TY - JOUR
AU - Medinsky, M. A.
AB - Abstract 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 Vmax 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. 1,3-butadiene, CYP2E1, genotoxicity, micronuclei, gas uptake 1,3-Butadiene (BD) is a volatile, flammable, colorless gas used in the production of resins and plastics such as butadiene and styrene rubber, nitrile rubber, and styrene-butadiene latex (Chemical Week, 1994). Human exposure to BD occurs primarily via inhalation. BD is emitted in automobile exhaust (U.S. EPA, 1990) and cigarette smoke (Brunnemann et al., 1990; Pelz et al., 1990). BD can enter the ambient air during storage and transport and has been detected in urban air (1–5 ppb) and air around industrial sites (19–30 ppb) (Cote and Bayard, 1990). BD is listed as one of the 189 hazardous air pollutants in the 1990 Clean Air Act Amendment (U.S. EPA, 1990). The epidemiology of BD has been previously reviewed (Himmelstein et al., 1997). Recent epidemiological studies on workers in North American styrene-butadiene rubber plants have shown an increased risk of leukemia in workers compared with the general population (Delzell et al., 1996). BD is carcinogenic in mice and rats, with mice being more sensitive to BD-mediated carcinogenicity than rats and exhibiting a vastly different tumor-site specificity (NTP, 1984NTP, 1993; Owen et al., 1990). Significant increases in lung tumors occur in mice at inhaled BD concentrations as low as 6.25 ppm. Cytochrome P450 2E1 (CYP2E1) is hypothesized to be the enzyme responsible for BD activation in vivo based on in vitro studies of BD metabolism. The metabolic activation of BD involves oxidation to the epoxide metabolite epoxybutene (EB), which is further oxidized to diepoxybutane (DEB) (Fig. 1). Csánady et al. (1992) found that the rate of BD oxidation correlated with the rate of metabolism of a known CYP2E1 substrate, suggesting that CYP2E1 was the primary isoform responsible for BD oxidation in human liver microsomes. Using both cDNA-expressed human P450 enzymes and correlation studies with human liver microsomal preparations, Duescher and Elfarra (1994) provided evidence for major roles of CYP2A6 and CYP2E1 in butadiene oxidation. These observations are consistent with a suggestion of Guengerich et al. (1991) that low-molecular-weight compounds such as BD are preferentially metabolized by CYP2E1. Metabolism of BD is suspected to be necessary for genotoxicity to occur after exposure to BD. The reactive metabolites EB and DEB are genotoxic in vitro and upon direct administration to mice and rats (Conner et al., 1983). DEB is suspected to be the putative carcinogen, since DEB is more mutagenic than EB (Cochrane and Skopek, 1994). EB and DEB have tested positive in several cytogenetic assays, both in vivo and in vitro, with genotoxic end points including chromosomal aberrations, increased micronuclei formation, and sister chromatid exchanges (Autio et al., 1994; Tice et al., 1987). BD appears to be a procarcinogen primarily metabolized to metabolites that act through a genotoxic mechanism. We hypothesized that oxidation of BD by CYP2E1 is required for genotoxicity to occur in vivo. This hypothesis was tested by quantifying Vmax, the maximum rate of the metabolism of BD, during inhalation exposures of mice pretreated with inhibitors of cytochromes P450, including CYP2E1. The frequency of micronuclei in the bone marrow cells of exposed mice was also determined. These studies are the first to describe the role of a specific CYP450 enzyme in the oxidation of BD in vivo. MATERIALS AND METHODS Chemicals. 1,3-Butadiene (>99% pure), 4`-6-diamidino-2-phenylindole (DAPI), and polyoxyethylenesorbitan monolaurate (Tween 20) were purchased from Aldrich Chemical Company (Milwaukee, WI). 1-Aminobenzotriazole (>98% pure), 7-pentoxyresorufin, and p-nitrophenol were purchased from Sigma Chemical Company (St. Louis, MO). 