Urinary mutagenicity on TA98 and YG1024 Salmonella typhimurium strains after a hamburger meal: influence of GSTM1 and NAT2 genotypes

Mutagenicity on TA98 and YG1024 Salmonella typhimurium strainsof pan–fried hamburger extracts and of 24 h post–mealurine from 32 non–smoking volunteers was evaluated. Eachparticipant in the study was GSTM1 and NAT2 genotyped. Aftercooking the meat showed mutagenic activity (mean ± SD)on strains TA98 and YG1024 of 114 ± 129 and 1437 ±1536 net revertants/g respectively. Twenty three of 32 urinesamples showed clear mutagenic activity (i.e. caused at leasta doubling of the number of spontaneous revertants) on the 0-acetyltransferaseoverproducing strain YG1024, while none of the post-meal 24h urine samples was clearly mutagenic on strain TA98. Total24 h post–meal YG1024–active urinary mutagens werewell correlated with the levels of mutagen intake with the meal(r² = 0.5977, F = 44.58, P < 0.01). In the group under studyGSTM1 genotypes did not influence urinary mutagenicity. Highlyexposed subjects (n = 15) with the NAT2–ss genotype showedsignificantly increased levels of urinary mutagenicity on strainYG1024 in comparison with NAT2-R subjects (mutagen intake-adjustedtotal 24 h mutagen excretion = 1.00 ± 0.29 versus 0.66± 0.32, Mann-Whitney U test, U = 12.5, P < 0.05).Our results suggest that the levels of urinary mutagens derivedfrom diets rich in heterocyclic aromatic amines, which are specificallydetected by the YG1024 Salmonella strain, are modulated by NAT2-dependentenzyme activity, slow acetylators having higher levels of mutagensin their urine. Subjects with the rapid acetylator genotype,who are known to be at risk for colon cancer, seem to be partiallyprotected with respect to the risk of bladder cancer. ⁴To whom correspondence should be addressed. Tel: 498216637; Fax: 498216621; Email: clonfero{at}uxl.unipd.it     

Temperature and time effects on mutagen production in cooked lamb meat

Basic extracts isolated from lamb meat treated at various temperatures were tested for mutagenicity in Salmonella typhimurium strain TA98 in the presence of S9 mix. Samples of ground lamb patties were cooked for 10 min per side at 100, 150, 200, 250 and 300 degrees C. Low cooking temperatures resulted in products with low levels of mutagenicity. At temperatures greater than 150 degrees C the mutagenic activity of the cooked meat increased to reach a maximum at 300 degrees C. In another series of experiments, lamb patties were cooked at 250 degrees C for 2, 4, 6, 8, 10 and 12 min. Short cooking times (rare products) caused no mutagenic activity and uncooked ground meat showed no activity. Prolonged cooking appeared to increase the mutagenicity of the products with a maximum value at 10 min. The results indicate that the formation of mutagens depends on both cooking temperature and cooking time. The level of mutagenicity tends to increase with the degree of charring.     

Effects of cigarette smoke and a heterocyclic amine,MeIQx on cytochrome P-450, mutagenic activation of various carcinogens and glucuronidation in rat liver

In order to elucidate the mechanism underlying enhancement by cigarette smoke (CS) of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced rat hepatocarcinogenesis, hepatic levels of cytochrome P-450 (CYP) enzymes, mutagenic activation of various carcinogens and UDP-glucuronyltransferase (UDPGT) activities were assayed in male F344 rats. Immunoblot analyses for microsomal CYP proteins revealed induction of CYP1A1 and constitutive CYP1A2 (2.3- to 2.7-fold), but not CYP2B1/2, 2E1 or 3A2, by CS exposure for 1, 12 or 16 weeks using a Hamburg type II smoking machine; the enhancement of CYP1A2 was 4.7-5.7 times that of CYP1A1. CS exposure also elevated the mutagenic activities of MeIQx and five other heterocyclic amines (HCAs) 1.4- to 3.7-fold, but not those of benzo[a]pyrene (BP) and aflatoxin B(1) in strain TA98 and N-nitrosodimethylamine, N-nitrosopyrrolidine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in strain TA100. In contrast, feeding 300 p.p.m. MeIQx in the diet for 1 or 16 weeks produced no significant alterations in the levels of these CYP species and mutagenic activities. However, i.g. administration of 50 or 100 mg/kg MeIQx in a single dose selectively increased CYP1A1 and 1A2 (2.6-fold) levels and mutagenic activities of five HCAs (1.7- to 3.3-fold), but not BP. On the other hand, feeding of MeIQx for 16 weeks enhanced UDPGT activities towards 4-nitrophenol and testosterone (2.9- and 1.5-fold, respectively), but not bilirubin, while CS exposure induced that towards 4-nitrophenol (1.6-fold); combined treatment with CS and MeIQx showed a summation effect on induction of UDPGT1A6 activity (3.5-fold). Consequently, these results demonstrate that CS and MeIQx have a bifunctional action, with similar induction patterns of specific CYP proteins, mutagenic activity and UDPGT activity. In conjunction with the finding of N-hydroxy-MeIQx being a poor substrate for rat liver UDPGT, our results clearly indicate that enhancement by CS of MeIQx-induced hepatocarcinogenesis in F344 rats can be attributed to an increase in metabolic activation of MeIQx by hepatic CYP1A2 during the initiation phase.     

