In vitro toxicity and genotoxicity assessment of disinfection by-products, organic N-chloramines.

Disinfection by-products (DBPs) are of concern to both water industries and health authorities. Although several classes of DBPs have been studied, and there are regulated safe levels in disinfected water for some, a large portion of DBPs are not characterized, and need further investigation. Organic N-chloramines are a group of DBPs, which can be formed during common disinfection processes such as chlorination and chloramination, but little is known in terms of their toxicological significance if consumed in drinking water. Only a few in vitro studies using bacterial assays have reported some genotoxic potential of organic N-chloramines, largely in the context of inflammatory processes in the body rather than exposure through drinking water. In this study, we investigated 16 organic N-chloramines produced by chlorination of model amino acids and amines. It was found that within the drinking water-relevant micromolar concentration range, four compounds were both cytotoxic and genotoxic to mammalian cells. A small reduction of cellular GSH was also observed in the treatment with these four compounds, but not of a magnitude to account for the cytotoxicity and genotoxicity. The results presented in this study demonstrate that some organic N-chloramines, at low concentrations that might be present in disinfected water, can be harmful to mammalian cells.

Flow-cytometric analysis of in vitro micronucleus formation: comparative studies with WIL2-NS human lymphoblastoid and L5178Y mouse lymphoma cell lines.

The development of a flow cytometry-based micronucleus (FCMMN) assay for measuring the micronucleus (MN) frequency in cells following exposure to test chemicals has potential for improving reproducibility and reducing turn-around time when compared with the traditional microscopy-based micronucleus method. A major drawback of the FCMMN assay is that a false-positive interpretation could result from the presence of large numbers of apoptotic or necrotic bodies in the measured sample. Although several studies have reported ways in which the FCMMN assay could be improved using different staining techniques or electronic gating strategies, to date none of these protocols are suitable for use as a screening assay. To reduce the interference from apoptosis, performing the FCMMN assay with an apoptosis-resistant cell line may be an alternative approach. This study reports the use of p53-mutated cell lines to minimise the interference found in the FCMMN assay. Two commonly used cell lines (WIL2-NS and L5178Y) were investigated by comparison of (1) cytotoxicity and micronucleus induction in the FCMMN assay following treatment with model genotoxicants and (2) apoptotic responses after exposure to inducers of apoptosis. Both cell lines were responsive to all genotoxicants, producing concentration-dependent results with respect to genotoxicity. WIL2-NS cells were found to be more tolerant to apoptosis induction than L5178Y cells. This characteristic could be beneficial to minimise the interference from apoptotic nuclei in the FCMMN genotoxicity-screening assay.