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Cytogenetic effects of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in rat peripheral lymphocytes in vitro and in vivo
Abstract
3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a potent direct-acting Salmonella mutagen found in wood pulp chlorination effluents and chlorinated drinking water. In cultured rat peripheral lymphocytes, MX induced significant dose-related increases in sister-chromatid exchanges (SCEs) and chromosome aberrations at doses of 20-60 micrograms/ml and of 60-80 micrograms/ml, respectively. MX produced primarily chromatid-type as opposed to chromosome-type aberrations. The peripheral lymphocytes of male and female rats exposed to MX by gavage 5 days a week for 14-18 weeks showed significant dose-related increases in SCEs at both levels of exposure (30 and 45-75 mg/kg) in both sexes. The present results demonstrate for the first time that MX is genotoxic in vivo.
... CHFs, especially MX, induce genetic alterations in numerous biological systems in vitro. In cultured rodent cells, they cause chromosomal aberrations (Meier et al., 1987a;Jansson et al., 1993;Mäki-Paakkanen et al., 1994;Harrington-Brock et al., 1995), sister chromatid exchanges (Brunborg et al., 1991;Jansson et al., 1993;Mäki-Paakkanen et al., 1994) and DNA damage (Brunborg et al., 1991). MX also induces DNA strand breaks (Chang et al., 1991;Nunn and Chipman, 1994;Marsteinstredet et al., 1997a) in cultured human cells. ...
... CHFs, especially MX, induce genetic alterations in numerous biological systems in vitro. In cultured rodent cells, they cause chromosomal aberrations (Meier et al., 1987a;Jansson et al., 1993;Mäki-Paakkanen et al., 1994;Harrington-Brock et al., 1995), sister chromatid exchanges (Brunborg et al., 1991;Jansson et al., 1993;Mäki-Paakkanen et al., 1994) and DNA damage (Brunborg et al., 1991). MX also induces DNA strand breaks (Chang et al., 1991;Nunn and Chipman, 1994;Marsteinstredet et al., 1997a) in cultured human cells. ...
... Furihata et al. (1992) observed DNA damage (measured by alkaline elution) in rat stomach after oral exposure to MX, whereas Brunborg et al. (1991) failed to detect any effect in several organs of mouse after oral and i.p. administration. MX caused sister chromatid exchanges in rat peripheral lymphocytes and kidney after oral administration (Jansson et al., 1993;Mäki-Paakkanen and Jansson, 1995). In mice exposed orally to MX, DNA damage (measured with the Comet assay) was observed in the liver and other organs (Sasaki et al., 1997), but unscheduled DNA synthesis (UDS) was not detected in liver cells (Nunn et al., 1997). ...
Chlorohydroxyfuranones (CHFs) are mutagenic disinfection by-products found in chlorine-treated drinking water. In the current study, the genotoxicity of four CHFs, 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA), 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF), 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), was determined. Two in vitro assays, the microscale micronucleus assay on L5178Y mouse lymphoma cells and the unscheduled DNA synthesis assay on a hepatocyte primary culture from Fisher F344 rats, were carried out. All four CHFs demonstrated genotoxic effects in both assays. In the two systems used, CMCF was the most genotoxic compound, followed by MCA, MX and MCF, respectively. This work was the first study of the DNA damaging properties of all four CHFs in two in vitro genotoxicity tests. These new data expand the range of genetic damages induced by this group of compounds.
... _________________________________________________________________________________________ DNA damage (alkaline elution, alkaline Brunborg et al., 1991; DNA unwinding assay, comet assay, Chang et al., 1991; unscheduled DNA synthesis) Holme et al., 1999;Le Curieux et al., 1999 Harrington-Brock et al., 1995; or mutations at the thymidine kinase locus) Hyttinen et al., 1996;Jansson and Hyttinen, 1994 Brunborg et al., 1991;Jansson et al., 1993;CHO cells (+) Mäki Harrington-Brock et al., 1995;Jansson et al., 1993;CHO cells (+) Meier et al., 1987a; L5178Y mouse lymphoma cells (+) Mäki-Paakkanen et al., 1994; Le Curieux et al., 1999 L5178Y mouse lymphoma cells (+) _________________________________________________________________________________________ a Positive result. b Positive, when MX was diluted in a buffered salt solution, but negative when MX was diluted in tissue culture medium (Matsumura et al., 1994). ...
... _________________________________________________________________________________________ DNA damage (alkaline elution, alkaline Brunborg et al., 1991; DNA unwinding assay, comet assay, Chang et al., 1991; unscheduled DNA synthesis) Holme et al., 1999;Le Curieux et al., 1999 Harrington-Brock et al., 1995; or mutations at the thymidine kinase locus) Hyttinen et al., 1996;Jansson and Hyttinen, 1994 Brunborg et al., 1991;Jansson et al., 1993;CHO cells (+) Mäki Harrington-Brock et al., 1995;Jansson et al., 1993;CHO cells (+) Meier et al., 1987a; L5178Y mouse lymphoma cells (+) Mäki-Paakkanen et al., 1994; Le Curieux et al., 1999 L5178Y mouse lymphoma cells (+) _________________________________________________________________________________________ a Positive result. b Positive, when MX was diluted in a buffered salt solution, but negative when MX was diluted in tissue culture medium (Matsumura et al., 1994). ...
... Increases in nuclear anomalies, including MN, have been observed in the duodenum of B6C3F1 mice after a single oral dose of MX and MN are apparent in peripheral blood lymphocytes of Han/Wistar rats after oral administration of MX on three consecutive days (Mäki-Paakkanen and . Increases in SCEs have been observed in peripheral blood lymphocytes and kidney cells of Han/Wistar rats after administration to MX by gavage on three consecutive days, and in peripheral lymphocytes of rats exposed to MX by gavage for 14-18 weeks (Jansson et al., 1993;Mäki-Paakkanen and Jansson, 1995). Other types of DNA damage (DNA single-strand breaks, alkalilabile sites, differential DNA repair) have been detected in a variety of tissues both in rats and mice after a single dose of MX (Furihata et al., 1992;Fekadu et al., 1994;Sasaki et al., 1997). ...
... MX is a direct-acting mutagen formed in chlorinated drinking water that has strong mutagenic activity in bacteria, accounting for about one-third to half of the total mutagenicity induced in bacteria by chlorinated tap water [72,73]. Although MX has been shown to induce chromosomal aberrations in in vitro studies conducted on Chinese hamster ovary cells [74] and on rat peripheral lymphocytes [75], the compound has shown weak or no mutagenicity in in vivo studies conducted on mammalian cells. In this study, the researchers demonstrated effective inhibition of MX using cysteamine, suggesting that direct chemical inactivation of the mutagen is the responsible mechanism. ...
Cysteamine is an aminothiol endogenously synthetized by human cells during the degradation of coenzyme A. Since the 1950s, this substance has been used in radiation protection, immunomodulation, and is the sole FDA-approved oral agent for the treatment of cystinosis. Many in vitro and in vivo studies recommend cysteamine as an effective anti-carcinogenic agent. In this article, we will review the preventive and therapeutic effects of cysteamine for 3 types of cancers: carcinomas, sarcomas and melanomas. Cysteamine has been shown to be effective in vitro and in vivo for the treatment of different cancer types generally leading to the significant reduction of lesions and/or the increase of survival time. Cysteamine has been shown to enhance the anti-melanoma effect of doxorubicin in vivo and cysteamine derivatives have shown anti-melanoma efficacy in animal models. Although the mechanisms of action are not fully understood, possible explanations on the pathophysiology of cell suppression by cysteamine are given : (i) free radical scavenging (ii) alteration of the tumor cell proliferation by affecting nucleic acid and protein synthesis or inhibition of DNA synthesis (iii) hormone regulation.
... On the other hand, widely used purification methods of water like H 2 O 2 and chlorination may cause formation of carcinogenic disinfection by-products (DBPs) because of the intensified pollution of water supplies to organic matters (Villanueva et al., 2014). In fact these widely used methods for disinfection of drinking water (Kusamaran et al., 2003) are responsible for more than half of positive Ames mutagenicity assessments in apparently purified waters (Jansson et al., 1993), (Wright et al., 2002). For these reasons cross sectional analysis of genotoxic and mutagenic compounds in drinking water by a battery of genotoxic methods has been considered recently (Abda et al., 2015). ...
