Azoreductase Research Articles

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.

A comparison of intraperitoneal and oral gavage administration in comet assay in mouse eight organs

One of the important advantages of the comet assay is its ability to detect genotoxicity in many different organs. Since the exposure route of the test compounds is likely to influence the genotoxicity detected in a given organ, it is an important factor to consider when conducting the assay. In this study, we compared the effects of numerous model compounds on eight organs when administered to mice by intraperitoneal (i.p.) injection and oral (p.o.) gavage. Groups of four mice were treated once i.p. or p.o. at the identical proportion of LD50 for each route, and the stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were sampled 3, 8, and 24 h after treatment. For 19 of the 20 tested mutagens with various modes of action, genotoxicity in some organs varied with treatment route; only the genotoxicity of methyl methane sulfonate was not affected. Treatment route, however, did not produce a qualitative difference in the genotoxicity of promutagens at the sites of conversion to ultimate mutagens, with aromatic hydrocarbons as the exception. When chemicals with positive responses in at least one organ were considered to be comet assay-positive, the administration route made no difference. Since azo reduction is mediated by azo reductase synthesized in the gastrointestinal wall and by gut microflora and i.p.-administered azo dyes bypass their activation site (colon), the administration route is expected to make a difference in their in vivo genotoxicity. Direct-acting mutagens are expected to affect the mucosa of the gastrointestinal tract when given p.o. For those mutagens, however, the administration route did not make a qualitative difference in gastrointestinal tract genotoxicity. Moreover, although the gastrointestinal mucosa is the first site to be exposed to p.o. administered agents, the peak times in the stomach tended to be the same as in most other organs. Based on those results, we concluded that the genotoxicity at high exposures was due to a systemic effect, and that both routes are acceptable for the comet assay when the liver and gastrointestinal organs are sampled, so long as appropriate dose levels for systemic exposure are selected for each route.

The disinfection by-products dichloro-, dibromo-, and bromochloroacetic acid impact intestinal microflora and metabolism in Fischer 344 rats upon exposure in drinking water.

Human consumption of chlorinated drinking water has been linked epidemiologically to bladder, kidney, and rectal cancers. The disinfection by-product (DBP) dichloroacetic acid is a hepatocarcinogen in Fischer 344 rats and B6C3F1 mice. The objective of this study is to determine the effect of the DBPs dichloro-, bromochloro-, and dibromoacetic acids (DCA, BCA, DBA) on intestinal microbial populations and their metabolism, with emphasis on enzymes involved in the bioactivation of procarcinogens and promutagens. One-month-old male Fischer 344 rats were provided water ad libitum containing 1 g/l DCA, BCA, or DBA for up to 5 weeks. At 1, 3, and 5 weeks of treatment, beta-glucuronidase (GLR), beta-galactosidase (GAL), beta-glucosidase (GLU), nitroreductase (NR), azoreductase (AR), and dechlorinase (DC) activities were determined in cecal and small and large intestinal homogenates. After 5 weeks of treatment, intestinal populations were enumerated on selective media. Cecal GAL (DCA, BCA, DBA) and GLR (DCA, DBA) activities were reduced after 1 and 3 weeks of treatment and GAL activity was elevated at 5 weeks (BCA). Large intestinal GAL (DCA, BCA) and GLU (DCA, BCA, DBA) activities were elevated after 5 weeks of treatment. Week 5 cecal AR (DCA, BCA, DBA), NR (DCA), and DC (DCA, DBA) activities were reduced. Even though some significant changes in intestinal populations were observed, use of selective media was not sensitive enough to explain fluctuations in enzyme activity. Haloacetic acids in the drinking water alter intestinal metabolism, which could influence bioactivation of promutagens and procarcinogens in the drinking water.

The function of cytoplasmic flavin reductases in the reduction of azo dyes by bacteria.

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an "azo reductase." The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic "azo reductases," which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.

