Effect of Carbamazepine and Acetaminophen Interaction Toxicity on Tissue Repair Response in Drug Induced Liver Injury (Dili) Model of Swiss

Abstract

Drug induced liver injury (DILI) is the major health problem that challenges not only the health care professionals but also the industries and regulatory authorities. It is the leading cause for acute liver failure and liver transplantation in western countries. There are several risk factors for the development of DILI, one of which is drug interaction. It is known that exposure to a combination of drugs might leads to exaggerated toxicity even though individual drug might be less toxic or nontoxic when used alone. Carbamazepine (CBZ) when used along with acetaminophen (APAP) results in enhanced liver toxicity. Although, the liver injury occurs at the beginning, the dynamic compensatory tissue repair response is stimulated, that determines the final outcome of the injury i.e. either survival or death of the animal. Clinically, CBZ and APAP interactions have been reported but preclinical data with respect to tissue repair response has not been evaluated. The aim of the present study was to determine the role of tissue repair response on interaction toxicity (CBZ and APAP) induced liver injury in female Swiss albino mice. Adult female Swiss Albino mice of 8-10 weeks of age were chosen and randomly divided into three different groups like- a) Control group b) CBZ treated, and c) CBZ+APAP treated. The control group of animals were given only vehicle (Corn Oil, p.o., for 5 days) for five days. The CBZ treated animals were given only CBZ for five days (300mg/kg, p.o.) where CBZ suspended in corn oil. The CBZ+APAP treated animals were given CBZ (300mg/kg, p.o.) for five days, immediately followed by a single dose of APAP on 5th day (300mg/kg in 0.25% tragacanth, p.o.). Before treatment, both CBZ and CBZ+APAP treated animals were further subdivided into different time points like - 0, 24, 36, 48, and 96 hrs. Whereas, the control group of animals were further subdivided into only three different time points like - 0, 36, and 96 hrs. Immediately after last dosing (day 5th), both blood and liver samples were collected at different time intervals like - 0, 24, 36, 48, 96 hrs for biochemical and histopathological analysis. Similarly for control animals, samples were collected at time points like - 0, 36, and 96 hrs. The blood samples collected were then utilized for estimation of serum parameters such as SGOT, SGPT and glucose. Liver tissues were utilized for glycogen estimation and histological evaluations.The CBZ and CBZ+APAP treated animals showed gradual rise in SGPT and SGOT level from 0 to 36 hrs time point. Thereafter, it started declining and showed nearly normal values at 96 hr time point. The rise in the SGOT and SGPT levels was found maximum at 36 hrs in both the groups. In comparison to CBZ group, the levels of SGPT was found significantly (P<0.05) higher at both 24 and 36 hrs time points, whereas level of SGOT was significantly (P<0.05) higher only at 36 hrs in CBZ+APAP group. The control group of animals did not show any elevation in enzyme parameters at any time point, suggesting vehicle per se did not cause any liver injury. Results demonstrated that the liver injury as indicated by the maximum elevation of SGPT and SGOT at 36 hrs after the treatment in both the groups. Injury was much higher in combination of CBZ and APAP as compared to CBZ alone, indicating higher liver damage in case of interaction. Serum glucose levels were estimated in all the groups. In case of control group there was no significant change but the CBZ and CBZ+APAP treated animals showed much variations at all the time points. Serum glucose level was found to be elevated initially at 24 hrs, thereafter the drastic fall observed at 36 hrs followed by increased level at later time points. These observations hints altered energy utilization in both the groups. The elevated level of glucose at 24 hrs was significantly higher than 0 hr time point in both the CBZ and CBZ+APAP treated animals. At 36 hrs time point both the CBZ and CBZ+APAP treated animals, the glucose level fallen down but in CBZ+APAP treated animals the glucose levels were significantly (P<0.05) lower than 0 hr. After this at later time point i.e. at 48 hrs glucose level started rising in both CBZ and CBZ+APAP treated group and at the end of 96 hrs animals attained nearly normal levels of glucose. Now, here the initial rise at 24 hr indicates the involvement of glycogenolysis which causes more of glucose release in serum as tissue repair might have started at 24 hrs which requires energy for proliferation. The fallen level of glucose at 36 hrs indicates the utilization of serum glucose for cell proliferation which was peaked at this time point. Unlike glucose, hepatic glycogen levels appeared significantly lower at 24 hrs in both CBZ and CBZ+APAP group because of its conversion to glucose and at later time points the glycogen levels started increasing. The control animals did not show any changes in glycogen level throughout study period further strengthens our liver injury data. The CBZ and CBZ+APAP treated animals showed decreased level of glycogen but in CBZ+APAP, the levels were significantly (P<0.05) lower at 24 than 0 hr time point and control animals. Initial decline in glycogen hints the conversion of glycogen to glucose which was required for tissue repair response. This fall in glycogen level at 24 hrs supported the increased level of serum glucose at 24 hr in both CBZ and CBZ+APAP treated groups. In comparison of both the groups it was seen that the utilization of serum glucose and depletion of glycogen levels were much higher in CBZ+APAP group, indicates requirement of more energy for tissue repair. Further, to confirm the tissue repair response, the histopathological study was performed for all the animals of control and treated groups. The control group of animals showed no changes in the histological appearance. The changes in the histology were observed in both CBZ and CBZ+APAP treated groups, where the cell injury was scored according to the damage observed. This evaluation demonstrated that the maximum damage was observed at 36 hr time in the CBZ treated and CBZ+APAP treated when compared to control group. Histological changes that reflect the injury were scored more in CBZ+APAP group. This indicates the liver injury was higher in case of CBZ+APAP than CBZ alone. Furthermore, the histology of liver tissue was checked for the appearance of the mitotic nuclei as a marker for cell proliferation. Mitotic cells were observed at 24 hrs and found in maximum numbers at 36 hrs time point in both CBZ and CBZ+APAP treated group. This histological data which suggested the maximum cells in mitotic phase at 36hr time point, further supported by the changes in the energy levels where maximum utilization of glucose was observed at 36 hrs confirming involvement of tissue repair response after liver injury. The extent of injury was decreased at 48 hrs as compared to 36 hrs time point and by 96 hrs damage was reversed nearly to normal in both the groups. The liver injury due to interaction toxicity [CBZ+APAP] was more as compared to CBZ alone and control animals. The tissue repair response was observed in both CBZ and CBZ+APAP treated animals but the energy utilization for tissue repair as demonstrated by the glucose and glycogen levels were much affected in CBZ+APAP treated animals. More utilization of glucose hints the higher energy requirement for compensatory tissue repair in CBZ+APAP treated animals. Additionally, the histological appearance showed maximum damage in CBZ+APAP group at 36 hrs when compared to CBZ alone group. Injury caused by interactive toxicity was reversed nearly to normal archistructure by 96 hrs which further confirms the role of tissue repair response. Based upon my present data, it was concluded that the liver injury was significantly higher in CBZ+APAP group, but the prompt and robust mitotic cell division regressed the progression of liver injury and thereby rescuing of the animals from interactive toxicity.