Diseases related to water impurities may present as major public health burdens. The present study aimed to assess the mutagenicity of drinking water from different zones of Tehran, and evaluate possible health risks through making tea with tea bags, by Ames mutagenicity test using TA 100, TA 98 and YG1029 strains. For this purpose, 450 water samples were collected over the period of July to December 2014 from 5 different zones of Tehran. Except for one sample, no mutagenic potential was detected during these two seasons and the MI scores were almost normal (≤1-1.6) in TA 100, TA 98 and YG1029 strains. Although no mutagenic effects were considered in TA 98 and TA 100 in the test samples of our three evaluated tea bag brands, one sample from a local company showed mutagenic effects in the YG1029 strain (MI=1.7-1.9 and 2) after prolonged (10-15 min.) steeping. Despite the mild mutagenic effect discovered for one of the brand, this cross sectional study showed relative safety of water samples and black tea bags in Tehran. According to the sensitivity of YG1029 to the mutagenic potential of water and black tea, even without metabolic activation by s9 fraction, this metabolizer strain could be considered as sensitive and applicable to food samples for quantitative analysis of mutagens.
... An increase in SCE frequency was also reported for other mammal species exposed to different mutagenic agents. In rats, SCE frequency also doubled (from 7.67 ± 2.91/cell to 22.00 ± 4.36/cell) after exposure to methyl methanesulfonate [70], while in bovines chloramphenicol caused a small but statistically significant effect on the basaline SCE frequency (6.37 ± 1.07/cell) [71]. ...
Sentinel species are useful tools for studying the deleterious effects of xenobiotics on wildlife. The large hairy armadillo (Chaetophractus villosus) is the most abundant and widely distributed mammal in Argentina. It is a long-lived, omnivorous, burrowing species, with fairly restricted home ranges. To evaluate the level of spontaneous genetic damage in this mammal, we determined the baseline values of several genotoxicity biomarkers. The study included 20 C. villosus adults of both sexes from eight pristine localities within its geographic distribution range. Genotoxicity analysis was performed on 72-h lymphocyte cultures, using mitomycin C as positive control. We obtained the baseline values of mitotic index (MI = 10.52 ± 0.30 metaphases/total cells, n = 20), chromosome aberrations (CA = 0.13 ± 0.22, n = 20), sister chromatid exchanges (SCE) = 6.55 ± 0.26, n = 6) and replication index (RI = 1.66, n = 6). MI and CA did not show significant differences (P > 0.05) among localities or between sexes. No significant differences in MI, CA, SCE, and RI (P > 0.05) were found between values from the pristine localities and historical data. There were significant differences in CA, SCE, and RI (P < 0.05) between lymphocyte cultures from pristine localities and those exposed to mitomycin C. We propose the large hairy armadillo as a sentinel organism for environmental biomonitoring of genotoxic chemicals due to its abundance, easy manipulation, well-known biology, the fact that it is usually exposed to different mixtures and concentrations of environmental contaminants, and the baseline values of genetic damage characterized by MI, CA, SCE and RI as biomarkers.
... Las muestras de sangre fueron obtenidas de un voluntario joven (25 años), saludable, no fumador, sin ningún tipo de tratamiento clínico, no deportista. Se adicionaron 1,0 ml de sangre venosa con heparina y 0,2 ml de fitohemaglutinina (PHA) a 8 ml de medio RPMI-1640, con 5 % (v/v) de suplemento de suero bovino fetal inactivado por calor (56 °C, 30 minutos), penicilina (100 U/ ml) y estreptomicina (100 µg/ml), y se cultivaron por 48 horas a 37 °C (30,31). Luego, la sangre se centrifugó y el precipitado de células se depositó en un gradiente de densidad de Hystopaque. ...
Introducción. Los mutágenos contenidos en mezclas complejas presentan interacciones desinergismo, aditivas o antagónicas. Se han desarrollado enfoques experimentales que permitandilucidar el responsable de las interacciones en la mezcla.
Objetivo. Desarrollar un diseño experimental para comprender los procesos que se llevan a caboentre los compuestos presentes en las mezclas complejas.
Materiales y métodos. Se expusieron linfocitos humanos a mezclas binarias de mutágenos B[a]P,DMBA, Trp-P-1 y MX durante una hora, con activación metabólica y sin ella. La viabilidad se evaluócon azul de tripano y, la genotoxicidad, con cometa alcalino.
Resultados. Ningún hidrocarburo tuvo efecto con furanona. Con S9 y sin él, se observó que sepresentaban interacciones tóxicas entre hidrocarburos. Se observó sinergismo sin S9 entre B[a]P yTrp-P-1 y, con actividad metabólica, entre DMBA y Trp-P-1. Sin S9 se observó interacción antagónicaentre Trp-P-1 y DMBA y, con S9, entre Trp-P-1 y MX y entre MX y DMBA. Se observó un incrementodependiente de la dosis en la longitud de la cola. Hubo daño genotóxico medio y aumento de lascélulas dañadas. Para todas las mezclas se pudo determinar la concentración mínima en la que seobservaban efectos adversos y solo para algunas se determinó la concentración máxima en la cual nose observaron efectos adversos.
Conclusión. Se hace un aporte para comprender los procesos que ocurren cuando en una mezclahay presentes, al menos, dos mutágenos y se valida un modelo de análisis que permite dilucidar elcompuesto que tiene efecto sobre otro. También, se demostró que según el tipo de compuestos en lamezcla, se tendrá o no un umbral de riesgo.
doi: http://dx.doi.org/10.7705/biomedica.v32i3.739
... The chlorohydroxyfuranones also exert various genotoxic effects in mammalian test systems, including DNA strand breaks, sister-chromatid exchanges, chromosome aberrations and gene mutations (Meier et al. 1987a;Brunborg et al. 1991;Chang rt al. 1991;Jansson et al. 1993;Mgki-Paakkanen et al. 1994;Matsumura et ul. 1994;Jansson & Hyttinen 1994;Harrington-Brock et ul. ...
: Epidemiological studies indicate an association between exposure to chlorinated drinking water and risk of intestinal cancer. In order to study this experimentally, we have examined the effects of 3,4-dichloro-5-hydroxy-2[5H]-furanone (mucochloric acid, MCA) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), mutagenic and genotoxic compounds in drinking water, on aberrant crypt foci and tumours in the intestines of male F344 rats and Balb/cA mice, and C57BL/6J-Min (multiple intestinal neoplasia)/+ mice of both sexes, in six independent experiments. In some experiments the effects of MCA and MX on aberrant crypt foci induced by the colon carcinogens 1,2-dimethylhydrazine or its metabolite azoxymethane were also studied. Neither MCA nor MX alone induced aberrant crypt foci or intestinal tumours when given in drinking water. With this route of exposure neither MCA nor MX, when given in combination with 1,2-dimethylhydrazine or azoxymethane, had any effect on the induction or growth of the aberrant crypt foci. Drinking water exposure of MX did not affect the number or growth of aberrant crypt foci or intestinal tumours in the Min/+ mice. When administered intrarectally MCA had a weak inducing effect on aberrant crypt foci in the colons of Balb/cA mice. Exposure to MCA and MX intrarectally apparently promoted the growth of aberrant crypt foci both in rats and mice, increasing the crypt multiplicity, aberrant crypts/aberrant crypt foci. Based on an overall evaluation of these experiments, the intestinal tract, at least in rats and mice, seems not to be a main target organ for effects of MCA or MX on preneoplastic or neoplastic development.
... Ce composé est un puissant mutagène qui est généralement responsable de 30 à 60 % de l'ensemble de l'activité mutagène directe de l'eau potable sur Salmonella typhimurium (KRONBERG et VARTIAINEN, 1988 ;. Les résultats négatifs obtenus avec le test micronoyau triton sur les échantillons d'eau en cours de potabilisation sont en accord avec la tendance généralement décrite dans la littérature concernant les effets génotoxiques du MX in vivo : ce composé n'induit pas de cassures monobrins de l'ADN dans le tractus gastrointestinal, le foie, le rein, les poumons, les testicules, la vessie ou la moelle osseuse de rat (BRUNBORG et al., 1991), ne démontre aucune activité clastogène dans le test micronoyau sur moelle osseuse de souris (MEIER et al., 1987b ;TIKKANEN et KRONBERG, 1990) ou dans le test micronoyau sur érythrocytes de triton (GAUTHIER, 1992). Quelques études plus récentes ont toutefois permis de démontrer l'existence d'une faible activité genotoxique du MX in vivo (FURIHATA et al., 1992 ;JANSSON et al., 1993). Comme le MX, les sous-produits de chloration de substances humiques seraient facilement détoxifiés in vivo. ...