Reduction of azo dyes by redox mediators originating in the naphthalenesulfonic acid degradation pathway of Sphingomonas sp. strain BN6

The anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 was analyzed. Aerobic conversion of 2-naphthalenesulfonate (2NS) by cells of strain BN6 stimulated the subsequent anaerobic reduction of the sulfonated azo dye amaranth at least 10-fold. In contrast, in crude extracts, the azo reductase activity was not stimulated. A mutant of strain BN6 which was not able to metabolize 2NS showed increased amaranth reduction rates only when the cells were resuspended in the culture supernatant of 2NS-grown BN6 wild-type cells. The same increase could be observed with different bacterial strains. This suggested the presence of an extracellular factor which was formed during the degradation of 2NS by strain BN6. The addition of 1,2-dihydroxynaphthalene, the first intermediate of the degradation pathway of 2NS, or its decomposition products to cell suspensions of the mutant of strain BN6 (2NS-) increased the activity of amaranth reduction. The presence of bacterial cells was needed to maintain the reduction process. Thus, the decomposition products of 1,2-dihydroxynaphthalene are suggested to act as redox mediators which are able to anaerobically shuttle reduction equivalents from the cells to the extracellular azo dye.

Localization of the Enzyme System Involved in Anaerobic Reduction of Azo Dyes by Sphingomonas sp. Strain BN6 and Effect of Artificial Redox Mediators on the Rate of Azo Dye Reduction

The effect of different artificial redox mediators on the anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 or activated sludge was investigated. Reduction rates were greatly enhanced in the presence of sulfonated anthraquinones. For strain BN6, the presence of both cytoplasmic and membrane-bound azo reductase activities was shown.

Investigations of Bifidobacterial Ecology and Oligosaccharide Metabolism in a Three-Stage Compound Continuous Culture System

Background: Several different types of in vitro fermentation systems are currently employed to investigate pro- and prebiotic activities in the human large intestinal microbiota, ranging from simple batch cultures, with or without stirring and pH control, to more complex models involving pH controlled single and multiple-component continuous culture systems. Methods: In this investigation, we used a three-stage continuous culture model to study the activities of colonic bacteria. This fermentation system reproduces several of the nutritional and environmental characteristics of the proximal large intestine (vessel 1) and the distal colon (vessels 2 and 3), and was validated using bacteriological, metabolic and chemical measurements made with intestinal material obtained from different regions of the large bowel. In this paper, we report studies on prospective probiotic effects of Bifidobacterium longum NCFB 2259 in relation to other bacterial populations, production of tyrosine and phenylalanine metabolites, and bacterial synthesis of enzymes involved in the formation of putatively genotoxic metabolites, including ß glucosidase (GS), arylsulphatase (AS), ß-glucuronidase (GN), nitroreductase (NR) and azoreductase (AR). Results: Bacterial activities at two different retention times were studied (31.1 and 68.4 h), which correspond to large intestinal transit times. At R = 31.4 h, significant probiotic effects were observed with respect to reductions in GS and GN, upon adding B. longum. However, despite the fact that this organism does not ferment aromatic amino acids or produce significant amounts of genotoxic enzymes, dysbiotic manifestations occurred in that both NR synthesis and dissimilatory tyrosine metabolism were stimulated. In contrast, at R = 68.4 h, GS formation increased between five and 20-fold, while GN and NR activities increased by a factor of two after adding the bifidobacterium. These data are reviewed in relation to potential health hazards that may be encountered with long-term probiotic administration. In the prebiotic experiments, the three-stage fermentation system was operated at R = 65 h. Oligofructose was added to VI to give an initial concentration of 30 grams per litre, when the system was in steady state, to study its effects on a number of experimental parameters including bifidogenicity, bacterial growth, fermentation product formation and mutagenicity. After addition of the oligosaccharide, a multiplicity of effects were observed in VI, where synthesis of NR and AR, bifidobacterial populations and overall fermentation processes were stimulated, although these influences progressively diminished in V2 and V3. Conclusions: These studies indicate that bacterial metabolism and putative beneficial consequences associated with the breakdown of readily fermentable prebiotics in the large intestine may in some circumstances be spatially and temporally limited to the proximal bowel.

