Insights into the toxicokinetics and toxicodynamics of 1,3-butadiene.

1,3 Butadiene (BD) is a colorless gas used in the production of synthetic rubber and plastics. BD is carcinogenic in rats and mice, however, there are striking species differences in cancer potency and spectrum of tumors, with mice being more susceptible to tumor induction than rats. Epidemiology studies suggest an excess incidence of leukemia in workers in the styrene-butadiene rubber industry. Consideration of mechanisms of BD carcinogenicity can provide insights into differences in cancer potency between rodents and serve to elucidate the extent to which BD exposure may cause cancer in humans. Mechanistic research in the areas of biochemical toxicology, molecular biology, molecular dosimetry, and susceptibility factors can impact BD cancer risk assessment for humans. This research has focused on quantitating species differences in the metabolism of BD and BD epoxides, defining molecular lesions produced by BD epoxides, identifying biomarkers for BD exposure to explore metabolic pathways in humans, and determining potential risk factors for sensitive subpopulations. BD is activated by P450 isozymes, including CYP2E1, to at least two genotoxic metabolites, epoxybutene (EB) and diepoxybutane (DEB). Dosimetry data from several laboratories on EB and DEB following inhalation exposure to BD indicate that blood concentrations of EB were four-eight-fold higher in mice compared with rats and that blood concentrations of DEB were 25-100-fold higher in mice than in rats. The higher levels of these two DNA-reactive metabolites in mice compared with rats probably contribute to the species differences in carcinogenic effects of BD between mice and rats. In vitro metabolism studies of BD in rats, mice, and human tissues indicate that there are significant quantitative species differences in the metabolic activation of BD to EB and DEB and the detoxication of EB and DEB. Activation/detoxication ratios calculated using in vitro kinetic constants reveal that ratios in mice were greater than in both rats and humans. In vitro data are consistent with in vivo dosimetry data and cancer potency for rodents, and suggest that humans may be at a decreased risk. Data on mutagenicity and mutational spectra of BD epoxides show mechanistic differences between EB- and DEB-induced mutational events suggesting involvement of DEB in the development of cancer. Concentrations of DEB that are genotoxic in vitro are within the range of concentrations measured in mice in vivo, whereas concentrations of EB that are genotoxic in vitro are ten-100-fold greater than concentrations observed in vivo. Characterization of molecular events indicate that EB-induced genotoxicity is due to point mutations and small deletions, while DEB induces point mutations, small deletions, and large-scale deletions involving many base pairs. The extent to which epoxybutanediol is involved in BD carcinogenesis is not known. Molecular dosimetry studies in rodents and humans have focused on urinary metabolites and DNA and hemoglobin adducts. Data from these studies are consistent with in vivo and in vitro metabolism data providing further support for the differences in metabolic activation and deactivation of BD and BD epoxides across species and the role of DEB in tumor development. Research on potential susceptibility factors points to other P450 isozymes, in addition to CYP2E1, that are involved in both the metabolic activation and mutagenicity of BD. Taken together, mechanistic data on BD toxicokinetics and toxicodynamics provide an integrated insight into critical steps in initiation of cancer, metabolites responsible for cancer, sensitive biomarkers for exposure, and potential risk factors for individual susceptibility. Available evidence suggests that BD is unlikely to be a human carcinogen at the low exposure concentrations currently encountered in the environment or workplace.

Sites and mechanisms for uptake of gases and vapors in the respiratory tract.

Inhalation is a common route by which individuals are exposed to toxicants. The air contains a multitude of gases and vapors that are brought into the respiratory tract with each breath. Depending upon the physical and chemical characteristics of the toxicant, the respiratory tract can be considered as a target organ in addition to a portal of entry. Sufficient information is not always available on the fate or effects of an inhaled gas or vapor. Two physiochemical principles, water solubility and reactivity, can be used to predict the site of uptake of gases and vapors in the respiratory tract and potential mechanisms for reaction with respiratory tract tissue and absorption into the blood. Four model compounds, formaldehyde, ozone, dibasic esters, and butadiene are discussed as examples of how knowledge of aqueous solubility and chemical reactivity can help toxicologists predict sites and mechanisms by which inhaled gases and vapors interact with respiratory tract tissues.

