Mutation Research/Genetic Toxicology and Environmental Mutagenesis
Assessing the genotoxicity of two commonly occurring byproducts of water disinfection: Chloral hydrate and bromal hydrate
Introduction
One of the most significant public health advances of the twentieth century was the adoption of drinking water disinfection in many countries [1]. This practice has sharply reduced the incidence of infectious diseases such as cholera, typhoid, and dysentery [2], [3]. After this dramatic success, disinfection practices have been introduced into swimming pools and other recreational water venues to ensure the elimination of pathogenic microorganisms and the prevention of waterborne disease outbreaks [4]. However, disinfection treatments result in the undesirable formation of chemical contaminants known as disinfection byproducts (DBPs), in consequence to reactions taking place between disinfectants and organic matter present in water [5], [6]. Exposure to DBPs in humans can take place through ingestion of drinking water or inhalation and dermal absorption during showering or swimming [7], [8], [9], [10]. Many studies have suggested associations between exposure to DBPs and adverse health effects. Increased incidence of asthma [11], bladder cancer [12], [13], and colorectal cancer [14] have been reported. Adverse pregnancy outcomes such as spontaneous abortions [15], stillbirth [16], and fetal growth restriction [17] have also been noted. To date, more than six hundred DBPs including trihalomethanes, haloacids, halonitriles, haloaldeydes, haloketones, halonitromethanes, haloamines, haloamides, haloalcohols, and halobenzoquinones have been identified in disinfected waters [9], [18], [19], [20], [21], [22], [23]. Several laboratory-controlled studies have been conducted to evaluate potential toxicities of DBPs providing evidence about cytotoxic, genotoxic, carcinogenic and teratogenic potentials [20], [24], [25], [26], [27], [28]. However, the toxicological data are limited to only a small fraction of identified DBPs. In consequence, many DBPs that have been detected in disinfected waters remain with unknown toxicological profiles. Chloral hydrate and bromal hydrate, the hydrated forms of trichloroacetaldehyde and tribromoacetaldehyde respectively, belong to the chemical class of haloacetaldehydes. This class of DBPs has been reported to be one of the most abundant DBP classes by weight [19], [25], [29], [30]. Occurrence studies have shown that the predominant trhihaloacetaldehyde in chlorinated waters is chloral hydrate, while bromal hydrate is the predominant trihaloacetaldehyde in chlorinated waters containing high levels of bromide [19], [31]. In a recent study, BH was detected as one of the degradation byproducts of benzophenone-3, a UV filter commonly used in sunscreens, in chlorinated swimming pools filled with seawater [32].
Toxicokinetic studies have shown that CH is rapidly absorbed after oral administration, and enters the liver where it undergoes extensive metabolism in rodents [33], [34] and in humans [35], [36]. Studies of the potential carcinogenicity of CH in mice have demonstrated that it is able to induce hepatocellular adenomas and carcinomas, and exposure to CH has been associated with increases in malignant lymphoma and adenoma of the pituitary gland [37], [38], [39]. However, there was still no persuasive evidence to connect chloral hydrate exposure and the development of cancers in humans [40]. CH was also found to induce significant aneugenic effects in mice [41]. Furthermore, micronuclei were produced in germ cells of male mice treated intraperitoneally with CH [42]. CH was also reported to be able to lead to chromosomal loss in mouse spermatids [43] and in human lymphocytes [44]. Nevertheless, most of the investigations incorporated only one or two in vitro assays [25], [45] and results from genotoxicity assessment of CH remain inconclusive. Concerning BH, although little is known about its toxicity, the U.S. Environmental Protection Agency (EPA) included this compound to the list of priority DBPs to be monitored in a nationwide occurrence study [46] due to anticipations of potential toxicity based on alarming structure-activity relationships [47]. To address this scarcity of data, we analyzed the genotoxicity of CH and BH using a battery of three genotoxicity assays, namely the Ames test, the comet assay, and the micronucleus assay. The use of a test battery is critical since no single genotoxicity test is capable of detecting all genotoxic mechanisms [48]. We performed the three assays in the absence and presence of the metabolic activation fraction S9 mix to assess the effects of metabolic reactions on the toxicity of the two compounds.
Section snippets
Chemicals
The identifiers and structures of CH and BH are shown in Table 1. CH (crystallized, ≥98%) was obtained from Sigma-Aldrich (China). BH was prepared by adding tribromoacetaldehyde (bromal, Sigma-Aldrich, UK, 97% purity), to ultrapure water and then recrystallizing the product from a small volume of water. Ultrapure water was produced from a Millipore water system (resistivity = 18.2 MΩ.cm). Before toxicological analyses, stock solutions were prepared in dimethyl sulfoxide (DMSO, Chromasolv plus,
The Ames test
We evaluated the capacity of CH and BH to induce mutations in DNA using the Ames test in four Salmonella tester strains: TA97a, TA98, TA100, and TA102. The assay was carried out in the absence and presence of exogenous metabolic activation (S9 mix). As shown in Table 2, CH did not exhibit mutagenic effects in the tested strains, unlike BH, which induced mutagenic activity in the strain TA100. The results of the regression analyses for the dose-response relationship of BH in the presence and
Discussion
Several epidemiologic studies have suggested that exposure to DBPs is associated with increased incidence of cancer, particularly bladder and colorectal cancers [12], [13], [14]. The analysis of genotoxicity of DBPs allows identifying the chemical species that could be responsible for carcinogenicity [48]. Genotoxicity testing detects carcinogens that are thought to act primarily via a mechanism involving direct genetic damage [48]. CH has been frequently detected as a predominantly occurring
Conclusions
Our findings show that CH did not induce any genotoxic effects using the Ames test, the comet assay, and the micronucleus test, while BH was mutagenic in the Salmonella assay and caused genomic damage in the comet assay but not in the micronucleus test. These results imply that the mechanism of genotoxicity of BH involves inducing mutations and DNA damage but not chromosomal aberrations. The genotoxicity of BH decreased in the presence of metabolic fractions suggesting the formation of less
Acknowledgment
T.M. acknowledges the French Ministry of Higher Education and Research for his doctoral scholarship.
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