pulses for 30 s each with 30 s interval between each pulse. Aliquots of mucosal homogenates were saved and quickly frozen for further analyses. c. Homogenate was then passed through four layers of cheesecloth and CaCl2 was added to the filtrate (to a final concentration of 10 mM), with constant stirring and the solution was left on ice for 15-20 min. d. The homogenate was then centrifuged at 5000 rpm (2000 x g) for 10 min in a Beckman J2M1 refrigerated centrifuge using a JA-17 rotor. e. The pellet was discarded and supernatant was centrifuged at 17000 rpm (35000 x g) in JA17 rotor for 30 min. f. Pellet was resuspended in a small volume (1-2 ml) of 50 mM sodium maleate, pH 6.8, buffer with four complete passes by a loose fitting dounce homogenizer (Wheaton, USA)

57

and centrifuged at 17000 rpm (35000 x g) for 30 min in a 15 ml corex tube using a JA-20 rotor. g. Pellet was resuspended in a small volume (1-2 ml) of 50 mM sodium maleate, pH 6.8, buffer with four complete passes by a loose fitting dounce homogenizer (Wheaton, USA) and centrifuged at 17000 rpm (35000 x g) for 30 min in a 15 ml corex tube using a JA-20 rotor. h. The white outer fluffy portion of the pellet was resuspended in 50 mM sodium maleate, pH 6.8, buffer leaving the dark brown center of pellet undisturbed (mitochondrial contamination). i. The suspension thus obtained was homogenized by hand held Douncer or passed through a gauge needle number 21. This BBM suspension was quickly frozen in small aliquots and used for enzyme analysis. 5. Enzyme Assays: 9 All enzymes were assayed at zero order kinetics unless otherwise specified. 9 The activities of each enzyme from various comparing groups were determined simultaneously under similar conditions by using same solutions to avoid day to day experimental variations. 9 One unit of enzyme activity is defined as the amount of enzyme required to catalyze the formation of 1 mmole of product per min or hour under the specified experimental conditions. 9 Specific activity is defined as the enzyme unit per mg protein. 9 The enzymes of carbohydrate metabolism, enzymatic and non-enzymatic antioxidant parameters were assayed in cortical, medullary and intestinal homogenates and marker enzymes of brush border membrane were measured in respective homogenates and BBMV. (A)

Assay of carbohydrate metabolism enzymes: The assays were carried out in kidney

{cortical homogenate (CH) and medullary homogenate (MH)}, intestine {mucosal homogenate and liver {liver homogenate (LH)}by measuring the extinction changes in a Cintra 5 (GBC, Scientific Equipment Pty. Australia) in a final volume of 3 ml at room temperature (28-30ºC). The net reaction rate was measured by difference of the extinction values obtained from addition of the substrate only and for actual enzymatic reaction following the addition of substrate. The molar 58

extinction coefficient of NADH and NADPH used in calculation of reaction rate was 6.22 mM1cm-1 at 340 nm. G6Pase and FBPase were assayed at 37ºC. (i)

Lactate dehydrogenase (L-Lactate: NAD oxidoreductase; LDH; E.C. 1.1.1.27): The

activity of LDH was measured by the method of Kornberg (1955). The reaction mixture, in a total volume of 3 ml, contained 150 mmoles Tris-HCl buffer pH 7.4; 10 mmoles MgCl2; 5 mmoles sodium pyruvate; 0.24 mmoles NADH and 4.0-6.0 mg protein. The activity was measured as decrease in absorbance, by pyruvate dependent NADH oxidation to NAD+ for 5 min at 340 nm. (ii)

Malate dehydrogenase (L-Malate: NAD oxidoreductase; MDH; E.C. 1.1.1.37): The

activity of MDH was measured by the method of Meyer et al., (1948). The reaction mixture, in a total volume of 3 ml, contained 100 mmoles Tris-HCl buffer, pH 7.4; 2.5 mmoles oxaloacetate (OAA, pH neutralized to 7.4); 0.24 mmoles NADH and 2.0-3.0 mg protein. The activity was measured as decrease in absorbance, by OAA dependent NADH oxidation to NAD+ for 5 min at 340 nm. (iii)

Malic enzyme (L-Malate: NADP oxidoreductase; ME; E.C. 1.1.1.40): ME was assayed

according to the method of Ochoa et al., (1948). The reaction mixture, in a total volume of 3 ml, contained Tris-HCl buffer, pH 7.4, 100 mmoles; 10 mmoles MnCl2; 5 mmoles L-malic acid (pH adjusted to 7.4); 0.24 mmoles NADP+ and 0.6-1.2 mg protein. The activity was measured as increase in absorbance, by monitoring the malic acid dependent reduction of NADP+ to NADPH for 5 min at 340 nm. (iv)

Glucose-6-phosphate dehydrogenase (D-Glucose-6-phosphate: NADP oxidoreductase;

G6PDH; E.C. 1.1.1.49): G6PDH was assayed by the method of Shonk and Boxer (1964). The reaction mixture, in a total volume of 3 ml, contained Tris-HCl buffer, pH 7.4, 150 mmoles; 10 mmoles MgCl2; 5 mmoles glucose-6-phosphate; 0.24 mmoles NADP+; and 0.6-1.2 mg protein. The activity was measured by monitoring increase in absorbance, by glucose-6-phosphate dependent reduction of NADP+ to NADPH for 5 min at 340 nm. (v)

Hexokinase (ATP:glucose phospho transferase; HK; E.C. 2.7.1.1.): It was assayed

according to the method of Crane and Sols (1953). The reaction mixture in a total volume of 1ml contained Tris-HCl buffer, pH 7.4, 50 mmoles; 2 mmoles KH2PO4; 10 mmoles MgCl2; 5 mmoles ATP; 2 mmoles glucose and 1-1.5 mg protein. The reaction was carried out at 37ºC and stopped 60

with 0.5 ml of 10% ZnSO4 and Ba(OH)2 each, after 60 min. The samples were centrifuged at 4000 rpm (Remi centrifuge, Mumbai, India) and the remaining glucose was estimated in the protein and phosphorylated derivatives free supernatant by the method of Nelson [1944]. A calibration curve was simultaneously prepared using a known concentration of glucose solution ranging between 5ȝJ (vi)

Glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; G6Pase; E.C.

3.1.3.9): It was assayed according to the method of Shull et al., (1956). The reaction mixture in a total volume of 1.5 ml contained Tris-HCl buffer pH 7.4, 50 mmoles; 10 mmoles MgCl2; 10 mmoles glucose-6-phosphate and 2-3 mg protein. The reaction was carried out at 37ºC and stopped with 1ml of 10% TCA after 60 min. The samples were centrifuged at 4000 rpm (Remi Centrifuge, Mumbai, India) and phosphorus was estimated in the protein free supernatant by the method of Tausky and Shorr [1953]. (vii)

Fructose-1,

6-Bisphosphatase

(D-fructose-1,6-bisphosphate-1-phosphohydrolase;

FBPase; E.C. 3.1.3.11): It was assayed according to the method of Freedland and Harper (1959). The reaction mixture in a total volume of 1.5 ml contained Tris-HCl buffer pH 8.4, 50 mmoles; 10 mmoles MgCl2; 12 mmoles cysteine-HCl; 10 mmoles fructose-1,6-diphosphate; 0.6-0.8 mg protein. The reaction was carried out at 37oC and stopped with 1 ml of 10% TCA after 60 min. The samples were centrifuged at 4000 rpm (Remi Centrifuge, Mumbai, India) and phosphate released was estimated in protein free supernatant by the method of Tausky and Shorr (1953). (B)

Assay of lysosomal marker enzymes:

(i)

Acid phosphatase (ACPase, E.C. 3.1.3.2): The activity of acid phosphatase (ACPase) in

cortical, medullary and intestinal homogenates was determined by the method of Verjee (1969). The reaction mixture contained 2.4 ml acetate buffer (0.05 M sodium acetate, pH 4.5) and 100 ml enzyme (35-40 mg). The reaction was started by the addition of 0.5 ml p-nitrophenyl phosphate (final concentration 0.8 mM in 3ml) and incubated for 15 min at 30oC. The reaction was stopped by adding 2ml, 2 N NaOH. A calibration curve of known concentrations of p-nitrophenol (0.050.4 mmoles) was prepared simultaneously. The yellow colour developed was read at 405 nm against a reagent blank.

61

(C)

Assay of marker enzymes of brush border membrane: The enzymes were assayed

simultaneously in kidney {cortical homogenate (CH) and cortical BBMV (CBBMV)}, intestine {mucosal homogenate (IH) and intestinal BBMV (IBBMV)} and liver {liver homogenate (LH)} under similar conditions by using same solutions to avoid day-to-day experimental variations. Aliquots of CH and CBBMV were diluted with 10 mM Tris-HCl buffer, pH 7.5 and those of IH and IBBMV with 5 mM Tris-HCl buffer, pH 7.5 to obtain suitable enzyme protein concentration. (i)

Alkaline phosphatase (AlkPase, E.C. 3.1.3.1): The activity of AlkPase in CH, IH and

respective BBMV was determined according to the method of Shah et al., (1979) as modified by Kempson et al., (1979). The reaction mixture contained 1.4 ml assay buffer (55 mM glycine; 36 mM NaCl and 45 mM NaOH, pH 10.5) and 100 ml enzyme protein (10-25 mg protein for CH, IH and 4-8 mg for BBMV). The reaction was started by adding 15 ml p-nitrophenyl phosphate (final concentration 5.8 mM in 1.5 ml) and incubated at 37ºC for the required time (5-20 min). The reaction was stopped by adding 50 ml of 5 N NaOH. A calibration curve was prepared simultaneously by using known concentration of p-nitrophenol (0.01-0.2 mmoles). The colour was read at 405 nm against a reagent blank. (ii)

g-Glutamyl transferase (GGTase, E.C. 2.3.2.2): The activity of GGTase in CH, IH and

respective BBMV was determined according to the method of Glossman and Neville (1972) as described by Kempson et al., (1985). The reaction was started by adding 100 ml enzyme protein (15-20 mg for CH; 100 mg for IH; 2-5mg for cortical BBMV and 50 mg for intestinal BBMV) to 1.9 ml substrate buffer (20 mM MgCl2, 2 mM g-glutamyl-p-nitroanilide, 4 mM glycylglycine, 100 mM Tris-base, pH 8.2) and incubated in a shaking water bath maintained at 37ºC for the required time (15-30 min). The reaction was stopped by adding 100 ml of 15 M acetic acid. A calibration curve of known concentration of standard p-nitroaniline (0.025-0.2 mmoles) was prepared simultaneously. The yellow colour developed was read at 405 nm against a reagent blank. (iii)

L-Leucine aminopeptidase (LAP, E.C. 3.4.11.2): This enzyme was assayed by the method

of Goldmann et al., (1976). The reaction was started by the addition of 100 ml cortical, medullary or intestinal homogenates or BBMV (40-60 mg protein for homogenate and 10-15 mg for BBMV) to 1.9 ml substrate buffer (50 mM sodium phosphate buffer, 0.33 mM L-leucine p-nitroanilide, pH 7.2) and incubated at 25ºC for the required time (15-30 min). The reaction was stopped with 100 ml of 15 M acetic acid. A calibration curve of standard p-nitroaniline (0.025-0.20 mmoles) was 62

also prepared simultaneously. The yellow color obtained was read at 405 nm against a reagent blank. (iv)

Sucrase (E.C. 3.2.1.48): Sucrase was assayed by the method of Goldstein and Lampen

(1975). The reaction mixture (0.3 ml) contained 50 ml of 0.1 M sodium maleate buffer, pH 6.0, 200 ml of diluted BBM or intestinal homogenate and 50 ml of 0.1 M sucrose solution. The assay mixture was incubated at 37ºC for 10 min and the reaction was terminated by addition of 200 ml of 0.2 M phosphate buffer, pH 7.0, immediately followed by heating in a boiling water bath. The tubes were cooled and 0.5 ml of distilled water was added. Glucose thus liberated was determined by adding 1 ml of 3, 5-dinitrosalicylic acid reagent and kept in a boiling water bath for 5 min, and 3 ml of distilled water was again added. A calibration curve was simultaneously prepared using known amounts of glucose (0.5-2.5 mmoles) and the colour was read at 540 nm against a reagent blank. (D)

Assay of enzymatic antioxidant parameters: The activities of the three key enzymes of the antioxidant defense system i.e. superoxide dismutase, catalase and

glutathione peroxidase were measured simultaneously in the homogenates of cortex, medulla, intestine and liver under similar conditions by using same solutions to avoid day-to-day experimental variations. (i)

Superoxide dismutase (SOD, E.C. 1.15.1.1): It was assayed by the method of Marklund

and Marklund (1974). To 0.08 ml of supernatant 2.82 ml of 0.05 mM Tris-succinate buffer, pH 8.2 was added, mixed well and incubated at 25ºC for 20 min. The reaction was started by adding 0.1 ml of 8 mM pyrogallol solution. Change in absorbance per minute was immediately recorded for the initial 3 min at 420 nm. A reference set, containing 0.08 ml distilled water instead of supernatant solution, was also run simultaneously. (ii)

Catalase (E.C. 1.11.1.6): This enzyme was assayed according to the method of Claiborne

[1985] as described by Giri et al., (1996). The assay mixture consisted of 1.95 ml of 0.05 M potassium phosphate buffer pH 7.0, 1 ml of 0.019 M hydrogen peroxide and 0.05 ml homogenate (50-100 µg protein) in a final volume of 3 ml. The decrease in absorbance at 240 nm was immediately noted after every 30 seconds for 3 min. Enzyme activity was calculated using the molar extinction coefficient of H2O2 (436 M-1 cm-1 at 240 nm). 63

(iii)

Glutathione peroxidase (GSH-Px, E.C. 1.11.1.9): The activity of GSH-Px was determined

by the method of Flohe aQG *XQ]OHU 7KH DVVD\ PL[WXUH FRQWDLQHG ȝO RI 0 potassium phosphate/1 mM EDTA buffer (pH 7.0), 50 ml of 1 mM of sodium azide, 50 ml of homogenate (100-200 mg protein), 100 ml glutathione reductase (0.24 U), and exactly 100 ml of 10 mM GSH. The mixture was preincubated for 10 min at 37º&7KHQȝORIP01$'3+ solution was added and the hydrogen peroxide independent consumption of NADPH was PRQLWRUHGIRUPLQ7KHRYHUDOOUHDFWLRQZDVVWDUWHGE\DGGLQJȝORISUHZDUPHGP0 hydrogen peroxide solution and the decrease in absorbance at 340 nm was monitored for 5 min. Enzyme activity was calculated using the molar extinction coefficient of NAPDH (‫ = ܭ‬6.22 x 103 M-1 cm-1). (E) Assay of non-enzymatic antioxidant parameters: (i)

Total-SH groups: Total-SH groups were determined by the method of Sedlak and Lindsay

(1968). To 0.4 ml of 10% tissue homogenate, 2.1 ml of 0.1 M Tris-HCl (pH 8.2), 0.5 ml of 10% SDS and 0.3 ml of 0.1 M EDTA were added. The reaction was incubated in a boiling water bath for 5 min. Then, 0.1 ml DTNB (40 mg/100 ml methanol) was added. After 30 min at room temperature, the absorbance was read at 412 nm. A calibration curve with different amounts of cysteine (20-160 nmoles) was constructed by the same procedure as described above and used to calculate the total-SH groups in the samples. (ii)

Lipid Peroxidation: Lipid peroxidation (LPO) in various tissue homogenates was

determined spectrophotometrically by the method of Ohkawa et al. [1979]. To 0.1 ml of tissue homogenate 1.5 ml of 20% trichloroacetic acid, 0.2 ml of 8% SDS, 0.7 ml of distilled water and 1.5 ml of 0.8% thiobarbituric acid were added and after mixing incubated at 95ºC for 20 min. After cooling to room temperature and centrifugation at 10,000 rpm for 10 min the absorbance of the supernatant was read at 532 nm a against reagent blank. Amount of malondialdehyde (MDA, end product of LPO) was calculated using the molar extinction coefficient of thiobarbituric acid (e = 1.56 x 105 M-1 cm-1). 6. Phosphate Transport: Measurment of inorganic phosphate (32Pi) uptake in BBMVs was carried out at 25ºC by the rapid filtration technique as described by Yusufi et al., (1994) either in the presence or absence of a Na64

gradient. Uptake was initiated by addition of 30 ml incubation medium (100 mM NaCl/KCl, 5 mM Tris-HEPES, pH 7.5, 5 mM K2HPO4) and radioactive substrate (32Pi) to 15 ml BBM suspension (50-PJSURWHLQ DQGLQFXEDWHGIRUWKHGHVLUHGWLPHLQWHUYDOVVHH³5HVXOWV´ $WWKHHQGRIWKH incubation period the uptake was stopped by rapid addition of 3 ml ice cold stop solution (delivered by a Cornwall Syringe type pipette) containing 135 mM NaCl, 10 mM sodium arsenate, 5 mM Tris-HEPES, pH 7.5, and filtered immediately through 0.45 mm DAWP millipore filters and washed 3 times with the same ice cold stop solution. Correction for non-specific binding to filters was made by subtracting from all data the value of the corresponding blank obtained by filtration of the incubation medium without vesicles. The radioactivity of the dried filters was measured by OLTXLG VFLQWLOODWLRQ FRXQWLQJ 3KDUPDFLD 6ZHGHQ ZLWK PO VFLQWLOODWLRQ IOXLG ³&RFNWDLO-7´ SRL, Mumbai, India). 7. Protein Estimation: Protein was measured by the method of Lowry et al., (1951) with minor modifications as described by Yusufi et al., (1983). The samples were made to 0.8 ml with 0.5% SDS. Then 2 ml alkaline FRSSHUUHDJHQWZDVDGGHGDQGDIWHUPLQLQFXEDWLRQDWURRPWHPSHUDWXUHPO)ROLQ¶VUHDJHQW (1 N) was added with brisk shaking and incubated for 30 min at room temperature. A calibration curve of standard BSA (5-80 mg) was prepared simultaneously. The blue colour obtained was read at 660 nm. 8. Estimation of Tissue Nitrite Concentrations: In biological systems conversion of NO in aqueous solution to nitrite and nitrate is thought to favour nitrite production [Butler et al., 1995]. It has been reported that nitrite is the only stable end-product of the autooxidation of NO in aqueous solution [Ignarro et al., 1993] and measurement of nitrite concentrations in the serum and tissue homogenates widely accepted as an index for NOS activity [Tucntan and Altug, 2004; Tunctan et al., 1998, 2000, 2003]. Therefore, concentrations of nitrite in tissue homogenates were measured by using the diazotization method based on the Griess reaction, which is an indirect assay for NO production [Tunctan et al., 1998]. Kidney, intestine and liver homogenates were centrifuged at 5000 g for 10 min. 500µl supernatant was removed and an equal volume of modified Griess reagent was added and the samples were incubated in the dark for 20 min. Standard curves were generated using 0-20 µM sodium nitrite. Absorbance was measured in standards and samples at 550 nm. Concentrations of nitrite were determined from the 65

standard curve, which was linear over the entire concentration range tested. Values were expressed as nanomols per gram tissue. 9. Statistical Analysis: Unless specified, all experiments were repeated at least four or five times to document reproducibility. All data are expressed as Mean ± SEM. Significance of difference in mean values were evaluated using either one-ZD\DQDO\VLVRIYDULDQFH$129$ IROORZHGE\DVWXGHQW¶VJURXS t-test for comparison between two mean values using SPSS 7.5 software. A probability level of p < 0.05 was selected as indicating statistical significance. Most of the changes between various groups were compared with control values for better understanding and clarity. However, specific differences and statistical significance between other groups were evaluated separately e.g. (i) SNP vs. FOSNP; SNP vs. FXOSNP, (ii) SNT vs. FOSNT; SNT vs. FXOSNT and (iii) ARG vs. FOARG; ARG vs. FXOARG

66

PART - I Â HYPOTHESIS I: Fish oil (FO) and IOD[VHHGRLO);2 HQULFKHGLQȦ-3 fatty acids would similarly protect against SNT induced nephrotoxic and other deleterious effects in different rat tissues. Â EXPERIMENTAL DESIGN I: As shown in Figure 14, initially four groups (viz Control, SNT, FOSNT and FXOSNT) of rats (10 rats/group) entered the study after acclimatization. They were fed on normal diet (control/SNT) or diet containing 15% fish oil (FOSNT), 15% flaxseed oil (FXOSNT). SNT (15 mg/kg bwt/d) was administered intraperitonealy in 0.9% saline daily for 10 days to the groups designated as SNT, FOSNT and FXOSNT. Control animals received an equivalent volume of normal saline for the same period. The rats were sacrificed 24 h after the last injection under light ether anesthesia. Blood was withdrawn from left jugular vein and serum was separated by centrifugation at 2000 x g for 10 min. Urine samples were collected for 4 h in standard metabolic cages a day before the sacrifice of rats. The kidneys, intestine and liver were extracted and processed for the preparation RIKRPRJHQDWHVDQGEUXVKERUGHUPHPEUDQHYHVLFOHV%%09 DVGHVFULEHGLQ³0HWKRGV´%RG\ weights of rats were recorded at the start and completion of experimental procedure. Â RESULTS I: Â Effect of dietary fish oil and flaxseed oil on SNT induced toxicity in serum and urinary parameters: In general the rats remained clinically well throughout the study. There was no significant difference in daily food intake and body weights between control and other experimental rats (data not shown). SNT treatment to control rats resulted in significant increase in serum creatinine (Scr), blood urea nitrogen (BUN), cholesterol, glucose and phospholipids but decrease in inorganic phosphate (Pi) compared to control rats (Table 1). These changes were associated with profound phosphaturia, proteinuria and glucosuria (Table 2) accompanied by decrease in creatinine clearance (Table 2). Feeding of FO or FXO diet to SNT administered (FOSNT and FXOSNT) rats resulted in significant reversal of various SNT elicited deleterious effects on serum and urine

67

parameters. Both FO and FXO diets prevented SNT-induced increase of Scr, BUN, glucose and cholesterol and decrease of serum Pi (Table 1). FO/FXO diets appear to greatly improve renal functions in a similar manner as evident by the increase of creatinine clearance and decrease in the excretion of protein, phosphate and glucose in the urine (Table 2). Â Effect of dietary fish oil and flaxseed oil on SNT induced alterations in metabolic enzyme activities in renal cortex, medulla, small intestine and liver: The main function of kidney i.e. reabsorption of various ions and solutes depends on the continuous energy supply as ATP which is generated by various metabolic pathways including glycolysis and oxidative metabolism. The acute renal failure produced by toxic insult results in reduced oxygen consumption due to damage caused to mitochondria and other organelles. The effect of SNT, FO, FXO and MO-diets and their combined treatment was determined on the activities of various enzymes of carbohydrate metabolism involved in glycolysis, TCA cycle, gluconeogenesis and HMP shunt pathway in various organs including kidney, small intestine and liver in rats (Table 3, 4, 5, 6). (a) Effect on carbohydrate metabolism in renal cortex: As shown in Table 3 and 4, SNT treatment to control rats significantly increased the activity of lactate dehydrogenase (LDH) but decreased malate dehydrogenase (MDH), hexokinase (HK); glucose-6-phosphatase (G6Pase) and fructose-1, 6-bisphosphatase (FBPase) activities in the renal cortex (Table 3, 4). When SNT treatment was extended to FO and FXO-fed rats, SNT-induced alterations in metabolic enzyme activities were not only prevented by FO and FXO diets, but G6Pase remained significantly higher in FOSNT and FXOSNT compared to control as well as SNT rats in the renal cortex. The effect of FO, FXO and SNT combined was also determined on glucose-6-phosphate dehydrogenase (G6PDH) and NADP-malic enzyme (ME), source of NADPH production needed in various anabolic reactions (Table 4). SNT treatment to control rats significantly increased G6PDH but decreased ME activity. SNT elicited increase of G6PDH activity was normalized to near control values by both FO and FXO diets. SNT induced decrease in ME activity, however was arrested by both FO and FXO diet.

69

70

48.69 ± 0.78* (+85%) 30.66 ± 0.65 (+16) 29.52 ± 0.33 (+12%)

1.28 ± 0.09* (+54%) 0.93 ± 0.76 (+12%) 0.99 ± 0.84* (+19%)

SNT

FOSNT

FXOSNT

80.76 ± 0.56 (-2%)

83.89 ± 0.87 (+1%)

97.27 ± 0.12* (+18%)

82.73 ± 0.92

(mg/dl)

Cholesterol

28.82 ± 0.54* (+19)

26.77 ± 0.88 (+10%)

35.36 ± 0.12* (+46%)

24.29 ± 0.09

(mg/dl)

Phospholipid

1.26 ± 0.66 (-6%)

1.44 ± 0.27 (+7%)

0.92 ± 0.76* (-32%)

81.54 ± 0.11 (-9%)

83.78 ± 0.46 (-7%)

112.11± 0.09* (+25%)

89.91 ± 0.19

(mg/dl)

ȝPROVPO

1.35 ± 0.52

Glucose

Phosphate

Results are Mean ± SEM for five different preparations.

6LJQLILFDQWO\GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\GLIIHUHQWIURP617DWSE\RQHZD\$129$ Values in parentheses represent percent change from control.

26.32 ± 0.11

0.83± 0.59

Control

BUN (mg/dl)

(mg/dl)

Creatinine

Groups

Group

Table 1: Effect of Fish oil (FO) and Flaxseed oil (FXO) on serum parameters with SNT treatment.

Table 3: Effect of Fish oil (FO) and Flaxseed oil (FXO) activities of HK, LDH and MDH in homogenates of a) cortex and b) medulla with SNT treatment. Enzyme Groups

HK

LDH

MDH

(Pmols/mg protein/h)



Control

64.21 ± 0.76

27.72 ± 0.01

94.71 ± 0.01

SNT

46.71 ± 0.29* (-27%)

35.61 ± 0.22* (+29%)

61.09 ± 0.61* (-35%)

FOSNT

59.87 ± 0.77* (-7%)

28.89 ± 0.77 (+4%)

97.61 ± 0.23 (+3%)

FXOSNT

67.78 ± 0.12 (+6%)

30.04 ± 0.07 (+8%)

90.66 ±0.96 (-4%)

Control

69.33 ± 0.39

40.91 ± 0.09

86.41 ± 0.13

SNT

46.21 ± 0.77* (-33%)

49.69 ± 0.06* (+21%)

64.21 ± 0.07* (-25%)

FOSNT

60.03 ± 0.73 (-13%)

43.87 ± 0.54 (+7%)

88.22 ± 1.35 (+2%)

FXOSNT

63.71 ± 0.45 (-8%)

42.87 ± 0.12 (+5%)

77.87 ± 0.66 (-10%)

a) Cortex

b) Medulla

Results (specific activities) are Mean ± SEM for five different preparations. *Significantly different from FRQWUROVLJQLILFDQWO\GLIIHUHQWIURP617DWSE\RQHZD\$129$9DOXHV in parentheses represent percent change from control.

72

needed in various anabolic reactions (Table 4). SNT treatment to control rats significantly increased G6PDH but decreased ME activity. SNT elicited increase of G6PDH activity was normalized to near control values by both FO and FXO diets. SNT induced decrease in ME activity, however was arrested by both FO and FXO diet. (b) Effect on carbohydrate metabolism in renal medulla: The activities of LDH, MDH, HK, G6Pase and FBPase in medullary homogenates were similarly affected by SNT treatment (Table 3, 4) as in the cortical homogenates. Feeding of FO and FXO containing diets to SNT treated rats prevented SNT induced alterations in various enzyme activities. Similar to cortex, G6PDH activity increased whereas ME activity decreased by SNT in the medulla (Table 4). FO and FXO diet to SNT treated rats decreased G6PDH activity and increased ME activity. (c) Effect on carbohydrate metabolism in small intestine: The effect of SNT and dietary oils was also determined on the enzymes of carbohydrate metabolism in mucosal homogenates from different experimental rats (Table 5, 6). SNT treatment to control rats caused significant increase in LDH (+40%) and hexokinase (HK) but decrease in MDH (-20%) activities. SNT caused significant decrease in gluconeogenic enzymes, G6Pase and FBPase in mucosal humogenates. The activities of LDH, G6Pase and to some extent FBPase remained significantly higher after SNT treatment to FO and FXO-fed rats compared to control rats, thus reversing the SNT-induced decrease in G6Pase and FBPase activities. SNT-induced decline in MDH activity was also restored to near control values by both FO and FXO-diets. The effect of SNT and dietary FO/FXO was also determined on G6PDH and malic enzyme (ME), sources of NADPH (Table 6). The activity of G6PDH significantly increased (+34%) but that of ME decreased (-21%) by SNT treatment compared to control rats. However, when SNT treatment was given to FO/FXO diet fed rats, SNT induced increase in G6PDH and decrease in ME activities were not observed indicating that both FO and FXO diet significantly prevented the alterations caused by SNT. (d)Effect on carbohydrate metabolism in liver: In addition to the kidney and intestine, the effect of SNT and FO/FXO combinations was also observed in rat liver homogenates. SNT treatment significantly increased the activities of LDH 73

(+62%), HK (+43%) and G6PDH (+30%) whereas decreased G6Pase, FBPase and ME activities in the liver (Table 5, 6). When SNT treatment was extended to FO-fed rats, SNT-induced decrease of metabolic enzyme activities was significantly prevented by FO and FXO diets alike except HK and MDH, whose activities remained significantly higher than control values. Â Effect of dietary fish oil and flaxseed oil on SNT induced alterations on marker enzymes of BBM and lysosomes in cortical/medullary homogenates and in isolated BBMV: To assess the structural integrity of certain organelles e.g., plasma membrane (BBM) and lysosomes, the effect of SNT alone and in combination with FO or FXO diets was determined on biomarker enzymes of BBM and lysosomes in the homogenates of renal cortex and medulla and isolated BBM preparations from renal cortex. a) Effect of SNT alone and with FO or FXO diet on biomarkers of BBM and lysosomes in cortical/medullary homogenates: The activites of DONDOLQHSKRVSKDWDVH$ON3DVH Ȗ-glutamyl transpeptidase (GGTase) and leucine aminopeptidase (LAP) and acid phosphatase were determined under different experimental conditions in the homogenates of renal cortex and medulla (Table 7). SNT treatment to control rats caused significant reduction in the specific activities of AlkPase (-35%), GGTase (-57%) and LAP (-56%) in cortical homogenate. The prior feeding of FO or FXO diet with SNT treatment prevented SNT elicited decrease in BBM enzyme activities. As can be seen from the data SNT induced decrease in BBM enzyme activities were similarly prevented by FO or FXO diet. The activity of acid phosphatase (ACPase) was also decreased (-54%) by SNT in cortical homogenates and FO/FXO diet was able to prevent the decrease in enzyme activity in a similar manner (Table 7). The activites of BBM enzymes similar to the cortex were also lowered in the medulla by SNT administration although to a lower extent than observed with cortex (Table 7). The consumption of FO/FXO enriched diet in combination with SNT treatment resulted in the reversal of SNT induced decrease in AlkPase (-19%), GGTase (-33%) and LAP (-42%) in the medulla. The activity of ACPase was not affected by SNT and SNT+FO. However, it was found to be lower in SNT+FXO rats (-18%) than in control rats.

74

Table 4: Effect of Fish oil (FO) and Flaxseed oil (FXO) on activities of G6Pase, FBPase, G6PDH and ME in homogenates of a) cortex and b) medulla with SNT treatment. Enzyme

G6Pase

FBPase

G6PDH

ME





Control

0.56 ± 0.02

1.91 ± 0.02

0.34 ± 0.02

1.42 ± 0.63

SNT

0.33 ± 0.07* (-41%)

1.57 ± 0.08* (-18%)

0.48 ± 0.02* (+42%)

1.15 ± 0.71* (-19%)

FOSNT

0.65 ± 0.08* (+16%)

1.82 ± 0.41 (-5%)

0.38 ± 0.77 (+11%)

1.55 ± 0.67 (+9%)

FXOSNT

0.68 ± 0.11* (+21%)

1.74 ± 0.23 (-9%)

0.39 ± 0.01* (+15%)

1.335 ± 0.34 (-6%)

1.61 ± 0.11

0.25 ± 0.03

1.02 ± 0.21

1.21 ± 0.05* (-25%)

0.31 ± 0.02* (+24%)

0.89 ± 0.01* (-13%)

1.84 ± 0.12 (+14%)

0.24 ± 0.09 (-4%)

0.94 ± 0.87 (-8%)

1.51 ± 0.86 (-6%)

0.20 ± 0.99 (-20%)

0.99 ± 0.71 (-3%)

Groups a) Cortex

b) Medulla Control SNT

FOSNT

FXOSNT

0.80 ± 0.03 0.53 ± 0.24* (-34%) 0.71 ± 0.12 (-11%) 0.86 ± 0.81 (+8%)

Results (specific activities) are Mean ± SEM for five different preparations. *Significantly GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\GLIIHUHQWIURP617DWSE\RQHZD\$129$ Values in parentheses represent percent change from control.

75

Table 6: Effect of Fish oil (FO) and Flaxseed oil (FXO) on activities of G6Pase, FBPase, G6PDH and ME in homogenates of a) intestine and b) liver with SNT treatment.