Inhibition of cytochrome P450 2E1 decreases, but does not eliminate, genotoxicity mediated by 1,3-butadiene.

1,3-Butadiene (BD), a rodent carcinogen, is metabolized to mutagenic and DNA-reactive epoxides. In vitro data suggest that this oxidation is mediated by cytochrome P450 2E1 (CYP2E1). In this study, we tested the hypothesis that oxidation of BD by CYP2E1 is required for genotoxicity to occur. Inhalation exposures were conducted with B6C3F1 mice using a closed-chamber technique, and the maximum rate of butadiene oxidation was estimated. The total amount of butadiene metabolized was then correlated with the frequency of micronuclei (MN). Three treatment groups were used: (1) mice with no pretreatment; (2) mice pretreated with 1,2-trans-dichloroethylene (DCE), a specific CYP2E1 inhibitor; and (3) mice pretreated with 1-aminobenzotriazole (ABT), an irreversible inhibitor of cytochromes P450. Mice in all 3 groups were exposed to an initial BD concentration of 1100 ppm, and the decline in concentration of BD in the inhalation chamber with time, due to uptake and metabolism of BD, was monitored using gas chromatography. A physiologically based pharmacokinetic model was used to analyze the gas uptake data, estimate V(max) for BD oxidation, and compute the total amount of BD metabolized. Model simulations of the gas uptake data predicted the maximum rate of BD oxidation would be reduced by 60% and 100% for the DCE- and ABT-pretreated groups, respectively. Bone marrow was harvested 24 h after the onset of the inhalation exposure and analyzed for frequency of micronuclei in polychromatic erythrocytes (MN-PCE). The frequency of MN-PCE per 1000 PCE in mice exposed to BD was 28.2 +/- 3.1, 19.8 +/- 2.5, and 12.3 +/- 1.9, for the mice with no pretreatment, DCE-pretreated mice and ABT-pretreated mice, respectively. Although inhibition of CYP2E1 decreased BD-mediated genotoxicity, it did not completely eliminate genotoxic effects. These data suggest that other P450 isoforms may contribute significantly to the metabolic activation of BD and resultant genotoxicity.

Analysis of respiratory exchange of methanol in the lung of the monkey using a physiological model.

A physiologically based pharmacokinetic (PBPK) model was developed for the monkey, to account for fractional systemic uptake of inhaled methanol vapors in the lung. Fractional uptake of inhaled [14C]-methanol was estimated using unreported exhaled breath time course measurements of [14C]-methanol from the D.C. Dorman et al. (1994, Toxicol Appl Pharmacol. 128, 229-238) lung-only exposure study. The cumulative amount of [14C]-methanol exhaled was linear with respect to exposure duration (0.5 to 2 h) and concentration (10 to 900 ppm). The model estimated that forty to eighty-one percent of the of inhaled [14C]-methanol delivered to the lung was taken into systemic circulation in female Cynomolgus monkeys exposed for two h to 10-900 ppm of [14C]-methanol. There was no apparent trend between the percent of inhaled [14C]-methanol absorbed systemically and the [14C]-methanol exposure concentration. Model simulations were conducted using a single saturable Michaelis-Menten equation with Vmaxc, the metabolic capacity set to 15.54 mg/kg/h and Km, the affinity constant, to 0.66 mg/l. The [14C]-methanol blood concentrations were variable across [14C]-methanol exposure groups and the PBPK model tended to over-predict systemic clearance of [14C]-methanol. Accounting for fractional uptake of inhaled polar solvents is an important consideration for risk assessment of inhaled polar solvents.

Effects of a thirteen-week inhalation exposure to ethyl tertiary butyl ether on fischer-344 rats and CD-1 mice.

