Investigations on epigenetic modifications in cell cycle regulatory genes in response to substance of abuse pesticides and drugs

Abstract

The present study was undertaken to investigate the effects of Acetaminophen, a commonly used analgesic and cypermethrin, a widely used pyrethroid pesticide on the liver function (ALT, AST, ALP), hepatic structure, gene regulation as well as methylation status of cell cycle regulatory genes (p53, CDKN1A, cdk2, CDK4). These genes were shortlisted through STRING as well as Cytoscape analysis for in vivo examination in APAP and cypermethrin liver toxicity. Intraperitoneal administration of APAP (300 mg Kg-1 b. wt.) and oral administration of 1/10 LD50 oral dose of cypermethrin (2.5 mg/kg) the test animals once in a week for eight weeks. BALB/c mice produced liver injury as evinced by increased ALT, AST and ALP levels. RTPCR analysis demonstrated a time dependent up-regulation in the expression of cell cycle regulatory genes viz. p53, p21 along with related apoptosis genes viz. Bax and caspase-3. There was a time dependent decrease in the expression of CDK4 and CDK2 genes. Apoptosis related BCL-2 gene saw decline in the expression in test groups whereas apoptosis was induced by activating Bax and Caspases 3. The APAP treatment groups did not show any methylation changes in the promoter regions of p53 and p21. The sequencing results of CYP treated DNA samples demonstrated methylation change in the p53 promoter region with methylation percentage of 45.5% in the test group as compared to 36.4 % in the control group as observed by BISMA sequencing analysis of Bisulphite treated DNA. The outcome of the study lays down strong foundations for exploiting DNA methylation as a predictor for possible long-term injury to substance of abuse. newline