Meat-related mutagens/carcinogens in the etiology of colorectal cancer

Diets containing substantial amounts of red or preserved meats may increase the risk of various cancers, including colorectal cancer. This association may be due to a combination of factors such as the content of fat, protein, iron, and/or meat preparation (e.g., cooking or preserving methods). Red meat may be associated with colorectal cancer by contributing to N-nitroso compound (NOC) exposure. Humans can be exposed to NOCs by exogenous routes (from processed meats in particular) and by endogenous routes. Endogenous exposure to NOCs is dose-dependently related to the amount of red meat in the diet. Laboratory results have shown that meats cooked at high temperatures contain other potential mutagens in the form of heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs). To investigate the role of these compounds, we have created separate databases for HCAs and PAHs, which we have used in conjunction with a validated meat-cooking food frequency questionnaire. The role of meat type, cooking methods, doneness levels, and meat-cooking mutagens has been examined in both case-control studies and prospective cohort studies, with mixed results. Here, we review the current epidemiologic knowledge of meat-related mutagens, and evaluate the types of studies that may be required in the future to clarify the association between meat consumption and colorectal cancer.     

Polymorphisms of Aspirin-Metabolizing Enzymes CYP2C9, NAT2 and UGT1A6 in Aspirin-Intolerant Urticaria

Acetyl salicylic acid (ASA) is metabolized by UDP-glucuronosyltransferase 1A6 (UGT1A6), cytochrome P4502C9 (CYP2C9), and N-acetyl transferase 2 (NAT2). Variations in the activities of these enzymes may modulate adverse ASA-related symptoms such as urticaria. We examined whether polymorphisms in the UGT1A6, CYP2C9, and NAT2 genes are related to ASA-intolerant urticaria (AIU). The genotypes of 148 subjects with AIU (AIU group) and 260 normal healthy control subjects (NC group) were analyzed with respect to the following single nucleotide polymorphisms: CYP2C9 -1188T>C and CYP2C9(*)3A1075C; UGT1A6 T181A A>G and UGT1A6 R184S A>C; and NAT2 9796A>T, NAT2 197G>A, NAT2 286G>A, NAT2 9601A>G, and NAT2 9306A>G. There were significant differences in the allele frequencies for the CYP2C9 polymorphisms between the two groups. The frequency of the minor allele CYP2C9 -1188T>C was significantly higher in the AIU group than in the NC group (P=0.005). The frequency of the variant genotype CC was higher in the AIU group compared with the controls in both the co-dominant (P=0.007) and recessive models (P=0.012). The frequency of haplotype 2 [CA] was also significantly higher in the AIU group in both the co-dominant (P=0.006) and dominant models (P=0.012). There was no significant difference in genotype frequencies for any of the UGT1A6 or NAT2 polymorphisms between the two groups. Clinical parameters did not differ according to genotype. These results suggest that the C allele of CYP2C9 -1188T>C may be associated with AIU.     

