The modulatory effect of aminothiols on the clastogenic activity of X-rays assayed by the vivo mouse micronucleus test

The modulatory effect of AET (S-2-aminoethylisothio-uronium bromide hydrobromide) and WR-2721 (S-2-/3-ami-nopropylamino/ethylphosphorothioic acid) on the clastogenic activity of X-rays was assessed by the in vivo mouse micronucleus test. The frequency of micronucleated polychromatic erythrocytes (MNPCEs) in the peripheral blood of adult male Swiss mice exposed to 5 Gy X-rays alone, or treated with AET or WR-2721, at a does of 200 mg/kg body weight, 15 or 30 minutes prior to X-irradiation, respectively, was determined during a fifteen-day period. The number of micronuclei increased on day 1 post-irradiation in X-irradiated mice and declined thereafter with the frequency of MNPCEs remaining lower in the thiol pre-treated mice. A more effective protection against the clastogenic activity of X-rays in the erythropoietic system was observed after WR-2721 administration than AET application.     

Hydroquinone increases the frequency of micronuclei in a dose-dependent manner in mouse bone marrow

The frequency of micronuclei (micronucleated polychromatic erythrocytes, MPCE and micronucleated normochromatic erythrocytes, MNCE) was studied at 12, 24 and 36 h post-treatment in the bone marrow of mice treated with 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 75 and 100 mg/kg body wt of hydroquinone (HQ). Treatment of mice with various doses of HQ resulted in a dose dependent increase in the frequency of both MPCE and MNCE at all the post-treatment time periods. The frequency of MPCE was significantly higher after administration of 3.125 mg/kg HQ at 24 h post-treatment, except 12 and 36 h, where a significant increase in the frequency of MPCE was observed only after administration of 6.25 mg/kg drug dose. Similarly, a significant increase in the frequency of MNCE was observed after 12.5 mg/kg HQ treatment at all the post-treatment time periods. The dose effect relationship between various HQ doses and MPCE and MNCE induction was linear and linear quadratic, respectively at all the post-treatment time periods. The PCE/NCE ratio declined in a dose dependent manner at all the post-treatment time periods and this decline was significant when compared to non-drug treated controls. The dose effect relationship was linear quadratic at all the post-treatment time periods studied.     

Effect of various concentrations of lead nitrate on the induction of micronuclei in mouse bone marrow

The frequency of micronuclei was evaluated in the bone marrow of mice of either sex administered with 0, 0.625, 1.25, 2.5, 5, 10, 20, 40 and 80 mg/kg b.wt of lead nitrate at 12, 24 and 36 h post-treatment. The frequency of micronucleated polychromatic erythrocytes (MPCE) and micronucleated normochromatic erythrocytes (MNCE) increased significantly at 12, 24 and 36 h after treatment with lead nitrate compared to non-drug treated controls. The frequency of micronuclei did not show a dose related increase and the elevation in the frequency of micronuclei was fluctuating type. One important observation which emerged from this study was that the male mice were more sensitive to the induction of micronuclei compared to female mice. This was evidenced by higher frequencies of MPCE in males than females at all the doses for all the post-treatment time periods. The lead nitrate treatment resulted in a spurt in the erythropoiesis as is evidenced by a significant increase in the ratios of polychromatic to normochromatic erythrocytes (P/N ratio) compared to non-drug treated controls at 12, 24 and 36 h post-treatment. The P/N ratio was significantly higher in females than males at 12 and 24 h post-treatment.     

Rhodopsin mutations as the cause of retinal degeneration. Classification of degeneration phenotypes in the model system Drosophila melanogaster

The modifying effect of treatment with vitamins C, E and beta-carotene on the clastogenic activity of gamma rays was investigated in mice. Damage in vivo was measured by the micronucleus assay in bone marrow polychromatic erythrocytes and exfoliated bladder cells. The vitamins were administered orally, either for five consecutive days before or immediately after irradiation with 2 Gy of gamma rays. The results show that pretreatment with vitamin E (100-200 mg/kg/day) and beta-carotene (3-12 mg/kg/day) were effective in protecting against micronucleus induction by gamma rays. Vitamin C depending on its concentration enhanced the radiation effect (400 mg/kg/day), or reduced the number of micronucleated polychromatic erythrocytes (50-100 mg/kg/day). Such effect was weekly observed in exfoliated bladder cells. The most effective protection in both tissues was noted when a mixture of these vitamins was used as a pretreatment. Administration of the all antioxidant vitamins to mice immediately after irradiation was also effective in reducing the radiation-induced micronucleus frequency. The data from the in vitro experiments based on the comet assay show that the presence of the vitamins in culture medium influences the kinetic of repair of radiation-induced DNA damage in mouse leukocytes.     