The 1990 Clean Air Act Amendments require that oxygenates be added to automotive fuels to reduce emissions of carbon monoxide and hydrocarbons. One potential oxygenate is the aliphatic ether ethyl tertiary butyl ether (ETBE). Our objective was to provide data on the potential toxic effects of ETBE. Male and female Fisher 344 rats and CD-1 mice were exposed to 0 (control), 500, 1750, or 5000 ppm of ETBE for 6 h/day and 5 days/wk over a 13-week period. ETBE exposure had no effect on mortality and body weight with the exception of an increase in body weights of the female rats in the 5000-ppm group. No major changes in clinical pathology parameters were noted for either rats or mice exposed to ETBE for 6 (rats only) or 13 weeks. Liver weights increased with increasing ETBE-exposure concentration for both sexes of rats and mice. Increases in kidney, adrenal, and heart (females only) weights were noted in rats. Degenerative changes in testicular seminiferous tubules were observed in male rats exposed to 1750 and 5000 ppm but were not seen in mice. This testicular lesion has not been reported previously for aliphatic ethers. Increases in the incidence of regenerative foci, rates of renal cell proliferation, and alpha2u-globulin containing protein droplets were noted in the kidneys of all treated male rats. These lesions are associated with the male rat-specific syndrome of alpha2u-globulin nephropathy. Increases in the incidence of centrilobular hepatocyte hypertrophy and rates of hepatocyte cell proliferation were seen in the livers of male and female mice in the 5000-ppm group, consistent with a mitogenic response to ETBE. These two target organs for ETBE toxicity, mouse liver and male rat kidney, have also been reported for methyl tertiary butyl ether and unleaded gasoline.

Quantitative and qualitative differences in the metabolism of 14C-1,3-butadiene in rats and mice: relevance to cancer susceptibility.

1,3-Butadiene (butadiene) is a potent carcinogen in mice, but not in rats. Metabolic studies may provide an explanation of these species differences and their relevance to humans. Male Sprague-Dawley rats and B6C3F1 mice were exposed for 6 h to 200 ppm [2,3-14C]-butadiene (specific radioactivity [sa] 20 mCi/mmol) in a Cannon nose-only system. Radioactivity in urine, feces, exhaled volatiles and 14C-CO2 were measured during and up to 42 h after exposure. The total uptake of butadiene by rats and mice under these experimental conditions was 0.19 and 0.38 mmol (equivalent to 3.8 and 7.5 mCi) per kg body weight, respectively. In the rat, 40% of the recovered radioactivity was exhaled as 14C-CO2, 70% of which was trapped during the 6-h exposure period. In contrast, only 6% was exhaled as 14C-CO2 by mice, 3% during the 6-h exposure and 97% in the 42 h following cessation of exposure. The formation of 14C-CO2 from [2,3-14C]-labeled butadiene indicated a ready biodegradability of butadiene. Radioactivity excreted in urine accounted for 42% of the recovered radioactivity from rats and 71% from mice. Small amounts of radioactivity were recovered in feces, exhaled volatiles and carcasses. Although there was a large measure of commonality, the exposure to butadiene also led to the formation of different metabolites in rats and mice. These metabolites were not found after administration of [4-14C]-1,2-epoxy-3-butene to animals by i.p. injection. The results show that the species differences in the metabolism of butadiene are not simply confined to the quantitative formation of epoxides, but also reflect a species-dependent selection of metabolic pathways. No metabolites other than those formed via an epoxide intermediate were identified in the urine of rats or mice after exposure to 14C-butadiene. These findings may have relevance for the prediction of butadiene toxicity and provide a basis for a revision of the existing physiologically based pharmacokinetic models.

A physiological model for tert-amyl methyl ether and tert-amyl alcohol: hypothesis testing of model structures.

