107 | Protective effects of hydroxytyrosol against dichlorodiphenilethylene-induced oxidative injury in liver cells

Vincenzo Migliaccio¹, Ilaria Di Gregorio¹, Martina Coletta¹, Giuliana Panico², Assunta Lombardi², Lillà Lionetti¹ | ¹Dept Chemistry and Biology “Adolfo Zambelli”, University of Salerno, Italy; ²Dept Biology, University Federico II, Napoli, Italy.

Exposure to environmental contaminants may compromise hepatic function by inducing oxidative stress and activating pathways involved in cell death. Consequently, nutritional strategies rich in antioxidant molecules play a key role in preserving liver cell homeostasis. Among phenolic compounds derived from olives, hydroxytyrosol (HT) stands out as a particularly powerful antioxidant compound. This study aimed to evaluate the potential protective action of HT, administered either alone or in combination with the environmental pollutant dichlorodiphenylethene (DDE), the main metabolite of the pesticide dichlorodiphenylethane (DDT), on human hepatocarcinoma cell line HepG2. Specifically, the effects on cell viability and morphology, oxidative stress levels, and mitochondrial structure/dysfunction were examined. Cells were treated for 24 hours with DDE (30 or 100 µM) and/or HT (50 or 100 µM). Cell viability was assessed through the MTT assay. Morphological changes, intracellular reactive oxygen species (ROS) production, and mitochondrial network alteration was evaluated by fluorescent microscopy. Apoptotic processes were quantified by measuring caspase-3 activity using a colorimetric assay. The activity of key antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) was measured by spectrophotometric methods. In addition, mitochondrial responses were further characterized by western blot analysis of SOD2 protein levels and the mitochondrial markers voltage-dependent anion channel (VDAC) and translocase of the outer membrane 20 (TOM20), both indicative of mitochondrial mass. Treatment with DDE at both tested concentrations (30 and 100 µM) produced significant decline in cell viability and induced marked morphological alterations, including progressive cytoplasm vacuolization. These effects were accompanied by elevated caspase-3 activity, mitochondrial fragmentation, and reduction in SOD2 expression. At the higher concentration, DDE also led to a pronounced reduction in mitochondrial content. With respect to antioxidant defenses, exposure to 30 µM DDE resulted in increased CAT activity, suggesting an adaptive cellular response to low-level toxic stress. In contrast, HT administration at 100 µM reduced cell viability without activating apoptotic pathways, pointing to a possible inhibitory effect on cell proliferation rather than direct cytotoxicity. Under the experimental conditions used, no significant variations in lipid accumulation were observed. HT treatment also stimulated the activity of antioxidant enzymes overall. Notably, combined exposure to DDE and HT exerted a protective effect, attenuating DDE-induced oxidative stress, apoptosis, and morphological damage, likely through the upregulation of SOD and the recovery of mitochondrial content. Overall, the results highlight the protective capacity of hydroxytyrosol against DDE-related cellular damage, mitochondrial impairment, and apoptotic cell death, further supporting the beneficial role of extra virgin olive oil polyphenols in safeguarding liver health.