Mutagenicity of metronidazole: presence of several active metabolites in human urine.

Mutagenic activity was found in the urine of 10 patients given therapeutic dosages of metronidazole orally or per vagina. Paper chromatographic separation revealed that mutagenicity in the urine was associated with unmodified metronidazole and at least four of its known urinary metabolites. Activity was also recovered in a region of the chromatogram heretofore not assigned to a metronidazole metabolite.

Carcinogen hemoglobin adducts, urinary mutagenicity, and metabolic phenotype in active and passive cigarette smokers.

In 100 healthy volunteers, we have studied the relationship between the type (air- or flue-cured) and number of cigarettes smoked and different biomarkers relevant to the risk of bladder cancer, including the levels of 4-aminobiphenyl (ABP) hemoglobin adduct (a marker of internal dose), urinary mutagenicity in Salmonella typhimurium TA98, and the N-acetylation phenotype (a marker of susceptibility). ABP is a potent bladder carcinogen that is N-acetylated as an overall detoxification step. Levels of the ABP hemoglobin adduct were higher in smokers of black tobacco (air-cured) than in smokers of blond tobacco (flue-cured), confirming our earlier study. In addition, "slow" acetylators had higher levels of the ABP hemoglobin adduct for the same type and quantity of cigarettes smoked. Urinary mutagenicity was also associated with quantity of cigarettes but not with the acetylation phenotype. Convex dose-response relationships were found between the amount smoked and ABP hemoglobin adduct levels or urinary mutagenicity. In 15 nonsmokers who reported exposure to environmental tobacco smoke, ABP hemoglobin adduct levels, unlike urinary mutagenicity, were found to be an aspecific exposure indicator.

Determination of benzo[a]pyrene diol epoxide-DNA adducts in white blood cell DNA from coke-oven workers: the impact of smoking.

We have undertaken a study among coke-oven workers to test the feasibility of an enzyme-linked immunosorbent assay with anti-trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene- DNA antibodies for monitoring occupational exposure to polycyclic aromatic hydrocarbons (PAH). Coke-oven workers are occupationally exposed to relatively high levels of PAH and are at increased risk for lung cancer. Three blood samples were collected from each of the 56 coke-oven workers exposed to PAH and 44 unexposed workers employed in a steel-rolling factory of the same plant. In addition, PAH levels were measured in ambient air by personal sampling, and the excretion of 1-hydroxypyrene in urine was also measured on 3 consecutive working days. All participants were interviewed regarding working conditions, personal hygiene, and smoking habits. The results showed that the coke-oven workers were exposed to substantial concentrations of atmospheric PAH (1-186 micrograms/m3), including benzo[a]pyrene (0.1-7.8 micrograms/m3) and pyrene (0.6-23.6 micrograms/m3). Both benzo[a]pyrene and pyrene were shown to be representative for the whole group of PAH. Forty-seven percent of the coke-oven workers had detectable levels of PAH-DNA adducts in their white blood cells, compared with 30% of the controls. In both groups, smokers had significantly higher levels of PAH-DNA adducts than did nonsmokers. At one site, we found the correlation positive between DNA adducts and the duration of exposure (r = .47, P = .005). Generally, the correlation was not significant between PAH-DNA adducts in blood and the concentration of PAH in the air and 1-hydroxypyrene in urine.

Effect of microsomal enzyme inducers on the urinary excretion pattern of mutagenic metabolites of the carcinogen 2,4-toluenediamine.

