Toxicity of Haloacetonitrile Mixtures in a Normal Tissue-Derived Human Cell Line: Are they additive, synergistic, or antagonistic?

Haloacetonitriles (HANs), a class of nitrogen-containing disinfection by-products found in treated drinking water, are cytotoxic and genotoxic to mammalian cells. However, most cell toxicity data has been ascertained using transformed or cancer-derived animal and human cell lines with an ambiguous relevance to human health. In this study, we evaluated the cytotoxicity of individual chloro-, bromo-, and iodo-acetonitrile (ClCH2CN, BrCH2CN, and ICH2CN) and their mixtures using normal tissue-derived human epithelium-derived RPE-1hTERT cells. The order for individual HAN cytotoxicity from most to least toxic was ICH2CN>BrCH2CN>>ClCH2CN with the inhibitory concentration that reduced the cell viability by 50% of the untreated cells (IC50) of 3.0, 8.7, and 219.8 µM, respectively. For HAN mixtures cytotoxicity from most to least toxic was BrCH2CN+ICH2CN>ICH2CN+ClCH2CN>ClCH2CN+BrCH2CN+ICH2CN>ClCH2CN+BrCH2CN with IC50 of 8.9, 9.9, 10.1 and 17.8 µM, respectively. The cytotoxicity of ClCH2CN+BrCH2CN was well predicted by both concentration addition (CA) and independent action (IA) models. The CA model overestimated the toxicity of the other three mixtures which indicates an antagonistic effect with a model deviation ratio of less than 2. The IA model predicted the cytotoxicity of BrCH2CN+ICH2CN and ClCH2CN+BrCH2CN+ICH2CN slightly better than the CA model. According to the Chou-Talalay method, all binary mixtures showed strong antagonistic cytotoxic effects, particularly at low concentrations. However, binary mixtures with ClCH2CN had a slight synergistic effect at high concentrations.