Enzyme

G6Pase

FBPase

G6PDH

ME





Control

1.81 ± 0.02

2.64 ± 0.12

0.62 ± 0.04

0.73 ± 0.19

SNT

1.62 ± 0.73 (-10%)

2.02 ± 0.79* (-23%)

0.83 ± 0.10* (+34%)

0.58 ± 0.06* (-21%)

FOSNT

1.98 ± 0.23 (+9%)

2.82 ± 0.56 (+7%)

0.58 ± 0.77 (-6%)

0.85 ± 0.62 (+16%)

FXOSNT

2.18 ± 0.41* (+20%)

2.39 ± 0.23 (-10%)

0.56 ± 0.01* (-10%)

0.77 ± 0.54 (+5%)

Control

0.56 ± 0.09

1.21 ± 0.03

0.37 ± 0.03

1.22 ± 0.24

SNT

0.39 ± 0.02* (-30%)

0.48 ± 0.02* (+30%)

1.35 ± 0.18 (-11%)

FOSNT

0.71 ± 0.12 * (+27%)

0.34 ± 0.34 (-8%)

1.44 ± 0.87 * (+18%)

FXOSNT

0.66 ± 0.81 * (+17%)

0.40 ± 0.79 (+8%)

1.16 ± 0.34 (-5%)

Groups a) Intestine

b) Liver

1.15 ± 0.12 (-5%) 1.44 ± 0.12 * (+19%) 1.53 ± 0.86 * (+26%)

Results (specific activities) are Mean ± SEM for five different preparations. * Significantly different frRPFRQWUROVLJQLILFDQWO\GLIIHUHQWIURP617DWSE\RQHZD\$129$9DOXHV in parentheses represent percent change from control

77

b) Effect of SNT and SNT plus FO and FXO diet on BBM marker enzymes in isolated BBMV: The effect of SNT, FO and FXO on BBM marker enzymes was further analyzed in BBMV preparations isolated from the renal cortex (Table 8, Figure 15). The data shows a similar activity pattern of BBM enzymes as observed in cortical homogenates. However the magnitude of the effects was much more pronounced in BBMV than in cortical homogenates. Activities of AlkPase (-67%), GGTase (-53%) and LAP (-51%) profoundly declined by SNT treatment as compared to control rats. Fish oil (FO) and flaxseed oil (FXO) dietary supplementation similar to the effect in the homogenates appeared to lower the severity of the SNT treatment. SNT-induced decrease in BBM enzyme activities was significantly prevented by dietary FO and to a much greater extent by FXO diet. Â Effect of dietary fish oil and flaxseed oil on SNT induced alterations on marker enzymes of BBM and lysosomes in mucosal homogenates and in isolated BBMV: The effect of SNT, FO and FXO was determined on BBM marker enzymes involved in terminal digestion and absorption in mucosal homogenates (Table 9) and in isolated BBMV preparations (Table 10, Figure 15) from rat small intestine. SNT treatment to control rats caused significant decrease in AlkPase, GGTase, LAP and sucrase in mucosal homogenates. SNT treatment also resulted in the decrease of ACPase activity. When SNT treatment was given to FO/FXO fed rats normalization of the activities of AlkPase, GGTase, and LAP appeared to be normalized whereas activity of sucrase and ACPase was slightly improved by FXO diet but not by FO diet. The effect of SNT, FO and FXO diets was also observed on BBM marker enzymes in isolated BBMV preparations. The results showed a similar activity pattern of various enzymes in the BBMV as observed in mucosal homogenates (Table 10, Figure 16). SNT treatment profoundly decreased the activities of all BBM enzymes but sucrase was elevated. FO/FXO diet caused marked increase in AlkPase and GGTase but LAP and sucrase activities were significantly normalized by both FO/FXO diets. SNT induced decrease in the BBMV and lysosomal marker enzymes was not only reversed by FO/FXO diets but the activities were restored to near control values.

78

Table 7: Effect of Fish oil (FO) and Flaxseed oil (FXO) on biomarkers of BBM and lysosomes in homogenates of a) cortex and b) medulla with SNT treatment. Enzyme Groups

AlkPase

GGTase

LAP

ACPase





Control

25.41 ± 0.51

34.62 ± 0.31

4.55 ± 0.29

12.21 ± 0.45

SNT

16.33 ± 1.24* (-35%)

14.21 ± 0.08* (-57%)

2.01 ± 0.62* (-56%)

5.61 ± 0.312* (-54%)

FOSNT

23.67 ± 0.56 (-7%)

25.36 ± 0.09* (-26%)

5.13 ± 0.48 (+13%)

10.63 ± 0.93 (-13%)

FXOSNT

20.71 ± 0.223* (-18%)

28.45 ± 0.43* (-18%)

3.87 ± 0.09* (-15%)

11.09 ± 1.08 (-9%)

11.121 ± 1.24

18.03 ± 0.05

4.143 ± 0.60

6.53 ± 0.13

SNT

9.038 ± 0.19* (-19%)

12.06 ± 0.22* (-33%)

2.38 ± 1.04* (-42%)

5.99 ± 0.82 (-8%)

FOSNT

13.765 ± 0.93* (+24%)

15.76 ± 0.46* (-13%)

3.34 ± 0.67 (-19%)

6.87 ± 0.77 (+5%)

FXOSNT

10.346 ± 0.38 (-7%)

17.45 ± 0.61 (-3%)

3.985 ± 0.88 (-4%)

5.33 ± 0.34* (-18%)

a) Cortex

b) Medulla Control

Results (specific activities) are Mean ± SEM for five different preparations. *Significantly GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\GLIIHUHQWIURP617DWSE\RQHZD\$129$9DOXHV in parentheses represent percent change from control.

79

Â Effect of dietary fish oil and flaxseed oil on SNT induced alterations on marker enzymes of BBM and lysosomes in liver homogenates: The effect of SNT and FO/FXO diet was also determined on marker enzymes of BBM and lysosomes in liver homogenates (Table 9). SNT treatment to rats resulted in extensive reduction in the specific activity of alkaline phosphatase (AlkPase) whereas other enzymes were not altered significantly in the liver homogenates. The feeding of FO and FXO-diet to SNT treated (FOSNT/FXOSNT) rats not only prevented SNT elicited changes, but the activities of GGTase, AlkPase and LAP were even increased by FO/FXO diet compared to control and much higher compared to SNT-rats. Â Effect of dietary fish oil and flaxseed oil on SNT induced alterations in antioxidant defense parameters in renal cortex and medulla: It is evident that reactive oxygen species generated by various toxicants are important mediators of cellular injury and pathogenesis of various diseases [Walker, 1999]. Primary components of oxidative stress and cellular injury response include elevation of lipid peroxidation (LPO), depletion of GSH and suppression of antioxidant enzymes [Banday et al., 2008b; Priyamvada et al., 2008, 2010]. To ascertain the role of antioxidant system in SNT-induced toxicity, the effect of SNT observed on oxidative stress parameters in various tissues. SNT enhanced lipid peroxidation (LPO) significantly and altered antioxidant enzymes both in cortex and medulla, albeit differently (Table 11). LPO measured in terms of malondialdehyde (MDA levels) significantly enhanced in the cortex (+46%) and medulla (+40%) to similar extent whereas total-SH declined in these tissue (-29% to -33%). SNT treatment caused marked increase in superoxide dismutase (SOD, +58%) and glutathione peroxidase (GSH-Px, +31%) activities but decrease in catalase (-16%) activity in the renal cortex. In medulla however the activity of SOD (-58%) and catalase (-28%) significantly decreased but the activitiy of GSH-Px (+45%) was significantly increased by SNT administration alone. 6LQFH GLHW VXSSOHPHQWHG ZLWK )2 HQULFKHG LQ Ȧ-3 fatty acids has been shown to reduce cyclosporine A and gentamicin induced nephrotoxicity parameters by strengthening antioxidant defense mechanism [Priyamvada et al., 2008, 2010], the protective effect of both FO and FXO HQULFKHGLQȦ-3 fatty acids was determined on SNT-induced oxidative stress parameters.

83

The results indicate that FO and FXO dietary supplementation was able to ameliorate the SNT induced oxidative damage in both renal cortex and medulla. SNT induced increase in LPO and decrease in total-SH was not observed by feeding FO or FXO diet to SNT-treated rats. The activity of SOD and catalase in the cortex and the activity of catalase and GSH-Px in the medulla respectively remained significantly higher in FO/FXO + SNT rats compared to SNT and/or control rats. The results indicate marked protection by both FO/FXO diet against SNT induced oxidative damage to renal tissues. Â Effect of dietary fish oil and flaxseed oil on SNT induced alterations in antioxidant defense parameters in small intestine: The effect of SNT, FO and FXO were also determined on various antioxidant parameters in mucosal homogenates (Table 12). SNT treatment caused marked increase in SOD (+160%), catalase (+55%) and GSH-Px (+28%). These changes were associated with significant increase in lipid peroxidation (+87%) measured in terms of MDA levels and decrease in total-SH (-21%). Both FO and FXO-diet partially prevented SNT-induced increase in SOD, catalase and GSH-Px activities. SNT induced increase in LPO and decrease in SH-content were respectively decreased and increased by FO/FXO diet. Â Effect of dietary fish oil and flaxseed oil on SNT induced alterations in antioxidant defense parameters in rat liver: The effect of SNT, FO and FXO-diet was also determined on various antioxidant parameters in liver homogenates (Table 12). SNT treatment to control rats caused marked increase in SOD (+34%) and GSH-Px (+55%) but decrease in catalase (-20%) activities. These changes were associated with significant increase in lipid peroxidation (+30%) and decrease in total-SH content (-19%). Both FO/FXO-diets prevented SNT-induced increase in SOD and GSH-Px activities whereas catalase was significantly lowered by FO and FXO diet when given in combination with SNT treatment. Total-SH and LPO were also normalized to near control values by both FO/FXO diet to SNT administered rats.

86

Â Effect of dietary fish oil and flaxseed oil on SNT induced alterations in Na-gradient dependent transport of 32Pi in BBMV isolated from renal cortex: The bulk of filtered Pi in the kidney is reabsorbed by its proximal tubule. The Na-gradient dependent {Naoutside (Nao)>Nainside (Nai)} transport of Pi in renal proximal tubule across its luminal brush border membrane (BBM) is an initial and regulatory step. The Pi is transported by secondary active transport mechanism, requires expenditure of energy in the form of ATP that is generated by cellular metabolism. The uptake of

32

Pi was determined in the presence and absence of Na-

gradient in the initial uphill phase (30s) and after equilibrium at 120min in BBM preparations. The rate of concentrative uphill uptake of 32Pi in the presence of a Na-gradient (NaCl in the medium) was markedly decreased by SNT treatment (Table 13). However, the uptake of

32

Pi at the

equilibrium phase (120min) when Nao=Nai was not significantly different between the two groups. Also Na-independent uptake (in the absence of a Na-gradient, when NaCl in the medium was replaced by KCl where Ko> Ki) of 32Pi at 30s and 120min was also not affected by SNT treatment indicating specific alterations only when Na-gradient was present. When SNT treatment was extended to FO and FXO feeding rats, SNT induced decrease in 32Pi transport was not observed. Moreover, the net uptake of

32

Pi remained significantly higher in FOSNT and FXOSNT rats

compared to control rats and much more higher compared to SNT treated rats. The effect was specific only in the presence of Na-gradient. The transport of Pi in the absence of Na-gradient (i.e. when Ko>Ki) and after 120 mins was not affected by any of these treatments. Â Effect of dietary fish oil and flaxseed oil on SNT induced alterations in nitrite concentrations in all tissues: Nitrite concentration is an important parameter in cases where nitrogen metabolites (nitrite, nitrate and NO) are being overproduced. Since nitrite, nitrate and NO are recycled back and forth, the amount of nitrite in the tissues reflects the amount of all these metabolites. As expected, exposure of SNT to rats increased the tissue concentrations of nitrite in all tissues (Figure 17), the accumulation of nitrite being greater in renal cortex (+78%), medulla (+51%) and intestine (+52%). This can be attributed to the fact that most of the ingested nitrate gets converted into nitrite by bacterial action in the gut. Also kidneys are the major filtration site for nitrite. When FO/FXO was given in combination with SNT, SNT-induced increase of tissue nitrite concentrations were

87

ameliorated to near control both by FO and FXO diet. The results indicate that either the nitrite was not accumulated in the tissues or it was degraded and washed out of the body.

90

DISCUSSION I A number of environmental contaminants and pharmaceutical agents have been shown to dramatically alter the structure and function of various tissues and produce multiple adverse effects in the liver, kidney, intestine and heart [Sanchez et al., 2001; Barbier et al., 2006]. These environmental variables are also known to influence the incidence and expression of various diseases including different forms of cancers, cardiovascular and renal diseases [Stochs et al., 2000; Fatima et al., 2004]. Nitrite is a widely used agent in the food processing and colouring industry. When used in combination with salts (in the form of SNT), nitrites serve as important antimicrobial agents in meat to inhibit the growth of bacterial spores that cause botulism, a deadly food-borne illness. Additionally, nitrites have been employed as a vasodilator or a circulatory depressant to relieve smooth muscle spasms and an antidote for treating cyanide poisoning [Karlik et al., 1995]. Although nitrites play vital roles in the normal functioning of the human body when present at their physiological concentrations but excessive exposure of nitrite has been shown to cause certain adverse effects. Perhaps the most common adverse effect of nitrite toxicity in humans is the formation of methemoglobin, a substance that interferes with the ability of red blood cells to carry oxygen when concentrations reach 30-40% of total hemoglobin concentration, which can be fatal [Starodubtseva et al., 1999]. Nitrites have been shown to react with certain amines in food to produce carcinogenic N-nitroso compounds, nitrosamines [Webb et al., 2004; Mirvish et al., 1980] many of which are known to cause cancer. A high dietary intake of nitrite, implicated as a risk factor for human cancer and formation of nitroso compounds in the stomach during inflammation has been directly correlated with the amount of nitrite in food and water [Zaldivar and Robinson, 1973; Hawksworth et al., 1974]. A number of biochemical changes, functional impairments and histopathological lesions have been observed in nitrite treated rats. SNT has also been documented to be mutagenic [Ohnuma et al., 1996] Several approaches utilizing different mechanisms have been attempted to reduce chemical and drug induced nephrotoxicity and other adverse effects. In past few years, much interest has been centered on the role of naturally occurring dietary substances for the control and prevention of various chronic diseases such as cancer and cardiovascular diseases [De Caterina et al., 1994; Kakar et al., 2008]. Omega-3 PUFA and antioxidant rich green tea have been shown to ameliorate many ailments [De Caterina et al., 1994; Kakar et al., 2008]. Several epidemiological and clinical 91

VWXGLHV KDYH VKRZQ WKDW Ȧ-3 fatty acids from marine sources like fish oil (FO) retard the progression of various forms of cancers, depression, arthritis, asthma, cardiovascular and renal disorders [De Caterina et al., 1994;

Kakar et al., @ 5HFHQWO\ Ȧ-3 fatty acids from plant

sources e.g. linolenic acid, showed similar beneficial health effects in growth and development and cardiovascular diseases [Nannicini et al., 2006; Vijaimohan et al., 2006]. Recently it has been GRFXPHQWHG WKDW Ȧ-3 fatty acids also protects against gentamicin and uranyl nitrate-induced nephrotoxicity [Priyamvada et al., 2008, 2010]. Since nitrites are a part of the very environment in which we live, it is virtually impossible to escape from their exposure and to eliminate them completely from the body to reduce the risk of various disorders. However attempts were made to reduce SNT-induced toxicity with the use of dietary vitamin E and selenium [Chow and Hong, 2002]. The present work was designed to study detailed mechanism of SNT-induced nephrotoxic and other adverse alterations and the possibOHSURWHFWLYHHIIHFWVRI)2DQG);2GLHWVHQULFKHGLQȦ-3 PUFA in preventing those alterations caused by SNT exposure. The results of the present study show that SNT administration produced a typical pattern of nephrotoxicity as indicated by marked increased in Scr, BUN accompanied by massive proteinuria, glucosuria and phosphaturia indicating that a considerable damage has occurred to renal tubules and in brush border membrane (BBM) by SNT administration. FO when given for 15 days prior to and during SNT administration prevented SNT induced deleterious effects in various blood/urine parameters and enzyme activities of various pathways. SNT-induced increase of Scr/BUN and hypercholesterolemia, phospholipidosis, proteinuria, glucosuria and phosphaturia were all absent in FO-fed SNT treated rats. SNT induced alterations in various serum and urine parameters were similarly prevented by FXO-diet when given together with SNT. This renoprotective effect was possible by the fact that both FO and FXO alone were able to increase serum phosphate and reduce serum cholesterol, glucose, phospholipids and BUN along with reduction in urinary excretion of Pi and proteins as shown earlier for GM and UN induced adverse effects. The reabsorption of Na+ ions by proximal tubular BBM is considered to be the major function of the kidney because the transport of other ions and various solutes depends directly or indirectly on Na+ reabsorption [Coux et al., 2001]. Since these transport depends on structural integrity of BBM 92

and available energy as ATP which is supplied by various metabolic pathways it is imperative that any alterations to these pathways caused by toxic insult would determine the rate of renal transport functions [Khundmiri et al., 2004; 2005; Fatima et al., @ $V VKRZQ LQ WKH ³5HVXOWV´ WKH activities of various enzymes in glycolysis, TCA cycle, gluconeogenesis and HMP shunt pathway were differentially altered by SNT treatment. SNT caused significant increase of LDH and G6PDH activities and decrease in the activities of MDH (TCA cycle); G6Pase, FBPase (gluconeogenesis) and ME in both renal cortex and medulla albeit to different extent. Although the actual rates of glycolysis and other pathways were not determined, however marked decrease in MDH activity indicates an impaired oxidative metabolism of glucose/fatty acids that will lead to lower ATP production due to mitochondrial dysfunction. These results indicate a shift in the energy metabolism alternatively from aerobic to anaerobic most likely due to mitochondrial dysfunction as evident by significant increase in LDH [Banday et al., 2008a]. The decrease in TCA cycle enzymes may have caused decrease in gluconeogenic enzymes. This can be explained by the fact that lower TCA cycle enzyme activities especially that of MDH will result in lower oxaloacetete production from malate which is required not only for the continuation of TCA cycle but also for gluconeogenesis. Thus SNT-induced toxicity appeared to be primarily due to renal mitochondrial damage similar to genatmicin/urany nitrate/ cisplatin induced toxicity [Priyamvada et al., 2008, 2010; Khan et al., 2009 a, b]. Dietary FO and FXO were also able to prevent SNT-induced decrease/increase in the activities of certain enzymes involved in carbohydrate metabolism both in renal cortex and medulla. These protective effects can be attributed to the fact that FO and FXO alone were shown to effectively enhance the activities of enzymes [Priyamvada et al., 2008, 2010]; resulting in overall improvement of carbohydrate metabolism in renal tissues similar to those reported in rat liver [Yilmaz et al., @ Ȧ-3 PUFA have been shown to coordinately regulate the expression of several enzymes involved in carbohydrate and lipid metabolism [Yoshida et al., 2001]. The structural and functional integrity of BBM and lysosomes was assessed by the status of their respective biomarker enzymes. SNT caused significant decrease in the activities of AlkPase, GGTase and LAP (BBM enzymes) in cortical homogenates and to a much greater extent in BBMV preparations. The decrease in BBM enzyme activities might have occurred due to loss of BBM enzyme and other components into lumen followed by their excessive excretion in the urine as 93

reported earlier [Banday et al., 2008b; Priyamvada et al., 2008, 2010]. A marked reduction observed in Na-dependent transport of 32Pi also supports SNT induced severe damage to BBM of renal proximal tubules. SNT also caused a significant decline in ACPase activity, indicating SNTinduced damage to lysosomes as shown by phospholipidosis [Laurent et al., 1990]. Taken together the present results show that SNT treatment indeed significantly altered the structural integrity and functional capacity of renal proximal tubules and especially its BBM and lysosomes as reflected by marked decrease in the activities of BBM enzymes: AlkPase, GGTase, LAP, Na-dependent transport of 32Pi across renal BBM, lysosomal enzyme ACPase and certain enzymes of carbohydrate metabolism respectively. It has been shown that dietary fatty acids incorporate in the cellular membranes thus altering the structural integrity and functional capacities of plasma membrane and other organelles leading to altered cellular metabolic activities and those of membrane associated enzymes [Thakkar et al., 2000; Carrillo-Tripp and Feller, 2005]. Dietary FO/FXO given together with SNT not only prevented SNT-induced decrease of BBM enzymes but the activity of AlkPase and/or GGTase remained higher in FOSNT and FXOSNT than control rats in the BBM. Both FO/FXO also resulted in significant increase of 32Pi transport across the renal BBM in SNT treated rats. Thus FO and FXO caused reversal of SNT induced alterations because FO/FXO alone have the intrinsic capability to significantly enhance AlkPase and GGTase activity and Pi transport by themselves suggestive of an overall improvement in renal BBM integrity, preventing the loss either by lessening the damage caused by SNT or by increasing the regeneration process or both. It appears from the data that SNT by lowering oxidative metabolism lowers ATP production whereas dietary FO/FXO supplementation increased ATP production by increasing the metabolic activities to support many cellular functions especially in the kidney. Further, FO/FXO-diets increased the UHQDO3LWUDQVSRUWVXJJHVWLYHRIȦ-3 PUFA induced Pi conservation by the kidneys that maintains and/or improves metabolic activities of not only kidney but also of other tissues. In contrast to kidney, SNT exerted differential effects on the enzymes of carbohydrate metabolism and BBM in the intestine and liver. As have been observed earlier in GM/UN/CP induced toxicity [Priyamvada et al., 2008, 2010; Khan et al., 2009 a, b] the activity of sucrase was significantly increased whereas AlkPase, GGTase and LAP decreased by SNT treatment in intestinal and liver homogenates. SNT also caused significant increase in LDH and G6PDH but decreased MDH, ME 94

and ACPase activities in mucosal and liver homogenates. Due to the down-regulation of LDH by SNT, anaerobic glycolysis remained the major source of energy as reported earlier for rat intestine [Farooq et al., 2004]. Thus when SNT treatment was extended to FO/FXO fed rats, the activities of almost all metabolic enzymes further improved. Reactive nitrogen oxide species (RNOS), are produced when NO reacts with either superoxide or oxygen. They were considered to be important mediators of SNT-induced nephrotoxicity [Wink et al., 1996 a, b]. RNOS produce cellular injury and necrosis via several mechanisms including peroxidation of membrane lipids, proteins and DNA [Wink et al., 1996 a, b]. A major cellular defense against free radicals is provided by SOD and catalase, which together convert superoxide radicals first to H2O2 and then to molecular oxygen and water. Other enzymes e.g. GSH-Px use thiol-reducing power of glutathione to reduce oxidized lipids and protein targets of RNOS. The present results show that SNT administration to control rats caused severe damage to renal tissues most likely by RNS generation as apparent by altered activities of above antioxidant enzymes and total-SH content that lead to increased lipid peroxidation (LPO). Enhanced LPO may have resulted in protein damage and inactivation of membrane bound enzymes either through direct attack by free radicals or through chemical modification by its end product (malondialdehyde) [Halliwell and Gutteridge, 1999]. Thus, observed increase in LPO could provide an additional explanation for decreased activities of BBM enzymes. SNT increased the activities of antioxidant enzymes in association with increased LPO and decreased total-SH in small intestine. In the liver, SNT increased LPO with concomitant decrease in total-SH. Further, SNT decreased the activity of catalase but increased the activity of SOD and GSH-Px in liver. The feeding of FO-diet to SNT treated rats prevented SNT-induced augmentation of LPO and suppression of antioxidant enzyme activities. Similar protection was also extended by FXO-diet. The protection against SNT induced renal dysfunction and decline in activities of BBM enzymes by FO and FXO can be attributed to their intrinsic biochemical and natural antioxidant properties. 7KH Ȧ-3 fatty acids possess antioxidant enhancing activity thereby raising the efficiency of antioxidant defence system [Ruiz-Gutierrez et al., 1999; Komatsu et al., 2003]. As can be seen from the results, feeding of FO and FXO alone caused significant normalization of SOD, catalase and GSH-Px activities accompanied by lower LPO values in renal tissues. Thus it 95

DSSHDUV )2 DQG );2 HQULFKHG LQ Ȧ-3 fatty acids enhanced resistance to free radical attack generated by SNT administration. Dietary FO supplementation has also been shown to strengthen antioxidant defense mechanism in the plasma of normal rats [Erdogan et al., 2004]. FXO may have elongated and further desaturated to EPA and/or DHA to exert similar protective effects as reported earlier against cylophasphamide and radiation induced oxidative stress [Bhatia et al., 2006; 2007]. Recently dietary fish oil has been shown to protect against acetaminophen (paracetamol)-induced hepatotoxicity [Speck and Lauterburgh, 1991], ethanol-induced gastric mucosal injury [Leung, 1992] in rats, a number of inflammatory diseases including lupus nephritis [Chandrasekar and Fernandes, 1994], IgA nephropathy [Donadio, 2001] and murine AIDS [Xi and Chen, 2000]. Recent studies also showed partial protection by dietary FO/w-3 fatty acids against GM//UN induced nephrotoxicity [Priyamvada et al., 2008, 2010]. Our results thus support the rationale that w-3 fatty acids enriched FO and FXO may be an effective dietary supplementation in the management of SNT nephrotoxicity and other pathologies in which antioxidant defense mechanism are decelerated. The present biochemical studies clearly demonstrate that SNT administration produces severe nephrotoxicity and causes profound damage to plasma membranes, mitochondria, peroxisomes etc. of renal proximal tubules. The enzymes of oxidative carbohydrate metabolism and gluconeogenesis; BBM, antioxidant defense mechanism and 32Pi transport capacity appeared to be severely affected by SNT treatment. In contrast, FO and FXO, major source of w-3 fatty acids by altering membrane fatty acid composition appeared to affect membrane organization and functions. Both FO and FXO appear to accelerate repair and/or regeneration of injured organelles e.g. mitochondria, peroxisomes and increased activity of TCA cycle and BBM as evident by increased activities of the enzymes of carbohydrate metabolism. Most importantly, by activating endogenous antioxidant defense mechanism FO and FXO provided protection from SNT induced free radical attack. FO/w-3 fatty acids targeted cell cycle regulation/cell signaling may also underlie its protective effects [Yusufi et al., 2003]. The underlying mechanisms by which SNT caused nephro/gastro/hepato-toxicity is not well understood. It has been documented that nitrite directly causes methemoglobinemia by reacting with hemoglobin and produces carcinogenic N-nitroso compounds after its interaction with amines in vivo [Mirvish, 1975]. Nitrite can also produce peroxynitrite (ONOO) by reacting with H2O2 96

[Carmichael et al., 1993]. Eventually nitrite is also converted to NO which can induce genotoxic, mutagenic, carcinogenic and apoptotic effects [Wink et al., 1991; Nguyen et al., 1992]. SNT inhibits enzymes of respiratory chain [Brown and Borutaite, 2002] but enhances glycolytic enzymes [Maletic et al., 2000] and some enzyme antioxidant enzymes viz. catalase and GSH-Px [Brown, 1995; Asahi, 1995] via its conversion to NO. Most of these effects are known to be mediated by RNOS generated from NO. Taken together, in addition to some direct effects, SNT has the ability to cause many more adverse effects by converting to NO as it is both a NO-donor and a NO-metabolite. In contrast, FO and FXO, major source of w-3 fatty acids by altering membrane fatty acid composition appeared to affect membrane organization and functions. Both FO and FXO appear to accelerate repair and/or regeneration of injured organelles e.g. mitochondria, peroxisomes and increased activity of TCA cycle and BBM as evident by increased activities of the enzymes of carbohydrate metabolism. Most importantly, by activating endogenous antioxidant defense mechanism FO and FXO provided protection from SNT induced free radical attack. FO/w-3 fatty acids targeted cell cycle regulation/cell signaling may also underlie its protective effects [Yusufi et al., 2003]. We conclude that SNT elicited deleterious nephrotoxic and other adverse alterations in rat kidney, intestine and liver by causing major damage to mitochondria, lysosomes, and basolateral/brush border membranes as reflected by significant decrease in the activities of specific biomarkers of these intracellular organelles. Most of these effects, if not all can be attributed to excessive NO or other nitrogen oxides production caused by SNT biotransformation. Dietary supplementation with fish/flaxseed oils dietV HQULFKHG LQ Ȧ-3 fatty acids caused significant improvement in nutrition/energy metabolism, BBM integrity, 32Pi transport capacity and antioxidant defenses and thus prevented SNT induced various deleterious effects. Based on our present observations and already known health benefits we propose that dietary fish/flaxseed oil supplementation may provide

a

cushion

for

a

prolonged

therapeutic

option

nephrotoxicity/gastrotoxicity/hepatotoxicity without harmful side effects.

97

against

SNT±induced

PART-II Â HYPOTHESIS II: Fish oil (FO) and IOD[VHHGRLO);2 HQULFKHGLQȦ-3 fatty acids would similarly protect against SNP induced deleterious effects in different rat tissues. Â EXPERIMENTAL DESIGN II: As shown in figure 18, initially four groups (viz Control, SNP, FOSNP and FXOSNP) of rats (10 rats/group) entered the study after acclimatization. They were fed on normal diet (control/SNP) or diet containing 15% fish oil (FOSNP), 15% flaxseed oil (FXOSNP). SNP (1.5 mg/kg bwt/d) was administered intraperitonealy in 0.9% saline daily for 5 days to the groups designated as SNP, FOSNP and FXOSNP. Control animals received an equivalent volume of normal saline for the same period. The rats were sacrificed 24 h after the last injection under light ether anesthesia. Blood was withdrawn from left jugular vein and serum was separated by centrifugation at 2000 x g for 10 min. Urine samples were collected for 4 h in standard metabolic cages a day before the sacrifice of rats. The kidneys, intestine and liver were extracted and processed for the preparation RIKRPRJHQDWHVDQGEUXVKERUGHUPHPEUDQHYHVLFOHV%%09 DVGHVFULEHGLQ³0HWKRGV´%RG\ weights of rats were recorded at the start and completion of experimental procedure. Â RESULTS II: Â Effect of dietary fish oil and flaxseed oil on SNP induced deleterious effects in serum and urine parameters: In general the rats remained clinically well throughout the study. There was no significant difference in daily food intake and body weights between control and other experimental rats (data not shown). SNP treatment to control rats resulted in significant increase in serum creatinine (Scr), blood urea nitrogen (BUN), cholesterol, glucose and phospholipids but decrease in inorganic phosphate (Pi) compared to control rats (Table 1). These changes where associated with profound phosphaturia, proteinuria and glucosuria (Table 2) accompanied by decrease in creatinine clearance (Table 2). Feeding of FO or FXO diet to SNP administered (FOSNP and FXOSNP) rats resulted in significant reversal of various SNP elicited deleterious effects on

98

serum and urine parameters. Both FO and FXO diets prevented SNP-induced increase of Scr, BUN, glucose and cholesterol and decrease of serum Pi (Table 1). FO/FXO diets appear to greatly improve renal functions in a similar manner as evident by the increase of creatinine clearance and decrease in the excretion of protein, phosphate and glucose in the urine (Table 2). Â Effect of dietary fish oil and flaxseed oil on SNP induced alterations in metabolic enzyme activities in renal cortex, medulla, small intestine and liver: The main function of kidney i.e. reabsorption of various ions and solutes depends on the continuous energy supply as ATP which is generated by various metabolic pathways including glycolysis and oxidative metabolism. The acute renal failure produced by toxic insult results in reduced oxygen consumption due to damage caused to mitochondria and other organelles. The effect of SNP, FO, FXO and MO-diets and their combined treatment was determined on the activities of various enzymes of carbohydrate metabolism involved in glycolysis, TCA cycle, gluconeogenesis and HMP shunt pathway in kidney, small intestine and liver (Table 3, 4, 5, 6). (a) Effect on carbohydrate metabolism in renal cortex: As shown in Table 3 and 4, SNP treatment to control rats significantly increased the activity of lactate dehydrogenase (LDH) and hexokinase (HK) but decreased malate dehydrogenase (MDH), glucose-6-phosphatase (G6Pase) and fructose-1, 6-bisphosphatase (FBPase) activities in the renal cortex (Table 3). When SNP treatment was extended to FO and FXO-fed rats, SNP-induced alterations in metabolic enzyme activities were not only prevented by FO and FXO diets, but G6Pase remained significantly higher in FOSNP and FXOSNP compared to control as well as SNP rats in the renal cortex. The effect of FO, FXO and SNP combined was also determined on glucose-6-phosphate dehydrogenase (G6PDH) and NADP-malic enzyme (ME), source of NADPH production needed in various anabolic reactions (Table 4). SNP treatment to control rats significantly increased G6PDH but decreased ME activity. SNP elicited increase of G6PDH activity was normalized to near control values by both FO and FXO diets. SNP induced ME activity decrease was arrested by both FO and FXO diet.

100

Table 3: Effect of Fish oil (FO) and Flaxseed oil (FXO) activities of HK, LDH and MDH in homogenates of a) cortex and b) medulla with SNP treatment.

Enzyme Groups

HK

LDH

MDH




Control

48.51 ± 0.93

28.12 ± 1.86

73.84 ± 1.83

SNP

59.71 ± 0.29* (+23%)

34.84 ± 1.89* (+23%)

51.30 ± 1.18* (-30%)

FOSNP

47.81 ± 0.77 (-1%)

33.11 ± 1.45* (+17%)

81.69 ± 2.21 (+11%)

FXOSNP

54.08 ± 0.92 (+11%)

27.28 ± 1.12 (-4%)

73.66 ± 2.10 (NA)

Control

72.34 ± 1.09

39.65 ± 0.80

74.19 ± 1.42

SNP

99.71 ± 0.67* (+38%)

47.46 ± 3.08* (+20%)

46.20 ± 1.35* (-38%)

FOSNP

80.03 ± 0.83 (+11%)

37.15 ± 1.91 (-7%)

76.86 ± 0.84 (+4%)

FXOSNP

75.41 ± 1.45 (+4%)

42.03 ± 0.89 (+6%)

66.41 ± 1.62 (-10%)

a) Cortex

b) Medulla


103

(b)Effect on carbohydrate metabolism in renal medulla: The activities of LDH, MDH, HK, G6Pase and FBPase in medullary homogenates were similarly affected by SNP treatment (Table 3) as in the cortical homogenates. Feeding of FO and FXO to SNP treated rats prevented SNP induced alterations in various enzyme activities. Similar to cortex, G6PDH activity increased whereas ME activity decreased by SNP in the medulla (Table 4). FO and FXO diet to SNP treated rats decreased G6PDH activity and increased ME activity. (c)Effect on carbohydrate metabolism in small intestine: The effect of SNP and dietary oils was also determined on the enzymes of carbohydrate metabolism in mucosal homogenates from different experimental rats (Table 5, 6). SNP treatment to control rats caused significant increase in LDH (+29%) and hexokinase (HK) but decrease in MDH activities. SNP caused significant decrease in gluconeogenic enzymes, G6Pase and FBPase in the intestine. The activities of LDH, G6Pase and to some extent FBPase remained significantly higher after SNP treatment to FO and FXO-fed rats compared to control rats reversing the SNPinduced decrease in G6Pase and FBPase activities. SNP-induced decline in MDH activity was also restored by both FO and FXO-diets. The effect of SNP and dietary FO/FXO was also determined on G6PDH and malic enzyme (ME), sources of NADPH (Table 6). The activity of G6PDH significantly increased (+29%) but that of ME decreased (-19%) by SNP treatment compared to control rats. However, when SNP treatment was given to FO/FXO diet fed rats, SNP induced increased in G6PDH and decrease in ME activities were not observed indicating that both FO and FXO diet significantly prevented the alterations caused by SNP. (d)Effect on carbohydrate metabolism in liver: In addition to the kidney and intestine, the effect of SNP and FO/FXO combinations was also observed in rat liver homogenates. SNP treatment significantly increased the activities of LDH (+35%) and HK (+59%) whereas decreased G6Pase, FBPase, ME and G6PDH activities in the liver (Table 5, 6). When SNP treatment was extended to FO-fed rats, SNP-induced decrease of metabolic enzyme activities was prevented by FO and FXO alike except HK and MDH, whose activities remained significantly higher than control by FXO.