Developmental regulation of the 3-methylcholanthrene- and dioxin-inducible CYP1A5 gene in chick embryo liver in vivo

Measurement of specific adducts to hemoglobin can be used to establish the dosimetry of electrophilic compounds and metabolites in experimental animals and in humans. The purpose of this study was to investigate the dose response for adduct formation and persistence in rats and mice during long-term low-level exposure to butadiene by inhalation. Adducts of 3,4-epoxy-1-butene, the primary metabolite of butadiene, with N-terminal valine in hemoglobin were determined in male B6C3F1 mice and male Sprague-Dawley rats following exposure to 0, 2, 10, or 100 ppm of 1,3-butadiene, 6 h/day, 5 days/week for 1, 2, 3, or 4 weeks. Blood samples were collected from groups of five mice and three rats at the end of each week during the 4 weeks of exposure and weekly for 3 weeks following the end of the 4-week exposure period. The increase and decrease, respectively, of the adduct levels during and following the end of the 4-week exposure followed closely the theoretical curve for adduct accumulation and removal for rats and mice, thereby demonstrating that the adducts are chemically stable in vivo and that the elimination follows the turnover of the red blood cells. The adduct level increased linearly with butadiene exposure concentration in the mice, whereas a deviation from linearity was observed in the rats. For example, after exposure to 100 ppm butadiene, the epoxybutene-hemoglobin adduct levels were about four times higher in mice than in rats; at lower concentrations of butadiene, the species difference was less pronounced. Blood concentrations of epoxybutene, estimated from hemoglobin adduct levels, were in general agreement with reported concentrations in mice and rats exposed by inhalation to 62.5 ppm. These studies show that adducts of epoxybutene with N-terminal valine in hemoglobin can be used to predict blood concentration of epoxybutene in experimental animals.     

Free radical induced respiratory muscle dysfunction

1,3-Butadiene (BD) is carcinogenic and mutagenic in B6C3F1 mice. We determined the lacI mutant frequency and mutational spectrum in spleen following inhalation exposure to BD at levels that are known to induce tumors. B6C3F1 lacI transgenic mice were exposed to air or to 62.5, 625, or 1250 ppm BD for 4 weeks (6 h/day, 5 days/week) and euthanized 14 days after the last exposure. BD increased the lacI mutant frequency in spleen at all levels of BD examined. In BD-exposed mice, an increased frequency of G:C-->A:T transitions occurred at non-5'-CpG-3' sites. Exposure to BD in B6C3F1 lacI transgenic mice also increased the frequency of base substitution mutations that occurred at A:T base pairs when compared to air controls. The increased frequency of specific mutations at G:C base pairs in spleen was not observed in our previous studies in bone marrow and indicates tissue-specific differences in the BD-induced mutational spectrum. These data demonstrate that in vivo transgenic mouse mutagenicity assays can identify tissue-specific mutagenicity and mutational spectrum responses of genotoxic carcinogens at exposure levels that are known to induce tumors.     

Genotoxic effect of triarylmethane dye--acid green 16 after chronic ethanol consumption in mice

Genotoxic effect and hepatic microsomal monooxygenase activities were assessed in mice treated with Acid Green 16 (single i.p. injection at dose 75 mg/kg) superimposed on prolonged ethanol consumption (10% solution in drinking water for 2-4 months). Treatment of mice with Acid Green 16 led to an increased frequency of micronucleated erythrocytes in bone marrow. In animals pretreated with ethanol the frequency of micronucleated erythrocytes, produced by Acid Green 16, was significantly higher. The changes in frequency of micronucleated erythrocytes were accompanied by the enhanced activity of microsomal monooxygenases manifested by higher activity of 7-ethoxycoumarin o-deethylase (the level of cytochrome P-450 was not altered). The obtained results showed that ethanol tended to increase the genotoxic effect of Acid Green 16. However, the slight inductive effect of ethanol on microsomal monooxygenases did not provide clear evidence that the genotoxic effect of Acid Green 16 was associated with ethanol stimulation of the metabolic activation of the dye in the liver.     