Dietary intake of meat and meat-derived heterocyclic aromatic amines and their correlation with DNA adducts in female breast tissue

It was the aim of this study to examine the association of the consumption of meat in general, meat prepared by different cooking methods and the dietary intake of heterocyclic aromatic amines (HCA) with the level of DNA adducts in the breast tissue of women undergoing reduction mammoplasty. Dietary intake of meat and HCA were assessed via questionnaire in 44 women undergoing reduction mammoplasty. DNA adduct analysis in breast tissue was performed by (32)P-postlabelling analysis. Spearman rank correlation coefficients (r) were calculated to examine the association of meat consumption and dietary HCA intake with tissue DNA adduct levels. A median DNA adduct level of 18.45 (interquartile range 12.81-25.65) per 10(9) nucleotides in breast tissue was observed; median HCA intake was 40.43 ng/day (interquartile range 19.55-102.33 ng/day). Total HCA intake (r = 0.33, P = 0.03), consumption of fried meat (r = 0.39, P = 0.01), beef (r = 0.32, P = 0.03) and processed meat (r = 0.51, P = 0.0004) were statistically significantly correlated with the level of DNA adducts in breast tissue. The detected DNA adducts could not be confirmed to be specific HCA-derived DNA adducts by comparison with external standards, using the (32)P-postlabelling assay. We observed strong correlations of dietary HCA intake and consumption of fried and processed meat with DNA adduct levels in breast tissue of 44 women. Since the detected DNA adducts were not necessarily specific only for HCA, it is possible that HCA intake is a surrogate of other genotoxic substances, such as polycyclic aromatic hydrocarbons, in meat prepared at high temperatures.     

Mutagenic activity of overnight urine from healthy non-smoking subjects

Urinary mutagenicity was evaluated in relation to environmental mutagen exposure (i.e., diet, indoor/outdoor activities, residential area etc.) on the day prior to sample collection, and also considering factors that contribute to the variability of Salmonella mutagenicity assay results. Overnight urine samples from 283 healthy non-smoking residents of northeast Italy (46% males, 20-62 years) were analyzed for mutagenicity on sensitive Salmonella typhimurium strain YG1024 with S9 mix employing the preincubation version of the plate incorporation assay (i.e., the Salmonella reverse mutation test). Urinary mutagenicity varied between 0.02 and 9.84 rev/ equiv. ml, and 7% of samples were positive (i.e., sample elicited a two-fold increase in revertants). There was an evident increase in mutagenicity in subjects with increased intake of mutagen-rich meals (n = 80) (P < 0.01 and positive urine 13% vs. 5%, P = 0.025). Indoor-exposed subjects (n = 65) also showed a higher percentage of positive urine (14% vs. 5%, P = 0.015). In particular, those subjects exposed to cooking fumes the previous evening (n = 28) revealed higher urinary mutagenicity (P = 0.035, positive urine 25% vs. 5%, P < 0.001) than non-indoor exposed. The sources of variability of the mutagenicity assay, mainly the histidine content of the urine concentrate (z = 4.06, P < 0.0001), and to a lesser extent bacterial inoculum size (z = 2.33, P = 0.019), also significantly influenced urinary mutagenicity values. In a linear multiple regression analysis, their effects were still significant (i.e., histidine content P = 0.026 and inoculum size P = 0.021), but the effects of diet, indoor exposure, and other environmental exposures (i.e., traffic and heating system exhausts, residential area) were not. It is concluded that the previous day's exposure to mutagen-rich meals and cooking fumes may influence the presence of mutagenic activity in the overnight urine of non-smoking subjects. This mutagenic activity, which remains in contact with bladder mucosa for several hours, could be considered risk factors for colorectal adenoma and possibly other cancers (i.e., bladder) in non-smokers. Accurate control of histidine content and bacterial inoculum size is strongly recommended when investigating the mutagenic activity of urine from non-smokers.     

Development and characterization of CHO repair-proficient cell lines for comparative mutagenicity and metabolism of heterocyclic amines from cooked food

