Mutagenicity of 1-nitropyrene metabolites from lung S9.

The mutagenicity of 1-nitropyrene metabolites from rabbit lung S9 incubates was evaluated using the Salmonella typhimurium plate incorporation assay with strain TA98, with and without Aroclor-induced rat liver S9. The following metabolites were isolated, identified and quantitated by HPLC: 1-nitropyrene -4,5- or -9,10-dihydrodiol (K-DHD), N-acetyl-1-aminopyrene ( NAAP ), 1-aminopyrene (1-AMP), 10-hydroxy-1-nitropyrene, 4-, 5-, 6-, 8- or 9-monohydroxy-1-nitropyrene (phenols) and 3-hydroxy-1-nitropyrene. The predominant metabolites formed by lung S9 incubates were K-DHD, 3-OH-1-nitropyrene and phenols. All of the metabolites were mutagenic in the absence of the exogenous rat liver S9 metabolic activation system, and several, including two unidentified metabolites were more potent than the parent 1-nitropyrene. The mutagenicity of 3 of the metabolites ( NAAP , 10-OH-1-nitropyrene and phenols) were enhanced by S9 while most of the other metabolites were less mutagenic in the presence of S9. These results indicate that lung tissue is capable of both oxidative and reductive metabolism which produced mutagenic metabolites, several of which were more potent than the parent compound, 1-NP.

Hepatic DNA adducts and production of mutagenic urine in 2,6-dinitrotoluene-treated B6C3F1 male mice.

The hepatocarcinogen 2,6-dinitrotoluene (2,6-DNT) is an intermediate in the chemical synthesis of 2,4,6-trinitrotoluene and polyurethane products and can contaminate the waste stream emitted by these industries. In this study, the production of mutagenic urine metabolites and the formation of hepatic DNA adducts is examined in the B6C3F1 male mouse. Animals were administered 50 mg/kg 2,6-DNT by gavage for 3 consecutive days. No body or liver weight effects were observed in treated animals. Following sacrifice, the livers were excised and DNA isolated for examination of 2,6-DNT-derived DNA adducts. During 2,6-DNT treatment, urine was collected, concentrated, and tested for mutagenicity in the Salmonella reversion bioassay. Mutagenic urine metabolites (469+/-53 revertants/ml urine) were excreted from B6C3F1 mice treated with 2,6-DNT and were comparable to results obtained for CD-1 mice and Fischer 344 rats. Two distinct hepatic DNA adducts (0.8+/-0.1 and 0.6+/-0.1 RAL/10(8) nucleotides) were detected in B6C3F1 mice by (32)P-postlabeling and thin layer chromatography which differed from the four adducts observed in hepatic DNA from 2,6-DNT-treated Fischer 344 rats.

Comparative gastrointestinal enzyme activity and activation of the promutagen 2,6-dinitrotoluene in male CD-1 mice and male Fischer 344 rats.

Comparative intestinal nitroreductase, azo reductase, beta-glucuronidase, dechlorinase and dehydrochlorinase activities in young male Fischer 344 rats and young male CD-1 mice were measured in vitro while the comparative biotransformation of 2,6-dinitrotoluene to mutagenic metabolites was determined in vivo. The mice, which exhibit a high spontaneous incidence of hepatomas, had markedly greater nitroreductase activity and metabolized significantly more 2,6-dinitrotoluene to mutagenic metabolites than did Fischer 344 rats, which show a low incidence of liver tumors. Results of this study indicate that species differences in the incidence of hepatomas may be influenced by microbial flora and/or the biotransformation of xenobiotics in the G.I. tract.

Role of the intestinal microbiota in the activation of the promutagen 2,6-dinitrotoluene to mutagenic urine metabolites and comparison of GI enzyme activities in germ-free and conventionalized male Fischer 344 rats.

After male germ-free and conventionalized Fischer 344 rats were administered per os (p.o.) 75 mg/kg 2,6-DNT, intestinal nitroreductase, beta-glucuronidase, and azo reductase activities were lower in the cecum and large intestine of germ-free animals. However, there was no significant difference in the small intestinal nitroreductase and azo reductase compared to the conventionalized counterparts. This indicated a potential mucosal source for the enzymes. Urines from germ-free rats (1144 +/- 64 revertants/ml) were less mutagenic than those from conventionalized animals (1467 +/- 171 revertants/ml) in Salmonella typhimurium strain TA98 without S9. In the presence of S9, urine from conventionalized animals (894 +/- 56 revertants/ml) was more mutagenic than that from germ-free rats (686 +/- 60 revertants/ml). The presence of the intestinal flora plays an important role in the activation of 2,6-DNT but other metabolic pathways, such as the small intestinal mucosal and/or hepatic enzymes, are present that can generate excreted genotoxicants.

