Binding of the strong bacterial mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) to bovine serum albumin.

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.     

Assay for nucleoside and nucleotide binding of a potent mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone

Deoxyadenosine, deoxyguanosine, deoxycytidine; thymidine and a dinucleotide, 2'-deoxycytidylyl-(3'-5')-2'-deoxythymidine (dCdT), were reacted with 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Nucleoside HPLC analysis revealed that the major products are the degradation products of MX and no stable MX-adducts were found. The same result was obtained with sodium borohydride reduction. The fluorescence spectra of deoxyguanosine-MX showed no fluorescence at neutral or alkaline pH, indicating no binding products to the N2-groups of deoxyguanosine.     

3-氯-4-二氯甲基-5-羟基-2(5氢)-呋喃酮对L-02细胞DNA损伤与ras基因表达的影响

目的　研究饮水氯化消毒副产物 3 氯 4 二氯甲基 5 羟基 2 (5氢 ) 呋喃酮 (MX)对人胚肝细胞 (L 0 2 )DNA损伤与ras基因表达的诱导 ,初步探讨MX致癌的分子机制。方法　以L 0 2细胞作为靶细胞 ,应用彗星试验 (SCGE)和原位杂交试验 (ISH)研究MX诱导L 0 2细胞DNA损伤与ras基因表达。MX设 10、3 0、10 0和 3 0 0 μmol L 4个剂量 ,二甲基亚砜 (DMSO ,10mL L)为溶剂对照 ,H2 O2 (3 0 0 μmol L)和B(a)P(2 0 0 μmol L)分别为SCGE和ISH的阳性对照。 结果　与溶剂对照相比 ,3 0、10 0和 3 0 0 μmol L的MX能明显增加L 0 2细胞的DNA迁移度 (P <0 0 5 ,P <0 0 1,P <0 0 0 1) ;同时也明显增加ras基因表达水平 (P <0 0 0 1,P <0 0 0 1,P <0 0 1)。MX在 10～ 10 0 μmol L的浓度范围内 ,诱导的ras基因表达水平和DNA迁移度呈正相关 (r =0 79)。结论　饮水氯化消毒副产物MX可诱导L 0 2细胞DNA损伤与ras基因表达 ,ras基因表达水平可能与DNA损伤程度有关。     

Induction of oxidative stress in murine cell lines by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX)

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), the potent bacterial mutagen produced during chlorination of drinking water, was tested for the induction of oxidative stress in two murine cell lines: NIH 3T3 (fibroblasts) and L929 (fibrosarcoma cells). Following 1 h MX treatment at concentrations between 100 and 1000 microM, cellular stress conditions were monitored by measuring reactive oxygen species formation (ROS) and reduced glutathione levels (GSH). The kinetics of ROS formation and GSH depletion was investigated from 10 min to 1 h. MX caused detachment of cells at 1000 microM in L929 cells and at 300 microM in NIH 3T3 cells but the viability of the cells, measured by the trypan blue assay, decreased only by 20 and 7%, respectively, in 1h. MX increased ROS production in L929 cells in a dose-dependent manner, by 120% at 500 microM of MX in 1 h. The maximum ROS production was attained already in 10min. In NIH 3T3 cells, the ROS production was slightly, but not statistically significantly stimulated at 200 microM between 20 and 60 min. Concomitantly, MX decreased the intracellular content of GSH dose-dependently in both cell lines, by 48% in L929 cells at 500 microM of MX and 32% in NIH 3T3 cells at 200 microM of MX in one hour. The majority of this GSH depletion had occurred in 10 min. These findings indicate that MX induces oxidative stress in mammalian cells in vitro though the sensitivity of cells may differ for this effect.     