SUMMARY Since the identification of organohalides in drinking water in 1974, several investigators hâve detected genotoxic activity in drinking water concentrâtes. It is now widely admitted that the observed genotoxidty 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 évaluation of the genotoxic potency of organohalogen ated compounds and of complex mixtures. In a wider context, the purpose is to identify a test or a bat- tery of tests that can contribute to the control of natural and drinking water
... Las muestras de sangre fueron obtenidas de un voluntario joven (25 años), saludable, no fumador, sin ningún tipo de tratamiento clínico, no deportista. Se adicionaron 1,0 ml de sangre venosa con heparina y 0,2 ml de fitohemaglutinina (PHA) a 8 ml de medio RPMI-1640, con 5 % (v/v) de suplemento de suero bovino fetal inactivado por calor (56 °C, 30 minutos), penicilina (100 U/ ml) y estreptomicina (100 µg/ml), y se cultivaron por 48 horas a 37 °C (30,31). Luego, la sangre se centrifugó y el precipitado de células se depositó en un gradiente de densidad de Hystopaque. ...
Introduction:
Mutagens contained in complex mixtures can present synergistic interactions, either additive or antagonistic. Therefore, development of experimental approaches is necessary to elucidate which is the responsible agent for the effect in the mixtures.
Objective:
An experimental design was developed that allowed an understanding of the processes between the compounds of complex mixtures.
Materials and methods:
Human lymphocytes were exposed to binary mixtures of the mutagens B[a]P, DMBA, Trp-P-1 and MX for 1 hour with or without S9. Viability was assessed with trypan blue dye and the genotoxicity by the comet assay.
Results:
All of the hydrocarbon showed an effect with furanone. With and without S9, the most toxic interactions were observed between hydrocarbons. Synergistic interaction was observed without S9 between B [a] P and Trp-P-1 and between DMBA and Trp-P-1 with metabolic activity. Without S9 antagonistic interaction was observed only between Trp-P-1+DMBA, and with S9 between Trp-P-1+MX and MX+DMBA. It observed an increase dose dependent in tail length. Half the cultures showed genotoxic damage and increased cell damage. For each mixture, minimum concentrations were determined at which adverse effects are observed; for some only the maximum concentration was determined at which no adverse effects are observed.
Conclusion:
The processes between mutagens present in a mixture have become better understood, and the results validated an analytical model that determined which component had an effect on another. The results also showed that the type of compounds in the mixture determined whether or not a risk threshold was present.
... MX also induced a wide variety of DNA damage in mammalian cells in vitro (Jansson & Hyttinen, 1994;Maki-Paakkanen & Hakulinen, 2008) including human cells (Chang, Daniel, & Deangelo, 1991) such as sister chromatid exchange (SCE), chromosomal aberrations (Hyttinen et al., 1996;Jansson et al., 1993), DNA strand breaks and different kinds of mutations (Hyttinen et al., 1996;Richardson et al., 2007), and other effects (King, Hester, Warren, & DeMarini, 2009 Several studies have concluded that a potential risk of cancer is associated with the consumption of chlorinated water (Cantor, 1997;Tao, Zhu, & Matanoski, 1999). IARC (1995) conducted research that concluded that a positive correlation exists between chlorinated water consumption and the development of kidney and bladder cancer. ...
Water used for human consumption may contain mutagens and carcinogens generated during the disinfection process with chlorine. In the study described in this article, the mutagenicity and genotoxicity of water samples taken from the San Cristobal treatment plant in Medellin, Colombia, were evaluated. Short-term mutagenic and genotoxic assays using the Ames test and comet assay, respectively, were employed to examine the genotoxic activity of the extracts of these water samples. Two samples were taken before and after the chlorination process. The treated water samples without chlorination did not show mutagenic effects using the Ames test, while the chlorinated samples produced mutagenic activity in both strains. A dose-response relationship for the comet assay was obtained only in the chlorinated samples. MX (3-chloro-4-[dichloromethyl]-5-hydroxy-2[5H]-furanone), E-MX ([E]-2-chloro-3-[dichloromethyl]-4-oxobutenoic acid), and some trihalomethanes were detected at low concentrations. These concentrations were enough, however, to cause detectable mutagenic and genotoxic activity in the extracts of chlorinated water samples.
Cancer is one of the leading causes of death worldwide. First-line treatments usually include surgery, radiotherapy, and/or systemic therapy. These methods can be associated with serious adverse events and can be toxic to healthy cells. Despite the new advances in cancer therapies, there is still a continuous need for safe and effective therapeutic agents. Cysteamine is an aminothiol endogenously synthetized by human cells during the degradation of coenzyme-A. It has been safely used in humans for the treatment of several pathologies including cystinosis and neurodegenerative diseases. Cysteamine has been shown to be a potent antimutagenic, anticarcinogenic, and antimelanoma in various in vitro and in vivo studies, but a review on these aspects of cysteamine’s use in medicine is lacking in the current literature. The efficacy of cysteamine has been shown in vitro and in vivo for the treatment of different types of cancer, such as gastrointestinal cancer, pancreatic cancer, sarcomas, hepatocellular carcinoma, and melanoma, leading to the significant reduction of lesions and/or the increase of survival time. Although the mechanisms of action are not fully understood, possible explanations are (i) free radical scavenging, (ii) alteration of the tumor cell proliferation by affecting nucleic acid and protein synthesis or inhibition of DNA synthesis, and (iii) hormone regulation. In conclusion, regarding the high safety profile of cysteamine and the current literature data presented in this article, cysteamine might be considered as an interesting molecule for the prevention and the treatment of cancer. Further clinical studies should be performed to support these data in humans.
Disinfection by-products of drinking water chlorination (DBP) have been suspect as capable of increasing human cancer risks since the 1970s. Laboratory and epidemiologic studies have suggested that the mutagenic activity of chlorinated drinking water, as determinded in bacterial assays, may indicate a carcinogenic potential of some DBPs. 3-chloro-4-(dichlorometyl)-5-hydroxy-2 (5H)-furanone (MX), a particularly genotoxic DBP, has been shown to be carcinogenic in rats. Reported concentration of MX in European, North American and Asian chlorinated drinking water have been very low (0.8-67 ng/l), but have nevertheless accounted for between 3% and 67% of the mutagenic activity of the samples studied. The objectives of this study were to determine whether drinking waters in regions with little usage of chlorine for disinfection purposes show any mutagenic activity, and whether they contain detectable quantities of MX. Concentrates of raw water and drinking water samples from 11 waterworks in Germany, where concentrates of chlorine used of drinking water disinfection are lower than in other countries, were examined. At least one sample from each waterworks showed both mutagenic activity and the presence of MX. Mutagenic activities, up to 750 net-revertants/litre, were considerably lower than values recorded in Scandinavia and elsewhere, and fell within the target range that Finnish authorities aim to establish by 2005 through modifications of drinking water processing methods. Four raw water samples (ground and surface water) showed traces of MX near the detection limit. In the chlorinated drinking water samples detectable MX concentrations ranged from 0.9 to 15.4 ng/l. A systematic survey is required to substantiate these findings. In the meantime, unresolved questions concerning possible adverse effects on health arising from mutagenic activity and MX in chlorinated drinking water should not lead to prejudice disinfection measures that are essential for microbial safety of drinking water.
3-Chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF), a strong bacterial mutagen reacts with cytidine in buffered aqueous solutions. The reaction products were separated by preparative HPLC C18 column chromatography and were structurally characterized by UV absorbance, 1H NMR and 13C NMR spectroscopy, and mass spectrometry. The main products were identified as 7-(β-D-ribofuranosyl)-4-carboxy-6-oxo-6,7-dihydro-4H- pyrimido[1,6-a]pyrimidine-3-carbaldehyde (cM1FA-Cyd), and 7-(β-D-ribofuranosyl)-6-oxo-6,7-dihydro-4H-pyrimido[1,6-a] pyrimidine-3-carbal-dehyde (M1FA-Cyd). The highest yields of cM 1FA-Cyd and M1FA-Cyd, being 12 and 1 mol % respectively, were obtained in the reaction performed at pH 4.6 and 37°C for 7 days. M1FA-Cyd and cM1FA-Cyd are structurally related to pfA-dR, the adduct previously identified in the reaction of 3-chloro-4-(dichloromethyl) -5-hydroxy-2(5H)-furanone (MX) or 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)- furanone (CMCF) with 2′-deoxyadenosine.
Water Chlorination: Essential Process or Cancer Hazard? Bull, R. J., Birnbaum L. S., Cantor, K., Rose, J. B., Butterworth, B. E., Pegram, R., and Tuomisto, J. (1995). Fundam. Appl. Toxicol. 28, 155-166.
