Effect of fuel zinc content on toxicological responses of particulate matter from pellet combustion in vitro.

Significant amounts of transition metals such as zinc, cadmium and copper can become enriched in the fine particle fraction during biomass combustion with Zn being one of the most abundant transition metals in wood combustion. These metals may have an important role in the toxicological properties of particulate matter (PM). Indeed, many epidemiological studies have found associations between mortality and PM Zn content. The role of Zn toxicity on combustion PM was investigated. Pellets enriched with 170, 480 and 2300 mg Zn/kg of fuel were manufactured. Emission samples were generated using a pellet boiler and the four types of PM samples; native, Zn-low, Zn-medium and Zn-high were collected with an impactor from diluted flue gas. The RAW 264.7 macrophage cell line was exposed for 24h to different doses (15, 50,150 and 300 µg ml(-1)) of the emission samples to investigate their ability to cause cytotoxicity, to generate reactive oxygen species (ROS), to altering the cell cycle and to trigger genotoxicity as well as to promote inflammation. Zn enriched pellets combusted in a pellet boiler produced emission PM containing ZnO. Even the Zn-low sample caused extensive cell cycle arrest and there was massive cell death of RAW 264.7 macrophages at the two highest PM doses. Moreover, only the Zn-enriched emission samples induced a dose dependent ROS response in the exposed cells. Inflammatory responses were at a low level but macrophage inflammatory protein 2 reached a statistically significant level after exposure of RAW 264.7 macrophages to ZnO containing emission particles. ZnO content of the samples was associated with significant toxicity in almost all measured endpoints. Thus, ZnO may be a key component producing toxicological responses in the PM emissions from efficient wood combustion. Zn as well as the other transition metals, may contribute a significant amount to the ROS responses evoked by ambient PM.

Safety evaluation of food contact paper and board using chemical tests and in vitro bioassays: role of known and unknown substances.

In vitro toxicological tests have been proposed as an approach to complement the chemical safety assessment of food contact materials, particularly those with a complex or unknown chemical composition such as paper and board. Among the concerns raised regarding the applicability of in vitro tests are the effects of interference of the extractables on the outcome of the cytotoxicity and genotoxicity tests applied and the role of known compounds present in chemically complex materials, such as paper and board, either as constituents or contaminants. To answer these questions, a series of experiments were performed to assess the role of natural substances (wood extracts, resin acids), some additives (diisopropylnaphthalene, phthalates, acrylamide, fluorescent whitening agents) and contaminants (2,4-diaminotoluene, benzo[a]pyrene) in the toxicological profile of paper and board. These substances were individually tested or used to spike actual paper and board extracts. The toxic concentrations of diisopropylnaphthalenes and phthalates were compared with those actually detected in paper and board extracts showing conspicuous toxicity. According to the results of the spiking experiments, the extracts did not affect the toxicity of tested chemicals nor was there any significant metabolic interference in the cases where two compounds were used in tests involving xenobiotic metabolism by the target cells. While the identified substances apparently have a role in the cytotoxicity of some of the project samples, their presence does not explain the total toxicological profile of the extracts. In conclusion, in vitro toxicological testing can have a role in the safety assessment of chemically complex materials in detecting potentially harmful activities not predictable by chemical analysis alone.

Test procedures for obtaining representative extracts suitable for reliable in vitro toxicity assessment of paper and board intended for food contact.

This paper describes the use of a suite of extraction procedures applicable to the assessment of the in vitro toxicity of paper/board samples intended for food-contact applications. The sample is extracted with ethanol, water, or exposed to modified polyphenylene oxide (Tenax) for fatty, non-fatty and dry food applications, respectively. The water extracts are directly suitable for safety assessment using in vitro bioassays. The ethanol extracts of the paper/board and of the exposed Tenax require pre-concentration to give acceptable sensitivity. This is because the in vitro bioassays can tolerate only a small percentage of added organic solvent before the solvent itself inhibits. The extraction procedures have been selected such that they mimic the foreseeable conditions of use with foods and that they are also fully compatible with the battery of in vitro biological assays for the safety assessment of the total migrate. The application of the extraction protocols is illustrated by the results for one of the many paper/board samples provided by the BIOSAFEPAPER project industrial platform members. The assessment indicated that this sample should not be considered as suitable for use with fatty foodstuffs but was suitable for dry and non-fatty foods. Information subsequently received from the manufacturer revealed that this was a non-food-grade product included in the project to test the capabilities of the bioassay procedures. The selection criteria for the test conditions and the suite of methods developed have been prepared in Comité Européen de Normalisation (CEN) format and is currently being progressed by CEN/TC172 as a European Standard.