104

Table 4: Effect of Fish oil (FO) and Flaxseed oil (FXO) on activities of G6Pase, FBPase, G6PDH and ME in homogenates of a) cortex and b) medulla with SNP treatment. Enzyme

G6Pase

FBPase

G6PDH

ME





Control

0.61 ± 0.01

1.73 ± 0.03

0.36 ± 0.01

1.55 ± 0.06

SNP

0.50 ± 0.01* (-18%)

1.35 ± 0.02* (-22%)

0.48 ± 0.01* (+33%)

1.18 ± 0.07* (-24%)

FOSNP

0.69 ± 0.01* (+13%)

2.44 ± 0.10* (+41%)

0.38 ± 0.01 (+5%)

1.58 ± 0.03 (+2%)

FXOSNP

0.63 ± 0.01 (+3%)

1.97 ± 0.05* (+14%)

0.35 ± 0.04 (-3%)

1.49 ± 0.02 (-4%)

Control

0.58 ± 0.01

1.32 ± 0.02

0.27 ± 0.01

1.02 ± 0.01

SNP

0.38 ± 0.01* (-34%)

0.81 ± 0.04* (-38%)

0.39 ± 0.08* (+44%)

0.75 ± 0.01* (-26%)

FOSNP

0.61 ± 0.11 (+5%)

1.40 ± 0.03 (+6%)

0.32 ± 0.01* (+18%)

1.15 ± 0.03 (+13%)

FXOSNP

0.61 ± 0.01 (+5%)

1.59 ± 0.01 (+20%)

0.29 ± 0.03 (+7%)

1.03 ± 0.04 (+1%)

Groups a) Cortex

b) Medulla

Results (specific activities) are Mean ± SEM for five different preparations. *Significantly GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\different from SNP at p < 0.05 by one way ANOVA. Values in parentheses represent percent change from control.

105

Table 5: Effect of Fish oil (FO) and Flaxseed oil (FXO) activities of HK, LDH and MDH in homogenates of a) intestine and b) liver with SNP treatment.

Enzyme

HK

LDH

MDH




Control

179.06 ± 5.09

87.19 ± 1.34

46.31 ± 1.01

SNP

207.51 ± 1.71* (+16%)

112.41 ± 0.89* (+29%)

29.23 ± 0.88* (-37%)

FOSNP

189.27 ± 2.87 (+6%)

86.07 ± 0.92 (-1%)

56.01 ± 1.66* (+21%)

FXOSNP

167.28 ± 1.34 (-7%)

78.04 ± 0.42 (-10%)

50.87 ± 0.96 (+10%)

Control

41.98 ± 0.84

47.09 ± 0.82

29.98 ± 0.68

SNP

66.97 ± 0.97* (+59%)

63.61 ± 1.37* (+35%)

18.77 ± 1.12 (-37%)

FOSNP

45.23 ± 1.03 (+8%)

37.17 ± 0.94* (-21%)

33.92 ± 0.75 (+13%)

FXOSNP

51.88 ± 0.55* (+24%)

50.58 ± 1.04 (+7%)

35.87 ± 0.92* (+20%)

Groups a) Intestine

b) Liver


106

Table 6: Effect of Fish oil (FO) and Flaxseed oil (FXO) on activities of G6Pase, FBPase, G6PDH and ME in homogenates of a) intestine and b) liver with SNP treatment.

Enzyme

G6Pase

FBPase

G6PDH

ME





Control

1.61 ± 0.12

2.18 ± 0.02

0.49 ± 0.01

0.85 ± 0.09

SNP

0.89 ± 0.17 (-45%)

1.62 ± 0.09* (-26%)

0.63 ± 0.09* (+29%)

0.69 ± 0.04* (-19%)

FOSNP

1.74 ± 0.23 (+8%)

2.52 ± 0.16 (+16%)

0.54 ± 0.07 (+10%)

0.78 ± 0.02 (-8%)

FXOSNP

1.88 ± 0.31 (+17%)

2.09 ± 0.03 (-4%)

0.38 ± 0.01* (-22%)

0.81 ± 0.04 (-5%)

Control

0.67 ± 0.05

1.43 ± 0.13

0.38 ± 0.03

1.09 ± 0.14

SNP

0.59 ± 0.02 (-12%)

0.29± 0.02* (-24%)

0.75 ± 0.08* (-31%)

FOSNP

0.76 ± 0.07 (+13%)

1.34 ± 0.12 (-6%)

0.34 ± 0.04 (-11%)

1.24 ± 0.07* (+14%)

FXOSNP

0.70 ± 0.01 (+4%)

1.30 ± 0.06 (-9%)

0.40 ± 0.09 (+5%)

1.16 ± 0.04 (+6%)

Groups a) Intestine

b) Liver

1.09 ± 0.12 (-24%)


107

Â Effect of dietary fish oil and flaxseed oil on SNP induced alterations on marker enzymes of BBM and lysosomes in cortical/medullary homogenates and in isolated BBMV: To assess the structural integrity of certain organelles e.g., plasma membrane (BBM) and lysosomes, the effect of SNP alone and in combination with FO or FXO diets was determined on biomarker enzymes of BBM and lysosomes in the homogenates of renal cortex and medulla and isolated BBM preparations from renal cortex. a) Effect of SNP alone and with FO or FXO diet on biomarkers of BBM and lysosomes in cortical/medullary homogenates: The activites of DONDOLQHSKRVSKDWDVH$ON3DVH Ȗ-glutamyl transpeptidase (GGTase) and leucine aminopeptidase (LAP) and acid phosphatase were determined under different experimental conditions in the homogenates of renal cortex and medulla (Table 7). SNP treatment to control rats caused significant reduction in the specific activities of GGTase (-24%) and LAP (-56%) in cortical homogenates. The prior feeding of FO or FXO diet with SNP treatment prevented SNP elicited decrease in BBM enzyme activities. As can be seen from the data SNP induced decrease in BBM enzyme activities were similarly prevented by FO or FXO diet. The activity of acid phosphatase (ACPase) was increased (+31%) by SNP in cortical homogenates and FO/FXO diet was able to restore the enzyme activity in a similar manner (Table 4). The activites of BBM enzymes similar to the cortex were also lowered in the medulla by SNP administration although to a lower extent (Table 7). The consumption of FO/FXO in combination with SNP treatment resulted in the reversal of SNP induced decrease in GGTase (-29%) and LAP (-48%) in the medulla. The activity of ACPase was increased (+46%) by SNP. b) Effect of SNP and SNP plus FO and FXO diet on BBM markers in isolated BBMV: The effect of SNP, FO and FXO on BBM marker enzymes was further analyzed in BBMV preparations isolated from the renal cortex (Table 8, Figure 19). The data shows a similar activity pattern of BBM enzymes as observed in cortical homogenates. However the magnitude of the effects was much more pronounced in BBMV than in cortical homogenates. Activities of AlkPase (-25%), GGTase (-13%) and LAP (-43%) profoundly declined by SNP treatment as compared to control rats.

108

Fish oil (FO) and flaxseed oil (FXO) dietary supplementation similar to the effect in the homogenates appeared to lower the severity of the SNP treatment. SNP-induced decrease in BBM enzyme activities was significantly prevented by dietary FO and to greater extent by FXO diet. Â Effect of dietary fish oil, flaxseed oil and corn oil on SNP induced alterations on marker enzymes of BBM and lysosomes in mucosal homogenates and in isolated BBMV: The effect of SNP, FO and FXO was determined on BBM marker enzymes involved in terminal digestion and absorption in mucosal homogenates (Table 9) and isolated BBMV preparations (Table 10, Figure 20) from rat small intestine. SNP treatment to control rats caused significant decrease in AlkPase, GGTase and LAP in mucosal homogenates. SNP treatment also resulted in the decrease of ACPase activity. When SNP treatment was given to FO/FXO fed rats normalization of the activities of AlkPase, GGTase, LAP and sucrase whereas ACPase was partially restored to control values. The effect of SNP, FO and FXO diets was also observed on BBM marker enzymes in isolated BBMV preparations. The results showed a similar activity pattern of various enzymes in the BBMV as observed in mucosal homogenates (Table 10, Figure 20). SNP treatment profoundly decreased the activities of all BBM enzymes, FO/FXO caused significant normalization in the values of AlkPase, GGTase and sucrase. SNP induced decrease in the BBMV and lysosomal marker enzymes was not only preserved by FO/FXO diets but the activities were restored to near control values. Â Effect of dietary fish oil and flaxseed oil on SNP induced alterations on marker enzymes of BBM and lysosomes in liver homogenates: The effect of SNP and FO/FXO diet was also determined on marker enzymes of BBM and lysosomes in liver homogenates (Table 9). SNP treatment to rats resulted in extensive reduction in the specific activity of GGTase and LAP whereas AlkPase and ACPase where increased in the liver homogenates. The feeding of FO and FXO-diet to SNP treated (FOSNP/FXOSNP) rats not only prevented SNP elicited changes, but the activities of GGTase and LAP were even increased by FO/FXO diet.

110

Table 8: Effect of Fish oil (FO) and Flaxseed oil (FXO) activities of biomarker enzymes of BBM in cortical BBMV with SNP treatment.

Enzyme

AlkPase

GGTase

LAP




117.38 ± 4.06

195.67 ± 4.09

35.54 ± 2.46

*

20.29 ± 1.03*

Groups

Control

169.61 ± 2.75

87.78 ± 1.92* (-25%)

SNP

106.65 ± 3.88

(-13%)

234.32 ± 0.12

(-43%)

39.04 ± 1.09

FOSNP (-9%)

(+19%)

104.06 ± 4.19

209.08 ± 1.83

(+10%)

43.07 ± 1.27

FXOSNP (-11%)

(+7%)

(+21%)

Results (specific activities) are Mean ± SEM for five different preparations. *Significantly GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\GLIIHUHQWIURP613DWSE\RQHZD\$129$ Values in parentheses represent percent change from control. Control

SNP

FOSNP

FXOSNP

Specific acivity

250 200 150 100 50 0 AlkPase

GGTase

LAP

Figure 19: Effect of FO and FXO on activities of AlkPase, GGTase and LAP in cortical BBM with SNP treatment. $ERYHILJXUHLVGUDZQXVLQJYDOXHVIURP7DEOH5HVXOWVȝPROVPJSURWHLQKRXU DUH0HDQ SEM for five different preparations. *Significantly GLIIHUHQW IURP FRQWURO VLJQLILFDQWO\ different from SNP: at p < 0.05 by one way ANOVA.

111

Â Effect of dietary fish oil and flaxseed oil on SNP induced alterations in antioxidant defense parameters in renal cortex and medulla: Primary components of oxidative stress and cellular injury response include elevation of lipid peroxidation (LPO), depletion of GSH and suppression of antioxidant enzymes [Priyamvada et al., 2008, 2010]. To ascertain the role of antioxidant system in SNP-induced toxicity, the effect of SNP observed on oxidative stress parameters. SNP enhanced lipid peroxidation (LPO) and significantly altered antioxidant enzymes both in cortex and medulla, albeit differently (Table 11). LPO measured in terms of malodialdehyde (MDA levels) significantly enhanced in the cortex (+42%) and medulla (+75%) to similar extent whereas total-SH declined in these tissue (-22% to -37%). SNP treatment caused marked increase in superoxide dismutase (SOD, +40%) but decrease in Glutathione peroxidase (GSH-Px, -29%) and catalase (-18%) activities in the renal cortex. In medulla however the activity of SOD (-87%), catalase (-35%) and GSH-Px (-42%) significantly decreased by SNP administration alone. Since diet supplemHQWHG ZLWK )2 HQULFKHG LQ Ȧ-3 fatty acids has been shown to reduce gentamicin/uranyl nitrate induced nephrotoxicity parameters by strengthening antioxidant defense mechanism [Priyamvada et al., 2008, 2010], the protective effect of both FO and FXO enriched in Ȧ-3 fatty acids was determined on SNP-induced oxidative stress parameters. The results indicate that FO and FXO dietary supplementation was able to ameliorate the SNP induced oxidative damage in both renal cortex and medulla. SNP induced increase in LPO and decrease in total-SH was not observed by feeding FO or FXO diet to SNP-treated rats. The activity of catalase and GSH-Px in the cortex and medulla remained significantly higher in FO/FXO + SNP rats compared to SNP rats. The results indicate marked protection by both FO/FXO diet against SNP induced oxidative damage to renal tissues. Â Effect of dietary fish oil and flaxseed oil on SNP induced alterations in antioxidant defense parameters in rat intestine: The effect of SNP, FO and FXO were determined on various antioxidant parameters in mucosal homogenates (Table 12). SNP treatment caused marked increase in SOD (+46%) but decrease in catalase (-36%) and GSH-Px (-39%). These changes were associated with significant increase in lipid peroxidation (+60%) measured in terms of MDA levels and decrease in total-SH (-23%). Both FO and FXO-diet partially 114

prevented SNP-induced alteration in SOD, catalase and GSH-Px activities. SNP induced increase in LPO and decreased SH-content were prevented by FO/FXO diet. Â Effect of dietary fish oil and flaxseed oil on SNP induced alterations in antioxidant defense parameters in rat liver: The effect of SNP, FO and FXO-diet was determined on various antioxidant parameters in liver homogenates (Table 12). SNP treatment to control rats caused marked decrease in SOD (-21%) and GSH-Px (-43%) activities. These changes were associated with significant increase in lipid peroxidation (+34%) and decrease in total-SH content (-40%). Both FO/FXO-diets prevented SNP-induced increase in SOD and GSH-Px activities whereas catalase was significantly elevated by FO and FXO diet when given in combination with SNP treatment. Total-SH and LPO were also normalized to near control values. Â Effect of dietary fish oil and flaxseed oil on SNP induced alterations in Na-gradient dependent transport of 32Pi in BBMV isolated from renal cortex: The bulk of filtered Pi in the kidney is reabsorbed by its proximal tubule. The Na-gradient dependent {Naoutside (Nao)>Nainside (Nai)} transport of Pi in renal proximal tubule across its luminal brush border membrane (BBM) is an initial and regulatory step. The Pi is transported by secondary active transport mechanism, requires expenditure of energy in the form of ATP that is generated by cellular metabolism. The uptake of

32

Pi was determined in the presence and absence of Na-

gradient in the initial uphill phase (30s) and after equilibrium at 120min in BBM preparations. The rate of concentrative uphill uptake of 32Pi in the presence of a Na-gradient (NaCl in the medium) was markedly decreased by SNP treatment (Table 13). However, the uptake of

32

Pi at the

equilibrium phase (120min) when Nao=Nai was not significantly different between the two groups. Also Na-independent uptake (in the absence of a Na gradient, when NaCl in the medium was replaced by KCl where Ko> Ki) of 32Pi at 30s and 120min was also not affected by SNP treatment indicating specific alterations only when Na-gradient was present. When SNP treatment was extended to FO and FXO feeding rats, SNP induced decrease in 32Pi transport was not observed. Moreover, the net uptake of

32

Pi remained significantly higher in FOSNT and FXOSNT rats

compared to control rats and much more higher compared to SNT treated rats. The effect was specific only in the presence of Na-gradient. The transport

117

Table 13: Effect of Fish Oil (FO) and Flaxseed oil (FXO) on uptake of 32Pi in brush border membrane vesicles (BBMV) from whole cortex with SNP treatment. (A) Na+-gradient dependent uptake (Na o>Nai) (pmol/mg protein) Group

30sec

120min

Control

1698.02 ± 54.72

SNP

725.26 ± 73.71* (-57%)

452.24 ± 24.41 (+27%)

FOSNP

2117.01 ± 36.49 (+25%)

410.73 ± 23.81 (+16%)

FXOSNP

2136.39 ± 59.63 (+26%)

428.14 ± 56.3 (+20%)

354.71 ± 48.97

(B) K+-gradient dependent uptake (Ko>Ki) (pmol/mg protein) Group Control

30sec

120min

142.25 ± 7.53

373.40 ± 2.31

SNP

173.36 ± 12.44 (+21%)

285.49 ± 20.39 (-23%)

FOSNP

162.08 ±21.29 (+14%)

367.39 ± 11.25 (-2%)

FXOSNP

155.86 ±10.08 (+10%)

413.02 ± 10.67 (+11%)

(C) Net Na+-gradient dependent uptake at 30 sec

Group

Na+-gradient dependent uptake

K+-gradient dependent uptake

Net uptake (Na+-K+)

Control

1698.02 ± 54.72

142.25 ± 7.53

1555.77 ± 35.64

SNP

725.26 ± 73.71

173.36 ± 12.44

551.90 ± 9.72* (-65%) 1954.93 ± 18.76 (+26%) 1980.53 ±19.25 (+27%)

FOSNP

2117.01 ± 36.49

162.08 ±21.29

FXOSNP

2136.39 ± 59.63

155.86 ±10.08


6LJQLILFDQWO\GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\GLIIHUHQWIURPSNP at p < 0.05 by one way ANOVA. Values in parentheses represent percent change from control.

118

Table 14: Effect of Fish oil (FO) and Flaxseed oil (FXO) on tissue nitrite concentrations in different tissue homogenates with SNP treatment. Groups Tissues

Cortex

Medulla

Intestine

Liver

(nanomols/gm tissue)




Control

21.72 ± 1.03

8.34 ± 0.63

33.14 ± 0.96

22.71 ± 1.07

SNP

30.36 ± 0.54* (+40%)

14.18 ± 0.83* (+70%)

47.01 ± 1.95* (+42%)

31.58 ± 0.67* (+39%)

FOSNP

24.36 ± 0.68* (+14%)

10.04 ± 0.77* (+20%)

39.06 ± 0.81* (+18%)

25.48 ± 0.42 (+12%)

FXOSNP

23.53 ± 0.93 (+8%)

11.18 ± 0.72* (+34%)

37.53 ± 1.75 (+13%)

26.70 ± 0.26* (+18%)

Results are Mean ± SEM for five different preparations. * Significantly different from control, VLJQLILFDQWO\GLIIHUHQWIURP613DWSE\RQHZD\$129$9DOXHVLQSDUHQWKHVHVUHSUHVHQW percent change from control

SNP

FOSNP

FXOSNP

Nitrite (nanomoles/gm tissue)

Control

Cortex

Medulla

Intestine

Liver

Figure 20: Effect of FO and FXO on tissue nitrite concentration with SNP treatment. Above figure is drawn using values from Table 14. Results (nanomoles/gm tissue) are Mean ± SEM for five different preparations. * Significantly different from control, significantly different from SNT: at p < 0.05 by one way ANOVA

119

of Pi in the absence of Na-gradient (i.e. when Ko>Ki) and after 120 mins was not affected by any of these treatments. Â Effect of dietary fish oil and flaxseed oil on tissue nitrite concentration after SNP treatment in different rat tissues: Nitrite is the predominant metabolite of nitric oxide in vivo therefore, the effect of SNP, FO and FXO on tissue accumulation of nitrite was determined by the Griess reagent assay as described in methods (Table 14; Figure 21). SNP significantly increased nitrite accumulation in all tissues however the effect was maximum in renal medulla. Both FO and FXO reduced the amount of nitrite in rat tissues. Thus we can say that feeding of FO/FXO successfully scavenged excess NO produced by SNP. Further we can also say that by improving renal function, renal clearance of nitrite was enhanced, which did not let nitrite accumulate in the tissues.

120

DISCUSSION II Nitric oxide (NO) has become one of the most important molecules in the area of biological and medical investigation in recent years [Kerwin et al., 1995; Moncada at al. 1991; Nathan, 1992]. NO is a ubiquitous, physiologically active molecule involved in many biological functions and disease states including vasodilation, endotoxic shock, sexual function, neurotransmission, cerebral ischemia and inflammation [Kerwin et al., 1995; Moncada at al. 1991; Nathan, 1992]. Beckman, 1991 and Schimdt et al., 1992have suggested that NO has a double-edged role in specialized tissues and cells. This means that NO is not only an important bioregulatory agent but may also be an endogenous cytotoxin, mutagenand/or carcinogen. Sodium nitroprusside (disodium pentacyanonitrosylferrate(2-) dihydrate, Na2Fe(CN)5NO*2H2O) comprises of a ferrous ion center complexed with five cyanide moieties and a nitrosyl group. SNP spontaneously generates NO, thus functioning as a prodrug [Moncada et al., 1991]. Sodium nitroprusside (SNP) is an NO donor compound that has been used as an anti-hypertensive agent since the 1920s [Scimdt et al., 1992]. Although the data are very limited, it has been shown that long-term use of nitrates as donors of nitric oxide has the potential to induce many pathophysiological conditions. It has been reported that SNP-induced toxicities are associated with increased generation of reactive oxygen species (ROS) [Bastianetto et al., 2000]. SNP has been documented to induce genotoxicity as assessed by the measurement of micronucleated lymphocytes [Andreassi et al., 2001] and to cause DNA strand breaks [Asaka et al., 1997]. SNP generates ROS during the redox cycling of nitroprusside [Ramakrishna et al., 1996]. It is converted to nitrite, NO, cyanide and oxygen free radicals such as superoxide and hydroxyl radicals [Ramakrishna et al., 1996]. Since SNP is a NO donor, it can also be converted to peroxynitrite (ONOO) and thus can cause mutagenesis [Lin et al., 1998]. The potential of SNP to induce apoptosis directly from NO liberation has been well established [Feldman et al., 1997]. Thus NO and its derivatives (e.g. ONOO) can induce a variety of toxic effects including DNA damage, inhibition of DNA synthesis, cell cycle arrest [Wink et al., 1991; Nguyen et al., 1992] and inhibition of respiratory chain [Brown and Borutaite, 2002]. On the other hand SNP has been shown to induce stimulation of glycolysis and glycolytic production of ATP [Maletic et al., 2000]. However it may not be sufficient to compensate decrease in ATP production due to inhibition of

121

oxidative phosphorylation. Hence total energy production was found to be decreased by SNP and SNP-induced NO formation. Thus, the mitochondria appears to be the primary site of SNP effects. Several approaches utilizing different mechanisms have been attempted to reduce chemical and drug induced nephrotoxicity and other adverse effects. In past few years, much interest has been centered on the role of naturally occurring dietary substances for the control and prevention of various chronic diseases such as cancer and cardiovascular diseases. Omega-3 PUFA have been shown to ameliorate many ailments [Simopoulos, 2001; Kromann and Green, 1980; Dyerberg et al., 1975; Bang et al., @5HFHQWVWXGLHVKDYHVKRZQWKDWȦ-3 fatty acids retard the progression of various forms of cancers, depression, arthritis, asthma, cardiovascular and renal disorders [De Caterina et al., 1994; Kakar et al., 2008]. Recently it has been documented tKDWȦ-3 fatty acids protects against gentamicin and uranyl nitrate-induced nephrotoxicity [Priyamvada et al., 2008, 2010]. Attempts were also made to reduce SNP-induced toxicity with the use of resveratrol and red wine due to their antioxidant properties [Bastianetto et al, 2000] and tea [Paquay JBG et al, 2000]. The present work was designed to study detailed mechanism of SNP-induced nephrotoxic alterations and other adverse effects and the possible mechanism by which FO/FXO consumption exerts their protective effect in ameliorating SNP induced nephrotoxicity. The results show that SNP administration produced a typical pattern of nephrotoxicity/hepatotoxicity/gastrotoxicity as indicated by increased Scr, BUN accompanied by massive proteinuria, glucosuria and phosphaturia indicating that significant kidney damage has occurred and that SNP administration has caused alterations in structure and function of renal proximal tubular membrane. FO when given for 15 days prior to and during SNP administration prevented SNP induced deleterious effects in various blood/urine parameters and enzyme activities of various pathways in different rat tissues. SNP-induced increase of Scr/BUN and proteinuria, glucosuria and phosphaturia were all absent in FO-fed SNP treated rats. SNP induced alterations in various serum and urine parameters were similarly prevented by FXO-diet when given together with SNP administration. This renoprotective effect was possible by the fact that both FO and FXO alone were able to increase serum phosphate, serum cholesterol, phospholipids, phosphate and reduce, glucose, and BUN along with reduction in urinary excretion of Pi and proteins as has been described elsewhere [Priyamvada et al., 2008, 2010] 122

The reabsorption of Na+ ions by proximal tubular BBM is considered to be the major function of the kidney because the transport of other ions and various solutes depends directly or indirectly on Na+ reabsorption [Coux et al., 2001]. Since these transport depends on structural integrity of BBM and available energy as ATP which is supplied by various metabolic pathways it is imperative that any alterations to these pathways caused by toxic insult would determine the rate of renal transport functions [Khundmiri et al., 2004; 2005; Fatima et al., 2005@ $V VKRZQ LQ WKH ³5HVXOWV´ WKH activities of various enzymes in glycolysis, TCA cycle, gluconeogenesis and HMP shunt pathway were differentially altered by SNP treatment. SNP caused significant increase of LDH and G6PDH activities and decrease in the activities of MDH (TCA cycle); G6Pase, FBPase (gluconeogenesis) and ME in both renal cortex and medulla albeit to different extent. Although the actual rates of glycolysis and other pathways were not determined, however marked decrease in MDH activity indicates an impaired oxidative metabolism of glucose/fatty acids that will lead to lower ATP production due to mitochondrial dysfunction. These results also indicate a shift in the energy metabolism alternatively from aerobic to anaerobic most likely due to mitochondrial dysfunction as evident by significant increase in LDH [Banday et al., 2008a]. The decrease in TCA cycle enzymes may have caused decrease in gluconeogenic enzymes. This can be explained by the fact that lower TCA cycle enzyme activities especially that of MDH will result in lower oxaloacetete production from malate which is required not only for the continuation of TCA cycle but also for gluconeogenesis. Thus SNP-induced toxicity appeared to be primarily due to renal mitochondrial damage similar to GM/UN/CP induced damage [Priyamvada et al., 2008, 2010; Khan et al., 2009 a, b]. Dietary FO and FXO were also able to prevent SNP-induced decrease/increase in the activities of certain enzymes involved in carbohydrate metabolism both in renal cortex and medulla. These protective effects can be attributed to the fact that both FO and FXO alone were able to effectively enhance the activities of enzymes [Priyamvada et al., 2008, 2010] resulting in overall improvement of carbohydrate metabolism in renal tissues similar to those reported in rat liver [Yilmaz et al., @Ȧ-3 PUFA have been shown to coordinately regulate the expression of several enzymes involved in carbohydrate and lipid metabolism [Yoshida et al., 2001]. Since BBM of renal proximal tubules and other intracellular organelles such as mitochondria and lysosomes are known targets of free radicals [Cronin and Henrich, 1996], the structural and functional integrity was assessed by the status of their respective biomarker enzymes. SNP caused 123

significant decrease in the activities of AlkPase, GGTase and LAP (BBM enzymes) in cortical homogenates and to a much greater extent in BBMV preparations. The decrease in BBM enzyme activities might have occurred due to loss of BBM enzyme and other components into lumen followed by their excessive excretion in the urine as reported earlier [Banday et al., 2008a; Priyamvada et al., 2008, 2010]. A marked reduction observed in Na-dependent transport of 32Pi also supports SNP induced severe damage to BBM of renal proximal tubules. However, SNP caused significant increase in ACPase activity both in the cortex and medulla. Taken together the present results show that SNP treatment indeed significantly altered the structural integrity and functional capacity of renal proximal tubules and particularly BBM and lysosomes as reflected by marked decrease in the activities of BBM enzymes: AlkPase, GGTase, LAP, Na-dependent transport of 32Pi across renal BBM and increase in lysosomal enzyme ACPase and certain specific alteration in the enzyme sof carbohydrate metabolism. It has been shown that dietary fatty acids incorporate in the cellular membranes altering structural integrity and functional capacity of plasma membrane and other organelles leading to altered cellular metabolic activities and those of membrane associated enzymes [Brasitus et al., 1985; Thakkar et al., 2000; Carrillo-Tripp and Feller, 2005]. Dietary FO/FXO given together with SNP not only prevented/retarded SNP-induced decrease of BBM enzymes but the activity of AlkPase and/or GGTase remained higher in FOSNP and FXOSNP than control rats in the BBM. Both FO/FXO also resulted in significant increase of 32Pi transport across the renal BBM in SNP treated rats. Thus FO and FXO caused reversal of SNP induced alterations because FO/FXO alone have the intrinsic capability to significantly enhance AlkPase and GGTase activity and Pi transport by themselves suggestive of an overall improvement in renal BBM integrity by FO/FXO diet thus, preventing the loss either by lessening the damage caused by SNP or by increasing the regeneration process or both [Masters, 1996; Ozgocmen et al., 2000]. It appears from the data that SNP by lowering oxidative metabolism lowers ATP production whereas dietary FO/FXO supplementation increased ATP production by increasing the metabolic activities to support many cellular functions especially in the kidney. Further, FO/FXO-GLHWVLQFUHDVHGWKHUHQDO3LWUDQVSRUWVXJJHVWLYHRIȦ-3 PUFA induced Pi conservation by the kidneys that maintains and/or improves metabolic activities of not only kidney but also of other tissues. In contrast to kidney, SNP exerted differential effects on the enzymes of carbohydrate metabolism and BBM in the intestine and liver. As observed earlier for GM/UN/CP induced toxicity 124

[Priyamvada et al., 2008, 2010; Khan et al.,2009 a, b] the activity of sucrase was significantly increased whereas AlkPase, GGTase and LAP decreased by SNP treatment in intestinal and liver homogenates. SNP also caused significant increase in LDH and G6PDH but decreased MDH, ME and ACPase activities in mucosal and liver homogenates. Due to the down-regulation of LDH by SNP, anaerobic glycolysis remained the major source of energy as reported earlier for rat intestine [Farooq et al., 2004]. Thus when SNP treatment was extended to FO/FXO fed rats, SNP induced decrease/increase were not observed and the activities of almost all metabolic enzymes further improved. Excessive production/accumulation of NO always has the ability to result in the formation of other reactive intermediates of nitrogen. Reactive nitrogen species (RNS) produce cellular injury and necrosis via several mechanisms including peroxidation of membrane lipids, proteins and DNA [Dawson and Dawson, 1996]. Several studies have shown that NO-induced toxicity is engendered by its reaction with superoxide radical to yield the highly toxic ROS peroxynitrite. Peroxynitrite then decomposes to form nitrogen dioxide and the hydroxyl free radical that leads to lipid peroxidation, protein oxidation and DNA damage [Dawson and Dawson, 1996]. A major cellular defense against free radicals is provided by SOD and catalase, which together convert superoxide radicals first to H2O2 and then to molecular oxygen and water. Other enzymes e.g. GSH-Px use thiol-reducing power of glutathione to reduce oxidized lipids and protein targets of RNS. The present results show that SNP administration to control rats caused severe damage to renal tissues most likely by RNS/ROS generation as apparent by altered activities of above antioxidant enzymes and total-SH content that resulted in increased lipid peroxidation. As consisitent with previous studies, catalase and GSH-Px were inhibited by SNP induced NO production [Brown, 1995; Asahi et al., 1995]. Enhanced LPO may result in protein damage and inactivation of membrane bound enzymes either through direct attack by free radicals or through chemical modification by its end product (malondialdehyde) [Halliwell and Gutteridge, 1999]. Thus, observed increase in LPO could provide an additional explanation for decreased activities of BBM enzymes and other proteins. SNP decreased the activities of antioxidant enzymes in association with increased LPO and decreased total-SH in small intestine similarly as observed in renal tissues. However, SNP induced increase in LPO was associated with concomitant decrease in total-SH most likely due to SNP induced decrease in the activity of SOD and GSH-Px in the liver. The feeding of FO-diet to SNP 125

treated rats prevented SNP-induced augmentation of LPO and suppression of antioxidant enzyme activities. Similar protection was also extended by FXO-diet. The protection against SNP induced renal dysfunction and decline in activities of BBM enzymes by FO and FXO can be attributed to their intrinsic biochemical and natural antioxidant properties. 7KH Ȧ-3 fatty acids possess antioxidant enhancing activity thereby raising the efficiency of antioxidant defence system [Ruiz-Gutierrez et al., 1999; Komatsu et al., 2003].

