Identification de composés génotoxiques dans les eaux de boisson

Depuis la mise en évidence de trihalométhanes dans les eaux potables en 1974, de multiples travaux ont démontré la présence de nombreux composés génotoxiques dans l'eau de boisson. L'eau potable obtenue à partir d'eau de surface subit un traitement incluant généralement une étape de chloration. Il est aujourd'hui largement admis que l'activité génotoxique des eaux de boisson provient principalement de la chloration des substances humiques, composés organiques naturels contenus dans l'eau brute et issus de la dégradation des déchets animaux et végétaux. Les très faibles concentrations en composés génotoxiques dans les eaux potables nécessitent la concentration des échantillons, procédé qui risque toutefois de modifier la génotoxicité. Plusieurs tests mettant en oeuvre des cellules procaryotes ou eucaryotes, des plantes ou des mammifères, ont permis de mettre en évidence les effets génotoxiques dans des eaux potables chlorées. L'identification des composés génotoxiques est réalisée au moyen des données de la spectrométrie de masse et de la spectroscopie UV ou RMN (proton ou carbone). Ces agents sont généralement non volatils, acides et polaires. Bien que certains composés inorganiques interviennent parfois, la majeure partie de la génotoxicité est attribuée aux agents organohalogénés (bromés ou/et chlorés), les principaux étant les trihalométhanes, acides acétiques, acétonitriles, cétones, et hydroxyfuranones. La fixation de normes contribue à limiter l'exposition des populations aux agents potentiellement dangereux. La qualité des eaux de boisson peut être accrue en utilisant une eau brute moins chargée en matière organique, et en améliorant le traitement chimique tout en veillant à conserver la qualité microbiologique de l'eau produite.In 1974, two independent studies - one in the Netherlands and the other in the United States - demonstrated the occurrence of trihalomethanes in drinking water. Following studies showed that these chemicals were common contaminants of drinking water and that chloroform, i.e. one of these trihalomethanes, was carcinogenic in rodents. Further investigations demonstrated that extracts of chlorinated drinking water induced significant mutagenicity in the Ames/Salmonella assay. In the present paper we will fist discuss the methods used to detect the genotoxic activity of drinking water and, then, the methods developed to identify the compounds responsible for this activity. After this, we will present the main genotoxic chemicals identified in drinking water, before finally considering several propositions to limit the exposure of populations to these genotoxic compounds.Drinking water is usually produced through a multistage process which includes one or several chlorination steps. It is now widely accepted that the genotoxic activity of drinking water mainly originates from the reaction of chlorine with humic substances present in raw water. Humic substances are natural organic matters (resulting from the degradation of plants and animal tissues) of very complex structure with most chemical functions arranged in aromatic rings or aliphatic chains. The identification of a genotoxic activity in drinking water usually requires concentration of the water samples. Even though such a process implies a probable qualitative/quantitative alteration of the constituents of water samples, the extremely low amounts of genotoxic compounds in drinking water require concentration steps. Among the many genotoxicity tests carried out, the Ames test (which detects reverse mutations in bacteria Salmonella typhimurium) is the assay which was the most frequently used in the field of drinking water mutagenicity. Other tests were performed on eucaryotic cells. Assays detecting micronuclei or chromosomal aberrations in plants, or mutations in mold, yeast, or maize enabled the detection of genotoxic effects of drinking water extracts. Tests on mammal cells also showed that drinking water extracts induced point mutations, sister chromatid exchanges, chromosomal aberrations and micronuclei. In vivo tests on aquatic organims such as newt or mussels demonstrated the micronuclei inducing effect of unconcentrated drinking water samples.Regarding the identification of the compounds responsible for the genotoxicity, it is obviously not possible to identify all of the thousands of chemicals that may be involved. But such a process is important in order to evaluate the specific genotoxicity and the risk associated with (at least) the main chemicals occurring in drinking water. The identification process usually follows three steps: 1. concentration of the sample can be performed using reverse osmosis, freeze drying, liquid-liquid extraction, and/or adsorption on non ionic resin followed by extraction with organic solvent; 2. the purification step uses one or a combination of chromatographic techniques (TLC, packed column liquid chromatography, HPLC or GC); 3. structural identification of the chemical is performed using data from mass spectrometry, and proton and carbon NMR, or UV spectroscopy. The analysis of the genotoxic compounds of drinking water showed that they are rather non-volatile, quite acid and not stable at high pH, rather polar, and with a mean molecular weigh around 200.Turning now to the identity of these compounds, it is considered that the genotoxicity of drinking water is mainly due to organohalogenated chemicals. Some inorganic chemicals (this class of chemicals is usually not recovered in drinking water extracts) which induce genotoxic or carcinogenic effects must, however, be recalled. Arsenic, nitrates, bromates and radon are natural or human-activity-related drinking water contaminants which are responsible for cancers in rodents or in humans. Among the many genotoxic or carcinogenic organohalogenated compounds identified in drinking water, the most abundant chemicals are chlorinated and/or brominated trihalomethanes. Other important groups of compounds are chlorinated and/or brominated derivatives of acetic acids, acetonitriles, ketones, phenolic compounds. The chlorinated hydroxyfuranones, although present at concentrations lower than 0.1 µg/l in drinking water, can be responsible for more than half of the Ames mutagenicity. MX, the most potent of these chlorohydroxyfuranones, has been submitted to intensive toxicological studies worldwide and was very recently identified as a potent carcinogen in rats.Now that the presence of genotoxic compounds in drinking waters is a well documented and accepted fact, the perspectives lies in the better identification of the impact of these drinking water contaminants. The development of more sensitive tests such as the Comet assay (detection of DNA strand breaks) or the 32P postlabelling assay (detection of DNA adducts) should be pursued. Moreover, the interaction between genotoxic compounds and DNA must be investigated more thoroughly, including the identification of adduct structures. More globally, it is of interest to better assess the impact of these agents on public health and on the occurrence of specific human cancers. At present, even though a few individual water contaminants are classified as human probable carcinogens, the chlorinated drinking water (in itself) is not considered as carcinogenic to humans. Exposure to these potentially harmful agents can be limited with 1. improving drinking water quality - i.e. decreasing the formation of genotoxins - by using raw water containing lower amounts of organic matter; and 2. modifying the water chemical treatment by using lower amounts of chlorine and/or combining chlorine with other disinfectants. The public health can also be protected by the setting of guidelines for drinking water: each compound identified as dangerous would be given a concentration threshold which should never be exceeded. The Environmental Protection Agency in the U.S.A. and the World Health Organisation are authorities setting such guidelines. Finally, we believe it is important to limit the concentration of genotoxic compounds in drinking water as much as possible, and one way to do so is to use chlorine in smaller amounts and in a more efficient way. But it is of paramount importance to keep in mind that the disinfection process (in which chlorine still plays a major role) and the providing of a microbiologically safe drinking water should never be jeopardized

