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Influence of Sodium Fluoride and Caffeine on the Kidney Function and Free-Radical Processes in that Organ in Adult Rats 1 EWA BIRKNER,1 E . WA GRUCKA-MAMCZAR, KRYSTYNA ZWIRSKA-KORCZALA,2 ˛ ,3 JOLANTA ZALEJSKA-FIOLKA,1 BARBARA STAWIARSKA-PIETA SL⁄ AWOMIR KASPERCZYK,*,1 AND ALEKSANDRA KASPERCZYK1
Departments of 1Biochemistry, 2Physiology, and Pathology, Silesian Medical University in Katowice, Jordana 19, 41-808 Zabrze, Poland e-mail:
[email protected]
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Received February 7, 2005; Revised April 22, 2005; Accepted May 5, 2005
ABSTRACT An experiment was carried out on Sprague–Dawley rats (adult males) that for 50 days were administered, in the drinking water, NaF and NaF with caffeine (doses, respectively: 4.9 mg of NaF/kg body mass/24 h and 3 mg of caffeine/kg body mass/24 h). Disturbances were noted in the functioning of kidneys, which were particularly noticeable after the administration of NaF with caffeine. Changes in the functioning of kidneys were also confirmed by such parameters as the level of creatinine, urea, protein, and calcium. Modifications of the enzymatic antioxidative system (superoxide dismutase, catalase, and glutathione peroxidase) and lipid peroxidation (malondialdehyde) were also observed. Changes in the contents of the above parameters as well as pathomorphological examinations suggest increased diuresis, resulting in dehydration of the rats examined. Index Entries: Fluorine; NaF; caffeine; kidneys; catalase; superoxide dismutase; glutathione peroxidase; malondialdehyde; arginase; aldolase.
INTRODUCTION The fluoride anion has the lowest atomic weight and the smallest radius of the halogens, which are indicative of easier penetration to cells. *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
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In soft tissues, the toxicity of fluorine might increase, as they do not contain such buffer factors as are present in bones, where apatite is considered the factor neutralizing fluoride ions (1). Fluoride ions are absorbed in the blood mainly from the digestive tract. The concentration of fluorine ions in blood plasma is low, because of efficient excretion of the fluoride by the kidneys and its capture by the osseous system (2). In 1994, Chen and Whitford (3) confirmed that caffeine causes increased concentration of fluoride ions in plasma. The authors found that 2 h after the administration of F– in a caffeine solution, the concentration of fluoride ions in plasma was higher than in the case of the administration of F– in water. The communications of recent years indicate an important role for fluorides in free-radical processes (4). In the organism, reactive oxygen species (ROS) could originate as a result of both the influence of external physical factors, such as ionizing or ultraviolet radiation, and endogenous transformations (5–7). Of particular importance is the intracellular generation of ROS, resulting from single-electrode oxygenation of many compounds and enzymatic reactions. In most biochemical transformations, the first substance to be generated is superoxide ions (O2.–), which is the source of the next reactive forms of oxygen (6,8,9). One of the most reactive oxidants is the hydroxyl radical (OH.), which might oxidize all biologically vital compounds present in the organism (5–9). The amount of ROS appearing in physiological conditions in cells and tissues depends on the balance between their synthesis and their removal by various enzymes and nonenzymatic substances demonstrating antioxidative properties (6,7). The antioxidative defense of the organism takes place in a number of stages. The first line of defense is formed by transition metals, preventing the formation of ROS, such as ferritin, transferin, and ceruloplasmin. The second line of defense is made up of compounds causing ROS inactivation. They contain antioxidative enzymes (superoxide dismutase [SOD], E.C. 1.15.1.1; glutathione peroxidase [GPX], E.C. 1.11.1.9; and catalase [CAT], E.C. 1.11.1.6), as well as micromolecular antioxidants (α-tocopherol, βcarotene, vitamin C, and glutathione). The third line of defense consists of repair systems for particles damaged by ROS (6,8). A disturbance of the pro-oxidative/antioxidative balance, labeled oxidative stress, could result from increased ROS or decreased concentration of antioxidants, or both processes happening simultaneously. The consequence is intensified peroxidation of membrane lipids, leading to changes in their physical–chemical properties and, in consequence, disturbances in transmembrane transport, activity of the respiratory chain, and signal conduction. The products of membrane lipid peroxidation include aldehydes, hydroxy-aldehydes, and ketones, as well as carbohydrates such as ethane and penthane. Among the most important ones is malondialdehyde (MDA), which is a recognized marker of oxidative stress. Aldehydes react mainly with amino groups of proteins, lipids, amino acids, and nitrogen Biological Trace Element Research
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bases present in nucleic acids, which changes the structures and functions of enzymes, transcription factors, and cyto-skeleton proteins (5,6,8,10,11). Free-radical processes participate in the etiopathogenesis of numerous diseases, including diseases of the kidney (12). On the basis of the above observations, as well as the fact that kidneys have an important function in maintaining the homeostasis in organism, we undertook an attempt to assess the kidney function in adult rats after a 50-d administration of combined doses of F– and caffeine, as well as to assess the free-radical processes in that organ.