The BIOSAFEPAPER project for in vitro toxicity assessments: preparation, detailed chemical characterisation and testing of extracts from paper and board samples.

Nineteen food contact papers and boards and one non-food contact board were extracted following test protocols developed within European Union funded project BIOSAFEPAPER. The extraction media were either hot or cold water, 95% ethanol or Tenax, according to the end use of the sample. The extractable dry matter content of the samples varied from 1200 to 11,800 mg/kg (0.8-35.5 mg/dm2). According to GC-MS the main substances extracted into water were pulp-derived natural products such as fatty acids, resin acids, natural wood sterols and alkanols. Substances extracted into ethanol particularly, were diisopropylnaphthalenes, alkanes and phthalic acid esters. The non-food contact board showed the greatest number and highest concentrations of GC-MS detectable compounds. The extracts were subjected to a battery of in vitro toxicity tests measuring both acute and sublethal cytotoxicity and genotoxic effects. None of the water or Tenax extracts was positive in cytotoxicity or genotoxicity assays. The ethanol extract of the non-food contact board gave a positive response in the genotoxicity assays, and all four ethanol extracts gave positive response(s) in the cytotoxicity assays to some extent. These responses could not be pinpointed to any specific compound, although there appeared a correlation between the total amount of extractables and toxicity.

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.

Investigation of co-genotoxic effects of radiofrequency electromagnetic fields in vivo.

We investigated the possible combined genotoxic effects of radiofrequency (RF) electromagnetic fields (900 MHz, amplitude modulated at 217 Hz, mobile phone signal) with the drinking water mutagen and carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Female rats were exposed to RF fields for a period of 2 years for 2 h per day, 5 days per week at average whole-body specific absorption rates of 0.3 or 0.9 W/kg. MX was given in the drinking water at a concentration of 19 microg/ml. Blood samples were taken at 3, 6 and 24 months of exposure and brain and liver samples were taken at the end of the study (24 months). DNA damage was assessed in all samples using the alkaline comet assay, and micronuclei were determined in erythrocytes. We did not find significant genotoxic activity of MX in blood and liver cells. However, MX induced DNA damage in rat brain. Co-exposures to MX and RF radiation did not significantly increase the response of blood, liver and brain cells compared to MX exposure only. In conclusion, this 2-year animal study involving long-term exposures to RF radiation and MX did not provide any evidence for enhanced genotoxicity in rats exposed to RF radiation.

Bromide affecting drinking water mutagenicity.

The effect of bromide on the mutagenicity of artificially recharged groundwater and purified artificially recharged groundwater after chlorine, ozone, hydrogen peroxide, permanganate, and UV treatments alone and in various combinations was studied. The highest mutagenicity was observed after chlorination, while hydrogen peroxide-ozone-chlorine treatment produced the lowest value for both waters. Chlorinated waters, which were spiked with bromide, had up to 3.7 times more mutagenic activity than waters without bromide after every preoxidation method. 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) was found to correspond as much as 76% of the overall mutagenicity in the waters not spiked with bromide. MX formation was found to be lower when the treated water contained bromide, implicating the formation of brominated MX analogues. Trihalomethane formation increased when the treated water contained bromide.

Systemic immunoresponses in mice after repeated exposure of lungs to spores of Streptomyces californicus.

Microbial growth in moisture-damaged buildings is associated with respiratory and other symptoms in the occupants. Streptomyces spp. are frequently isolated from such buildings. In the present study, we evaluated the responses of mice after repeated exposure to spores of Streptomyces californicus. Mice were exposed via intratracheal instillation to six doses (at 7-day intervals) of the spores of S. californicus, originally isolated from the indoor air of a moisture-damaged building, at three dose levels (2 x 10(3), 2 x 10(5), and 2 x 10(7) spores). Inflammation and toxicity, including changes in cell populations in the lungs, lymph nodes, and spleen, were evaluated 24 h after the last dosage. The exposure provoked a dose-dependent inflammatory cell response, as detected by the intense recruitment of neutrophils, but the numbers of macrophages and lymphocytes in the airways also increased. The cellular responses corresponded to the dose-dependent increases in inflammation- and cytotoxicity-associated biochemical markers (i.e., levels of albumin, total protein, and lactate dehydrogenase) in bronchoalveolar lavage fluid. The spore exposure increased the number of both activated and nonactivated T lymphocytes. Also, the amounts of CD3(-) CD4(-) and unconventional CD3(-) CD4(+) lymphocytes in the lung tissue were augmented. Interestingly, the spore exposure decreased cells in the spleen. This effect was strongest at the dose of 2 x 10(5) spores. These results indicate that the spores of S. californicus are capable of provoking both immunostimulation in lungs (inflammation) and systemic immunotoxicity, especially in the spleen. The immunotoxic effect resembled that caused by chemotherapeutic agents, originally isolated from Streptomyces spp. Thus, S. californicus must be considered a microbial species with potential to cause systemic adverse health effects in occupants of moisture-damaged buildings.