In order to understand the role of repair and metabolism in the mutagenicity of heterocyclic amines from cooked foods, we previously developed the nucleotide excision repair-deficient CHO 5P3NAT2 cell line engineered to coexpress the mouse CYP1A2 and human N-acetyltransferase genes. In the present study, we have made a matched repair-competent cell line by mutagenizing 5P3NAT2 cells with ethyl methanesulfonate and selecting for resistance to cytotoxicity by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). In the differential cytotoxicity (DC) assay, 4 out of 15 clones showed no cytotoxic effect with IQ at the highest dose (30 microg/ml) tested, in contrast to repair-deficient 5P3NAT2 cells, which showed approximately 100% cytotoxicity at 0.3 microg/ml. Subsequently, these IQ-resistant clones were examined for resistance to killing by UV irradiation. All four IQ-resistant clones, which show resistance to UV similar to that of repair-proficient AA8 cells, still express both the CYP1A2 and N-acetyltransferase genes. Sequence analysis of CXPD cDNA from the 5P3NAT2R9 clone revealed an A:T-->G:C reversion event at the site of the UV5 mutation. This base change results in reversion of the codon 116 tyrosine in UV5 cells back to the original cysteine in AA8 cells, thereby restoring wild-type CXPD activity and repair function. In contrast to 5P3NAT2 cells, the repair-proficient 5P3NAT2R9 revertant cell line shows little IQ-induced cell killing, and dramatically lower levels of induced mutation at the adenine phosphoribosyltransferase (Aprt) gene locus over the range of 2-40 microg/ml IQ. This matched pair of repair-proficient/deficient cell lines can provide insight not only into the genotoxicity of heterocyclic amine dietary carcinogens such as IQ and PhIP, but also into the effects of nucleotide excision repair on the ultimate mutagenicity of these compounds.     

Evidence for the presence of mutagenic arylamines in human breast milk and DNA adducts in exfoliated breast ductal epithelial cells

Aromatic and heterocyclic amines are ubiquitous environmental mutagens present in combustion emissions, fried meats, and tobacco smoke, and are suspect human mammary carcinogens. To determine the presence of arylamines in breast tissue and fluid, we examined exfoliated breast ductal epithelial cells for DNA adducts and matched human milk samples for mutagenicity. Breast milk was obtained from 50 women who were 4-6 weeks postpartum, and exfoliated epithelial-cell DNA was evaluated for bulky, nonpolar DNA adducts by (32)P-postlabeling and thin-layer chromatography. Milk was processed by acid hydrolysis, and the extracted organics were examined in the standard plate-incorporation Ames Salmonella assay using primarily strain YG1024, which detects frameshift mutations and overexpresses aryl amine N-acetyltransferase. DNA adducts were identified in 66% of the specimens, and bulky adducts migrated in a pattern similar to that of 4-aminobiphenyl standards. The distribution of adducts did not vary by NAT2 genotype status. Of whole milk samples, 88% (22/25) had mutagenic activity. Among the samples for which we had both DNA adduct and mutagenicity data, 58% (14/19) of the samples with adducts were also mutagenic, and 85% (11/13) of the mutagenic samples had adducts. Quantitatively, no correlation was observed between the levels of adducts and the levels of mutagenicity. Separation of the milk showed that mutagenic activity was found in 69% of skimmed milk samples but in only 29% of the corresponding milk fat samples, suggesting that the breast milk mutagens were moderately polar molecules. Chemical fractionation showed that mutagenic activity was found in 67% (4/6) of the basic fractions but in only 33% (2/6) of acidic samples, indicating that the mutagens were primarily basic compounds, such as arylamines. Although pilot in nature, this study corroborates previous findings of significant levels of DNA adducts in breast tissue and mutagenicity in human breast milk and indicates that breast milk mutagens may be moderately polar basic compounds, such as arylamines.     

Cyclopenta[cd]fluoranthene and its precursors in combustion exhausts: a survey of their bacterial mutagenic activity

Cyclopenta[cd]fluoranthene (1) and 3-ethynylfluoranthene (2) have both recently been identified in combustion exhausts. In this study, their mutagenic activities were compared to that of fluoranthene (3), one of the most abundant polycyclic aromatic hydrocarbons (PAHs) in combustion exhausts, in the Salmonella/microsome reversion assay (Ames assay) using S. typhimurium strain TA98. The mutagenicity of 1 was modest in comparison to other active cyclopenta PAHs. Unexpectedly, 2 was mutagenic both with and without exogenous metabolic activation (rat liver S9). Furthermore, cyclopenta[cd]fluoranthene-3,4-epoxide (6) was synthesized in order to evaluate its role as the ultimate mutagenic active form of 1. The epoxide 6 was a direct-acting mutagen. In addition, a pyrolysate containing a mixture of 1 (85%), 2 (2%), and 3 (13%) obtained by flash vacuum thermolysis of 3-(1-chloroethenyl)fluoranthene (2a) at 1,050 degrees C was also mutagenic, but a significant mutagenic response was detected only in the presence of S9 activation. The results of this study indicate that 1 and 2 can contribute to the mutagenic activity of combustion exhausts.     

