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Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada (P.G.F., B.M., K.S.C.); Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California (R.M.B., M.A.S.); and Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan (L.H.L., D.A.P.)
(Received April 10, 2005; accepted June 24, 2005)
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Abstract |
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). The National Toxicology Program (1990) has estimated that 3.5 million workers are exposed annually to TCE in the United States. Although the current occupational exposure limit for TCE is 50 ppm (American Conference of Governmental Industrial Hygienists, 2004), workers are still exposed to TCE under conditions where adequate protective measures are not in place (National Institute for Occupational Safety and Health, 1978; Forkert et al., 2003). As a result of its versatility and widespread use, TCE has become a prevalent soil and groundwater contaminant (Davidson and Beliles, 1991).
The major target organs of TCE exposure are the liver, kidney, and lung (for review, see Lash et al., 2000). Substantial data have accrued to affirm that an obligate step in the toxicities induced by TCE is its bioactivation to reactive metabolites capable of binding covalently to tissue constituents, including proteins. The metabolism of TCE is mediated through conjugative and oxidative pathways. Although P450 enzymes are present in the kidney, the nephrotoxic effects of TCE are mediated via the glutathione conjugation pathway. Initial conjugation with glutathione, which takes place mainly in the liver, produces S-(1,2-dichlorovinyl)glutathione, and subsequent metabolic processes, including transport and biotransformation, lead to the formation of S-(1,2-dichlorovinyl)-L-cysteine, a metabolite that has been shown to be nephrotoxic in rats (Elfarra and Anders, 1984; Elfarra et al., 1986; Dekant et al., 1990). However, the predominant pathway of TCE metabolism is oxidation via the cytochrome P450 system, mainly by CYP2E1, although other P450 enzymes, including CYP1A1/2, CYP2B1/2, and CYP2C11/6, have also been implicated (Nakajima et al., 1990, 1992; Guengerich et al., 1991).
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; Dekant et al., 1986). Dichloroacetyl chloride subsequently undergoes decomposition to form dichloroacetic acid (DCA), which can also be formed from dechlorination of TCA (for review, see Lash et al., 2000). Thus, exposure to TCE and its metabolism results in the generation of several metabolites, including chloral, CH, dichloroacetyl chloride, TCA, TCOH, and DCA. It has been proposed that TCA mediates the hepatotoxicity of TCE (Bull, 2000), whereas TCA and DCA are responsible for its hepatocarcinogenic effects (Larson and Bull, 1992). Although it has been difficult to identify the reactive species responsible for a specific toxic effect, there is general agreement that TCA, TCOH, and DCA mediate the toxic and/or carcinogenic effects of TCE (Pastino et al., 2000).
Although the liver is the primary tissue in which TCE oxidation occurs, oxidative metabolism also takes place in other tissues such as the lung, where injury is selectively manifested in Clara cells of the bronchiolar epithelium (Forkert et al., 1985; Forkert and Birch, 1989). The pneumotoxic lesion is associated with TCE metabolism to reactive intermediates that bind covalently to lung proteins. In this investigation, we have undertaken studies to test the hypothesis that cytochrome P450 enzymes, including CYP2E1, CYP2F2, and CYP2B1, catalyze the bioactivation of TCE. Kinetic studies were performed to determine the relative affinities for TCE metabolism to CH using incubations of TCE with recombinant rat and human CYP2E1 (rCYP2E1), recombinant rat CYP2B1 (rCYP2B1), recombinant rat CYP2F4 (rCYP2F4), or recombinant mouse CYP2F2 (rCYP2F2). Unlike CYP2E1 and CYP2B1, which are present in liver at high levels, the CYP2F enzyme resides primarily in lung tissue and is expressed as only a single member in each of the species studied: human (2F1) (Nhamburo et al., 1989); mouse (2F2) (Ritter et al., 1991); goat (2F3) (Wang et al., 1998); and rat (2F4) (Baldwin et al., 2005). Alterations of immunoreactive CYP2E1, CYP2F2, and CYP2B1 proteins and associated enzyme catalytic activities were evaluated in lung microsomes from TCE-treated mice. Formation of CH was also determined in incubations of lung microsomes from mice and humans. Our results demonstrated that rat and human rCYP2E1, rCYP2F, and rCYP2B1 were all capable of mediating TCE metabolism to CH, although rat rCYP2E1 exhibited greater affinity than rCYP2F, rCYP2B1, or human rCYP2E1. Moreover, markedly higher rates of CH production were observed in murine than in human lung.
