George W. Lucier

Association between CYP1A1 genotype, mRNA expression and enzymatic activity in humans.

Genetic susceptibility factors may play a role in determining adverse effects of exposure to environmental toxins. As a preliminary step to a molecular epidemiological study in a population exposed to 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD), we investigated 20 healthy Caucasian volunteers with a set of putative susceptibility markers including a CYP1A1 Msp I restriction fragment length genetic polymorphism (RFLP), CYP1A1 mRNA expression, and ethoxyresorufin-O-deethylase (EROD) activity in cultured and mitogen-activated blood lymphocytes. Both basal (p = 0.008) and induced (p = 0.0001) EROD activity was significantly higher among persons with a mutation in one or both alleles of the CYP1A1 gene (variant CYP1A1 genotype). Induction in vitro by TCDD significantly increased EROD activity in both variant and wild-type CYP1A1 subjects; however, the absolute increase was greater in subjects with variant genotypes. An additive interaction between genotype and TCDD induction was suggested. Expression of CYP1A1 mRNA, both basal and induced, did not vary significantly across the genotypes.

Effect of dose on the absorption and excretion of [14C]benzene administered orally or by inhalation in rats and mice

The effect of dose on the absorption and excretion of [14C]benzene was studied using 13-week old male F344/N rats, Sprague-Dawley rats, and B6C3F1 mice. Gastrointestinal absorption of benzene administered by gavage was greater than 97% in these species for doses between 0.5 and 150 mg benzene/kg body wt. At oral doses below 15 mg/kg, greater than 90% of the 14C excreted was in the urine as nonethylacetate extractable material. Above 15 mg/kg, in both rats and mice, an increasing percentage of the administered benzene was exhaled unmetabolized, suggesting saturation of metabolic pathways. Above 50 mg/kg, total metabolites (as determined by 14C in the urine, feces, and carcass after 2 days) were not linearly related to administered dose. Total metabolites per unit body weight was equal in F344/N rats and B6C3F1 mice at gavage doses up to 50 mg/kg; however, total metabolites in mice did not increase at higher doses. For inhalation exposures, the percentage of inhaled benzene that was absorbed and retained during a 6-hr exposure decreased from 33 +/- 6% (mean +/- standard deviation) to 15 +/- 9% in rats, and from 50 +/- 15 to 10 +/- 2% in mice as the exposure concentration was increased from approximately 26 to 2600 micrograms/liter (10 to 1000 ppm at 615 Torr, 23 degrees C). Total metabolite formation was exponentially related to the benzene exposure concentration with one-half the maximal amount of metabolite formation occurring at 220 micrograms/liter (84 ppm) for B6C3F1 mice and 650 micrograms/liter (250 ppm) for F344/N rats. Total metabolites were higher in mice than in rats at any of the vapor concentrations used due mainly to the higher amount inhaled by mice. Saturation of overall metabolism in mice but not in rats at high doses by both routes of administration indicates species differences in metabolism of benzene.

Microsomal rat liver UDP glucuronyltransferase: Effects of piperonyl butoxide and other factors on enzyme activity

Activation of rat liver microsomal UDP glucuronyltransferase by detergent or ageing was not accompanied by a change in K m for 1-naphthol or UDPGA. Divalent cations (Mg 2+ , Mn 2+ , and Co 2+ ) stimulated glucuronic acid conjugation of 1-naphthol and p -nitrophenol in both unactivated and activated microsomes, however, Zn 2+ strongly inhibited UDP glucuronyltransferase. A total activation factor of 30-fold resulted when both Triton X-100 and MgCl 2 were added to the incubation medium. Triton X-100 and MgCl 2 also stimulated UDP glucuronyltransferase from rabbit liver microsomes with a total activation factor of 20-fold. The UDP glucuronyltransferases conjugating 1-naphthol, testosterone, and a dieldrin metabolite were inhibited in vitro by insecticide synergist chemicals. Piperonyl butoxide had I 50 values of 7.3×10 −6 m and 9.0×10 −5 m for testosterone and 1-naphthol glucuronyltransferase, respectively. Inhibition of enzyme from detergent-activated microsomes was considerably less than that of enzyme from unactivated or aged microsomes. Pretreatment of male or female rats with piperonyl butoxide for 3 days resulted in a two-fold induction of the UDP glucuronyltransferase conjugating 1-naphthol and p -nitrophenol. 1-Naphthol and p -nitrophenol were shown to be glucuronidated by the same enzyme by of the mixed substrate method, divalent cation requirements, activation by detergent or ageing, submicrosomal distributions, levels of induction by piperonyl butoxide, and gravitational studies. This work was greatly facilitated by the use of a new, rapid, and sensitive radioassay for UDP glucuronyltransferase which is described here.