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

The subchronic (14–18 wk) toxicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a mutagenic by-product in chlorinated drinking water, was evaluated in Wistar rats. In a range-finding study, MX was administered daily for 14 days by gavage in deionized water to male rats (five animals per group) at doses of 12.5, 25, 50, 100 or 200 mg/kg body weight. The doses above 50 mg/kg were lethal and three out of five animals also died during treatment at 50 mg/kg. The range-finding study was repeated with doses of 5, 10 and 20 mg MX/kg, given on 5 days a week, to both males and females (10 animals per group). These doses were not overtly toxic but caused several changes in plasma clinical chemistry at 10 and 20 mg MX/kg in comparison with the controls. These included increased urea, creatinine and bilirubin and decreased inorganic phosphate and potassium in females and increased cholesterol in males. In the subchronic toxicity study, rats (15 per group) were given MX by gavage, on 5 days a week, at doses of 0 (controls) or 30 mg/kg (low dose) for 18 wk, or, in the high-dose group, at doses increasing from 45 to 75 mg/kg over 14 wk. The high dose was finally lethal (two males and one female died) and caused hypersalivation, wheezing respiration, emaciation and tangled fur in animals. The body weights of the high-dose males decreased by 15%, and food consumption was decreased by 15 to 20%, but the water consumption increased by 15% to 60%. Plasma cholesterol and triglycerides were elevated and urine excretion was increased. Urine specific gravity was decreased and the relative weights of the liver and kidneys were increased in both sexes at both doses in comparison with the controls. At both doses, duodenal hyperplasia occurred in males and females, and slight focal epithelial hyperplasia in the forestomach was observed in males. Splenic atrophy and haemosiderosis were seen in two high-dose females, and epithelial cell atypia in the urinary bladder of one high-dose male and female. The frequency of bone marrow polychromatic erythrocytes with micronuclei was slightly increased in low-dose males. The results indicate that repeated administration of MX disturbs the fluid-electrolyte balance and induces diuresis, causes mucosal hyperplasia in the gastro-intestinal tract as a local effect, and affects lipid metabolism.     

Enhancement of 3-methylcholanthrene-induced neoplastic transformation by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone in the two-stage transformation assay in C3H 10T1/2 cells

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)furanone (MX) is a mutagenic by-product found in chlorinated drinking water. It is a multi-site carcinogen in Wistar rats although the mechanisms of action of the carcinogenesis remain unresolved. We evaluated the ability of MX to promote development of transformation foci in a two-stage cell transformation assay in vitro. C3H 10T1/2 mouse embryonic fibroblasts were exposed to 3-methylcholanthrene (MC, 5 microg/ml) in the initiation phase and to MX (0.5, 1 or 2 microg/ml) or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, the positive control, 0.3 microg/ml) during the promotion phase of the assay on dishes. In other experiments, the cells were exposed to MX (0.5, 5 or 10 microg/ml) only in the initiation phase. At the end of the assay (6 weeks from the start of the assay), the transformation foci were counted and scored after fixation and staining of the cells. MC increased the total number of transformation foci per dish and the number of malignant type III foci, and TPA further promoted this phenomenon. When MX was added during the promotion phase in the MC-initiated cells, it promoted the development of the transformation foci in a dose-dependent manner. MX alone (added as an initiator) also slightly increased the development of the foci, including the malignant forms (type II and III), but the effect was not dose-dependent. In contrast to MC-induced foci, TPA did not promote the development of MX-initiated foci, it even decreased their number. The results suggest that MX may also have potential to promote tumor development.     

Potent inhibition of gap junctional intercellular communication by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in BALB/c 3T3 cells