Chlorine has been successfully used for the control of waterborne infectious disease for nearly a century. In the 1970s it was found that chlorine reacted with natural organic matter present in surface waters to produce disinfection by-products (DBP). Concern focused initially on the trihalomethanes (THM), but a wide variety of DBPs are now known to result from chlorination. Chlorination of drinking water has been one of the most effective public health measures ever undertaken. There are a number of alternatives to chlorination that are in active use in many parts of the world, but the risks associated with their by-products are even less well established than for chlorination. Moreover, the use of these alternatives vary in their effectiveness and some require greater sophistication in their application. This can mean less protection to public health as a result of inappropriate application and control. Therefore, hazards associated with the use of such a clearly beneficial process as chlorination must be carefully considered not only in an absolute sense, but also in the context of alternative approaches for producing a safe drinking water. The key question is whether the hazards associated with by-products have been sufficiently well established to warrant regulations that will undoubtedly have both positive and negative impacts on the public health. This symposium examined the toxicological and epidemiological data on chemical hazards associated with chlorination and attempted to measure this hazard against competing microbial risks. The first presentation discussed the available analytical epidemiological studies. A second presentation dealt with the importance of chlorination to the prevention of waterborne infectious disease. Pharmacokinetic, mechanistic, and modeling information on the prototypical DBP, chloroform, were discussed and contrasted with data on brominated THMs to determine if it was scientifically appropriate to regulate THMs as a single toxicological class. The fifth presentation dealt with the carcinogenic properties of a potent mutagen that is produced by chlorination. The final presentation discussed the haloacetates, carcinogenic DBPs whose concentrations approach and occasionally exceed those of the THMs. Clearly, there is a need to carefully weigh these different types and sometimes competing risks when considering the delivery of drinking water to ever-increasing populations for which there are finite sources of fresh water.
Mutagen X (MX), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone is one of the most potent directing acting mutagen ever tested in SAL TA100 assay. Although MX analogues have been synthesized, tested for mutagenicity and modeled by structure-activity relationship (SAR) methods, the mechanism of interaction of these compounds with DNA to produce their remarkable mutagenic potency remains unresolved. MX exists as an equilibrium mixture of both ring and open form in water. This equilibrium is very fast for Ames test. Because the mixture is not separable by experimental methods, it is not clear which one is really responsible for the observed mutagenicity. There have been many debates that which one is really responsible for the observed mutagenicity. We used ab initio methods for the MX analogues. It seems both ring and open form could react with DNA bases as electrophiles. However, every open form has consistently lower LUMO energy than corresponding ring form. It is reasonable to assume that the major reaction will go through via open form for MX analogues. This suggest that the open form is more likely really mutagenic.
Neben Abwässern aus der Industrie entstehen auch bei anderen technischen Prozessen Abwässer, die gentoxische Substanzen enthalten können. Dabei handelt es sich sowohl um punktförmige Quellen wie z.B. dem Bergbau, Kraftwerken oder Anlagen zur Abfall- bzw. Abwasserentsorgung als auch um diffuse Emissionen aus Landwirtschaft und Verkehr. Obwohl eine Vielzahl von mutagenen Verbindungen bekannt ist, war es in konkreten Fällen noch nicht möglich, die mutagene Aktivität von Wässern bestimmten Substanzen zuzuordnen. Bei der Chlorierung und Ozonierung im Rahmen der Trinkwasseraufbereitung können ebenfalls Stoffe mit mutagenem Potential entstehen. Dieses Gesundheitsrisiko für den menschen kann derzeit noch nicht abschließend beurteilt werden.
We tested the genotoxicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in the mouse in 6 organs (liver, lung, kidney, brain, spleen, and bone marrow) and in the mucosa of stomach, jejunum, ileum, colon, and bladder using the alkaline single-cell gel electrophoresis (SCG) (Comet) assay modified by us. Mice were sacrificed 1, 3, 6, and 24 h after oral administration of the mutagen at 100 mg/kg. MX yielded statistically significant DNA damage in the liver, kidney, lung, and brain and in all the mucosa samples. While DNA damage persisted in the gastrointestinal and urinary tract for 6–24 h after a single oral dosing, it peaked in the liver at 1 h and returned to almost the control level at 3 h. Our present results suggest that MX is genotoxic for various mouse organs, but not for the hematopoietic system, and that the alkaline SCG assay with a homogenization technique can be used to predict genotoxicity in the gastrointestinal and urinary tracts.
Various mutagens and carcinogens are formed during the chlorine disinfection of raw drinking water. Among them, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), exhibits a high mutagenic activity against Salmonella typhimurium TA100 without S9 mix, which is comparable to aflatoxin B1. Recently, it has been demonstrated that MX induces stomach tumor to rats in the 2-stage carcinogenicity test using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In this review, we summarize the distribution of MX in several water samples including drinking water and its chemical, biological and genotoxic properties.
The brominated hydroxyfuranones sructurally related to the potent mutagen MXnamely3-chloro-4-(bromochlorome-thyl)-5-hydroxy-2(5H)-furanone (BMX-1), has been synthesized and characterized. The mutagenic activity of this bromohydro-xyfuranone together with those of its analogs BMX-2 and BMX-3 has been assessed by employing the Ames test with the Salmonella typhimurium strains TA98 and TA100. The mutagenic potencies in TA100 without S9 metabolic activation were 22.05 ± 3.15 revertants/ng for BMX-1, 28.64 ± 2.65 revertants/ng for BMX-2, and 37.29 ± 5.73 revertants/ng for BMX-3, whereas in the TA98 strain without metabolic activation, they were six- to ninefold lower. Studies carried out on the stability of these halogenated furanones suggest that the mutagenic activity observed is more related to the cyclic form of these derivatives.
This is the first report of clastogenic effects of chlorinated hydroxyfuranones (CHFs) in plants. Two byproducts of water chlorination, 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) and 3,4-dichloro 5-hydroxy-2[5H]-furanone (MA) induced a dose dependent increase of micronuclei (MN) in pollen mother cells of Tradescantia when doses up to 100 μg MX and 500 μg MA were applied directly to the inflorescences. In contrast, exposure of the stems in aqueous solutions containing up to 1 mg/I MX and 10 mg/I MA did not cause a positive response.
Dissolved humic substances, which are mostly fulvic acids, impart colour to natural waters. Colour from humic substances is especially evident in waters from upland peats. Chlorination of such waters may result in the formation of bacterial mutagens; hence there is a need for methods for the removal of the coloured materials prior to chlorination. A column system of Beringite (a calcined Palaeozoic schist) has been used in the present study for this purpose. Results show that although Beringite can sorb fulvic acids (and thus mutagenic precursors) from water its sorption capacity is low. Methods were investigated for the desorption from Beringite of the sorbed mutagen precursors. Sodium carbonate removed approximately 72% of the sorbed humic material and was the most effective of the chemical agents used to regenerate the Beringite. Tetrasodium pyrophosphate displaced approximately 65% of the colour, but it formed a complex with the Beringite which solidified. Sodium hydroxide and sodium tetraborate were ineffective desorbing agents.
Three brominated analogues of the highly mutagenic drinking water micropollutant 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) have been synthesized from MX by halogen exchange reaction.
Hypochlorite treatment of a simulated food-processing mixture produces 3,4-dichloromaleimide and 3,3-dichloro-4-(dichloromethylene)-2,5-pyrrolidinedione (C5HCl4NO2). The tetrachloro compound and two analogs, which can be synthesized from citraconic anhydride and itaconic anhydride, are direct-acting Ames mutagens in Salmonella typhimurium TA100 tester strain. These novel five-carbon cyclic imides are structurally similar to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), the principal mutagenic compound present in paper pulp bleaching liquors. Molecular structure analysis of the mutagens was based on X-ray crystallography, 13C NMR, and mass spectrometry of synthetic chlorinated imides with identical mass spectra and gas chromatographic retention indices. The tetrachloroimide accounts for much of the mutagenicity of the dichloromethane-extractable pH 2 fraction from chlorination of a simulated food-processing system consisting of chicken frankfurters. In the Ames TA100 tester strain it has a molecular mutagenicity of 1450 revertants/nmol without microsomal activation, making it the second most potent mutagen reported from a chlorination process. Keywords: Poultry chiller water; bacterial mutagens; chlorine disinfection byproducts; chlorinated imides
ABSTRACT3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is known as a by-product of wood pulp manufacture and a contaminant of chlorinated drinking water. Since our previous studies (Teramoto et al., 1998, 1999) demonstrated in a micromass in vitro test a strong inhibitory effect of MX on rat embryo cell differentiation, the potential teratogenicity was investigated in this study by using a suspension organ culture system. Twelve-day mouse embryo palatal explants were cultivated for 72 hr in the MX-containing medium at a concentration of 0, 1, 10, 100 or 300 μg/ml and examined for closure of the palatal shelves. All control explants showed almost complete closure of the palatal shelves. Similar results were also obtained in the MX-treated explants at concentrations up to and including 100 μg/ml. Immunohistochemistry revealed no difference between the control and MX-treated explants in distribution of PCNA-and TUNEL-positive cells in the palatal mesenchyme and medial edge epithelium, respectively. When the MX concentration was raised to 300 μg/ml, palatal shelves remained wide open. However, histopathology revealed extensive pyknosis of the mesenchymal cells and loss of the epithelium. These results may indicate that MX is cytotoxic against the mouse palate at a high concentration, and that it has no cleft-palate inducing effects in mice.