Utilisation de trois tests de génotoxicité pour l'étude de l'activité génotoxique de composés organohalogénés, d'acides fulviques chlorés et d'échantillons d'eau (non concentrés) en cours de traitement de potabilisation

Il est admis aujourd'hui que la génotoxicité identifiée dans les extraits d'eau potable provient principalement de l'action du chlore sur la matière organique naturelle qui donne naissance à des dérivés organohalogénés.Dans le présent travail, nous avons comparé la sensibilité de trois essais de génotoxicité (SOS chromotest, test d'Ames-fluctuation et test micronoyau triton) lors de l'étude de composés organohalogénés, d'acides fulviques chlorés et d'échantillons d'eau (non concentrés) en cours de traitement de potabilisation.Les composés organohalogénés étudiés sont 4 trihalométhanes, 5 acétonitriles et 5 chloropropanones identifiés dans l'eau potable ou dans des solutions de substances humiques chlorées. Les résultats obtenus révèlent que le SOS chromotest est globalement le moins sensible des trois essais et que le test d'Ames-fluctuation et le test micronoyau triton permettent généralement de détecter les plus faibles concentrations de composés génotoxiques. Les essais ont également permis de démontrer que la nature des substituants halogénés (brome ou chlore), le nombre et la position des atomes de chlore influencent notablement la génotoxicité des composés organohalogénés.Toutefois, les résultats obtenus indiquent qu'aucun des trois tests réalisés n'est suffisant à lui seul pour détecter l'ensemble des produits génotoxiques. Ces observations confirment la nécessité de réaliser une batterie de tests qui mette en oeuvre divers types cellulaires et différents systèmes de métabolisation, et détecte divers évènements de génotoxicité.Les travaux portant sur les solutions concentrées d'acides fulviques chlorés montrent l'intérêt des essais sur bactéries (particulièrement le test d'Ames-fluctuation) pour la détection rapide de l'activité génotoxique de ces solutions.L'étude concemant les échantillons d'eau prélevés à différents niveaux d'une station de potabilisation, et analysés sans concentration préalable, indique que le test d'Ames- fluctuation est le seul capable de détecter une activité génotoxique dans les échantillons non concentrés étudiés. On montre, conformément à la littérature, que l'activité mutagène observée résulte de la chloration de l'eau.Since the identification of organohalides in drinking water in 1974, several investigators have detected genotoxic activity in drinking water concentrates. It is now widely admitted that the observed genotoxicity originates mainly from the reaction of chlorine on natural organic matter contained in the raw water, which leads to the formation of organohalogenated compounds.The aim of this study is to show the benefit of three short-term assays for the evaluation of the genotoxic potency of organohalogenated compounds and of complex mixtures. In a wider context, the purpose is to identify a test or a battery of tests that can contribute to the control of natural and drinking water genotoxicity.The three genotoxicity assays carried out during this work were:- the SOS chromotest, a primary DNA damage in vitro assay on Escherichia coli; - the Ames- fluctuation test, a point mutation in vitro assay on Salmonella typhimurium; - and the newt micronucleus test, a chromosomal aberration in vivo assay on the amphibian Pleurodeles waltl. These assays display a valuable advantage: the water samples under study can be analyzed without concentration prior to testing. Thus, the different concentration procedures, which may modify the original genotoxicity of the water samples, are avoided.A previous study on seven reference genotoxic chemicals had indicated that the SOS chromotest was never the most sensitive of the three tests (for a given chemical, the most sensitive assay is defined as the test which detects the lowest concentration inducing a significant genotoxic effect). On the contrary, the Ames-fluctuation test proved to be the most sensitive for compounds showing direct genotoxic activity, and the newt micronucleus test the most sensitive for chemicals with indirect genotoxic effects. None of the assays was the most sensitive for every substance analyzed. These observations suggested the need to implement a battery of tests using several cell types, different metabolization systems and detecting several genotoxicity events. This earlier study also showed, in accord with several results in the literature, that the Ames-fluctuation test (in liquid medium) demonstrated a better sensitivity than the