Robert W. Chadwick

Dehydrogenation: A previously unreported pathway of lindane metabolism in mammals

Abstract This study presents evidence for the dehydrogenation of lindane by a hepatic microsomal mixed-function oxidase system. Preliminary investigation established that the incubation of lindane with rat liver homogenates produces a chlorinated, nonpolar compound identified as hexachlorocyclohexene. Differential centrifugation resulted in the sedimentation of most of the dehydrogenase activity in the microsomal fraction. Optimum in vitro assay conditions were established and it was found that the dehydrogenase system required molecular oxygen and reduced pyridine nucleotide coenzyme for maximum activity. Inhibition by SKF 525-A and CO suggested that the enzyme was cytochrome P-450 dependent. Lack of inhibition by cyanide indicated that the cytochrome b5 desaturase system was probably not involved. Pretreatment of rats with DDT, which stimulates lindane metabolism, also induced significantly higher dehydrogenase activity. Both the in vivo and in vitro metabolism of hexachlorocyclohexene produced previously identified lindane metabolites. The existence of a cytochrome P-450 dependent mixed-function oxidase which catalyzes the dehydrogenation of lindane has not previously been reported and may be of importance in the metabolism of other xenobiotics.

Comparative effect of pretreatment with phenobarbital, Aroclor 1254, and β-naphthoflavone on the metabolism of lindane☆☆☆

Abstract An attempt was made to distinguish different patterns of microsomal enzyme induction by phenobarbital, β-naphthoflavone, and Aroclor 1254 on the biotransformation and excretion of the organochlorine insecticide lindane. Treated groups of six weanling female Sprague-Dawley rats, individually housed in metabolism cages, received diets containing either 500 ppm Aroclor 1254, 356 ppm phenobarbital, or 418 ppm β-naphthoflavone. After 1 week all animals, except one group of controls, were dosed p.o. with 1.89 mg lindane (containing 1.63 μCi [U- 14 C]lindane). Twenty-four hours later the rats were sacrificed and urine, feces, expired air, and tissue samples were taken for analysis of radioactivity. Hepatic cytochrome P -450 content, microsomal phospholipid content, and the enzyme activity involved in the dehydrogenation of lindane, the dechlorination of lindane, and the hydroxylation of the intermediate hexachlorocyclohexene were determined in vitro . Moreover, the effect of pretreatment on the excretion of radioactivity and the distribution of eight lindane metabolites was examined. Even though the rate of lindane metabolism was unchanged by the β-naphthoflavone pretreatment, results of the study indicated that all three pretreatments significantly altered lindane metabolism. The pretreatments differed from one another in that they selectively altered specific metabolic pathways.

The identification of three previously unreported lindane metabolites from mammals

Abstract Previously unreported metabolites from the urine of rats treated with lindane have been identified as configurational isomers of 2,4,5,6- and 2,3,4,6-tetrachloro-2-cyclohexen-1-ol. In addition, an intermediate metabolite from the incubation of lindane with liver preparations, under N2, has been identified as the configurational isomer γ-3,4,5,6-tetrachlorocyclohex-1-ene. The pathways leading to these metabolites appear to have an important role in the metabolism of lindane by mammals.

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

Abstract 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 32 P-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.

Effects of age and obesity on the metabolism of lindane by black a/a, yellow Avy/a, and pseudoagouti Avy/a phenotypes of (YS x VY) F1 hybrid mice.

Lindane (gamma-hexachlorocyclohexane) has been shown to produce hepatomas in some strains of mice but not in others. Genetic factors and/or altered metabolism may play a role in the susceptibility to lindane-induced hepatomas. This study reports the effect of age and obesity on the comparative metabolism and disposition of lindane in obese yellow Avy/a and in lean pseudoagouti Avy/a and black a/a phenotypes of (YS x VY) F1 hybrid female mice at 8, 17, 30, 56, and 86 wk of age. At 24 h prior to sacrifice the mice were dosed p.o. with 18 mg lindane (containing 55 microCi [U-14C]lindane/kg). Aging altered the biotransformation of lindane such that while the excretion of lindane and its metabolites declined, the proportion of conjugated and polar metabolites increased. Tissue storage was elevated in older animals. In the yellow Avy/a mice, which are known to have a predisposition to the formation of hepatomas, there was accelerated and prolonged growth, reduced metabolite excretion, a greater proportion of conjugated metabolites, and higher dechlorinase activity compared to that of their pseudoagouti Avy/a and black a/a siblings.

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.

Effect of lindane on intestinal nitroreductase, azoreductase, β-glucuronidase, dechlorinase, and dehydrochlorinase activity

Abstract It has been reported that pretreatment of rats with the organochlorine insecticide γ-hexachlorocyclohexane (lindane) significantly increases nitroreductase activity as well as the biotransformation of parathion to aminoparathion in the intestinal tract of rats. Such an interaction could affect the metabolism, toxicity, and risk assessment of many environmental nitro compounds that become toxic, mutagenic, or carcinogenic upon reduction of their nitro groups. This study was conducted to determine: (i) the effect of lindane on the activity of five microbial enzymes in the intestinal tract; (ii) possible regional differences in the effect of lindane; (iii) whether the enzymes selected for study were exclusively of microbial origin; and (iv) how lindane produced the effect. Weanling male and female Fischer 344 rats received daily po injections of 20 mg/kg lindane in peanut oil for 2 or 5 weeks while controls received the vehicle. After 4 weeks, half the remaining treated animals were transferred to isolation cubicles and received antibiotics in addition to their regular treatment. At autopsy, the small intestine and cecum were aseptically excised, homogenized, and incubated with a dimethyl sulfoxide mixture of p,p′ -DDT, 3,4-dichloronitrobenzene, p -nitrophenyl β- d -glucuronide, and methyl orange. After 2 weeks, lindane produced significantly higher β-glucuronidase activity in the small intestine and significantly greater dechlorinase and azoreductase activity in the cecum. After 5 weeks, lindane significantly increased nitroreductase and azoreductase activity in the small intestine. Coadministration of antibiotics with lindane markedly reduced the microbial flora in both the small intestine and cecum. However, whereas enzyme activity in the cecum of the treated rats was decimated, enzyme activity in the small intestine of these animals was reduced to a level not significantly below that of the controls. Thus, while increased enzyme activity in the small intestine of the lindane-treated rats appears to be of microbial origin, substantial amounts of mucosal enzyme may also be present in this region.

Saturation of lindane metabolism in chronically treated (YS × 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.

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)

Use of γ‐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.