2,4-Toluenediamine [(TDA) CAS: 95-80-7] was administered to rats pretreated with the microsomal enzyme inducers phenobarbital (PB), beta-naphthoflavone (beta NF), or 3-methylcholanthrene (MCA). The 24-hour urines of male F344 rats were examined for their mutagenic potency by means of the Salmonella assay, with the Aroclor 1254-pretreated rat liver S-9 fraction as an activating system. No revertants were found with TDA or its urinary metabolites in the absence of the S-9 fraction. In the presence of S-9, the number of revertants increased as the concentration of TDA or its urinary metabolites increased. The urinary metabolites, generated after the microsomal enzyme inducers (PB, beta NF, MCA), had increased mutagenic activity as compared with the controls (saline, corn oil). In the presence of beta-glucuronidase (beta G), increased numbers of TA98 revertants were noted in the urine of rats pretreated with PB, saline, or corn oil. Addition of sulfatase did not alter the number of TA98 revertants. Conversely, beta G treatment of urine from rats pretreated with MCA or beta NF led to a decrease in the number of TA98 revertants as compared to levels in urine without beta G. Addition of known urinary metabolites of TDA, such as 4-acetylamino-2-aminobenzoic acid or 2,4-diacetylaminobenzoic acid, to beta NF-pretreated rat urine had no inhibitory effect on the mutagenicity in the absence of beta G. However, in the presence of beta G, the inhibitory effect was similar to that noted with beta NF-pretreated rat urine. Upon separation of urinary metabolites (beta NF-pretreated rat urine) into free, conjugated, and water-soluble forms, the maximum number of TA98 revertants was associated with the free ethyl acetate-extractable fraction, which accounted for the total mutagenic activity associated with the original volume of urine. Conjugated metabolites showed much less mutagenic activity, and an inhibitory principle was associated with the water-soluble fraction.

Detection of low levels of urinary mutagen excretion by chemotherapy workers which was not related to occupational drug exposures.

Urine specimens from a total of 26 subjects who were either nurses, pharmacists, or pharmacy technicians engaged in the preparation, handling, or administration of cancer chemotherapeutic agents were analyzed for the presence of mutagenic substances. Assays were performed using bacterial strains TA98 and TA100 in the Salmonella/mammalian microsomal mutagenicity assay developed by Ames et al. (Mutat. Res., 31: 347-364, 1975). Findings were compared with results from similar assays of urine specimens for 38 hospital personnel not exposed to cancer chemotherapeutic agents. There was no evidence of an association between occupational exposure to chemotherapy drugs and the presence of mutagenic substances that could be detected by this assay procedure in either specimens of filter-sterilized urine or extracts of urine concentrated with XAD-2 resins. An association was observed, however, between smoking and increased urinary excretion of mutagens. None of the observed associations was changed substantially by statistical adjustment for the occupational category of the subject (nurse or pharmacist), hospital of employment, or values of concurrent solvent controls for the mutagenesis assays. Associations with occupational exposures were not changed by controlling for smoking history. In addition to the large increases for smokers, testing of extracts of urine from nonsmokers with bacterial strain TA98 yielded mutagenicity values that averaged about 50% higher than values for solvent controls. Similar small increases were observed in previous published reports of human urine mutagenicity assays using tester strains TA1538 and TA98. We found little evidence to suggest that the small increases observed for nonsmokers were associated with technical factors such as the presence in the extracts of histidine or other substances promoting bacterial growth or contamination of the specimens during collection or extraction procedures. Since it appears that technical factors can be excluded, we believe that the increases were associated with urinary excretion of low levels of mutagen by a high proportion of subjects tested. The lack of an association of mutagenicity with occupational exposure to chemotherapeutic drugs may have been due to protective measures at the hospitals surveyed and suggests that, with appropriate procedures, these agents can be administered in a manner such that human exposure cannot be detected using this approach.(ABSTRACT TRUNCATED AT 400 WORDS)

Urinary mutagen levels in smokers.

Urinary mutagen levels were measured over an 8-week period in a group of 13 smokers. Individual urinary mutagen levels were observed to be relatively constant and while there was a general correlation between mutagen excretion levels and cigarette consumption, individuals' excretion levels could not be predicted on the basis of either the numbers of cigarettes smoked or the tar content of the cigarettes.

Urinary mutagens in humans after fried pork and bacon meals.

Urinary mutagenic activity on Salmonella typhimurium strain TA1538 with S9 was determined after morning meals of fried pork and bacon. Both in fasting and non-fasting subjects a very marginal elevation of urinary mutagenic activity was observed, accounting for a small fraction only of the total amount of mutagens ingested.

Investigations on the detection of mutagenic activity of beef extract in rats after oral administration.

After oral administration of beef extract the body fluids of Aroclor-treated and untreated rats were investigated for mutagenicity using the Salmonella/microsome test. In the stomach contents, the bile and the urine of the animals, mutagenic activity was discovered after S-9 activation. Although the mutagenic substances must have been transported by the blood stream to the excreting organs no increased mutagen-induced his+ revertants were observed in venous blood. Direct-acting mutagens were not detected in the tested body fluids, either in the Aroclor-treated rats or in the untreated ones.