3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) causes complete inhibition of rat embryonic midbrain (CNS) cell differentiation in the micromass in vitro test when applied at a concentration of 5 μg/ml under conditions where MX is rapidly degraded in culture medium with a half-life of 56 min. This study investigated whether or not degradation products of MX have inhibitory effects on CNS cell differentiation following pre-incubation of MX in culture medium for 0.5, 1 or 2 hr. When MX was pre-incubated for 0.5 hr, the mean number of differentiated foci was 0.2 against 62.5 for the control. However, the number increased to 44.7 when pre-incubation time was extended to 2 hr. These results suggest that MX, but not its degradation products, is a teratogen in vitro. MX manifested almost complete inhibitory effects on CNS cell differentiation by 0.5 hr of exposure.
The brominated hydroxyfuranones sructurally related to the potent mutagen MXnamely3-chloro-4-(bromochlorome-thyl)-5-hydroxy-2(5H)-furanone (BMX-1), has been synthesized and characterized. The mutagenic activity of this bromohydro-xyfuranone together with those of its analogs BMX-2 and BMX-3 has been assessed by employing the Ames test with the Salmonella typhimurium strains TA98 and TA100. The mutagenic potencies in TA100 without S9 metabolic activation were 22.05 ± 3.15 revertants/ng for BMX-1, 28.64 ± 2.65 revertants/ng for BMX-2, and 37.29 ± 5.73 revertants/ng for BMX-3, whereas in the TA98 strain without metabolic activation, they were six- to ninefold lower. Studies carried out on the stability of these halogenated furanones suggest that the mutagenic activity observed is more related to the cyclic form of these derivatives.
3-Chloro-4-(dichloromethyl)-5-hydroxy-5H-furan-2-one (Mutagen X, MX) was synthesized in six steps from commercially-available and inexpensive starting materials (27% overall yield). This synthesis enables the preparation of MX analogs and does not require the use of chlorine gas, as do previously reported methods.
The synthesis of three brominated analogues of the environmental mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), namely, 3-chloro-4-(bromochloromethyl)-5-hydroxy-2(5H)-furanone (BMX-1), 3-chloro-4-(dibromomethyl)-5-hydroxy-2(5H)-furanone (BMX-2) and 3-bromo-4-(dibromomethyl)-5-hydroxy-2(5H)-furanone (BMX-3), by employing a simple procedure from a common precursor, is described.
3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a by-product of wood pulp manufacture and a contaminant of chlorinated drinking water, was investigated for potential teratogenicity using the micromass in vitro test system. Twelve-day rat embryo midbrain (central nervous system, CNS) and limb bud (LB) cells were exposed to MX at concentrations of 1, 2, 5, or 10 microg/ml in the culture medium with or without S9 mix. Under the experimental conditions, the amount of MX rapidly declined in the culture medium with a half-life of 56 min. Nevertheless, differentiation of CNS and LB cells was significantly inhibited at concentrations of 2 microg/ml or more, when the cells were exposed to MX in the absence of S9 mix. The estimated IC50 was approximately 3 microg/ml for both CNS and LB cell cultures. On the other hand, exposure of CNS and LB cells to MX along with S9 mix did not reduce the number of differentiated foci at any concentrations tested. These results suggest that MX may be a potential direct-acting in vitro teratogen.
3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) was tested without exogenous activation in L5178Y/TK+/(-)-3.7.2C mouse lymphoma cells for mutation at the thymidine kinase locus and for clastogenicity. At a concentration of 0.75 micrograms/ml, the induced mutant frequency was 1027 per 10(6) survivors (survival = 11%). A concentration-related increase of large and small colony mutants was observed, but the majority of the MX induced mutants formed small colonies, consistent with the positive clastogenic response that was observed. MX primarily induced chromatid breaks and rearrangements (30 chromatid and 4 chromosome aberrations per 100 cells) at the 0.75 microgram/ml dose. These studies indicate that MX induces a broad spectrum of genetic damage.
The aim of this study was to investigate DNA damage in human dermal fibroblasts from a healthy subject and from a subject affected by Turner's syndrome that were exposed for 24 h to radiofrequency (RF) radiation at 900 MHz. The RF-radiation exposure was carried out alone or in combination with 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a well-known environmental mutagen and carcinogen produced during the chlorination of drinking water. Turner's syndrome fibroblasts were also exposed for a shorter time (1 h). A signal similar to that emitted by Global System for Mobile Communications (GSM) mobile phones was used at a specific absorption rate of 1 W/kg under strictly controlled conditions of temperature and dosimetry. To evaluate DNA damage after RF-radiation exposure alone, the alkaline comet assay and the cytokinesis-block micronucleus assay were used. In the combined-exposure experiments, MX was given at a concentration of 25 microM for 1 h immediately after the RF-radiation exposure, and the effects were evaluated by the alkaline comet assay. The results revealed no genotoxic and cytotoxic effects from RF radiation alone in either cell line. As expected, MX treatment induced an increase in DNA migration in the comet assay, but no enhancement of the MX-induced DNA damage was observed in the cells exposed to RF radiation.
Chlorinated hydrophilic macromolecular humic acids (CHMA) (0.07-3.0 mg per plate) were mutagenic in Salmonella typhimurium strain TA100. In contrast, the chlorinated derivatives did not induce unscheduled DNA synthesis (nuclear incorporation of [3H]thymidine) in cultured rat hepatocytes even after depletion of intracellular glutathione with buthionine sulphoximine (0.001 mM) and at concentrations of CHMA up to 3.0 mg per plate eliciting cytotoxicity. Glutathione depletion did however potentiate cytotoxicity and hepatocyte glutathione concentrations were lowered by CHMA treatment indicating reactivity of CHMA in the cell.
The mutagenic activity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), which is formed during chlorination of drinking water, was effectively inhibited by sulfhydryl compounds such as cysteine, cysteamine, glutathione, dithiothreitol and 2-mercaptoethanol. Preincubation of 0.5 micrograms MX with 15 micrograms cysteine (molar ratio 1:37) in a phosphate buffer (pH 6.0-8.0) at 37 degrees C for 15 min prior to exposure of bacterial cells depleted the mutagenic activity of MX. Together with the result showing a change in the UV spectra, it is suggested that sulfhydryl compounds inactivate MX by direct chemical interaction before MX induces DNA damage. On the other hand, a variety of antioxidants other than the sulfhydryl compounds showed no inhibitory effects. Investigation using structural analogs of cysteine revealed that the thiol moiety was indispensable for antimutagenic activity and the amino moiety appeared to enhance the MX-inactivating reaction of the SH group.
3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent direct-acting Salmonella mutagen found in chlorinated drinking water, was tested in Chinese hamster ovary (CHO) cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase locus to 6-thioguanine resistance (TGr). MX treatment of CHO cells for 3 h at 37 degrees C resulted in significant dose-related increases in mutant frequency. The lowest observed effective dose was 2.5 micrograms/ml, where the cloning efficiency estimated on the day after treatment was not affected. The relationship between the dose of MX and the frequency of TGr mutants was approximately linear over the range of 0-5 micrograms/ml with an estimated slope (+/- 95% confidence limits) of 7.2 +/- 2.6 mutants per 10(6) clonable cells per microgram/ml.
Three short-term assays (the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test) were carried out to evaluate the genotoxicity of five chlorinated propanones identified in several chlorinated waters (monochloropropanone, 1,1-dichloropropanone, 1,3-dichloropropanone, 1,1,1-trichloropropanone and 1,1,3-trichloropropanone). In the SOS chromotest, all the compounds except monochloropropanone were found to induce primary DNA damage in Escherichia coli. With the fluctuation test, all five chloropropanones showed mutagenic activity on Salmonella typhimurium strain TA100. The newt micronucleus assay detected a clastogenic effect on the peripheral blood erythrocytes of Pleurodeles waltl larvae only for 1,3-dichloropropanone and 1,1,3-trichloropropanone. Moreover, two structure-activity relationships are noticeable: (1) chloropropanones with chlorine substituents on both carbon positions (1,3-DCP and 1,1,3-TCP) are by far more genotoxic than chloropropanones substituted only on one carbon position (1,1-DCP and 1,1,1-TCP); (2) the increase of the number of chlorine substituents decreases the mutagenic activity (fluctuation test) of the chlorinated propanones studied.
