Individual and mixture toxic effects of monohaloacetonitriles to a human cell model
Creators: name: Jayawardana, Amarasinghe Senadeerage Dona Prabhani Thilina Kumari
Publisher: name: Graduate Studies
Year: 2023
Subjects: subject: Environmental Sciences
subject: Toxicology
subject: Chemistry--Analytical

description: Haloacetonitriles (HANs) are an emerging class of nitrogen-containing disinfection by-products (DBPs) and have been reported up to a maximum of 41 µg/L in treated drinking water. There is a growing interest in HANs because they are more cyto- and genotoxic than the regulated carbon-based DBPs and have been recently reported as key drivers in overall drinking water toxicity. However, most data presented thus far has focused on monitoring cell toxicity in animal and human cancer-derived cell models with an uncertain relevance to human health. Additionally, those studies have not addressed the possibility of HAN degradation in cell culture conditions. Therefore, the observed effects may come from HANs or HAN-degradation products. In this study I aimed to assess the stability of HANs in cell culture media and the toxic effects of DBPs to a normal tissue-derived human cell model. First, the stability of 8 HANs was evaluated under cell culture conditions used in in vitro toxicological studies (37°C, pH=7.4, for 3 days). Mono-HANs were stable during the incubation period; however, the stability of di- and tri-HANs declined significantly. Di- and tri-HANs transformed into their corresponding haloacetamides and haloacetic acids (HAAs), which are less cytotoxic than HANs. To obtain a more accurate measurement of HAN toxicity, it is recommended to consider changing cell culture media for di- and tri-HANs daily. Then, individual mono-HANs and their mixtures were evaluated for chronic cytotoxicity to a normal tissue-derived human cell model. Cytotoxicity was measured using cell viability and cell growth. HAN toxicity increased in the order of ClCH2CN &lt;&lt; BrCH2CN &lt; ICH2CN. At lower concentrations, the mixture of BrCH2CN+ICH2CN had the highest combined cytotoxicity. ClCH2CN demonstrated additive/synergistic effects in the presence of BrCH2CN and ICH2CN. Those results confirmed the previous conclusions from Chinese hamster ovary cell-based assays: cytotoxicity of HANs increased in the order of ClCH2CN &lt;&lt; BrCH2CN &lt; ICH2CN. In addition, the cytotoxicity of mono-HAAs and their binary and tertiary mixtures were also evaluated. From the tested HAAs, ClCH₂COOH was the least cytotoxic compound. BrCH₂COOH and ICH₂COOH had similar cytotoxicity. ClCH₂COOH + BrCH₂COOH had the least combined cytotoxicity, and ClCH₂COOH + IAA had the highest combined cytotoxicity. Afterward, the experimental results were compared with toxicity models to evaluate the type of toxic interactions (i.e., additivity, synergism, and antagonism). This study provided a primary understanding of the DBPs as mixtures. Lastly, cell metabolites were extracted and identified after cell exposure to CH3CN. The lowest concentration that negatively affects cells was determined using the non-monotonic dose-response curves for the reduction of cell viability and growth of RPE-1hTERT cells. From these experiments, 1 mM CH3CN was chosen as the non-cytotoxic concentration to treat cells for the metabolomic study. Then, two solvent mixes were evaluated to identify the solvent mix that extracted the most metabolic features. A solvent mix of 80% methanol and 20% H2O was selected. Cells treated with CH3CN for 72 h did not induce a significant change in the metabolic profile of RPE-1hTERT cells. To my knowledge, this is the first attempt to characterize the toxicity of HANs and HAAs to a normal tissue-derived human cell model. Mono-HANs' and mono-HAAs' toxicity to RPE-1hTERT cells increased with the change of halogen substitute from chlorine to bromine/iodine. Although the combined effects of mono-HANs were antagonistic, the combined effects of mono-HAAs were synergistic, implying that HANs and HAAs may affect different cellular mechanisms. Moreover, to the best of my understanding, this study is the first to measure the cytotoxicity of CH3CN to a normal tissue-derived human cell model. The observation of this study concludes that exposure to a lower dose (&lt; 1mM) of CH3CN does not cause cytotoxic effects to RPE-1hTERT cells nor significantly alter the metabolic profile of RPE-1hTERT cells.