Potentiation of 2,6-dinitrotoluene genotoxicity in Fischer 344 rats by pretreatment with coal tar creosote.

Pretreatment of male Fischer 344 rats for 5 wk with coal tar creosote, a coal distillation product that is widely used as a wood preservative, potentiated the excretion of urinary mutagens in 2,6-dinitrotoluene (DNT) treated rats. Creosote increased the bioactivation of DNT to significantly greater levels of urinary genotoxic metabolites and/or formed DNA adducts in the liver. A significant increase in the excretion of mutagenic DNT metabolites was observed after the first week of creosote treatment, peaked at wk 3, and then decreased by 33% after 5 wk of treatment. Nevertheless, there was a significant increase (66%) in the formation of DNT-derived DNA adducts in the livers of rats treated with DNT plus creosote at wk 5. Increased cecal beta-glucuronidase activity and reduced small intestinal nitroreductase activity may play roles in the bioactivation of DNT. The excretion of mutagenic DNT metabolites supplies useful information about the bioactivation of DNT; it does not provide a useful index of DNT-derived hepatic DNA adduct formation. Such interactions could be important to predictive risk assessment because the overall cancer risk of such chemical mixtures may exceed the sum of the component risks.

Possible errors in assay for beta-glycosidase activity.

Cecal homogenates were assayed for the enzymes beta-glucosidase, beta-glucuronidase, and beta-galactosidase. Anaerobic incubation with the addition of excess 3,4-dichloronitrobenzene, a substrate for nitroreductase, significantly increased the detection of the beta-glycosidase enzymes' activities.

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.

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.

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.

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, beta-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 beta-glucuronidase activity decreased. By 4 weeks, treated and untreated animal intestinal enzyme activities were indistinguishable.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Effect of lindane on nitroreductase and dechlorinase enzyme activity in the gastrointestinal tract.

A previously reported acceleration of parathion metabolism in the gastrointestinal (GI) tract of lindane-pretreated rats could have been due to either a prolonged residence time of parathion or increased GI nitroreductase activity or both. Thus to determine the effect on GI nitroreductase and dechlorinase activity, 20 mg/kg lindane or 535 mg/kg neomycin were administered daily, by gavage, to weanling F-344 rats. Enzyme activity in the small intestine and cecum were assayed after 2 weeks and 5 weeks of treatment. Neomycin treatment inhibited the activity of both enzymes in the cecum but had no significant effect on enzyme activity in the small intestine, suggesting the presence of mucosal nitroreductase and dechlorinase in the small intestine. In contrast, lindane, which had no effect on enzyme activity in the cecum, significantly increased nitroreductase activity in the small intestine after treatment for 5 weeks. This increased nitroreductase may account for the previously reported lindane-parathion interaction and could influence the metabolism, toxicity, and risk assessment of many other environmental nitro-compounds that become toxic, mutagenic or carcinogenic upon reduction of their nitro-groups.

Effect of lindane on hormonal control of reproductive function in the female rat.

The effect of the gamma isomer of 1,2,3,4,5,6-hexachlorocyclohexane, lindane, on reproductive function in the female rat was examined in two experiments. In the first experiment, chronic treatment with 0, 5, 10, 20, and 40 mg/kg lindane delayed vaginal opening and disrupted ovarian cyclicity until approximately 110 days of age. Thereafter, regular ovarian cycles were present in the majority of females in all dose groups. When killed on the day of vaginal proestrus, the females receiving the two higher doses of lindane had smaller pituitary and uterine weights, lower serum and pituitary luteinizing hormone (LH) and prolactin, and higher pituitary follicle stimulating hormone (FSH) concentrations than the oil-treated control females. Serum estrogen concentrations were not different from controls in the 5 and 20 mg/kg groups, significantly greater than the controls in the 10 mg/kg group, and significantly less than the controls in the group receiving 40 mg/kg. In a second experiment, the uterine weight and pituitary hormone response of 28-day-old, lindane-treated females to a 10-micrograms injection of estradiol benzoate (EB) were investigated. The uteri of the lindane-treated prepubertal females were smaller than controls at 30 hr after EB injection. Furthermore, the EB-induced increase in serum luteinizing hormone, observed at 30 hr after EB injection, was lower in the lindane-treated animals. Similarly, the reduction in pituitary LH, FSH, and prolactin induced by EB was not as great in the lindane-treated animals as in the controls. Serum estrogen concentrations in the lindane-treated animals were not different from controls. These data indicate that lindane may effectively block the response of estrogen-dependent tissues to this ovarian steroid hormone and that this apparent antiestrogenic effect of lindane is responsible for the disturbances observed in the neuroendocrine control of ovarian function in the rat.