The strong bacterial mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) was tested for the induction of mutation at the Na/K ATPase locus to ouabain resistance (OuaR), sister-chromatid exchanges (SCEs), and chromosome aberrations (CAs) in Chinese hamster ovary (CHO) cells without metabolic activation. MX increased the frequency of OuaR mutants in CHO cells when the cells were treated with it in PBS (effective dose range 2-3 micrograms/ml) or in medium (McCoy's 5A) without serum (effective dose range 20-30 micrograms/ml). MX also induced SCEs in CHO cells, at 0.19-1.5 microgram/ml, exposure in PBS; at 6-24 micrograms/ml, exposure in medium; and at 3-24 micrograms/ml, exposure in medium plus 2.5% fetal calf serum. The maximum induction of SCEs was about 1.5-2.5-fold compared with control level, irrespective of exposure conditions (PBS, medium or medium plus serum). The most pronounced genotoxic effect of MX was observed in CAs (100% aberrant cells at the dose level of 4 micrograms/ml, exposure in PBS) which were mainly of the chromatid type. In general, MX was more toxic to CHO cells treated in PBS compared with exposure in medium or medium plus 2.5% serum.
3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) induced DNA strand-breakage (measured by fluorometric analysis of DNA unwinding) in human white blood cells at sub-cytotoxic doses (1.0-1000 microM; 60 min exposure). Although a dose-dependent decrease in glutathione levels was observed with MX, this is not necessarily the causative factor in the observed DNA damage, since no strand breakage was seen on depletion of cellular glutathione to 23% of control by diethylmaleate. In addition, the strand scission does not appear to be mediated by elevation of intracellular calcium as no MX-induced release of calcium stores was observed. Strand breakage may, however, be Ca(2+)-dependent as evidenced by inhibition following deprivation of Ca2+ by Quin-2. Chlorinated fulvic acids (> 3 micrograms/ml) also depleted glutathione and induced strand breaks at sub-cytotoxic doses (up to 300 micrograms/ml) on prolonged exposure (60 min). The unchlorinated material was, however, equally able to cause DNA strand breakage (without glutathione depletion). MX appears, therefore, to be only one of a number of components of chlorinated humic substances able to induce DNA strand breakage.
Three short-term assays (the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test) were carried out to evaluate the genotoxicity of six halogenated acetonitriles identified in chlorinated waters (monochloro-, dichloro-, trichloro-, monobromo-, dibromo- and bromochloroacetonitrile). With the SOS chromotest, three of the chemicals studied (dichloro-, dibromo- and bromochloroacetonitrile) were found to induce primary DNA damage in Escherichia coli PQ37. In the Ames-fluctuation test, all the compounds except dibromoacetonitrile showed mutagenic activity on Salmonella typhimurium strain TA100. The newt micronucleus assay detected a clastogenic effect on the peripheral blood erythrocytes of Pleurodeles waltl larvae for all the six haloacetonitriles studied. Moreover, two structure-activity relationships were noted: (1) the genotoxic activity of haloacetonitriles containing bromine substituents appeared higher than the corresponding chlorinated acetonitriles and (2) the clastogenic activity of the chlorinated acetonitriles increased with the number of chlorine substituents.
The cytotoxicity of the, in Salmonella, potent mutagenic compound, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and its structural analogue 3,4-dichloro-5-hydroxy-2[5H]-furanone (mucochloric acid, MCA), was studied in freshly isolated rainbow trout hepatocytes and gill epithelial cells by determining 86Rb-leakage and decrease in fluorescence intensity in calcein AM-loaded cells. The acute toxicity of the compounds to Daphnia magna was studied by determining the concentration causing immobilization of the organism. MX proved to be more toxic than MCA both in the cellular assays and in the acute toxicity test with D. magna. MX was more toxic to hepatocytes than to gill epithelial cells. The uptake of [14C]MX was also much more efficient in hepatocytes than in gill epithelial cells. The uptake of [14C]MX in hepatocytes was not inhibited by taurocholic acid in excess, indicating that MX is not taken up by the carrier complex responsible for the uptake of taurocholate in the hepatocytes. Both the acute toxicity to D. magna and cytotoxicity of MX and MCA was rather low (EC50 values > 0.1 mM) and we conclude that it is very unlikely that MX and MCA at concentrations occurring in recipients receiving chlorination effluents from pulp mills or chlorinated domestic sewage, as regards their acute toxicity, implies a risk for aquatic animals.
Activities of the xenobiotic metabolizing enzymes were measured in the liver, kidney, duodenum and lung microsomes and cytosol fractions of Wistar rats after subchronic administration of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent bacterial mutagen in chlorinated drinking water. MX was administered by gavage at the dose level of 30 mg/kg for 18 weeks (low dose), or at the dose level which was raised gradually from 45 mg/kg for 7 weeks via 60 mg/kg for 2 weeks to a clearly toxic dose of 75 mg/kg for 5 weeks (high dose). Microsomal and cytosolic preparations were made and the activities of 7-ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-dealkylase (PROD), NADPH-cytochrome-c-reductase, UDP-glucuronosyltransferase (UDPGT) and glutathione-S-transferase (GST) were measured. Kidneys were affected most. A dose-dependent decrease was observed in EROD (90% in males, 80% in females at the high dose) and in PROD (58% in females at the high dose) in kidneys. An increase was, however, detected in kidney NADPH-cytochrome-c-reductase (66% in females at high dose), UDPGT (89% in males and 97% in females at high dose) and GST activities (56% in males and 50% in females at high dose). MX caused only a few changes in the enzyme activities of the liver. The EROD activity was decreased 25% to 37%, both in the livers of males and females, but the total content of P450s was not altered. Hepatic GST activity was elevated in females in a dose-dependent manner (31% and 44%). GST activity was elevated in duodenum in females (59%) at the high dose. There were no marked changes in the enzyme activities in the lungs. MX was a weak inhibitor of EROD activity both in the liver and kidney microsomes in vitro, decreasing the EROD activity by 53% and 43%, respectively at the concentration of 0.9 mM. The results indicate that MX decreases the activity of phase I metabolism enzymes, but induces phase II conjugation enzyme activities, particularly in kidneys in vivo. It is possible that these changes contribute to metabolism of MX in kidneys and renders them susceptible to MX in the course of repeated exposure.
The acid, mutagenic compounds present in chlorinated drinking water have caused concern about the potential cancer risk of drinking-water mutagenicity. In this study, past exposure to drinking water mutagenicity was assessed for the years 1955 and 1970 in 56 Finnish municipalities, using the historical information on water quality and treatment. Cases of leukemia, lymphomas, and cancers of the liver, pancreas, and soft tissue were derived from the Finnish Cancer Registry for two periods: 1966-1976 and 1977-1989. Relative risk was estimated in an additive Poisson regression model, adjusting for age, gender, social class, urbanity, and time period. In an ordinary municipality that was supplied with mutagenic drinking water (3,000 net rev/l), the observed exposure-response relationship indicated a relative risk of 1.1-1.3 for lymphomas and 1.1-1.2 for pancreatic cancer, compared with municipalities in which nonmutagenic drinking water was consumed.
We reported previously that 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)- furanone (MX), a chlorine disinfection by-product in drinking water, induces sister-chromatid exchanges (SCEs) in the peripheral lymphocytes of male and female rats after subchronic exposure. In the present study, we found that the peripheral lymphocytes of male rats exposed to MX (25-150 mg/kg) by gavage on three consecutive days showed a significant dose-related increase in chromosomal damage measured as micronuclei, in addition to SCEs. Moreover, MX produced a significant dose-related increase in SCEs in the kidney cells of the exposed rats. The magnitude of the genotoxic responses observed was relatively weak. The finding is, however, consistent with the known pharmacokinetic distribution of MX in the rat after oral dosing.