1,2-trans-Dichloroethylene (>95% pure) was purchased from Fluka Scientific Company (New York, NY). The CREST serum (antikinetochore antibody) and the antikinetochore antibody fluoresceinated rabbit anti-human IgG were purchased from Antibodies Incorporated (Davis, CA). Animals. This study was conducted under federal guidelines for the use and care of laboratory animals and was approved by the CIIT Institutional Animal Care and Use Committee. Animals were housed in a humidity- and temperature-controlled, HEPA-filtered, mass air-displacement room in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. Male B6C3F1 mice, age 10–12 weeks at the time of delivery, were obtained from Charles River Breeding Laboratories (Raleigh, NC). Mice were housed, one per cage, in polystyrene shoe-box cages with cellulose bedding (ALPHA-dri; Shepherd Specialty Papers, Kalamazoo, MI) and filter top lids. Rodents were provided with NIH-07 rodent chow (Ziegler Bros., Gardener, PA) and deionized, filtered water ad libitum. Lighting was on a 12-h light-dark cycle. Rodents were acclimated to the animal facility for 2 weeks prior to use. Animals were housed in the animal facility as part of an ongoing surveillance program for parasitic, bacterial, and viral infections and were pathogen-free throughout the study. Butadiene Gas Uptake Studies Rates of in vivo metabolism of BD were quantitated and compared for treatment groups, using the closed-chamber, gas-uptake experimental technique (Andersen et al., 1980; Gargas et al., 1986). When using this technique, experimental animals are placed in a closed recirculating chamber with a known initial concentration of test chemical; this technique is in contrast to studies in which animals are exposed to constant concentrations of chemical for a specific duration of time. Since it is a closed system, the concentration in the chamber declines as the animals inhale and absorb the BD, and this decline can be monitored by gas chromatography. The gas-uptake exposure system was operated as described by Gargas et al. (1986). Briefly, groups of 5 mice were placed in a 3.0-liter glass chamber with recirculating air and allowed to acclimate for 10–30 min prior to onset of the exposure. Oxygen was monitored and maintained at a level of 19–21%. Carbon dioxide was removed with Sodasorb (W.R. Grace & Co., Atlanta, GA). BD was injected into the chamber to achieve the targeted initial concentration of approximately 1100 ppm. This dose was selected because it was previously demonstrated to induce quantifiable levels of micronuclei (Cunningham et al., 1986). The decline of BD concentration in the chamber was monitored by gas chromatography every 8–10 min by means of an automatic gas sampling loop. Air samples were analyzed using a gas chromatograph (Hewlett-Packard Model 5890 Series II) equipped with a flame ionization detector and a Tenax 35/60 column, 7 ft × 1/8 in. Analytical conditions were as follows: oven temperature, isothermal at 160°C; detector temperature, 200°C; injector temperature, 200°C; carrier gas (He) flow rate, 17.5 ml/min; hydrogen flow rate, 30 ml/min; and air flow rate, 400 ml/min. The retention time for BD under these conditions was 0.9 min. BD concentrations in the chamber were determined by comparison to a standard curve prepared in 10-L Tedlar bags containing BD gas in known concentrations. Loss of chemical from the system was determined prior to every exposure and was less than 2%. Three closed-chamber gas uptake studies were conducted using B6C3F1 mice: (1) mice that were not pretreated, (2) mice pretreated with ABT (100 mg/kg, ip, 12 and 24 h prior to exposure), and (3) mice pretreated with DCE (0.2 mg/kg, ip, 2 h prior to exposure). ABT is an irreversible inhibitor of cytochromes P450 (Ortiz de Montellano and Mathews, 1981), and DCE is an irreversible inhibitor of CYP2E1 (Mathews et al., 1997; Thornton-Manning et al., 1994). A physiologically based pharmacokinetic (PBPK) model for BD originally published by Medinsky et al. (1994) was used to analyze the gas uptake data. The compartments in this PBPK model included slowly and rapidly perfused tissue groups, lung, liver, and fat. The model was adapted for gas uptake by addition of a chamber compartment. Physiological, biochemical, and chemical-specific parameters were those used by Medinsky et al. (1994) (Table 1). For each of the 3 groups, model simulations were based on the assumption that the fraction of the total metabolic capacity of the liver and lung would vary depending upon pretreatment with the different enzyme inhibitors or no pretreatment. To describe this mathematically, a new parameter, F, representing the fraction of metabolic capacity remaining, was introduced into the model. This parameter was multiplied by the Michaelis-Menten kinetics equation describing BD metabolism in the liver and the lung. For example, the Michaelis-Menten equation describing the CYP450 mediated oxidation