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Ashif Iqubal et al. / Journal of Pharmacy Research 2016,11(1),39-47

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Review Article ISSN: 0974-6943

Various rodent models for inducing Hepatotoxicity and evaluating Hepatoprotective drugs a

Ashif Iqubal a, Mohammad Kashif Iqubal b and Syed Ehtaishamul Haque a* Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard-110062, New Delhi, India. b Shri Gopichand College of Pharmacy, Baghpat, U.P ,India. Received on:16-09-2016; Revised on: 09-10-2016; Accepted on: 07-01-2017

ABSTRACT Liver is the principal organ for maintaining the body’s internal environment. There is no substitute for the liver function. Its major influence is on the flow of nutrients and control of carbohydrate, protein and fats metabolism. Liver undergo various kinds of stress and hepatocytes get damaged in presence of toxins resulting into acute liver disease. Acute liver disease when remain undiagnosed progresses into chronic liver disease (CLDs) and become life threatening. Autoimmune disorders, viral infection, metabolic disorder and alcohol abuse are some common driving factors for CLDs. Advanced stage of CLDs can’t be managed & only option left is organ transplantation. Therefore it is very important to understand the molecular mechanism of hepatic damage. Rodents are routinely being used by researchers for preclinical studies but they possess distinct immune system and have different metabolic rates for hepatic homeostasis. Nevertheless, traditional as well as newer animal models (genetically modified models) mimic certain attributes of CLDs. Thus in this review we have discussed traditional animal models as well as recent advances in animal models. KEYWORDS:Autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, Lieber- De- Carli model, NAFLDs, animal models. INTRODUCTION: Liver fibrosis, more often called liver cirrhosis is regarded as a final pathway of chronic liver disease as fibrosis and cirrhosis progress into hepatocellular carcinoma [1]. The rats were first used to investigate the cellular and molecular mechanism of fibrosis but later on mice were used, as mice model respond better to the stimulus of fibrosis. CCL4, dimethylnitrosamine (DMN) and thioacetamide (TAA) is common noninvasive (chemical) models whereas bile duct ligation (BDL) and portal vein ligation (PVL) are the invasive animal models used for fibrosis [1]. Genetically engineered mice (GEM) have emerged as a powerful biological tool for analysis of gene function. GEM technology has helped the researchers to insert an exogenous gene or delete endogenous genes[2]. Since last decade combination of the genetic model with pro-fibrotic chemicals such as CCL4, TAA & DEN are commonly used in the laboratory[2]. Various animal models available for studying liver complications are as follows.

causes liver fibrosis[1]. According to WHO, till date more than 200 million people suffers frominfection world wide out of which 200 thousands dies annually[1]. Praziquantel is most effective therapy, still 30 % of patients develop fibrosis[1]. Mice once exposed to schistosomia mansoni, rapidly develop granulomas in the hepatocyte. Parasite initiates the immunological response in portal tract. This pathology in mice closely resembles fibrosis in human [3]. Thus this model is widely used for understanding the mechanism of fibrosis and developing novel therapy against fibrosis[4].

ANIMALMODELFOR AUTOIMMUNE HEPATITIS (AIH): AIH is a potentially severe chronic liver disease in which there is abnormal elevation of serum transaminase, gammaglobuline & circulating antibodies[5]. AIH usually occurs in women and diagnosed at the end stage, hence there is limited information available regarding ANIMAL MODELFOR EXPERIMENTAL HEPATIC FIBROSIS: the cellular and molecular mechanism[5]. Once the disease is identified, therapeutic implementation results into severe adverse effects. Schistosomiasis model: Therefore animal models are generally used to explore the Schistosomiasis is a parasitic disease caused by schistosomia and pathophysiology of disease and helps in identifying drugs that can be used with minimum side effects[6]. *Corresponding author. Dr. Syed Ehtaishamul Haque Assistant Professor, Department of Concanavalin A (Con A): Pharmacology, Faculty of Pharmacy, Jamia Con A is a Plant lectin obtained from jack beans. Con A selectively Hamdard, New Delhi- 110062, India. binds with mannose residue of glycoprotein and initiate the activation Journal of Pharmacy Research Vol.11 Issue 1 January 2017