The genotoxic effects of three chlorohydroxyfuranones (CHFs), 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), 3-chloro-4-(chloromethyl)-5-hydroxy-2[5H]furanone (CMCF) and 3,4,-dichloro-5-hydroxy-2[5H]furanone (MCA), which are formed as byproducts of water disinfection with chlorine, were investigated in bacterial differential DNA repair assays in vitro and in animal-mediated assays in vivo. As indicators of DNA damage, E. coli K-12 strains were used that differ in their repair capacity (uvrB/recA vs. uvr+/rec+). Liquid incubation of the compounds without metabolic activation caused a pronounced reduction of the viability of the repair-deficient strain relative to the repair-proficient wild-type strain. The order of potency of genotoxic activity in vitro (dose range 0.004-10 micrograms/ml) was MX > CMCF > MCA. Addition of mouse S-9 mix or bovine serum albumin to the incubation mixtures resulted in an almost complete loss of the activity of all three test compounds. In the animal-mediated assays, mixtures of the indicator bacteria were injected intravenously into mice which were subsequently treated with the test compounds (200 mg/kg b.w.). Two hours later, the cells were recovered from various organs and the relative survival frequencies determined. Under these conditions, all three compounds caused pronounced genotoxic effects, MX and CMCF being stronger genotoxins than MCA. The strongest effects were consistently found in the gastrointestinal tract, but statistically significant DNA damage was also observed in indicator cells recovered from lungs, liver, spleen and kidneys. In a further experiment, the effects of lower doses of MX (4.3, 13 and 40 mg/kg) were investigated. In these experiments dose-dependent effects were measured in all organs. CMCF and MA caused only marginal effects at 40 mg/kg except in the stomach where approximately a 50% reduction of relative survival frequency was observed with CMCF. The results of these animal-mediated assays indicate that (i) all three CHFs cause genotoxic effects in the living animal, and (ii) the potencies of the three compounds observed under in vivo conditions are not commensurate with their extremely high activities measured in vitro. One possible explanation for the weaker responses observed in the animal-mediated assays might be that CHFs are inactivated by non-specific protein binding.
1 Rat liver 9,000 g supernatant protected against the mutagenic effect of chlorinated hydrophilic macromolecular humic acids (CHMA) in Salmonella typhimurium strain TA100.
2 Protection against mutagenicity of CHMA was mediated by glutathione and was partially dependent on glutathione S-transferase activity.
3 In contrast to the above findings, CHMA showed lower mutagenicity in Salmonella typhimurium and Escherichia coli strains of bacteria that are deficient in glutathione compared to their mutagenicity in parental (glutathione-rich) bacterial strains.
4 Glutathione-deficient cells do not provide test systems with elevated sensitivity for the detection of mutagenic chlorinated humic substances.
The purpose of this study was to investigate the relationship between exposure to mutagenic drinking water and cancers of the gastrointestinal and urinary tract.
Past exposure to drinking water mutagenicity was assessed in 56 Finnish municipalities for the years 1955 and 1970. The cases of bladder, kidney, stomach, colon, and rectum cancers were derived from two periods (1967 to 1976 and 1977 to 1986). Age, sex, social class, urban living, and time period were taken into account in the Poisson regression analysis.
Statistically significant exposure-response association was observed between exposure and incidence of bladder, kidney, and stomach cancers. In an ordinary municipality using chlorinated surface water, this exposure would indicate a relative risk of 1.2 for bladder cancer and of 1.2 to 1.4 for kidney cancer compared with municipalities where nonmutagenic drinking water was consumed.
The acidic mutagenic compounds present in drinking water may play a role in the etiology of kidney and bladder cancers, but, because the results are based on aggregate data, they should be interpreted with caution.
Investigations were carried out on 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent direct-acting bacterial mutagen found in chlorinated tap water, for the potential to induce 6-thioguanine (6-TG) resistant mutations in cultured Chinese hamster V79 cells. The mutagenicity of MX was manifested when cells were treated with MX in Hanks balanced salt solution, but not in serum-free Eagle's minimal essential medium. A 12-fold higher mutation frequency over control was obtained at 12.5 micrograms/ml MX. In addition, the inhibitory effect of MX on metabolic cooperation between cocultivated 6-TG- sensitive (6-TGs) and -resistant (6-TGr) V79 cells was investigated. Recovery of 6-TGr cells significantly increased at 0.8-2.0 micrograms/ml MX due to impaired intercellular communication with 6-TGs cells. MX is believed to exert tumor-promoting and mutagenic activity in mammalian cells.
Toxic effects and excretion in urine of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), the potent mutagenic compound in chlorinated drinking water, was evaluated in male Wistar rats by the up-and-down method. MX was dosed by gavage in deionized water at doses between 200 mg/kg and 600 mg/kg, for one animal at a time, and effects were observed for 14 days. Urine was collected in metabolism cages up to 72 h after dosing for chemical analysis of MX in urine. The animals receiving 200 mg/kg did not display clear clinical signs but at higher doses the signs of ill effects included dyspnea, laborious, wheezing and gasping breathing, decreased spontaneous motor activity, ataxia, nostril discharges, catalepsia and cyanosis. In necropsy bronchi contained foamy liquid and the lungs appeared edematous and spongy. The stomach cavity was expanded due to accumulation of fluid and gas and the gastrointestinal tract from stomach to caecum was reddish. Microscopically, the main target organ of toxicity was the gastrointestinal tract (diffuse congestion and necrosis in the mucosa). Signs of toxicity were recorded also in lungs (slight edema) and kidneys (dilated tubules, thin tubular epithelium, brownish tubular and interstitial concretion). The LD50 in 48 h was 230 mg/kg. Only 0.03-0.07% of the dose (200 mg/kg or 300 mg/kg) was excreted in urine as intact MX. The results indicate that at high doses MX has a strong local irritating effect in the gastrointestinal tract and it probably increases liquid permeability in lungs. MX may also cause tubular damage in kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)
The assessment of past exposure is a prerequisite to all epidemiological studies on drinking water and cancer. In this study the past exposure assessment of drinking water carcinogenicity was done in terms of the drinking water mutagenicity estimated from historical water parameters and compared with the methods used previously in past exposure assessments in studies on drinking water and cancer. The method was applied in 56 municipalities in Finland. The comparison of different methods in past exposure assessment suggests some advantages for the method presented as it allows a quantitative exposure assessment based on historical information on drinking water mutagenicity. Nevertheless the relevance of the method is with respect to the role of mutagenicity in carcinogenicity and the water type in question.
The mutagenicities of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX, compound 1), 3-chloro-4-(dichloromethyl)-2(5H)-furanone (RMX, compound 6), and 2-(dichloromethyl)-3,3-dichloropropenal (TCB, compound 7) were determined in the same assay and in repetitive determinations using Salmonella typhimurium (TA 100) without microsomal fraction activation. In addition, the mutagenicity of 2-methyl-3,3-dichloropropenal (compound 8) was assayed in the same manner although not simultaneously with MX, RMX, and TCB. This study was undertaken to ascertain the role of open- and closed-ring forms of MX in the mutagenicity of MX. MX proved to be roughly 100 times more mutagenic than the open-ring analogue TCB and 10 times more mutagenic than the closed-ring analogue RMX. Compound 8 was inactive. Assay stability of the three active compounds in Vogel-Bonner medium at 38 degrees C was estimated as the chemical half-life values by following the change in UV absorbance at selected wave lengths. Half-life values were 10.7, 2.6, and 2.8 hr, respectively, for MX, RMX, and TCB. The enhanced mutagenicity of MX relative to RMX and TCB is attributed to the intrinsic mutagenicity of MX and its greater stability is judged to play only a minor role. Moreover, the greater mutagenicity of the closed-ring analogue RMX relative to the open-ring analogue TCB points to the ring form of MX as the active species even though the open form of MX is predominant under assay conditions.
Starting from tetrachloroacetone 1 and (carbomethoxymethylene)triphenylphosphorane 4, the title compound 16 was prepared in five steps in 44% yield. Spectroscopic and chromatographic data indicated that this material was identical to a potent mutagen isolated from kraft pulp chlorination effluent. Various mutant Salmonellatyphimurium strains (TA 98, 100, and 1537) behaved similarly when incubated with either 16 or the isolated mutagen.
A procedure for the synthesis of (14)C-MX using bromoacetic-2-(14)C acid as the starting material was developed. Use of the radiolabeled product in a distribution study in the rat indicated that the majority of the radioactivity was cleared in feces (47%) and urine (33%) within 48 hours. The parent compound was not detected in blood samples taken after 48 hours or in the feces and urine samples.
The strong Ames test mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2-(5H)-furanone (MX) was found to occur in chlorinated drinking and humic waters in sub μg L−1 concentrations. Quantitation by GC/MS using Selective Ion Monitoring in parallel with Ames tests of waters indicated that this furanone accounted for 15 – 30% of the Ames test mutagenicity of both a drinking water and a humic water.