Effect of S-methyl cysteine sulphoxide and its metabolite methyl methane thiosulphinate, both occurring naturally in Brassica vegetables, on mouse genotoxicity

S-methyl cysteine sulphoxide (SMCSO) and its metabolite methyl methane thiosulphinate (MMTSO), both naturally occurring compounds present in Brassica vegetables, were investigated for their putative ability to inhibit benzo[a]pyrene (B[a]P)-induced genotoxicity in ICR mice. The mouse bone marrow micronucleus assay was used as an indicator of in vivo genotoxicity. Doses of 0.5 mmol SMCSO and 0.05mmol MMTSO per kg body weight significantly inhibited the formation of B[a]P-induced micronucleated polychromatic erythrocytes (MPCEs) by 31 and 33%, respectively, compared with control mice. Two higher doses of MMTSO (0.5 and 1.0 mmol/kg body weight) administered to mice displayed severe acute toxicity. The inhibition of experimental genotoxicity by these two organosulphur compounds present in Brassica may, in part, be responsible for the anticarcinogenic effect of these vegetables.     

Genotoxicity of Farbasol and its component: 4-ethyltoluene

A combination of assays for gene mutations in Salmonella typhimurium TA97a, TA98, TA100 and TA102 strains with and without rat liver activation, and for micronucleus and sister chromatid exchange (SCE) in bone marrow cells of Imp:Balb/c mice was used to provide data on the mutagenic and genotoxic properties of the mixture of aromatic solvents, known under the trade name of Farbasol. In addition, 4-ethyltoluene (the main ethylmethylbenzenic component of Farbasol) was also tested for muta- and genotoxicity. The results revealed that neither Farbasol nor 4-ethyltoluene induced an increased reverse mutation in bacterial cells or the formation of micronucleated polychromatic erythrocytes in bone marrow. However, those compounds were found to be active as sister chromatid exchange (SCE) agents.     

Effect of ultrasonicated carboxymethylglucan on cyclophosphamide induced mutagenicity

Carboxymethylglucan (CMG) with ultrasonically lowered molecular weight (0.89 x 10(5)) was administered either intraperitoneally, intravenously or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei in mouse bone marrow was evaluated. Both parenteral (intraperitoneal and intravenous) and oral administration of CMG decreased the clastogenic effect of CP. The protective effect induced by intravenous and intraperitoneal administration was concentration-dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg body weight. With the lower dose of CMG a 2-h interval was necessary between intravenous CMG administration and CP injection. Oral pretreatment of mice with CMG decreased statistically significantly the frequency of micronuclei in polychromatic erythrocytes of the bone marrow. The fact that ultrasonically depolymerized CMG was effective also on oral administration is indicative of the passage of smaller CMG molecules through the wall of the gastrointestinal tract.     