Ames test (in agar solid medium).The first part of the present study involved testing the genotoxicity of 14 organo- halogenated compounds identified in drinking water samples or in chlorinated humic matter samples. The chemicals studied were four chlorinated and/or brominated trihalomethanes (trichloro-, bromodichloro-, chlorodibromo- and tribromomethane), five chlorinated or brominated acetonitriles with one, two or three halogens (monochloro-, dichloro-, trichloro-, monobromo- and dibromoacetonitrile) and five chlorinated propanones with one, two or three substitutions on one or two carbon atoms (monochloro-, 1,1-dichloro-, 1,3-dichloro-, 1,1,1-trichloro- and 1,1,3- trichloropropanone). Although the SOS chromotest was the most sensitive for 3 of the 14 substances analyzed, the results confirmed that this test was globally the least sensitive; the Ames-fluctuation test and the newt micronucleus test remained the most efficient assays. It is interesting to note that the Ames-fluctuation test appeared the most sensitive for all the chloropropanones tested and the newt micronucleus test, for all the haloacetonitriles analyzed. Moreover, several structure-activity relationships were demonstrated: the nature of the halogenated substituents (bromine or chlorine), the number and, above all, the position of chlorine atoms strongly influenced the genotoxicity of the organohalides studied.In the second part of the work we analyzed the effects of complex mixtures containing several organohalogenated compounds: the three tests were performed on two chlorinated fulvic acids of different origin. Pornic fulvic acid was extracted from a surface water reservoir used to produce drinking water in Vendée (France) and Pinail fulvic acid came from a forest pond near Poitiers (France). The total organic carbon was about 1 g/l in the solution subjected to chlorination and the molar chlorination ratio was 1.5 Cl2/C. The results showed the advantage of tests using bacteria: the Ames fluctuation test was the only assay able to detect the genotoxicity of both chlorinated fulvic acids; the SOS chromotest detected the genotoxic effect of only one of the chlorinated fulvic acids (Pinail). In contrast, the newt micronucleus test did not show any genotoxicity of the chlorinated fulvic acids. However, it must be pointed out that, as insufficient fulvic acid was available, the genotoxic potency of these solutions on the newt was not tested under adequate conditions (e.g., subchronic concentrations were not studied). Nevertheless, the concentrations of fulvic acid analyzed were very close to those found in the aquatic environment.The last part of the study attempted to approximate environmental and human exposure conditions: the three tests were performed on four water samples taken at several stages of a drinking water treatment plant. These samples were analyzed for genotoxicity in the three test systems without preconcentration. The plant studied is characterized by the following treatment steps: - coagulation-flocculation; - chlorination at 6 g Cl2/m3; - sand filtration; - ozonation at 1.9 g O3/m3; - final chlorination at 1.4 g Cl2/m3 before sending the treated water into the distribution system. The four samples were taken:1. before any treatment (raw water, 10 to 13 mg total organic carbon per liter), 2. between the sand layer filter and the ozonator (chlorinated water), 3. after the ozonator (ozonated water), 4. and at the end of the treatment process (treated water). The results obtained confirmed the advantage of the Ames fluctuation test, which was the only assay able to detect a genotoxic activity in the unconcentrated water samples studied. Regarding the influence of the different chemical treatments on the mutagenicity observed, it was demonstrated that the first chlorination step led to the formation of direct-acting mutagens. The treatment with ozone, at the rate used, did not significantly modify the mutagenicity of the samples that had previously been chlorinated. Similarly, the second chlorination step did not significantly increase the direct mutagenicity detected.Practically, our study indicated that the Ames-fluctuation test is the only assay, among the three performed that is able to contribute efficiently to the control of drinking water genotoxicity. In this context, the benefit of the SOS chromotest appears only in case of accidental pollution: indeed, it is the only test able to yield results within 24 hours. The implementation of the newt micronucleus test could be useful for the control of natural or drinking water genotoxicity in case of extensive pollution

