ELUCIDATION OF UNDERLYING MOLECULAR MECHANISMS INVOLVED IN BISPHENOL A INDUCED CARDIOTOXICITY AND NEUROTOXICITY

Abstract

Exposure to environmental toxicants such as Bisphenol A (BPA) has raised serious health issues globally particularly in developing countries. BPA an endocrine disruptor, is ubiquitously used in the manufacturing of canned food, feeding bottles and different daily life products. BPA-generated reactive oxygen species can lead to several diseases including cardiotoxicity. However, the endpoints stimulated in BPA induced cardiotoxicity and neurotoxicity yet need to be investigated. The current study aimed to investigate the underlying molecular pathways which may contribute in revealing the protective effects of Pistacia integerrima against BPA induced oxidative stress. Herein, adult Sprague Dawley rats were administrated (subcutaneously) with BPA (100 μg/kg BW, 1 mg/kg BW, and 10 mg/kg BW), 200mg/kgBW P. integerrima, and melatonin (4 mg/kg BW) for 16 days of BPA. Our results showed BPA exposure significantly increased the oxidative stress as demonstrated by increased free radicals (ROS), TBARs level, and decreased antioxidant enzymes including superoxide dismutase and catalase in heart, liver, kidney and brain tissues. Present results of western blotting and qRTPCR showed the increased expression of p53, PUMA and Drp1, while downregulation of Ubc13 in heart and brain tissues of BPA treated groups whereas the levels were reversed upon treatment with P. integerrima. The role of BPA in tissue apoptosis was further confirmed by the increased level of P-p53, cytochrome c and disrupted cellular architecture whereas the P. integerrima has shown its ameliorative potential by mitigating the adverse effects of BPA. Moreover, the lipid, and liver markers profile has also revealed the therapeutic potential of P. integerrima by maintaining the levels in the normal range. However, melatonin has also manifested the normalized expression of apoptotic markers, biochemical markers, and tissue architecture. MicroRNAs are the key regulators in various cellular mechanisms and have the potential to develop new therapeutic strategies. Therefore, the study also sought to investigate the role of miRNA-15a-5p and miRNA-214-3p in BPA induced cardiotoxicity and neurotoxicity. Different bioinformatics tools including TargetScan database were used to find the putative target genes of the miRNAs. The qRT-PCR analysis showed the upregulation of miRNA-15a-5p in the BPA administered groups. However, the expression of the putative target genes Bcl-2 and Mfn-2 was significantly downregulated at both transcriptional and translational level in the disease groups. The significant downregulation of miRNA-214-3p and upregulation of its putative target genes, cdip1, BNIP3 and PKCD in the disease groups suggests the role of miRNA-214-3p in BPA Abstract xvii induced toxicity. However, the treatment with P. Integerrima and melatonin ameliorated the toxic effects of BPA. Conclusively, the data suggest that P. Integerrima may be a potential candidate for the treatment of BPA induced toxicity by neutralizing the oxidative stress through miRNA mediated p53/PUMA/Drp-1/Bcl-2 signalling pathway. The second aim of the present study was to elucidate the highly selective antioxidative potential of synthetic tetra aniline polymers Es-37 and L-37 against BPAinduced cardiac cellular impairments (toxicity). In our results, BPA administration significantly elevated ROS, altered p53, cytochrome c, Bcl-2, and particularly miRNA- 15a-5p expression; however: these changes were notably averted and reversed by Es- 37 and L-37 treatment. Additionally, molecular docking of synthetic polymers L-37 and Es-37 with three proteins (p53, Cytochrome c, and Bcl-2) validated that L-37 has a greater binding affinity with the target proteins compared to Es-37, with the highest binding values reported for the enzymatic protein cytochrome c. Thus, our findings suggest that both synthetic polymers Es-37 and L-37 have the potential to scavenge free radicals, boost-up antioxidant enzyme activities, and avert (BPA-induced) toxicity, thus, may serve as cardioprotective agents. Concurrently, our finding suggests that miRNA-15a-5p overexpression is associated with oxidative stress and coincides with cardiotoxicity; thus, it may be used as a diagnostic marker in BPA-meditated cardiac pathologies. Keeping in view the findings, our research provides a platform for the development of miRNA-based therapeutic and diagnostic strategies against BPAinduced toxicity. After the elucidation of the toxic effects of BPA in cardiac and brain tissues, the current study also aimed to investigate the effect of BPA in breast cancer progression. Breast cancer is an abnormal division of breast cells. BPA, an environmental toxicant, is identified as an emerging risk factor for breast cancer development. However, to the best of our knowledge, no previous study has investigated the BPA levels in breast cancer patients in Pakistan. The present study sought to explore the role of BPA in tumor growth among the Pakistani population. As an endocrine-disrupting chemical (EDC), BPA has more significant potential to initiate tumorigenic events in breast tissue by generating oxidative stress. The level of BPA in the serum samples of breast cancer patients was significantly higher than control. Histological analysis of breast cancer tissue samples revealed distinct subtypes of tumor, such as ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). There was a significant increase in ROS level while a significant decrease in the levels of superoxide dismutase and catalase enzymes in malignant breast tissue samples as Abstract xviii compared to control tissue samples. We found upregulated expression of p53, ZEB1 and WNT1 genes at mRNA level in malignant breast tissue samples. p53 protein expression in malignant breast tissue samples was also enhanced at the translational level. Conclusively, the current findings suggest a relationship between BPA and the progression of breast cancer, which may open new horizons of research for the development of future therapeutic strategies