These

observations were recently confirmed by Priyamvada et al., 2008, 2010]. As can be seen from the results, feeding of FO and FXO alone caused significant normalization of SOD, catalase and GSH-Px activities accompanied by lower LPO values in renal tissues. Thus it DSSHDUV )2 DQG );2 HQULFKHG LQ Ȧ-3 fatty acids enhanced resistance to free radical attack generated by SNP administration similiarly as demonstrated in lupus nephritis and other pathologies [Chandrasekar and Fernandes, 1994; Donadio, 2001] LQFOXGLQJ FURKQ¶V GLVHDVH ulcerative colitis and gastric ulceration [Manjari and Das, 2000; Barbosa et al., 2003; Bhattacharya et al., 2006]. Dietary FO supplementation has also been shown to strengthen antioxidant defense mechanism in the plasma of normal rats [Erdogan et al., 2004]. FXO must have elongated and further desaturated to EPA and/or DHA to exert similar protective effects as reported earlier in protection against cylophasphamide and radiation induced oxidative stress [Bhatia et al., 2006; 2007]. Recently dietary fish oil has been shown to protect against acetaminophen (paracetamol)-induced hepatotoxicity [Speck and Lauterburgh, 1991], ethanol-induced gastric mucosal injury [Leung, 1992] in rats, a number of inflammatory diseases including lupus nephritis [Chandrasekar and Fernandes, 1994], IgA nephropathy [Donadio, 2001] and murine AIDS [Xi and Chen, 2000]. Recent reports also showed partial protection by dietary FO/Z-3 fatty acids against GM/cyclosporine/UN induced nephrotoxicity [Ali and Bashir, 1994; Thakkar et al., 2000; Priyamvada et al., 2008, 2010] however, mechanism involved was not studied in detail. Our results thus support the rationale that Z-3 fatty acids enriched FO and FXO may be an effective dietary supplementation in the management of SNP induced nephrotoxicity and other pathologies in which antioxidant defense mechanism are decelerated. The present biochemical studies clearly demonstrate that long-term SNP administration produces severe nephrotoxicity and causes profound damage to plasma membranes, mitochondria, 126

peroxisomes etc. of renal proximal tubules. The enzymes of oxidative carbohydrate metabolism and gluconeogenesis; BBM, antioxidant defense mechanism and 32Pi transport capacity appeared to be severely affected by SNP treatment. Most of the SNP induced effects can be attributed to NO because SNP is a potential NO-donor. In contrast, FO and FXO, major source of Z-3 fatty acids by altering membrane fatty acid composition appeared to affect membrane organization and functions. Both FO and FXO appear to accelerate repair and/or regeneration of injured organelles e.g. mitochondria, peroxisomes and increased activity of TCA cycle and BBM as evident by increased activities of the enzymes of carbohydrate metabolism. Most importantly, by activating endogenous antioxidant defense mechanism FO and FXO provided protection from SNP induced free radical attack. FO/Z-3 fatty acids targeted cell cycle regulation/cell signaling may also underlie its protective effects [Yusufi et al., 2003]. We conclude that while SNP elicited deleterious nephrotoxic/hepatotoxic/gastrotoxic effects by causing severe damage to renal mitochondria, brush border membrane and other organelles and by suppressing antioxidant defense mechanism, dietary supplementation with fish/flaxseed oil HQULFKHGLQȦ-3 fatty acids caused improvement in nutrition/energy metabolism, BBM integrity, 32

Pi transport capacity and antioxidant defenses and thus prevented SNP induced various

deleterious effects. Since SNP is a NO donor, most of these effects can be attributed to NO induced RNOS formation. Based on our present observations and already known health benefits we propose that dietary fish/flaxseed oil supplementation may provide a cushion for a prolonged therapeutic option against SNP nephropathy and other adverse alterations without harmful side effects.

127

and urine parameters. Both FO and FXO diets prevented ARG-induced increase of Scr, BUN, glucose and renal functions in a similar manner as evident by the increase of creatinine clearance and decrease in the excretion of protein, phosphate and glucose in the urine (Table 2). Â Effect of dietary fish oil and flaxseed oil on ARG induced alterations in metabolic enzyme activities in renal cortex, medulla, small intestine and liver: The main function of kidney i.e. reabsorption of various ions and solutes depends on the continuous energy supply as ATP which is generated by various metabolic pathways including glycolysis and oxidative metabolism. The acute renal failure produced by toxic insult results in reduced oxygen consumption due to damage caused to mitochondria and other organelles. The effect of ARG, FO, FXO and MO-diets and their combined treatment was determined on the activities of various enzymes of carbohydrate metabolism involved in glycolysis, TCA cycle, gluconeogenesis and HMP shunt pathway in kidney, small intestine and liver (Table 3, 4, 5, 6). (a) Effect on carbohydrate metabolism in renal cortex: As shown in Table 3 and 4, ARG treatment to control rats significantly increased the activity of lactate dehydrogenase (LDH) and hexokinase (HK) but decreased malate dehydrogenase (MDH), glucose-6-phosphatase (G6Pase) and fructose-1, 6-bisphosphatase (FBPase) activities in the renal cortex (Table 3, 4). When ARG treatment was extended to FO and FXO-fed rats, ARGinduced alterations in metabolic enzyme activities were not only prevented by FO and FXO diets, but MDH remained significantly higher in FOARG compared to control as well as ARG rats in the renal cortex. The effect of FO, FXO and ARG combined was also determined on glucose-6-phosphate dehydrogenase (G6PDH) and NADP-malic enzyme (ME), source of NADPH production needed in various anabolic reactions (Table 4). ARG treatment to control rats significantly increased G6PDH but decreased ME activity. ARG elicited increase of G6PDH activity was normalized to near control values by both FO and FXO diets. ARG induced ME activity decrease was arrested by both FO and FXO diet.

130

Table 3: Effect of Fish oil (FO) and Flaxseed oil (FXO) activities of HK, LDH and MDH in homogenates of a) cortex and b) medulla with L-Arginine (ARG) treatment.

Enzyme Groups

HK

LDH

MDH




Control

88.09 ± 0.76

23.35± 0.45

83.74 ± 0.16

ARG

107.91 ± 0.08* (+23%)

27.69 ± 0.29* (+19%)

63.81 ± 0.15* (-24%)

FOARG

97.47 ± 0.37 (+11%)

25.48 ± 0.35 (+9%)

98.74 ± 0.14* (+18%)

FXOARG

101.28 ± 0.12* (+15%)

24.21 ± 0.28 (+4%)

93.83 ± 0.17 (+12%)

Control

42.63 ± 0.69

37.63 ± 0.12

86.57 ± 0.27

ARG

53.21 ± 0.77* (+25%)

43.92 ± 0.51* (+17)

70.02 ± 0.19* (-19%)

FOARG

40.03 ± 0.70 (-6%)

39.17 ± 0.08 (+4%)

85.25 ± 0.41 (-2%)

a) Cortex

b) Medulla

41.31 ± 0.43 89.21 ± 0.17 39.61 ± 0.16 (-7%) (+3%) (+10%) Results (specific activities) are Mean ± SEM for five different preparations.

6LJQLILFDQWO\GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\GLIIHUHQWIURPARG at p < 0.05 by one way ANOVA. Values in parentheses represent percent change from control. FXOARG

133

(b)Effect on carbohydrate metabolism in renal medulla: The activities of LDH, MDH, HK, G6Pase and FBPase in medullary homogenates were similarly affected by ARG treatment (Table 3, 4) as in the cortical homogenates. Feeding of FO and FXO to ARG treated rats prevented ARG induced alterations in various enzyme activities. Similar to cortex, G6PDH activity increased whereas ME activity decreased by ARG in the medulla (Table 4). FO and FXO diet to ARG treated rats decreased G6PDH activity and increased ME activity (c)Effect on carbohydrate metabolism in small intestine: The effect of ARG and dietary oils was also determined on the enzymes of carbohydrate metabolism in mucosal homogenates from different experimental rats (Table 5, 6). ARG treatment to control rats caused significant increase in LDH (+26%) but decrease in MDH activities. ARG caused significant decrease in gluconeogenic enzyme, G6Pase in the intestine. The activity of G6Pase remained significantly higher after ARG treatment to FO and FXO-fed rats compared to control rats reversing the ARG-induced decrease in its activity. ARG-induced decline in MDH activity was also restored by both FO and FXO-diets. The effect of ARG and dietary FO/FXO was also determined on G6PDH and malic enzyme (ME), sources of NADPH (Table 6). The activity of G6PDH significantly increased (+24%) but that of ME decreased (-39%) by ARG treatment compared to control rats. However, when ARG treatment was given to FO/FXO diet fed rats, ARG induced increased in G6PDH and decrease in ME activities were not observed indicating that both FO and FXO diet significantly prevented the alterations caused by ARG. (d)Effect on carbohydrate metabolism in liver: In addition to the kidney and intestine, the effect of ARG and FO/FXO combinations was also observed in rat liver homogenates. ARG treatment significantly increased the activities of LDH (+41%), HK (+35%) and G6PDH (+50%) whereas decreased G6Pase, MDH and ME activities in the liver (Table 5, 6). When ARG treatment was extended to FO-fed rats, ARG-induced decrease of metabolic enzyme activities was prevented by FO and FXO.

134

Table 4: Effect of Fish oil (FO) and Flaxseed oil (FXO) on activities of G6Pase, FBPase, G6PDH and ME in homogenates of a) cortex and b) medulla with L-Arginine (ARG) treatment.

Enzyme

G6Pase

FBPase

G6PDH

ME





Control

0.66 ± 0.93

1.99 ± 0.62

0.35± 0.84

1.38 ± 1.65

ARG

0.57 ± 0.25* (-14%)

1.61 ± 0.34* (-19%)

0.43 ± 0.99* (+22%)

0.98 ± 0.13* (-28%)

FOARG

0.68 ± 0.01 (+3%)

2.16 ± 0.08 (+9%)

0.33 ± 0.02 (-6%)

1.25 ± 0.63 (-9%)

FXOARG

0.62 ± 0.08 (-7%)

2.26 ± 0.07 (+14%)

0.31 ± 0.01 (-11%)

1.34 ± 0.03 (-3%)

Control

0.69 ± 0.42

1.58 ± 0.80

0.27 ± 0.36

1.02 ± 0.02

ARG

0.56 ± 0.71* (-19%)

1.37 ± 0.45 (-13%)

0.38 ± 0.32* (+40%)

0.68 ± 0.78* (-33%)

FOARG

0.76 ± 0.08 (+10%)

1.54 ± 0.06 (-3%)

0.28 ± 0.02 (+4%)

0.89 ± 0.06 (-13%)

FXOARG

0.73 ± 0.02 (+6%)

1.62 ± 0.05 (+3%)

0.31 ± 0.34 (+15%)

0.86 ± 0.91 (-16%)

Groups a) Cortex

b) Medulla

Results (specific activities) are Mean ± SEM for five different preparations. *Significantly GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\GLIIHUHQWIURP$5*DWSE\RQHZD\$129$ Values in parentheses represent percent change from control.

135

Table 5: Effect of Fish oil (FO) and Flaxseed oil (FXO) activities of HK, LDH and MDH in homogenates of a) intestine and b) liver with L-Arginine (ARG) treatment.

Enzyme

HK

LDH

MDH




Control

188.56 ± 2.09

71.29 ± 0.14

38.41 ± 0.22

ARG

213.50 ± 1.38 (+13%)

89.83 ± 1.13* (+26%)

33.42 ± 0.27* (-13%)

FOARG

179.50 ± 0.37 (-5%)

78.408 ± 0.23 (+10%)

37.41 ± 0.24 (-3%)

FXOARG

201.78 ± 0.93 (+7%)

75.61 ± 0.19 (+6%)

39.13 ± 0.23 (+2%)

Control

49.33 ± 0.94

55.33 ± 0.225

36.98± 0.24

ARG

66.48 ± 1.17* (+35%)

77.82 ± 0.07* (+41)

28.63 ± 0.18* (-22%)

FOARG

45.03 ± 0.53 (-9%)

61.17 ± 0.86 (+11%)

32.65 ± 0.17 (-11%)

Groups a) Intestine

b) Liver

44.61 ± 0.15 31.74 ± 0.18 63.45 ± 0.93 (+15%) (-14%) (-10%) Results (specific activities) are Mean ± SEM for five different preparations.

6LJQLILFDQWO\GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\ different from ARG at p < 0.05 by one way ANOVA. Values in parentheses represent percent change from control. FXOARG

136

Table 6: Effect of Fish oil (FO) and Flaxseed oil (FXO) on activities of G6Pase, FBPase, G6PDH and ME in homogenates of a) intestine and b) liver with L-Arginine (ARG) treatment.

Enzyme

G6Pase

FBPase

G6PDH

ME

(Pmol/mg protein/h)

(Pmol/mg protein/h)

(Pmol/mg protein/h)

(Pmol/mg protein/h)

Control

1.89 ± 0.08

2.43 ± 0.292

0.59 ± 0.98

6.39 ± 0.47

ARG

1.53 ± 0.46* (-19%)

2.13 ± 0.64 (-12%)

0.73 ± 0.02* (+24%)

3.91 ± 0.03* (-39%)

FOARG

1.99 ± 0.78 (+5%)

2.65 ± 0.23 (+9%)

0.58 ± 0.27 (-1%)

6.21 ± 0.23 (-3%)

FXOARG

1.87 ± 0.21 (-1%)

2.77 ± 0.97 (+14%)

0.49 ± 0.34* (-17%)

6.78 ± 0.02 (+6%)

12.84 ± 0.09

45.84 ± 0.18

0.32 ± 0.07

0.93 ± 0.21

*

Groups a) Intestine

b) Liver

Control

*

ARG

9.65 ± 0.01 (-25%)

41.64 ± 0.05 (-9%)

0.48 ± 0.18 (+50%)

0.76 ± 0.09* (-18%)

FOARG

11.39 ± 0.43 (-11%)

46.98 ± 1.42 (+2%)

0.38 ± 0.36* (+19%)

0.89 ± 0.01 (-4%)

FXOARG

11.60 ± 0.88 (-10%)

48.23 ± 0.45 (+5%)

0.31 ± 0.02 (-3%)

1.04 ± 0.02 (+12%)

Results (specific activities) are Mean ± SEM for five different preparations. *Significantly GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\GLIIHUHQWIURP$5*DWSE\RQHZD\$129$9DOXHV in parentheses represent percent change from control.

137

Â Effect of dietary fish oil and flaxseed oil on ARG induced alterations on marker enzymes of BBM and lysosomes in cortical/medullary homogenates and in isolated BBMV: To assess the structural integrity of certain organelles e.g., plasma membrane (BBM) and lysosomes, the effect of ARG alone and in combination with FO or FXO diets was determined on biomarker enzymes of BBM and lysosomes in the homogenates of renal cortex and medulla and isolated BBM preparations from renal cortex. a) Effect of ARG alone and with FO or FXO diet on biomarkers of BBM and lysosomes in cortical/medullary homogenates: The activites of DONDOLQHSKRVSKDWDVH$ON3DVH Ȗ-glutamyl transpeptidase (GGTase) and leucine aminopeptidase (LAP) and acid phosphatase were determined under different experimental conditions in the homogenates of renal cortex and medulla (Table 7). ARG treatment to control rats caused significant reduction in the specific activities of AlkPase (-28%), GGTase (-15%) and LAP (-29%) in cortical homogenate. The prior feeding of FO or FXO diet with ARG treatment prevented ARG elicited decrease in BBM enzyme activities. As can be seen from the data ARG induced decrease in BBM enzyme activities were similarly prevented by FO or FXO diet. The activity of acid phosphatase (ACPase) was also decreased (-17%) by ARG in cortical homogenates and FO/FXO diet was able to prevent the decrease in enzyme activity in a similar manner (Table 7). The activites of BBM enzymes similar to the cortex were also lowered in the medulla by ARG administration (Table 7). The consumption of FO/FXO in combination with ARG treatment resulted in the reversal of ARG induced decrease in GGTase (-22%) and LAP (-40%) in the medulla. The activity of ACPase was also lowered by ARG (-16%). However it was found to be higher in ARG+FO rats (+19%) compared with the control rats. b) Effect of ARG and ARG plus FO and FXO diet on BBM markers in isolated BBMV: The effect of ARG, FO and FXO on BBM marker enzymes was further analyzed in BBMV preparations isolated from the renal cortex (Table 8, Figure 23). The data shows a similar activity pattern of BBM enzymes as observed in cortical homogenates. However the magnitude of the effects was much more pronounced in

138

Table 7: Effect of Fish oil (FO) and Flaxseed oil (FXO) on biomarkers of BBM and lysosomes in homogenates of a) cortex and b) medulla with L-Arginine (ARG) treatment.

Enzyme Groups

AlkPase

GGTase

LAP

ACPase





Control

23.35 ± 0.62

32.43 ± 0.21

4.62 ± 0.22

11.12 ± 0.02

ARG

16.81 ± 0.62* (-28%)

24.93 ± 0.58* (-27%)

3.26 ± 0.12* (-29%)

9.18 ± 0.39* (-17%)

FOARG

26.51± 1.11 (+14%)

48.93 ± 0.62* (+51%)

4.88 ± 0.91* (+6%)

9.82 ± 0.08 (-11%)

FXOARG

28.22 ± 0.60* (+21%)

39.99 ± 0.35 (+23%)

4.512 ± 0.17 (-2%)

10.29 ± 0.75 (-7%)

17.83 ± 0.44

12.11 ± 0.56

4.03 ± 0.60

8.29 ± 0.13

ARG

15.74 ± 0.79 (-12%)

9.67 ± 0.35* (-20%)

2.38 ± 0.64* (-40%)

6.99 ± 0.52* (-16%)

FOARG

23.73 ± 0.53* (+33%)

14.34 ± 0.46* (+18%)

3.78 ± 0.61 (-6%)

9.87 ± 0.77* (+19%)

FXOARG

19.39 ± 0.38 (+9%)

14.85 ± 0.68* (+23%)

3.43 ± 0.80 (-15%)

8.61 ± 0.34 (+4%)

a) Cortex

b) Medulla Control

Results (specific activities) are Mean ± SEM for five different preparations. *Significantly GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\GLIIHUHQWIURP$5*DWSE\RQHZD\$129$9DOXHV in parentheses represent percent change from control.

139

Table 8: Effect of Fish oil (FO) and Flaxseed oil (FXO) activities of biomarker enzymes of BBM in cortical BBMVs with L-Arginine (ARG) treatment. Enzyme Groups

AlkPase

GGTase

LAP




107.64 ± 0.420 57.63 ± 0.22* (-47%)

237.24 ± 2.01 129.63 ± 3.52* (-45%)

33.62 ± 0.29 20.01 ± 0.86* (-40%)

FOARG

113.86 ± 1.04 (+6%)

219.89 ± 2.63 (-7%)

36.63 ± 0.29 (+9%)

FXOARG

124.65 ± 0.21 (+16%)

229.54 ± 1.68 (-3%)

38.01 ± 0.48 (+13%)

Control ARG

Results (specific activities) are Mean ± SEM for five different preparations. * Significantly GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\GLIIHUHQWIURP$5*DWSE\RQHZD\$129$9DOXHV in parentheses represent percent change from control.

Control

ARG

FOSNT

FXOSNT

Specific acivity

300 250 200 150 100 50 0 AlkPase

GGTase

LAP

Figure 23: Effect of FO and FXO on activities of AlkPase, GGTase and LAP in cortical BBM with L-Arginine (ARG) treatment. $ERYHILJXUHLVGUDZQXVLQJYDOXHVIURP7DEOH5HVXOWVȝPROVPJSURWHLQKRXU DUH0HDQ 6(0 IRU ILYH GLIIHUHQW SUHSDUDWLRQV 6LJQLILFDQWO\ GLIIHUHQW IURP FRQWURO VLJQLILFDQWO\ different from ARG: at p < 0.05 by one way ANOVA. 140

BBMV than in cortical homogenates. Activities of AlkPase (-47%), GGTase (-45%) and LAP (40%) profoundly declined by ARG treatment as compared to control rats. Fish oil (FO) and flaxseed oil (FXO) dietary supplementation similar to the effect in the homogenates appeared to lower the severity of the ARG treatment. ARG-induced decrease in BBM enzyme activities was significantly prevented by dietary FO and to greater extent by FXO diet. Â Effect of dietary fish oil and flaxseed oil on ARG induced alterations on marker enzymes of BBM and lysosomes in mucosal homogenates and in isolated BBMV: The effect of ARG, FO and FXO was determined on BBM marker enzymes involved in terminal digestion and absorption in mucosal homogenates (Table 9) and isolated BBMV preparations (Table 10, Figure 24) from rat small intestine. ARG treatment to control rats caused significant decrease in AlkPase, GGTase and LAP in mucosal homogenates. ARG treatment also resulted in the decrease of ACPase activity. When ARG treatment was given to FO/FXO fed rats normalization of the activities of AlkPase, GGTase, LAP and sucrase whereas ACPase was partially restored to control values. The effect of ARG, FO and FXO diets was also observed on BBM marker enzymes in isolated BBMV preparations. The results showed a similar activity pattern of various enzymes in the BBMV as observed in mucosal homogenates (Table 10, Figure 24). ARG treatment profoundly decreased the activities of all BBM enzymes but sucrase which was elevated, FO/FXO caused marked increase in AlkPase and GGTase whereas LAP and sucrase activities were significantly normalized by both FO/FXO diets. ARG induced decrease in the BBMV and lysosomal marker enzymes was not only preserved by FO/FXO diets but the activities were restored to near control values. Â Effect of dietary fish oil and flaxseed oil on ARG induced alterations on marker enzymes of BBM and lysosomes in liver homogenates: The effect of ARG and FO/FXO diet was also determined on marker enzymes of BBM and lysosomes in liver homogenates (Table 9). ARG treatment to rats resulted in extensive reduction in the specific activity of alkaline phosphatase (AlkPase), GGTase LAP and ACPase in the liver homogenates. The feeding of FO and FXO-diet

141

Table 10: Effect of Fish oil (FO) and Flaxseed oil (FXO) activities of biomarker enzymes of BBM in intestinal BBMVs with L-Arginine (ARG) treatment.

Enzyme Groups

AlkPase

GGTase

LAP

Sucrase





Control

37.33 ± 0.34

11.24 ± 0.54

33.24 ± 0.38

273.14 ± 4.05

ARG

20.11 ± 0.13* (-46%)

8.39 ± 1.31* (-25%)

17.29 ± 0.51* (-48%)

297.55 ± 3.36 (+9%)

FOARG

34.63 ± 0.49 (-7%)

11.98 ± 0.83 (+7%)

34.67 ± 0.43 (+4%)

239.63 ± 1.93 (-12%)

FXOARG

38.78 ± 0.98 (+4%)

12.56 ± 0.52 (+11%)

32.01 ± 0.61 (-4%)

233.19 ± 1.87 (-15%)

Results (specific activities) are Mean ± SEM for five different preparations. * Significantly GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\GLIIHUHQWIURP$5*DWSE\RQHZD\$129$9DOXHV in parentheses represent percent change from control.

Control

ARG

FOSNT

FXOSNT

Specific acivity

400 300 200 100 0 AlkPase

GGTase

LAP

Sucrase

Figure 24: Effect of FO and FXO on activities of AlkPase, GGTase and LAP in intestinal BBM with L-Arginine (ARG) treatment. $ERYHILJXUHLVGUDZQXVLQJYDOXHVIURP7DEOH5HVXOWVȝPROVPJSURWHLQKRXU DUH0HDQ ± SEM for five different preparations. * 6LJQLILFDQWO\GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\ different from ARG: at p < 0.05 by one way ANOVA. 143

to ARG treated (FOARG/FXOARG) rats not only prevented ARG elicited changes, but the activities of GGTase and AlkPase were even increased by FO diet. Â Effect of dietary fish oil and flaxseed oil on ARG induced alterations in antioxidant defense parameters in renal cortex and medulla: It is evident that reactive oxygen species generated by various toxicants are important mediators of cellular injury and pathogenesis of various diseases [Walker 1999]. Primary components of oxidative stress and cellular injury response include elevation of lipid peroxidation (LPO), depletion of GSH and suppression of antioxidant enzymes [Priyamvada et al., 2008, 2010]. To ascertain the role of antioxidant system in ARG-induced toxicity, the effect of ARG observed on oxidative stress parameters. ARG enhanced lipid peroxidation (LPO) and significantly altered antioxidant enzymes both in cortex and medulla, albeit differently (Table 11). LPO measured in terms of malodialdehyde (MDA levels) significantly enhanced in the cortex (+43%) and medulla (+51%) to similar extent whereas total-SH declined in these tissue (-16% to -26%). ARG treatment caused marked increase in superoxide dismutase (SOD, +68%) but decrease in Glutathione peroxidase (GSH-Px, -37%) activity in the renal cortex. In medulla however the activity of SOD (-36%) and GSH-Px (-16%) significantly decreased by ARG administration alone. The protective eIIHFWRIERWK)2DQG);2HQULFKHGLQȦ-3 fatty acids was determined on ARGinduced oxidative stress parameters .The results indicate that FO and FXO dietary supplementation was able to ameliorate the ARG induced oxidative damage in both renal cortex and medulla. ARG induced increase in LPO and decrease in total-SH was not observed by feeding FO or FXO diet to ARG-treated rats. The activity of GSH-Px and catalase in the cortex and the activity of catalase and GSH-Px in the medulla respectively remained significantly higher in FO/FXO + ARG rats compared to ARG and/or control rats. The results indicate marked protection by both FO/FXO diet against ARG induced oxidative damage to renal tissues. Â Effect of dietary fish oil and flaxseed oil on ARG induced alterations in antioxidant defense parameters in rat intestine: The effect of ARG, FO and FXO were determined on various antioxidant parameters in mucosal homogenates (Table 12). ARG treatment caused marked increase in SOD (+35%) but decrease in catalase (-22%). These changes were associated with 144

significant increase in lipid peroxidation (+30%) measured in terms of MDA levels and decrease in total-SH (-21%). Both FO and FXO-diet partially prevented ARG-induced alterations in SOD, catalase and GSH-Px activities. ARG induced increase in LPO and decreased SH-content were prevented by FO/FXO diet. Â Effect of dietary fish oil and flaxseed oil on ARG induced alterations in antioxidant defense parameters in rat liver: The effect of ARG, FO and FXO-diet was determined on various antioxidant parameters in liver homogenates (Table 12). ARG treatment to control rats caused marked decrease in catalase (-22%) and GSH-Px (-17%) activities. These changes were associated with significant increase in lipid peroxidation (+35%) and decrease in total-SH content (-21%). Both FO/FXO-diets prevented ARG-induced decrease in catalase and GSH-Px activities when FO and FXO diets were given in combination with ARG treatment. Total-SH and LPO were also normalized to near control values. Â Effect of dietary fish oil and flaxseed oil on ARG induced alterations in Na-gradient dependent transport of 32Pi in BBMV isolated from renal cortex: The bulk of filtered Pi in the kidney is reabsorbed by its proximal tubule. The Na-gradient dependent {Naoutside (Nao)>Nainside (Nai)} transport of Pi in renal proximal tubule across its luminal brush border membrane (BBM) is an initial and regulatory step. The Pi is transported by secondary active transport mechanism, requires expenditure of energy in the form of ATP that is generated by cellular metabolism. The uptake of

32

Pi was percent change from control determined in the

presence and absence of Na-gradient in the initial uphill phase (30s) and after equilibrium at 120min in BBM preparations. The rate of concentrative uphill uptake of 32Pi in the presence of a Na-gradient (NaCl in the medium) was markedly decreased by ARG treatment (Table 13). However, the uptake of 32Pi at the equilibrium phase (120min) when Nao=Nai was not significantly different between the two groups. Also Na-independent uptake (in the absence of a Na gradient, when NaCl in the medium was replaced by KCl where Ko> Ki) of 32Pi at 30s and 120min was also not affected by ARG treatment indicating specific alterations only when Na-gradient was present. When ARG treatment was extended to FO and FXO feeding rats, ARG induced decrease in transport was not observed.

147

32

Pi

Table 13: Effect of Fish Oil (FO) and Flaxseed oil (FXO) on uptake of 32Pi in brush border membrane vesicles (BBMV) from whole cortex with L-Arginine (ARG) treatment. (A) Na+-gradient dependent uptake (Na o>Nai) (pmol/mg protein) Group

30sec

120min

Control

1631.09 ± 17.06

334.78 ± 10.89

ARG

1232.83 ± 75.37* (-25%)

397.21 ± 13.02 (+19%)

FOARG

1711.64 ± 69.79 (+5%)

344.81 ± 7.99 (+3%)

FXOARG

1637.20 ± 61.29 (NA)

368.22 ± 2.41 (+10%)

(B) K+-gradient dependent uptake (Ko>Ki) (pmol/mg protein) Group Control

30sec 191.05 ± 6.93

120min 323.93 ± 2.85

ARG

200.79 ± 3.50 (+5%)

324.65 ± 0.61 (NA)

FOARG

194.76 ± 5.85 (+2%)

345.11 ± 2.55 (+6%)

FXOARG

192.70 ± 0.30 (+1%)

302.28 ± 2.83 (-7%)

(C) Net Na+-gradient dependent uptake at 30 sec

Group

Na+-gradient dependent uptake

K+-gradient dependent uptake

Net uptake (Na+-K+)

Control

1631.09 ± 17.06

191.05 ± 6.93

1440.04 ± 13.76

ARG

1232.83 ± 75.37

200.79 ± 3.50

FOARG

1711.64 ± 69.79

194.76 ± 5.85

FXOARG

1637.20 ± 61.29

192.70 ± 0.30

1032.04 ± 10.09* (-28%) 1516.88 ± 18.93 (+5%) 1444.50 ± 14.75 (NA)


6LJQLILFDQWO\GLIIHUHQWIURPFRQWUROVLJQLILFDQWO\GLIIHUHQWIURPARG at p < 0.05 by one way ANOVA. Values in parentheses represent percent change from control.

148

Table 14: Effect of Fish oil (FO) and Flaxseed oil (FXO) on tissue nitrite concentrations in different tissue homogenates with L-Arginine (ARG) treatment.

Tissues Groups

Cortex

Medulla

Intestine

Liver

(Nanomols/gm tissue)




Control

17.23 ± 0.56

10.12 ± 0.84

35.61 ± 1.17

17.81 ± 0.27

ARG

21.16 ± 0.64* (+23%)

13.68 ± 0.53* (+35%)

45.61 ± 0.92* (+28%)

21.88 ± 0.47* (+23%)

FOARG

18.34 ± 0.61 (+6%)

10.04 ± 0.57 (NA)

38.16 ± 0.84 (+7%)

20.08 ± 1.02 (+13%)

FXOARG

18.57 ± 1.03 (+8%)

11.07 ± 0.32 (+9%)

37.43 ± 1.62 (+5%)

18.83 ± 0.76 (+6%)

Results are Mean ± SEM for five different preparations. * Significantly different from control, VLJQLILFDQWO\ GLIIHUHQW IURP $5* DW S E\ RQH ZD\ $129$ 9DOXHV LQ SDUHQWKHVHV represent percent change from control

Nitrite (nanomoles/gm tissue)

Control

ARG

FOARG

FXOARG

50 40 30 20 10 0 Cortex

Medulla

Intestine

Liver

Figure 24: Effect of FO and FXO on tissue nitrite concentration with L-Arginine (ARG) treatment. Above figure is drawn using values from Table 14. Results (nanomols/gm tissue) are Mean ± 6(0 IRU ILYH GLIIHUHQW SUHSDUDWLRQV 6LJQLILFDQWO\ GLIIHUHQW IURP FRQWURO VLJQLILFDQWO\ different from ARG: at p < 0.05 by one way ANOVA. 149

Â Effect of dietary fish oil and flaxseed oil on tissue nitrite concentration after ARG treatment in different rat tissues: Nitrite concentration is an important parameter in cases where nitrogen metabolites (nitrite, nitrate and NO) are being overproduced. Since nitrite, nitrate and NO are recycled back and forth, the amount of nitrite in the tissues reflects the amount of all three metabolites. As expected, exposure of ARG to rats increased the tissue concentrations of nitrite in all tissues (Figure 25), the accumulation of nitrite being greater in renal medulla (+35%) and intestine (+28%). This can be attributed to the fact that most of the ingested nitrate gets converted into nitrite by bacterial action in the gut. Also kidneys are the major filtration site for nitrite. When FO/FXO was given in combination with ARG, both FO and FXO partially improved the values to near control in renal cortex, medulla and intestine, whereas in the liver complete amelioration was observed (Table 14). When FO/FXO was given in combination with ARG, ARG-induced increase of tissue nitrite concentrations were ameliorated to near control both by FO and FXO diet. The results indicate that either the nitrite was not accumulated in the tissues or it was degraded and washed out of the body.

