* Chemical Industry Institute of Toxicology, 6 Davis Drive, P.O. Box 12137, Research Triangle Park, North Carolina 27709-2137; and
Curriculum in Toxicology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina.
Received September 8, 1998; accepted January 25, 2000
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ABSTRACT |
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Key Words: 1,3-butadiene; CYP2E1; genotoxicity; micronuclei; gas uptake.
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INTRODUCTION |
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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.
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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.
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MATERIALS AND METHODS |
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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 1012 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 1030 min prior to onset of the exposure. Oxygen was monitored and maintained at a level of 1921%. 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 810 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 x 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).
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This equation, and the equation describing CYP450-mediated oxidation of BD in the lung, were multiplied by F:
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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.
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RESULTS |
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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: Boehringer Ingelheim Pharmaceuticals, Toxicology and Safety Assessment, 900 Ridgebury Rd, P.O. Box 368, Ridgefield, CT 06877-0368.
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