The oxygenate tert-amyl methyl ether (TAME) is a gasoline fuel additive used to reduce carbon monoxide in automobile emissions. To evaluate the relative health risk of TAME as a gasoline additive, information is needed on its pharmacokinetics and toxicity. The objective of this study was to use a physiologically-based pharmacokinetic (PBPK) model to describe the disposition of TAME and its major metabolite, tert-amyl alcohol (TAA), in male Fischer-344 rats. The model compartments for TAME and TAA were flow-limited. The TAME physiological model had 6 compartments: lung, liver, rapidly perfused tissues, slowly perfused tissues, fat, and kidney. The TAA model had 3 compartments: lung, liver, and total-body water. The 2 models were linked through metabolism of TAME to TAA in the liver. Model simulations were compared with data on blood concentrations of TAME and TAA taken from male Fischer-344 rats during and after a 6-hour inhalation exposure to 2500, 500, or 100 ppm TAME. The PBPK model predicted TAME pharmacokinetics when 2 saturable pathways for TAME oxidation were included. The TAA model, which included pathways for oxidation and glucuronide conjugation of TAA, underpredicted the experimental data collected at later times postexposure. To account for biological processes occurring during this time, three hypotheses were developed: nonspecific binding of TAA, diffusion-limited transport of TAA, and enterohepatic circulation of TAA glucuronide. These hypotheses were tested using three different model structures. Visual inspection and statistical evaluation involving maximum likelihood techniques indicated that the model incorporating nonspecific binding of TAA provided the best fit to the data. A correct model structure, based upon experimental data, statistical analyses, and biological interpretation, will allow a more accurate extrapolation to humans and, consequently, a greater understanding of human risk from exposure to TAME.

Influence of gender and acetone pretreatment on benzene metabolism in mice exposed by nose-only inhalation.

Benzene (BZ) requires oxidative metabolism catalyzed by cytochrome P-450 2E1 (CYP 2E1) to exert its hematotoxic and genotoxic effects. We previously reported that male mice have a two-fold higher maximum rate of BZ oxidation compared with female mice; this correlates with the greater sensitivity of males to the genotoxic effects of BZ as measured by micronuclei induction and sister chromatid exchanges. The aim of this study was to quantitate levels of BZ metabolites in urine and tissues, and to determine whether the higher maximum rate of BZ oxidation in male mice would be reflected in higher levels of hydroxylated BZ metabolites in tissues and water-soluble metabolites in urine. Male and female B6C3F, mice were exposed to 100 or 600 ppm 14C-BZ by nose-only inhalation for 6 h. An additional group of male mice was pretreated with 1% acetone in drinking water for 8 d prior to exposure to 600 ppm BZ; this group was used to evaluate the effect of induction of CYP 2E1 on urine and tissue levels of BZ and its hydroxylated metabolites. BZ, phenol (PHE), and hydroquinone (HQ) were quantified in blood, liver, and bone marrow during exposure and postexposure, and water-soluble metabolites were analyzed in urine in the 48 h after exposure. Male mice exhibited a higher flux of BZ metabolism through the HQ pathway compared with females after exposure to either 100 ppm BZ (32.0 2.03 vs. 19.8 2.7%) or 600 ppm BZ (14.7 1.42 vs. 7.94 + 0.76%). Acetone pretreatment to induce CYP 2E1 resulted in a significant increase in both the percent and mass of urinary HQ glucuronide and muconic acid in male mice exposed to 600 ppm BZ. This increase was paralleled by three- to fourfold higher steady-state concentrations of PHE and HQ in blood and bone marrow of acetone-pretreated mice compared with untreated mice. These results indicate that the higher maximum rate of BZ metabolism in male mice is paralleled by a greater proportion of the total flux of BZ through the pathway for HQ formation, suggesting that the metabolites formed along this pathway may be responsible for the genotoxicity observed following BZ exposure.

A comparison of physiologically based pharmacokinetic model predictions and experimental data for inhaled ethanol in male and female B6C3F1 mice, F344 rats, and humans.