150

DISCUSSION III L-Arginine (ARG) is a semi-essential, proteinogenic amino acid [Rose, 1938] that was discovered in mammalian protein by Hedin in 1895. Since 1886 it has been recognized as a naturally occurring molecule [Schulze, 1886]. It is involved in two major metabolic pathways, one of them is the urea cycle and the other being nitric oxide synthase (NOS) pathway by which ARG is converted to NO and L-citrulline [Moncada and Higgs, 2002; Sidney and Morris, 2002]. ARG and NO affect the cardiovascular system as endogenous anti-atherogenic molecules that protect the endothelium, modulate vasodilatation, and interact with the vascular wall and circulating blood cells [Boger et al., 1996; Cooke and Dzau, 1997; Cooke, 2004; Li et al., 2006; Napoli et al., 2006]. Together, they can function in the brain as noradrenergic, noncholinergic neuro-transmitters in learning and memory, synaptic plasticity, and neuroprotection [Bohme et al., 1993; Paakkari and Lindsberg, 1995]. They can influence the immune system too by playing a key role in regulating inflammatory processes [Potenza et al., 2001] and redox stress. They can also modulate the metabolism of glucose and insulin activity as natural constituents from diets and regulate neurogenesis [Lobgena et al., 2006]. Alternatively, L-Arginine may enhance the synthesis of potentially detrimental NO from iNOS which countered the neuroprotective effects of iNOS inhibitor in several studies [Luiking et al., 2004; Bronte and Zanovello, 2005]. Increased production of NO has been described in various human cancers, that might contribute to tumour development by favouring neo-angiogenesis, tumour metastasis, resistance to DNA-damaging drugs, and tumour-related immunosuppression [Bogdan, 2001; Xu et al., 2000]. Mizinuma et al., (1984) were the first who studied the effect of an excessive dose of ARG on different tissues of rats. High doses of ARG were linked with pancreatitis along with multi-organ failure [Mizinuma et al., 1984]. The extra pancreatic manifestations such as circulatory, pulmonary, renal and hepatic failure contributed significantly to the morbidity and mortality of pancreatitis [Steinberg and Tenner, 1994; Renner et al., 1985; Karomgani et al., 1992]. It has been reported that high doses of ARG caused widespread morphological changes [Kishino, 1984]. The liver cells showed slight vacuolar degeneration [Hegyi et al., 2004]. The kidney contained eosinophilic compositions in the proximal convoluted tubules [Hegyi et al., 2004]. Adipose tissues around the pancreas showed fat necrosis [Hegyi et al., 2004]. The weight of different organs viz. liver, kidney, spleen and thymus were not changed 151

however, the weight of the pancreas was found to be doubled [Hegyi et al., 2004]. Motoo et al., (2000) examined the effect of ARG on cellular morphology, PAP expression and apoptosis in rat pancreatic acinar cells. The growth of AR-2J cells were inhibited in a dose dependent manner. ARG-induced acute pancreatitis has been studied in much greater detail than in other organs [Hegyi et al., 2004]. It has been shown that oxidative stress development also includes remote organs such as liver and kidney during ARG-induced acute pancreatitis [Hegyi et al., 2004; Czako et al., 2000a]. The MDA concentration was found to be significantly increased in the liver, kidney along with the pancreas. It was associated with significantly reduced endogenous scavengers, Mnn and Cu, Zn-SOD and glutathione peroxidase (GSH-Px) both in the liver and in the kidney [Hegyi et al., 2004]. Catalase was shown to be significantly increased in the liver whereas significantly decreased in the kidney [Hegyi et al., 2004]. The oxidative stress was observed concomitant with histological damage to the liver, pancreas and renal proximal convoluted tubules [Hegyi et al., 2004]. Recently, the use of naturally occurring dietary substances for prevention of various chronic diseases and against various toxicities is gaining interest. Omega-3 fatty acid rich fish oil and polyphenol rich green tea are shown to have several beneficial health effects against various pathologies including cancer, CVD and immune inflammatory disorders [Simopoulos, 1991; Higdon and Frei, 2003; Kakar et al., 2008]. Watkins et al., (2007) have also suggested the WKHUDSHXWLF XVH RI Ȧ-3 PUFA and flavonoids against inflammation that is mediated by environmental pollutants. In view of the above, the present work was undertaken to test the hypothesis whether dietary fish RLO DQG IOD[VHHG RLO ULFK LQ Ȧ-3 FA would be able to protect against ARG induced nephrotoxicity/gastrotoxicity/hepatotoxicity. The effects of ARG treatment and dietary FO and FXO were determined on various nephrotoxicity parameters and on the enzymes of carbohydrate metabolism, brush border membrane and oxidative stress in rat kidney and intestine. $V FDQ EH VHHQ IURP WKH ³5HVXOWV´ D VLQJOH GRVH RI $5* SURGXFHG D W\SLFDO SDWWHUQ RI nephrotoxicity manifested by increased serum creatinine and elevated BUN accompanied by polyuria, proteinuria, glucosuria, phosphaturia and decreased creatinine clearance indicating that a significant damage to kidney has occurred by ARG exposure. Both FO and FXO-diet given 15 d prior to and during ARG administration prevented ARG-induced alterations in various 152

serum/urine parameters. ARG-induced increase of Scr/BUN, phospholipidosis, proteinuria and glucosuria were all absent in FO/FXO fed ARG treated rats. Thus, the results clearly indicated that ARG -induced alterations in serum/urine parameters were prevented similarly by both FO and FXO-diets. The preventive effects of FO and FXO-diet on ARG treated rats were made possible because of increase in serum Pi, glucose and decrease in serum creatinine, BUN by FO/FXO alone. A positive balance of cholesterol and phospholipids (essential membrane components) in serum may facilitate the repair and regeneration of the membrane as required after ARG exposure. The activities of BBM marker enzymes were determined to examine the structural and functional damage caused by ARG to BBM integrity and any protection against ARG actions exerted by FO/FXO-GLHWV$VVKRZQLQWKH³5HVXOWV´WKHDFWLYLWLHVRI$ON3DVH**7DVHDQG/$3PDUNHGO\ decreased by ARG treatment to control rats in renal cortical BBM preparations. However, the activities of AlkPase and GGTase were significantly increased by FO/FXO-diet alone. Thus FO/FXO-diets to ARG treated rats were able to prevent the decrease in the activities of these enzymes. Dietary fatty acids are known to be incorporated in the membrane causing changes in the lipid composition which in turn affect the biophysical characteristics of the membrane and the activities of certain membrane associated enzymes [Stenson et al., 1989; Kaur et al., 1996; Thakkar et al., 2000; Carrillo-Tripp M and Feller, 2005]. Thus by the virtue of their influence on membrane ph\VLRORJ\GLHWDU\ Ȧ-3 PUFA might have played an important role in reducing the PHPEUDQHSHUWXUELQJHIIHFWVRI$5*$VLPLODUSRVLWLYHLPSDFWRIȦ-3 PUFA against cyclosporine A induced changes in membrane structure and function has been reported earlier [Thakkar et al., 2000]. The reabsorption of Na+ by renal proximal tubular BBM is considered to be the major function of the kidney because the transport of other ions and various solutes depend directly or indirectly on Na+ reabsorption [Coux et al., 2001; Khundmiri et al., 2005]. Since these transports depend on the structural integrity of BBM and available energy as ATP provided by various metabolic pathways it is imperative that any alterations to BBM integrity and metabolic pathways caused by toxic insult or otherwise would determine the rate of renal transport functions [Khundmiri et al., @$VVKRZQLQWKH³5HVXOWV´WKHDFWLYLWLHVRIYDULRXVHQ]\PHVLQYROYHGLQJO\FRO\VLV TCA cycle, gluconeogenesis and HMP shunt pathway were significantly affected by ARG treatment albeit differently. Taken together, ARG significantly increased the activity of HK and 153

LDH (glycolysis) whereas the activity of and MDH (TCA cycle) significantly decreased. The marked decrease in MDH activity indicates an impaired oxidative metabolism of glucose and decreased ATP production and hence depressed renal transport function. Although the actual rates of glycolysis and other pathways were not determined, the increased activity of LDH along with simultaneous decline in TCA cycle enzymes suggest a shift in energy production from mitochondria to

anaerobic glycolysis due to mitochondrial damage caused by ARG. The

decreased activities of gluconeogenic enzymes: FBPase and G6Pase may be the result of decrease in TCA cycle enzyme activities. This can be further explained by the fact that the reduced activities of TCA cycle enzymes especially that of MDH may have reduced the production of oxaloacetate from malate which is required not only for the continuation of TCA cycle but also for gluconeogenesis. The oxidative conversion of glucose or glucose-6-phosphate to 6phosphogluconate by G6PDH of HMP shunt pathway, was also lowered in part due to mitochondrial dysfunction by ARG. FO/FXO diet normalized the activities of the metabolic enzymes when they were given to ARG treated rats prevented ARG -induced decrease of various enzymes involved in glucose degradation and production. To further correlate BBM integrity and ATP production by metabolic activity of the kidney the results indicate that ARG caused reduction in the enzyme activities of oxidative metabolism and BBM whereas FO and FXO-diet increased most of them, would lead to decrease/increase production of ATP by ARG and FO/FXO respectively. Thus, both situations would affect transport functions of the kidney. Indeed, Na-dependent transport of Pi across renal cortical brush border membrane was significantly declined by ARG exposure whereas increased by FO/FXO-diets. ARG -induced decrease in Pi transport was also prevented by FO/FXO-diets when given together (FOARG /FXOARG), indicating a positive correlation between ARG and FO/FXO actions on NaPi transport. It has been reported that some toxicants including certain drugs and heavy metals exert their effect by inducing the generation of ROS/RNS. However, redox stress can occur as a result of either increased ROS/RNS generation or decreased antioxidant defense or both. Of them SOD, catalase and GSH-Px constitute the main components of the antioxidant defense system. The present results demonstrate that ARG exposure causes increase in the activities of SOD and decrease in catalase

154

and GSH-Px activity accompanied by increased LPO and decreased total-SH in renal cortex whereas all antioxidant enzymes were depressed in renal medulla. The feeding of both FO and FXO-diets with ARG treatment prevented ARG -induced increase in LPO and decrease in total-SH and antioxidant enzymes activities remained normal in FO/FXOARG treated compared to control rats. Taken together, the present observations clearly demonstrate that FO/FXO-diets were able to prevent many of the adverse alterations caused by ARG in metabolic, BBM and antioxidant enzymes in the kidney. Dietary FO/FXO have been shown to improve antioxidant defenses in a variety of pathophysiologic situations where the oxidant/antioxidant defense mechanism are decelerated [Xi and Chen; 2000; Barbosa et al., 2003; Erdogan et al., 2004; Bhattacharya et al., 2003; 2006; Bhatia et al., 2006; 2007]. Thus the changes in the cell antioxidant status and membrane fatty acid composition could significantly alter the ability of the cell to cope with ARG-induced toxicity. ARG and FO/FXO-diets and their combinations produced similar biochemical effects in the intestine and liver as observed in the kidney. The activity of LDH significantly increased but MDH, G6Pase, FBPase, G6PDH and ME activities significantly decreased by ARG exposure. ARG caused profound decrease in the activities of BBM enzymes: AlkPase, GGTase, and LAP in the intestinal/liver homogenates and BBM preparations. The feeding of FO/FXO-diet significantly increased the activities of all metabolic enzymes which were lowered by ARG exposure. The effect of ARG on metabolic enzymes was largely ameliorated by FO and FXO-diet alike. The activities of BBM enzymes in general remained significantly higher in FO/FXO-ARG treated rats compared to control rats. Effect of ARG and FO/FXO was also observed on various parameters of oxidative stress in rat intestine/liver. ARG caused significant increase in SOD activity but the activities of catalase and GSH-Px markedly decreased in both intestine and liver. LPO, an indicator of tissue injury/oxidative stress was significantly increased whereas total-SH declined. Collectively, these results show that ARG also caused marked oxidative damage to intestinal mucosa and liver and that ARG -induced damage to a large extent was reversed by FO and FXOdiets as reflected by the improvement in oxidative stress parameters. The mechanism by which ARG causes these effects is not fully understood. Accumulating evidence suggests that oxygen free radicals [Varga et al., 1997a, b; Czako et al., 1998, 2000a, b], NO [Takacs et al., 2002a], inflammatory mediators [Takacs et al., 1996, 2002b; Rakonczay et al., 155

2003; Czako et al., 2000b] all have a key role in the development of ARG-mediated alterations. In general NO is generated from ARG by an enzymatic pathway (NO synthase, NOS) originally demonstrated in vascular endothelial cells [Gross and Wolin, 1995]. Under physiological conditions, constitutive NOS (eNOS) results in a low level of NO while in different inflammatory processes inducible NOS (iNOS) produces larger quantities of NO in various cell types. The activity of eNOS was shown to be increased early (24-48 hrs) then declined thereafter at 48 hrs [Hegyi et al., 1997] whereas iNOS activity was found to be increased after 24 and 48 hrs vs control and remained higher thereafter [Hegyi et al., 2004]. It has been suggested that both the NOS forms play important role in the development of ARG-induecd toxic alterations [Hegyi et al., 2004]. It has been demonstrated that NO derived from ARG is a potential source of redox stress. It can be quickly cleared through reacting with superoxide (O2-) to generate peroxynitrite (ONOO) while cells are in a pro-oxidative state. As a highly reactive species, ONOO can react via homolytic or heterolytic cleavage and, generate secondary constituents of nitroxidative stress and highly reactive oxygen/nitrogen species (ROS/RNS) including NO2+, NO2-, and OH radicals. The present results show that ARG caused increase in LPO and decrease in SH content, whereas the activities of antioxidant defense system e.g. SOD, GSH-Px and catalase were perturbed in the kidney, liver and intestine although variably. The results confirmed previous studies which showed that ARG caused morphological changes in the liver, kidney and some other tissues including pancreas which were demonstrated to be mediated by perturbation in the oxidative stress parameters [Hegyi et al., 2004]. In contrast, FO and FXO, major source of w-3 fatty acids by altering membrane fatty acid composition appeared to affect membrane organization and functions. Both FO and FXO appear to accelerate repair and/or regeneration of injured organelles e.g. mitochondria, peroxisomes and increased activity of TCA cycle and BBM as evident by increased activities of the enzymes of carbohydrate metabolism. Most importantly, by activating endogenous antioxidant defense mechanism FO and FXO provided protection from ARG induced RNOS production. We conclude that while ARG elicited deleterious nephrotoxic and other adverse effects by causing severe damage to renal mitochondria, brush border membrane and other organelles and by suppressing antioxidant defense mechanism, dietary supplementation with fish/flaxseed oil HQULFKHGLQȦ-3 fatty acids caused improvement in nutrition/energy metabolism, BBM integrity, 32Pi transport capacity and antioxidant defenses and thus prevented ARG induced various 156

deleterious effects. ARG induced uncontrolled NO production and mediators such as ONOO and other RNOS appear to cause most of ARG-induced deleterious effects and FO/FXO elicited increased antioxidants counter these alterations. Based on our present observations and already known health benefits we propose that dietary fish oil supplementation may provide a cushion for a prolonged therapeutic option against ARG induced various adverse effects without harmful side effects.

157

SUMMARY & CONCLUSION Besides oxygen, carbon and hydrogen, nitrogen is an essential element that constitutes life. It is generally consumed as nitrate (NO3±) or nitrite (NO2±). Changes in patterns of agricultural practice, food processing and industrialization have impacted accumulation of nitrates/nitrites in the environment. Nitrate is abundantly present in the soil and water [Hill, 1991]. It is a valuable fertilizer and is used extensively in the farming practice. Livestock and poultry as well as urban sewage treatment also contribute nitrogenous wastes to the soil. Bacteria present in the soil, water, feed and foods are capable of utilizing these nitrogenous compounds to synthesize nitrates (and nitrites) de novo via heterotrophic nitrification (and nitrate reduction) [Tannenbaum et al., 1978]. Nitrates and nitrites, used in combination with salt, serve as important antimicrobial agents in meat to inhibit the growth of bacterial spores that cause botulism, a deadly food-borne illness. Nitrites are also use as preservatives and for flavoring and fixing color in a number of red meat, poultry, and fish products. Additionally, nitrites have been employed as a vasodilator, or a circulatory depressant to relieve smooth muscle spasm, and an antidote for cyanide poisoning [Nickerson, 1970], and potassium nitrate has been used therapeutically as a diuretic agent. Other precursors or sources of nitrite include N-containing foods, nitrogen containing drugs/chemicals (e.g. nitroglycerin,

an

anti-microbial

agent

and

coronary

dilator;

sodium

nitroprusside,

Na2[Fe(CN)6NO], an anti-hypertensive agent and nitric oxide (NO) [Chow and Hong, 2002]. Nitric oxide (NO), which is synthesized in vivo from L-arginine (ARG), can be oxidized to nitrite or nitrate. NO is also a component of cigarette smoke and automobile exhaust. Thus, man and animals are subjected to significant nitrate and nitrite levels in foods, feed, soil and water as well as those formed in vivo. Nitrites and nitrates formed from nitrogenous sources from microorganisms in saliva, intestine, however, are the major source of human exposure under normal conditions [White, 1975; Tannenbaum et al., 1978]. Nitrates are generally not toxic however, ingestion of large amounts of nitrates was found to be associated with methemoglobinemia, anemia, severe gastroenteritis and nephritis [Dollahite and Rowe, 1974]. Any toxicity linked with nitrate was found to be due to its bioconversion to nitrite by the bacteria in the soil, mouth or stomach [Duncan et al., 1995; Lundberg and Govoni, 2004]. The major health concerns with nitrite are the risk of methemoglobinaemia and its potential carcinogenic, mutagenic and cytotoxic effects [Ger et al., 1996, Magee and Bames, 1956; Spencer 158

et al., 2000; Sun et al., 2006]. A high dietary intake of nitrite has been implicated as a risk factor for human cancer, and formation of nitroso-compounds in the stomach during inflammation is correlated with nitrate in food and water [Ames, 1983; Mirvish, 1995]. A number of biochemical changes, functional impairments and histipathological lesions have been observed in nitrite-treated rats [Abuharfeil et al., 2001]. It has now been documented that reactions of H2O2 with nitrite and of superoxide with NO have a common intermediate-peroxynitrite (ONOO-) [Carmichael et al., 1993], which is responsible for nitration of proteins, DNA strand breaks and mutations, and with interference of protein functions [Sampson et al., 1998]. Moreover in acidic conditions, nitrite is spontaneously decomposed to NO that can be reconverted back to nitrite [Lundberg et al., 2008]. Sodium nitroprusside (SNP) is widely used as an anti-hypertensive agent in clinical practice. SNP has been documented to induce genotoxicity [Andreassi et al., 2001], mutagenictiy [Asaka et al., 1997; Lin et al., 1998] and apopotosis [Feldman et al., 1997]. Recent studies have shown that SNP alters the mitochondrial energy generation processes. It has been shown to irreversibly inhibit the respiratory chain [Brown and Borutaite, 2002] but stimulates glycolysis [Maletic et al., 2000] thereby decreasing total energy production. L-Arginine (ARG) is the substrate for the different nitric oxide synthase (NOS) isoforms which are responsible for the production of NO n vivo. ARG itself has very moderate toxicity, whatever harmful effects it manifests is mainly due to the overproduction of NO. An excessive dose of ARG was linked with pancreatitis along with multiorgan failure [Mizinuma et al., 1984]. It has been reported that high doses of ARG causes widespread morphological changes in the liver, kidney and adipose tissues [Kishino, 1984; Hegyi et al., 2001]. Nitric oxide is a double-edged sword as it can be both cytoprotective and cytotoxic. Produced by mammalian cells at an appropriate magnitude and tempo, it serves as a key signaling molecule in physiological processes as diverse as host-defense, neuronal communication, and vascular regulation [9-11]. On the other hand, excessive and unregulated NO synthesis has been implicated as causal or contributing to pathophysiological conditions including many lethal and debilitating human disorders: arthritis, atherosclerosis, cancer, diabetes, numerous degenerative neuronal diseases, stroke, and myocardial infarction to name a few [Culotta and Koshland, 1992, Gross and Wolin, 1995]. Recently both nitrates and nitrites are recognized as key oxidation products of NO, which can be generated in the tissues by a number of enzymatic and nonenzymatic reactions [Lundberg et al., 2008 review]. Therefore, they should now be viewed as storage pools for NO-like bioactivity, thereby complementing the NO synthase (NOS)-dependent 159

pathway. The recognition of this mammalian nitrogen cycle (nitrate±nitrite±NO) has led researchers to explore the role of nitrate and nitrite in physiological and pathophysiological processes where NO is known to play a key role. The last two decades have witnessed a major drift in the interests of the scientific community towards providing better means to containing the health risks of the human race. The century old chemotherapies against various disorders have never been a success, albeit not a total failure. Such therapies have a major drawback of side effects that give rise to unseen disorders that emerge as a new challenge. From ancient times, the physicians and scholars in Asia have understood that food has both preventive and therapeutic value and is an integral part of human health. The concept of chemo-prevention of certain diseases using naturally occurring substances that can be included in the diet consumed by human populations is gaining attention. Of late, renewed interest and vigor KDVEHHQVKRZHUHGRQWKHUROHRIRPHJDIDWW\DFLGV)LVKRLOHQULFKHGLQ Ȧ-3 PUFA (EPA and DHA) provide one such dietary source of biologically active components that has been shown to be co-preventative and co-therapeutic in a wide variety of ailments [Doughman et al., 2007]. Early clues to the possible importance of unsaturated fatty acids especially polyunsaturated fatty acids (PUFA) came from studies showing that young humans and experimental animals experienced impaired growth whHQ DOO IDWW\ DFLGV ZHUH UHPRYHG IURP WKH GLHW 6PDOO DPRXQWV RI Ȧ- ĮOLQROHQLF DFLG RU Ȧ-6 (linoleic acid) fatty acids prevented that impaired growth and thus were WHUPHG DV ³HVVHQWLDO´ >%XUU DQG %XUU @ +LJKO\ XQVDWXUDWHGSRO\XQVDWXUDWHG )$ (HUFA/PUFA) were relatively found to be several-fold more effective [Turpeinen, 1937]. )XUWKHU VWXGLHV KDYH UHYHDOHG WKDW ERWK Ȧ- DQG Ȧ-6 PUFA have numerous health benefits. +RZHYHU Ȧ-3 PUFA especially from fish/marine foods were found to be more effective in ORZHULQJLQFLGHQFHRIYDULRXVSDWKRORJLHVFRPSDUHGWRȦ-6 PUFA rich diets [Simopoulos, 2001]. This stems from the observations of the different prevalence of coronary heart disease and other chronic diseases (including psoriasis, bronchial asthma, diabetes mellitus and thyrotoxicosis) in the Greenland Inuit (Eskimo) population relative to Western populations [Kromann and Green, @Ȧ-3 PUFA (e.g. EPA and DHA) from marine fish and mammals were indicated as the main dietary factor responsible for such differences [Dyerberg et al., 1975; Bang et al., 1976]. The consumption of fish has now been linked with the rate of depression among various populations. In countries where people eat the least fish the rate of depression is highest, and vice versa 160

[Hibbeln, 1998; Small, 2002]. This correlation holds true across the world. The most dramatic change in what we eat has happened in the past century, with industrialization and development of the food industry, the intake of saturated fat, trans fat and especially Ȧ-6 enriched refined oils have HQRUPRXVO\LQFUHDVHGZKHUHDVWKHFRQVXPSWLRQRIȦ-3 PUFA has considerably declined. These dietary imbalances in fat intake, in fact, are prime cause of modern sufferings like cancer, hypertension, diabetes, depression, cardiovascular and renal disorders. 5ROH RI ILVK RLOȦ-3 PUFA has been extensively investigated and has received a great deal of attention in recent times as therapeutic options in a variety of clinical situations ranging from CVD to hyperlipidemia, cancer, inflammatory and immune disorders, respiratory diseases, depression and diabetes [De Caterina et al., 1994;

Kakar et al., @ /DWHO\ Ȧ-3 PUFA from certain

plants/seeds e.g. walnut, canola and most notably from flaxseed (linseed) showed many similar healWK EHQHILWV DV GHPRQVWUDWHG E\ Ȧ-3 PUFA from fish/fish oil [Nannicini et al., 2006; Vijaimohan et al., 2006]. $OWKRXJKȦ-3 PUFA have been shown as deterrents for environmental pollutants, studies on their possible beneficial effects against drug/chemical induced nephrotoxicity and gastrointestinal toxicity are very limited [Watkins et al., 2007; Priyamvada et al., 2008, 2010]. Considering SURIRXQG EHQHILFLDO KHDOWK HIIHFWV RI Ȧ-3 PUFA from marine or plant foods against various pathologies the present work embodied in this thesis was undertaken to study the detailed mechanisms of NO donors/metabolites induced nephrotoxic alterations and possible mechanism RISURWHFWLRQSUHYHQWLRQRIȦ-3 fatty acids enriched fish oil (FO) and/or flaxseed oil (FXO) in the diets against NO donors/metabolites induced nephropathies. Useful sources of NO for study in animal models are its essential amino acid substrate L-arginine and pharmacological donors like sodium nitroprusside (SNP). To study the role of NO metabolites, sodium nitrite (SNT) was used. In extension, the effects of NO donors/metabolites and FO/FXO were also studied in small intestine and liver, which are primary targets of toxic insult. In view of numerous beneficial health HIIHFWVRIȦ-38)$³ZHK\SRWKHVL]HGWKDWGLHWDU\)2DQG);2HQULFKHGLQȦ-3 PUFA would be able to prevent/reduce NO donors/metabolites induced nephrotoxic and other adverse effects in WKHUDWNLGQH\LQWHVWLQHDQGOLYHU´ To address this hypothesis the present work was undertaken to study the detailed biochemical events/cellular response/mechanism of NO donors/metabolites induced nephrotoxic and other 161

DGYHUVH HIIHFWV LQ UDW NLGQH\ LQWHVWLQH DQG OLYHU 7KH HIIHFWV RI GLHWDU\ Ȧ-3 PUFA was also determined to observe any protection provided by them against NO donors/metabolites nephrotoxicity. The following parameters were determined: (a)

Certain biochemical parameters in serum/urine.

(b)

The activities of certain enzymes of carbohydrate metabolism involved in glycolysis, TCA

cycle, gluconeogenesis and HMP-shunt pathway in renal cortex and medulla, small intestine and liver. (c)

The activities of BBM and lysosomal marker enzymes in renal cortical and mucosal

homogenates and BBM marker enzymes in isolated BBM preparations from renal cortex and intestinal mucosa. (d)

The transport of 32Pi in renal cortical BBM.

(e)

The enzymatic and non-enzymatic parameters of antioxidant defense system in renal cortex

and medulla, small intestine and liver. # THE RESULTS OBTAINED ARE SUMMARIZED AS FOLLOWS: PART I: EFFECT OF FISH OIL (FO) AND FLAXSEED OIL (FXO) ON SODIUM NITRITE (SNT) INDUCED NEPHROTOXIC AND OTHER ADVERSE EFFECTS (a)

Serum/Urine Parameters:

SNT treatment to control rats resulted in significant increase in serum creatinine (Scr) and blood urea nitrogen (BUN), but decrease in cholesterol, glucose, phospholipids and inorganic phosphate (Pi) as compared to control rats. These changes where associated with profound phosphaturia, proteinuria and glucosuria, accompanied by decrease in creatinine clearance. Feeding of FO or FXO diet to SNT administered rats resulted in significant reversal of various SNT elicited deleterious effects on serum and urine parameters. (b) Enzymes of carbohydrate metabolism: (i) SNT treatment to rats significantly increased the activity of lactate dehydrogenase (LDH) but decreased malate dehydrogenase (MDH), hexokinase (HK); glucose-6-phosphatase (G6Pase) and fructose-1, 6-bisphosphatase (FBPase) activities in the renal cortex. When SNT treatment was 162

extended to FO and FXO-fed rats, SNT-induced alterations in metabolic enzyme activities were not only prevented by FO and FXO diets, but G6Pase remained significantly higher in FOSNT and FXOSNT compared to control as well as SNT rats in the renal cortex. SNT treatment to control rats significantly increased G6PDH but decreased ME activity. SNT elicited increase of G6PDH activity was normalized to near control values by both FO and FXO diets. SNT induced ME activity decrease was arrested by both FO and FXO diet. (ii) The activities of LDH, MDH, HK, G6Pase and FBPase in medullary homogenates were similarly affected by SNT treatment (Table 3a) as in the cortical homogenates. Feeding of FO and FXO to SNT treated rats prevented SNT induced alterations in various enzyme activities. Similar to cortex, G6PDH activity increased whereas ME activity decreased by SNT in the medulla (Table 3b). FO and FXO diet to SNT treated rats decreased G6PDH activity and increased ME activity. (iii) The effect of SNT and dietary oils was also determined on the enzymes of carbohydrate metabolism in mucosal homogenates from different experimental rats. SNT treatment to rats caused significant increase in LDH (+40%) and hexokinase (HK) but decrease in MDH activities. SNT caused significant decrease in gluconeogenic enzymes, G6Pase and FBPase in the intestine. The activities of LDH, G6Pase and to some extent FBPase remained significantly higher after SNT treatment to FO and FXO-fed rats compared to control rats reversing the SNT-induced decrease in G6Pase and FBPase activities. SNT-induced decline in MDH activity was also restored by both FO and FXO-diets. The activity of G6PDH significantly increased (+34%) but that of ME decreased (-21%) by SNT treatment compared to control rats. However, when SNT treatment was given to FO/FXO diet fed rats, SNT induced increased in G6PDH and decrease in ME activities were not observed indicating that both FO and FXO diet significantly prevented the alterations caused by SNT. (iv) SNT treatment significantly increased the activities of LDH (+62%), HK (+43%) and G6PDH (+30%) whereas decreased G6Pase, FBPase and ME activities in the liver. When SNT treatment was extended to FO-fed rats, SNT-induced decrease of metabolic enzyme activities was prevented by FO and FXO alike except HK and MDH, whose activities remained significantly higher than control by FXO.

163

(c) Marker enzymes of BBM and lysosomes: L 7KH DFWLYLWHV RI DONDOLQH SKRVSKDWDVH $ON3DVH Ȗ-glutamyl transpeptidase (GGTase) and leucine aminopeptidase (LAP) and acid phosphatase were determined under different experimental conditions in the homogenates of renal cortex and medulla. SNT treatment to control rats caused significant reduction in the specific activities of AlkPase (-35%), GGTase (-57%) and LAP (-52%) in cortical homogenate. The prior feeding of FO or FXO diet with SNT treatment prevented SNT elicited decrease in BBM enzyme activities. The activity of acid phosphatase (ACPase) was also decreased (-45%) by SNT in cortical homogenates and FO/FXO diet was able to prevent the decrease in enzyme activity in a similar manner. The consumption of FO/FXO in combination with SNT treatment resulted in the reversal of SNT induced decrease in AlkPase (-19%), GGTase (-33%) and LAP (-42%) in the medulla. The activity of ACPase was not affected by SNT and SNT+FO. However it was found to be lower in SNT+FXO rats (-18%) compared with the control rats. (ii) The effect of SNT, FO and FXO on BBM marker enzymes was further analyzed in BBMV preparations isolated from the renal cortex. The data shows a similar activity pattern of BBM enzymes as observed in cortical homogenates. However the magnitude of the effects was much more pronounced in BBMV than in cortical homogenates. Activities of AlkPase (-67%), GGTase (-53%) and LAP (-51%) profoundly declined by SNT treatment as compared to control rats. Fish oil (FO) and flaxseed oil (FXO) dietary supplementation similar to the effect in the homogenates appeared to lower the severity of the SNT treatment. SNT-induced decrease in BBM enzyme activities was significantly prevented by dietary FO and to greater extent by FXO diet. (iii) SNT treatment to control rats caused significant decrease in AlkPase, GGTase and LAP in mucosal homogenates. SNT treatment also resulted in the decrease of ACPase activity. When SNT treatment was given to FO/FXO fed rats normalization of the activities of AlkPase, GGTase, LAP and sucrase whereas ACPase was partially restored to control values. The effect of SNT, FO and FXO diets was also observed on BBM marker enzymes in isolated BBMV preparations. The results showed a similar activity pattern of various enzymes in the BBMV as observed in mucosal homogenates. SNT treatment profoundly decreased the activities of all BBM enzymes but sucrase which was elevated, FO/FXO caused marked increase in AlkPase and GGTase whereas LAP and sucrase activities were significantly normalized by both FO/FXO 164

diets. SNT induced decrease in the BBMV and lysosomal marker enzymes was not only preserved by FO/FXO diets but the activities were restored to near control values. (iv) The effect of SNT and FO/FXO diet was also determined on marker enzymes of BBM and lysosomes in liver homogenates. SNT treatment to rats resulted in extensive reduction in the specific activity of alkaline phosphatase (AlkPase) whereas other enzymes were not altered significantly in the liver homogenates. The feeding of FO and FXO-diet to SNT treated (FOSNT/FXOSNT) rats not only prevented SNT elicited changes, but the activities of GGTase, AlkPase and LAP were even increased by FO/FXO diet. (d) Enzymatic and non-enzymatic antioxidant defense parameters: (i) SNT enhanced lipid peroxidation (LPO) and significantly altered antioxidant enzymes both in cortex and medulla, albeit differently. LPO measured in terms of malodialdehyde (MDA levels) significantly enhanced in the cortex (+46%) and medulla (+40%) to similar extent whereas totalSH declined in these tissue (-29% to -37%). SNT treatment caused marked increase in superoxide dismutase (SOD, +58%) and Glutathione peroxidase (GSH-Px, +30%) activities but decrease in catalase (-16%) activity in the renal cortex. In medulla however the activity of SOD (-58%) and catalase (-28%) significantly decresed but in the activitiy of GSH-Px (+45%) was significantly increased by SNT administration alone. FO and FXO dietary supplementation was able to ameliorate the SNT induced oxidative damage in both renal cortex and medulla. (ii) The effect of SNT, FO and FXO were determined on various antioxidant parameters in mucosal homogenates. SNT treatment caused marked increase in SOD (+160%), catalase (+55%) and GSH-Px (+28%). These changes were associated with significant increase in lipid peroxidation (+87%) measured in terms of MDA levels and decrease in total-SH (-21%). Both FO and FXOdiet partially prevented SNT-induced increase in SOD, catalase and GSH-Px activities. SNT induced increase in LPO and decreased SH-content were prevented by FO/FXO diet. (iii) The effect of SNT, FO and FXO-diet was determined on various antioxidant parameters in liver homogenates. SNT treatment to control rats caused marked increase in SOD (+34%) and GSH-Px (+55%) activities but decrease in catalase (-20%). These changes were associated with significant increase in lipid peroxidation (+30%) and decrease in total-SH content (-37%). Both FO/FXO-diets prevented SNT-induced increase in SOD and GSH-Px activities whereas catalase 165

was significantly lowered by FO and FXO diet were given in combination with SNT treatment. Total-SH and LPO were also normalized to near control values. (e) Na+ dependent transport of 32Pi in the BBMV isolated from renal cortex: The rate of concentrative uphill uptake of

32

Pi in the presence of a Na-gradient (NaCl in the

medium) was markedly decreased by SNT treatment. However, the uptake of

32

Pi at the

equilibrium phase (120 min) when Nao=Nai was not significantly different between the two groups. Also Na-independent uptake (in the absence of a Na gradient, when NaCl in the medium was replaced by KCl where Ko> Ki) of

32

Pi at 30s and 120min was also not affected by SNT

treatment indicating specific alterations only when Na-gradient was present. When SNT treatment was extended to FO and FXO feeding rats, SNT induced decrease in

32

Pi transport was not

observed. (f) Tissue concentration of nitrite: Exposure of SNT to rats increased the tissue concentrations of nitrite in all tissues, the accumulation of nitrite being greater in renal cortex (+78%) and intestine (+52%). When FO/FXO was given in combination with SNT, both FO and FXO partially improved the values to near control in renal cortex, medulla and intestine, whereas in the liver complete amelioration was observed (Table 14).