Genetic effects of 1,3-butadiene and associated risk for heritable damage

A summary of the results of the studies conducted in the EU Project "Multi-endpoint analysis of genetic damage induced by 1,3-butadiene and its major metabolites in somatic and germ cells of mice, rats and man" is presented. Results of the project are summarized on the detection of DNA and hemoglobin adducts, on the cytotoxic and clastogenic effects in somatic and germinal cells of mice and rats, on the induction of somatic mutations at the hprt locus of experimental rodents and occupationally exposed workers, on the induction of dominant lethal mutations in mice and rats, and on heritable translocations induced in mice, after exposure to butadiene (BD) or its major metabolites, butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). The primary goal of this project was to collect experimental data on the genetic effects of BD in order to estimate the germ cell genetic risk to humans of exposure to BD. To achieve this, the butadiene exposure are based on data for heritable translocations and bone marrow micronuclei induced in mice and chromosome aberrations observed in lymphocytes of exposed workers. A doubling dose for heritable translocations in human germ cells of 4900 ppm/h is estimated, which, assuming cumulative BD exposure over the sensitive period of spermatogenesis, corresponds to 5-6 weeks of continuous exposure at the workplace to 20-25 ppm. Alternatively, the rate of heritable translocation induction per ppm/h of BD exposure is estimated to be approximately 0.8 per million live born, compared to a spontaneous incidence of balanced translocations in humans of approximately 800 per million live born. These estimates have large confidence intervals and are only intended to indicate orders of magnitude of human genetic risk. These risk estimates are based on data from germ cells of BD-exposed male mice. The demonstration that clastogenic damage was induced by DEB in preovulatory oocytes at doses which were not ovotoxic implies that additional studies on the response of mammalian female germ cells to BD and its metabolites are needed. The basic assumption of the above genetic risk estimates is that experimental mouse data obtained after BD exposure can be extrapolated to humans. Several points exist in the present report and in the literature which contradict this assumption: (1) the level of BMO-hemoglobin adducts was significantly elevated in BD-exposed workers; however, it was considerably lower than would have been predicted from comparable rat and mouse exposures; (2) the concentrations of the metabolites DEB and BMO were significantly higher in mouse than in rat blood after BD exposure. Thus, while metabolism of BD is qualitatively similar in the two species, it is quantitatively different; (3) no increase of HPRT mutations was shown in 19 workers exposed on average to 1.8 ppm of BD, while in a different population of workers from a US plant exposed on average to 3.5 ppm of BD, a significant increase of HPRT variants was detected; and (4) data from cancer bioassays and cancer epidemiology suggest that rat is a more appropriate model than mouse for human cancer risk from BD exposure. However, the dominant lethal study in rats gave a negative result. At present, we do not know which BD metabolite(s) may be responsible for the genetic effects even though the bifunctional alkylating agent DEB is the most likely candidate for the induction of clastogenic events. Unfortunately, methods to measure DEB adducts in hemoglobin or DNA are only presently being developed. Despite these several uncertainties the use of the mouse genetic data is regarded as a justifiable and conservative approach to human genetic risk estimation given the considerable heterogeneity observed in the biotransformation of BD in humans.     

Comparison of glycoprotein components, tryptophan, lipid peroxidation and antioxidants in borderline and severe hypertension and myocardial infarction

Between 1957 and 1993, the Siberian Chemical Complex (Tomsk-7) located in the Tomsk region (Russia) regularly discharged radioactive liquid wastes into the Tom River which resulted in an extensive contamination of large territories with long lived radionuclides such as cesium-137 and strontium-90. In the summers of 1996 and 1997, Research Team of Siberian Medical University conducted biodosimetry and cytogenetic monitoring of pikes (Esox lucius) caught in the Tom River at various distances downstream from the Siberian Chemical Complex (SCC) using the micronucleus test and the gamma spectroscopy. Our findings demonstrated that the difference in frequency of micronucleated erythrocytes between the radiation-exposed fish caught downstream from the SCC and the controls was statistically significant (P < 0.01). Moreover, we found a good correlation between radiocesium concentration and micronucleated erythrocyte frequency in pikes. It was found that both the micronucleated erythrocyte frequency in pike blood and the level of the pike radiocesium concentration depended on the age of pikes. The micronucleated erythrocyte frequency gradually increased from the 1-year-old pikes to the over 20-year-old pikes. On the other hand, the average level of radiocesium concentration gradually increased from the 1-year-old pikes only up to the 10-year-old pikes. There is no correlation between radiocesium concentration and micronucleated erythrocyte frequency in the over 10-year-old pikes.     

Cytogenetic investigations on mice treated with lead

Dietary lead at different dose levels was given to female C57B1 mice for periods up to 3 months. No severe chromosome or chromatid aberrations were observed at any dose level and the frequency of chromatid gaps increased significantly only at the highest doses. (0.5 and 1% of lead in the diet). The number of micronuclei in polychromatic erythrocytes also remained at control levels after lead treatment, whereas known mutagenic agent such as Myleran and Mitomycine C raised their incidence markedly. Taking into account also the data in the literature, it is concluded that lead per se probably has no mutagenic action on mammals.     