A comparison of transgenic rodent mutation and in vivo comet assay responses for 91 chemicals.

A database of 91 chemicals with published data from both transgenic rodent mutation (TGR) and rodent comet assays has been compiled. The objective was to compare the sensitivity of the two assays for detecting genotoxicity. Critical aspects of study design and results were tabulated for each dataset. There were fewer datasets from rats than mice, particularly for the TGR assay, and therefore, results from both species were combined for further analysis. TGR and comet responses were compared in liver and bone marrow (the most commonly studied tissues), and in stomach and colon evaluated either separately or in combination with other GI tract segments. Overall positive, negative, or equivocal test results were assessed for each chemical across the tissues examined in the TGR and comet assays using two approaches: 1) overall calls based on weight of evidence (WoE) and expert judgement, and 2) curation of the data based on a priori acceptability criteria prior to deriving final tissue specific calls. Since the database contains a high prevalence of positive results, overall agreement between the assays was determined using statistics adjusted for prevalence (using AC1 and PABAK). These coefficients showed fair or moderate to good agreement for liver and the GI tract (predominantly stomach and colon data) using WoE, reduced agreement for stomach and colon evaluated separately using data curation, and poor or no agreement for bone marrow using both the WoE and data curation approaches. Confidence in these results is higher for liver than for the other tissues, for which there were less data. Our analysis finds that comet and TGR generally identify the same compounds (mainly potent mutagens) as genotoxic in liver, stomach and colon, but not in bone marrow. However, the current database content precluded drawing assay concordance conclusions for weak mutagens and non-DNA reactive chemicals