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Ashif Iqubal et al. / Journal of Pharmacy Research 2016,11(1),39-47 of lymphocytes, T cells, CD4 + T cells, NKT cells and kuffers cells which closely resembles AIH[7]. In this model TNF-alpha/gamma, IL2, IL-4 act as pathogenic cytokines whereas IL-10 act as protective cytokines[8]. The drawback of this model includes the lack of circulating antibodies which are commonly seen in AIH in humans[1]. Therefore this model is preferably used in primary screening of AIH drugs[1]. TGF- 1 -/- MICE on Balb/c background: TGF -1 contribute to maintain the immune homeostasis and thus exhibit variety of anti-inflammatory activities therefore mice lacking TGF –1 develop severe inflammatory lesions in multiple organs within 3-5 weeks of age[9]. Thus this model mimics many aspects of AIH. Still, this model is not an ideal choice for AIH because mice usually die within a few weeks after the development of AIH. However but this model gives an insight of genetic deficiencies that causes AIH[10]. NTx-PD-1 mice: Regulatory T cells (Tregs) are a special type of T cells that suppress the hyper immune system and maintain tolerance to autoimmunity[11] Neonatal thymectomy (NTx) when done on programmed death (PD) mice, develop hepatitis that mimics the human AIH and mice develop antinuclear antibodies that further elevate the level of serum ALT & AST[11]. ANIMAL MODEL FOR PRIMARY BILIARY CIRRHOSIS (PBC): Primary biliary cirrhosis is more often seen in women that men (women: men = 2:1) with unknown etiology[12]. PBC is diagnosed by the destruction of the intrahepatic bile duct epithelial cell. In PBC patient, antimitochondrial antibodies (AM-Ab) are seen in serum that reacts with pyruvate dehydrogenase complex (PDC) and damages the inner lipoyl domain of E2 subunit[1]. PBC at its advanced stage progress into fibrosis. Clinically ursodeoxy cholic acid (UDCA) is considered as best therapeutic regimen for PBC as UDCA reduces the biochemical markers and additionally have anti-apoptotic, anti-inflammatory, antioxidant and induces hypercholeratic effect[13]. SPONANIOUS MOUSE MODEL FOR PBC: NOD.c3c4 mice: It was first developed model for PBC in which B6 and B10 derived insulin dependent diabetic region were integrated into the non-obese diabetic (NOD) mice[14] that developed antibodies for E2 same as seen in human PBC[14]. When immunological–histopathological studies done for PBC induced mice, CD3+, CD4+ CD8+ T cells were spotted in biliary epithelium[14]. Reduction in T regulatory cells found