Ethanol is added to unleaded gasoline as an oxygenate to decrease carbon monoxide automobile emissions. This introduces inhalation as a new possible route of environmental exposure to humans. Knowledge of the pharmacokinetics of inhaled ethanol is critical for adequately assessing the dosimetry of this chemical in humans. The purpose of this study was to characterize the pharmacokinetics of inhaled ethanol in male and female B6C3F1 mice and F344 rats and to develop a physiologically based pharmacokinetic (PBPK) model for inhaled ethanol in mice, rats, and humans. During exposure to 600 ppm for 6 hr, steady-state blood ethanol concentrations (BEC) were reached within 30 min in rats and within 5 min in mice. Maximum BEC ranged from 71 microM in rats to 105 microM in mice. Exposure to 200 ppm ethanol for 30 min resulted in peak BEC of approximately 25 microM in mice and approximately 15 microM in rats. Peak BEC of about 10 microM were measured following exposure to 50 ppm in female rats and male and female mice, while blood ethanol was undetectable in male rats. No sex-dependent differences in peak BEC at any exposure level were observed. Species-dependent differences were found following exposure to 200 and 600 ppm. A blood flow limited PBPK model for ethanol inhalation was developed in mice, rats, and humans which accounted for a fractional absorption of ethanol. Compartments for the model included the pulmonary blood and air, brain, liver, fat, and rapidly perfused and slowly perfused tissues. The PBPK model accurately simulated BEC in rats and mice at all exposure levels, as well as BEC reported in human males in previously published studies. Simulated peak BEC in human males following exposure to 50 and 600 ppm ranged from 7 to 23 microM and 86 and 293 microM, respectively. These results illustrate that inhalation of ethanol at or above the concentrations expected to occur upon refueling results in minimal BEC and are unlikely to result in toxicity.

Pharmacokinetics of methanol and formate in female cynomolgus monkeys exposed to methanol vapors.

The 1990 Clean Air Act Amendments contain mandates for reduced automotive emissions and add new requirements for the use of alternative fuels such as methanol to reduce certain automotive pollutants. Methanol is acutely toxic in humans at relatively low doses, and the potential for exposure to methanol will be increased if it is used in automotive fuel. Formate is the metabolite responsible for neurotoxic effects of acute methanol exposure. Since formate metabolism is dependent on folate, potentially sensitive folate-deficient subpopulations, such as pregnant women, may accumulate formate and be at higher risk from low-level methanol exposure. Our objective was to determine the pharmacokinetics of 14C-methanol and 14C-formate in normal and folate-deficient monkeys after exposure to 14C-methanol vapors at environmentally relevant concentrations: below the threshold limit value (TLV), at the TLV of 200 parts per million (ppm), and above the TLV. Four normal adult female cynomolgus monkeys were individually anesthetized with isoflurane, and each was exposed by endotracheal intubation to 10, 45, 200, or 900 ppm 14C-methanol for 2 hours. Concentrations of the inhaled and exhaled 14C-methanol, blood concentrations of 14C-methanol and 14C-formate, exhaled 14C-carbon dioxide (14CO2), and respiratory parameters were measured during exposure. After exposure, 14C-methanol and 14CO2 exhaled, 14C-methanol and 14C-formate excreted in urine, and 14C-methanol and 14C-formate in blood were quantified. The amounts of exhaled 14C-methanol and 14CO2, blood concentrations of 14C-methanol and 14C-formate, and 14C-methanol and 14C-formate excreted in urine were linearly related to methanol exposure concentration. For all exposures, blood concentrations of 14C-methanol-derived formate were 10 to 1000 times lower than endogenous blood formate concentrations (100 to 200 mM) reported for monkeys and were several orders of magnitude lower than levels of formate known to be toxic. Since the metabolism of formate in primates depends on the availability of tetrahydrofolate, the same four monkeys were next placed on a folate-deficient diet until folate concentrations in red blood cells consistent with moderate folate deficiency (29 to 107 ng/mL) were achieved. Monkeys were then reexposed to the highest exposure concentration, 900 ppm 14C-methanol, for a similar 2-hour period, and again the pharmacokinetic data described above were obtained. Even with a reduced folate status, monkeys exposed to 900 ppm methanol for 2 hours had peak concentrations of methanol-derived formate that were well below the endogenous levels of formate. Although these results represent only a single exposure and therefore preclude broad generalizations, they do suggest the body contains sufficient folate stores to effectively detoxify small doses of methanol-derived formate from exogenous sources, such as those that might occur during normal use of automotive fuel.

Disposition of butadiene epoxides in Sprague-Dawley rats.