RAFFINOSE AND LACTIC ACID BACTERIA INFLUENCE CAECAL FERMENTATION AND SERUM CHOLESTEROL IN RATS

The effects of diets containing raffinose with or without Lactobacillus acidophilus plus Streptococcus thermophilus were investigated in rats. Raffinose induced a decrease in body weight. Caecal weight was increased by feeding raffinose plus lactic acid bacteria (LAB). Raffinose ingestion increased the caecal concentration of lactobacilli but decreased numbers of streptococci. Concurrent administration of raffinose plus LAB decreased the staphylococci and clostridia numbers. Caecal pH was significantly reduced by feeding raffinose or raffinose plus LAB. Adding LAB to a raffinose containing diet induced a decrease in azo reductase activity of the caecal contents. Raffinose, with or without LAB, increased total volatile fatty acids (VFA) production. Caecal butyrate was higher with the raffinose and LAB diet than with raffinose alone or control diets. Although total triglycerides in plasma was lowered by raffinose, this sugar did not affect concentrations of plasma cholesterol or high density lipoproteins (HDL). However, a significant increase in the HDL cholesterol was observed in response to a concurrent administration of raffinose plus LAB. Copyright © 1996 Elsevier Science Inc.

Bacterial Reduction of Azo Compounds as a Model Reaction for the Degradation of Azo-Containing Polyurethane by the Action of Intestinal Flora

Abstract Two types of model compounds containing an azo aromatic group were synthesized, and their reduction kinetics in an anaerobic culture of intestinal flora was investigated for insight into the degradation mechanism of the azo-containing polyurethane which can be specifically degraded by the action of azo reductases which are released by intestinal floras. A water-soluble azo compound, 3,3′ -azodibenzenemethanol (ADM), was readily reduced into its hydrazo form and then to the amine form, 3-aminobenzenemethanol (ABM). The reduction rate from azo to hydrazo was much faster than that from hydrazo to amine. In the case of a water-insoluble azo compound, 3,3′-azodibenzenemethanol bis(N-phenylcarbamate) (ADM-PC), in which the hydroxy groups of ADM were capped by N-phenylcarbamoyl groups, the reduction was stopped in the first step, and only the hydrazo form was produced. These data indicated that the azo-containing polyurethane which is hydrophobic in nature should be degraded by the azo-hydrazo reduction of the azo groups without the chain breakage occurring.

Peculiar toxicity mechanism of paraquat in Escherichia coli

Disinfection of drinking water has been one of the greatest public health successes. Numerous halogenated disinfection by-products (DBPs) occur and chronic ingestion has been associated with an increased risk for colorectal cancer in human populations. Because the intestinal microbiota can bioactivate xenobiotics, studies have been performed to examine the effects of individual DBPs on intestinal microbial metabolism. No studies have been conducted on a defined mixture of DBPs to determine if there is an enhancement of response to a mixture. Ten-week-old male Long-Evans rats were treated in their drinking water for 17 weeks with 0.4 g/l potassium bromate, 1.8 g/l chloroform, 0.7 g/l bromodichloromethane (BDCM), 0.07 g/l 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), or a mixture of the four chemicals or distilled water. Cecal nitroreductase (NR), azoreductase (AR), dechlorinase (DC), β-glucuronidase (GLR), β-galactosidase (GAL), and β-glucosidase (GLU) were assayed. No change in GLU or GLR activity was detected after treatment. BDCM treatment reduced DC and GAL activities and elevated NR and AR activity. GAL, AR, and NR activities were significantly different after treatment with bromate, chloroform, BDCM, and MX, but not the mixture. DC activity after chloroform-, MX-, or BDCM-treatment was significantly below control levels. The present study shows that changes in intestinal microbial metabolism do occur after treatment with individual and a mixture of DBPs but the changes were not additive in the mixture group.

In vivo evaluation of a colon-specific drug delivery system: an absorption study of theophylline from capsules coated with azo polymers in rats.

Azo polymers based upon 2-hydroxyethyl methacrylate, methyl methacrylate, and methacrylic acid, and containing N,N'-bis [(methacryloyloxyethyl)oxy(carbonylamino)]azobenzene as azo aromatic agent were evaluated in vivo as coating for colon-specific drug delivery. The gastrointestinal absorption of theophylline from capsules coated with the azo polymers was examined in the proximal part of the small intestine and the cecum of male Wistar rats. The capsules were surgically inserted in the region of interest. The plasma concentration of the drug was higher when the capsules were inserted in the cecum as compared to the small intestine. The appearance of theophylline in the plasma when capsules were administered in the small intestine can be attributed to simple diffusion of the drug through the swollen polymer coating. Release and absorption from the cecum is the combined results of diffusion and degradation of the azo polymer coatings by bacterial azo reductase.