Mutagenic extracts from kraft pulp chlorination-stage effluents were fractionated, and the distribution of mutagenicity was determined by the Ames test with tester strain TA 100. Most of the mutagenicity was caused by nonvolatile compounds extractable with ethyl acetate. Strong acids accounted for the principal part of the mutagenicity. The mutagenic components could be concentrated to a narrow band by silica thin-layer chromatography and reverse-phase high-pressure liquid chromatography. The results provide further support for the conclusion that the previously identified hydroxyfuranone (C5H3O3Cl3) is a major TA 100 mutagen in chlorination-stage effluents. The isolation of this compound, its Ames mutagenicity, and some of its chemical properties are described.
The pharmacokinetics of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) was evaluated after a single oral or intravenous administration in the rats using 14C-labelled compound. Twenty to 35% of the dose was absorbed into circulation from the gastrointestinal tract as assessed from the excretion in urine. The mean elimination half-life of the radioactivity in blood (T1/2 k10) was 3.8 hr. Traces of radioactivity remained in the blood for several days. The tissues lining the gastrointestinal and urinary tract, kidneys, stomach, small intestines and urinary bladder contained the highest radioactivity. The activity declined slowest in the kidneys. Urine was the main excretion route. Seventy-seven % of the total amount excreted appeared in urine in 12 hr and 90% in 24 hr. No radioactivity was exhaled in air suggesting that elimination through respiration did not occur. After an intravenous administration of 14C-MX, the T1/2 k10, was much longer, 22.9 hr, and the total elimination half-life (T1/2 beta), 42.1 hr. The results indicate that MX is absorbed from the gastrointestinal tract to a considerable degree and it is excreted in urine very rapidly. A fraction of MX or its metabolites is retained in blood for a longer period of time. The pharmacokinetics of MX does not suggest extensive cumulation of MX in tissues after continuous exposure.
Binding of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) to bovine serum albumin (BSA) was studied. MX bound mainly reversibly to BSA but, for a minor part, also irreversibly. It was possible to extract the main part of the reversibly bound MX with ethyl acetate and the extractable compound was chromatographically identical to MX. The affinity-binding characteristics of the interaction with albumin were K = 1.6 x 10(7) M-1, n = 3.4. Furthermore, mutagenicity studies indicated that reversibly bound MX remained mutagenic but that irreversibly bound MX was no longer mutagenic in the Ames test. These results suggest that the binding of MX to albumin is an important factor for both the toxicological effects and the toxicokinetics of MX.
The potent bacterial mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]- furanone) (MX), which is formed during chlorination of drinking water and accounts for about one third of the Ames mutagenicity of tap water, has been studied with respect to its genotoxicity in vitro and in vivo. Treatment with 30-300 microM MX (1 h) induced DNA damage in a concentration-dependent manner in suspensions of rat hepatocytes, as measured by an automated alkaline elution system. The effect was similar in hepatocytes from PCB-induced and uninduced rats. DNA damage was induced in V79 Chinese hamster cells and in isolated rat testicular cells, at the same concentration level as in hepatocytes. Pretreating testicular cells with diethylmaleate, which depletes 85% of cellular glutathione, had no significant effect on the DNA damage induced by MX. The treatment conditions used in the alkaline elution experiments were not cytotoxic to any of the cell types used, as determined by trypan blue exclusion. V79 cells exposed to 2-5 microM MX (2 h) showed an increased frequency of sister-chromatid exchanges (SCE) whereas no significant effect on HGPRT mutation induction was observed. Higher concentrations (greater than 10 microM, 2 h) apparently blocked cell division. The data indicate that MX can react directly with DNA or that MX is metabolized to an ultimate mutagen via some enzyme which is common in mammalian cells. The in vivo experiments showed no evidence of genotoxicity after intraperitoneal (18 mg/kg, 1 h) or oral (18, 63 or 125 mg/kg, 1 h) administration of MX, as measured by alkaline elution, in any of the following organs: the pyloric part of the stomach, the duodenum, colon ascendens, liver, kidney, lung, bone marrow, urinary bladder and the testes. In conclusion, MX is a direct-acting genotoxicant in vitro but no in vivo genotoxicity was detected.
Two chlorinated hydroxylated furanones, 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) and 3,4-(dichloro)-5-hydroxy-2[5H]-furanone (MA), are bacterial mutagens and they are also byproducts of chlorine disinfection, and frequent contaminants of drinking water. In this work MX is shown to induce nuclear anomalies in the gastrointestinal tract of the B6C3F1 mouse. The other chlorohydroxy-furanone, MA, gives suggestive evidence of activity. In this bioassay MX was approximately equivalent in potency to epichlorohydrin (ECH) but was much less potent than methylnitrosourea (MNU). The latter two chemicals are confirmed rodent gastrointestinal tract carcinogens. The duodenum was the most sensitive tissue responding with both increased numbers of nuclear anomalies per mouse and increased incidence of animals presenting the nuclear aberrations 24 hr after a single oral dose of 0.37 mmol/kg-1 of MX. MA also induced a significant increase in duodenal nuclear anomalies, but only at the highest dose (0.46 mmol/kg-1). The proximal colon and forestomach responded to MX but not MA. This is the first study demonstrating that chlorohydroxyfuranones are capable of inducing nuclear toxicity in vivo. However, it is clear, for MX at least, that its potency in the gastrointestinal tract nuclear anomalies assay is not commensurate with its extreme bacterial mutagenicity. Since the gastrointestinal tract tissues are directly exposed to orally administered genotoxins, one possible explanation for the weak response observed in this study could be that mammalian cells can effectively detoxify chlorohydroxyfuranones.
3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) was detected by gas chromatography/mass spectrometry in drinking water samples from 3 locations in the U.S.A., and also in a chlorinated humic acid solution. MX appears to account for a significant proportion of the mutagenicity of these samples, as measured in the Ames test using strain TA100 without metabolic activation. Studies on recovery of MX from spiked water samples by XAD-2/8 resin adsorption/acetone elution indicated that sample acidification prior to resin adsorption was essential to the effective recovery of MX. The stability of MX in aqueous solution was pH and temperature dependent. At 23 degrees C the order of stability, based on persistence of mutagenic activity was found to be: pH 2 greater than pH 4 greater than pH 8 greater than pH 6. The half-life at pH 8 and 23 degrees C was 4.6 days. One of the degradation products has been tentatively identified as 2-chloro-3-(dichloromethyl)-4-oxo-2-butenoic acid, an open form of MX which appears to be in the "E" configuration. Overall, these results suggest that MX is formed during water chlorination as a result of reaction of chlorine with humic substances, and that a substantial fraction of the MX formed is likely to persist throughout the distribution system.
The Ames mutagenicity and the concentration of the strong Ames mutagen 3-chloro-4-(dichloro-methyl)-5-hydroxy-2(5H)-furanone (MX) and its geometric isomer E-2-chloro-3-(dichloromethyl)-4-oxobutenoic acid (E-MX), derived from chlorination of humus, were determined in XAD extracts of tap water collected from 26 localities in Finland. The 23 tap waters treated with disinfectants gave a positive response in strain TA100. MX and E-MX were detected in all extracts exhibiting mutagenicity with the exception of 3 extracts of marginal activity. MX accounted for 15-57% (average 33%) of the observed mutagenicity. The concentration of E-MX was slightly lower than the corresponding concentration of MX. Linear correlations were observed between mutagenicity and concentration of MX and E-MX, with correlation coefficients of 0.894 for MX and 0.910 for E-MX.
3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) was found to be a direct-acting mutagen in the Ames test for strains TA1535, TA1538, TA92, TA97, TA98, TA100 and TA102. The highest mutagenic response (approximately 13,000 revertants/nmol) was seen in strain TA100. The TA100 response was six- to tenfold higher than in TA98, TA97, and TA102, and 100- to 500-fold higher than in TA1535, TA92, and TA1538. The addition of a 9,000 x g supernatant fraction (S-9) from livers of polychlorinated biphenyl-treated rats, along with cofactors for NADPH generation, resulted in a 90% reduction in the TA100 mutagenicity. MX induced chromosomal aberrations in Chinese hamster ovary cells after 6-8 hr exposure without S-9 at a dose as low as 4 micrograms/ml, and after 2 hr exposure with S-9 at a dose of 75 micrograms/ml. The oral dose of MX lethal to 50% (LD50) in Swiss-Webster mice was determined to be 128 mg/kg. MX did not induce micronuclei in mouse bone marrow when administered by oral gavage at doses up to 70% of the LD50.
Peripheral blood lymphocytes from 32 male rotogravure workers with daily exposure to toluene were studied for chromosome aberrations and sister chromatid exchange. Neither of these two cytogenetic parameters differed significantly from the correspondong frequencies in 15 unexposed control subjects. However, a significant increase in sister chromatid exchange was observed among smokers, both exposed and occupationally unexposed, compared to nonsmoking referents.