Genetic effects of benzene and radiation in ICR and X/Gf mice.

X/Gf mice (a tumor-resistant strain) were compared with ICR mice (moderately tumor-sensitive) for their sensitivity to chromosomal damage caused by benzene, cyclophosphamide (CP), benzo(a)pyrene (BP) and radiation. There was no difference between strains in the level of micronucleus formation caused by BP, CP or radiation. Although X/Gf mice metabolized somewhat less of the dose of benzene per weight than ICR mice, and had somewhat higher levels of genetic damage, it is not known whether X/Gf mice would be measurably more resistant to benzene carcinogenicity. Short-term genotoxicity tests are used as indicators of initiation, therefore, equal sensitivity to a set of standard clastogens suggests that tumor resistance in X/Gf mice is a function of later stages of carcinogenesis.

Detection of mutagenic activity in human urine following fried pork or bacon meals.

Mutagenic activity has been demonstrated in urine of human subjects after ingestion of fried pork or bacon. Activity was detected with Salmonella strains TA1538 and TA98 particularly, in the presence of liver homogenate S9, and was not enhanced by prior incubation of urine with beta-glucuronidase. Chemical and biological characteristics of the urine activity closely resemble those found in extracts of fried pork and bacon which also increase the frequency of sex-linked recessive mutations in Drosophila melanogaster. Microwave-cooked meat neither contained extractable mutagenic activity, nor contributed to urinary mutagenicity, possible due to the paucity of browning reactions in meat cooked under these conditions. If the urine and meat factors are chemically identical, then approximately one-third of the food activity is recovered from the urine. These results show that mutagenic factors, generated during cooking of pork and bacon, are ingested and absorbed and are subject to urinary clearance in biologically detectable quantities. It is possible that the potential for genetic toxicity in humans of these and related factors has been underestimated.

Urinary excretion of frameshift mutagens in rats caused by passive smoking.

Several male Wistar rats were individually placed in a chamber resembling a room provided with minimal air flow. They were exposed separately to the main- and sidestream smoke of a commercial brand of cigarettes smoked by a smoking machine. Exposure to both sidestream and mainstream smoke of at least two cigarettes resulted in significant excretions of frameshift mutagens in urine within 24 h, detected by the bacterial microtiter fluctuation test with Salmonella typhimurium TA1538. Doubling exposure to the mainstream smoke resulted in similar quantitative mutagenic activities. Doubling exposure to the sidestream smoke resulted in reduced water intake by the animals and thus toxic effects of the urine concentrates on the test bacteria.

Stability of the mutagenicity in stored cigarette smokers' urine and extract.

Urine from cigarette smokers was analyzed for the effect upon mutagenic activity when stored for as long as 175 days. Frozen aliquots of urine were thawed out at various time points in the study and prepared for bioassay. These urine extracts were not bioassayed immediately, but rather refrozen until all of the unprocessed urine samples had eventually been prepared for bioassay. All extracts were obtained using cyanopropyl solid phase extraction techniques. At the end of 175 days, all extracts were bioassayed using a microsuspension assay of Salmonella typhimurium TA98. Urine from smokers was found to be mutagenic (14.4-30.9 revertants/ml equivalent) while a control set of urine from non-smokers was not. Data from the storage study when analyzed by analysis of variance techniques indicated no statistical loss of mutagens occurred over the 175-day period although near significance was observed (P = 0.054). This near significance was the result of decreasing mutant response as storage time increased for two of the higher doses tested.

Correlations between urinary nicotine or cotinine and urinary mutagenicity in smokers on controlled diets.