Increase in neuropsychiatric symptoms after occupational exposure to low levels of styrene

The results of this study suggest that exposure to styrene below the current Swedish permissible exposure limit of 20 ppm induces neurotoxic effects expressed as an increased number of neuropsychiatric symptoms. Twenty men exposed to styrene at a plastics factory participated. The reference group included 20 non-exposed men matched for age, working schedule, and physical work load. Exposure to styrene during one workday was assessed by personal air monitoring and biological monitoring. To evaluate the physical work load the pulse(heart) rate was measured. One week before the study each man completed a neuropsychiatric symptom questionnaire containing 16 items. Also 17 questions regarding acute symptoms of local irritation and symptoms of the central nervous system were presented after the psychometric tests were performed. The tests were simple reaction time, colour word vigilance, and symbol digit. A follow up with regard to the symptoms among the exposed men was done after their summer vacation, about two to five weeks after their last exposure. The mean eight hour time weighted average (TWA) concentration of styrene in air, measured by passive dosimetry was 8.6 ppm (range 0.04-50.4 ppm). The exposed men had significantly more symptoms than the referents although there were no significant differences for the psychometric tests. At the follow up the exposed men reported fewer symptoms. This study indicates that symptoms are earlier indicators of adverse effects than complex tests and underlines the importance of regular follow up of people exposed to styrene (and probably organic solvents in general).     

Retrograde migration of ureteral calculi

Aspergillus parasiticus NRRL-2999 was inoculated into meat mixtures with curing salts and into yeast extractsucrose (YES) and sucrose-ammonium salts (SAS) broth with and without curing salts to determine if the presence of curing salts significantly affected growth and aflatoxin production by the mold. The effect of individual curing salts or curing salt mixtures on growth and toxin elaboration by the aspergillus was substrate dependent. When YES broth contained 100 ppm of NaNO2, 2% NaCl, or 1 or 2% NaCl plus 200 ppm of NaNO2 or 200 ppm of NaNO3, growth and/or aflatoxin production was depressed. Biosynthesis of aflatoxin B1 was enhanced by presence of 1 and 4% NaCl in YES broth. The SAS broth containing only NaCl or NaCl combined with nitrite or nitrate yielded less aflatoxin than did control broth or no aflatoxin at all. When compared to the control, an increase in growth and amount of aflatoxin occurred in SAS broth which contained 200 ppm of NaNO3. Sausages containing 100 and 200 ppm NaNO2 and no NaCl supported more mold growth and aflatoxin production than did control sausage with 3% NaCl and 100 ppm of NaNO2. Addition of 2 and 3% NaCl and no nitrite to sausage resulted in less aflatoxin than in control sausage.     

Inhalation toxicity of 1,6-hexanediamine dihydrochloride in F344/N rats and B6C3F1 mice

1,6-Hexanediamine (HDA) is a high production volume chemical which is used as an intermediate in the synthesis of paints, resins, inks, and textiles and as a corrosion inhibitor in lubricants. Two- and 13-week studies of the toxicity of the dihydrochloride salt of HDA (HDDC) were conducted in male and female Fischer 344/N rats and B6C3F1 mice using whole-body inhalation exposure. Both species were evaluated for histopathologic and reproductive effects, and rats were examined for clinical chemistry and hematologic changes. In the 2-week inhalation studies, animals were exposed to 10-800 mg HDDC/m3, 6 hr per day. All rats, all female mice, and two of five male mice in the high-exposure group died before the end of the study. Surviving mice in this group had a dose-dependent depression in body weight gain. Clinical signs were primarily related to upper respiratory tract irritation and included dyspnea and nasal discharge in both species. Treatment-related histopathologic lesions included inflammation and necrosis of the laryngeal epithelium of both species and the tracheal epithelium of mice, as well as focal inflammation and ulceration of the respiratory and olfactory nasal mucosa. In the 13-week inhalation studies, animals were exposed to HDDC at concentrations of 1.6-160 mg/m3 for 6 hr per day, 5 days per week. In addition to the base study groups, a supplemental group of rats at each exposure level was included to assess the effect of HDDC on reproduction. No treatment-related changes in organ weights or organ-to-body-weight ratios occurred in rats, and no treatment-related clinical signs or gross lesions were seen in either species. Chemical-related microscopic lesions were limited to the upper respiratory tract (larynx and nasal passages) in the two highest exposure groups and were similar in both species. These lesions included minimal to mild focal erosion, ulceration, inflammation, and hyperplasia of the laryngeal epithelium, in addition to degeneration of the olfactory and respiratory nasal epithelium. HDDC caused no significant changes in sperm morphology or vaginal cytology and no significant adverse effects on reproduction in rats or mice. Hematologic and clinical chemistry changes in rats were minor and sporadic and were not accompanied by related histologic findings. HDDC did not increase the frequency of micronucleated erythrocytes in mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Toxicokinetics of inhaled 2-butoxyethanol and its major metabolite, 2-butoxyacetic acid, in F344 rats and B6C3F1 mice