1,2-Epoxybutene (BMO) and diepoxybutane (BDE) are metabolic products of 1,3-butadiene in rodents. Both BMO and BDE are suspect in the development of tumors in rats and mice. To understand the distribution and elimination of these compounds in the absence of the rate-limiting production from butadiene, the pharmacokinetics of BMO and BDE in blood were determined in adult male Sprague-Dawley rats following intravenous administration. All animals were dually cannulated in these studies. For the BMO studies, rats were dosed with 71, 143, or 286 mumol/kg BMO (n = 3 for each dose group). For the BDE studies, rats were dosed with 523 mumol/kg BDE (n = 3). All animals tolerated the BMO and BDE doses without grossly observable adverse effects. Blood was drawn at predetermined time points and extracted in methylene chloride. BDE and BMO concentrations were quantitated by gas chromatography or gas chromatography/mass spectrometry. The BMO distribution half-lives were short and ranged from 1.4 min at the lowest dose to 1.8 min at the highest dose. Volume of distribution at steady state ranged from 0.53 +/- 0.17 to 0.59 +/- 0.31 l/kg. Systemic clearances ranged from 67 +/- 17 to 114 +/- 20 ml/min per kg. The terminal elimination half-lives were also short and ranged from 5.7 to 8.5 min among the doses. The pharmacokinetic parameters after an i.v. dose of 523 mumol/kg BDE were a distribution half-life of 2.7 min, terminal elimination T1/2 of 14 min, volume of distribution at steady state of 0.73 +/- 0.06 l/kg, and systemic clearance of 76 +/- 8 ml/min per kg. These pharmacokinetic parameters demonstrate the similarity between disposition of the two epoxides in rats, that include a rapid distribution after i.v. administration into a small extravascular body compartment as well as a rapid elimination from blood. These pharmacokinetic data provide useful blood clearance information for assessing the critical physiological and biochemical determinants underlying the disposition of butadiene epoxides.

Physiologically based pharmacokinetic modeling of 1,3-butadiene, 1,2-epoxy-3-butene, and 1,2:3,4-diepoxybutane toxicokinetics in mice and rats.

1,3-Butadiene (BD) is a more potent tumor inducer in mice than in rats. BD also shows striking differences in metabolic activation, with substantially higher blood concentrations of 1,2:3,4-diepoxybutane (butadiene diepoxide; BDE) in BD-exposed mice than in similarly exposed rats. The objective of this study was to develop a single mechanistic model structure capable of describing BD disposition in both species. To achieve this objective, known pathways of 1,2-epoxy-3-butene (butadiene monoepoxide; BMO) and BDE metabolism were incorporated into a physiologically based pharmacokinetic model by scaling rates determined in vitro. With this model structure, epoxide clearance was underestimated for both rats and mice. Improved simulation of blood epoxide concentrations was achieved by addition of first-order metabolism in the slowly perfused tissues, verified by simulation of data on the time course for BMO elimination after i.v. injection of BMO. Blood concentrations of BD were accurately predicted for mice and rats exposed by inhalation to constant concentrations of BD. However, if all BD was assumed to be metabolized to BMO, blood concentrations of BMO were overpredicted. By assuming that only a fraction of BD metabolism produces BMO, blood concentrations of BMO could be predicted over a range of BD exposure concentrations for both species. In vitro and in vivo studies suggest an alternative cytochrome P-450-mediated pathway for BD metabolism that does not yield BMO. Including an alternative pathway for BD metabolism in the model also gave accurate predictions of blood BDE concentrations after inhalation of BD. Blood concentrations of BMO and BDE observed in both mice and rats are best explained by the existence of an alternative pathway for BD metabolism which does not produce BMO.

Neurogenic inflammation: with additional discussion of central and perceptual integration of nonneurogenic inflammation.