directly proportional to the protection of NOD-c3c4 mice from PBC[15]. Therefore this model confirms the role of T cells in the pathogenesis of in PBC[16]. dn TGF-RII mice: Currently this model is regarded as the most useful animal model for PBC. TGF-â1 knockout mice confirm the role of TGF-â1 in activation and proliferation of PBC[12]. One drawback of this model is the development of severe immunological abnormalities that result in high rate of mortality[17]. IL-2R - /- MICE: This model is yet another model for PBC. Generally, IL-2Rá is important for development and expression of CD4+ and CD25+ T regulatory cells[18] which suppress the autoimmunity, hence the lack of CD 25+ in IL-2R -/- mice lead to portal inflammation and PBC[19]. Ae2a, b -/- mice: Ae2a, b mediates the Cl/HCO3- exchange across the plasma membrane and thus crucial for maintenance of intracellular pH [20]. Ae2a, b -/- mice resembles some of the clinical features of human PBC, such as immunological abnormalities and hepatobiliary alteration[20]. Further Ae2a, b -/- mice shows an elevated level of CD8+ cells and reduced level of T regulatory cells, suggesting this model as a potential pharmacological target to modulate Treg cells[20]. Chemical xenobiotics immunised mice: There is strong evidence for the effect of bacterial toxin and xenobiotics in the pathophysiology of liver disorder[21]. Exposure of 2-octynoic acid (2A) induces hepatic damage and has a structure similar to PDC-E2, thus initiate the symptom of PBC[22]. ANIMAL MODEL FOR PRIMARY SCLEROSING CHOLANGITIS (PSC): PSC is a progressive cholestatic disorder that is common in male[13] . PSC is characterized by inflammation and fibrosis of intra and extra intrahepatic bile duct[23]. Perinuclear-anti neutrophil cytoplasmic Ab are seen in PSC, justifying it as an auto-immunised disorder[24]. Till date only UDCA have shown positive clinical outcome . Some of the animal models used to study PSC are Trinitrobenzene model (TNB), alpha-napthyliso thiocyanate (á-NIT) model and lithocholic acid model[25]. Mdr 2-/-: Multidrug-resistant gene function as coding the canalicular phospholipids flipase that participate in the transportation of biliary phospholipids into the canalicular cell membrane[26]. Therefore the

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Ashif Iqubal et al. / Journal of Pharmacy Research 2016,11(1),39-47 lack of Mdr genes in Mdr2-/- mice makes the mice devoid of biliary ANIMAL MODEL FOR HBV: phospholipids transportation that results in accumulation of non- HBV infection has affected more than 360 million people across the micellar bound free bile acid. This process initiate the injury of the globe[30]. There is always a possibility for development of liver bile duct and progress to HCC[27], thus Mdr mice model let the cirrhosis and hepatic carcinoma in a patient of HBV. HBV consist of researcher understand the basic pathophysiology of PSC and effect partially double strand DNA of size 3.2 Kb. HBV DNA template act of hepatoprotective agent in PSC patient[12]. for both replication and transcription[35]. There are many viruses of DDC Diet: 3, 5 Diethoxycarbonyl- 1, 4 dihydro colloidal (DDC) is well-established model for alcoholic and non-alcoholic steatohepatitis, metabolic liver disease and chronic cholestatic liver disorders[28]. DDC feeding causes cholangitis, periductular fibrosis and finally biliary fibrosis which mimic the etiology of PSC[28]. Therefore DDC model is extensively used for understanding the basic etiology of PSC and to test the novel; therapeutics in PSC[29]. ANIMAL MODEL OF HCV INFECTION: Till date hepatitis infection have infected more than 170 million people across the globe[1]. HCV is an enveloped positive strand RNA virus of molecular weight 9.6 kb. RNA-dependent RNA polymerase mediates the replication. RNA of HCV virus is devoid of proofreading so there is always the higher rate of mutation[1]. There is no vaccine available for , however combination of pegylated interferon and ribavirin is used clinically. This combination, apart from therapeutic activity, exerts higher adverse effect which is sometime fetal[30]. Over the years, good number of transgenic mice constitutively over expressed one or more HCV proteins in order to study their on liver pathology. Despite some contradictory results, three features of HCV pathogenesis could be demonstrated with these HCV transgenic mouse models[31]. Inducible-HCV transgenic mice: Transgenic mice model has been used since a long time but a major challenge encountered with transgenic mice model for expressing HCV protein is immunotolerance towards HCV protein. Therefore transgenic mice were developed that allows the expression of core HCV protein using Cre/IoxP switching system[32]. This new model additionally shows that transgenic mice expressing the core HCV protein, are resistant to Fas programmed cell death (apoptosis) in liver[33], thus indicating that HCV virus can maintain infection in the antiviral environment. This model also shows the role of NK cells in expression of core protein during infection[34]. Xenograft model: Transgenic mice model, inspite of many modifications, lack efficient mechanism of HCV infection. Therefore xenograft model of human hepatocytes in mice is used as alternate to transgenic mice model[1].