of BD in the liver is:  \[\frac{V_{max}\ {\star}\ C_{BD}}{K_{m}\ +\ C_{BD}}\] where Vmax is the maximum rate of metabolism of BD in the liver by oxidation, Km is the apparent affinity of BD for the enzyme, and CBD is the free concentration of BD in the liver. This equation, and the equation describing CYP450-mediated oxidation of BD in the lung, were multiplied by F:  \[F{\star}\ {[}\frac{V_{max}\ {\star}\ C_{BD}}{K_{m}\ +\ C_{BD}}{]}\ \] where F is a dimensionless number having allowable values from 0 to 1. The case where F = 0 simulates no metabolism, i.e., complete inhibition of CYP450-mediated oxidation of BD. By contrast, the case where F =1 represents metabolism with no inhibition. Values of F between 0 and 1 represent various degrees of metabolism, with (1 – F)*100 equal to the percent inhibition. Optimal values of F were obtained for each pretreatment group using a value of F = 1 as a starting point for optimization, using Simusolv Version 3.0 software (Dow Chemical Co., Midland, MI) on a VAX4000 computer. The amount of BD metabolized per treatment group was found by integrating the Michaelis-Menten equation for hepatic and lung metabolism, from 0 to 24 h, the time when the mice were sacrificed. These amounts were plotted against micronuclei frequencies to determine if a correlation existed. Linear [y = b0 + b1x] and quadratic [y = b0 + b2x2] regression models were fit to the micronuclei frequencies. A p value of < 0.05 for lack-of-fit test meant that the curve was not appropriate for the data set. Determination of Micronuclei Induction in Bone Marrow Animals were euthanized by CO2 asphyxiation 24 h after the start of exposures. This time period was necessary to allow the bone marrow cells to divide. For the micronucleus assay, the adhering soft tissue and epiphyses of both femurs were removed. Marrow was aspirated from the bone and transferred to centrifuge tubes containing 5 ml of fetal calf serum. Preparations of bone marrow cells were made by placing 250 μl of the aspirate onto glass slides using a cytocentrifuge (Cytospin 2, Shandon, Sewickley, PA). After the slides were allowed to air-dry, they were fixed in absolute methanol for 15 min, air-dried again, and stored desiccated. The bone marrow samples were stained with acridine orange (Hayashi et al., 1983). Briefly, acridine orange was prepared in a 1:20 solution with phosphate-buffered saline (PBS), and slides were stained for approximately 5 min. The slides were then rinsed with PBS and cover-slipped. Under a fluorescent microscope, polychromatic erythrocytes appear orange-red, and micronuclei appear yellowish-green, allowing for quantification of micronuclei. The data generated from the micronucleus assay were analyzed using a one-way analysis of variance (ANOVA) with a Tukey-Kramer test post hoc (Leavens et al., 1997). Differences that were significant at p < 0.05 were considered statistically significant. For control of bias, all slides were randomized and coded prior to scoring. Micronucleated polychromatic erythrocytes in the bone marrow were also analyzed for the presence of kinetochores using an antibody labeling method described by Eastmond and Tucker (1989). Micronuclei can be formed by exclusion of either whole chromosomes or chromatin fragments during cell division. Micronuclei that contain kinetochores also contain centromeric DNA regions of the chromosomes and would result from the exclusion of the entire chromosome from the nucleus. Micronuclei that lack kinetochores most likely result from chromosome breakage events. Thus this method can be used to determine the mechanism of micronuclei formation. Briefly, slides were placed in PBS-0.1% Tween for 5 min. Excess fluid was drained and 50 μl of the 50% PBS-0.1% Tween:antikinetochore antibody solution was placed on each slide. Slides were cover-slipped and placed in a humidified box at 37°C for 1 h. Cover slips were removed, and slides were rinsed twice in PBS-0.1% Tween for 2 min. Excess fluid was then drained, and 50 μl of fluoresceinated rabbit anti-human IgG-PBS-0.5% solution was placed on each slide. Slides were incubated, rinsed, and drained as above. Two drops of DAPI solution were added onto the slide using a pasteur pipette, and the slide was cover-slipped. In the anitkinetochore antibody assay, the slides were also randomized and coded prior to scoring. Statistical analysis of the anitkinetochore antibody assays was