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.

Comparable DNA and chromosome damage in Chinese hamster ovary cells by chlorohydroxyfuranones.

Chlorinated drinking water contains several chlorohydroxyfuranone (CHF) by-products whose contribution to cancer risk is not presently known. 3,4-Dichloro-5-hydroxy-2(5H)-furanone (MCA), 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF), and 3- chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) were studied for the induction of DNA damage, using the alkaline single-cell gel (SCG)/comet assay, and for chromosome damage, using sister-chromatid exchange (SCE) and chromosome aberration (CA) tests, in Chinese hamster ovary (CHO) cells. 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), the known genotoxic chlorination by-product and a rat carcinogen, was used as a reference chemical. The SCG analyses were done using concentrations that caused little or no cytotoxicity compared to that of the concurrent control cultures. In the cytogenetic tests, the CHFs were tested up to maximum cytotoxicity. MX and MCA were the most cytotoxic of the compounds in CHO cells followed by CMCF and MCF. All of the CHFs induced DNA damage, SCEs and CAs (mainly chromatid-type breaks and exchanges) in a concentration-related manner, with the exception that MCA was a weak inducer of SCEs. There were no significant differences between the lowest concentration of MX, MCA, and CMCF to cause DNA damage (SCG assay). Based on comparisons of the slopes of regression lines, MX was somewhat more potent than either MCA or CMCF, and MCF was clearly less potent than the other three compounds in the assay. The order of potency was MX > CMCF > MCA > MCF in inducing SCEs and MX > MCA > CMCF > MCF in inducing CAs. The data show that there are differences in the potency of genotoxicity among the CHFs tested. In many cases, however, the extent of maximum effect observed was comparable between the compounds. The results suggest that besides MX other CHFs should be considered in the evaluation of genotoxic risks associated with the consumption of chlorinated drinking water.

No consistent pattern of mutations in p53 and ras genes in liver tumors of rat treated with the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX).

The frequency of point mutations in p53 (exons 4-7) and in Ki-ras, Ha-ras, and N-ras (exons 1 and 2) and the expression of p53 protein were evaluated in the liver tumors of Wistar rats of a 104-week carcinogenicity study on 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a chlorine disinfection by-product in drinking water. Mutations were analyzed in 16 hepatocellular adenomas, 7 hepatocellular carcinomas, 23 cholangiomas, and 2 cholangiocarcinomas of the MX-treated animals and one hepatocellular carcinoma and cholangiocarcinoma in control animals using PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) or PCR-TGGE (temperature gradient gel electrophoresis) and direct sequencing. The expression of the p53 protein (wild-type and mutated protein) was detected by immunohistochemistry (CM5 antibody). The expression of p53 and that of the proliferating cell nuclear antigen (PCNA, 19 A2) were also evaluated in livers of female animals exposed to MX for 1 week, 3 weeks, or 18 weeks. Altogether, four mutations were found in p53 in three tumors, in two hepatocellular adenomas, and one cholangiocarcinoma, all in females receiving the highest MX dose (6. 6 mg/kg/day) of the study. Three of the mutations were G:C --> A:T transitions and one was an A:T --> T:A transversion. The mutations were scattered at different codons and positions of the codon. One hepatocellular adenoma contained two p53 mutations. All cholangiomas and cholangiocarcinomas, but no hepatocellular adenomas and carcinomas, overexpressed the p53 protein. MX treatment did not induce p53 expression at any age in the liver or alter the expression of the PCNA in the liver of younger animals. The p53 protein was overexpressed in hyperplastic bile ducts in aged rats but not in bile ducts of younger rats (up to 24 weeks). No mutations were observed in either Ki-ras, Ha-ras, or N-ras of the liver tumors. These data suggest that point mutations in p53, Ki-ras, Ha-ras, and N-ras are not involved in the MX-induced liver carcinogenesis in rats.