In vitro characterization of DNA adducts formed by foundry air particulate matter.

This study is part of an ongoing investigation of biomarkers in iron foundry workers exposed to polycyclic aromatic compounds. Foundry workers with the highest exposures had elevated levels of DNA adducts in their white blood cells in previous studies. The purpose of this study was to characterize the nature of DNA reactive chemicals in foundry air samples through incubating the foundry filter extract with DNA and activation enzymes. Calf thymus DNA was incubated with foundry filter extract and activated by either rat liver activation mixture (S9 mix) or xanthine oxidase. A complex pattern of adducts was observed on thin-layer chromatography (TLC) by the 32P-postlabeling assay. Two selected polycyclic aromatic hydrocarbons (PAHs)--1-NP-and anti(+/-)benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide [anti(+/-) BPDE]-DNA adducts--were used as marker compounds in characterizing the postlabeled DNA adducts by TLC combined with high-performance liquid chromatography (HPLC). After an initial separation of DNA adducts by TLC, individual spots were isolated and separated further on HPLC. HPLC analysis and spiking with anti(+/-)BPDE-DNA standard confirmed the co-migration of the anti(+/-)BPDE-DNA standard with one PAH adduct formed by the S9 mix-activated DCM extract in calf thymus DNA.

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.

Mutagenicity of HPLC-fractionated urinary metabolites from 2,4,6-trinitrotoluene-treated Fischer 344 rats.

The production and storage of explosives has resulted in the environmental accumulation of the mutagen 2,4,6-trinitrotoluene (TNT). In order to characterize the production of mutagenic urinary metabolites, 6-week old male Fischer 344 rats were administered 75 mg of TNT/kg or DMSO vehicle by gavage. The animals were placed into metabolism cages, and urine was collected for 24 hr. Following filtration, metabolites in the urine were deconjugated with sulfatase and beta-glucuronidase and concentrated by solid phase extraction. The eluate was fractionated by reverse-phase high-performance liquid chromatography (HPLC) using acetonitrile/water, and the fractions, were solvent exchanged in DMSO by nitrogen evaporation. Each HPLC fraction was bioassayed in strains TA98, TA98NR, TA100, and TA100NR without metabolic activation using a microsuspension modification of the Salmonella histidine reversion assay. Fractions 3, 5-18, 21, 22, and 24-26 contained mutagens detected by strain TA98. In the nitroreductase-deficient strain TA98NR, some mutagenic activity was lost; however, fractions 3, 6, 9-11, 15, and 25 clearly contained direct-acting mutagens. Fewer fractions were positive in strain TA100 (9-16, 19, 20, and 25) with less activity observed in the nitroreductase deficient strain TA100NR (fractions 3, 12, 14, 15, and 25). Although some mutagenic activity coeluted with known TNT metabolite standards, there were still many unidentified mutagenic peaks.

Atrazine treatment potentiates excretion of mutagenic urine in 2,6-dinitrotoluene-treated Fischer 344 rats.

Atrazine (ATZ), an s-triazine herbicide, is a widespread environmental contaminant. The hepatocarcinogenic component of technical grade dinitrotoluene, 2,6-dinitrotoluene (2,6-DNT, 19.5%), is a byproduct of trinitrotoluene synthesis and is found at production sites. This study explores the effect of ATZ treatment on the bioactivation of the promutagen, 2,6-DNT. Male Fischer 344 rats (5 weeks old) were administered 50 mg/kg of ATZ by gavage for 5 weeks. At 1, 3, and 5 weeks, both DMSO-control and ATZ-pretreated rats were treated p.o. with 75 mg/kg of 2,6-DNT and were housed in metabolism cages for urine collection. Sulfatase- and beta-glucuronidase-treated, concentrated urine was bioassayed for urinary mutagens in a microsuspension modification of the Salmonella assay with and without metabolic activation. No significant change in mutagen excretion was observed in ATZ-treated rats; however, an elevation in direct-acting urine mutagens from rats receiving ATZ and 2,6-DNT at weeks 1 (359 +/- 68 vs. 621 +/- 96 revertants/ml) and 5 (278 +/- 46 vs. 667 +/- 109 revertants/ml) of treatment was observed. The increase in production of urinary mutagens was accompanied by an elevation in small intestinal nitroreductase activity. Increases in large intestinal nitroreductase and beta-glucuronidase were observed after 5 weeks. There was no apparent effect of ATZ following 5 weeks of treatment on the production of 2,6-DNT-derived hepatic DNA adducts. ATZ treatment modifies intestinal enzymes responsible for promutagen bioactivation, and potentiates the excretion of mutagenic urine in 2,6-DNT-treated animals.

