Genotoxicity investigation of organic n-chloramines.

Organic N-chloramines have long been recognised as disinfection by-products (DBPs) found in both chlorinated and chloraminated water, but have gained little attention from water authorities in the past. However, in recent years studies have shown that organic N-chloramines are molecules involved in inflammation and several chronic diseases including cancers. A recent study (Bull et al., 2006) has suggested that organic N-chloramines can be potential health risks but due to a lack of available toxicological information toxicity studies of compounds in this group have been recommended as a priority in DBPs research. The aim of this study was to investigate genotoxicity of individual organic N-chloramines utilising a mammalian cell-based genotoxicity assay to help determine which compound(s) should be subject to further in vivo studies. The flow cytometry-based micronucleus (FCMN) assay was optimised and validated for use as a rapid screening for genotoxicity of organic N-chloramine candidates. A number of assay validations were conducted on two mammalian cell lines (WIL2-NS and L5178Y) using model genotoxicants with various modes of action. Comparative studies on these two cell lines showed that WIL2-NS cells were suitable for the FCMN assay and therefore selected for use in all studies described in this thesis. For the genotoxicity investigation of organic N-chloramines, 16 compounds were synthesised by chlorination of amine precursors. At least 3 concentrations (in μM range) were subjected to screening for genotoxicity using the validated FCMN assay and confirmed by microscopic counting of micronuclei. This study found that of the 16 compounds, 4 were genotoxic to WIL2-NS cells by both FCMN and microscopy based MN assay. Oxidative stress was hypothesised as a possible genotoxic mechanism of these compounds and also was investigated in this study. Following exposure to the 4 genotoxic organic N-chloramines, it was found that although there was a small reduction of cellular glutathione the change in lipid peroxidation was not observed. This suggested that oxidative stress is unlikely to be a mechanism involved in genotoxicity of these organic N-chloramines. The final part of this research demonstrated an application of using the optimised FCMN assay to identify genotoxic DBP precursors in Australian water. We collaborated with Curtin University, Western Australia on this aspect. Highly coloured surface water was collected, concentrated, and fractionated based on molecular weight (MW) of the organic contents by researchers at Curtin University. Eight MW fractions (pre- and post chlorination) were tested for genotoxicity using the FCMN assay. No genotoxicity was observed in all pre-chlorinated MW fractions while significant genotoxicity was seen in chlorinated products of several fractions of medium to high MW. This result indicated that these fractions contain materials that are precursors to genotoxic DBPs and may lead to future studies such as characterisation of the genotoxic DBP precursors for their removal prior to the disinfection process.Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 201