Research Projects

The shift from phthalates like di(2-ethylhexyl) phthalate (DEHP) to alternative plasticizers such as diisononyl cyclohexane-1,2-dicarboxylate (DINCH) and di(2-ethylhexyl) adipate (DEHA) aims to reduce reproductive toxicity. Despite their widespread use, limited data exist on their safety, particularly concerning male reproductive health. Real-world exposures often involve combinations of plasticizers, yet the potential synergistic or additive effects of DINCH and DEHA remain unexplored. This study evaluates the reproductive toxicity of DINCH, DEHA, and their combination while assessing the protective efficacy of Ayurvedic antioxidants. The research begins with ex vivo screening of Ayurvedic antioxidants—lycopene, ellagic acid, genistein, curcumin, quercetin, gallic acid, ascorbic acid, withanolide A, chlorogenic acid, and bacoside A. Primary testicular cell suspensions from untreated mice will be exposed to environmentally relevant concentrations of DINCH, DEHA, their combination, and DEHP as a positive control. ROS levels (via DCFDA assay) and cell viability (via MTT assay) will identify effective antioxidants. Two top-performing antioxidants will proceed to in vivo studies. In the in vivo phase, male Swiss albino mice will be exposed to DINCH, DEHA, and their combination at RfD-based doses, with DEHP as a positive control. Co-treatment with antioxidants will assess their efficacy in mitigating reproductive toxicity. Biochemical assays will measure antioxidant enzyme activities (catalase, GST, GPx, SOD), lipid peroxidation (MDA), and glutathione levels. ROS generation and apoptosis will be assessed in testicular cells and spermatozoa using H2-DCFDA and AO-EtBr staining. Sperm parameters, including count, motility, viability, and morphology, will be microscopically evaluated. To elucidate molecular mechanisms, immunofluorescence will detect Nrf2 nuclear translocation, while western blotting will quantify proteins involved in oxidative stress (Nrf2, HO-1, NQO-1, NOX-4), lipid peroxidation (4-HNE), DNA oxidative damage (8-OHdG), apoptosis (Bax, Bcl-2, Caspase-3, Caspase-9), and steroidogenesis (StAR, CYP11A1, 3β-HSD, CYP17A1). ELISA will quantify serum testosterone, LH, and FSH, while histological analyses using hematoxylin-eosin staining will examine testicular structural changes. Mass spectrometry will confirm tissue accumulation of DINCH, DEHA, and antioxidants. This integrated approach addresses critical gaps in understanding DINCH and DEHA toxicity, especially their combined effects on male reproduction at realistic exposures. By leveraging the therapeutic potential of Ayurvedic antioxidants, the study bridges traditional knowledge with cutting-edge toxicological research. The findings will provide actionable insights into mitigating oxidative stress and reproductive dysfunction, shaping regulatory policies and offering innovative solutions to safeguard male reproductive health in an increasingly plasticized world.