Modulation of 2,6-dinitrotoluene genotoxicity by alachlor treatment of Fischer 344 rats.

Due to its widespread use as a preemergent herbicide, alachlor has been detected as a groundwater contaminant. The procarcinogen, 2,6-dinitrotoluene (DNT), a by-product of the munitions industry and a precursor to polyurethane production, is found in the manufacturing waste stream. This study explores the effect of alachlor treatment on the bioactivation of DNT by examining urine mutagenicity, intestinal enzymes, and hepatic DNA adducts to detect changes in metabolism. Five-week-old male rats were treated daily by gavage with 50 mg/kg of alachlor for up to 5 weeks while control animals received an equal volume of peanut oil. At 1, 3, and 5 weeks following the initial alachlor dose, animals were administered p.o. 75 mg/kg DNT or DMSO. Urine was collected for 24 hr in metabolism cages. Following incubation with sulfatase and beta-glucuronidase, urines were individually concentrated by C-18 solid phase extraction, dried under N2, and prepared for bioassay in Salmonella typhimurium strain TA98 with and without metabolic activation. Urine from peanut oil- and alachlor-treated rots was not mutagenic. Even though calf thymus DNA-alachlor adducts formed in vitro, no hepatic DNA adducts were detected in vivo in these two treatment groups. Interestingly, a significant increase in excretion of mutagenic urine from DNT-treated rats was observed following 3 weeks of alachlor treatment in the absence of S9 (690 +/- 130 vs. 339 +/- 28 revertants/ml) which corresponded to increased DNT-related hepatic DNA adduct formation (5.90 +/- 0.88 adducts/10(8) nucleotides vs. 10.56 x +/- 0.59 adducts/10(8) nucleotides [relative adduct level (RAL)]). Elevation in the production of mutagenic urine from control and treated animals was linked to increases in intestinal nitroreductase and beta-glucuronidase activities; however, the only significant alachlor-related effects were an increase in small intestinal 1-week beta-glucuronidase and 5-week dehydrochlorinase activities. The increased urine mutagenicity and hepatic DNA adduct formation indicates that alachlor has a transient effect on DNT bioactivation that apparently is unrelated to intestinal bioactivation.

Potentiation of 2,6-dinitrotoluene genotoxicity in Fischer-344 rats by pretreatment with Aroclor 1254.

Pretreatment of Fischer 344 rats for 5 weeks with Aroclor 1254, a commercial mixture of polychlorinated biphenyls, potentiated the genotoxicity of 2,6-dinitrotoluene (DNT), a component of an industrial chemical used in the production of polyurethane foams. This interaction resulted from Aroclor 1254-mediated bioactivation of DNT to markedly greater levels of the genotoxic metabolites, that were excreted in urine and formed DNA adducts in the liver. A significant increase in the excretion of mutagenic urinary DNT metabolites was observed after the first week of Aroclor 1254 treatment, peaked at week 2 and then declined by nearly 25% at week 4. Nevertheless, by week 5, there was almost a 4-fold increase in the formation of hepatic DNA adducts. Significantly elevated hepatic metabolism and increased beta-glucuronidase in the small intestine and cecum, at 4 weeks, may account for the increased adducts and decreased urinary mutagens. Altered nitroreductase activity, reduced pH, and changes in the microfloral population may also play a role in the effect of Aroclor 1254 on the bioactivation of DNT. Such chemical interactions could be important to predictive risk assessment because the overall cancer risk of the mixture would exceed that determined by the current guidelines for chemical mixtures.

Mutagenicity of derivatives and metabolites of 1-nitropyrene: activation by rat liver S9 and bacterial enzymes.