Comparison of in vivo and in vitro methods for assessing the effects of repeated dosing with carbon tetrachloride on the hepatic drug-metabolizing enzyme system.

The effect of 7 daily i.p. injections of 0, 2, 20, or 200 microliters/kg carbon tetrachloride on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay, employing lindane (gamma-hexachlorocyclohexane), and by a battery of in vitro enzyme assays. The data in this study indicated that repeated administration of CCl4 for 7 days significantly increased phase I and phase II reactions in vivo and in vitro. Though there were differences between the responses of the in vivo and in vitro assays, this is the first report of increased hepatic drug-metabolizing enzyme activity from repeated treatment with CCl4.

Comparison of in vivo and in vitro methods for assessing the effects of carbon tetrachloride on the hepatic drug-metabolizing enzyme system.

The effect of a single i.p. injection of 0, 20, 200, and 1000 microliter/kg carbon tetrachloride on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay employing lindane (gamma-hexachlorocyclohexane) and by a battery of in vitro enzyme assays. The data in this study indicated that carbon tetrachloride had a biphasic influence on the phase I reactions with the lowest dose inducing a significant increase in enzyme activity while the highest dose produced significant inhibition. Significant CCl4-induced reductions in glucuronyltransferase and sulfotransferase activities were also observed while the effect on glutathione-S-transferase was ambiguous. The in vivo and in vitro assays showed good agreement.

Possible antiestrogenic activity of lindane in female rats.

During chronic peroral (PO) treatment of weanling, female Fischer 344 rats with daily injections (0.069 mmol/kg) of either 1,1'-(2,2,2-trichloroethylidene) bis [4-chlorobenzene] (p,p'-DDT), 2,4-dichlorophenoxy acetic acid (2,4-D), or gamma-hexachlorocyclohexane (lindane), the lindane treatment induced a significant 20% increase in body weight after 110 days. Further investigation with 0, 5, 10, 20, and 40 mg/kg lindane confirmed a significant increase in average body weight gain at the two highest doses after ten weeks of treatment. Significantly greater food consumption was observed, and the Lee index indicated that lindane treatment induced obesity. In addition to obesity, lindane caused a delay in vaginal opening, disrupted estrous cycling, reduced pituitary and uterine weight, and elevated food consumption during proestrus (when appetite is normally suppressed by estradiol). These responses suggest that, by inducing alterations in the reproductive function of the female rat and by interfering with hormonal regulation of energy balance, lindane may be antiestrogenic rather than estrogenic as previously proposed.

Comparability of in vitro and in vivo methods for the determination of alterations in drug metabolism.

The above series of experiments taken in toto suggest the usefulness of lindane as a model substrate for studying the effects of a variety of compounds on drug metabolism in vivo. Excellent correlations were observed in comparison with the in vitro measurements both qualitatively and quantitatively. Unlike some of the other compounds discussed lindane offers some distinct advantages. One is that because the metabolites can be monitored in the urine, it is non-invasive in nature. A second is that a number of mixed function oxidase pathways (phase I reactions) can be determined at the same time. This would be of great importance if the effect of a compound is rather selective and does not alter the single pathway measured in the metabolism of other substrates which have been suggested as model compounds. However, the tradeoff is obviously the need for more analytical work. A third advantage is the ability of the system to detect changes in conjugative or phase II reactions at the same time. Further studies will be necessary with all of these model substrates to detect their usefulness and their limitations.

Comparison of in vivo and in vitro methods for assessing the effects of bromobenzene on the hepatic-metabolizing enzyme system.