performed using the Fisher's exact test to compare the frequencies of kinetochore-positive cells between control and treated slides. Sufficient material (bone marrow) was collected from each animal to allow for both analyses (antikinetochore antibody and acridine orange staining). Five slides per animal were made. Generally, 2 slides were used for the acridine orange staining procedure and one slide was used for the antikinetochore staining. Micronuclei frequencies were determined independently for each staining procedure. Effect of DCE Pretreatment on Microsomal CYP450 Activity and Induction of Micronuclei To determine the effect of DCE treatment on microsomal CYP450 activity and the induction of micronuclei, livers and bone marrow were harvested from animals that were not exposed to BD. B6C3F1 mice pretreated with DCE (0.2 mg/kg, ip, 2 h prior to euthanasia) as well as a parallel group of control animals that received no pretreatment were used. Liver microsomes were prepared following standard procedures (Csánady et al., 1992). Activities of CYP2E1, CYP1A1/2, CYP2A5, and CYP2B1 were quantitated in microsomal samples using several standard assays: p-nitrophenol hydroxylase activity (PNP, CYP2E1), 7-ethoxyresorufin o-dealkylation (EROD, CYP1A1/2), 7-pentoxyresorufin o-dealkylation (PROD, CYP2B1), and coumarin 7-hydroxylase activity (CYP2A5). PNP activity was measured according to Reinke et al. (1985) using 1 μM p-nitrophenol and expressed as nmol/mg/min. EROD activity was assayed according to Burke et al. (1985) using 1.7 μM ethoxyresorufin and expressed as pmol/mg/min. PROD activity was assayed according to Lubet et al. (1985) by using 1.7 μM pentoxyresorufin and expressed as pmol/mg/min. Coumarin 7-hydroxylase activity was measured as described by van Iersel et al. (1994) using 10 μM coumarin and expressed as pmol/mg/min. Total microsomal protein content was determined by the Lowry assay (Lowry et al., 1951) using bovine serum albumin as a standard. Cytochrome P450 spectra were obtained using the spectrophotometric method described by Omura and Sato (1964). Data generated from the CYP450 activity assays were analyzed for statistically significant differences using Student's t-test. Bone marrow samples from mice pretreated with DCE (0.2 mg/kg, ip, 2 h prior to exposure) but not exposed to BD and mice that received neither BD nor DCE were prepared as previously described to assess micronuclei and the presence of kinetochores. The data were also analyzed using a one-way ANOVA with a Tukey-Kramer test post hoc. Differences were considered statistically significant at p < 0.05. For control of bias, all slides were randomized and coded prior to scoring. RESULTS The gas uptake curves from the BD exposures are shown in Figure 2. The value of F was equal to 0.988 ± 0.067 in the untreated animals exposed to BD; this corresponds to Vmax values of 333 and 21.3 μmole/kg/h for liver and lung BD oxidation, respectively. Following inhibition of CYP2E1 by pretreatment with DCE, the value of F was 0.4 ± 0.004; this corresponds to Vmax values of 135 and 8.64 μmole/kg/h for liver and lung BD oxidation, respectively. The value of F for the ABT-pretreated group was 0 ± 0.018, indicating virtual elimination of all BD oxidative metabolism. All gas-uptake curves were fit with the same Km value of 2.0 μM for liver oxidation and 5.01 μM for lung oxidation. The use of the specific CYP2E1 inhibitor DCE significantly reduced CYP2E1 activity, as measured using the isoform-specific substrate p-nitrophenol. Approximately 83% of all CYP2E1 activity was eliminated (Fig. 3). DCE pretreatment had no effect on the activity of other isoforms examined, using specific probes for other CYP450 isoforms (Fig. 3). The frequency of micronuclei, used as a measure of BD-mediated genotoxicity, was reduced by pretreatment with inhibitors (Table 2). Mice pretreated with DCE and exposed to BD displayed decreased frequency of micronuclei. However, micronuclei were not decreased to the level found in unexposed, unpretreated (naive) mice or in unexposed mice pretreated with DCE. In contrast, mice pretreated with ABT and exposed to BD displayed levels of micronuclei similar to naive mice. Pretreatment with DCE, without exposure to BD, did not alter the frequency of micronuclei relative to that of naive animals. These data suggest that metabolism of BD is needed for BD-mediated genotoxicity to occur but yield no