Evaluation of interaction and combined toxicity of polyaromatic hydrocarbons (PAHs) and metals using human cell-based bioassays

Abstract

Mixed contamination of polyaromatic hydrocarbons (PAHs) and metals are ubiquitous in the environment. Some of the individual PAHs are known as human carcinogens, and metals are associated with various systemic toxicity and cancers in humans. Although the individual toxicity of PAHs and metals are well documented, our present state of knowledge about the adverse health effects of these mixtures is very limited in terms of exposure associated toxicity in humans, the underlying mechanism of toxicity and predicting the toxicity of mixtures. Due to this, their risk assessment has been mostly carried out based on their individual toxicity data. This research work investigated the interaction and combined toxicity of four PAHs (benzo[a]pyrene (B[a]P), naphthalene, phenanthrene, and pyrene) and three metals (arsenic, cadmium and lead) in HepG2 cells. The mixture of PAHs and metals were evaluated for their combined effects on the cytotoxicity, oxidative stress response, genotoxicity, cell cycle parameters, and AhR (aryl hydrocarbon receptor) activity. The toxic endpoints were selected based on the knowledge of the individual toxicity of PAHs and metals. A human liver cell line (HepG2 cell line) was selected as a test system due to its inherent metabolic capacity and is well established for toxicological research. The individual and binary to quaternary mixtures of B[a]P and metals were found to be toxic to HepG2 cells. The combination index (CI)-isobologram method predicted the response of synergism, additivity or antagonism at different effect levels of mixtures (IC10 to IC90). In general, all the mixtures displayed synergism and antagonism at low and high effect levels, respectively. The CI method could be better for predicting the mixture toxicity compared to those of concentration addition (CA) and independent action (IA) model. Since oxidative stress is one of the common modes of action of metals and PAHs, the individual and mixtures of PAHs and metals were evaluated for their effects on the Nrf2 antioxidant response pathway (an indicator of oxidative stress response) in the ARE reporter-HepG2 cells. Both individual and binary to seven-component mixtures of PAHs and metals activated the Nrf2 antioxidant response pathway. The mixture effect is well predicted by the CA model with the exception of underestimation of few mixture combinations. Based on the effects of mixtures of PAHs and metals on cytotoxicity and oxidative stress response, studies were conducted to define the relationship between oxidative stress parameters (reactive oxygen species (ROS) generation and total glutathione (GSH) level) and the observed cytotoxicity in HepG2 cells. The binary mixtures of metals and mixtures of B[a]P with metals showed significant interaction on the cytotoxicity. All the six mixtures increased the ROS production, but the total GSH level was increased or decreased depending on the concentrations and types of mixtures, respectively. The results emphasised the significant role of oxidative stress parameters in the mixture toxicity of B[a]P and metals. Some PAHs and metals are known carcinogens. A flow cytometry based micronucleus (MN) test was used to evaluate the genotoxicity of mixtures of PAHs and metals in HepG2 cells. In addition, the effects of mixtures on cell cycle parameters and the AhR activity were also evaluated. The mixture studies were designed to evaluate the effects of metals and/or PAHs on MN formation of B[a]P. In general, the mixture of metals or PAHs increased or decreased the genotoxicity of B[a]P, respectively. The observed genotoxic effect of mixtures of PAHs and metals is well correlated with the changes in cell cycle parameters and AhR activation. Soil is the major sink for both PAHs and metals. Studies with spiked and contaminated soil samples were conducted to understand the toxicity of the bioaccessible fraction of PAHs and metals in soil. The soil samples were spiked with metals or B[a]P and aged for a period of three months. The bioaccessibility and toxicity were assessed using the Unified BARGE method (UBM) method and HepG2 cell based bioassays (cytotoxicity and oxidative stress response), respectively. A higher % of bioaccessibility was obtained for metals and but not for B[a]P. The bioaccessible fraction of metals in the UBM extract was not toxic to HepG2 cells, however the individual or mixtures of metals induced the oxidative stress response at this concentration in the Nrf2 antioxidant assay. In conclusion, the toxicity of selected mixtures of PAHs and metals was determined using human cell based bioassays. In this project, various bioassays, including HepG2 cell based ARE-Nrf2 antioxidant response pathway and AhR-CAFLUX assays, and flow cytometry based MN test were standardised for mixture studies. The observed or predicted interactions among these mixtures vary depending on the toxic endpoints, concentrations, and the number of individual chemicals that are present in the mixtures. Therefore, the traditional CA or IA model for health risk assessment may over or underestimate the risk of mixtures. Further mechanistic studies will be useful to understand the observed effects in various toxic endpoints.