Oxidative stress and DNA damage induced by a drinking-water chlorination disinfection byproduct 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in mice

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a water chlorine disinfection byproduct, can induce DNA damage (e.g., modification of nucleotides and DNA strand breaks) and subsequent DNA repair in vitro. However, the underlying mechanism(s) how DNA damage is induced by MX is unknown. We hypothesized that MX may cause oxidative stress that leads to DNA damage in vivo. In the present study, we exposed groups of mice to MX at concentrations of 0 (solvent control), 11 (low), 33 (medium) and 99 (high) mg/kg b.w. by single intraperitoneal injection. After treating the mice for 3h, we detected cellular levels of malondialdehyde (MDA) and glutathione (GSH) to assess oxidative stress in the target cells. In addition, we also evaluated DNA damage using single cell gel electrophoresis (SCGE or Comet assay). We found that the levels of DNA damage in all cell types were correlated positively with levels of MDA but negatively with levels of GSH (P<0.05 for all). Also, there were negative correlations between levels of MDA and GSH (r=-0.995 for liver cells, -0.916 for kidney cells, -0.975 for intestine cells, respectively; P<0.05 for all but kidney cells). Our findings suggest that MX may induce DNA damage by the mechanism of causing cellular oxidative stress as measured by increased MDA and decreased GSH, at least in mice.     

Developmental toxicity evaluation of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in Wistar rats

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a genotoxic chlorination by-product in drinking water. There is some evidence that it has developmental toxic effects in vitro but its potential to cause developmental effects in vivo is not known. The developmental effects were evaluated in Wistar rats. Rats (22-26 dams per dose group) were administered MX by gavage at the dose levels of 3, 30, or 60 mg/kg in water on gestation days 6-19. Control animals received plain water. Clinical signs, body weight, and food and water consumption were recorded for the dams. On gestation day 20, a cesarean section was performed and the ovaries anduterine contents of the dams were examined and the liver, kidneys, spleen, and thyroid glands weighed. The fetuses of all dose groups were weighed, sexed, and observed for external and skeletal malformations and the fetuses of the two highest dose groups were evaluated for visceral malformations. The highest dose, 60 mg/kg of MX, was slightly toxic to the dams. It decreased the corrected body weight gain of dams by 32% and the water consumption by 16-17%. Kidney and liver weights were slightly increased. MX did not affect the number of implantations nor did it cause any resorptions. The body weights of fetuses were not significantly affected. MX did not cause external malformations or skeletal anomalies. Two fetuses at 60 mg/kg and one fetus at 30 mg/kg had major visceral malformations (persistent truncus arteriosus, diaphragmatic hernia, dilated aorta with a stenosis of pulmonary arteries) and two minor artery abnormalities were observed in those animals. The frequency of unilateral displaced testis was slightly higher (9.2%) in the 60-mg/kg dose group than in controls (1.6%). Since the abnormalities did not form a consistent pattern and occurred most at maternally toxic dose, we conclude that MX can be regarded as non-teratogenic.     

Rapid inactivation of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent mutagen in chlorinated drinking water,by sulfhydryl compounds

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 μg MX with 15 μg cysteine (molar ratio 1:37) in a phosphate buffer (pH 6.0–8.0) at 37°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.     

Genotoxic effects of the chlorinated hydroxyfuranones 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone and 3,4-dichloro-5-hydroxy-2[5H]-furanone in Tradescantia micronucleus assays

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.     

Ames mutagenicity and concentration of the strong mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and of its geometric isomer E-2-chloro-3-(dichloromethyl)-4-oxo-butenoic acid in chlorine-treated tap waters

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.     

DNA damage induced by the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in mammalian cells in vitro and in mice

3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) formed during chlorination of water containing natural organic substances, is a very potent bacterial mutagen. Recently, tumours at multiple sites were reported in rats given MX-containing drinking water. We have investigated the genotoxicity of MX in mammalian cells exposed in vitro and in vivo using alkaline filter elution to detect DNA single-strand breaks and/or alkali-labile sites (SSBs). Concentrations as high as 100 and 300 microM MX were required to induce detectable levels of SSBs in the HL-60 cells. If MX treatment was carried out in the presence of DNA repair inhibitors (AraC plus hydroxyurea), the sensitivity of the assay to detect MX-induced SSBs was increased by a factor of 100. The presence of serum proteins during exposure resulted in a minor reduction of the MX-induced DNA damage in HL-60 cells at the lowest MX concentrations. In primary cultures of testicular cells as well as in resting human peripheral blood mononuclear cells (PBMC), a slightly increased level of SSBs was observed at MX-concentrations above 30 microM, this effect was not further increased by repair inhibitors. In LLC-PK1 renal proximal tubular epithelial cells and in growth stimulated human peripheral PBMC, increased SSBs were detected at MX concentrations as low as low as 3-10 microM and higher using repair inhibitors, and at 10 times higher concentrations without repair inhibitors. No dose dependent DNA damage was detected in the liver, kidney, spleen and colon of male B6C3F1 mice administrated high doses of MX (40 and 80 mg kg-1). Moderately increased and dose dependent SSBs were detected in the liver and kidney in the presence of DNA repair inhibitors during MX treatment, but no such increase was observed in the spleen and colon.     