A physiological model for simulation of benzene metabolism by rats and mice

Studies conducted by the National Toxicology Program on the chronic toxicity of benzene indicated that B6C3F1 mice are more sensitive to the toxic effects of benzene than are F344 rats. A physiological model was developed to describe the uptake and metabolism of benzene in rats and mice and to determine if the observed differences in toxic effects could be explained by differences in the pathways for metabolism of benzene or by differences in uptake of benzene. Major pathways for elimination of benzene included metabolism to hydroquinone glucuronide or hydroquinone sulfate, phenyl glucuronide or phenyl sulfate, muconic acid, and prephenyl mercapturic acid or phenyl mercapturic acid. Model simulations for total benzene metabolized and for profiles of benzene metabolites were conducted for oral or inhalation exposure and compared to data for urinary excretion of benzene metabolites after exposure of rats and mice to [14C]- or [3H]-benzene by inhalation or gavage. Results for total amount of benzene metabolized, expressed per kilogram body weight, indicated that for inhalation exposure concentrations up to 1000 ppm, mice metabolized at least two to three times as much benzene as did rats. Simulations of oral exposure to benzene resulted in more benzene metabolized per kilogram body weight by rats at oral exposures of greater than 50 mg/kg. Patterns of metabolites formed after either route of exposure were very different for F344/N rats and B6C3F1 mice. Rats primarily formed the detoxification metabolite, phenyl sulfate. Mice formed hydroquinone glucuronide and muconic acid in addition to phenyl sulfate. Hydroquinone and muconic acid are associated with pathways leading to the formation of the putative toxic metabolites of benzene. Metabolic rate parameters, Vmax and Km, were very different for hydroquinone conjugate and muconic acid formation compared to formation of phenyl conjugates and phenyl mercapturic acids. Putative toxication pathways could be characterized as high affinity, low capacity whereas detoxification pathways were low affinity, high capacity. Model simulations suggested that for both rats and mice at lower exposure concentrations hydroquinone and muconic acid represented a larger fraction of the total benzene metabolized than at higher exposure concentrations where detoxification metabolites were predominant. Preferential production of a putative toxic metabolite at low exposure concentrations may have important implications in risk assessment for benzene.

Animal models of human response to dioxins.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent member of a class of chlorinated hydrocarbons that interact with the aryl hydrocarbon receptor (AhR). TCDD and dioxinlike compounds are environmentally and biologically stable and as a result, human exposure is chronic and widespread. Studies of highly exposed human populations show that dioxins produce developmental effects, chloracne, and an increase in all cancers and suggest that they may also alter immune and endocrine function. In contrast, the health effects of low-level environmental exposure have not been established. Experimental animal models can enhance the understanding of the effects of low-level dioxin exposure, particularly when there is evidence that humans respond similarly to the animal models. Although there are species differences in pharmacokinetics, experimental animal models demonstrate AhR-dependent health effects that are similar to those found in exposed human populations. Comparisons of biochemical changes show that humans and animal models have similar degrees of sensitivity to dioxin-induced effects. The information gained from animal models is important for developing mechanistic models of dioxin toxicity and critical for assessing the risks to human populations under different circumstances of exposure.

Nature of the enhancement of hepatic uridine diphosphate glucuronyltransferase activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats

Abstract After single low-level oral doses of 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) to rats, hepatic microsomal p -nitrophenol (PNP) glucuronyltransferase activity was elevated approximately 6-fold, whereas the hepatic glucuronyltransferase conjugating testosterone or estrone was unaffected. Solubilized and purified PNP glucuronyltransferase and steroid glucuronyltransferases from control and TCDD-treated rats exhibited the same relative activities (TCDD:control) as when the enzymes were bound to the endoplasmic reticulum. Elevation of PNP glucuronyltransferase was still evident 73 days after a single oral dose of 25 μg TCDD/kg. Female rats were more susceptible to TCDD actions on liver microsomal PNP glucuronyltransferase than males. The effects of TCDD treatment on PNP glucuronyltransferase appeared to be related to increased amounts of liver enzyme for the following reasons: (1) K m values for PNP and UDPGA were unchanged by TCDD treatments; (2) the magnitude of the TCDD-induced increase of PNP glucuronyltransferase activity was the same whether enzyme activity was measured in the presence or absence of Mg 2+ or Triton X-100; (3) TCDD, when added in in vitro , had no detectable effect on enzyme activity; (4) TCDD treatment of rats did not change total hepatic microsomal phospholipid or cholesterol contents: (5) pH optima were unaffected by TCDD treatment; (6) solubilization of enzyme was not accompanied by a change in the TCDD induction effect: and (7) actinomycin D appeared to block the initial phase of induction.

