Toxicity of Haloacetonitrile Mixtures to a Normal Tissue-Derived Human Cell Line: Are They Additive, Synergistic, or Antagonistic?

Abstract

Haloacetonitriles (HANs)─a class of nitrogen-containing disinfection byproducts found in treated drinking water─are cytotoxic and genotoxic to mammalian cells. However, most cell toxicity data have been ascertained by using transformed animal- or cancer-derived human cell lines. 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-1^hTERT 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 2.52 ± 0.19, 7.24 ± 0.68, and 190 ± 18.5 μM, respectively. For HAN mixtures, cytotoxicity from most to least toxic was BrCH₂CN+ICH₂CN > ICH₂CN+ClCH₂CN ≈ ClCH₂CN+BrCH₂CN+ICH₂CN > ClCH₂CN+BrCH₂CN with a total IC₅₀ of 4.65 ± 0.71, 8.12 ± 1, 7.91 ± 0.64, and 13.6 ± 2.04 μM, respectively. The cytotoxicity of all four mixtures at IC₅₀ was well predicted by both concentration addition (CA) and independent action (IA) models, which confirmed additivity effects. However, the Chou–Talalay method (CT) showed antagonistic cytotoxic effects. The difference could primarily stem from the different threshold criteria of each model for additivity, synergy, and antagonism, leading to different conclusions. Results indicate that evaluating cumulative mixture toxic effects with CA, IA, and CT can improve the overall confidence of the analysis.