The mutagenicity and activation requirements of purified synthetic derivatives and potential metabolites of 1-nitropyrene have been characterized in the Ames plate incorporation assay with the Salmonella tester strains TA98, TA98NR and TA98/1,8-DNP6, in the presence or absence of exogenous metabolic activation provided by Aroclor-induced rat liver S9. All the compounds tested (1-aminopyrene, N-acetyl-1-aminopyrene, N-hydroxy-N-acetyl-1-aminopyrene, 3-hydroxy-1-nitropyrene, 6-hydroxy-1-nitropyrene, and 8-hydroxy-1-nitropyrene) exhibited mutagenic activity under one or more assay conditions. 1-Nitropyrene was metabolized to 3-hydroxy-1-nitropyrene, 6- or 8-hydroxy-1-nitropyrene, 1-aminopyrene, N-acetyl-1-aminopyrene and other unidentified products (including some bound to protein) by an S9 preparation analogous to that used for exogenous metabolic activation in the Ames assay. 1-Nitropyrene and 3-hydroxy-1-nitropyrene were activated primarily by the 'classical' nitroreductase, while the other compounds, particularly in the presence of S9 metabolic activation, were dependent on transesterification for expression of their mutagenicity.

Bacterial mutagenicity of aceanthrylene: a novel cyclopenta-fused polycyclic aromatic hydrocarbon of low molecular weight.

Aceanthrylene, a non-alternant cyclopenta-fused hydrocarbon, was shown to be weakly mutagenic without S9 and strongly mutagenic with S9 in the Ames Salmonella plate incorporation assay. The compound was most active in strain TA100 (35 revertants/nmole in the presence of 0.3 mg of S9 protein), and less active in strains TA98, TA1537 and TA1538 (20, 10 and 3.1 rev/nmole respectively, + S9). Strain TA1535 was unresponsive, suggesting that this compound induces frameshift mutations rather than base-pair substitutions. The mutagenic potency of aceanthrylene is consistent with predictions of its activity based on the relatively large delocalization energy (delta E deloc/beta = 0.931) of the carbonium ion which would result from oxirane ring opening of the 1,2-epoxide, a potential active metabolite.

Mutagenicity in lung of big Blue((R)) mice and induction of tandem-base substitutions in Salmonella by the air pollutant peroxyacetyl nitrate (PAN): predicted formation of intrastrand cross-links.

Peroxyacetyl nitrate (PAN) is a ubiquitous air pollutant formed from NO(2) reacting with acetoxy radicals generated from ambient aldehydes in the presence of sunlight and ozone. It contributes to eye irritation associated with photochemical smog and is present in most urban air. PAN was generated in a chamber containing open petri dishes of Salmonella TA100 (gas-phase exposure). After subtraction of the background mutation spectrum, the spectrum of PAN-induced mutants selected at 3.1-fold above the background mutant yield was 59% GC-->TA, 29% GC-->AT, 2% GC-->CG, and 10% multiple mutations - primarily GG-->TT tandem-base substitutions. Using computational molecular modeling methods, a mechanism was developed for producing this unusual tandem-base substitution. The mechanism depends on the protonation of PAN near the polyanionic DNA to release NO(2)(+) resulting in intrastrand dimer formation. Insertion of AA opposite the dimerized GG would account for the tandem GG-->TT transversions. Nose-only exposure of Big Blue((R)) mice to PAN at 78ppm (near the MTD) was mutagenic at the lacI gene in the lung (mutant frequency +/-S.E. of 6.16+/-0.58/10(5) for controls versus 8.24+/-0.30/10(5) for PAN, P=0.016). No tandem-base mutations were detected among the 40 lacI mutants sequenced. Dosimetry with 3H-PAN showed that 24h after exposure, 3.9% of the radiolabel was in the nasal tissue, and only 0.3% was in the lung. However, based on the molecular modeling considerations, the labeled portion of the molecule would not have been expected to have been bound covalently to DNA. Our results indicate that PAN is weakly mutagenic in the lungs of mice and in Salmonella and that PAN produces a unique signature mutation (a tandem GG-->TT transversion) in Salmonella that is likely due to a GG intrastrand cross-link. Thus, PAN may pose a mutagenic and possible carcinogenic risk to humans, especially at the high concentrations at which it is present in some urban environments.

Colonization and clearance of environmental microbial agents upon intranasal exposure of strain C3H/HeJ mice.