family hepadnaviridae which closely resembles HBV. Woodchucks HBV (WHBV) & duck HBV (DHBV) models provide sufficient information about the mode of infection and onset of cirrhosis and HCC[36]. HBV transfected mice: Since transgenic mice were not found to be suitable for HBV model since HBV infection are not permeable in transgenic mice, therefore transfected mice models were developed [1]. To develop this model two methods were used (a) Use of adenovirus vector to develop HBV DNA in mice (b) Hydrodynamic injection of HBV DNA[37]. In both of these models, DNA is not integrated into host genome of transfected mice, therefore, this model is used for the study of antiviral drugs[38]. A common drawback of transfected mouse models is the relatively short duration of viral replication, which is limited by the host immune response against viral vectors[37]. Trimeric mouse model: This is a valid model for both HCV & HBV[39]. In this model, mouse model is irradiated to break down their immune system and again the immune system is reconstituted using Severe combined immunodeficiency (SCID) method[40]. The mouse is then transplanted with ex vivo HBV-infected liver fragment of human beneath the capsule (capsule). This transplanted human HBV hepatocytes remain functional for several weeks[40]. This model is mostly used for shortterm screening of antiviral drugs[40]. ANIMAL MODEL FOR NAFLDs: NAFLDs is define as the presence of steatosis in absence of alcohol consumption. NASH is a further progression of NAFLDs which is more often characterized by steatosis, lobular inflammation, hepatocellular ballooning and fibrosis[41]. NAFLD/NASH are the most common form of liver disorder in older people associated with metabolic syndrome[41]. Till date there are few animal models that mimic the NAFLDs. Genetic model, dietary model and the combination of a genetic nutritional factor are used to access the cellular and molecular mechanism of NAFLDs[1]. GENETIC MODEL: Genetic model such as Ob/ob mice, db/db mice (fa/fa mice) are randomly used in the laboratory to induce NAFLDs.


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Ashif Iqubal et al. / Journal of Pharmacy Research 2016,11(1),39-47 Ob/ob mice model: Leptin is the well-known hormone that regulates the hunger, energy uptake and energy expenditure[42] . Whenever there is a mutation in gene encoding leptin (Ob/ob mice, leptin-deficient mice) or mutation at leptin receptor (leptin resistant receptor), mice become hyperphagic, obese, hyperglycemic, inactive and insulin resistant[42]. These characteristics mimic the pathophysiology of NAFLD and this model suggests the role of leptin in this pathology [43]. Some other genetic models used for NAFLDs are kk-Ay-/- mice, PPAR-á /- mice and PTEN -/- mice[1].

combination of HFD model with MCD model mimics the exact etiology of human NAFLDs/NASH[53]. Some other model with the similar outcome is combinations are LDL+ HFD (oxidized Low-density lipoprotein + high-fat diet) and GTH (Gold thioglucoside) + HFD[1].

DIETRY MODEL FOR INDUCING NAFLDS:

Liquid diet model (Lieber- De- Carli model): Consumption of alcohol in drinking water is a most relevant animal model for ALD as it is cheapest and convenient model [2]. A challenge for this model is an aversion of rodents to smell and test of ethanol which limits the administration of excess of ethanol more than once in a single dose[55]. This model is regarded as an excellent tool for understanding the early stage of injury in ALD[1] . Recently 10 days model have been introduced in which 6.3% v/v ethanol and 28% ethanol derived calories in low carbohydrate were given[56] . In the first 7 days mice begin to loose weight and increase in ALT & AST is observed. This model induces more severe hepatocellular injury than classical liquid diet model but failed to induce inflammation and necrosis, as evident by histopathological studies done[56]. This model was further modified with ethanol, 5% of the diet and single gavage of ethanol (chronic binge model) for 10 days. This modified model induced marked fatty liver injury with hepatocyte inflammation and necrosis[57].