Liver tumor-promoting activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in TCDD-sensitive and TCDD-resistant rat strains.

Risk assessment of dioxins is currently based on induction of liver tumors in rats. The toxicity of dioxins is characterized by large sensitivity differences among animal species and even strains of the same species, which complicates the risk assessment. The significance of these differences in dioxin-induced carcinogenicity is not known. We therefore studied the liver tumor-promoting activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the sensitive Long-Evans (L-E) and the resistant Han/Wistar (H/W) rats differing >1000-fold in their sensitivity to the acute lethaity of TCDD. Female rats were partially hepatectomized, initiated with nitrosodiethylamine, and treated with TCDD for 20 weeks. Altered hepatic foci (AHF) were stereologically quantitated using glutathione S-transferase P as a marker. AHF were significantly (P < 0.001) and dose dependently increased in L-E rats at 10 and 100 ng/kg/day, but in H/W rats only at 1000 ng/kg/day and above, indicating a remarkable (approximately 100-fold) sensitivity difference between L-E and H/W rats. The same sensitivity difference but 10-fold less foci were observed between nonhepatectomized/noninitiated L-E and H/W rats. Induction of AHF was related to hepatotoxicity but not to cytochrome P4501A1 activity in the liver. Liver TCDD concentrations were similar in both strains. H/W rats are exceptionally resistant to induction of AHF by TCDD, and the resistance is associated with an altered transactivation domain of the aryl hydrocarbon receptor. Genetic differences may account for significant interindividual/intraspecies sensitivity differences in dioxin-induced carcinogenesis. Understanding the role of transactivation domain of the aryl hydrocarbon receptor in carcinogenesis is therefore likely to improve dioxin risk assessment.

Effects of gestational exposure to a video display terminal-like magnetic field (20-kHz) on CBA/S mice.

Possible adverse effects of magnetic fields (MFs) on reproduction have been an open question. To verify the embryo-lethal effect of pulsed MF of the type emitted by video display terminals (VDTs) reported previously in CBA/S mice, a developmental toxicity study was conducted in animals of the same origin. Mated CBA/S mice (80-86 pregnant animals per group) were exposed to a 20-kHz MF with sawtooth waveform continuously from gestational day 0-18. The flux density of the vertical MF was 15 microT peak-to-peak (150 mG). This field was previously reported to increase the number of resorptions in CBA/S mice. On gestational day 18, the dams were killed and blood and bone marrow samples were taken for hematology and micronuclei analysis, respectively. The number of corpora lutea was counted and the content of the uterus examined. There were no statistically significant differences in maternal or fetal body weights, number of corpora lutea, implantations, resorptions, dead and live fetuses, or external and skeletal malformations. MF did not alter the number of blood cells or cause micronuclei in bone marrow erythrocytes in the dams. The mean number of resorptions was slightly but not statistically significantly, higher in the MF group than in controls. The results do not indicate marked developmental, hematological, or clastogenic effects of 20-kHz MFs.

Effects of low-frequency magnetic fields on fetal development in CBA/Ca mice.

Effects of alternating magnetic fields (MFs) on the embryonic and fetal development in CBA/Ca mice were studied. Mated females were exposed continuously to a sinusoidal 50 Hz (13 microT or 0.13 mT root mean square) or a sawtooth 20 kHz (15 microT peak-to-peak) MF from day 0 to day 18 of pregnancy for 24 h/day until necropsied on day 18. Control animals were kept under the same conditions without the MF. MFs did not cause maternal toxicity. No adverse effects were seen in maternal hematology and the frequency of micronuclei in maternal bone marrow erythrocytes did not change. The MFs did not increase the number of resorptions or fetuses with major or minor malformations in any exposure group. The mean number of implantations and living fetuses per litter were similar in all groups. The corrected weight gain (weight gain without uterine content) of dams, pregnancy rates, incidences of resorptions and late fetal deaths, and fetal body weights were similar in all groups. There was, however, a statistically significant increase in the incidence of fetuses with at least three skeletal variations in all groups exposed to MFs. In conclusion, the 50 Hz or 20 kHz MFs did not increase incidences of malformations or resorptions in CBA/Ca mice, but increased skeletal variations consistently in all exposure groups.

Association between the clastogenic effect in peripheral lymphocytes and human exposure to arsenic through drinking water.