information regarding the potential mechanism for formation of micronuclei. The frequency of kinetochore-negative micronuclei (MN-) increased between naive mice and mice exposed to BD, indicating that BD is a clastogen. The frequency of MN- decreased in mice exposed to BD following inhibition of CYP2E1 by DCE, suggesting that the clastogenic response was reduced. There was no significant change in the frequency of kinetochore-positive micronuclei (MN+), indicating that we were unable to detect anuegenic damage above background. We correlated the total amount of BD metabolized with the frequency of MN (Fig. 4). For the MN and MN- correlations, the linear model was rejected by the lack-of-fit test (lack-of-fit p = 0.004 and 0.002, respectively). Statistical tests were consistent with a quadratic relationship between the total amount of BD metabolized and the frequency of MN and MN- (lack-of-fit tests could not be rejected, p = 0.3 and 0.15 for MN and MN-, respectively). Statistical tests were consistent with a linear relationship between the total amount of BD metabolized and the frequency of MN+ (lack-of-fit test could not be rejected, p = 0.3). DISCUSSION The metabolism of BD is hypothesized to be mediated by CYP2E1. This metabolism results in the formation of the monoepoxide epoxybutene (EB). EB is also hypothesized to be metabolized by CYP2E1, resulting in the formation of the diepoxide diepoxybutane (DEB). In addition to these processes, EB and DEB are substrates for epoxide hydrolase, can be conjugated with glutathione, and can be hydrolyzed by nonenzymatic processes, all of which serve as detoxication reactions. DEB is genotoxic in vitro at concentrations lower than those required by EB. Therefore, if the putative genotoxic metabolite of BD is EB or DEB, then understanding the mechanism of formation of these metabolites and the induction of micronuclei by either or both metabolites will be required, in order to better characterize potential risk factors relevant to the development of an accurate and complete scenario of BD-mediated toxicity. In humans, 6 CYP450 isoforms have been shown to date to be involved in the activation of procarcinogens: 1A1, 1A2, 1B1, 2A6, 2E1, and 3A4 (Guengerich and Shimada, 1991). Cytochrome P450 2E1 is the ethanol-inducible isoform of CYP450 and is considered to be among the six most important CYP450s. The CYP2E family of CYP450 enzymes, and specifically CYP2E1, is generally conserved in regulation and function between vertebrates (Nelson et al., 1996). Most substrates of CYP2E1 are low-molecular-weight procarcinogens such as benzene, vinyl chloride, and chloroform (Guengerich et al., 1991). This isoform has received a great deal of attention recently due to the discovery of several genetic polymorphisms in the cyp2e1 gene in humans that may be related to oxidative capacity (Hu et al., 1997; Kato et al., 1995). Therefore the degree to which CYP2E1 in particular is responsible for BD oxidative metabolism and hence genotoxicity in mice is germane to the evaluation of this enzyme as a potential human risk factor for BD exposure. Csánady et al. (1992) suggested that the correlation between the rate of chlorzoxazone hydroxylation, a CYP2E1-mediated reaction, and BD oxidation was weaker than that reported for other substrates, suggesting the involvement of other P450 isoforms in the oxidation of BD in vitro. Further, Duescher and Elfarra (1994) found that both CYP2A6 and CYP2E1 activity correlated with the rates of BD oxidation in vitro. In addition, the authors stated that the activity of CYP2A6 predominated at high concentrations, while the activity of CYP2E1 may predominate at low concentrations. Seaton et al. (1995) also used several cDNA-expressed human CYP450 isoforms to address the role of CYP450s in the oxidation of EB to DEB; these studies indicate that CYP2E1 and CYP3A4 are the hepatic isoforms involved in this oxidation in vitro. In humans, in vitro data suggests that CYP2E1-mediated oxidation of BD may predominate at low concentrations. These data, coupled with the extensive in vivo and in vitro data, indicate that metabolites of BD may be the putative carcinogen(s). We tested the hypothesis that CYP2E1-mediated oxidation of BD is required for genotoxicity to ensue following BD exposure. Statistical tests indicated a strong positive correlation between the amount of BD metabolized and the frequency of MN and MN-. Elimination