Induction of TK mutations in human lymphoblastoid TK6 cells by the rat carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX)

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a chlorine disinfection by-product in drinking water, is carcinogenic in rats and genotoxic in mammalian cells in vitro. In the current study, the mechanism of genotoxicity of MX in human lymphoblastoid TK6 cells was investigated by use of the Comet assay, the micronucleus test, and the thymidine kinase (TK) gene-mutation assay. MX induced a concentration-dependent increase in micronuclei and TK mutations. The lowest effective concentrations in the MN test and the TK gene-mutation assay were 37.5μM and 25μM, respectively. In the Comet assay, a slight although not statistically significant increase was observed in the level of DNA damage induced by MX in the concentration range of 25-62.5μM. Molecular analysis of the TK mutants revealed that MX induced primarily point mutations or other small intragenic mutations (61%), while most of the remaining TK mutants (32%) were large deletions at the TK locus, leading to the hemizygous-type loss-of-heterozygosity (LOH) mutations. These findings show that aside from inducing point mutations, MX also generates LOH at the TK locus in human cells and may thus cause the inactivation of tumour-suppressor genes by LOH.     

Studies on the potent bacterial mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone: aqueous stability, XAD recovery and analytical determination in drinking water and in chlorinated humic acid solutions

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.     

No effects of radiofrequency radiation on 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone-induced tumorigenesis in female Wistar rats

This study evaluated possible effects of radiofrequency (RF) radiation on tumorigenesis induced by the mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) given in drinking water. Female Wistar rats aged 7 weeks at the beginning of the experiments were randomly divided into four groups of 72 animals: a cage-control group and three MX-exposed groups (a daily average dose of 1.7 mg MX/kg body weight for 104 weeks), of which two were exposed to 900 MHz pulsed RF radiation and the third served as a sham-RF-radiation group. The RF-radiation groups were exposed 2 h per day, 5 days per week for 104 weeks at nominal whole-body average SARs of 0.3 W/kg and 0.9 W/kg. Complete histopathology was performed on the rats of the three MX-exposed groups. The tumor types and incidences observed in the MX-exposed animals were similar to those reported earlier in MX-exposed female Wistar rats. RF radiation did not statistically significantly affect mortality or organ-specific incidence of any tumor type. The only statistically significant difference was an increase in the combined frequency of vascular tumors of the mesenteric lymph nodes in the high-RF-radiation group compared to the sham-RF-radiation group. However, additional histopathological analysis of the cage-control animals suggested that this difference was due to unusually low frequency of this type of tumor in the sham-RF-radiation group rather than a high frequency in the high-RF-radiation group. With respect to non-neoplastic findings, statistically significant differences between the RF-radiation groups and the sham-RF-radiation group were observed only for single findings in the lacrimal glands, lungs, liver and skin. Such changes are commonly seen in aged rats and were considered to be unrelated to RF radiation. The results of the present study do not support co-carcinogenic effects of low-level long-term RF-radiation exposure in rats.     

Formation of reactive oxygen species in L929 cells after exposure to 900 MHz RF radiation with and without co-exposure to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone

The aim of this study was to investigate the induction of reactive oxygen species in murine L929 fibrosarcoma cells exposed to radiofrequency (RF) radiation at 900 MHz, with or without co-exposure to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent environmental carcinogen produced during chlorination of drinking water. Both continuous-wave and GSM mobile phone signals were applied for 10 or 30 min at specific absorption rates of 0.3 and 1 W/kg. Simultaneous sham exposures were performed for each exposure condition. MX treatment was performed at a subtoxic level of 500 microM, and the RF-field exposure was carried out during the first 10 or 30 min of the chemical treatment. The formation of reactive oxygen species was followed soon after the exposure and at different harvesting times until 1 h after RF-field treatment. The studied provided no indication that 900 MHz RF-field exposure, either alone or in combination with MX, induced formation of reactive oxygen species under any of the experimental conditions investigated. In contrast, exposure to MX resulted in a statistically significant increase in the formation of reactive oxygen species for all the treatment durations investigated, confirming that MX is an inductor of oxidative stress in L929 cells.     

