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  • dihydrofolate reductase DEN has been reported to induce a re

    2018-11-06

    DEN has been reported to induce a relentless generation of free radicals in the liver, which in turn augment the demand of antioxidant enzymes. Subsequently, it ends in oxidative stress and initiation of carcinogenesis. Single dose of DEN injection on 14th and 28th day reduced body weight and noticeably increased the ratio of liver to overall body weight in comparison with normal rat. Depletion in food intake and therefore, the reduction of body weight gain noticed in DEN-treated animals, could be predominantly due to loss from skeletal muscle and adipose tissue as already shown, and it could be considered as an ancillary indication of the diminishing hepatic function following exposure to DEN. In addition, evaluation of the liver/body weights ratio was used to scrutinize potential changes in the liver size, but no changes were found. In the present investigation, the hoisted levels of marker enzyme in serum and liver are determinations of cellular damage and loss of functional integrity of the cell membrane due to DEN administration. Treatment with Carvacrol (15mg/kg) brought back these dihydrofolate reductase to near routine level by possibly preserving the functional integrity of the hepatocytes, showing its defense action against DEN induced Hepatotoxicity. It was already reported that, the dosage of 25mg/kg bodyweight of Carvacrol pretreatment resulted in the protection against thioacetamide-induced liver toxicity by attenuation of oxidative stress, inflammation, and apoptosis [41]. Though, previous studies by Nafees et al., showed 25mg/kg body weight of Carvacrol as a maximum protective dose, our studies suggesting that even 30mg/kg body weight of Carvacrol had similar protective effective as like in 15mg/kg bodyweight of Carvacrol (based on dosage fixation study in figure). So we fixed up a dose of 15mg/kg body weight of Carvacrol as a maximum efficacy with minimum dosage and we used this concentration as standard concentration for further experimental studies. GR plays a key role in cellular defense against oxidative stress by arresting accumulation of GSSG and thus maintaining the redox state. The decreased level of GR activity after DEN administration possibly reflects conformity to oxidative conditions. The reduction in the specific activities of antioxidant enzymes and drug metabolizing enzyme, after DEN administration may be associated to their inhibition by DEN through immediate interaction with the enzyme molecules or modification of the post-transcriptional and post-translational steps in the enzyme synthesis. DEN, an indirect acting carcinogen that requires metabolic activation to yield an ultimate carcinogen. It is widely accepted that metabolic activation of nitrosamines by Cytochrome P450 enzymes to reactive electrophiles is required for their cytotoxic, mutagenic and carcinogenic activity. It has been reported that DEN after its metabolic biotransformation promutagenic adducts that may initiate liver carcinogenesis. Carvacrol treated animals showed a significant decrease in the activities of these enzymes when compared with group 2 DEN-induced animals. Detoxification of activated procarcinogen and thereby protection against carcinogenic injury occurs in the presence of Phase II drug metabolizing enzymes. One of the important pathways is GSH conjugation catalyzed by GST that is involved in the removal of the proximate and ultimate carcinogens through formation of more water soluble and non electrophilic detoxification products. GST and UDP-GT enzymes are significantly decreased in liver injured animals when compared with normal control animals, this might be resulted because of enhanced covalent binding of DEN metabolites to cellular DNA and thereby carcinogenesis. Upon Carvacrol treatment liver injured rats showed a significant increase in the activities of these enzymes. A significant DEN-induced hepatotoxicity hoisted levels of Serum AST and ALT and a fall in their levels after 30 days. GGT is one of the important marker of liver injury, and their elevation disclose cholestasis and bile duct necrosis. As DEN causes liver injury, there was a significant increase in the activities of these enzymes in DEN-induced (group 2) animals when compared to control rats (Tables 1 and 2). On the other hand, the activities of these marker enzymes were significantly decreased in rats treated with Carvacrol (group 4) when compared to group 2 animals. Administration of DEN has been described to generate more LPO products like Malondialdehyde. Such discovery corroborate with our results as we had seen the elevated level of LPO in DEN-treated rats (Fig. 7), which was significantly lowered on Carvacrol treatment. Natural antioxidants are capable of preventing ROS production, and thereby, it detracts the intracellular oxidative stress. GSH is an important naturally occurring cellular non-enzymatic antioxidant and is needed to maintain the normal reduced state of the cells and to counteract ROS thereby reducing the oxidative stress. GSH depletion ultimately stimulates oxidative stress, with a cascade of effects so that influencing functional and structural coherence of cell and organelle membrane. The Carvacrol treated rats administered with DEN showed significant raise in the levels of hepatic GSH when compared to DEN-treated animals (Table 3). This increase in GSH levels could have suppressed DEN-induced LPO. The decrease of GSH levels and GPx and catalase activities indicates the extremity of the oxidative stress-induced during the exposure to DEN. Interestingly, Carvacrol counteracted the hepatic oxidative damage by preventing the reduction of these parameters provoked by DEN. Enhancement of liver LDH activity, a notable marker of hepatocytes injury, indicates a non-detailed alteration in the plasma membrane integrity and permeability and/or may be due to its overproduction by tumor cells [10]. From our observations, it can be concluded that Carvacrol may counteract the oxidative stress induced by DEN in rats by preventing LPO through scavenging of free radicals or by enhancing the activity of antioxidants, which could detoxify free radicals. Thus, the present study highlights the protective effect of Carvacrol on N-Nitrosodiethylamine induced oxidative stress, which may be involved in the free radical scavenging mechanism.