We describe the association between structural chromosome aberrations (CAs) and parameters of exposure to arsenic among 42 individuals exposed to arsenic through well waters in Finland. The median concentration of arsenic in the wells was 410 microg/l, the total arsenic concentrations in urine (As-tot) was 180 microg/l, and in hair 1.3 microg/g, for current users (n = 32) of contaminated wells. Urinary arsenic species and CAs were also analyzed in eight control individuals from the same village who consumed water which contained arsenic <1.0 microg/l (detection limit). Increased arsenic exposure, indicated best by increased concentrations of arsenic species (inorganic arsenic, methylarsonic acid (MMA), dimethylarsinic acid (DMA)) in urine, was associated with increased frequency of CAs. The increased urinary ratio of MMA/As-tot and the decreased ratio of DMA/As-tot were associated with increased CAs when all aberration types, including gaps, were considered. Associations between CAs and arsenic exposure indicators were stronger among current users than among persons who had stopped using the contaminated well water for 2-4 months before sampling (ex-users, n = 10). Furthermore, there was a positive but not statistically significant association between CAs and arsenic in hair among the current users, but not among the ex-users, who still had relatively high arsenic concentrations in hair. The results suggest that the effect observed in the present study reflects relatively recent arsenic exposure.

Genotoxicity of dihydroabikoviromycin, a secondary metabolite of Streptomyces anulatus.

The potential genotoxicity of dihydroabikoviromycin was assessed in bacterial and sister-chromatid exchange (SCE) test systems. Direct cytotoxicity was also assayed using bioluminescence methods to screen for differences in cell viability among different tumour cell lines following exposure to the drug. Differential killing tests with Escherichia coli WP2 and its repair-deficient derivative CM871 indicated that a functional DNA repair system was protective against the action of dihydroabikoviromycin, implying that this compound causes some form of DNA damage and is almost certainly therefore genotoxic. Dose-dependent reversion from His- to His+ with dihydroabikoviromycin was observed in the Ames test with Salmonella typhimurium TA100, but not in frameshift tester strain TA98. Dihydroabikoviromycin also induced the sfiA gene, as indicated by beta-galactosidase induction in an SOS-chromotest with E. coli PQ37 strain. A dose-related increase in SCEs by dihydroabikoviromycin was observed in CHO cells. Growth of tumour cells was also suppressed by dihydroabikoviromycin at a dose of 10 micrograms/ml.

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 md/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.

Analysis by fluorescence in situ hybridization with a mouse gamma satellite DNA probe of isolated micronuclei induced in mice by two clastogens and two spindle poisons.

The presence of centromeric DNA was studied in micronuclei isolated from the blood of male ddY mice after five weekly intraperitoneal injections of mitomycin C (MMC), 1-beta-D-arabinofuranosylcytosine (Ara-C), colchicine (COL) or vinblastine sulfate (VBL). In agreement with our earlier findings, about half of the micronuclei isolated from vehicle control mice showed centromere signals as analyzed by fluorescence in situ hybridization (FISH) with a mouse major (gamma) satellite DNA probe. In an earlier experiment with mice acutely exposed to the same chemicals, the clastogens MMC and Ara-C did not reduce the proportion of micronuclei with centromere signals. In the present study, however, MMC and Ara-C decreased the proportion of micronuclei with centromeres. In contrast, the spindle poisons COL and VBL increased the proportion of micronuclei that contained centromeres.

Cytogenetic effects in the peripheral lymphocytes and kidney cells of rats exposed to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) orally on three consecutive days.

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.

Mutagenicity in vitro of 3,4-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid), a chlorine disinfection by-product in drinking water.

The mutagenicity of chlorinated humic drinking waters is accounted for mainly by a single contaminant, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), as assessed in Salmonella typhimurium strain TA100. In the present study 3,4-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid, MA), another drinking water contaminant much less potent as a mutagen in TA100 than MX, was tested in Chinese hamster ovary (CHO) cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (hprt) locus to 6-thioguanine resistance (TGr). Unexpectedly, MA induced TGr mutants in CHO cells with a potency comparable to that reported previously for MX. In subsequent experiments with S. typhimurium, the presence of pKM101 plasmid in strain TA100 increased susceptibility to the mutagenicity of MA, but much less than to that of MX, relative to the parental strain TA1535 lacking pKM101. The difference between the two compounds in TA100 thus appears to be due to a higher enhancement of the mutagenicity of MX than that of MA by pKM101 mediated error-prone DNA repair.