Methionine-choline deficient diet (MCD): MCD is a classical dietary model for NASH/NAFLD. This model contains 40% sucrose and 10% of fat but is deficient in choline and methionine, which are crucial for mitochondrial â oxidation and formation of VLDLV [44]. Therefore this model initiates the steatohepatitis with pericellular and pericentral fibrosis with elevation in ROS[45]. Rats or mice fed with the MCD diet show significant loss of body weight (up to 10% in 10 weeks) and reduction in plasma triglyceride and cholesterol levels. Serum levels of insulin, leptin and glucose are also decreased, serum adiponectin remains unchanged or increased and the animals are peripherally insulin sensitive, although they exhibit hepatic insulin resistance[46]. High fat diet (HFD): HFD model is more reliable for rat than mice. This model constitutes 70% of calorie from fat, 11% from carbohydrate and 18% of calorie from protein[47]. When a rat is given HFD, it rapidly gains weight, become obese and reflects the pathophysiology of NAFLDs[48]. The increase in ALT & AST is seen with consumption of HFD. Additionally, cholestasis and inflammation are also reported in this model[42]. This model takes comparatively long time in the induction of NAFLDS/NASH but in general consumption of HFD induces mild to moderate liver injury[49]. Frutose: Consumption of excess of fructose in the form of corn syrup which is generally found in soft drinks induces NAFLDs/NASH[50]. Metabolism of fructose starts the de novo lipogenesis and inhibition of mitochondrial â oxidation. This result in hepatic steatosis with hyperglycemia, obesity and insulin resistance[51]. Fructose also promotes intestinal bacterial overgrowth, leading to increased endotoxin levels in the portal blood, subsequent Kupffer cell activation and inflammation in the liver [52]. Excess of fructose consumption also elevates the level of ROS [53]. Therefore

ANIMAL MODEL OFALCOHOLIC LIVER DISEASE: Heavy consumption of alcohol leads to alcoholic liver disease (ALD). Pathology of ALD ranges from simple cholestasis to inflammation, necrosis, fibrosis, cirrhosis and HCC[54] . Some of the common animal model use in the laboratory for induction of ALD are as follow.

Internal ethanol infusion (Tsukamoto-French Model): All the model we have seen for ALD, cannot induce injury beyond the fatty liver, therefore another model was developed with a hypothesis that rodent have a higher rate of metabolism for alcohol than human and require a higher concentration of alcohol in blood in a shorter duration of time[58]. A catheter is implanted into the stomach and ethanol is infused through the catheter into stomach[59]. This model, therefore, induces hepatocyte injury in a shorter period of time and mimic the exact same etiology as that of human ALD[60]. Further, if carbonyl iron is added to this infusion, and even more severe form of necrosis and inflammation is produced[61] . This model thus proves the role of TNF-, TGF- and activation of kuffer cells in the pathophysiology of ALD[62]. ANIMAL MODEL FOR HEPATOCELLULAR CARCINOMA: Hepatic carcinoma is growing exponentially and new research is needed for better treatment regimen. Here we summarize some of the carcinogenic substance used for developing such models to study