The Working Group on Neurogenic Inflammation proposed 11 testable hypotheses in the three domains of neurogenic inflammation, perceptual and central integration, and nonneurogenic inflammation. The working group selected the term people reporting chemical sensitivity (PRCS) to identify the primary subject group. In the domain of neurogenic inflammation, testable hypotheses included: PRCS have an increased density of c-fiber neurons in symptomatic tissues; PRCS produce greater quantities of neuropeptides and prostanoids than nonsensitive subjects in response to exposure to low-level capsaicin or irritant chemicals; PRCS have an increased and prolonged response to exogenously administered c-fiber activators such as capsaicin; PRCS demonstrate augmentation of central autonomic reflexes following exposure to agents that produce c-fiber stimulation; PRCS have decreased quantities of neutral endopeptidase in their mucosa; exogenous neuropeptide challenge reproduces symptoms of PRCS. In the domain of perceptual and central integration, testable hypotheses included: PRCS have alterations in adaptation, habituation, cortical representation, perception, cognition, and hedonics compared to controls; the qualitative and quantitative interactions between trigeminal and olfactory systems are altered in PRCS; higher integration of sensory inputs is altered in PRCS. In the domain of nonneurogenic inflammation, testable hypotheses included: increased inflammation is present in PRCS in symptomatic tissues and is associated with a heightened neurosensory response; PRCS show an augmented inflammatory response to chemical exposure. The working group recommended that studies be initiated in these areas.

Mechanistic considerations in benzene physiological model development.

Benzene, an important industrial solvent, is also present in unleaded gasoline and cigarette smoke. The hematotoxic effects of benzene in humans are well documented and include aplastic anemia, pancytopenia, and acute myelogenous leukemia. However, the risks of leukemia at low exposure concentrations have not been established. A combination of metabolites (hydroquinone and phenol, for example) may be necessary to duplicate the hematotoxic effect of benzene, perhaps due in part to the synergistic effect of phenol on myeloperoxidase-mediated oxidation of hydroquinone to the reactive metabolite benzoquinone. Because benzene and its hydroxylated metabolites (phenol, hydroquinone, and catechol) are substrates for the same cytochrome P450 enzymes, competitive interactions among the metabolites are possible. In vivo data on metabolite formation by mice exposed to various benzene concentrations are consistent with competitive inhibition of phenol oxidation by benzene. In vitro studies of the metabolic oxidation of benzene, phenol, and hydroquinone are consistent with the mechanism of competitive interaction among the metabolites. The dosimetry of benzene and its metabolites in the target tissue, bone marrow, depends on the balance of activation processes such as enzymatic oxidation and deactivation processes such as conjugation and excretion. Phenol, the primary benzene metabolite, can undergo both oxidation and conjugation. Thus the potential exists for competition among various enzymes for phenol. Zonal localization of phase I and phase II enzymes in various regions of the liver acinus also impacts this competition. Biologically based dosimetry models that incorporate the important determinants of benzene flux, including interactions with other chemicals, will enable prediction of target tissue doses of benzene and metabolites at low exposure concentrations relevant for humans.

Reduction of benzene metabolism and toxicity in mice that lack CYP2E1 expression.

Transgenic CYP2E1 knockout mice (cyp2e1-/-) were used to investigate the involvement of CYP2E1 in the in vivo metabolism of benzene and in the development of benzene-induced toxicity. After benzene exposure, absence of CYP2E1 protein was confirmed by Western blot analysis of mouse liver samples. For the metabolism studies, male cyp2e1-/- and wild-type control mice were exposed to 200 ppm benzene, along with a radiolabeled tracer dose of [14C]benzene (1.0 Ci/mol) by nose-only inhalation for 6 hr. Total urinary radioactivity and all radiolabeled individual metabolites were reduced in urine of cyp2e1-/- mice compared to wild-type controls during the 48-hr period after benzene exposure. In addition, a significantly greater percentage of total urinary radioactivity could be accounted for as phenylsulfate conjugates in cyp2e1-/- mice compared to wild-type mice, indicating the importance of CYP2E1 in oxidation of phenol following benzene exposure in normal mice. For the toxicity studies, male cyp2e1-/-, wild-type, and B6C3F1 mice were exposed by whole-body inhalation to 0 ppm (control) or 200 ppm benzene, 6 hr/day for 5 days. On Day 5, blood, bone marrow, thymus, and spleen were removed for evaluation of micronuclei frequencies and tissue cellularities. No benzene-induced cytotoxicity or genotoxicity was observed in cyp2e1-/- mice. In contrast, benzene exposure resulted in severe genotoxicity and cytotoxicity in both wild-type and B6C3F1 mice. These studies conclusively demonstrate that CYP2E1 is the major determinant of in vivo benzene metabolism and benzene-induced myelotoxicity in mice.