; Interpretation: In general, SNT caused severe adverse effects on various serum/urine parameters and on the enzymes of carbohydrate metabolism, BBM and antioxidant defense system in rat kidney and intestine. FO and/or FXO diet similarly protected against GMinduced nephrotoxic and other adverse effects most likely due to their intrinsic biochemical and antioxidant properties.

166

PART II: EFFECT OF FISH OIL (FO) AND FLAXSEED OIL (FXO) ON SODIUM NITROPRUSSIDE (SNP) INDUCED NEPHROTOXIC AND OTHER ADVERSE EFFECTS (a) Serum/urine parameters: SNP treatment to control rats resulted in significant increase in serum creatinine (Scr), blood urea nitrogen (BUN), cholesterol, glucose and phospholipids but decrease in inorganic phosphate (Pi) compared to control rats. These changes where associated with profound phosphaturia, proteinuria and glucosuria accompanied by decrease in creatinine clearance (Table 2). Feeding of FO or FXO diet to SNP administered (FOSNP and FXOSNP) rats resulted in significant reversal of various SNP elicited deleterious effects on serum and urine parameters. (b) Enzymes of carbohydrate metabolism: (i) SNP increased the activity of lactate dehydrogenase (LDH) and hexokinase (HK) but decreased malate dehydrogenase (MDH), glucose-6-phosphatase (G6Pase) and fructose-1, 6-bisphosphatase (FBPase) activities in the renal cortex. When SNP treatment was extended to FO and FXO-fed rats, SNP-induced alterations in metabolic enzyme activities were prevented by FO and FXO diets alike. SNP treatment to control rats significantly increased G6PDH but decreased ME activity. SNP elicited increase of G6PDH activity was normalized to near control values by both FO and FXO diets. SNP induced ME activity decrease was arrested by both FO and FXO diet. (ii) The activities of LDH, MDH, HK, G6Pase and FBPase in medullary homogenates were similarly affected by SNP treatment as in the cortical homogenates. Feeding of FO and FXO to SNP treated rats prevented SNP induced alterations in various enzyme activities. Similar to cortex, G6PDH activity increased whereas ME activity decreased by SNP in the medulla (Table 4). FO and FXO diet to SNP treated rats decreased G6PDH activity and increased ME activity. (iii) SNP treatment to control rats caused significant increase in LDH (+29%) and hexokinase (HK) but decrease in MDH activities. SNP caused significant decrease in gluconeogenic enzymes, G6Pase and FBPase in the intestine. The activities of LDH, G6Pase and to some extent FBPase remained significantly higher after SNP treatment to FO and FXO-fed rats compared to control rats reversing the SNP-induced decrease in G6Pase and FBPase activities. SNP-induced decline in MDH activity was also restored by both FO and FXO-diets. The activity of G6PDH significantly 167

increased (+29%) but that of ME decreased (-19%) by SNP treatment compared to control rats. However, when SNP treatment was given to FO/FXO diet fed rats, SNP induced increased in G6PDH and decrease in ME activities were not observed indicating that both FO and FXO diet significantly prevented the alterations caused by SNP. (iv) SNP treatment significantly increased the activities of LDH (+35%) and HK (+59%) whereas decreased G6Pase, FBPase, ME and G6PDH activities in the liver. When SNP treatment was extended to FO-fed rats, SNP-induced decrease of metabolic enzyme activities was prevented by FO and FXO alike except HK and MDH, whose activities remained significantly higher than control by FXO. (c) Marker enzymes of BBM and lysosomes: (i) SNP treatment to control rats caused significant reduction in the specific activities of GGTase (-24%) and LAP (-56%) in cortical homogenates. The prior feeding of FO or FXO diet with SNP treatment prevented SNP elicited decrease in BBM enzyme activities. The activity of acid phosphatase (ACPase) was increased (+31%) by SNP in cortical homogenates and FO/FXO diet was able to restore the enzyme activity in a similar manner. The activites of BBM enzymes similar to the cortex were also lowered in the medulla by SNP administration although to a lower extent. The consumption of FO/FXO in combination with SNP treatment resulted in the reversal of SNP induced decrease in GGTase (-29%) and LAP (-48%) in the medulla. The activity of ACPase was increased (+46%) by SNP. (ii) Activities of AlkPase (-25%), GGTase (-13%) and LAP (-43%) profoundly declined by SNP treatment as compared to control rats in renal BBM. Fish oil (FO) and flaxseed oil (FXO) dietary supplementation similar to the effect in the homogenates appeared to lower the severity of the SNP treatment. SNP-induced decrease in BBM enzyme activities was significantly prevented by dietary FO and to greater extent by FXO diet. (iii) SNP treatment to control rats caused significant decrease in AlkPase, GGTase and LAP in mucosal homogenates. SNP treatment also resulted in the decrease of ACPase activity. When SNP treatment was given to FO/FXO fed rats normalization of the activities of AlkPase, GGTase, LAP and sucrase whereas ACPase was partially restored to control values. SNP treatment profoundly decreased the activities of all BBM enzymes, FO/FXO caused significant normalization in the 168

values of AlkPase, GGTase and sucrase. SNP induced decrease in the BBMV and lysosomal marker enzymes was not only preserved by FO/FXO diets but the activities were restored to near control values. (iv) SNP treatment to rats resulted in extensive reduction in the specific activity of GGTase and LAP whereas AlkPase and ACPase where increased in the liver homogenates. The feeding of FO and FXO-diet to SNP treated (FOSNP/FXOSNP) rats prevented SNP elicited changes. (d) Enzymatic and non-enzymatic antioxidant defense parameters: (i) SNP enhanced lipid peroxidation (LPO) and significantly altered antioxidant enzymes both in cortex and medulla, albeit differently. LPO measured in terms of malodialdehyde (MDA levels) significantly enhanced in the cortex (+42%) and medulla (+75%) to similar extent whereas totalSH declined in these tissue (-22% to -37%). SNP treatment caused marked increase in superoxide dismutase (SOD, +40%) but decrease in Glutathione peroxidase (GSH-Px, -29%) and catalase (18%) activities in the renal cortex. In medulla however the activity of SOD (-87%), catalase (35%) and GSH-Px (-42%) significantly decreased by SNP administration alone. SNP induced increase in LPO and decrease in total-SH was not observed by feeding FO or FXO diet to SNPtreated rats. The activity of catalase and GSH-Px in the cortex and medulla remained significantly higher in FO/FXO + SNP rats compared to SNP rats. (ii) SNP treatment caused marked increase in SOD (+46%) but decrease in catalase (-36%) and GSH-Px (-39%). These changes were associated with significant increase in lipid peroxidation (+60%) measured in terms of MDA levels and decrease in total-SH (-23%). Both FO and FXOdiet partially prevented SNP-induced alteration in SOD, catalase and GSH-Px activities. SNP induced increase in LPO and decreased SH-content were prevented by FO/FXO diet. (iii) SNP treatment to control rats caused marked decrease in SOD (-21%) and GSH-Px (-43%) activities. These changes were associated with significant increase in lipid peroxidation (+34%) and decrease in total-SH content (-40%). Both FO/FXO-diets prevented SNP-induced increase in SOD and GSH-Px activities whereas catalase was significantly elevated by FO and FXO diet when given in combination with SNP treatment. Total-SH and LPO were also normalized to near control values.

169

(e) Na+ dependent transport of 32Pi in the BBMV isolated from renal cortex: The rate of concentrative uphill uptake of

32


medium) was markedly decreased by SNP treatment (Table 13). However, the uptake of

32

Pi at

the equilibrium phase (120 min) when Nao=Nai was not significantly different between the two groups. When SNP treatment was extended to FO and FXO feeding rats, SNP induced decrease in 32

Pi transport was not observed.

(f) Tissue concentration of nitrite: Nitrite is the predominant metabolite of nitric oxide in vivo therefore. The effect of SNP, FO and FXO on tissue accumulation of nitrite was determined by the Griess reagent assay as described in methods. SNP significantly increased nitrite accumulation in all tissues however the effect was maximum in renal medulla. Both FO and FXO reduced the amount of nitrite in rat tissues.

; Interpretation: SNP treatment caused severe damage to the kidney and intestine as reflected by increase in serum creatinine and BUN and decrease in biomarker BBM enzymes. There appeared to be a shift from aerobic to anaerobic metabolism most likely due to SNP induced mitochondrial damage. SNP also increased oxidative stress as indicated by increased LPO and decreased total-SH levels associated with imbalances in the activities of antioxidant enzymes. FO/FXO-diet prevented/retarded SNP-induced alterations in various biochemical parameters and enzyme activities most likely due to their incorporation in cellular membranes and antioxidant properties.

PART III: EFFECT OF FISH OIL (FO) AND FLAXSEED OIL (FXO) ON L-ARGININE (ARG) INDUCED NEPHROTOXIC AND OTHER ADVERSE EFFECTS (a) Serum/Urine Parameters: ARG treatment to control rats resulted in significant increase in serum creatinine (Scr), blood urea nitrogen (BUN), glucose and phospholipids but decrease in inorganic phosphate (Pi) compared to control rats. These changes where associated with profound phosphaturia, proteinuria and glucosuria accompanied by decrease in creatinine clearance. Feeding of FO or FXO diet to ARG 170

administered (FOARG and FXOARG) rats resulted in significant reversal of various ARG elicited deleterious effects on serum and urine parameters. (b) Enzymes of carbohydrate metabolism: (i) ARG treatment to control rats significantly increased the activity of lactate dehydrogenase (LDH) and hexokinase (HK) but decreased malate dehydrogenase (MDH), glucose-6-phosphatase (G6Pase) and fructose-1, 6-bisphosphatase (FBPase) activities in the renal cortex (Table 3, 4). When ARG treatment was extended to FO and FXO-fed rats, ARG-induced alterations in metabolic enzyme activities were prevented by FO and FXO diets. ARG treatment to control rats significantly increased G6PDH but decreased ME activity. ARG elicited increase of G6PDH activity was normalized to near control values by both FO and FXO diets. ARG induced ME activity decrease was arrested by both FO and FXO diet. (ii) The activities of LDH, MDH, HK, G6Pase and FBPase in medullary homogenates were similarly affected by ARG treatment as in the cortical homogenates. Feeding of FO and FXO to ARG treated rats prevented ARG induced alterations in various enzyme activities. Similar to cortex, G6PDH activity increased whereas ME activity decreased by ARG in the medulla. FO and FXO diet to ARG treated rats decreased G6PDH activity and increased ME activity (iii) ARG treatment to control rats caused significant increase in LDH (+26%) but decrease in MDH activities. ARG caused significant decrease in gluconeogenic enzyme, G6Pase in the intestine. The activity of G6Pase remained significantly higher after ARG treatment to FO and FXO-fed rats compared to control rats reversing the ARG-induced decrease in its activity. ARGinduced decline in MDH activity was also restored by both FO and FXO-diets. The activity of G6PDH significantly increased (+24%) but that of ME decreased (-39%) by ARG treatment compared to control rats. However, when ARG treatment was given to FO/FXO diet fed rats, ARG induced increased in G6PDH and decrease in ME activities were not observed. (iv) ARG treatment significantly increased the activities of LDH (+41%), HK (+35%) and G6PDH (+50%) whereas decreased G6Pase, MDH and ME activities in the liver (Table 5, 6). When ARG treatment was extended to FO-fed rats, ARG-induced decrease of metabolic enzyme activities was prevented by FO and FXO.

171

(c) Marker enzymes of BBM and lysosomes: (i) ARG treatment to control rats caused significant reduction in the specific activities of AlkPase (-28%), GGTase (-27%) and LAP (-29%) in cortical homogenate. The prior feeding of FO or FXO diet with ARG treatment prevented ARG elicited ecrease in BBM enzyme activities. As can be seen from the data ARG induced decrease in BBM enzyme activities were similarly prevented by FO or FXO diet. The activity of acid phosphatase (ACPase) was also decreased (-17%) by ARG in cortical homogenates and FO/FXO diet was able to prevent the decrease in enzyme activity in a similar manner. The activities of BBM enzymes similar to the cortex were also lowered in the medulla by ARG administration (Table 7). The consumption of FO/FXO in combination with ARG treatment resulted in the reversal of ARG induced decrease in GGTase (-22%) and LAP (40%) in the medulla. The activity of ACPase was also lowered by ARG (-16%). However it was found to be higher in ARG+FO rats (+19%) compared with the control rats. (ii) Activities of AlkPase (-47%), GGTase (-45%) and LAP (-40%) profoundly declined by ARG treatment as compared to control rats in renal BBM. Fish oil (FO) and flaxseed oil (FXO) dietary supplementation similar to the effect in the homogenates appeared to lower the severity of the ARG treatment. (iii) ARG treatment to control rats caused significant decrease in AlkPase, GGTase and LAP in mucosal homogenates. ARG treatment also resulted in the decrease of ACPase activity. When ARG treatment was given to FO/FXO fed rats normalization of the activities of AlkPase, GGTase, LAP and sucrase whereas ACPase was partially restored to control values. ARG treatment profoundly decreased the activities of all BBM enzymes but sucrase which was elevated in intestinal BBM, FO/FXO caused marked increase in AlkPase and GGTase whereas LAP and sucrase activities were significantly normalized by both FO/FXO diets. ARG induced decrease in the BBMV and lysosomal marker enzymes was not only preserved by FO/FXO diets but the activities were restored to near control values. (iv) ARG treatment to rats resulted in extensive reduction in the specific activity of alkaline phosphatase (AlkPase), GGTase LAP and ACPase in the liver homogenates. The feeding of FO and FXO-diet to ARG treated (FOARG/FXOARG) rats not only prevented ARG elicited changes, but the activities of GGTase and AlkPase were even increased by FO diet. 172

(d) Enzymatic and non-enzymatic antioxidant defense parameters: (i) To ascertain the role of antioxidant system in ARG-induced toxicity, the effect of ARG observed on oxidative stress parameters. ARG enhanced lipid peroxidation (LPO) and significantly altered antioxidant enzymes both in cortex and medulla, albeit differently. LPO measured in terms of malodialdehyde (MDA levels) significantly enhanced in the cortex (+43%) and medulla (+51%) to similar extent whereas total-SH declined in these tissue (-16% to -26%). ARG treatment caused marked increase in superoxide dismutase (SOD, +68%) but decrease in Glutathione peroxidase (GSH-Px, -37%) activity in the renal cortex. In medulla however the activity of SOD (-36%) and GSH-Px (-16%) significantly decreased by ARG administration alone. ARG induced increase in LPO and decrease in total-SH was not observed by feeding FO or FXO diet to ARG-treated rats. The activity of GSH-Px and catalase in the cortex and the activity of catalase and GSH-Px in the medulla respectively remained significantly higher in FO/FXO + ARG rats compared to ARG and/or control rats. (ii) ARG treatment caused marked increase in SOD (+35%) but decrease in catalase (-22%). These changes were associated with significant increase in lipid peroxidation (+30%) measured in terms of MDA levels and decrease in total-SH (-21%). Both FO and FXO-diet partially prevented ARGinduced increase in SOD, catalase and GSH-Px activities. ARG induced increase in LPO and decreased SH-content were prevented by FO/FXO diet. (iii) ARG treatment to control rats caused marked decrease in catalase (-22%) and GSH-Px (-17%) activities. These changes were associated with significant increase in lipid peroxidation (+35%) and decrease in total-SH content (-21%). Both FO/FXO-diets prevented ARG-induced decrease in catalase and GSH-Px activities when FO and FXO diets were given in combination with ARG treatment. Total-SH and LPO were also normalized to near control values. (e) Na+ dependent transport of 32Pi in the BBMV isolated from renal cortex: The rate of concentrative uphill uptake of

32


medium) was markedly decreased by ARG treatment. However, the uptake of

32

Pi at the

equilibrium phase (120 min) when Nao=Nai was not significantly different between the two groups. Also Na-independent uptake (in the absence of a Na gradient, when NaCl in the medium was replaced by KCl where Ko> Ki) of 32Pi at 30s and 120min was also not affected by ARG treatment 173

indicating specific alterations only when Na-gradient was present. When ARG treatment was extended to FO and FXO feeding rats, ARG induced decrease in 32Pi transport was not observed. (f) Tissue concentration of nitrite: ARG administration to rats increased the tissue concentrations of nitrite in all tissues, the accumulation of nitrite being greater in renal medulla (+35%) and intestine (+28%). When FO/FXO was given in combination with ARG, both FO and FXO partially improved the values to near control in renal cortex, medulla and intestine, whereas in the liver complete amelioration was observed.

; Interpretation: In general, ARG caused severe adverse effects on various serum/urine parameters and on the enzymes of carbohydrate metabolism, BBM and antioxidant defense system in rat kidney and intestine. FO and/or FXO-diet similarly protected against ARGinduced nephrotoxic and other adverse effects most likely due to their intrinsic biochemical and antioxidant properties.

174

CONCLUSION: In conclusion the results of the present studies demonstrate that SNT/SNP/ARG elicited deleterious nephrotoxic effects by causing major damage to mitochondria, lysosomes and in particular to BBM of renal proximal tubule, intestinal mucosa and liver as reflected by significant decrease in the activities of their specific biomarker enzymes, confirming some of the previous morphologic and biochemical observations that showed these organelles as prime NO donors/metabolites targets. NO donors/metabolites seem to enhance glycolytic enzyme LDH in various tissues in order to increase energy dependence on glycolysis due to mitochondrial damage and depressed TCA cycle enzymes. Gluconeogenic enzymes also appeared to decrease also due to mitochondrial dysfunction as oxalate produced by MDH is not available. Serum Pi and renal Pi transport was also decreased suggesting a decrease in metabolic activity. These nephrotoxic and other adverse effects appeared to be mediated in part due to NO donors/metabolites elicited oxidative/nitrosative damage. In agreement with previous studies except for few direct effects, SNT/SNP/ARG appear to cause many of these deleterious effects either by conversion to NO or by its uncontrolled excessive production, as supported by increased tissue levels of nitrite as NO is rDSLGO\ FRQYHUWHG WR QLWULWH 'LHWDU\ Ȧ-3 PUFA enriched fish oil and flaxseed oil to a larger extent prevented NO donors/metabolites induced nephrotoxicity parameters by enhancing nutrition/energy metabolism and 32Pi transport capacity, by strengthening antioxidant defense PHFKDQLVP DQG E\ DOWHULQJ PHPEUDQH RUJDQL]DWLRQIOXLGLW\SHUPHDELOLW\ Ȧ-3 PUFA enriched FO/FXO diets leads to an enhancement in the metabolic processes. This enhancement leads to increase in the demand of Pi, which in turn leads to increased utilization, as Pi is required in the synthesis of membranes such as endoplasmic reticulum, mitochondria and plasma membrane in RUGHUWRVXSSRUWKLJKHUPHWDEROLFDFWLYLWLHV7KHUHVXOWVDOVRVXJJHVWWKDWȦ-3 PUFA strengthen the biological antioxidant defense system thereby reducing oxidative/nitrosative damage induced by NO donors/metabolites. ,WLVDSSDUHQWWKDWȦ-3 PUFA is a source of wide range of nutraceuticals that are digested, absorbed and metabolized and that they exert their effects at the cellular level. Fish/flaxseed oil HQULFKHGLQȦ-3 PUFA provide a dietary source of biologically active components that has been shown to be co-preventative and co-therapeutic in a wide variety of ailments. Based on our present observations and already known numerous health benefits, ZHSURSRVHWKDWȦ-3 PUFA enriched 175

FO/FXO may provide a cushion for a prolonged therapeutic option against drug/chemical induced nephropathy without harmful side effects. Similar protective effects of FO and FXO provide a much needed relief for millions of vegetarians who do not eat fish/fish oil for religious or other reasons.

176

BIBLIOGRAPHY Abrahamsson T, Brandt U, Marklund SL, Sjoqvist. Vascular bound recombinant extracellular superoxide dismutase type C protects against the detrimental effects of superoxide radicals on endothelium-dependent arterial relaxation. Circ Res 1992; 70: 264-271. Abuharfeil N, Sarsour E, Hassuneh M. The effect of sodium nitrite on some parameters of the immune system. Food Chem Toxicol 2001; 39: 119-124. Addiscott TM. Fertilizers and nitrate leaching. In Agricultural Chemicals and the Environment, Issues in Environmental Science and Technology. [RE Hester and RM Harrison, editors]. Cambridge: Royal Society of Chemistry 1996; 5: 1±26. Akin A and Sumer S. The mutagenic effects of sodium nitrite and monosodium glutamate used as food additives demonstrated by the Salmonella microsome test system. Mikrobiyoloji bulteni 1991; 25: 94±107. Ali BH and Bashir AA. Effect of fish oil treatment on gentamicin nephrotoxicity in rats. Ann Nutr Metabl 1994; 38: 336-339. Allman MA, Pena MM, Pang D. Supplementation with flaxseed oil versus sunflowerseed oil in healthy young men consuming a low fat diet: effects on platelet composition and function. Eur J Clin Nutr 1995; 49: 169-178. Ames BN. Dietary carcinogens and anticarcinogens: oxygen radicals and degenerative diseases. Science 1983; 221: 1256-1264. Andreassi MG et al. Chronic long-term nitrate therapy: possible cytogenetic effect in humans? Mutagenesis 2001; 16: 517±521. Anti M et al. Effects of different doses of fish oil on rectal cell proliferation in patients with sporadic colonic adenomas. Gastroenterol 1994; 107: 1892-1894. Aperia AC and Liebow AA. Implications of urine PO2 for renal medullary blood flow. Am J Physiol 1964; 206: 499-504. $ULDV ,0 %D\HU ) -DFRE\ :% 6FKDFKWHU ' 6FKDIULW] '$ ³7KH /LYHU´ %LRORJ\ DQG Pathobiology, 3rd ed. Raven Press, New York, 1994.

177

Arivarasu NA, Fatima S, Mahmood R. Effect of cisplatin on brush border membrane enzymes and antioxidant systems of rat intestine. Life Sci 2007; 81: 393-398. Aronson WJ, Glaspy JA, Reddy ST, Reese D, Heber D, Bagga D. Modulation of n-3 and n-6 polyunsaturated fatty acid ratios with dietary fish oils in men with prostrate cancer. Urology 2001; 58: 283-288. Asahi M et al. Inactivation of Glutathione Peroxidase by Nitric Oxide implication for cytotoxicity. The J of Biol Chem 1995, 270: 21035-21039. Asaka M, Takeda H, Sugiyama T, Kato M. What role does Helicobacter pylori play in gastric cancer? Gastroenterology 1997; 113: S56±S60. Aukland K and Krog J. Influence of various factors on urine oxygen tension in the dog. Acta Physiol Scand 1961; 52: 350-365. Aukland K and Krog J. Renal oxygen tension. Nature 1960; 188: 671. Aukland K. Hemoglobin oxygen saturation in the dog kidney. Acta Physiol Scand 1962; 56: 315323. Baggio B, Budakovic A, Priante G, Gambaro G, Manzato E, Khan S. Dietary fatty acid supplementation modulates the urinary excretion of calcium and oxalate in the rat. Insight into calcium lithogenesis. Nephron 2002; 91: 486-491. Baker JE et al. Nitrite confers protection against myocardial infarction: role of xanthine oxidoreductase, NADPH oxidase and K(ATP) channels. J Mol Cell Cardiol 2007; 43: 437±444. Balagura-Baruch S, Burich RL, King VF. Effects of alkalosis on renal citrate metabolism in dogs infused with citrate. Am J Physiol 1973; 225: 385-388. Banday AA, Farooq N, Priyamvada S, Yusufi ANK, Khan F. Time dependent effects of gentamicin on the enzymes of carbohydrate metabolism, brush border membrane and oxidative stress in rat kidney tissues. Life Sci 2008a; 82: 450-459. Banday AA, Priyamvada S, Farooq N, Yusufi ANK, Khan F. Effect of uranyl nitrate on enzymes of carbohydrate metabolism and brush border membrane in different kidney tissues. Food Chem Toxicol 2008b; 46: 2080-2088.

178

Bang HO, Dyerberg J, Hjoorne N. The composition of food consumed by Greenland Eskimos. Acta Med Scand 1976; 200: 69-73. %DQN10XW]%)$\QHGMLDQ+67KHUROHRI³OHDNDJH´of tubular fluid in anuria due to mercury poisoning. J Clin Invest 1967; 46: 695-704. Barbier O, Jacquillet G, Tauc M, Cougnan M, Poujeol P. Effect of heavy metals on, and handling by kidney. Nephron Physiol 2005; 99: 105-110. Barbosa DS, Cecchini R, El Kadri MZ, Rodriguez MA, Burini RC, Dichi I. Decreased oxidative stress in patients with ulcerative colitis supplemented with fish oil omega-3 fatty acids. Nutrition 2003; 19: 837-842. Barbul. Physiology and pharmacology of arginine. New York: Elsevier Science Publishing Co; 1990. Bartlett GR. Colorimetric assay methods for free and phosphorylated glyceric acids. J Biol Chem 1959; 234: 469-471. Basch E et al. Flax and Flaxseed Oil (Linum usitatissimum): A Review by the Natural Standard Research Collaboration. J Soc Integr Oncol 2007; 5: 92-105. Basnakian AG, Kaushal GP, Shah SV. Apoptotic pathways of oxidative damage to renal tubular epithelial cells. Antioxid Redox Signal 2002; 4: 915-924. Bastianetto S, Zheng WH, Quirion R. Neuroprotective abilities of resveratrol and other red wine constituents against nitric oxide-related toxicity in cultured hippocampal neurons. British Journal of Pharmacology 2000: 131: 711-720. Beckman J, Carson M, Smith CD, Koppenol WH. ALS, SOD and peroxynitrite. Nature 1993; 364: 584. Beckman JS. The double-edged role of nitric oxide in brain function and superoxide-mediated injury. J Dev Physiol 1991; 15: 53±59. Belluzzi A, Brignola C, Campieri M, Pera A, Boschi S, Miglioli M. Effect of an enteric-caoted ILVKRLOSUHSDUDWLRQRQUHODSVHVLQ&URKQ¶VGLVHDVH. The N Eng J Med 1996; 334: 1557-1560.

179

Benjamin, N. et al. Stomach NO synthesis. The first suggestion of NOS-independent NO generation from inorganic nitrite and in vitro demonstration of its role in gastric host defence. Nature 1994; 368: 502 Berquin IM, Edwards IJ, Chen YQ. Multi-targeted therapy of cancer by omega-3 fatty acids. Cancer Lett 2008; 269(2): 363-377. Bhatia AL, Manda K, Patni S, Sharma AL. Prophylactic action of linseed (Linum usitatissimum) oil against cyclophosphamide-induced oxidative stress in mouse. J Med Food 2006; 9: 261-264. Bhatia AL, Sharma A, Patni S, Sharma AL. Prophylactic effect of flaxseed oil against radiationinduced hepatotoxicity in mice. Phytother Res 2007; 21: 852-859. Bhatia AL, Sharma A, Patni S, Sharma AL. Prophylactic effect of flaxseed oil against radiationinduced hepatotoxicity in mice. Phytother Res 2007; 21: 852-859. Bhattacharya A, Ghosal S, Bhattacharya SK. Effect of fish oil on offensive and defensive factors in gastric ulceration in rats. Prostaglandins Leukot Essent Fatty Acids 2006; 74: 109-116. Bhattacharya A, Lawrence RA, Krishnan A, Zaman K, Sun D, Fernandes G. Effect of dietary n-3 and n-6 oils with and without foor restricition on activity of antioxidant enzymes and lipid peroxidation in livers of cyclophosphamide treated auto-immune prone NZB/W female mice. J Am Coll Nutr 2003; 22: 388-399. Birnboim HC and Privora H. Depletion of intracellular glutathione reduces mutations by nitric oxide-donating drugs. Nitric Oxide 2000; 4: 496±504. Bitterman N and Bitterman H. J Appl Physiol 1998; 84: 1633-1638. Bjelke E. Epidemiologic Studies of Cancer of the Stomach Colon and Rectum. Scand J Gastroenterol 1974; 9(31): 1-235. Bjorne, H. H. et al. Nitrite in saliva increases gastric mucosal blood flow and mucus thickness. J Clin Invest 2004; 113: 106±114. Bode-Boger SM. Influence of L-arginine on development of atherosclerosis: what is the therapeutically assured? Dtsch Med Wochenschr 2005; 130(11): 593-8. Bogdan C. Nitric oxide and the immune response. Nature Immunol 2001; 2: 907±916.

180

Boger RH, Bode-Boger SM, Frolich JC. The L-arginine ± nitric oxide pathway: Role in atherosclerosis and therapeutic implications. Atherosclerosis 1996; 127: 1-11. Boger RH, L-Arginine improves vascular function by overcoming deleterious effects of ADMA, a novel cardiovascularrisk factor. Altern Med Rev 2005; 10(1): 14-23. Bohme GA et al. Altered synaptic plasticity and memory formation in nitric oxide synthase inhibitor-treated rats. Proc Natl Acad Sci USA 1993; 90: 9191-9194. Bonjour JP and Caverzasio JHC. Phosphate transport in the Kidney. Rev Physiol Biochem Pharmacol 1984; 100: 161-214. Boughton-Smith

NK

et

al.

Nitric

oxide

synthase

activity

in

ulcerative

colitis and Crohn's disease. Lancet 1993; 342: 338-340. Bourre JM. Where to find omega-3 fatty acids and how feeding animals with diet enriched in omega-3 fatty acids to increase nutritional value of derived products for human: what is actually useful? J Nutr Health Aging 2005; 9: 232-242. Boveris A, Costa LE, Poderoso JJ, Carreras MC, Cadenas E. Regulation of mitochondrial respiration by oxygen and nitric oxide. Ann N Y Acad Sci 2000; 899: 121±135. Brasitus TA, Davidson NO, Schachter D. Variations of dietary triacylglycerol saturation alter the lipid composition and fluidity of rat intestinal plasma membranes. Biochim Biophys Acta 1985; 812: 469-472. Brasitus TA, Schachter D, Mamouneas TG. Functional interactions of lipids and proteins in fat intestinal microvillus membranes. Biochemistry 1979; 18: 4136-4144. Bronte V and Zanovello P. regulation of immine responses by L-arginine metabolism. Nature 2005; 5: 641-654. Bronte V and Zanovello P. Regulation of immune responses by L-arginine metabolism. Nat Rev Immunol 2005; 5(8): 641-54. Brown GC and Borutaite V. Nitric oxide inhibition of mitochondrial respiration and its role in cell death. Free Radic Biol Med 2002; 33: 1440-1450. Brown GC. Reversible Binding and Inhibition of Catalase by Nitric Oxide. European Journal of Biochemistry 1995; 232(1): 188-191. 181

Bruijns E. Treatment of kidney stones with ammonium nitrate. PhD Thesis, University of Nijmegen 1982. Bryan NS et al. Cellular targets and mechanisms of nitros(yl)ation: an insight into their nature and kinetics in vivo. Proc Natl Acad Sci USA 2004; 101: 4308±4313. Bryan NS et al. Nitrite is signalling molecule and regulator of gene expression in mammalian tissue. Nature Chem Biol 2005; 1: 290±297. Budhoski L, Challis RAJ, New Scholme EA. Effect of starvation on the maximal activities of some glycolytic and citric acid cycle enzymes and glutaminase in mucosa of the small intestine of the rat. Biochem J 1982; 206: 169-172. Burch HB et al. Distribution along the rat nephron of three enzymes of gluconeogenesis in acidosis and starvation. Am J Physiol 1978; 35: F246-F253. Burnett AL, Lowenstein CJ, Bredt DS, Chang CSK, Cynder SH. Nitric oxide: a physiologic mediator of penile erection. Science 1992; 257:401-403. Burney S, Niles JC, Dedon PC, Tannenbaum SR. DNA damage in deoxynucleosides and oligonucleotides treated with peroxynitrite. Chem Res Toxicol 1999; 12: 513±520. Burr GO and Burr MM. The nature and role of the fatty acids essential in nutrition. J Biol Chem 1930; 86: 587-621. Burr ML et al. Effects of changes in fat, fish and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART). The Lancet 1989; 2: 757-761. Butler AR, Flitney FW, Williams DLH. NO, nitrosonium ions, nitroxide ions and iron-nitrosyls in Biology: A chemists perspective. Trends Pharmacol Sci 1996; 16: 18-22. Caballero B. Fortification, supplementation and nutrient balance. Eur J Clin Nutr 2003; 57: S76S78. Carlsson S, Wiklund NP, Engstrand L, Weitzberg E, Lundberg JO. Effects of pH, nitrite, and ascorbic acid on nonenzymatic nitric oxide generation and bacterial growth in urine. Nitric Oxide 2001; 5: 580±586. Carmichael AJ, Steel-Goodwin L, Gray B, Arroyo CM. Reactions of reactive oxygen species studied by EPR and spin trapping. Free Rad Res Commun 1993; 19: S1-S16. 182

Carrie I, Clement M, De Javel D, Frances H, Bourre JM. Learning deficits in first generation OF1 mice deficient in (n-3) polyunsaturated fatty acids do not result from visual alteration. Neurosci Lett 1999; 266: 69-72. Carrillo-Tripp M and Feller SE. Evidence for a mechanism by which omega-3 polyunsaturated lipids may affect membrane protein function. Biochemistry 2005; 44: 10164-10169. Castro L, Rodriguez M, Radi R. Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide. J Biol Chem 1994; 269: 29409-29415. Cave WT. Dietary n-3 (Z-3) Polyunstaurated fatty acid, effects on animal tumorigenesis. FASEB J 1991; 5: 2160-2166. Ceccatelli S, Hulting AL, Zhang X, Gustafsson L, Villar M, Hokfelt T. Nitric oxide synthase in the rat anterior pituitary gland and the role of nitric oxide in regulation of luteinizing hormone secretion. Proc Natl Acad Sci USA 1993; 90: 11292-11296. Chandrasekar B and Fernandes G. Decreased pro-inflammatory cytokines and increased DQWLR[LGDQWHQ]\PHJHQHH[SUHVVLRQE\Ȧ-3 lipids in murine lupus nephritis. Biochem Biophys Res Comm 1994; 200: 893-898. Cherla G. Role of L-arginine in the pathogenesis and treatment of renal disease. J Nutr 2004; 134(10): 2801S-2806S. Chilton FH, Rudel LL, Parks JS, Arm JP, Seeds MC. Mechanisms by which botanical lipids affect inflammatory disorders. Am J Clin Nutr 2008; 87: 498S-503S. Chow CK and Hong CB. Dietary vitamin E and selenium and toxicity of nitrite and nitrate. Toxicology 2002; 180: 195-207. Claiborne A. Catalase activity. In: CRC Handbook of Oxygen Radical Research, Greenwald RA (editor), CRC Press, Boca Raton, FL: 1985; 283-284. Cleeter MWJ, Cooper JM, Darley-Usmar VM, Moncada S, Schapira AHV. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide: Implications for neurodegenerative diseases. FEBS Lett 1994; 345: 50-54. Cohen JJ. Is the function of the renal papilla coupled exclusively to an anaerobic pattern of metabolism? Am J Physiol 1979; 236: F423-F433. 183

Comly HH. Cyanosis in infants caused by nitrates in well water. Journal of the American Medical Association 1945; 129: 112± 116. Connor WE. Importance of n-3 fatty acids in health and disease. Am J Clin Nutr 2000; 71: 171S175S. Cooke JP and Dzau VJ. Nitric oxide synthase: role in the genesis of vascular disease. Annu Rev Med 1997; 48: 489-509. Cooke JP. The pivotal role of nitric oxide for vascular health. Can J Cardiol 2004; 20(B): 7B-15B. Coquette A, Berna N, Vandenbosch A, Rosdy M, Poumay Y. Differential expression and release of cytokines by an in vitro reconstructed human epidermis following exposure to skin irritant and sensitizing chemicals. Toxicology in Vitro 1999; 13: 867±877. Correa P, Haenszel W, Cuello C, Tannenbaum S, Archer M. A Model for Gastric Cancer Epidemiology. Lancet 1975; 2: 58-59. Correa P, Sasano N, Stemmerman GN, Haenszel W. Pathology of Gastric Carcinomain Japanese Population : Comparisons between Miyagi Prefecture, Japan and Hawaii. J Natl Cancer Inst 1973; 51: 1449-1457. Cosby K et al. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nature Med 2003; 9: 1498±1505. Coux G, Trurumper L, Elias MM. Cortical Na+-K+ ATPase activity, abundance and distribution after in vivo renal ischemia without reperfusion in rats. Nephron 2001; 89: 82-89. Crane RK and Sols A. The association of particulate fractions of brain and other tissue homogenates. J Biol Chem 1953; 203: 273-292. Cronin RE and Henrich WL. Toxic nephropathy. In: The kidney, Brenner BM and Rector FC (editors), WB. Saunders Co. 1996; 2: 1680-1711. Cronin RE, Brown DM, Simonsen R. Protection by thyroxine in nephrotoxic acute renal failure. Am J Physiol 1986; 25: F408-F416.