Nonenzymatic formation of 4,5-dimethyl-3-hydroxy-2(5H)-furanone in foodstuffs

The mechanism of formation of 4,5-dimethyl-3-hydroxy-2/5H/-furanone in some food-stuffs was investigated. The compound was found to result from condensation of acetaldehyde under oxidative conditions. The intermediate product of the condensation is 2-oxobutanoic acid.     

(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone reduces corrosion from Desulfotomaculum orientis

(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the red marine alga Delisea pulchra was found previously to inhibit the growth, swarming and biofilm formation of Gram-positive bacteria (Ren et al., 2002, Lett Appl Microbiol 34: 293-299). In the present study, the Gram-positive sulphate-reducing bacterium (SRB), Desulfotomaculum orientis, was used to study the inhibition of mild steel corrosion due to the addition of furanone. The weight loss from batch coupon experiments incubated with 40 microg x ml(-1) furanone was reduced fivefold compared with samples that lacked furanone. Analysis of the metal surface with environmental scanning electron microscopy further confirmed the protection afforded by the addition of furanone. In agreement with the corrosion inhibition, most probable number (MPN) analysis showed that 20 and 40 microg x ml(-1) furanone inhibited 58% and 96% of the D. orientis growth respectively. Hence, furanone has the potential to inhibit microbial-induced corrosion related to Gram-positive bacteria.     

The formation mechanism by yeast of 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone in Miso

The mechanism of the formation of 4-hydroxy-2(or 5)-ethyl-5-(or 2)-methyl-3(2H)-furanone (HEMF) with yeast under caltivation in a medium containing amino-carbonyl reactants of ribose and glycine was investigated using stable isotopes of the corresponding compounds. It was confirmed that the skeleton of the five-membered ring and the methyl group of the side chain of HEMF was formed from ribose, and that the ethyl group was derived from the glucose metabolite by yeast. The formation of HEMF was confirmed when acetaldehyde as the glucose metabolite and a cell-free extract from yeast were added to the medium containing amino-carbonyl reactants. These results suggest that the role of yeast in HEMF formation is not only to provide the glucose metabolite, but also in combining the amino-carbonyl reactants with the glucose metabolite.     

3-(4-Methyl-3-pentenyl)-2(5H)-furanone, alpha,alpha-acariolide and 4-(4-methyl-3-pentenyl)-2(5H)-furanone, alpha,beta-acariolide: new monoterpene lactones from the astigmatid mites, Schwiebea araujoae and Rhizoglyphus sp. (Astigmata: Acaridae)

A new monoterpene lactone from the acarid mite, Schwiebea araujoae, was elucidated without its isolation by GC/FT-IR and GC/MS analyses to be 3-(4-methyl-3-pentenyl)-2(5H)-furanone (1) and tentatively named as alpha,alpha-acariolide. The structure of 1 was identified by its synthesis from alpha-bromo-gamma-butyrolactone via 4 reaction steps. The synthesized compound gave the same GC/MS and GC/FT-IR spectra as those of the natural product. The other monoterpene lactone was likewise elucidated from the unidentified Rhizoglyphus mite to be 4-(4-methyl-3-pentenyl)-2(5H)-furanone (2) and named as alpha,beta-acariolide; it was also identified by its synthesis in 5 reaction steps from the same butyrolactone as the starting material. GC/MS and GC/FT-IR spectra of the preparation were identical to those of the natural product.     

Inhibition of biofilm formation and swarming of Escherichia coli by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone

The quorum-sensing disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) of the alga Delisea pulchra was found to inhibit the swarming motility of Escherichia coli completely at 13 microg cm-2 (also at 20 microg ml-1) but did not inhibit its growth rate at 13-52 microg cm-2 or from 20 to 100 microg ml-1. Swimming was not inhibited by the furanone at 20-40 microg ml-1. In addition, confocal scanning laser microscopy revealed that this furanone at 60 microg ml-1 inhibited the biofilm formation of E. coli, as it decreased its thickness by 55%, reduced the number of water channels and decreased the percentage of live cells by 87%. This suggests that natural furanone may be used as a new method to control bacterial biofilms that does not involve toxicity. Furanone at 10 microg ml-1 also inhibited by 3300-fold the quorum sensing of Vibrio harveyi via autoinducer 1 (AI-1) and inhibited by 5500-fold that of V. harveyi via of autoinducer 2 (AI-2) as well as inhibited by 26-600-fold the quorum sensing of E. coli via AI-2; hence, this furanone is a non-specific intercellular signal antagonist.