Detection of mutagenic activity in the urine of rodents treated with p-rosaniline

p-Rosaniline was fed to male and female Fischer 344 rats and B6C3F1 mice at doses of 1,000 and 2,000 ppm for male rats and 500 and 1,000 ppm for female rats and mice of both sexes. Urine was collected overnight at 1-wk intervals over a 4-wk treatment period and frozen until its use in the mutagenicity assay. The neat urine was tested in triplicate without S-9 on Salmonella tester strains TA98, TA100, TA1535, and TA1537 at 0.75, 0.5, 0.2, and 0.05 ml per plate. When sufficient urine was available, samples were tested on TA100 in the presence of S-9. Either urine samples were pretreated for 18 hr at 37 degrees C with beta-glucuronidase, or the deconjugating enzyme was added to the top agar at the time of plating in the mutagenicity assay (non-pretreatment). Direct-acting mutagenic activity was detected on TA98 in the urine from male mice, but only when using the non-pretreatment deconjugation method. No direct-acting mutagenic activity was detected in the urine of male and female rats and female mice; however, in the presence of S-9, mutagenic activity was observed in the urine of male rats and in the urine of male and female mice regardless of the deconjugation method used. The non-pretreatment method was superior for detecting direct acting mutagenic activity, and the pretreatment method was superior for detecting mutagenic activity requiring metabolic activation by S-9.     

Mammary gland carcinogenesis by food-derived heterocyclic amines: metabolism and additional factors influencing carcinogenesis by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

The heterocyclic amines (HCAs) are a family of mutagenic/carcinogenic compounds found in cooked meats. Several HCAs are mammary gland carcinogens in rats. Of these compounds, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the major one present in the human diet. This report reviews the studies on rat mammary gland carcinogenesis by HCAs; discusses what is currently known regarding mechanisms of mammary gland carcinogenesis of PhIP, especially the significance of metabolic processing; and further highlights the evidence for the possible role of PhIP in human breast cancer.     

Mutagenic and cytotoxic action of heated pork meat extracts in human diploid fibroblasts.

Extracts from lean pork heated at 200 degrees C have a strong mutagenic activity in the Ames Salmonella assay (strain TA98 +S9). The formation of mutagenicity is highly temperature dependent, thus an extract heated at 100 degrees C is not mutagenic in this system. This paper shows that the 200 degrees C extract also causes mutations at the hprt locus in normal human fibroblasts, as demonstrated by a dose-dependent increase of 6-thioguanine resistant mutants. The mutation frequencies were increased 6 and 13 times, respectively, for extract concentrations corresponding to 100 and 200 mg meat/ml medium. Heterocyclic amines, previously shown to be present in the 200 degrees C extract are conceivably responsible for at least part of the observed mutagenicity. The extracts prepared at 100 degrees C had no significant effect on the mutant frequency in human fibroblasts. A pronounced, dose-dependent, decrease in cell survival was observed with both the 100 and the 200 degrees C extracts. The nature of the cytotoxic components is not clear and might be different in the two extracts.     

Urinary and serum mutagenicity studies with rats implanted with depleted uranium or tantalum pellets

During the 1991 Persian Gulf War several US military personnelwere wounded by shrapnel fragments consisting of depleted uranium.These fragments were treated as conventional shrapnel and werenot surgically removed to spare excessive tissue damage. Uraniumbioassays conducted over a year after the initial uranium injuryindicated a significant increase in urine uranium levels abovenatural background levels. The potential mutagenic effects ofdepleted uranium are unknown. To assess the potential mutageniceffects of long-term exposure to internalized depleted uranium,Sprague-Dawley rats were implanted with depleted uranium andtheir urine and serum were evaluated for mutagenic potentialat various times after pellet implantation using the Ames Salmonellareversion assay. Tantalum, an inert metal widely used in prostheticdevices was used for comparison. Enhancement of mutagenic activityin Salmonella typhiurium strain TA98 and the Ames II^TM mixedstrains (TA7001–7006) was observed in urine samples fromanimals implanted with depleted uranium pellets. In contrast,urine samples from animals implanted with tantalum did not showa significant enhancement of mutagenic activity in these strains.In depleted uranium-implanted animals, urine mutagenicity increasedin a dose- and time-dependent manner demonstrating a strongpositive correlation with urine uranium levels (r = 0.995, P< 0.001). There was no mutagenic enhancement of any bacterialstrain detected in the sera of animals implanted with eitherdepleted uranium or tantalum pellets. The results suggest thaturanium content in the urine is correlated with urine mutagenicityand that urinary mutagenicity might be used as a biomarker todetect exposure to internalized uranium. ⁵To whom correspondence should be addressed. Tel: +1 301 295 9232; Fax: +1 301 295 6503; Email: millera{at}mx.afrri.usuhs.mil     