The effect of daily i.p. injections of 0, 0.05, 0.5 and 5.0 mmol/kg bromobenzene for 1 week on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay employing lindane (gamma-hexachlorocyclohexane) and by a battery of in vitro enzyme assays. The data in this study indicated that repeated pretreatment with bromobenzene for 1 week stimulated a dose-related increase in phase I reactions while inducing phase II reactions at the high dose (5 mmol/kg bromobenzene). The in vivo and in vitro assays showed good agreement.

Saturation of lindane metabolism in chronically treated (YS x VY) F1 hybrid mice.

The organochlorine insecticide lindane (gamma-hexachlorocyclohexane) induces hepatomas in select strains of mice including two of three phenotypic classes of (YS X VY) F1 hybrid mice. In contrast, lindane does not induce hepatomas in rats and other strains of mice. It has been suggested that variations in the biotransformation of lindane may play a role in the different susceptibility of rodents to lindane-induced hepatomas. This study reports the effect of chronic treatment with 160 ppm dietary lindane on the comparative metabolism and disposition of this insecticide in obese yellow Avy/a, lean pseudoagouti Avy/a, and lean black a/a phenotypes of (YS X VY) F1 hybrid female mice at 17, 30, 56, and 86 wk of age. At 24 h prior to necropsy, all mice were dosed po with 18 mg lindane (containing 55 muCi [U-14C]lindane)/kg. Urine, feces, and expired air were sampled for analysis. Data indicated that metabolism of lindane and excretion of its metabolites by these mice differ significantly from those of rats that are resistant to lindane-induced hepatomas. Treatment of the mice with 160 ppm lindane in the diet appeared to saturate the elimination pathways and resulted in an increased tissue burden of the insecticide and its metabolites in the older animals. Results indicate that differences in lindane metabolism and disposition observed in the (YS X VY) F1 hybrid mice were associated with chronic lindane treatment, aging, and obesity but not with genotype.

A comparison of in vitro and in vivo methods for evaluating alterations in hepatic drug metabolism following mercuric chloride administration.

Mercuric chloride was administered once i.p. to female Fischer-344 rats at doses of 0, 0.2, 0.6 and 1.8 mg/kg. Although there were no alterations in the urinary excretion of lactate dehydrogenase, significant elevations in the activities of urinary (U) alkaline phosphatase, glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) indicated that mercuric chloride was nephrotoxic. There was no evidence of hepatotoxicity as hepatic glucose-6-phosphatase and serum sorbitol dehydrogenase were essentially unaffected by mercuric chloride administration. The activities of ethylmorphine demethylase, hexobarbital oxidase and aldrin epoxidase determined in vitro were not inhibited by mercuric chloride although aniline hydroxylase activity was decreased. Of the four phase-II reactions measured, only the glucuronidation of chloramphenicol was diminished by treatment with mercuric chloride. Results from the in vivo studies on the metabolism of lindane, which indicated no change in the excretion of free or conjugated metabolites, were in close agreement with the in vitro data suggesting that the nephrotoxic effects of mercuric chloride do not alter the urinary excretion of the model substrate lindane.

Use of gamma-hexachlorocyclohexane (lindane) to determine the ontogeny of metabolism in the developing rat.

The compound lindane (gamma-hexachlorocyclohexane) has been used to study the ontogeny of metabolism in the developing Fischer 344 rat. The distribution and metabolic fate of lindane at 2, 9, 16, and 23 d of age was investigated following subcutaneous administration of lindane at 20 mg/kg containing 0.5 microCi [U-14C]lindane in peanut oil. Groups of 10 pups (5 male and 5 female) were sacrificed at 4-h intervals during the 24-h period following dosing. Adrenals, blood, brain, heart, lung, liver, and kidneys were analyzed for radioactivity. Urine samples were analyzed for radioactivity and metabolites of lindane. There was a significant age-dependent increase in the metabolism of lindane in the rat. High levels of radioactivity in the lung and increased reductive dechlorination suggest that the lung may play a greater role in metabolism of lindane by young rats. Oxidative phase I reactions increased significantly, while anaerobic reductive dechlorination of lindane to 4-chlorophenylmercapturic acid decreased significantly with age. Phase II sulfate and glutathione conjugations decreased significantly and glucuronide conjugation increased significantly with age. Metabolism and excretion of lindane appear to parallel development of the hepatic enzymes involved in phase I and phase II reactions.