Cigarette smokers have been reported to void urine that is more mutagenic, as measured in the Salmonella/microsome assay, than urine voided by nonsmokers. Several previous studies have attempted to correlate indices of tobacco smoke exposure (e.g. nicotine, cotinine, tar intake) with urinary mutagenicity, with conflicting results. These studies generally involved small numbers of smokers and did not carefully control diet, which is known to affect urinary mutagenicity markedly. Our objective was to conduct a controlled study to determine clearly if there were a correlation between urinary nicotine, cotinine, or nicotine + cotinine and urinary mutagenicity and to determine if nicotine or its major metabolite plays a role in the mutagenicity of urine from cigarette smokers. We used a large number of smokers (31), each of whom smoked both a tobacco-burning cigarette and a tobacco-heating cigarette on consecutive weeks, and we prepared and served identical diets to all subjects. Nicotine and cotinine concentrations were determined in small aliquots from urine samples collected over 24 hr, and the remaining urine sample was extracted and concentrated on XAD-2 resin for mutagenicity assays in the Salmonella/microsome test. Nicotine, cotinine, and nicotine + cotinine were statistically correlated with mutagenicity of urine from smokers of the tobacco-burning cigarette, but there was no correlation between nicotine, cotinine, or nicotine + cotinine and mutagenicity of urine from smokers of the tobacco-heating cigarettes. Thus, although urinary nicotine and cotinine concentrations correlate with urinary mutagenicity in smokers of tobacco-burning cigarettes, the present results indicate that nicotine and its metabolite are not responsible for the mutagenicity of smokers' urine.

Urine mutagenicity and biochemical parameters as markers of exposure to petroleum pitch using a rat model.

A petroleum pitch sample collected in a carbon electrode factory was studied using a series of in vivo assays for genotoxicity and enzymatic induction capability. Rats were treated with the petroleum derivative in three doses: 100, 50, and 10 mg/kg body weight. The treatment produced a rapid excretion of mutagenic substances in the urines of the first 24 hr only in rats treated with high doses (100 and 50 mg/kg). No faecal mutagenic activity was observed. Analyses of urinary thioethers showed that urinary metabolites derived from the compounds present in the pitch-sample at the lowest dose-administered (10 mg/kg) were eliminated primarily as cysteine conjugates. The pitch sample was found to be a good inducer of pulmonary and hepatic aryl hydrocarbon hydroxylase, especially after a 50 mg/kg dose. Urinary D-glucaric acid content was always statistically increased in treated animals compared with controls, confirming the enzymatic induction activity. Hepatic glutathione-S-transferase activity increased following treatment with 50 and 10 mg/kg doses.

Effect of pentachlorophenol on the activation of 2,6-dinitrotoluene to genotoxic urinary metabolites in CD-1 mice: a comparison of GI enzyme activities and urine mutagenicity.

2,6-Dinitrotoluene (2,6-DNT) and pentachlorophenol (PCP) are used for industrial purposes and are found in the environment as hazardous contaminants. Because concurrent exposure to both compounds can occur, it is of interest to determine if organochlorine compounds potentiate the effect of nitroaromatic chemicals. CD-1 mice were treated with PCP (42.8 mg/kg) for 4 weeks. On weeks 1, 2, and 4 after the initial PCP dose, mice were treated p.o. with 2,6-DNT (75 mg/kg) and 24 hr urines were collected. After concentration, the urines were tested for their mutagenic activity in Salmonella typhimurium strain TA98 without metabolic activation in a microsuspension bioassay. A significant increase (P less than .05) in mutagenicity was observed in urines from mice treated with 2,6-DNT alone and in combination with PCP. By week 4, mice that received both 2,6-DNT and PCP excreted urine that was more mutagenic than that from animals which received only 2,6-DNT. At weeks 2 and 4, mice were sacrificed and intestinal enzyme activities (nitroreductase, azo reductase, beta-glucuronidase, dechlorinase, and dehydrochlorinase) were quantitated. The enhanced genotoxicity observed in urines from 2,6-DNT/PCP-treated mice coincided with a decrease in nitroreductase and an increase in beta-glucuronidase activities in the small intestine.

Is there influence of smoking on the mutagenicity of follicular fluid?

The mutagenicity of follicular fluid was examined in 24 patients, 12 smoking and 12 nonsmoking, who were treated in an in vitro fertilization program. The Salmonella microsome assay was used. It was found that the mutagenicity of follicular fluid was not influenced by the number of cigarettes smoked. Urine samples of smoking in vitro fertilization (IVF) patients however showed a dose-dependent elevation of the mutagenicity.

The appearance of mutagens in urine of rats after the administration of benzidine and some other aromatic amines.

