The binary, ternary and quaternary mixture toxicity of benzo[a] pyrene, arsenic, cadmium and lead in HepG2 cells

Polycyclic aromatic hydrocarbons (PAHs) and heavy metal/loid(s) are common environmental pollutants. Toxicological interaction data on benzo[a] pyrene (B[a] P) and heavy metal/loid(s) are lacking. In this study, we have determined the combined toxicity of B[a] P, arsenic (As), cadmium (Cd) and lead (Pb) in HepG2 cells. The binary, ternary and quaternary mixture toxicity of B[a] P and heavy metal/loid(s) was predicted by using the combination index (CI)-isobologram method. This method is useful to predict the quantitative nature of an interaction between chemicals at different effect (inhibitory concentration) levels from 0.1 to 99% using computerised quantitation. A total of 11 mixtures including six binary mixtures, four ternary and one quaternary mixtures of B[a] P and heavy metal/loid(s) were evaluated for their interactions. The cytotoxicity of individual and multi-component mixtures was evaluated by MTS assay. The selected concentrations for the individual dose response study were 0-100 mu M - B[a] P; 0-40 mu M - Cd; 0-400 mu M - As and Pb. The individual dose response results showed that all four chemicals were toxic to liver cells with Cd being the most potent toxicant. Mixtures of B[a] P and heavy metal/loid(s) were prepared based on their individual D-m concentration using a 1 : 1 ratio and exposed to HepG2 cells. By using the CI-isobologram method, the predicted interactions between these chemicals were synergism, additivity or antagonism at different effect levels. All the mixtures except the ternary mixture of B[a] P + As + Pb displayed synergism at a lower effect level (IC10-IC30), and additivity, synergism or antagonism at 50-90% effect levels. Among these mixtures, mixtures of heavy metal/loid(s) (both binary and ternary combinations) and a quaternary mixture of B[a] P + As + Cd + Pb showed a strong synergistic response at lower effect levels compared to other mixtures. The predicted interaction response by the CI method was compared with classical models of concentration addition and independent action. The CI method displayed an improved prediction power compared to classical models. The predicted synergistic interaction between B[a] P and heavy metal/loid(s) may have important implications in the human health risk assessment of these mixed chemical mixtures at contaminated sites.