of CYP2E1-mediated oxidation of BD by pretreatment with DCE reduced the frequency of MN and MN-, although not to the level of unexposed controls. In contrast, inhibition of all P450 isoforms by ABT treatment eliminated all BD oxidation; consequently, no increases in MN and MN- frequencies were observed. Taken together, these data suggest a role for other P450 isoforms in BD metabolism and resultant genotoxicity. Inhibition of target enzymes is one established method for the determination of CYP450-dependent metabolism. To date, several inhibitors of CYP2E1 have been utilized (Brady et al., 1991; Chang et al., 1994). However, the complete elimination of CYP2E1 has not been achieved by the use of these inhibitors. Recently, 1,2-trans-dichloroethylene (DCE) was demonstrated to be an effective inhibitor of CYP2E1 (Lilly et al., 1998; Mathews et al., 1998). In acetone-induced rat liver microsomes treated with DCE, p-nitrophenol metabolism was reduced 78% in the treated group. DCE is an attractive alternative to previously used inhibitors because of the low IC50 value, rapid elimination, and multiple options for exposure routes. The use of DCE as a specific inhibitor of CYP2E1 in the experiments presented here, along with confirmation that additional isoforms of CYP450 were not affected by this pretreatment, will be valuable in the study of the toxicity of compounds that may be preferentially metabolized by CYP2E1. As compared with other cytogenetic assays, there are many advantages of quantifying micronuclei as an end point of genotoxicity, including the ease and quickness of analysis, the nonrequirement of metaphase cells, and the variety of cell types that can be utilized. Different mechanisms can be involved in the formation of micronuclei, including clastogenesis (chromosomal breakage) and spindle disruption (aneuploidogenesis). Molecular methods, such as the use of DNA probes or antikinetochore antibodies, have been used to determine the origin of micronuclei induction. Using fluorescence in situ hybridization (FISH) with centromere-specific probes to investigate the nature of micronuclei induced by BD in the bone marrow of mice, researchers have shown that exposure to BD, EB, and DEB results in predominantly clastogenic effects and only a weak anuegenic response in mice (Xiao et al., 1996). An assay was reported for the identification of aneuploidy-inducing agents using an antikinetochore antibody (Eastmond and Tucker, 1989). This assay is based upon the assumption that a micronucleus containing a kinetochore presumably contains the centromere and whole chromosome and therefore arose via an aneuploidogenic mechanism. Previous analysis of micronuclei using FISH in samples from rodents exposed to 1,3-butadiene and metabolites has been described (Xiao et al., 1996). These data demonstrated that BD exposure decreased centromere-positive micronuclei, demonstrating that BD exposure functions as a strong clastogen. Our results with the antikinetochore antibody assay are comparable to the work of Xiao et al. (1996) in mice and Xi et al. (1997) in human cell culture, in that BD exposure was shown to be a strong clastogen in all three cases. Our data expand upon previous findings by illustrating that non-CYP2E1-mediated oxidation of BD to reactive epoxide metabolites is responsible for approximately one-half the clastogenic effect of BD exposure. Metabolic transformation of other environmental chemicals such as benzene and the requirement of this metabolism for toxicity have been studied by other researchers. In the case of benzene, experiments involving mice that lacked a functional cyp2e1 gene were utilized to test this requirement (Valentine et al., 1996). Those experiments conclusively demonstrated that CYP2E1 is the primary isoform responsible for the oxidative metabolism of benzene and subsequent toxicity. Mice lacking a functional cyp2e1 gene demonstrated negligible metabolism and toxicity after exposure to benzene. The expression of CYP2E1 in humans will more than likely become a key risk factor in human health risk assessments for benzene. By contrast, the data presented here suggest other isoforms of CYP450 may be in part responsible for the metabolism of BD and BD-mediated genotoxicity. Based on our studies, these enzymes may be responsible for approximately one-half of the genotoxicity, as measured by micronuclei induction, produced by BD metabolites. Consequently, while CYP2E1 may play an important role in the oxidation of BD to DNA-reactive metabolites, an understanding of the extent to which additional enzymes are involved is necessary to completely characterize these enzymes as potential risk factors for BD toxicity in humans. TABLE 1 Physiological Parameters in the Model Used to Analyze Gas-Uptake Data Parameter  Value   Note. Values taken Medinsky et al. (1994).  