The chlorohydroxyfuranones (CHFs) MX [3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone], MCA [3,4-dichloro-5-hydroxy-2(5H)-furanone], CMCF [3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone], and MCF [3-chloro-4-methyl-5-hydroxy-2(5H)-furanone] are genotoxic disinfection by-products of drinking water chlorination. MX, MCA, and MCF also promote foci formation in the two-stage cell transformation assay. The cellular mechanisms underlying this apparent promotional effect are not known. In the present study, the effects of MX, MCA, CMCF, and MCF on gap junctional intercellular communication (GJIC) were measured in BALB/c 3T3 cells using the scrape loading dye technique. The effect of MX on apoptosis in the same cell line was explored by assaying caspase-3-like protease activity. All the four CHFs inhibited GJIC after 30 min exposure in a dose-dependent fashion but there was a marked difference in the ranges of their active concentrations. MX was almost as potent an inhibitor of GJIC (inhibition at nanomolar concentrations) as 12-O-tetradecanoylphorbol-13-acetate (TPA) (positive control), while MCA was 10 times weaker, CMCF 10,000 times weaker, and MCF 20,000 times weaker than MX. After prolonged exposure periods (up to 6 h), GJIC recovered somewhat upon MX and MCA exposures, the inhibition of GJIC by MCF remained constant but CMCF showed an irreversible increasing inhibitory effect. MX caused apoptosis as a "window" effect at concentrations 2000-4000-fold higher than those needed to inhibit GJIC. The results indicate that MX is a potent inhibitor of GJIC in BALB/c 3T3 cells and this inhibition might be one mechanism by which MX can promote malignant foci formation. MCA also has a specific potential to inhibit GJIC whereas MCF and CMCF affected GJIC at concentrations, similar to those evoking genotoxicity in vitro.     

Toxic effects and excretion in urine of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in the rat after a single oral dose

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. Data also indicate that after an oral dose only traces of MX are excreted in urine as intact compound, suggesting that MX is subjected to intense metabolism before excretion, even at lethal doses. Received: 14 December 1993/Accepted: 28 February 1994     

Assessment of the genotoxicity of the rat carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in rat liver epithelial cells in vitro.

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a disinfection by-product in chlorinated drinking water, is a multisite carcinogen in rats. One main target organ is the liver. The mechanism of the tumorigenicity was evaluated by testing the genotoxicity of MX in rat liver epithelial cell line cells. In the studies, the single cell gel/Comet assay and the hypoxanthine phosphoribosyl transferase locus assay to 6-thioguanine resistance were used. MX induced a dose-related genotoxic response in the comet assay. The lowest effective concentration was 120 microM when the exposure was in medium plus supplements and 3.75 microM when the exposure was in phosphate-buffered salt solution. MX also increased the frequency of TG(r) mutants, when the cells were treated in phosphate-buffered salt solution, at a concentration range of 2.3-9.2 microM. The present results show for the first time that MX causes DNA damage and gene mutations in rat liver epithelial cells, the target cells of MX's tumorigenicity in rats. We have earlier shown that MX also inhibits gap junctional intercellular communication in the same cells. The genotoxic effects were induced starting at about 60 times higher concentration, in identical exposure conditions, compared with the lowest concentration of MX causing the tumor promoter effect.     

Identification of adducts derived from reactions of (1-chloroethenyl)oxirane with nucleosides and calf thymus DNA

(1-Chloroethenyl)oxirane is a major mutagenic metabolite of chloroprene, an important large-scale petrochemical used in the manufacture of synthetic rubbers. The reactions of (1-chloroethenyl)oxirane with 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, thymidine, and calf thymus DNA have been studied in aqueous buffered solutions. The adducts from the nucleosides were isolated by reversed-phase HPLC, and characterized by their UV absorbance and (1)H and (13)C NMR spectroscopic and mass spectrometric features. The reaction with 2'-deoxyguanosine gave one major adduct, N7-(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGI), and eight minor adducts which were identified as diastereoisomeric pairs of N1-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyguanosine (dGII, dGIII), N3,N7-bis(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGIV, dGV), N7,N9-bis(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGVI, dGVII), and N1,N7-bis(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGVIII, dGIX). The reaction of 2'-deoxyadenosine with (1-chloroethenyl)oxirane gave two adducts: N1-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (dAI) and N(6)-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (dAII). The adduct dAII was shown to arise via a Dimroth rearrangement of adduct dAI. The HPLC analyses of the reaction mixtures of (1-chloroethenyl)oxirane with 2'-deoxycytidine and thymidine showed the formation of one major product in each reaction. The adduct from 2'-deoxycytidine was identified as N3-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyuridine (dCI) derived by alkylation at N-3 followed by deamination. The adduct from thymidine was identified as N3-(3-chloro-2-hydroxy-3-buten-1-yl)-thymidine (TI). Reaction of (1-chloroethenyl)oxirane with calf thymus DNA gave all of the adducts observed from the individual nucleosides except dGII and dGIII. However, there was selectivity for the formation of dGI and dCI. The adduct levels in DNA were 9,630 (dGI), 240 (dCI), 83 (dAI), 6 (dAII), and 28 (TI) pmol/mg DNA, respectively. The preferred formation of dCI may be relevant to chloroprene mutagenesis.     