184

Culcasi M, Lafon-Cazal M, Pietri S, Bockaert I. Glutamate receptors induce a burst of superoxide via activation of nitric oxide synthase in arginine-depleted neurons. J Biol Chem 1994; 269: 1258912593. Culotta E and Koshland DE. NO news is good news. Science 1992; 258: 1862±1865. Cylwik D, Mogielnicki A, Buczko B. L-arginine and cardiovascular system. Pharmacol Rep 2005; 57(1): 14-22. Czako L et al. Involvement of oxygen-derived free radicals in L-arginine-induced acute pancreatitis. Dig Dis Sci 1998; 43: 1770-1777. Czako L et al. Oxidative stress in distant organs and the effects of allopurinol during experimental acute pancreatitis. Int J Pancreatol 2000a; 27: 209-216. Czako L et al. The pathogenesis of L-arginineinduced acute necrotizing pancreatitis: inflammatory mediators and endogenous cholecystokinin. J Physiol Paris 2000b; 94: 43-50. '¶$PLFR*7KHFRPPRQHVWJORPHUXORQHSKULWLVLQWKHZRUOG,J$QHSKURSDWK\Q J Med 1987; 245: 709-727. Darley-Usmar VM, Hogg N, O'Leary VJ, Wilson MT, Moncada S. The Simultaneous Generation of Superoxide and Nitric Oxide Can Initiate Lipid Peroxidation in Human Low Density Lipoprotein. Free Rad Res Commun 1992; 17: 9-20. Davis BC and Kris-Etherton PM. Achieving optimal essential fatty acid status in vegetarians: current knowledge and practical implications. Am J Clin Nutr 2003; 78: 640S-646S. Dawson and Dawson. Nitric oxide neurotoxicity. J Chem Neuroanat 1996; 10: 179-190 De Caterina R, Endres S, Kristensen SD, Schmidt EB. n-3 fatty acids and renal diseases. Am J Kid Dis 1994; 24: 397-415. Deng HX et al. Amyotrophic lateral sclerosis and structural defects in Cu, Zn superoxide dismutase. Science 1993; 261: 1047-1051. Djousse L et al. Relation between dietary linolenic acid and coronary artery disease in the National Heart, Lung, and Blood Institute Family Heart Study. Am J Clin Nutr 2001; 74: 612-619. Dollahite JW and Rowe LD. Nitrate and nitrite intoxication in rabbits and cattle. Southwest Vet 1974; 27: 246-248. 185

Donadio JV Jr, Larson TS, Bergstralh EJ, Grande JP. A randomized trial of high ±dose compared with low-dose omega-3 fatty acids in severe IgA nephropathy. J Am Soc Nephrol 2001; 12: 791799. Donadio JV. Omega-3 polyunsaturated fatty acids: a potential new treatment of immune reanl disease. Mayo Clin Proc 1991; 66: 1018-1028. Donadio JV. The emerging role of omega-3 polyunsaturated fatty acids in the management of patients with IgA nephropathy. J Ren Nutr 2001; 11: 122-128. Donahoe WE. Cyanosis in infants with drinking water as a cause. Paediatrics 1949; 3: 308±311. Doughman SD, Krupanidhi S, Sanjeevi CB. Omega-3 fatty acids for nutrition and medicine: considering microalgae oil as a vegetarian source of EPA and DHA. Curr Diabetes Rev 2007; 3: 198-203. Duncan C et al. Chemical generation of nitric oxide in the mouth from the enterosalivary circulation of dietary nitrate. Nature Med 1995; 1: 546±551. Dupont J et al. Food uses and health effects of corn oil. J Am Coll Nutr 1990; 9: 438-470. Dyerberg J and Bang HO. A hypothesis on the development of acute myocardial infarction in Greenlanders. Scand J Clin Lab Invest 1982; 161: 7-13. Dyerberg J, Bang HO, Hjorne N. Fatty acid composition of the plasma lipids in Greenland Eskimos. Am J Clin Nutr 1975; 28: 958-966 Endo H, Takahashi K, Kinoshita N, Baba T. Production of Gastric and EsophagealTumors in Rats by Methylnitrosocyanamide, a Possible Candidate of Etiologic Factors for Human Gastric Cancer. Proc Japan Acad 1974; 50: 497-502. Engler MB, Engler MM. Docosahexaenoic acid ± induced vasorelaxation in hypertensive rats: mechanisms of action. Biol Res Nurs 2000; 2: 85-95. Erdogan H et al. Effect of fish oil supplementation on plasma oxidant/antioxidant status in rats, Prostaglandin Leukot Essent Fatty Acids 2004; 71: 149-152. Evan AP, Vincet GH, Schwartz GJ. Development of solute transport in rabbit proximal tubule. II. Morphologic segmentation. Am J Physiol 1983; 245: F391-F407. 186

Farooq N, Priyamvada S, Arivarasu NA, Salim S, Khan F, Yusufi ANK. Influence of Ramadantype fasting on enzymes of carbohydrate metabolism and brush border membrane in small intestine and liver of rat used as a model. Br J Nutr 2006; 96: 1087-1094. Farooq N, Priyamvada S, Khan F, Yusufi ANK. Time dependent effect of gentamicin on enzymes of carbohydrate metabolism and terminal digestion in rat intestine. Hum Exp Toxicol 2007; 26: 17. Farooq N, Yusufi ANK, Mahmood R. Effect of fasting on enzymes of carbohydrate metabolism and brush border membrane in rat intestine. Nutr Res 2004; 24: 407-416. Fatima S, Arivarasu NA, Banday AA, Yusufi ANK, Mahmood R. Effect of potassium dichromate on renal brush border membrane enzymes and phosphate transport in rats. Hum Exp Toxicol 2005; 24: 631-638. Fatima S, Yusufi ANK, Mahmood R. Effect of cisplatin on renal brush border membrane enzymes and phosphate transport. Hum Exp Toxicol 2004; 23: 547-554. Feldman PL, Griffith OW, Stuehr DJ. The surprising life of nitric oxide. Chem Eng News 1993; 71: 26-39. Feldmann G. Liver apoptosis. J Hepatol 1997; 26: 1-11. Fernandis G and Venkatraman JT. Role of omega-3 fatty acids in health and disease. Nutr Res 1993; 13: S19-S45. Flohe L, Gunzler W. Assay of glutathione peroxidase. In: Methods Enzymol, Colowick SP and Kaplan NO (editors). New York: Academic Press 1984; 105: 114-121. Foulon T et al. Effects of fish oil fatty acids on plasma lipids and lipoproteins and oxidantantioxidant imbalance in healthy subjects. Scand J Clin Lab Invest 1999; 59: 239-248. Francois H and Danielle C. Functional segmentation of the nephron. In: The Kidney: Physiology and Pathophysiology, Seldin DW and Giebisch G (editors). Raven Press, New York, 1985; pp. 519-529. Freedland RA and Harper AE. Metabolic adaptions in higher animals V. The study of metabolic pathways by means of metabolic adaptions. J Biol Chem 1959; 234: 1350-1354.

187

Gago B, Lundberg JO, Barbosa RM, Laranjinha J. Red wine-dependent reduction of nitrite to nitric oxide in the stomach. Free Radic Biol Med 2007; 43: 1233±1242. Galli F, Rossi R, Simplicio P, Floridi A, Canestrari F. Protein thiols and glutathione influence the nitric oxide-dependent regulation of the red blood cell metabolism. Nitric Oxide 2002; 6: 186-199. Galli F, Rovidati S, Ghibelli L, Canestrari F. S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase decreases the enzyme affinity to the erythrocyte membrane. Nitric Oxide 1998; 2: 17-27. Gamoh S et al. Chronic administration of docosahexaenoic acid improves reference memory ± related learning ability in young rats. Neuroscience 1999; 93: 237-241. Ganapathy V, Miyamoto Y, Tirruppathi C, Leibach FH. Peptide transport across the animal cell plasma membrane: recent developments. Indian J Biochem Biophys 1991; 28: 317-323. Garle MJ, Bull S, Willshaw A, Ward RK, Clothier RH. Nitrite synthesis in a murine macrophage cell line stimulated by products released from irritant-treated normal human keratinocytes. Toxicology in Vitro 1996; 10: 265±271. Gaston B, Drazen JM, Loscalzo J, Stamler JS. The biology of nitrogen oxides in the airways. Am J Respir Crit Care Med 1994; 149: 538-551. Ger J, Kao H, Shih TS, Deng JF. Fatal toxic methemoglobinemia due to occupational exposure to methyl nitrite. Clin Med J 1996; 57: S78. Giri U, Iqbal M, Athar M. Porphyrin -mediated photosensitization has a weak tumor promoting activity in mouse skin: possible role of in-situ generated reactive oxygen species. Carcinogenesis 1996; 17: 2023-2028. Gladwin MT et al. The emerging biology of the nitrite anion. Nature Chem Biol 2005; 1: 308±314. *ORVVPDQQ+DQG1HYLOOH'0Ȗ-glutamyl transferase in kidney brush border membranes. FEBS Lett 1972; 19: 340-344. Goaz PW and Biswell HA. Nitrite reduction in whole saliva. J Dent Res 1961; 40: 355±365. Godber BL et al. Reduction of nitrite to nitric oxide catalyzed by xanthine oxidoreductase. J Biol Chem 2000; 275: 7757±7763.

188

Goldman DR, Schlesinger H, Segal S. Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells. Biochim Biophys Acta 1976; 419: 251-260. Goldstein A and Lampen JO. Beta-D-fructofuranoside fructohydrolase from yeast. Methods Enzymol 1975; 42: 504-511. Goldstein DJ et al. Fish oil ameliorates renal injury and hyperlipidemia in the Milan normotensive rat model of focal glomerulosclerosis. J Am Soc Nephrol 1995; 6: 1468-1475. Grande JP and Donadio JV. n-3 Polyunsaturated fatty acid in the treatment of patients with IgA nephropathy, In: Medicinal fatty acids in inflammation, Kremer JM (editor), Birkhauser Veklag Basel/Switzerland, 1998; 125-140. Grande JP and Donadio JV. Role of dietary fish oil supplementation in IgA nephropathy: mechanistic implications. Minerva Urol Nephrol 2001; 53: 201-209. Granger DL, Hibbs JB Jr, Perfect JR, Durack DT. Specific amino acid (L-arginine) requirement for the microbiostatic activity of murine macrophages. J Clin Invest 1987; 81:1129-1136. Green SJ, Meltzer MS, Hibbs JB Jr, Nacy CAl. Activated macrophages destroy intracellular. Leishmania major amastigotes by an L-arginine- dependent killing mechanism. J Immunol 1990; 144: 278- 283. Greene EL and Paller MS. Oxygen free radicals in acute renal failure. Min Elect Metab 1991; 17: 124-132. Grisham MB et al. Effects of neutrophil derived oxidants on intestinal permeability, electrolyte transport and epithelial cell viability. Inflammation 1990; 14: 531-542. Gross SS and Wolin MS. Nitric oxide: Pathophysiological mechanisms. Annu Rev Physiol1995; 57: 737±769. Grosser O, Lewis FT, Murrich JP. In: Human Embryology, Keibal F and Mall FP (editors). Philadelphia Lippinocott, 1992; 11: 291. Guder WG. Regulation der Glukoneogenese der Niere. Habil.-Schrift, Munich, LudwigMaximaillians-Univ. Munchen, 1973.

189

Haddad LY, Pataki G, Hu P, Galliani C, Beckman JS, Matalon S. Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J Clin Invest 1994; 94: 24072413. Haenszel W, Kurihara M, Segi M, Lee RKC. Stomach Cancer among Japanese in Hawaii. J Nail Cancer Inst 1972; 49: 969-988. Halliwell B and Dizdaroglu M. The measurement of oxidative damage to DNA by HPLC and GC/MS techniques. Free Rad Res Commun 1992; 16: 75-87. Halliwell B and Gutteridge JMC. Free Radic Biol Med (3rd ed.) Oxford: Clarendon Press. 1999. Halliwell B. Free radicals and antioxidants. A personal view. Nutr Rev 1994; 52: 253-265. Hammerman MR and Schwab SJ. Phosphate transport in the kidney. Studies with isolated membrane vesicles. In: Epithelial Calcium and Phosphate Transport: Molecualr and cellular aspects, Bronner F and Peterlik M (editors), New York: Liss, 1984; pp. 325-330. Hardman WE. Omega-3 fatty acids to augment cancer therapy. J Nutr 2002; 132: 3508S-3512S. Harper CR and Jacobson TA. The fats of life: the role of omega-3 fatty acids in the prevention of coronary heart disease. Arch Intern Med 2001; 161: 2185-2192. Harris KP, Lefkowith JB, Klahr S, Schreiner GF. Essential fatty acid deficiency ameliorates acute renal dysfunction in the rat after the administration of the aminonucleoside of puromycin. J Clin Invest 1990; 86: 1115-1123. Harris WS. Alpha-linolenic acid: A gift from land? Circulation 2005; 111: 2872-2874. Harris WS. n-3 fatty acids and serum lipoproteins: human studies. Am J Clin Nutr 1997; 65: 1645S1654S. Hawksworth G, Hill MJ, Gordillo G, Cuello C. Possible Relationship between Nitrates, Nitrosamines, and Gastric Cancer in Southwest Colombia. In: P. Bogovski, E. A. Walker, and W. Davis (eds.), N-Nitroso Compounds in the EnvironmentLyon. France: International Agency for Research on Cancer. 1974; 9: 229- 234. Hedin SG. Eine methode das lysin zu isolieren, nebst einigen Bemerkungen uber das lysatinin. Z Physiol Chem 1895; 21: 297-305.

190

Hegyi P, Takacs T, Jarmay K, Nagy I, Czako L, Lonovics J. Spontaneous and cholecystokininoctapeptide-promoted regeneration of the pancreas following L-arginine-induced pancreatitis in rat. Int J Pancreatol 1997; 22: 193-200. Hegyi P. L-arginine-induced experimental pancreatitis. World J Gastroenterology 2004; 10(14): 2003-2009. Hems DA and Gaja G. Carbohydrate metabolism in isolated perfused rat kidney. Biochem J 1972; 128: 412-426. Hibbeln J. Fish consumption and major depression. The Lancet 1998; 351: 1213. Hibbs JB Jr, Taintor RR, Vavrin Z. Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite. Science 1987a; 235: 473-476. Hibbs JB Jr, Vavrin Z, Taintor RR. L-arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells. J Immunol 1987b; 138: 550-565. Higdon JV and Frei B. Tea catechins and polyphenols: health effects, metabolism and antioxidant functions. Crit Rev Food Sci Nutr 2003; 43: 89-143. Hill MJ. Nitrates and nitrites in food and water in relation to human disease. In Nitrates and Nitrites in Food and Water. Chichester: Ellis Horwood. 1991; 163±193. Hoffman RA, Langrehr JM, Billiar TR, Curran TR, Simmons RL. Alloantigen-induced activation of rat splenocytes is regulated by the oxidative metabolism of L-arginine. J Immunol 1990; 145: 2220-2226. Hoffman RA, Langrehr JM, Simmons RL. Nitric oxide synthesis: a consequence ofalloimmune interaction. Xeno 1994; 2: 5-7. Holm M, Olbe L, Fandriks L. Intragastric CO2 and nitric oxide participate in the regulation of peptone-induced gastrin release in humans. Scand J Gastroenterol 2000; 35: 1260±1265. Holman RT. Nutritional and metabolic interrelationships between fatty acids. Fed Proc 1964; 23: 1062-1067. Holman RT. The slow discovery of the importance of Z3 essential fatty acids in human health. J Nutr 1998; 128: 427S-433S. 191

Hopfer U, Nelson K, Perotto J, Isselbacher KJ. Glucose transport in isolated brush border membrane from rat small intestine. J Biol Chem 1973; 248: 25-32. Hopfer U. Kinetics of sodium ion dependent D-glucose transport. Am J Physiol 1978; 234: F89F96. Huie RE and Padmaja S. The reaction of NO with superoxide. Free Radic Res Commun 1993; 18:195-199. Iacono JM, Puska P, Dougherty RM. Studies on the effect of dietary fat on blood pressure. Ann Clin Res 1984; 16: 116-125. Iadecola C, Zhang F, Xu X. Inhibition of inducible nitric oxide synthase ameliorates cerebral ischemic damage. American Journal of Physiology 1995; 268: R286-R292. Ide T et al. Comparative effects of perilla and fish oils on the activity and gene expression of fatty acid oxidation enzymes in rat liver. Biochim Biophys Acta 2000; 1485: 23-25. Ignarro LJ, Fukuto JM, Griscavage JM, Rogers NE, Byrns RE. Oxidation of nitric oxide in aqueous solution to nitrite but not nitrate: comparison with enzymatically formed nitric oxide from LArginine. Proc Natl Acad Sci USA 1993; 90: 8103-8107. Ignarro LJ. Endothelium-derived nitric oxide: Pharmacology and relationship to the actions of organic esters. Pharm Res 1989; 6: 651±659. Ishiwata H, Tanimura A, Ishidate M. Nitrite and nitrate concentrations in human saliva collected from salivary ducts. J Food Hyg Soc Jpn 1975; 16: 89±92. Jeong JK, Juedes MJ, Wogan GN. Mutations induced in the supF gene of pSP189 by hydroxyl radical and singlet oxygen: relevance to peroxynitrite mutagenesis. Chem Res Toxicol 1998; 11: 550±556. Jobgena WS, Friedb SK, Fuc WJ, Meiningerd CJ, Wu G. Regulatory role for the arginine±nitric oxide pathway in metabolism of energy substrates. J Nutr Biochem 2006; 17: 571-588. Juedes MJ and Wogan GN. Peroxynitrite-induced mutation spectra of pSP189 following replication in bacteria and in human cells. Mutat Res 1996; 349: 51±61. Jung KH et al. Early intravenous infusion of sodium nitrite protects brain against in vivo ischemiareperfusion injury. Stroke 2006; 37: 2744±2750. 192

.DKO 5³/LYHU´ ,Q 0DUTXDUGW+6FKDIHU6* 0F&OHOODQ5':HOVFK)(GV Toxicology 1st edition NY: Academic Press, 1999; 273-296. Kakar P, Watson T, Gregory YH. New approaches to therapy with omega-3 fatty acids. Curr Atheroscler Rep 2008; 10: 79-87. Kakar P, Watson T, Gregory YH. New approaches to therapy with omega-3 fatty acids. Curr Atheroscler Rep 2008; 10: 79-87. Karlik W et al. The influence of nitrite, nitrate and nitric oxide on ammonia use and urea production in primary cultures of sheep hepatocytes. Toxicology in Vitro 1995; 9: 711±716. Karomgani I, Porter KA, Langevin RE, Banks PA. Prognostic factors in sterile pancreatic necrosis. Gastroenterology 1992; 103: 1636-1640. Karupiah G, Xie Q-W, Buller RML, Nathan C, Duarte C, MacMicking, J D. Inhibition of viral replication by interferon-y-induced nitric oxide synthase. Science 1993; 261:1445-1448. Kaur H and Halliwell B. Evidence for nitric oxide-mediated oxidative damage in chronic inflammation. Nitrotyrosine in serum and synovial fluid from rheumatoid patients. FEBS Lett 1994; 350: 9-12. Kaur M, Kaur J, Ojha S, Mahmood A. Dietary fat effects on brush border membrane composition and enzyme activites in rat intestine. Ann Nutr Metab 1996; 40: 269-276. Kelley DS, Branch LB, Love JE, Taylor PC, Rivera YM, Iacono JM. Dietary D-linolenic acid and immunocompetence in humans. Am J Clin Nutr 1991; 53: 40-46. Kempson SA and Dousa TP. Current concepts of regulation of phosphate transport in reanl proximal tubules. Biochem Pharmacol 1986; 35: 721-726. Kempson SA, Kim JK, Northrup TE, Knox FG, Dousa TP. Alkaline phosphatase in adaptation to low dietary phosphate intake. Am J Physiol Endocrinol Metab 1979; 237: E465- E473. Kempson SA, Turner ST, Yusufi AN, Dousa TP. Actions of NAD+ on renal brush border transport of phosphate in vivo and in vitro. Am J Physiol 1985; 249: F948-955. Kerwin JF Jr, Lancaster JR Jr, Feldman PL. Nitric oxide: a new paradigm for second messengers. J Med Chem 1995; 38: 4343±4362. 193

Kessler M, Acuto O, Storelli C, Murer H , Muller M, Semenza G. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems. Biochim Biophys Acta 1978; 50: 136-154. Khan SA, Priyamvada S, Khan S, Khan MW, Farooq N, Yusufi AN. Protective effect of green tea extract on Gentamicin induced nephrotoxicity and oxidative damage in rat kidney. Pharm Res 2009a; 59: 254-262. Khan SA, Priyamvada S, Khan MW, Khan S, Farooq N and Yusufi AN. Studies on the protective effect of green tea against cisplatin induced nephrotoxicity. Pharm Res 2009b; 60: 382-391. Khundmiri SJ et al. Effect of ischemia and reperfusion on enzymes of carbohydrate metabolism in rat kidney. J Nephrol 2004; 17: 377-383. Khundmiri SJ et al. Effect of reperfusion on sodium dependent phosphate transport in renal brush border membranes. Biochim Biophys Acta 2005; 1716: 19-28. Kielar M et al. New possibilities in treatment of atherosclerosis--increase of nitric oxide synthesis by L-arginine supplementation. Pol Merkuriusz Lek 2004; 17(102): 656-60. Kim HK and Choi H. Dietary alpha-linolenic acid lowers postprandial lipid levels with increase of eicosapentaenoic and docosahexaenoic acid contents in rat hepatic membrane. Lipids 2001; 36: 1331-1336. Kinsella JE, Lokesh B, Stone RA. Dietary n-3 polyunsaturated fatty acids and amelioration of cardiovascular disease: possible mechanisns. Am J Clin Nutr 1990; 52: 1-28. Kishino Y, Kawamura S. Pancreatic damage induced by injecting a large dose of arginine. Virchows Arch B Cell Pathol Incl Mol Pathol 1984; 47: 147-155. Klahr S and Morrissey J. L-arginine as a therapeutic tool in kidney disease. Semin Nephrol 2004; 24(4): 389-94. Klenk E and Mohrhauer H. Studies on the metabolism of polyenoic fatty acids in the rat. Hoppe Seylers Z Physiol Chem 1960; 320: 218-232.

194

Kogteva GS and Bezuglov VV. Unsaturated fatty acids as endogenous bioregulators. Biochem (Mosc) 1998; 63: 4-12. Komatsu W, Ishihara K, Murata M, Saito H, Shinohara K. Docosahexaenoic acid suppresses nitric oxide production and inducible nitric oxide synthase expression in interferon-gamma plus lipopolysaccharide-stimulated murine macrophages by inhibiting the oxidative stress. Free Radic Biol Med 2003; 34: 1006-1016 Kornberg A. Lactic dehydrogenase of muscle. In: Methods Enzymol, Colowick SP and Kaplan NO (editors). New York: Academic Press, 1955; 1: 441-443. Kozlov AV, Dietrich B, Nohl H. Various intracellular compartments cooperate in the release of nitric oxide from glycerol trinitrate in liver. Br J Pharmacol 2003; 139: 989 ±997. Kris-Etherton PM et al. Fish consumption, fish oil, omega-3 fatty acids and cardiovascular diseases. Circulation 2002; 106: 2747-2757. Kromann N and Green A. Epidemiological studies in the Upernavik District, Greenland. Acta Med Scand 1980; 208: 401-406. Kubes P and Granger DN. Nitric oxide modulates microvascular permeability. Am J Physiol 1992; 262: H611-H615. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator ofleukocyte adhesion. Proc Natl Acad Sci U S A 1991; 88: 4651-4655. Lancaster JR Jr, Langrehr JM, Bergonia HA, Murase N, Simmons RL, Hoffman RA. EPR detection of heme and nonheme iron-containing protein nitrosylation by nitric oxide during rejection of rat heart allograft. J Biol Chem 1992; 267: 10994-10998. Larsson S, Kulin M, Ingelman-Sundberg M, Wolk A. Dietary long chain n-3 fatty acids for the prevention of cancer. Am Soc Clin Nutr 2004; 79: 935-945. Laurent G, Kishore BK, Tulkens PM. Aminoglycoside induced renal phospholipidosis and nephrotoxicity. Biochem Pharmacol 1990; 40: 2383-2392. Leaf A. Historical overview of n-3 fatty acids and coronary heart disease. Am J Clin Nutr 2008; 87: 1978S-1980S.

195

Lee JB, Vance VK, Cahill Jr GF. Metabolism of 14C-labelled substrates by rabbit kidney cortex and medulla. Am J Physiol 1962; 203: 27-36. Leung FW. Fish oil protects against ethanol-induced gastric mucosal injury in rats. Digest Dis Sci 1992; 37:636-637. Levinson SA and MacFate RP. Clinical Laboratory Diagnosis (7th ed.), Philadelphia, Lee and Febiger, 1969; pp- 413. Li G, Regunathan S, Barrow CJ, Eshraghi J, Cooper R, Reis DJ. Agmatine: an endogenous clonidine-displacing substance in the brain. Science 1994; 263(5149): 966-9. Li XA, Everson W, Smart EJ. Nitric oxide, caveolae, and vascular pathology. Cardiovasc Toxicol 2006; 6: 1-13. Lichtenberger LM, Kurinets A. Phosphatidylcholine-associated aspirin accelerates healing of gastric ulcers in rats. Digest Dis Sci 1998; 43: 786-790. Lin W et al. Study on DNA strand breaks induced by sodium nitroprusside, a nitric oxide donor, in vivo and in vitro. Mutat Res 2000; 466: 187±195. Lin W, Xue H, Liu S, He Y, Fu J, Zhou Z. Genotoxicity of nitric oxide produced from sodium nitroprusside. Mutat Res 1998; 413: 121±127. Lipton SA et al. A redox based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature 1993; 364: 626-632. Lise B, Nadine B, Marie-Marcelle T. Heterogeneity of nephron anatomy. Kid Int 1987; 31: S25S39. Liu RH and Hotchkiss JH. Potential genotoxicity of chronically elevated nitric oxide: A review. Mutat Res 1995; 339: 73±89. Lorgeril de M and Salen P. Alpha-linolenic acid and coronary heart disease. Nutr Metab Cardiovasc Dis 2004; 14: 162-169. Low H. Nitroso compounds: safety and public health. Archives of Environmental Health 1974; 29: 256±260.

196

Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein determination with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. Lu P et al. Nitrite-derived nitric oxide by xanthine oxidoreductase protects the liver against ischemia-reperfusion injury. Hepatobiliary Pancreat Dis Int 2005; 4: 350±355. Luiking YC, Poeze M, Dejong CH, Ramsay G, Deutz NE. Sepsis: an arginine deficiency state? Crit Care Med 2004; 32: 2135±2145. Lundberg JO and Govoni M. Inorganic nitrate is a possible source for systemic generation of nitric oxide. Free Radical Biol Med 2004; 37: 395±400. Lundberg JO et al. Urinary nitrite: more than a marker of infection. Urology 1997; 50: 189±191. Lundberg JO, Weitzberg E, Cole JA, Benjamin N. Nitrate, bacteria and human health. Nature Rev Microbiol 2004; 2: 593±602. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therpeutics. Nature 2008; 7: 156-167. Lundberg JO, Weitzberg E, Lundberg JM, Alving K. Intragastric nitric oxide production in humans: measurements in expelled air. Gut 1994; 35: 1543±1546. Magee PH and Barnes JM. The production of malignant primary heptic tumors in the rat by feeding dimethylnitrosamine. Br J Cancer 1956; 10: 114±122. Malawista SE, Montgomery RR, Van Blaricom G. Evidence for reactive nitrogen intermediates in killing of staphylococci by human neutrophil cytoplasts. J Clin Invest 1992; 90: 631-636. Maleque A, Endou H, Koseki C, Sakai F. Nephron heterogeneity. Gluconeogenesis from pyruvate in rabbit nephron. FEBS Lett 1980; 116: 154-156. Maletic SD, Dragicevic LJM, Zikic RV, Stajn AS, Kostic MM. Effects of nitric oxide donor, isosorbide dinitrate, on energy metabolism of rat reticulocytes. Physiol Res 1999a; 48: 417-427. Maletic SD, Dragicevic-Djokovic LJM, Jakovljevic VLJ, Milovanovic DR, Rosic MA, Kostic MM. Energy metabolism alterations in rat reticulocytes under the influence of molsidomine. Exp Clin Cardiol 1999b; 4: 152-158.

197

Maletic SD, Dragicevic-Djokovic LJM, Zikic RV, Stajn AS, Milenkovic P, Kostic MM. Effects of nitric oxide donors on energy metabolism of rat erythrocytes. J Environ Pathol Toxicol Oncol 2000; 19: 383-390. Maletic SD, Kostic MM. Effects of nitroglycerin on energy metabolism of rat reticulocytes. J Physiol Pharmacol 1999; 50: 75-87. Mallozzi C, Di Stasi AM, Minetti M. Peroxynitrite modulates tyrosine-dependent signal transduction pathway of human erythrocyte band 3. FASEB J 1997; 11: 1281-1290. Mandel LJ and Balaban RS. Stoichiometry and coupling of active transport of oxidative metabolism in epithelial tissues. Am J Physiol 1981; 240: F357-F371. Mane J et al. Effect of L-arginine on the course of experimental colitis. Clin Nutr 2001; 20(5): 415-22. Manjari V and Das UN. Effect of polyunsaturated fatty acids on dexamethasone-induced gastric mucosal damage. Prostaglandins Leukot Essent Fatty Acids 2000; 62: 85-96. Mantzioris E, James MJ, Gibson RA, Cheland LG. Dietary substitution with an ALA rich vegetable oil increases EPA concentrations in tissues. Am J Clin Nutr 1994; 59: 1304-1307. Marcel YL, Christiansen K, Holman RT. The preferred metabolic pathway from linoleic acid to arachidonic acid in vitro. Biochim Biophys Acta 1968; 164: 25-34. Marinetti, GV. Chromatographic separation identification and analysis of phosphatides. J Lipid Res 1962; 3: 1-20. Marklund S and Marklund G. Involvement of the superoxide anion radical in the auto oxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974; 47: 469-474. Mason J. The pathophysiology of ischaemic acute renal failure. A new hypothesis about the initiation phase. Renal Physiol 1986; 9: 129-147. Massary SG and Fleisch H. Renal handling of phosphate. New York: Plenum Medical Book. Company, New York-London, 1980. Masters C. Omega-3 fatty acids and the peroxisome. Mol Cell Biochem 1996; 165: 83-93.