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Ashif Iqubal et al. / Journal of Pharmacy Research 2016,11(1),39-47 anti-cancer drugs. These carcinogenic substances are classified in Thioacetamide: two categories depending on their action to induce tumor. (1) Geotaxis Thioacetamide is a well known hepatic toxin and when administered chemicals, which act at genetic level and induces structural changes at dose of 0.02-0.05 % of water induces fibrosis in 10-15 weeks[80]. and leads to mutation leading to hepatic cancer (2) Chemicals which lacks effect on DNA but promote tumor formation in dose-dependent CONCLUSION: manner. Experimental animal models help the researcher to study and understand the pathophysiology and underlying cause of various Diethylnitrosamine (DEN) : chronic disorders. Similarly, animal models play a crucial role in preDEN induces tumor in organism-organ specific manner as in mice clinical study and in new drug development. Initially traditional animal DEN induces tumor in liver, pancreas, GI tract, Respiratory tract and models were used in which hepatotoxicity was induced using various in lungs[63] . DEN mainly act by alkylating DNA strand and it is toxins like CCL4, DMN and TAA, but it was observed that in human hydroxylated to alpha-hydroxyl nitrosamine[64 and this bioconversion CLD develops in decades as it is a slow process but in rodents it may is mediated by Cyt P450 enzyme which is highly present in take less time (months) to develop as they have shorter life span. hepatocytes[65]. Some time DEN causes oxidative stress leading to Some researcher also reported that when higher doses of hepatotoxins the generation of reactive oxygen species and causes mutation. In were used to reduce the time, elevation in inflammation and rate of mouse DEN induces tumor through H-ras proto oncogenes mortality were found. Also metabolic process of rodents is much activation[66]. further DEN induces tumor on sex basis, as male have faster and some time becomes a point of concern because agents 100% chance to develop tumor whereas female has only 70% chance responsible for NAFLDs and ALD in humans may behave abruptly to develop the tumor[67]. DEN at dose of 5-90 mg/kg ip in 15 days old in rodents. Nevertheless, traditional animal models have helped the mice induces tumor within 45-105 weeks[68] whereas in 6-week dose researchers to understand basic etiology of various CLDs. Now- aregimen of DEN ( 3 weeks administration of 75 mg/kg and 3 weeks days GMMs (gene knockout mice model) are used which are superior administration of 100 mg/kg) tumor in develop the 52 weeks[69]. to traditional models and imitate the disease etiology with humans. Finally pathogenesis of CLDs in humans is a complex process and Peroxisome proliferators (PPs): often involves various tissues, organs or system. It is difficult to These agents cause hepatomegaly and first tested in 1975[70]. Some draw inference using single animal models (traditional or GMMs), common PPS are bezafibrate, fenofibrateB[71], clofibrate[71]. PPs mainly hence often combination of traditional and genetic models are used act by activating PPARá which is responsible for expression of many to express the pathology of CLDs. genes including those of oncogenes[72]. Conflict of interest: None Aflatoxin B (AFB): AFB is derived from fungus Aspergillus flavium [73] and it REFERENCES: metabolized into 8, 9 epoxide which selectively binds with guanine 1. Yan Liu1, Christoph Meyer, Chengfu Xu, Honglei Weng, residue and induces mutation leading to HCC[74] it has been seen Claus Hellerbrand, Peter ten Dijke and Steven Dooley. that 7 days old mice when exposed to 8 mg/kg of AFB for 52 weeks, Animal models of chronic liver diseases; Am J Physiol develops a well defined tumor is induced[75]. Gastrointest Liver Physiol. 2012; 28:654-658. 2. Hayashi H and Sakai T. Animal models for the study of liver CCL4 : fibrosis: new insights from knockout mouse models. Am J Carbon tetrachloride: It is one of the most common carcinogenic Physiol Gastrointest Liver Physiol. 2011; 300:729-738. agent that induces tumor by causing lipid peroxidation[76-77], membrane 3. Boros DL. Immunopathology of Schistosoma mansoni damage and causes secretion of cytokines & pro-inflammatory factors infection. Clin Microbiol Rev. 1989; 2:250-269. from kuffers cells[78]. Administration of CCL4 with alcohol induces 4. Wilson MS, Mentink-Kane MM, Pesce JT, Ramalingam TR, tumor in 104 weeks[78]. Thompson R, and Wynn TA. Immunopathology of schistosomiasis. Immunol Cell Biol. 2007; 85:148-154. Choline-deficient diet (CDD): 5. Manns MP, Czaja AJ, Gorham JD, Krawitt EL, Mieli-Vergani Choline deficient diet initially causes steatosis[79] and at later stage G, Vergani D, and 1298 Vierling JM. Diagnosis and [79] [79] mass of oval cells are formed due to oxidation of DNA fragment . management of autoimmune hepatitis. Hepatology. 2010; 51: 2193-1299. Journal of Pharmacy Research Vol.11 Issue 1 January 2017

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Source of support: Nil, India, Conflict of interest: None Declared


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