Microbial succession and intestinal enzyme activities in the developing rat.

The succession of gut bacteria and selected intestinal enzyme activities in developing 7-35-d-old rats was studied. Aerobes and anaerobes were identified as members of four broad major bacterial groups, i.e. Gram-positive rods, Gram-positive cocci, Gram-negative rods and obligate anaerobes. The enzyme activities of nitro and azo reductases, beta-glucuronidase, dechlorinase and dehydrochlorinase were determined by anaerobic incubation of intestinal homogenates with 3,4-dichloronitrobenzene, methyl orange, p-nitrophenyl-beta-D-glucuronide, and p,p'-DDT respectively. Nitroreductase and azo reductase activities increased significantly with the appearance of anaerobes in the large intestine. No increase in either nitroreductase or azo reductase activities in the small intestine was found. The early and high level of beta-glucuronidase activity in the small and large intestines coincided with high numbers of coliforms recovered in 7 and 14 d animals. Dehydrochlorinase activity appeared early but was undetectable at both 21 and 28 d. Its activity increased at 35 d. Dechlorinase activity was variable in development. The rapid changes in the microbial flora and intestinal enzyme activities may influence the susceptibility of pre-pubescent rats to a variety of toxicants. Therefore, age-dependent toxicity may be important in the risk assessment of some environmental chemicals.

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.

The relation between swelling properties and enzymatic degradation of azo polymers designed for colon-specific drug delivery.

Copolymers of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), and terpolymers of HEMA, MMA, and methacrylic acid (MA) were synthesized in the presence of N,N'-bis(methacryloyloxyethyloxycarbonylamino)azobenzene (B(MOEOCA)AB) and evaluated as coating materials for colonic targeting. The release of ibuprofen, a model drug, from capsules coated with the azo polymers was investigated in vitro. The release medium was made up of sonicated rat cecal content, benzyl viologen, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and nicotine amide dinucleotide phosphate (NADP) in phosphate buffer (pH 6.8, 0.05M). The drug-release profiles indicate that the degradation of the azo polymer coatings depends on their degree of swelling, due to a higher accessibility of the azo bonds for the bacterial azo reductase. The best results were obtained with azo polymers containing MA: 98.7 (+/- 1.1) % of ibuprofen was released after 19 hours residence in the release medium, while only 26.2 (+/- 4.9) % in the control experiment. These findings demonstrate that azo polymers are promising materials for delivering drugs selectivity to the colon.

2,4,5-Trichlorophenoxyacetic acid influence on 2,6-dinitrotoluene-induced urine genotoxicity in Fischer 344 rats: Effect on gastrointestinal microflora and enzyme activity

2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) and 2,6-dinitrotoluene (2,6-DNT) are hazardous chemicals that have potential harmful effects. 2,6-DNT is recognized as a hepatotoxicant while 2,4,5-T, a component of Agent Orange, is also suspect. 2,6-DNT requires both oxidative and reductive metabolism to elicit genotoxic effects. To determine what effect 2,4,5-T had on 2,6-DNT metabolism, intestinal enzymes, microbial populations, and urine mutagenicity were examined during 2,4,5-T treatment. Weanling Fischer 344 male rats were treated daily with 54.4 mg/kg 2,4,5-T by gavage for 4 weeks. One, two, and four weeks after the initial 2,4,5-T dose, rats were administered (po) 2,6-DNT (75 mg/kg) and urine was collected for 24 hr in metabolism cages. Azo reductase, nitroreductase, β-glucuronidase, dechlorinase, and dehydrochlorinase activities were examined concurrently. Treatment of rats for 1 week reduced the transformation of 2,6-DNT to mutagenic urinary metabolites. This was accompanied by a decrease in the fecal anaerobic microorganisms. The elimination of Lactobacillus fermentum from the small intestine and cecum of treated animals accompanied a significant increase in oxygen-tolerant lactobacilli and other unidentified aerobic microorganisms. However, there were no significant alterations in the intestinal enzyme activities examined. By 2 weeks of 2,4,5-T treatment, microbiota and urine genotoxicity returned to the levels observed in control animals. This trend continued for the duration of the experiment. After 2 weeks, while cecal nitroreductase and azo reductase activities increased, small intestinal β-glucuronidase activity decreased. By 4 weeks, treated and untreated animal intestinal enzyme activities were indistinguishable. The transient increase in azo reductase and nitroreductase after treatment with 2,4,5-T for 2 weeks may have been counteracted by the reduced β-glucuronidase activity, thus resulting in no change in 2,6-DNT-derived urine mutagenicity. However, other enviromental chemicals, unaffectd by β-glucuronidase, potentially could be activated by 2,4,5-T exposure.