Environmental dissemination of biotechnology agents is becoming a common practice. Most applications use historically innocuous species; however, potential health effects of individual products are not scrutinized unless they contain genetically engineered microorganisms. In order to investigate possible health concerns, four surrogate microbial agents were studied in vivo. Male C3H/HeJ (endotoxin-resistant) mice were administered intranasally (i.n.) with approximately 10(7) Pseudomonas aureofaciens, Burkholderia cepacia, P. fluorescens, or P. putida. To determine clearance of the dosed bacterial strains, lungs, small intestine, large intestine, cecum, mesenteric lymph nodes (MLN), spleen, and liver were homogenized individually, plated, and dilutions inoculated onto selective media. Pseudomonas fluorescens and P. putida were eliminated from the lungs by 2 d posttreatment, and P. aureofaciens was not detected in the lungs by 5 d posttreatment. Burkholderia cepacia was reisolated from the lungs and cecum for the experimental duration (14 d). Translocation to extraintestinal sites (MLN, spleen, and liver) also occurred. Burkholderia cepacia was recovered from the MLN for 10 d after treatment of mice. Pulmonary exposure to several bacterial strains resulted in unexpected mortality. Pseudomonas aureofaciens was lethal at the lowest dose (8.26 x 10(6) CFU/ mouse), while P. fluorescens and B. cepacia were fatal at higher doses (6.15 x 10(8) CFU/mouse and 1.34 x 10(8) CFU/mouse, respectively). By using the model described in this study, human safety issues can be more easily addressed and evaluated.

Oral treatment of Fischer 344 rats with weathered crude oil and a dispersant influences intestinal metabolism and microbiota.

When oil is spilled into aquatic systems, chemical dispersants frequently are applied to enhance emulsification and biological availability. In this study, a mammalian model system was used to determine the effect of Bonnie Light Nigerian crude oil, weathered for 2 d with continuous spraying and recirculation, and a widely used dispersant, Corexit (Cx) 9527, on intestinal microbial metabolism and associated populations. To determine the subchronic dose, concentrated or diluted (1:2, 1:5, 1:10, 1:20) Cx9527 or oil was administered by gavage to Fischer 344 rats and the effect on body weight was determined. Next, rats were treated for 5 wk with oil, dispersant, or dispersant + oil. Body and tissue weights, urine mutagenicity, and the impact on the intestinal microflora and three microbial intestinal enzymes linked to bioactivation were determined in the small and large intestines and cecum. Two tested dispersants, Cx9527 and Cx9500, were toxic in vitro (1:1,000 dilution), and oil was not mutagenic in strains TA98 and TA100(+/-S9). None of the treated rats produced urine mutagens detected by TA98 or TA100. Undiluted dispersant was lethal to rats, and weight changes were observed depending on the dilution, whereas oil generally was not toxic. In the 5-wk study, body and tissue weights were unaffected at the doses administered. Small-intestinal levels of azoreductase (AR), beta-glucuronidase (BG), and nitroreductase (NR) were considerably lower than cecal and large-intestinal activities at the same time point. A temporal increase in AR activity was observed in control animals in the 3 tissues examined, and large-intestinal BG activity was elevated in 3-wk controls. No significant changes in cecal BG activity were observed. Oil- or dispersant-treated rats had mixed results with reduced activity at 3 wk and elevated activity at 5 wk compared to controls. However, when the dispersant was combined with oil at 3 wk, a reduction in activity was observed that was similar to that of dispersant alone. One-week nitroreductase activity in the small intestine and cecum was unaffected in the three treatment groups, but elevated activity was observed in the large intestines of animals treated with oil or dispersant. The effect of the combination dose was not significantly different from the control value. Due to experimental error, no 3- or 5-wk NR data were available. By 5 wk of treatment, enterobacteria and enterococci were eliminated from ceca of oil-treated rats. When oil was administered in combination with dispersant, an apparent protective effect was observed on the enterococci and lactose-fermenting and nonfermenting enterobacteria. A more detailed analysis at the species level revealed qualitative differences dependent on the treatment. This study suggests that prolonged exposure of mammals to oil, dispersant, or in combination impacts intestinal metabolism, which ultimately could lead to altered detoxification of oil constituents and coexposed toxicants.

Detection of DNA adducts in developing CD4+ CD8+ thymocytes and splenocytes following in utero exposure to benzo[a]pyrene.