Petroleum distillates suppress in vitro metabolic activation: higher [S-9] required in the Salmonella/microsome mutagenicity assay

To determine if standard conditions used in the Salmonella/mammalian microsome mutagenicity assay could reliably screen complex petroleum samples, two high-boiling (700-1,070 degrees F) distillates and their separated aromatic fractions were tested. The initial mutagenic activities were inconsistent with the samples' known polyaromatic hydrocarbon (PAH) contents and observed potencies in a dermal carcinogenesis bioassay. A significant mutagenic response was observed only at S-9 concentrations 5 to 10 times higher than those used in the standard assay, supporting the use of elevated levels of S-9 in the Salmonella/microsome assay to assess the carcinogenic potential of petroleum-derived materials. All four samples masked the expected mutagenic activity of added PAHs (benzo[a]pyrene and perylene). Data suggested that petroleum distillates suppress the functional efficacy of the S-9; possible mechanisms are discussed.     

Correlation between meteorological conditions and mutagenicity of airborne particupate samples in a tropical monsoon climate area from Kaohsiung City,Taiwan

Kaohsiung is a city of 1.5 million located in the southern part of Taiwan. It has a serious air pollution problem mainly attributable to much industrial and commercial activity. In order to estimate the effects of traffic, season, and meteorological conditions on the mutagenicity of Kaohsiung City's urban ambient particulate matter, 624 airborne particulate samples were collected on a weekly basis from 12 locations for an entire year. The mutagenic potential of acetone extracts of air samples was evaluated by the Salmonella/microsomal test with S. typhimurium TA98 in the presence and absence of S9 mixtures. The air samples from November 1990 showed the highest direct and indirect mutagenicity among the 12 months, whereas those from June and July 1991 had the lowest direct and indirect mutagenic activity, respectively. The mutagenicity showed a good correlation with amounts of the acetone extractable matter of airborne particulates. The meteorological conditions, monthly mean precipitation, and wind speed also showed a good correspondence with mutagenicity. Wind direction and temperature had a moderate relationship. The major mutagenic fractions of air samples that had the highest mutagenic activity in a month were purified using Sephadex LH-20 column chromatography, and the contents of PAHs, 1-NP, and DNPs were analyzed by HPLC. The characteristic concentration ratios of PAHs indicated that, for the main pollution sources of airborne particulates from Kaohsiung city, the mobile sources were more important than the stationary ones. The total amounts of 1-NP and DNPs in airborne particulates seemed to correspond to their mutagenicity. Although the total amounts of 1-NP and DNPs in the air samples correlated with their mutagenicity, the major mutagenic chemicals in the airborne particulate samples from Kaohsiung City need further investigation. © 1994 Wiley-Liss, Inc.     

Comparative mutagenicity of a coal combustion fly ash extract in Salmonella typhimurium and Chinese hamster ovary cells

The dichloromethane extract of a coal combustion fly ash sample obtained from an experimental fluidized bed coal combustor was tested for mutagenicity in Salmonella typhimurium and cultured Chinese hamster ovary (CHO) cells. The extract was directly mutagenic in S typhimurium strain TA98 and the nitroreductase deficient strains TA98NR and TA98/1,8DNP6. The mutagenicity observed in TA98NR and TA98/1,8DNP6 was lower than that in TA98. Addition of exogenous Aroclor 1254-induced rat liver supernatant (liver S9) decreased the bacterial mutagenicity of the extract. A different mutagenic response was observed in CHO cells. In the absence of liver S9, although the extract was cytotoxic to CHO cells, no significant mutagenicity was observed. Addition of exogenous liver S9 decreased the cytotoxicity and increased the mutagenicity at both Na+-K+-ATPase and hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene loci in CHO cells. Using gas chromatography/mass spectrometry (GC/MS) and tandem quadruple mass spectrometry, a number of polynuclear aromatic hydrocarbons (PAHs) and nitrated PAHs (nitro-PAHs) were tentatively identified and quantitated. A possible explanation of the difference in bacterial and mammalian mutagenicity of the extract is that the bacterial mutagenicity was induced by the nitro-PAHs that are potent bacterial mutagens and mammalian mutagenicity was induced by both PAHs and nitro-PAHs that are promutagens.     