198

Meyer AH, Kornberg A, Grisolia S, Ochoa S. Enzymic mechanism of oxidation reactions between malate or isocitrate and pyruvate. J Biol Chem 1948; 174: 961-977. Miller MJS et al. Role of inducible nitric oxide synthase expression and peroxynitrite formation in guinea pig ileitis. Gastroenterology 1995; 109: 1475±1483. Mirvish SS, Bulay O, Runge RG, Patil K. Study of the carcinogenicity of large doses of dimethylnitrosamine, N-nitroso-l-proline, and sodium nitrite administered in drinking water to rats. J Natl Cancer Inst 1980; 64: 1435±42. Mirvish SS. Formation of N-nitroso compounds: chemistry, kinetics, and in vivo occurrence. Toxicol Appl Pharmacol 1975; 31: 325±351. Mirvish SS. Role of N-nitroso compounds (NOC) and N-nitrosation in etiology of gastric, esophageal, nasopharyngeal and bladder cancer and contribution to cancer of known exposures to NOC. Cancer Lett 1995; 93: 17-48. Mizunuma T, Kawamura S, Kishino Y. Effects of injecting excess arginine on rat pancreas. J Nutr 1984; 114: 467-471. Moncada S and Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993; 329: 20022012. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991; 43: 109-42. Montague PR, Gancayco CD, Winn MJ, Marchase RB, Friedlander MJ. Role of NO production in NMDA receptor-mediated neurotransmitter release in cerebral cortex. Science 1994; 263:973-977. Montori VM, Wollan PC, Farmer A, Dinneen SF. Fish oil supplementation in type 2 diabetes. Diabetes Care 2000; 23: 1407-1415. Morgan DM. Polyamines, arginine and nitric oxide. Biochem Soc Trans 1994; 22(4): 879-83. Moro MA et al. Paradoxical fate and biological action of peroxynitrite on human platelets. Proc Natl Acad Sci USA 1994; 91: 6702-6706. Mostad IL, Bjerve KS, Bjorgass MR, Lydersen S, Grill V. Effects of n-3 fatty acids in subjects with type 2 diabetes: reduction of insulin sensitivity and time dependent alteration from carbohydrate to fat metabolism. Am J Clin Nutr 2006; 84: 540-550. 199

Motoo Y, Taga K, Su SB, Xie MJ, Sawabu N. Arginine induces apoptosis and gene expression of pancreatitis-associated protein (PAP) in rat pancreatic acinar AR4-2J cells. Pancreas 2000; 20: 61-66. Mouratoff GJ, Carroll NV, Scott EM. Diabetes mellitus in Eskimos. JAMA 1967; 199: 107-112. Mozaffarian D. Does alpha-linolenic acid intake reduce the risk of coronary heart disease? A review of the evidence. Altern Ther Health Med 2005; 11: 24-30. Mulligan SM, Hevel JM, Marletta MA, Ward PA. Tissue injury caused by deposition of immune complexes is L-arginine dependent. Proc Natl Acad Sci USA 1991; 88: 6338-6342. Nagababu E, Ramasamy S, Abernethy DR, Rifkind JM. Active nitric oxide produced in the red cell under hypoxic conditions by deoxyhemoglobin-mediated nitrite reduction. J Biol Chem 2003; 278: 46349±46356. Nakaki T and Kato R. Beneficial circulatory effect of L-arginine. Jpn J Pharmacol 1994; 66(2): 167-71. Nakaki T, Nakayama M, Kato R. Inhibition by nitric oxide and nitric oxide-producing vasodilators of DNA synthesis in vascular smooth muscle cells. Eur J Pharmacol 1990; 1898: 347-353. Nannicini F, Sofi F, Avanzi G, Abbate R, Gensini GF. Alpha-linolenic acid and cardiovascular diseases omega 3 fatty acids beyond eicosapentaenoic acid and docosahexaenoic acid. Minerva Cardioangiol 2006; 54: 431-442. Napoli C, de Nigris F, Williams-Ignarro S, Pignalosa O, Sica V, Ignarro LJ. Nitric oxide and atherosclerosis: An update. Nitric Oxide 2006; 15: 265-279. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992; 6: 3051±3064. National Toxicology Program,. NTP Toxicology and Carcinogenesis Studies of SODIUM NITRITE (CAS NO. 7632-00-0) Drinking Water Studies in F344/N Rats and B6C3F1 Mice. National Toxicology Program technical report series 2001; 495: 1±274. Neuringer M. Infant vision and retinal function in studies of dietary long-chain polyunsaturated fatty acids: methods, results, and implications. Am J Clin Nutr 2000; 71: 256S-267S.

200

Nguyen T, Brunson D, Crespi CL, Penman BW, Wishnok JS, Tannenbaum SR: DNA damage and mutation in human cells exposed to nitric oxide in vitro. Proc Natl Acad Sci USA 1992; 89: 30303034. Nickerson M. In: Goodman, L.S., Gilm1an, A. (Eds.), The Pharmacological Basis of Therapeutics, 4th edition. London, MacMillan, 1970; 745-763. Nohl H, Staniek K, Sobhian B, Bahrami S, Redl H, Kozlov AV. Mitochondria recycle nitrite back to the bioregulator nitric monoxide. Acta Biochim Pol 2000; 47:913±921. Nussler A, Drapier J-C, Renia L, Pied , Miltgen S, Gentilini M, Mazier D. L-arginine-dependent destruction of intrahepatic malaria parasites in response to tumor necrosis factor and/or interleukin 6 stimulation. Eur J Immunol 1991; 21: 227-230. Nussler AK and Billiar TR. Inflammation, immunoregulation, and inducible nitric oxide synthase. J Leukoc Biol 1993; 54: 171-178. Ochoa S, Mehler AH, Kornberg A. Biosynthesis of dicarboxylic acids by carbon dioxide fixation I. Isolation and properties of an enzyme from pigeon liver catalyzing the reversible oxidative decarboxylation of L-malic acid. J Biol Chem 1948; 174: 979-1000. Ockermann PA. Glucose-6-phosphatase in human jejunal mucosa properties demonstrating the specific character of the enzyme activity. Biochim Biophys Acta 1965; 105: 22-23. Oestergaard L. et al. Diminished NO release in chronic hypoxic human endothelial cells. Am J Physiol Heart Circ Physiol 2007; 293: H2894±H2903. O'Hara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide production. J Clin Invest 1993; 91: 2541-2551. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric aciShull KH, Ashmore J, Mayer J. Hexokinase, glucose-6-phosphatase and phosphorylase levels in hereditarily obese, hyperglycemic mice. Arch Biochem Biophysics 1956; 62: 210-216. Ohnuma S, Hirooka K, Hemmi H, Ishida C, Ohto C, Nishino T. Conversion of product specifcity of archaebacterial geranylgeranyl-diphosphate synthase. Identification of essential amino acid residues for chain length determination of prenyltransferase reaction. Journal of Biological Chemistry 1996; 271: 18831±18837. 201

Ohshima H and Bartsch H. Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutat Res 1994; 305: 253±264. Ozgocmen S, Atalay Catal S, Ardicoglu O, Kamanli A. Effect of omega-3 in the management of fibromylgia syndrome. Int J Clin Pharm 2000; 38: 362-363. Paakkari I and Lindsberg P. Nitric oxide in the central nervous system. Ann Med 1995; 27: 369377. Paquay JBG, Haenen GRMM, Stender G, Wiseman SA, Tijburg LBM, Bast A. Protection against Nitric Oxide Toxicity by Tea. J Agric Food Chem 2000; 48(11): 5768±5772. Paschos GK, Magkos F, Panagiotakos DB, Votteas V, Zampelas A. Dietary supplementation with flaxseed oil lowers blood pressure in dyslipidaemic patients. Eur J Clin Nutr 2007; 61: 1201-1206. Petersson J et al. Dietary nitrate increases gastric mucosal blood flow and mucosal defense. Am J Physiol Gastrointest Liver Physiol 2007; 292: G718±G724. Pitts RF and Macleod MB. Metabolism of blood glucose by the intact functioning kidney of the dog. Kid Int 1975; 7: 130-136. Pitts RF. Production of CO2 by the intact functioning kidney of the dog. Med Clin N Amer 1975; 59: 507-518. Porter GA and Bennett WM. Nephrotoxic acute renal failure due to common drugs. Am J Physiol 1981; 241: F1-F8. Posta TL, Gebauer SK, Kris-Etherton P. Dietary omega-3 fatty acid intake and cardiovascular risk. Am J Cardiol 2006; 98: 3i-18i. Potenza MA, Nacci C, Mitolo-Chieppa D. Immunoregulatory effects of L-arginine and therapeutical implications. Curr Drug Targets Immune Endocr Metabol Disord 2001; 1: 67-77. Prasad K. Flaxseed: a source of hypocholesterolemic and antiatherogenic agents. Drugs News Perspect 2000; 13: 99-104. Priyamvada S et al. Studies on the protective effect of dietary fish oil on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney. Prostaglandins Leukot Essent Fatty Acids 2008; 78(6): 369-81.

202

Priyamvada S et al. Studies on the protective effect of dietary fish oil on uranyl nitrate-induced nephrotoxicity and oxidative damage in rat kidney. Prostaglandins Leukot Essent Fatty Acids 2010; 82(1): 35-44 Rachmilewitz D et al. Peroxynitrite-induced rat colitis: A new model of colonic inflammation. Gastroenterology 1993; 105: 1681-1688. Radi R, Beckman JS, Bush KM, Freeman BA: Peroxynitrite oxidation of sulfhydryls: The cytotoxic potential of superoxide and nitric oxide. J Biol Chem 1991; 266: 4244-4250. Radomski MW, Palmer RMJ, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 1987; ii: 1057-1058. Rajendran VM, Barry JA, Kleinman JG, Ramaswamy K. Proton gradient dependent transport of glycine in rabbit renal brush border membrane vesicles. J Biol Chem 1987; 262: 14974-14977. Rajfer J, Aronson WJ, Bush PA, Dorey FJ, Ignarro LJ. Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission. N Engl J Med 1992; 326: 90-94. Rakonczay Z et al. NF-kappaB activation is detrimental in arginine-induced acute pancreatitis. Free Radic Biol Med 2003; 34: 696-709 Ramakrishna RDN and Cederbaum AI. Generation of reactive oxygen species by the redox cycling of nitroprusside. Biochim Biophys Acta 1996; 1289: 195±202. Ramaswamy K, Harig JM, Wilson D, Rajendran VM. Nutrient and electrolyte transport in human small intestinal brush border membrane vesicles. Indian J Biochem Biophys 1991; 28: 340-345. Rapoport SM: The Reticulocyte. CRC Press, Boca Raton, Florida, 1986. Rassaf T et al. Nitrite reductase function of deoxymyoglobin: oxygen sensor and regulator of cardiac energetics and function. Circ Res 2007; 100: 1749±1754. 5D\QHU7(DQG+RZH35&3XULILHGȦ-3 fatty acids retard the development of proteinuria in saltloaded hypertensive rats. J Hypertens 1995; 13: 771-780. Renner IG, Savage WT, Pantoga JL, Renner VJ. Death due to acute pancreatitis. A retrospective analysis of 405 autopsy cases. Dig Dis Sci 1985; 10: 1005-1008.

203

Rhoden EL, Lucas ML, Pereira-Lima L, Rhoden CR, Souto CA. Effects of L-arginine on the kidney levels of malondialdehyde in rats submitted to renal ischaemia-reperfusion. BJU Int 2001; 88(3): 273-277. Rodriguez J et al. Chemical nature of nitric oxide storage forms in rat vascular tissue. Proc Natl Acad Sci USA 2003; 100: 336±341. Rose WC. The nutritional significance of the amino acids. Physiol Rev 1938; 18: 109-136. Ruiz-Gutierrez V, Perez-Espinosa A, Vazquez CM, Santa ±Maria C. Effects of dietary fats (fish, olive and high-oleic acid sunflower oils) on lipid composition and antioxidant enzymes in rat liver. Br J Nutr 1999; 82: 233-241. Salomon P, Kornbluth AA, Janowitz HD. Treatment of ulcerative colitis with fish oil n-3 omega fatty acid an open trial. J Clin Gastroenterol 1990; 12: 157-161. Sampson JB, Ye Y, Rosen H, Beckman JS. Myeloperoxidase and horseradish catalyze tyrosine nitration in proteins from nitrite and hydrogen peroxide. Arch Biochem Biophys 1998; 356: 207213. Sanchez DZ, Belles M, Albina ML, Sirvent JJ, Domingo JL. Nephrotoxicity of simultaneous exposure to mercury and uranium in comparison to individual effects of these metals in rats. Biol Trace Elem Res 2001; 84: 139-154. Sandhu JK and Birnboim HC. Mutagenicity and cytotoxicity of reactive oxygen and nitrogen species in the MN-11 murine tumor cell line. Mutat Res 1997; 379: 241±252. Santamaria. Nitrate in vegetables: toxicity, content, intake and EC regulation. J of Sci of Food and Agri 2006; 86(8): 10-17. Schmidt HH, Warner TD, Murad F. Double-edged role of endogenous nitric oxide. Lancet 1992; 339: 986. Schmitz J, Preiser H, Maestracci D, Ghosh BK, Cerda JJ, Crane RK. Purification of the human intestinal brush border membrane. Biochim Biophys Acta 1973; 328: 98-112. Schultz SG. Sodium coupled solute transport by small intestine: A status report. Am J Physiol 1977; 233: E249-E254. Schulze E and Steiger E. Uber das Arginin. Z Physiol Chem 1886; 11: 43-65. 204

Schuman EM and Madison DV. A requirement for the intercellular messenger nitric oxide in longterm potentiation. Science 1991; 254: 1503-1506. Schumann K, Elsenhans B, Richter E. Gastrointestinal tract. In: Toxicology, Marquardt H, Schafer SG, McClellan R and Welsch F (editors). New York: Academic Press, 1999; pp. 573. Sedlak J and Lindsay RH. Estimation of total protein bound and non protein bound SH groups in WLVVXHZLWK(OOPDQ¶VUHDJHQWAnal Biochem 1968; 25: 192-205. Sen NP, Smith DC, Schwinghamer L. Formation of Nnitrosamines from secondary amines and nitrate in human and animal gastric juice. Food and Cosmetics Toxicology 1969; 7: 301± 307. Seo T, Blaner WS, Deekelbaum RJ. Omega-3 fatty acids: molecular approaches to optimal biological outcomes. Curr Opin Lipidol 2005; 16: 11-8. Shah SV, Kempson SA, Northrup TE, Dousa TP. Renal adaptation to a low phosphate diet in rats. J Clin Invest 1979; 64: 955-966. Sherwood L. Human Physiology: From cells to systems. 3rd Ed. Boston: Wadsworth, 1997. Shiva S et al. Ceruloplasmin is a NO oxidase and nitrite synthase that determines endocrine NO homeostasis. Nature Chem Biol 2006; 2: 486±493. Shiva S et al. Nitrite augments tolerance to ischemia/ reperfusion injury via the modulation of mitochondrial electron transfer. J Exp Med 2007; 204: 2089±2102. Shonk CC and Boxer GE. Enzyme patterns in human tissues I. Methods for determination of glycolytic enzymes. Cancer Res 1964; 24: 709-721. Shull KH, Ashmore J, Mayer J. Hexokinase, glucose-6-phosphatase and phosphorylase levels in hereditarily obese, hyperglycemic mice. Arch Biochem Biophysics 1956; 62: 210-216. Siddiqui RA, Jenski LJ, Harvey KA, Wiesehan JD, Stillwell W, Zaloga GP. Cell-cycle arrest in Jurkat Leukaemic cells: a possible role for docosahexaenoic acid. Biochem J 2003; 371: 621-629. Sidney M and Morris Jr. Regulation of enzymes of the urea cycle and arginine metabolism. Annu Rev Nutr 2002; 22: 87-105. Sigrist-Nelson K, Sigrist H, Bercovici T, Gitler C. Intrinsic proteins of the intestinal microvillus membrane. Iodonaphthylazide labeling studies. Biochim Biophys Acta 1977; 468: 163-176. 205

6LPRSRXORV$3.LIHU550DUWLQ5(HGLWRUV ³Health effects of polyunsaturated fatty acids in seafoods´2UODQGR)/$FDGHPLF3UHVV Simopoulos AP. Omega-3 fatty acids in wild plants nuts and seeds. Asia Pac J Clin Nutr 2002b; 11: S163-S173. Simopoulos AP. Human requirement for n-3 polyuinsaturated fatty acids. Poult Sci 2000; 79: 961970. Simopoulos AP. Omega-3 fatty acids in health and disease and in growth and development. Am J Clin Nutr 1991; 54: 438-463. Simopoulos AP. The Mediterranean diets: what is so special about the diet of Greece? The scientific evidence. J Nutr 2001; 131: 3065S-3073S. Sladek SM, Regenstein AC, Lykins D, Rober JM. Nitric oxide synthase activity in pregnant rabbit uterus decreases on the last day ofpregnancy. Am J Obstet Gynecol 1993; 169: 1285-1291. Small MF. The happy fat. New Scientist 2002; 34-37. Smith-Barbaro PA and Pucak GJ. Dietary fat and blood pressure. Ann Intern Med 1983; 98: 828831. Sobko T et al. Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite. Nitric Oxide 2005; 13: 272±278. Speck RF, Lauterburgh BH. Fish oil protects mice against acetaminophen hepatotoxocity in vivo. Hepatol 1991; 13: 557-561. Spencer JM, Whiteman M, Jenner A, Halliwell B. Nitrite-induced deamination and hypochloriteinduced oxidation of DNA in intact human respiratory tract epithelial cells. Free Radic Biol Med 2000; 28(7): 1039-1050. Spiegelhalder B, Eisenbrand G, Preussman R. Influence of dietary nitrate on nitrite content of human saliva: possible relevance to in vivo formation of N̻nitroso compounds. Food Cosmet Toxicol 1976; 14: 545±548. Stamler JS, Singel DJ, Loscalzo J. Biochemistry of nitric oxide and its redox-activated forms. Science 1992; 258: 1898-1902.

206

Starodubtseva MN, Ignatenko VA, Cherenkevich SN. Damage to erythrocytes initiated by the interaction of nitrite ions with hemoglobin. Biofizika 1999; 44: 1068±1972. Steinberg W and Tenner S. Acute pancreatitis. N Eng J Med 1994; 330: 1198-1220. Steinhausen M and Parekh N. Principles of acute renal failure. In: Nephrology, R. R. Robinson (editor). New York: Springer-Verlag, 1984, 1: 702-710. Stenson WF, Seetharam B, Talkad V, Pickett W, Dudeja P, Brasitus TA. Effects of dietary fish oil supplementation on membrane fluidity and enzyme activity in rat small intestine. Biochem J 1989; 263: 41-45. Stochs SJ, Bagchi D, Hassoun E, Bagchi M. Oxidative mechanisms in the toxicity of chromium and cadmium ions. J Environ Pathol Toxicol and Oncol 2000; 19: 201-213. Stuehr DJ and Nathan CF. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 1989; 169: 1543±1555. Sun J, Aoki K, Wang W, Guo A, Misumi J. Sodium nitrite-induced cytotoxicity in cultured human gastric epithelial cells. Toxicol In Vitro 2006; 20: 1133±1138. Tak Yee AW. Molecular and cellular responses to oxidative stress and changes in oxidationreduction imbalance in the intestine. Am J Clin Nutr 1999; 70: 557-565. Takacs T et al. Comparative effects of water immersion pretreatment on three different acute pancreatitis models in rats. Biochem Cell Biol 2002b; 80: 241-251. Takacs T et al. The role of nitric oxide in edema formation in L-arginine-induced acute pancreatitis. Pancreas 2002a; 25: 277-282. Takacs T, Czako L, Jarmay K, Farkas G Jr, Mandi Y, Lonovics J. Cytokine level changes in Larginine-induced acute pancreatitis in rat. Acta Physiol Hung 1996; 84: 147-156. Tannenbaum SR et al. Nitrite in human saliva. Its possible relationship to nitrosamine formation. J Natl Cancer Inst 1974; 53: 79±84. Tannenbaum SR, Frett D, Young VR, Land PD, Bruce WR. Nitrite and nitrate are formed by endogenous synthesis in the human intestine. Science 1978; 200: 1487-1489. Tausky HH and Shorr EA. A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem 1953; 202: 675-685. 207

Teitelbaum JE and Walker WA. Review: the role of omega 3 fatty acids in intestinal inflammation. J Nutr Biochem 2001; 12: 21-32. Thakkar RR et al. Docosahexaenoic acid reverses cyclosporin-A induced changes in membrane tructure and function. Biochim Biophys Acta 2000; 1474: 183-195. Thomson ABR, Keelan M, Garg M, Clandinin MT. Dietary effects of Z3-fatty acids on intestinal transport function. Can J Physiol Pharmacol 1988; 66: 985-992. Thomson ABR, Keelan M, Garg ML, Clandinin MT. Influence of dietary fat composition on intestinal absorption in the rat. Lipids 1989; 24: 494-501. Thurau K. Renal Na+ reabsorption and O2 uptake in dogs during hypoxia and HCTZ infusion. Proc Soc Exp Biol Med 1961; 19: 714-717. Tinoco J. Dietary requirements and functions of alpha-linolenic acid in animals. Prog Lipid Res 1982; 21: 1-45. Tischner R, Planchet E, Kaiser WM. Mitochondrial electron transport as a source for nitric oxide in the unicellular green alga Chlorella sorokiniana. FEBS Lett 2004; 576: 151±155. Torelli G, Mella E, Faelli A, Constantini S. Energy requirements for sodium reabsorption in the in vivo rabbit kidney. Am J Physiol 1986; 211: F576-F580. Tripatara P et al. Nitrite-derived nitric oxide protects the rat kidney against ischemia/reperfusion injury in vivo: role for xanthine oxidoreductase. J Am Soc Nephrol 2007; 18: 570±580. Tsujikawa T et al. Clinical importance of n-3 fatty acid rich diet and nutritional education for the PDLQWHQDQFHRIUHPLVVLRQLQ&URKQ¶VGLVHDVHJ Gastroenterol 2000; 35: 99-104. Tunctan B and Altug S. The use of nitric oxide synthase inhibitors in inflammatory diseases: A novel class of anti-inflammatory agents. Curr Med Chem Anti-Inflammatory & Anti-Allergy Agents 2004; 3: 271-301. Tunctan B, Uludag O, Altug S, Abacioglu N. Effects of nitric oxide synthase inhibition in lipopolysaccharide-induced sepsis in mice. Pharmacol Res 1998; 38: 405-411. Tunctan B, Uludag O, Altug S, Abacioglu N. Time-dependent variations in serum nitrite, 6-ketoprostaglandin FĮ and thromboxane B2 levels induced by lipopolysaccharide in mice. Biol Rhythm Res 2000; 31: 499-514. 208

Tunctan B, Uludag O, Altug S, Demirkay B, Abacioglu N. Effects of cyclooxygenase inhibitors on nitric oxide production and survival in a mice model of sepsis. Pharmacol Res, 2003; 48: 3748. Turpeinen O. Further studies on the unsaturated fatty acids essential in nutrition. J Nutr 1937; 15: 351-366. 7]DQDNDNLVHWDO³/LYHU$VVLVW'HYLFHV´$QQXDO5HYLHZRI%LRFKHPLFDO(QJLQHHULQJ 2000; 2: 607632. Uauy R, Hoffman DR, Peirano P, Birch DG, Birch EE. Essential fatty acids in visual and brain development. Lipids 2001; 36: 885-895. Udo Erasmus. Fats That Heal, Fats that kill. ISBN: 9780920470381. Books Alive: Publisher, 1993. Ullrich KJ, Caspasso G, Rumrich G, Papavassiliou F, Kloss SC. Coupling between proximal tubular transport processes. Studies with ouabain, SITS, and HCO3 free solutions. Pflugers Arch 1977; 368: 245-252. Ullrich KJ, Rumrich G, Kloss S. Specificity and sodium dependence of the active sugar transport in the proximal convolution of the rat kidney. Pflugers Arch 1974; 351: 35-48. Walker PD, Barri Y, Shah SV. Oxidant mechanisms on gentamicin nephrotoxicity. Ren Fail 1999; 21: 433-442. Walker R. The metabolism of dietary nitrites and nitrates. Biochem Soc Trans 1996; 24(3): 780± 785. Valsta LM, Salminen I, Aro A, Mutanen M. Alpha-linolenic acid in rapeseed oil partly compensates for the effect of fish restriction on plasma long chain n-3 fatty acids. Eur J Clin Nutr 1996; 50: 229-235. Van Der Vliet. A et al. Interactions of peroxynitrite with human plasma and its constituents: oxidative damage and antioxidant depletion. Biochem J 1994; 303: 295-301. Varga IS et al. Oxidative stress changes in L-arginine-induced pancreatitis in rats. Pancreas 1997b; 14: 355-359.

209

Varga IS, Matkovics B, Hai DQ, Kotorman M, Takacs T, Sasvari M. Lipid peroxidation and antioxidant system changes in acute Larginine pancreatitis in rats. Acta Physiol Hung 1997a; 85: 129-138. Watkins BA, Hannon K, Ferruzzi M, Li Y. Dietary PUFA and flavonoids as deterrents for environmental pollutants. J Nutr Biochem 2007; 18: 196-205. Watkins BA, Hannon K, Ferruzzi M, Li Y. Dietary PUFA and flavonoids as deterrents for environmental pollutants. J Nutr Biochem 2007; 18: 196-205. Webb A et al. Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemiareperfusion damage. Proc Natl Acad Sci USA 2004; 101: 13683±13688. Weinberg JM, Davis JA, Abarzua M, Smith RK, Kunkel R. Ouabain-induced lethal proximal tubule cell injury is prevented by glycine. Am J Physiol Renal Physiol 1990; 258: F346-F355. Weitzberg E and Lundberg JO. Nonenzymatic nitric oxide production in humans. Nitric Oxide 1998; 2: 1±7. Weller R et al. Nitric oxide is generated on the skin surface by reduction of sweat nitrate. J Invest Dermatol 1996; 107: 327±331. Wennmalm A et al. Nitric oxide synthesis and metabolism in man. Ann NY Acad Sci 1994; 714: 158±164. Verjee ZHM. Isolation of three acid phosphatases from wheat germ. Eur J Biochem 1969; 9: 439444. White JW. Relative significance of dietary source of nitrate and nitrite. J Am Food Chem 1975; 23: 886-891. Vijaimohan K, Jainu M, Sabitha KE, Suramaniyam S, Anandhan C, Shyamala Devi CS. Beneficial effects of alpha linolenic acid rich flaxseed oil on growth performance and hepatic cholesterol metabolism in high fat diet rats. Life Sci 2006; 79: 448-454. Wilcox CS et al. Nitric oxide synthase in macula densa regulates glomerular capillary pressure. Proc NatI Acad Sci USA 1992; 89:11993-11997.

210

Williams D, Verghese M, Walker LT, Boateng J, Shackelford L, Chawan CB. Flax seed oil and flax seed meal reduce the formation of aberrant crypt foci (ACF) in azoxymethane-induced colon cancer in Fisher 344 male rats. Food Chem Toxicol 2007; 45: 153-159. Willis AL. The eicosanoids: an introduction and overview. In: CRC Handbook of Eicosanoids: prostaglandin and related lipids, Willis AL (editor) 1987 CRC Press Inc., Boca Raton, Florida. 1987; 1: 3-46. Wink DA et al. DNA deaminating ability and genotoxicity of nitric oxide and its progenitors. Science 1991; 254: 1001-1003. Wink DA et al. The chemical biology of NO. Insights into regulation, protective and toxic mechanisms of nitric oxide. Curr Top Cell Regul 1996b; 34: 159±187. Wink DA, Darbyshire JF, Nims RW, Saavedra JE, Ford PC. Reactions of the bioregulatory agent nitric oxide in oxygenated aqueous media: Determination of the kinetics for oxidation and nitrosation by intermediates generated in the nitric oxide/oxygen reaction Chem Res Toxicol 1993; 6: 23-27. Wink DA, Grisham M, Mitchell JB, Ford PC. Direct and indirect effects of nitric oxide. Biologically relevant chemical reactions in biology of NO. Methods Enzymol 1996a; 268: 12±31. Witztum JL and Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest 1991; 88: 1785-1792. Woodhall PB, Tisher CC, Simonton CA, Robinson RR. Relationship between paraaminohippurate secretion and cellular morphology in rabbit proximal tubules. J Clin Invest 1978; 61: 1320-1329. Vorum H, Jessen H, Jorgesnesen KE, Sheikh MI. Mechanism of transport of L-alanine by luminal membrane vesicles from pars recta of rabbit proximal tubule. FEBS Lett 1988; 227: 35-38. Vosgen W. Curing - are nitrite and nitrate necessary or superfluous as curing substances. Fleishwirtschaft 1992; 72: 1675-1678. Xi S and Chen LH. Effects of dietary fish oil on tissue glutathione and antioxidant defense enzymes in mice with murine AIDS. Nutr Res 2000; 20: 1287-1299.

211

Xu W, Liu L, Smith GC, Charles G. Nitric oxide upregulates expression of DNA-PKcs to protect cells from DNA-damaging anti-tumour agents. Nature Cell Biol 2000; 2: 339±345. Yagil Y. Acute effect of cyclosporine on inner medullary blood flow in normal and postischemic rat kidney. Am J Physiol Renal Physiol 1990; 258: F1139-F1144. Yerushalmi HF et al. Role of polyamines in arginine-dependent colon carcinogenesis in ApcMin/+ mice. Molecular Carcinogenesis 2006; 45(2): 93-105. Yilmaz HR, Songur A, Ozyurt B, Zararsiz I, Sarsilmaz M. The effect of n-3 polyunsaturated fatty acids by gavage on some metabolic enzymes of rat liver. Prostaglandin Leukot Essent Fatty Acids 2004; 71: 131-135. Yoshida H, Ikeda I, Tomooka M, Imaizumi K, Seto, A, Tsuji H. Effect of dietary seal and fish oils on lipid metabolism in hamsters. J Nutr Sci Vitaminol (Tokyo) 2001; 47: 242-247. Yusufi ANK and Dousa TP. Studies on rabbit kidney brush border membranes: relationship between phosphate transport, alkaline phosphatase and NAD. Minerva Elect Metab 1987; 13: 397404. Yusufi ANK et al. Differential effects of low-dose docosahexaenoic acid and eicosapentaenoic acid on the regulation of mitogenic signaling pathways in mesangial cells. J Lab Clin Med 2003; 141: 318-329. Yusufi ANK, Low MG, Turner ST, Dousa TP. Selective removal of alkaline phosphatase from renal brush border membrane and sodium dependent brush border membrane transport. J Biol Chem 1983; 258: 5695-5701. Yusufi ANK, Murayama N, Gapstur SM, Szczepanska-Konkel M, and Dousa TP. Differential properties of brush border membrane vesicles from early and late proximal tubules of rat kidney. Biochim Biophys Acta 1994; 1191: 117-132. Zaldivar R and Robinson HZ. Epidemiological Investigation on Stomach Cancer Mortality in Chileans: Association with Nitrate. Fertilizei Z Krebforsch 1973; 80: 289-295. Zamaria N. Alteration of polyunsaturated fatty acid status and metabolism in health and disease. Reprod Nutr Dev 2004; 44: 273-282.

212

Zetterquist H. The ultrastructural organization of the columnar absorbing cells of the mouse jejunum. Monograph Aktieobolaget Godvil, Stockholm 1956. Zhang F, Casey RM, Ross ME, Iadecola C. Aminoguanidine ameliorates and L-arginine worsens brain damage from intraluminal middle cerebral artery occlusion. Stroke 1996; 27(2): 317-23. Zhu ZR, Mannisto JAS, Pietinene P. Fatty acid composition of breast adipose tissue in breast cancer patients and patients with benign breast disease. Nutr Cancer 1995; 24: 151-160. Zimmerman HJ. Hepatotoxicity. Disease-A-Month-Series. 1993; 39: 675-787. Zlatkis A, Zak B, Boyle AJ. A new method for the direct determination of serum cholesterol. J Lab Clin Med 1953; 41: 486-492.

213

Peer ±reviewed articles that materialized from this doctoral work 1. Khan MW, Priyamvada S, Khan SA, Khan S, Naqshbandi A, Yusufi ANK. Protective effect of Ȧ-3 polyunsaturated fatty acids (PUFA) on L-arginine induced nephrotoxicity and oxidative damage in rat kidney. Human and Experimental Toxicology 2012;31(10) 1022-1034. ISSN: 09603271. 2. Khan MW, Priyamvada S, Khan SA, Khan S, Naqshbandi A, Yusufi ANK. Protective effect of Ȧ-3 polyunsaturated fatty acids (PUFA) on Sodium nitroprusside induced nephrotoxicity and oxidative damage in rat kidney. Human and Experimental Toxicology 2012; 31(10) 1035-1049 ISSN: 0960-3271. 3. Md. Wasim Khan, Natarajan Anzabaghan Arivarasu, Shubha Priyamvada, Sara A Khan, Sheeba Khan, ANK, Yusufi. Studies on the protective effect of polyunsaturated fatty acids on sodium nitrite induced nephrotoxicity in rats. (2013) Journal of Functional Foods 2013; 5(2) 956-967. ISSN: 1756-4646.

Conference Proceedings that materialized from this doctoral work 1. Md. Wasim Khan, Shubha Priyamvada, Sheeba Khan, Sara A Khan, A.N.K. Yusufi. Effect of sodium nitrite on enzymes of carbohydrate metabolism, brush border membrane, oxidative stress and phosphate transport in rat kidney. International Symposium on the Predictive Preventive and Mechanistic Mutagenesis and XXXIII EMSI Annual Meeting, 2008. 2. Khan MW, Priyamvada S, Khan S, Khan SA, Yusufi ANK. ProtHFWLYH HIIHFW RI Ȧ-3 polyunsaturated fatty acids on sodium nitrite induced nephrotoxicity and oxidative damage in kidney. National symposium on Recent advances in Biochemistry and Allied Sciences. Department of Biochemistry, Aligarh Muslim University, Aligarh, India, 2008. 3. Md. Wasim Khan, Shubha Priyamvada, Sheeba Khan, Sara Anees Khan, Ahad Noor Khan Yusufi. Effect of sodium nitropruside on enzymes of carbohydrate metabolism, brush border membrane, oxidative stress and phosphate transport in rat kidney. SFRR-International Satellite Symposium, AMU, Aligarh, India 2009.