Investigation of In-vitro effects of Phyllanthus amarus on carmustine hepatotoxicity in AML-12 cell line

Background and Aim: Carmustine is an antineoplastic agent used in the treatment of glioma, glioblastoma multiforme, medulloblastoma, astrocytoma, multiple myeloma, Hodgkin and non-Hodgkin lymphoma. During treatment with carmustine, transient elevations in serum transaminases, hepatotoxicity, and hepatic veno-occlusive disease may occur in 5-20% of patients. It is aimed to reduce cytotoxicity by using P. amarus, which is known to have a hepatoprotective effect, together with carmustine. Material and Methods: Our study was planned as four groups: Control, Carmustine, PA, Carmustine+PA in alpha mouse liver 12 cell line. Substance concentrations to be applied to cell lines were determined by the 3-(4,5-dimethyliazol-2-yl)-2,5 diphenyl tetrozolium bromide method. In order to determine oxidative stress, superoxide dismutase, catalase and glutathione expression levels were measured by Real Time PCR. In order to evaluate the role of apoptosis pathways in the hepatotoxicity caused by carmustine and the effects of P. amarus on this damage, Real-Time PCR analysis used caspase-3, caspase-9, apoptotic protease activating factor 1, Bax, B-cell lymphoma-2, p53, second mitochondria-derived activator of caspases/ direct inhibitor of apoptosis-binding protein with low pI and apoptotic protease activating factor 1, topoisomerase 1, topoisomerase 2 gene expressions were examined. Results: Increased levels of superoxide dismutase (p<0.0001), catalase (p<0.0001) and glutathione (p<0.0001) in the Carmustine group compared to the control showed that antioxidant defense systems were activated. The increase in the expression of apoptosis protease activating factor-1 (p<0.0001) and Bax (p<0.0001) showed that apoptosis was triggered by the intrinsic pathway, and the increase in caspase-3, accompanied by the absence of caspase-9, showed that apoptosis was also triggered by the intrinsic pathway. While p53 increase (p<0.0001) in the PA group is an indicator of DNA damage, Topoisomerase 1 (p<0.0001) and Topoisomerase 2 (p<0.0001) increases are an indicator of this damage. While the increase in Bax (p<0.0001), apoptotic protease activating factor 1 (p<0.0001) and second mitochondria-derived activator of caspases/ direct inhibitor of apoptosis-binding protein with low pI (p<0.0001) indicates that apoptosis is triggered, caspase-9 and caspase-3 activation did not occur. This revealed to us the partial protective effect of P. amarus. The increase in glutathione (p<0.0001) and catalase (p<0.0001) levels in the carmustine+Phyllanthus group indicates that the antioxidant system is activated, and Topoisomerase 1 (p<0.0001) mRNA levels are increased. The increase may be the result of stress on DNA. Second mitochondria-derived caspase activator/Low pI (p<0.05), BAX (p<0.0001), caspase-3 (p<0.0001) increased and B-cell lymphoma-2 (p<0.001) The decrease can be interpreted as the cells going to apoptosis via the intrinsic pathway. Conclusion: It was determined P. amarus had a partially protective effect on hepatotoxicity caused by Carmustine. However, studies on different doses and application times for P. amarus are needed. Keywords: Apoptosis, carmustine, hepatotoxic, oxidative stress, P. amarus Financial Support: The present study was supported by the Research Fund of Trakya University. Project No: TUBAP-2022/03