2-Butoxyethanol (2BE) is used extensively in the production of cleaning agents and solvents. It is primarily metabolized in the liver to 2-butoxyacetic acid (2BAA), which is believed to be responsible for 2BE toxicities associated with hemolysis of red blood cells. The objective of the study was to characterize the systemic disposition of 2BE and 2BAA in rats and mice during 2-year 2BE inhalation toxicity studies. Male and female F344 rats and B6C3F1 mice (6-7 weeks old) were exposed to target 2BE concentrations of 0, 31.2, 62.5, or 125 ppm (rats), or 0, 62.5, 125, or 250 ppm (mice), by whole-body inhalation for 6 h/day, 5 days/week for up to 18 months. Postexposure blood samples were collected after 1 day, 2 weeks, and 3, 6, 12, and 18 months of exposure. Postexposure 16-h urine samples were collected after 2 weeks and 3, 6, 12, and 18 months of exposure. A separate set of mice was kept in the control chamber and exposed to 2BE for 3 weeks when they were approximately 19 months old. Postexposure blood samples were collected after 1 day and 3 weeks of exposure and 16-h urine samples were collected after 2 weeks of exposure from these aged mice. Blood samples were analyzed for both 2BE and 2BAA and urine samples were analyzed for 2BAA using GC/MS, and their kinetic parameters were estimated through the curve-fitting method using SAS. Systemically absorbed 2BE was rapidly cleared from blood (t1/2-RAT < 10 min; t1/2-MOUSE < 5 min after the 1-day exposure) independent of exposure concentration. Proportional increases in AUC2BE relative to increases in exposure concentration indicated linear 2BE kinetics. In contrast, the rate of 2BAA elimination from blood decreased as the exposure concentration increased. Nonproportional increases in AUC2BAA also indicated that 2BAA is eliminated following dose-dependent, nonlinear kinetics. Overall, mice eliminated both 2BE and 2BAA from blood faster than rats. Sex-related differences in 2BAA elimination were most significant with rats, in that females were less efficient in clearing 2BAA from the blood. Differences in renal excretion of 2BAA are possibly responsible for the sex-related difference in the 2BAA blood profiles in rats. As exposure continued, the rates of elimination for both 2BE and 2BAA decreased in both species, resulting in longer residence times in the blood. When 19-month-old naive mice were exposed to 125 ppm, 2BE was rapidly cleared from the systemic circulation, exhibiting clearance profiles similar to young mice. However, old mice eliminated 2BAA from blood > 10 times slower than young mice after 1-day of exposure. This delayed elimination of 2BAA in old mice was less obvious after 3 weeks of exposure, suggesting that there might be other factors in addition to the age of animals that could influence the apparent difference in 2BAA kinetics between old and young mice. It was concluded that the elimination kinetics of 2BE and 2BAA following repeated 2BE exposure appear to be dependent on species, sex, age, time of exposure, as well as the exposure concentration.     

Dose-dependent suppression of toluene metabolism by isopropyl alcohol and methyl ethyl ketone after experimental exposure of rats

In order to examine possible suppression of toluene metabolism due to coexposure to other solvents, female Wistar rats were exposed for 8 h to toluene alone (at 50 or 100 ppm), or in combination with either methyl ethyl ketone (at 50, 100, 200 or 400 ppm) or isopropyl alcohol (at 50, 100, 200, 400, 800 or 1600 ppm). Urine samples were collected for 24 h after initiation of each exposure, and subjected to analysis for two toluene metabolites, hippuric acid and o-cresol, both by HPLC. The excretion of hippuric acid, a major metabolite, was not modified when the concentrations of methyl ethyl ketone or isopropyl alcohol were low, i.e. 100 ppm or below, whereas it was reduced when methyl ethyl ketone or isopropyl alcohol concentrations were twice or more times higher than that of toluene. There were no changes in any cases in excretion of o-cresol, a minor metabolite. The observation after coexposure to methyl ethyl ketone or isopropyl alcohol at low concentration is in line with the negative interaction between toluene and methyl ethyl ketone as well as between toluene and isopropyl alcohol after occupational exposures at low concentrations. Metabolic interaction may take place when the exposure intensity is high, as observed in the present study and also after experimental exposure of volunteers to toluene and m-xylene, or occupational exposure to benzene and toluene.