Identification of Reproduction-Specific Genes Associated with Maturation and Estrogen Exposure in a Marine Bivalve Mytilus edulis

Background: While it is established that vertebrate-like steroids, particularly estrogens (estradiol, estrone) and androgens (testosterone), are present in various tissues of molluscs, it is still unclear what role these play in reproductive endocrinology in such organisms. This is despite the significant commercial shellfishery interest in several bivalve species and their decline. Methodology/Principal Findings: Using suppression subtraction hybridisation of mussel gonad samples at two stages (early and mature) of gametogenesis and (in parallel) following controlled laboratory estrogen exposure, we isolate several differentially regulated genes including testis-specific kinases, vitelline lysin and envelope sequences. Conclusions: The differentially expressed mRNAs isolated provide evidence that mussels may be impacted by exogenous estrogen exposure

Quantitative Analysis of Histone Modifications: Formaldehyde Is a Source of Pathological N6-Formyllysine That Is Refractory to Histone Deacetylases

Aberrant protein modifications play an important role in the pathophysiology of many human diseases, in terms of both dysfunction of physiological modifications and the formation of pathological modifications by reaction of proteins with endogenous electrophiles. Recent studies have identified a chemical homolog of lysine acetylation, N[superscript 6]-formyllysine, as an abundant modification of histone and chromatin proteins, one possible source of which is the reaction of lysine with 3′-formylphosphate residues from DNA oxidation. Using a new liquid chromatography-coupled to tandem mass spectrometry method to quantify all N[superscript 6]-methyl-, -acetyl- and -formyl-lysine modifications, we now report that endogenous formaldehyde is a major source of N[superscript 6]-formyllysine and that this adduct is widespread among cellular proteins in all compartments. N[superscript 6]-formyllysine was evenly distributed among different classes of histone proteins from human TK6 cells at 1–4 modifications per 10[superscript 4] lysines, which contrasted strongly with lysine acetylation and mono-, di-, and tri-methylation levels of 1.5-380, 5-870, 0-1400, and 0-390 per 10[superscript 4] lysines, respectively. While isotope labeling studies revealed that lysine demethylation is not a source of N[superscript 6]-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N[superscript 6]-formyllysine, with use of [[superscript 13]C,[superscript 2]H[subscript 2]]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources. Inhibitors of class I and class II histone deacetylases did not affect the levels of N[superscript 6]-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10%) with a peptide substrate containing the formyl adduct. These data suggest that N[superscript 6]-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary modification

Biomarker measurements in Trifolium repens and Eisenia fetida to assess the toxicity of soil contaminated with landfill leachate : a microcosm study

International audienceTo assess the toxicity of a soil contaminated with landfill leachate, biomarker measurements in two species living in close contact with the soil, i.e. a plant species Trifolium repens and an animal species Eisenia fetida, were conducted. Briefly, both species were studied after simultaneous exposure conducted in microcosms. The organisms were exposed to soil supplemented with pure leachate, leachate diluted to 50 % ; leachate diluted to 25 % and without leachate. After a 10 weeks exposure period, we observed an increase in the Olive Trail Moment in T. repens, compared to the reference, for 50 % and pure leachate. The response observed appears to be dose-dependent and linear in our experimental conditions. Addition of the leachate to the reference soil induced an increase in Cd-Metallothionein-coding mRNA quantity in E. fetida. In addition, expression level of another gene implied in detoxification and coding Phytochelatin synthase was significantly induced in worms exposed to the reference soil spiked with the leachate, regardless presence of T. repens. Thus, T. repens and E. fetida can be used in a complementary manner to assess soil quality. Sensitivities of the test species yield sensitive bioassays as both species responded at low doses despite the buffering effect of the soil