Metabolism of butadiene by mice, rats, and humans: a comparison of physiologically based toxicokinetic model predictions and experimental data.

1,3-Butadiene is a carcinogen in rats and mice, with mice being substantially more sensitive than rats. Our recent research is directed toward obtaining a better understanding of the cancer risk of butadiene in humans by evaluating species-dependent differences in the formation of the toxic metabolites epoxybutene and diepoxybutane. The recent data include in vitro studies on butadiene metabolism using tissues from humans, rats, and mice as well as experimental data and physiological model predictions for butadiene in blood and butadiene epoxides in blood, lung, and liver after exposure of rats and mice to inhaled butadiene. The findings suggest that humans would be more like rats and less like mice regarding the formation of butadiene epoxides. These research findings permit a reassessment of some default options that are used in carcinogen risk assessments. The research approach employed can be a useful strategy for developing mechanistic and toxicokinetic data to supplant default assumptions used in carcinogen risk assessments.

Physiologically based pharmacokinetic modeling of blood and tissue epoxide measurements for butadiene.

In vitro and in vivo butadiene (BD) metabolism data from laboratory animals were integrated into a rodent physiologically based pharmacokinetic (PBPK) model with flow- and diffusion-limited compartments. The resulting model describes experimental data from multiple sources under scenarios such as closed chamber inhalation and nose-only flow-through inhalation exposures. Incorporation of diurnal glutathione (GSH) variation allows accurate simulation of GSH changes observed in air control nose-only exposures and BD exposures. An isolated tissue model based on rate parameters determined in vitro predicts the decrease in epoxide concentrations in intact animals during the time lag between exsanguination and tissue removal for tissues capable of epoxide biotransformation, providing a better indication of in vivo dosimetry. Further refinements of the model are required relative to model predictions of an important BD metabolite, diepoxybutane.

Development of physiologically based pharmacokinetic model for methyl tertiary-butyl ether and tertiary-butanol in male Fisher-344 rats.

Methyl tertiary-butyl ether (MTBE) and its metabolite tertiary-butanol (TBA) both cause renal tumors in chronically exposed male rats. Knowledge of the kinetic behavior of MTBE and TBA in rats and its comparison to the kinetics of these chemicals in humans will aid in assessing human risk. The objective of this study was to develop a physiologically based pharmacokinetic (PBPK) model for MTBE and TBA in rats that will form the basis for a human model. Physiological parameters such as blood flows, tissue volumes, and alveolar ventilation were obtained from the literature. Chemical-specific parameters such as the solubility of MTBE and TBA in blood and selected tissues and metabolic rate constants to describe whole-body metabolism of MTBE in rats were measured using vial equilibration and gas uptake techniques, respectively. MTBE metabolism was described in the model as occurring through two saturable pathways. The model was able to predict gas uptake data (100 to 2000 ppm starting concentrations) and levels of MTBE in blood of rats exposed to MTBE by inhalation (400 to 8000 ppm, 6 hr), i.v. (40 mg/kg), and oral (40 or 400 mg/kg) administration. Two different models to describe the dosimetry of TBA in a rat were tested for their ability to predict TBA blood levels after MTBE exposure. TBA blood levels were predicted best at low MTBE exposure concentrations using a two-compartment model. The pharmacokinetics of TBA appear to be far more complex than those of MTBE, and additional experimental data on TBA distribution and elimination will be necessary to refine the submodel. With a quantitative description of the important determinants of MTBE and TBA dosimetry understood, a better assessment of the potential toxic and cancer risk for humans exposed to MTBE can be made.