Effects of multi-component mixtures of polyaromatic hydrocarbons and heavy metal/loid(s) on Nrf2-antioxidant response element (ARE) pathway in ARE reporter-HepG2 cells

Abstract

Exposure to polyaromatic hydrocarbons (PAHs) and heavy metal/loid(s) has been demonstrated to induce an oxidative stress response in mammalian cells. The combined effect of PAHs and heavy metal/loid(s) on the oxidative stress response has not been reported extensively. The Nrf2 antioxidant response pathway plays an important role in cellular antioxidant defense against oxidative stress-induced cell damage. In this study, we have determined the combined effect of four PAHs (benzo[a]pyrene (B[a]P), naphthalene (Nap), phenanthrene (Phe) and pyrene (Pyr)) and three heavy metal/loid(s) (arsenic (As), cadmium (Cd) and lead (Pb)) on the Nrf2 antioxidant pathway using the ARE reporter-HepG2 cell line. The mixture study was carried out for binary, ternary, quaternary and seven-component combinations of PAHs and heavy metal/loid(s). Initially, individual dose responses for the PAHs (B[a]P, Nap, Phe and Pyr) and heavy metal/loid(s) (As, Cd and Pb), as well as their respective concentrations that induced an induction ratio of 1.5 (EC_IR1.5), were determined. The luciferase assay system was used to quantify the induction of the Nrf2 antioxidant pathway. The individual dose response study showed that both PAHs and heavy metal/loid(s) activated the Nrf2 antioxidant pathway in ARE reporter-HepG2 cells. Among these chemicals, Cd was the most potent inducer, followed by B[a]P and As. Based on the individual dose response findings, PAHs and heavy metal/loid(s) were mixed at equipotent ratios using a fixed concentration ratio, and the effects of the mixtures of PAHs and heavy metal/loid(s) (binary to seven-component) on the Nrf2 antioxidant pathway were determined. The mixture effects were predicted by using the concentration addition (CA) model. Overall, the results showed that the multi-component mixtures of PAHs and heavy metal/loid(s) induced an oxidative stress response in ARE reporter-HepG2 cells, and that the CA model is an appropriate model to predict the interaction effect of these selected mixtures. A human cell line-based reporter gene assay system was successfully used to determine the mixture effects of two groups of common contaminants on oxidative stress response pathway.