aAlveolar ventilation, cardiac output and metabolic rates are given for a hypothetical 1-kg animal.  bBlood flows and organ volumes are expressed as a fraction of total blood flow or body weight.  Alveolar ventilationa  41 L/h/kg   Cardiac outputb  41& L/h/kg   Body weight  Actual values from experiment   Blood flow (fraction of cardiac output)b     Liver  0.25    Fat  0.09    Lung  1.0    Slowly perfused tissues  0.15    Richly perfused tissues  0.51   Organ volumes (fraction of body weight)b     Liver  0.0624    Fat  0.1    Lung  0.005    Slowly perfused tissues  0.7    Richly perfused tissues  0.0226   Tissue:air partition coefficients, butadiene     Blood  1.35    Liver  1.35    Lung  1.47    Muscle  4.01    Fat  19.2   In vivo rate constants for metabolism, butadiene     Liver (oxidation)       Vmax  338 μmol/h/kg     Km  2.0 μmol/L    Lung (oxidation)       Vmax  21.6 μmol/h/kg     Km  5.01 μmol/L  Parameter  Value   Note. Values taken Medinsky et al. (1994).  aAlveolar ventilation, cardiac output and metabolic rates are given for a hypothetical 1-kg animal.  bBlood flows and organ volumes are expressed as a fraction of total blood flow or body weight.  Alveolar ventilationa  41 L/h/kg   Cardiac outputb  41& L/h/kg   Body weight  Actual values from experiment   Blood flow (fraction of cardiac output)b     Liver  0.25    Fat  0.09    Lung  1.0    Slowly perfused tissues  0.15    Richly perfused tissues  0.51   Organ volumes (fraction of body weight)b     Liver  0.0624    Fat  0.1    Lung  0.005    Slowly perfused tissues  0.7    Richly perfused tissues  0.0226   Tissue:air partition coefficients, butadiene     Blood  1.35    Liver  1.35    Lung  1.47    Muscle  4.01    Fat  19.2   In vivo rate constants for metabolism, butadiene     Liver (oxidation)       Vmax  338 μmol/h/kg     Km  2.0 μmol/L    Lung (oxidation)       Vmax  21.6 μmol/h/kg     Km  5.01 μmol/L  View Large TABLE 2 Micronucleated Polychromatic Erythrocytes (MN-PCE) and Kinetochore-Positive and Kinetochore-Negative MN-PCE in Bone Marrow of Male B6C3F1 Mice Exposed to Butadiene Treatmenta  MN/1000 (AO)b  MN/1000 (AKAB)c  MN-/1000d  MN+/1000e  aNaive, animals neither pretreated with inhibitors nor exposed to BD; DCE, animals pretreated with 1,2-trans-dichloroethylene (DCE), a CYP2E1 inhibitor; BD +ABT, animals pretreated with 1-aminobenzotriazole (ABT), a CYP450 inhibitor, and exposed to 1100 ppm BD; BD + DCE, animals pretreated with DCE and exposed to 1100 ppm BD; BD, animals not pretreated and exposed to 1100 ppm BD.  bMN frequencies determined by acridine orange (AO) staining.  cMN frequencies determined by the antikinetochore antibody assay (AKAB).  dMN–, micronuclei that do not contain kinetochores, determined from the AKAB antibody.  eMN+, micronuclei that contain kinetochores, determined from the AKAB antibody.  fStatistically different (p < 0.05) from all other groups.  Naive  9.87 ± 2.1  10.1 ± 1.9  5.2 ± 1.7  4.9 ± 0.7   DCE  11.5  ± 2.0  10.8 ± 2.0  5.0 ± 0.9  5.8 ± 0.8   ABT + BD  12.3  ± 1.9  11.5 ± 2.2  6.4 ± 1.0  5.1 ± 0.6   DCE + BD  19.8  ± 2.5f  21.4 ± 2.0f  13.7 ± 1.3f  7.7 ± 0.6   BD  28.2  ± 3.1f  29.0 ± 3.0f  21.3 ± 1.2f  7.7 ± 0.3  Treatmenta  MN/1000 (AO)b  MN/1000 (AKAB)c  MN-/1000d  MN+/1000e  aNaive, animals neither pretreated with inhibitors nor exposed to BD; DCE, animals pretreated with 1,2-trans-dichloroethylene (DCE), a CYP2E1 inhibitor; BD +ABT, animals pretreated with 1-aminobenzotriazole (ABT), a CYP450 inhibitor, and exposed to 1100 ppm BD; BD + DCE, animals pretreated with DCE and exposed to 1100 ppm BD; BD, animals not pretreated and exposed to 1100 ppm BD.  bMN frequencies determined by acridine orange (AO) staining.  cMN frequencies determined by the antikinetochore antibody assay (AKAB).  dMN–, micronuclei that do not contain kinetochores, determined from the AKAB antibody.  eMN+, micronuclei that contain kinetochores, determined from the AKAB antibody.  fStatistically different (p < 0.05) from all other groups.  