Degradation of azo dyes by algae

The degradation of azo dyes by algae was evaluated and it was found that certain algae can degrade a number of azo dyes to some extent. The reduction rate appears to be related to the molecular structure of the dyes and the species of algae used. The azo reductase of algae is responsible for degrading azo dyes into aromatic amine by breaking the azo linkage. The aromatic amine is then subjected to further metabolism by algae. It is proposed in this paper that in stabilization ponds, algae can play a direct role in the degradation of azo dyes, rather than only providing oxygen for bacterial growth.

Potentiation of 2,6-dinitrotoluene genotoxicity in Fischer 344 rats by pretreatment with pentachlorophenol

The organochlorine pesticide, pentachlorophenol, a potent sulfotransferase inhibitor, reportedly reduces the binding of 2,6-dinitrotoluene, an industrial hepatocarcinogen to hepatic DNA by 95% after a single i.p. injection. Activation of 2,6-dinitrotoluene to genotoxic metabolites involves enzymes in both the liver and the intestinal flora. Since pentachlorophenol also has bactericidal activity and induces hepatic mixed function oxidase activity after longer treatment, the effect of pentachlorophenol on intestinal enzyme activity and the biotransformation of 2,6-dinitrotoluene to genotoxic metabolites was studied after 1, 2, 4, and 5 weeks of treatment. Male Fischer 344 rats were dosed daily, by gavage, with either 20 mg/kg pentachlorophenol or the peanut oil vehicle. After 1, 2, 4, and 5 weeks, select control and treated animals were injected p.o. with 75 mg/kg 2,6-dinitrotoluene and transferred to metabolism cages, where urine was collected for 24 hr and tested for mutagenic activity by the Ames Salmonella typhimurium reversion assay. At 2 and 4 weeks, six control and six treated animals were sacrificed and nitroreductase, azo reductase, β-glucuronidase, dechlorinase, and dehydrochlorinase activities were analyzed in homogenates of the small intestine, large intestine, and cecum. At 5 weeks, hepatic DNA adduct formation was assayed by the 32P-postlabeling of DNA. Results from this study indicated that pentachlorophenol accelerated the biotransformation of 2,6-dinitrotoluene to genotoxic metabolites and potentiated the formation of 2,6-dinitrotoluene-induced DNA adducts in the liver. This is the first report of a chemical interaction leading to increased DNA adduct formation and indicates that chemical interactions could be important to risk assessment since they alter the relationship between exposure, dose, and the effect of genotoxicants.

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.

Degradation of azo dyes by cell‐free extract from Pseudomonas stutzeri

The degradation of several azo dyes by the cell‐free extract from Pseudomonas stutzeri has been studied. The compounds 2‐carboxy‐4′‐dimethylaminoazobenzene, 4‐carboxy‐4′‐dimethylaminoazobenzene, 2‐carboxy 4′‐die thy lam inoazobenzene, 2‐met hoxycarbonyl‐4′‐dime t h y lam inoazobenzene, 4‐dime t h y lam inoazobenzene sulphonic acid, and 2‐hydroxy‐1 ‐naphthaleneazobenzenesulphonic acid were appreciably degraded by the cell‐ f ree extract. The cell‐ f ree extract was quite specific for 2‐ca rboxy‐4 ′‐diet h y lam inoazo benzen e, 2 met hoxyca rbonyI4′‐dime t h y lam inoazo benzene, and 2‐ca rboxy4′‐dime t h y lam inoazo benzene, which a reequal in their redox potential. Thus, it is suggested that the substrate specificity is characterised by a log Pdependence. The presence of azo reductase in the cell‐free extract was alsoconfirmed by electrophoresis.

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, β-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.