Mutagenicity study on pyrazole, seven pyrazole derivatives, and two nitroimidazoles with the L-arabinose resistance test of Salmonella typhimurium

The mutagenicity of pyrazole and seven pyrazole derivatives (4-nitropyrazole, 4-bromopyrazole, 1-methyl-4-nitropyrazole, 3,5-dimethyl-4-nitropyrazole, 1-methyl-4-bromopyrazole, 4,4'-dinitro-1, 1'-methylene-dipyrazole and 4,4'-dibromo-1,1'-methylene-dipyrazole) has been investigated with the L-arabinose forward mutation assay of Salmonella typhimurium. Two nitroimidazoles (1-methyl-5-nitroimidazole and metronidazole) were included as reference drugs. The mutagenicity of each chemical was determined by both preincubation and liquid tests, in the presence or absence of S9 microsomal fraction. The mutagenic response was expressed as the absolute number of L-arabinose resistant mutants growing in selective plates, supplemented with traces of D-glucose. Strain BA13 with a wildtype lipopolysaccharide barrier was used as a comparison to the deep rough derivative BA9. No mutagenic effect was detected with pyrazole and two of its derivatives, 1-methyl-4-bromopyrazole and 4,4'-dibromo-1,1'-methylene-dipyrazole. The other five pyrazole derivatives were mutagenic to different degrees, although their mutagenic potencies were always considerably lower than those of the two nitroimidazoles. The results suggest that 4-nitropyrazoles, as well as 4,4'-dinitro-1, 1'-methylene-dipyrazoles, should be investigated further as alternatives to, or even substitutes for, the currently used nitroimidazoles.     

Recombinant human P450 forms 1A1, 1A2, and 1B1 catalyze the bioactivation of heterocyclic amine mutagens in Escherichia coli lacZ strains

Three recombinant human P450 enzymes, forms 1A1, 1A2, and 1B1, were coexpressed with NADPH-cytochrome P450 reductase in an E. coli lacZ strain suitable for detection of the mutagenicity of heterocyclic and aromatic amines. The resulting strains expressed the recombinant P450 holoenzymes at high levels. MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline) was activated effectively by P450 1A2, weakly by P450 1A1, and not detectably by P450 1B1. MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) were activated by all three enzymes, with form 1A2 the most effective. These strains facilitate analysis of the substrate specificity of human P450 forms that participate in the metabolic activation of carcinogens.     

Stability and inactivation of mutagenic drugs and their metabolites in the urine of patients administered antineoplastic therapy

Urine samples from patients administered mutagenic antineoplastic drugs are mutagenic in the Ames assay, and hence may pose a genotoxic hazard to hospital personnel or family members caring for the patient. The urine samples in the present study were tested for mutagenicity in several strains of Salmonella typhimurium that were uvr negative (TA98, TA100) or positive (TA102, UTH8413, UTH8414), and were analyzed for the presence of drugs and their metabolites using high-pressure liquid chromatography (HPLC). Urine samples from cancer patients were kept at room temperature and their mutagenicity as well as the chemical stability of the drugs was tested for a period of 14 days. It was observed that, in general, the urine remained mutagenic for the 14-day period while the parent compound degraded within the first seven days. An exception was cisplatin, which was chemically stable as platinum, but the urine decreased in mutagenicity with time. This decrease was probably the result of ligand exchange with the platinum. Inactivation methods were developed to reduce the genotoxic hazard posed by the mutagenic compounds in the urine. Cisplatin was inactivated by complexing with sodium diethyldithiocarbamate (DDTC). Oxidation of urine containing mitomycin C and doxorubicin (sodium thiosulfate must be added to urine containing doxorubicin) with 5.25% sodium hypochlorite solution (bleach) results in mutagenic inactivation. Urine containing cyclophosphamide and its metabolites was oxidized with alkaline potassium permaganate and the active degradation products trapped with sodium thiosulfate. Both chemical and mutagenic assays are necessary to determine the reduction of risk. Methods of inactivation of mutagenic urine developed in this study are both effective and practical for the reduction of exposure to genotoxic hazards.