Disposition of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5h)-furanone (mx) in b6c3f1 mice and f344 rats

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a mutagenic by-product of chlorination of drinking water, particularly where the water contains humic matter. MX has been estimated to account for 50% of the mutagenic activity in some drinking water. A bioassay in rats demonstrated an increased tumor incidence, primarily in liver and thyroid glands. This study was designed to provide disposition/metabolism information in mice to evaluate the necessity of a National Toxicology Program chronic bioassay and to provide data for female rats. Radioactivity was rapidly absorbed and excreted near equally in urine (42-54%) and feces (40-51%) 72 h following oral administration of (14)C-labeled MX at single doses from 0.2 to 20 mg/kg to male and female mice and female rats. A larger percentage (71-73%) of MX-derived radioactivity was excreted in urine after an iv dose (0.2 mg/kg) in both female rats and male mice. Most MX-derived radioactivity was excreted within the first 24 h postdosing. MX was transformed to urinary and biliary metabolites. A major extremely polar urinary metabolite was tentatively identified as 1-hydroxy-1,2,2-ethanetricarboxylic acid. This metabolite is likely transformed from the MX degradation product 2-hydroxy-3-formyl-4-oxo-2-butenoic acid. Oral administration produced highest tissue/blood ratios in the following order: forestomach (>100), glandular stomach, intestine, and kidney. Intravenous administration resulted in high, prolonged levels of radioactivity in blood compared to oral dosing. Therefore, MX disposition appears to be dominated by its chemical reactivity with highest concentrations of radioactivity being found at the site of administration.     

Urine mutagenicity and biochemical effects of the drinking water mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), following repeated oral administration to mice and rats

Mutagenicity analysis of urine from rats treated by oral gavage with MX at a dose of 64 mg/kg for 14 days revealed that only 0.3% of the administered compound was excreted in a genotoxically active form. At lower doses, mutagenicity was not detectable. No evidence of micronucleus induction in peripheral blood erythrocytes was observed in mice treated similarly. These findings indicate that MX is extensively detoxified in vivo and is unlikely to cause genetic damage in systemic tissues except at relatively high doses where detoxification pathways become saturated. In a separate experiment, significant depressions were observed in -glucaric acid and thioether excretion and in levels of several liver enzymes involved in xenobiotic metabolism. The mechanism for these metabolic alterations and their relevance to the in vivo metabolism of the compound require further investigation.     

Identification of O2-substituted pyrimidine adducts formed in reactions of 4-(acetoxymethylnitrosamino)- 1-(3-pyridyl)-1-butanone and 4-(acetoxymethylnitros- amino)-1-(3-pyridyl)-1-butanol with DNA