MATERIALS AND METHODS The experiment was carried out using 18 rats (male) of the Sprague–Dawley breed, 4.5 mo old, from the Central Farm for Experimental Animals of the Silesian Medical University in Katowice. For the purpose of the experiment, the rats were divided into three groups of six animals, which were kept separately in metal–plastic cages. The natural day–night cycle was maintained for the animals. The rats had unlimited access to standard fodder. The first group of rats were kept on distilled drinking water; they constituted the control (group I). The second group of animals was given sodium fluoride solution to drink, in doses of 4.9 mg F–/kg body mass/24 h (group II), whereas the third group was given a solution of sodium fluoride combined with caffeine to drink, in doses respectively of 4.9 mg F–/kg body mass/24 h and 3 mg caffeine/kg body mass/24 h (group III). The experiment lasted 50 d. Then the rats were intraperitoneally anesthetized, using thipental at a dose of 30 mg/rat. Blood serum was taken for examination. In the serum, the following were determined: 1. The concentration of creatinine, using the diagnostic kit Biochemtest (by POCH Gliwice, Poland; cat. no. 178196149). 2. The concentration of protein, using the diagnostic kit MerckBiotrol (France; cat. no. A01394). 3. The concentration of urea, using the diagnostic kit Biochemtest (by POCH Gliwice, Poland; cat. no. 178226141). 4. The concentration of calcium, using the diagnostic kit Biochemtest (by POCH Gliwice, Poland; cat. no. 178280145). 5. The activity of SOD and its isoenzymes. The enzyme activity was determined in kidney homogenates, according to Oyanagui (13). Superoxide anionic radical is produced in the reaction of xanthine with O2.–. With the participation of xanthine oxidase it reacts with hydroxylamine, producing a nitrosol ion. The nitrosol ion then binds with naphthalenediamine and sulfaniline acid, giving a colorful product, the concentration of which is directly proportional to the amount Biological Trace Element Research
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of superoxide anionic radical generated. The enzymatic activity is expressed in nitric units (NU) per milligram of protein; 1 NU represents 50% inhibition by SOD of the nitrosol ion formation under the conditions of the method. The total activity of SOD was determined, as well as that of the isoenzymes mitochondrial (Mn-SOD) and cytosol (ZnCu-SOD), using KCN as the inhibitor of the cytosol isoenzyme. The activity of catalase. Catalase was determined in kidney homogenates, using the kinetic method by Aebi (14), utilizing a 50-mM buffer of Tris-HCl (pH 7.4) and perhydride. The absorbance was read at a wavelength of 240 nm, and the kinetics of changes in absorbance was followed every 30 s over a period of 2 min. Enzymatic activity has been presented as International Units per milligram of protein. The activity of glutathione peroxidase. The enzyme was determined in kidney homogenates, according to the Paglia method (15). The method consists of the reaction of reduced glutathione (GSH), in the presence of peroxidase, with t-butyl superoxide. The glutathione (GSSG) oxidized in that reaction is then regenerated in the presence of glutathione reductase and NADPH. The activity of GPX has been determined as the number of µmols of NADPH used for the regeneration of GSH within 1 min, recalculated per milligram of protein (IU/mg protein). The concentration of MDA. The concentration of MDA was determined in kidney homogenates, utilizing its reaction with thiobarbituric acid, according to Ohkawa (16). The readout was made using a fluorimeter by Shimadzu at wavelengths of 515 nm (absorbance) and 522 nm (emission). The fluorimetric readout, in contrast to the spectrophotometric one (at a wavelength of 532 nm) is more specific; it is not disturbed by interference from hemoglobin or bile pigments. The concentration of MDA was read from a standard curve, using 1,1,3,3tetraethoxypropane as the standard, and expressed in micromoles, recalculated per gram of protein. The activity of arginase. The activity of arginase was determined in kidney homogenates, applying the colorimetric method. The method consists of deproteinization of examined material and then the determination of urea in the examined material, at a wavelength of 460 nm, using the diagnostic kit Biochemist (by POCH Gliwice, Poland). The activity of aldolase. The activity of fructose-1,6-diphosphate aldolase was determined in kidney homogenates by applying the colorimetric method, using the fructose-1,6diphosphate aldolase as the substrate, determining the colorful product of triose reactions at the wavelength of 570 nm, in accordance with the Krawczynski method.
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Histopathological Examinations Kidneys taken for pathomorphological examinations were fixed in 10% formalin obtained by mixing 40% formaldehyde with water, in the proportion 1 : 9. Pathomorphological changes in rat kidneys were assessed on the basis of specimens made using the ordinary paraffin technique, stained with hematoxylin and eosin. Pathomorphological changes were assessed using light microscope, with magnifications of 100, 200, 400, and 600. Microphotographies were taken by means of a Docuval microscope with an attachment for taking photographs by Carl Zeiss Jena.
Statistical Analysis Statistical analysis was performed with Statistic 6.0 PL software. Statistical methods included mean, standard deviation (SD), and standard error of the mean (SEM). The Kruskal–Wallis analysis of variance (ANOVA) test was used for multiple comparisons of data. Additional statistical comparisons were made by the Mann–Whitney U-test. A value of p
