Role of Tissue Repair in Carbamazepine induced Liver Injury Model of Male Swiss Albino Mice

Abstract

Liver is the main site of drug and toxicant metabolism because of drug-metabolizing enzymes are present on liver. This has made liver a prime target for drug-induced injury. Numerous studies of drug induced liver injury (DILI) have established the existence of 2 distinct stages of chemical induced toxicity. Stage - I being the inflictive stage in which toxic chemicals initiate injury through well-established mechanisms, tempered via the net effect of bio-activation and detoxification processes. While stage - II is the progression/ regression phase of injury corresponding with the absence/ presence of compensatory tissue repair, respectively, this determines the final outcome of toxicity which is either death or survival. Recently a study by Higuchi et al. (2012) reported an in-vivo model of carbamazepine (CBZ) induced liver injury in male BALB/c mice explaining the mechanisms involved in CBZ induced liver injury. Being a recent model, role of liver regeneration was not reported tilldate. So, the objectives of the present study are, a) To test weather another strain of mice is also susceptible for CBZ induced liver injury? b) To study the role of tissue repair in CBZ induced liver injury model. Swiss albino mice of 8-10 weeks of age were divided in two groups like – a) Vehicle control (corn oil), and b) CBZ treated (CBZ suspended in corn oil). The development of model using CBZ was adapted from Higuchi et al. (2012) by dose optimization, when we used. By adopting reported dosing protocol of five days (400mg/kg, p.o. – for 4 days and 800 mg/kg, p.o. on 5th day), which caused 50% mortality in our studies, suggesting that the strain is less resilient to CBZ toxicity. So, the dose was reduced to 350mg/kg, p.o. for 4 days & 700 mg/kg, p.o. on 5th day, which also caused 33% mortality. We observed that animals surviving after first dose also survived after last lethal dose due to autoprotection phenomenon. Hence, a dose which was initially lower and the final a toxic dose was selected. In our study, treatment with CBZ for 5 days (300mg/kg, p.o. – for 4 days and 800 mg/kg, p.o. on 5th day) was given. After 5th day of administration both blood and liver tissues were collected at different time intervals for assessing liver injury and tissue repair response. Serum was used to estimate levels of SGPT, SGOT and glucose. The tissue was analyzed for glycogen levels and histopathology. Studies have reported that SGPT and SGOT enzymes are obtained in highest concentration in liver tissue and are specific markers whose levels elevate during liver injury. In our study mice administered with vehicle did not cause any elevation of SGPT or SGOT levels at any time point. Whereas, in CBZ treated mice levels were elevated as early as at 0 hr (5th Day) and increased up to 48 hr and thereafter declined gradually by 96 hr. This pattern of SGPT levels suggested that the progression of injury took place till 48 hr and then hindered by an opposing force which was a simultaneous process with injury. At 96 hr time point SGPT levels were nearly comparable to 0 hr time point, indicating liver injury is reversing nearly normal. Similar pattern was observed in SGOT levels which strengthened our observation. Toxicokinetics of CBZ indicate that on repeated administration, it gets excreted from the body within the first 24 hr by conjugation reaction mediated by phase 2 drug metabolizing enzymes. But our time course of injury suggests that the liver injury increases and progresses well beyond 24 hr when there is no parent drug or its metabolite in the body. This indicated that the progression of injury initiated by CBZ, takes place in toxicantindependent fashion. Detailed mechanism of CBZ induced liver injury is yet to be studied. However, three possible explanations for progression of injury have been reported. They are like - (1) Contribution of inflammatory cells (2) Production of free radicals, and (3) Leakage of degradative enzymes from the dying and injured cells. Further supporting data comes from histopathological analysis which is the direct measurement of liver injury on ground basis. Hence, we analyzed the photomicrographs of H&E stained liver sections for assessing liver injury. In the histopathological analysis the liver tissue from the vehicle control group showed normal liver archistructure. In contrast the CBZ treated groups showed a pattern of remarkable centrilobular necrosis. This may be because, CYP3A4 enzyme is highly expressed around central vein that causes more amount of CBZ to metabolize to toxic 3-OH CBZ causing injury in spherical shape around central vein. Thus, by evaluating the size of sphere the extent of injury was determined. At 0 and 12 hr the injury was not significant but distinct evidences of necrosis and apoptosis were visible at 24 hr with signs of pyknosis, hemorrhage and vacuolization which indicated accelerated injury. Extensive centrilobular necrosis started at 36 hr with infiltration of inflammatory cells and eosinophilic hepatocytes which was spread toward the mid-zonal area. Liver-cell columns were broken up with crumbling, indicating degeneration of cells. Necrosis was very prominent and almost the same for both the groups between 36 and 48 hr which indicated that injury was maximized during this period. This data advocated the pattern of SGPT and SGOT. Reports are available which shows that it is possible to analyze newly divided cells (mitotic cells) with H&E staining. Hence the sections were scanned and the mitotic cells were found as early as at 24 hr time point. They further increased at later time point like 36 and 48 hr after CBZ challenge. These indicated that these newly divided cells prevented the progression of liver injury after 48 hr time point and restored liver archistructure to nearly normal by 96 hr time point possibly by the process of liver regeneration. Furthermore, apoptotic bodies were also appearing during 72 and 96 hr time point suggesting a programmed cell proliferation control process, which might have ceased the proliferation process. Further to assess tissue repair and injury, levels of serum glucose and liver glycogen were estimated. Glucose and glycogen are hormonally regulated by hepatocytes and studies have shown that during liver regeneration hepatic energy status is compromised, suggesting that liver regeneration demands energy in terms of glucose. In our study, mice administered with vehicle did not cause significant change in glucose level. Whereas, CBZ treated mice glucose levels were elevated up to 24 hr showing that liver regeneration process demands energy in terms of glucose; and then serum glucose levels declined at 36 and 48 hr showing energy consumption in the process of cell division. This was supported by the data of liver glycogen where CBZ treated mice showed decrease in glycogen level as early as at 0 hr (5th day) and it continued up to 48 hr. The possible mechanism for its decline till 48 hr could be the conversion of glycogen to glucose (glycogenolysis) for satisfying the demand of energy required for cell proliferation process. This was already reinforced by glucose pattern whose levelsincreased up to 24 hr and declined at 36 and 48 hr. Levels of both glucose and glycogen were stabilized by 96 hr suggesting that the proliferation process stopped by this time. So based upon biochemical and histopathological data, we conclude that, a) male Swiss albino strain is also susceptible to CBZ induced liver injury, and b) Both prominent and robust mitotic cell division lead to tissue repair response which prevented progression of CBZ mediated liver injury and thereby rescued male Swiss albino mice from death.