Tumor necrosis factor involvement in 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated endotoxin hypersensitivity in C57BL/6J mice congenic at the Ah locus

An experimental model of endotoxin-induced release of tumor necrosis factor-alpha (TNF) into the serum of C57BL/6J mice congenic at the Ah locus was used to investigate the effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on TNF production. TCDD exposure of Ah-responsive mice (Ahbb) resulted in a dose-dependent increase in the concentration of TNF in the serum of endotoxin-exposed mice, with a significant increase observed at a dose of 10 micrograms/kg TCDD. At a dose of 500 micrograms/kg TCDD, Ahbb mice demonstrated a 46-fold increase in serum TNF levels compared to control. In contrast, congenic Ah-receptor deficient mice (Ahdd) did not show a significant increase in serum TNF levels until exposed to 150 micrograms/kg TCDD, and the maximum response was an 8-fold increase over control. These data suggest that increased TNF production may be responsible for endotoxin hypersensitivity in TCDD-treated mice and that the Ah locus mediates this response.

Inhibition of acute TCDD toxicity by treatment with anti-tumor necrosis factor antibody or dexamethasone

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) acute toxicity is characterized in part by a wasting syndrome with depletion of adipose tissue. Tumor necrosis factor (TNF) induces a similar response during chronic infection. The similarities of these toxic effects led to a hypothesis that TNF plays a role in TCDD acute toxicity. To test this hypothesis pharmacologic doses of an antibody specific for murine TNF and the potent anti-inflammatory agent Dexamethasone (DEX) were used to inhibit TCDD toxicity in mice. TNF antibody treatment resulted in a 54% reduction in TCDD-mediated mortality while DEX treatment, a glucocorticoid agonist that inhibits transcription of TNF, reduced mortality by 92%. Cyp 1A1 induction, the most commonly measured TCDD-mediated response, was not blocked by DEX, demonstrating separation of this biochemical effect from acute toxic responses to TCDD. These data suggest that TCDD-mediated changes in the TNF pathway may be an important mechanism for acute TCDD toxicity.

Dose-dependent elevation of Ah receptor binding by TCDD in rat liver

Changes in [3H]TCDD binding to unoccupied liver Ah receptor were examined following chronic or acute administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to adult female Sprague-Dawley rats. Chronic biweekly administration of TCDD equivalent to 0, 10, 30, or 100 ng/kg/day TCDD for 22 weeks caused dose-dependent increases in liver TCDD concentration, aryl hydrocarbon hydroxylase (AHH) induction, and [3H]TCDD binding to unoccupied cytosolic Ah receptor sites. Maximal increases (twofold) of cytosolic receptor binding occurred at an estimated dose of slightly greater than 30 ng/kg/day. The increase in [3H]TCDD binding was half-maximal at an estimated dose of approximately 17 ng/kg/day which produced a liver concentration of 1.5 ppb TCDD. Cytosolic [3H]TCDD binding in control and treated animals sedimented mainly in the 8-9 S region of sucrose density gradients with a minor peak sedimenting in the 4-5 S region. Binding was markedly elevated in the 8-9 S region of cytosols from the TCDD-induced rats; however, TCDD treatment had no affect on [3H]TCDD binding in the 4-5 S region. Saturation and Scatchard analyses of Ah receptor binding showed no apparent changes in Kd following chronic TCDD treatment; however, a twofold increase in the number of unoccupied Ah receptor binding sites was observed. Neither aging nor ovariectomy significantly changed measurable cytosolic Ah receptor binding in control animals. When adult female rats were administered a single dose of TCDD (6 micrograms/kg) an initial drop (approximately 40%) in cytosolic receptor binding was observed at 30 and 60 min, followed by a steady increase in binding up to 250% of controls 9 days after TCDD treatment.

Differences in the metabolism and disposition of inhaled [3H]benzene by F344N rats and B6C3F1 mice

Benzene is a potent hematotoxin and has been shown to cause leukemia in man. Chronic toxicity studies indicate that B6C3F1 mice are more susceptible than F334/N rats to benzene toxicity. The purpose of the studies presented in this paper was to determine if there were metabolic differences between F344/N rats and B6C3F1 mice which might be responsible for this increased susceptibility. Metabolites of benzene in blood, liver, lung, and bone marrow were measured during and following a 6-hr 50 ppm exposure to benzene vapor. Hydroquinone glucuronide, hydroquinone, and muconic acid, which reflect pathways leading to potential toxic metabolites of benzene, were present in much greater concentrations in the mouse than in rat tissues. Phenylsulfate, a detoxified metabolite, and an unknown water-soluble metabolite were present in approximately equal concentrations in these two species. These results indicate that the proportion of benzene metabolized via pathways leading to the formation of potentially toxic metabolites as opposed to detoxification pathways was much higher in B6C3F1 mice than in F344 rats, which may explain the higher susceptibility of mice to benzene-induced hematotoxicity and carcinogenicity.