Metabolic hydroxylation of the methyl group of the tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) results in the formation of intermediates that can alkylate DNA. Similarly, metabolic hydroxylation of the 2'-position of the tobacco specific carcinogen N'-nitrosonornicotine gives DNA alkylating intermediates. The resulting pyridyloxobutyl and pyridylhydroxybutyl adducts with dGuo have been characterized, but there are no reports of pyrimidine adducts. Therefore, in this study, we investigated the reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH(2)OAc) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (NNALCH(2)OAc) with DNA, dCyd, and dThd. NNKCH(2)OAc and NNALCH(2)OAc are stable precursors to the products formed upon metabolic methyl hydroxylation of NNK and NNAL. Analysis by LC-ESI-SIM of enzyme hydrolysates of DNA that had been allowed to react with NNKCH(2)OAc and NNALCH(2)OAc demonstrated the presence of major adducts with dCyd and dThd. The dCyd adducts were thermally unstable, releasing 4-HPB (18) or 4-hydroxy-1-(3-pyridyl)-1-butanol (25) upon treatment at 100 degrees C, pH 7.0. The dThd adducts were stable under these conditions. The dCyd adduct of NNALCH(2)OAc was characterized by its MS and UV and by conversion upon neutral thermal hydrolysis to the corresponding Cyt adduct, which was identified by MS, UV, and NMR. The dCyd and Cyt adducts of NNKCH(2)OAc were similarly characterized. The dThd adduct of NNKCH(2)OAc was identified by MS, UV, and NMR. Treatment of this adduct with NaBH(4) gave material, which was identical to that produced upon reaction of NNALCH(2)OAc with DNA or dThd. These data demonstrate that the major pyrimidine adducts formed in the reactions of NNKCH(2)OAc with DNA are O(2)[4-(3-pyridyl)-4-oxobut-1-yl]dCyd (26) and O(2)[4-(3-pyridyl)-4-oxobut-1-yl]dThd (30) while those produced from NNALCH(2)OAc are O(2)[4-(3-pyridyl)-4-hydroxybut-1-yl]dCyd (28) andO(2)[4-(3-pyridyl)-4-hydroxybut-1-yl]dThd (31). Levels of these pyrimidine adducts of NNKCH(2)OAc in DNA were substantially greater than those of the dGuo adducts of NNKCH(2)OAc, based on MS peak area. Furthermore, 26 was identified as a major 4-HPB releasing adduct of NNKCH(2)OAc. These results suggest that pyrimidine adducts of tobacco specific nitrosamines may be important contributors to their mutagenic and carcinogenic activity.     

MX [3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone], a drinking-water carcinogen, does not induce mutations in the liver of cII transgenic medaka (Oryzias latipes)

Mutagenicity assays with Salmonella have shown that 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), a drinking-water disinfection by-product, is a potent mutagen, accounting for about one-third of the mutagenic potency/potential of chlorinated drinking water. The ability of MX to induce mutations was investigated in the liver of medaka (Oryzias latipes), a small fish model, utilizing the cII transgenic medaka strain that allows detection of in vivo mutations. Methylazoxymethanol acetate (MAMAc), a carcinogen in medaka, served as a positive control. Fish were exposed to MX at 0, 1, 10, or 30 mg/L for 96 h, whereas the MAMAc exposures were for 2 h at 0, 0.1, 1, or 10 mg/L. Both exposures were conducted under static water conditions and with fasted medaka. Following exposure, fish were returned to regular culture conditions to allow mutation expression for 15 or 40 d for MX or for 15 or 32 d for MAMAc. Mutations were not induced in medaka exposed to MX for 96 h. However, a concentration- and time-dependent increase in mutations was observed from the livers of fish exposed to 1 and 10 mg/L MAMAc. In conclusion, mutation induction was not observed in the livers of cII medaka exposed to MX for 96 h; however, studies are planned to examine mutation induction in the gills and skin to explore the possibility that MX-induced DNA damage occurs primarily in the tissues of initial contact.     

