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.

Ascorbic acid requirements and metabolism in relation to organochlorine pesticides.

Those organochlorine pesticides which possess both high lipoid solubility and high resistance to biodegradation are prone to accumulation in animal tissues and produce relatively long-term effects as toxicants. Such compounds, typified by DDT, Dieldrin, and Lindane, are profound inducers of hepatic microsomal enzymes, including parts of the glucuronic acid and ascorbic acid biosynthetic pathways. Consequently, administering such pesticides to rats in accompanied by enhanced formation and excretion of D-glucuronic acid and L-ascorbic acid, or D-glucaric acid in the case of guinea pigs. Secondarily, the efficiency in biodegrading the pesticides is reduced in ascorbic-acid-deficient guinea pigs with correspondingly greater residue accumulation in tissue. This would aggravate chronic toxic effects of the compounds. Finally, the capacity of the liver to adapt to the presence of such toxicants through enhanced microsomal enzymatic levels appears to be sensitive to its ascorbate status. Impaired enzyme induction is apparent quite early during ascorbic acid depletion in guinea pigs. The enhanced turnover of ascorbate produced by such pesticides, the poor enzymatic adaptation to them during ascorbate depletion and the dependency of the oxidase system upon adequate ascorbate, all point to the central significance of ascorbate status in the liver, and possibly other tissues, as a determinant of their chronic toxicity.

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.

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.

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.

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.

Comparison of in vivo and in vitro methods for assessing effects of allyl alcohol on the liver.

Allyl alcohol was administered intraperitoneally (i.p.) to female Fischer 344 rats at doses of 0, 3, 10 and 30 mg/kg daily for 7 days. Plasma sorbitol dehydrogenase was minimally elevated. No dose-related changes were observed in hexobarbital oxidation, aniline hydroxylation, or ethylmorphine demethylation. Aldrin epoxidation was slightly elevated. Naphthol glucuronidation and glutathione-S-transferase activity with 1,2-dichloro-4-nitrobenzene were increased. Results from in vivo studies on the metabolism of lindane were in close agreement with the in vitro measurements suggesting that daily treatment for one week with allyl alcohol at doses of 3, 10 and 30 mg/kg has no significant effect on phase I pathways, has a selective effect on phase II pathways and, under the conditions of this experiment, has minimal hepatotoxic effects in these rats.

Comparison of in vitro methods and the in vivo metabolism of lindane for assessing the effects of repeated administration of ethanol on hepatic drug metabolism.

In vitro methods of assessing alterations in drug metabolism and the measurement of lindane metabolites in urine were compared for their ability to determine the influence of ethanol on drug metabolism. Ethanol was administered intraperitoneally (i.p.) to young adult female rats daily for 7 days at doses of 0.12, 0.60 and 3.0 ml/kg. No alterations were observed in ethylmorphine demethylation, hexobarbital oxidation or glucuronyltransferase. Aniline hydroxylation was decreased at the high dose level and aldrin epoxidation was increased at the intermediate dose. In vivo only the high dose of ethanol produced significant changes with significant increases observed for the oxidation of lindane to alcohol metabolites, the glucuronidation of the alcohol but not the chlorophenol metabolites, and glutathione conjugation. The latter increase was also observed in vitro. The in vivo and in vitro data suggest a minimal effect of ethanol on drug metabolism at low levels of administration.

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

The effect of daily i.p. injections of 0, 1, 10 and 80 mg/kg phenobarbital 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-HCH) and by a battery of in vitro enzyme assays. Comparison of the dose-response curves of the in vivo and in vitro assays indicated that urinary metabolites of lindane provided a good index of phenobarbital-induced change in both phase I and phase II reactions.

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.

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.

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.

Investigation of HCB as a metabolite from female rats treated daily for six days with lindane.

The biotransformation of lindane to hexachlorobenzene (HCB) by male rats was recently reported. Since HCB has been widely detected in human milk samples, and since the transplacental transfer of HCB to the fetus has been demonstrated in several species, the metabolism of lindane to HCB in female rats was investigated. Young adult female Fischer 344 rats were dosed p.o. with either 20 mg lindane/kg/day or an equivalent volume of the peanut oil vehicle. Feces samples were collected daily for two consecutive 4-hr intervals and a 16-hr interval. Twenty-four hours after the final treatment, all rats were sacrificed and adipose tissue samples were excised at necropsy. Extracts of fat and feces samples were analyzed by gas-liquid chromatography (GLC) on column packings of different polarity. Results of this study indicated that no significant biotransformation of lindane to HCB occurred in the female Fischer 344 rat.

Bioisomerization of lindane in rats.

The major environmental problem associated with the use of gamma-hexachlorocyclohexane (gamma-HCH, lindane) has been the appearance of the more oncogenic alpha- and beta- isomers as terminal residues in nature. To account for these residues it was suggested that gamma-hexachlorocyclohexane had been bioisomerized to the more stable alpha- and beta- isomers. In this study the effect of dose and duration of treatment on the proposed bioisomerization of gamma-hexachlorocyclohexane in the rat was investigated. Weanling female Sprague-Dawley rats were randomly assigned to one of four groups receiving Purina Lab Chow fortified with 0, 130, 215, or 350 ppm gamma-HCH. Six animals from each group were sacrificed after 1, 2, 4, 8, 16, and 24 weeks of treatment. Twenty-four hours prior to sacrifice all rats received a single oral dose of gamma-HCH in peanut oil. There were no significant differences in food consumption or body weights, and no deaths occurred throughout the study. The in vitro dechlorinase activity of the treated rats was significantly higher after 1, 4, and 24 weeks of treatment. Except at 4 weeks after treatment began, the liver/body weight ratios of the rats fed diets containing 350 ppm and 215 ppm lindane were significantly greater than the controls; while those receiving 130 ppm lindane were significantly greater than the controls after 1 and 2 weeks of treatment. No beta-HCH was detected in any of the samples analyzed throughout the study. The levels of alpha-HCH found in the adipose tissue after 24 weeks of treatment could be accounted for by trace contamination of the lindane used in this study. There was a negative correlation between the hepatic content of alpha-, gamma-, and sigma-HCH and duration of treatment. It was concluded that bioisomerization does not play a significant role in the metabolism of lindane by rats.

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.

Chlorobenzene-impaired lindane metabolism and the effect of pretreatment with chlorobenzene, lindane, or chlorobenzene plus lindane.

The storage and metabolism of lindane (gamma-HCH) was studied in the female rat after the administration of a hepatotoxic dose of chlorobenzene. Impaired lindane metabolism was observed following a challenge dose of 1.12 g chlorobenzene/kg. The data indicated that a hepatotoxic dose of chlorobenzene (CB) selectively impaired certain pathways, such as dehydrochlorination and the direct hydroxylation of lindane, to a greater extent than others, such as the dehydrogenation and dechlorination of lindane. Pretreatment with a subtoxic level of chlorobenzene produced: (1) significant increases in the dehydrogenation of lindane, (2) significant increase in the excretion of conjugated metabolites, (3) significant increases in the excretion of metabolites derived from the dehydrogenation of lindane through hexachlorocylohexene, gamma-HCCH, (4) significant improvement in the excretion of metabolites derived from CB-impaired dehydrochlorination of lindane as well as from the CB-impaired hydroxylation of lindane, and (5) significant reduction in the level of unaltered lindane stored in the adipose tissue. Repeated pretreatment with a subtoxic level of chlorobenzene offered significant protection against the reduction in lindane metabolism produced by the single hepatotoxic dose of chlorobenzene. Pretreatment with gamma-HCH alone was not as effective against the hepatotoxic effect of CB on lindane metabolism.