Naive  9.87 ± 2.1  10.1 ± 1.9  5.2 ± 1.7  4.9 ± 0.7   DCE  11.5  ± 2.0  10.8 ± 2.0  5.0 ± 0.9  5.8 ± 0.8   ABT + BD  12.3  ± 1.9  11.5 ± 2.2  6.4 ± 1.0  5.1 ± 0.6   DCE + BD  19.8  ± 2.5f  21.4 ± 2.0f  13.7 ± 1.3f  7.7 ± 0.6   BD  28.2  ± 3.1f  29.0 ± 3.0f  21.3 ± 1.2f  7.7 ± 0.3  View Large FIG. 1. View largeDownload slide Structure of 1,3-butadiene (BD) and its putative carcinogenic metabolites epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB). The scheme denotes the hypothesized role of CYP2E1 in this metabolic process. FIG. 1. View largeDownload slide Structure of 1,3-butadiene (BD) and its putative carcinogenic metabolites epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB). The scheme denotes the hypothesized role of CYP2E1 in this metabolic process. FIG. 2. View largeDownload slide Disappearance of 1,3-butadiene from a closed chamber holding male B6C3F1 mice (n = 5/treatment group) at an initial 1,3-butadiene concentration of 1100 ppm. The data (1,3-butadiene chamber concentrations at 10-min sampling intervals, represented by symbols) were simulated (smooth curves) by optimizing the parameter F in the PBPK model, where F represents the fraction of metabolism remaining after pretreatments. Model simulations of the gas uptake data predicted that BD oxidation would be reduced by 60% (F = 0.4) and 100% (F = 0), for the DCE-pretreated and ABT-pretreated groups, respectively. The value of F for animals not pretreated was 0.988. FIG. 2. View largeDownload slide Disappearance of 1,3-butadiene from a closed chamber holding male B6C3F1 mice (n = 5/treatment group) at an initial 1,3-butadiene concentration of 1100 ppm. The data (1,3-butadiene chamber concentrations at 10-min sampling intervals, represented by symbols) were simulated (smooth curves) by optimizing the parameter F in the PBPK model, where F represents the fraction of metabolism remaining after pretreatments. Model simulations of the gas uptake data predicted that BD oxidation would be reduced by 60% (F = 0.4) and 100% (F = 0), for the DCE-pretreated and ABT-pretreated groups, respectively. The value of F for animals not pretreated was 0.988. FIG. 3. View largeDownload slide Activity of CYP2E1, CYP2A5, CYP1A1/2, and CYP2B1 in microsomes isolated from male B6C3F1 mice pretreated with 1,2-trans-dichloroethylene inhibitor of CYP2E1 (gray bars), or untreated (black bars). *Significantly lower than control microsomal activity (p < 0.05). FIG. 3. View largeDownload slide Activity of CYP2E1, CYP2A5, CYP1A1/2, and CYP2B1 in microsomes isolated from male B6C3F1 mice pretreated with 1,2-trans-dichloroethylene inhibitor of CYP2E1 (gray bars), or untreated (black bars). *Significantly lower than control microsomal activity (p < 0.05). FIG. 4. View largeDownload slide Frequency of micronucleated polychromatic erythrocytes (MN-PCE) expressed as a function of the amount of 1,3-butadiene (BD) metabolized. The amount of BD metabolized per treatment group was found by integrating the Michaelis-Menten equations describing both lung and liver metabolism from 0 to 24 h, the time at which the animals were sacrificed. The squares represent the frequency of MN-PCE as a function of the total amount of BD metabolized. The triangles represent the frequency of MN-PCE arising via an aneugenic mechanism (MN+) as a function of the total amount of BD metabolized. The circles represent the frequency of MN-PCE arising via a clastogenic mechanism (MN–) as a function of the amount of BD metabolized. Each point represents the mean ± SD of data from 5 animals. FIG. 4. View largeDownload slide Frequency of micronucleated polychromatic erythrocytes (MN-PCE) expressed as a function of the amount of 1,3-butadiene (BD) metabolized. The amount of BD metabolized per treatment group was found by integrating the Michaelis-Menten equations describing both lung and liver metabolism from 0 to 24 h, the time at which the animals were sacrificed. The squares represent the frequency of MN-PCE as a function of the total amount of BD metabolized. 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TI - Inhibition of Cytochrome P450 2E1 Decreases, but Does Not Eliminate, Genotoxicity Mediated by 1,3-Butadiene
JF - Toxicological Sciences
DO - 10.1093/toxsci/55.2.266
DA - 2000-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/inhibition-of-cytochrome-p450-2e1-decreases-but-does-not-eliminate-g0QNvTUhWG
SP - 266
EP - 273
VL - 55
IS - 2
DP - DeepDyve
ER -