Environmental carcinogen exposure may play an important role in the incidence of cancer in children. In addition to environmental pollutants, maternal smoking during pregnancy may be a contributing factor. Major carcinogenic components of cigarette smoke and other combustion by-products in the environment include polycyclic aromatic hydrocarbons (PAH). Mouse offspring exposed during midpregnancy to the PAH, benzo[a]pyrene (B[a]P), show significant deficiencies in their immune functions, observed in late gestation which persist for at least 18 months. Tumor incidences in these progeny are 8 to 10-fold higher than in controls. We have demonstrated a significant reduction in thymocytes (CD4+ CD8+, CD4+ CD8+ Vbeta8+, CD4+ CD8+ Vgamma2+) from newborn and splenocytes (CD4+ CD8+) from 1-week-old mouse progeny exposed to B[a]P in utero. To investigate possible causes of the observed T cell reduction, we analyzed the thymocytes and splenocytes from progeny and maternal tissues for the presence of B[a]P-DNA adducts. Adducts were detected in maternal, placental and offspring lymphoid tissues at day 19 of gestation, at birth and 1-wk after birth. The presence of B[a]P-DNA adducts in immature T cells may, in part, explain the previously observed T cell immunosuppression and tumor susceptibility in mice exposed to B[a]P in utero. The effects of DNA lesions on progeny T cells may include interference with normal T-cell development. These results provide a possible explanation for the relationship between maternal smoking during pregnancy and childhood carcinogenesis.

In vitro methylation of inorganic arsenic in mouse intestinal cecum.

The capacity of mouse intestinal cecal microflora to methylate inorganic arsenicals (iAs) was examined in vitro under conditions of restricted bacterial growth. Cecal contents incubated under anaerobic conditions at 37 degrees C for 21 hr methylated up to 40% of either 0.1 microM arsenite (iAsIII) or 0.1 microM arsenate (iAsV). Methylarsenic (MAs) was the predominant metabolite; however, about 3% of either substrate was converted to dimethylarsenic (DMAs). Over the first 6 hr, the rate of methylation was several times greater for iAsIII than for iAsV. There was a 3-hr delay in the production of methylated metabolites from iAsV, suggesting that reduction of iAsV to iAsIII before methylation could be rate limiting. Over the concentration range of 0.1 to 10 microM of iAsIII or iAsV, there was an approximately linear increase in the production of MAs and DMAs. There was evidence of saturation or inhibition of methylation at 100 microM of either substrate. Substrate concentration had little effect on MAs/DMAs ratio. Incubation of cecal contents at 0 degrees C abolished methylation of either arsenical. Under aerobic or anaerobic conditions, cecal tissue homogenates produced little MAs or DMAs from either arsenical. Addition of potential methyl group donors, L-methionine and methylcobalamin, into cecal contents significantly increased the rate of methylation, especially for iAsV. Addition of glutathione, but not L-cysteine, had a similar effect. Selenite, a recognized inhibitor of iAs methylation in mammalian tissues, inhibited methylation of either substrate by cecal contents. These data suggest that cecal microflora are a high capacity methylation system that might contribute significantly to methylation of iAs in intact animals.

Metabolism of 1-nitro[14C]pyrene in vivo in the rat and mutagenicity of urinary metabolites.

The metabolic fate of the bacterial mutagen, environmental pollutant and potential carcinogen 1-nitropyrene (NP) has been investigated in the rat. Over half of an i.p. dose (10 mg/kg) of 1-nitro[14C]pyrene was excreted within 24 h of dosing, 15% of the dose in urine and 40% in the faeces. After 96 h greater than 80% of the dose had been recovered. The urinary and fecal metabolites of NP were separated and quantitated by h.p.l.c., then identified by high resolution gas chromatography/mass spectrometry (h.r.g.c./m.s.) and comparison with synthetic reference compounds, where available. Very little (less than 5%) of the dose was excreted unchanged. Urinary metabolites were all excreted in conjugate form, mainly with glucuronic acid. Among the principal metabolite fractions identified were 3-hydroxy-1-nitropyrene and 8-hydroxy-1-nitropyrene (already known as hepatic in vitro metabolites of 1-nitropyrene) and the hitherto unreported metabolites 6-hydroxy-N-acetyl-1-aminopyrene and 8-hydroxy-N-acetyl-1-aminopyrene. Mutagenic activity was detected, by means of the Ames Salmonella (strain TA 98) plate incorporation assay, in the urine of rats dosed with NP. This mutagenicity, unlike that of NP itself, required exogenous metabolic activation. It was predominantly associated with 6-hydroxy-N-acetyl-1-aminopyrene and with the nitropyrene phenols (specific mutagenicity 600 and 700 rev/nmol respectively in the presence of 0.6 mg of S9 protein per plate). The majority of the residual metabolites were polar, refractory to enzymic hydrolysis, and of low mutagenicity. The major proportion of the 14C in feces was not extractable or amenable to enzymic hydrolysis; the extractable fecal metabolites were similar in nature to those in urine.