Absorption of 3-chloro-4-(dichloromethyl)-5-hydroxy-2-[5H] furanone (MX) through rat small intestine in vitro

The intestinal absorption of 3-chloro-4-(dichloromethyl)-5-hydroxy-2-[5H] furanone (MX), a highly mutagenic furanone found in chlorinated waters, was studied using an in vitro everted rat gut sac system, using reverse mutation in Salmonella typhimurium to detect mutagens transported from the mucosal to the serosal compartments. Absorption was measurable, but limited, with significant increase in bacterial revertants (serosal compartment) noted at a dose of 50 μg/ml MX (mucosal compartment, p < 0.05). Gut sac incubation with MX and glutathione (GSH, 1.0 mM) resulted in no detectable absorption of mutagens. Preincubation with diethylmaleate to deplete mucosal GSH resulted in increased absorption of MX-derived mutagens compared to controls (a significant induction of revertant colonies was noted at a dose of 25μg/ml MX p < 0.05). Gut sac incubation with chlorinated fulvic acids resulted in no detectable absorption of mutagens. In vitro studies to assess the possibility of β-lyase activation of the postulated MX-GSH conjugate showed no mutagenic activation.     

Altered enzyme activities of xenobiotic biotransformation in kidneys after subchronic administration of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) to rats

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.     

Formation of acrolein adducts with 2'-deoxyadenosine in calf thymus DNA

Acrolein is a ubiquitous environmental contaminant that has been found to be mutagenic in prokaryotic and eukaryotic cells. In the present study, we examined the reactions of acrolein with 2'-deoxyadenosine and calf thymus single- and double-stranded DNA in aqueous buffered solutions at physiological conditions. The deoxynucleoside adducts were isolated by reversed-phase liquid chromatography, and their structures were determined by their UV absorbance, mass spectrometry, and 1H and 13C NMR spectroscopy. The reaction of 2'-deoxyadenosine with acrolein resulted in the formation of four structurally different adducts (dAI, dAII, dAIII, dAIV). The structures of the novel acrolein adducts, dAIII and dAIV, were assigned as 3-[N(6)-(2'-deoxyadenosinyl)]propanal (dAIII) and 9-(2'-deoxyribosyl-6-(3-formyl-1,2,5,6-tetrahydropyridyl)purine (dAIV), respectively. The adduct dAIII was found to arise via a Dimroth rearrangement of adduct dAI, while the adduct dAIV was shown to be formed upon further reaction of acrolein with dAIII. In the reaction of acrolein with calf thymus DNA, all studied 2'-deoxyadenosine-acrolein adducts were observed. For the first time, it is shown that the N(6)-adduct and the adducts which are derived from two acrolein units are formed in calf thymus DNA.     

Effect on mutagenicity of the stepwise removal of hydroxyl group and chlorine atoms from 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone: 13C NMR chemical shifts as determinants of mutagenicity

The response of mutagenicity to the stepwise replacement of chlorine atoms and the hydroxyl group by hydrogen in 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX, 1) was determined in several assays by using Salmonella typhimurium tester strain (TA100). In all, eight MX derivatives were assayed. Several were studied together in at least one assay. In addition to MX, the seven included 3-chloro-4-(dichloromethyl)-2(5H)-furanone (RMX, 2), 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (3), 3-chloro-4-(chloromethyl)-2(5H)-furanone (4), 4-(chloromethyl)-5-hydroxy-2(5H)-furanone (5), 4-chloromethyl-2(5H)-furanone (6), and 4-(dichloromethyl)-2(5H)-furanone (8). Compounds 1-6 were mutagenic. Compound 8 gave erratic results. 4-(Acetoxymethyl)-2(5H)-furanone (11) was nonmutagenic. The largest drop in mutagenicity amounted to a factor of about 10(2) for the replacement of the hydroxyl group or a C-3 chlorine atom from 3. Other replacements of the hydroxyl group or a C-3 or C-6 chlorine atom amounted to mutagenicity diminished by a factor of only 10. On the basis of the rates of UV spectral changes under assay conditions, chemical half-life values (ct 1/2) for 1-6 and 8 were estimated as indicators of compound stability. However, mutagenicity differences were shown to result principally from the intrinsic mutagenicities of the six compounds 1-6 rather than from differences in stability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetics in Rat of 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a Drinking Water Mutagen,after a Single Dose

Abstract: 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 (T_1/2 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 T_1/2 k₁₀, was much longer, 22.9 hr, and the total elimination half-life (T_1/2 β), 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.