The mutagenicity of urine from rats treated with benzidine or 5 other arylamines (0.25 mmol/kg; i.p.) was studied using the Ames-assay. It was found that samples of urine collected for 24 h after the administration of the carcinogens, benzidine, 4-aminobiphenyl and 2-aminonaphthalene, showed significantly mutagenic activity, whereas no mutagenicity was observed in urine after treatment with 3,3'-5,5'-tetramethylbenzidine, 2-aminobiphenyl and 1-aminonaphthalene. Mutagenic activities were dependent on the use of either hepatic S-9 Mix or cytosol as the activating enzyme preparation. The addition of beta-glucuronidase enhanced mutagenicity, except for 2-aminonaphthalene. The appearance of mutagens in urine was studied at varying doses of benzidine and at different time-intervals after the administration. The different excretion patterns found after the activation either with S-9 Mix or with cytosolic enzyme(s) suggest the presence in urine of different types of mutagenic products.

Methods for measuring mutagenicity in urine of rats dosed with [14C]di(2-ethylhexyl)phthalate.

Di(2-ethylhexyl)phthalate (DEHP) is extensively used as a plasticizer for vinyl plastic articles. It has been found to be positive in an NCI rodent bioassay but has generally given negative results in in vitro genotoxicity tests. We therefore decided to test the urine of rats fed [14C]DEHP for mutagenic activity in the Ames Salmonella test. The recovery of radioactivity from the urine of rats dosed with [14C]DEHP was examined by solvent extraction and XAD-2 resin absorption procedures. Both of these procedures were inadequate for quantitative recovery of urinary metabolites required for subsequent mutagenicity testing using the Ames Salmonella/microsome procedure. Recoveries of less than 5% were observed using standard solvent extraction techniques whereas the XAD-2 adsorption technique gave about 67% at high resin/urine ratios. Treatment of the urine with beta-glucuronidase/aryl sulfatase did not affect these recoveries. The direct urine plating procedure represents a viable alternative to the above concentration procedures for this phthalate ester. The effects of L-histidine and the beta-glucuronidase/aryl sulfatase preparation on the background reversion frequencies of the Ames tester strains is discussed.

Bacterial mutagenicity testing of urine from rats dosed with 2-ethylhexanol derived plasticizers.

Di-(2-ethylhexyl)phthalate (DEHP) produced hepatocellular carcinomas in rodents at high doses in a NTP/NCI bioassay. DEHP has not shown evidence of genotoxic activity in in vitro mutagenicity tests. We extended these studies by examining the mutagenicity of urine from rats dosed with DEHP, 2-ethylhexanol (2-EH), and several other 2-EH derived plasticizers, i.e. di-(2-ethylhexyl)adipate (DEHA), di-(2-ethylhexyl)terephthalate (DEHT) and tri-(2-ethylhexyl)trimellitate (TEHT). A modified Ames Salmonella/microsome assay was used to determine mutagenicity. Urine was pooled from male Sprague--Dawley rats dosed daily for 15 days with 2000 mg/kg of each test substance with the exception of 2-EH which was given at 1000 mg/kg. Direct plating procedures were used to determine the presence of mutagens in urine. Urine from rats dosed with 8-hydroxyquinoline was used as a positive control. There was no evidence that mutagenic substances were excreted in the urine by rats dosed with either DEHP, DEHA, DEHT, TEHT or 2-EH as determined in the presence or absence of rat liver microsomes, and with or without treatment with beta-glucuronidase/aryl sulfatase. Our findings indicate that the above test compounds were not converted to urinary metabolites that were mutagenic. These observations provide no evidence for a genotoxic mechanism for DEHP carcinogenicity in rodents.

Urinary mutagenicity after controlled exposure to environmental tobacco smoke (ETS).

20 non-smokers on a defined diet low in polycyclic aromatic hydrocarbons (PAH) were exposed to environmental tobacco smoke (ETS) in an unventilated room for 8 h. The urinary mutagenicity in the 24-h urine samples as tested with the Salmonella (TA98) microsome assay did not significantly increase after exposure to either 10 ppm CO or 20-25 ppm CO. We conclude that exposure of non-smokers to ETS does not lead to an increase in their urinary mutagenicity, provided the exposure conditions are within a realistic range.