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Materials and Methods |
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). The CYP2B1 monoclonal antibody (mAb 2-66-3) was donated by Dr. H. V. Gelboin (National Cancer Institute, Bethesda, MD). Recombinant rat CYP2F4, mouse CYP2F2, and rat NADPH-cytochrome P450 reductase were donated by Dr. Alan R. Buckpitt (University of California, Davis, CA). Animal Treatment. Male CD-1 mice, weighing 25 to 28 g, were purchased from Charles River Canada (St. Constant, QC, Canada). The animals were housed in an accredited facility (Canadian Council on Animal Care) with a 12-h light/dark cycle and free access to water and food (Mouse Diet 5015; PMI Nutrition International, Inc., St. Louis, MO). Mice were acclimatized to laboratory conditions for at least 1 week before being used in an experiment. For immunoblotting and enzyme catalytic activity studies, mice were treated with 750 mg/kg (i.p.) TCE in corn oil and sacrificed at 5, 15, and 30 min or 1, 2, and 4 h after treatment. Control mice were treated with equivalent volumes of the vehicle.
Incubation of TCE with Recombinant P450 Enzymes. Incubations with TCE were performed with recombinant P450 enzymes that were coexpressed with NADPH-cytochrome P450 reductase, with the exception of rCYP2F4 and rCYP2F2. Reaction mixtures in a final volume of 250 µl of 50 mM Tris-HCl buffer, pH 7.4, contained 10 pmol of rat rCYP2E1, 15 pmol of human rCYP2E1, or 10 pmol of rat rCYP2B1 and 0.05 to 5.0 mM TCE in 0.4% (v/v) acetonitrile and were preincubated at 37°C for 3 min. The reaction was initiated by the addition of 1.0 mM NADPH, and the incubations were continued for an additional 20 min. The reaction was terminated by freezing in liquid nitrogen. Reaction mixtures containing 5 pmol of rCYP2F4 or 5 pmol of rCYP2F2, NADPH-cytochrome P450 reductase (10 U/pmol P450), and 2 mM 3-[(-cholamidopropyl)dimethyl-amino]-1-propanesulfonate in 0.1 M Na2HPO4, pH 7.4, were preincubated at 4°C for 2 h. NADPH-cytochrome oxidoreductase was added to the incubations containing rCYP2F4 or rCYP2F2, because the recombinant enzymes were not coexpressed with the reductase. The reductase was purified from rat liver, and specific activity was determined using procedures described previously (Shultz et al., 2001). After preincubation, an NADPH-generating system (2 U of glucose-6-phosphate dehydrogenase, 15 mM glucose 6-phosphate, 2 mM NADP, and 1 mM MgCl2) was added and brought to a final volume of 250 µl. TCE concentrations of 0.25 to 3.0 mM in 0.4% (v/v) acetonitrile were added, and the incubation was carried out for 20 min at 37°C. Reactions were terminated by freezing in liquid nitrogen.
Preparation of Microsomes. Human lung tissue (10-50 g) was obtained from Kingston General Hospital (Kingston, ON, Canada) from consenting patients undergoing surgical lobectomies. The protocol for the studies in human lung was approved by the Human Ethics Committee of Queen's University. Tissue distant from the primary lesions was surgically excised, placed on ice, transferred immediately to a biohazard facility, sectioned into smaller pieces, frozen in liquid nitrogen, and stored at -80°C. Human lung microsomes were prepared by differential centrifugation using procedures described previously (Forkert et al., 2001). Murine lung microsomes were prepared as described in previous studies (Simmonds et al., 2004b), with minor modifications. Lung tissues from 10 mice were pooled for each microsomal sample, whereas human lung tissue was processed as individual samples. Lung tissue was minced and homogenized in 4 volumes of ice-cold phosphate-buffered KCl (139 mM KCl, 100 mM K2HPO4, and 1.5 mM EDTA, pH 7.4). The homogenate was subjected to centrifugation at 12,500g for 20 min at 2°C. The supernatant was then centrifuged at 105,000g for 60 min. The pellet was resuspended in buffer with approximately three strokes of the pestle and centrifuged at 105,000g for 60 min. The final pellet was resuspended in buffer. Aliquots of lung microsomes were frozen in liquid nitrogen and stored at -70°C. Microsomal protein concentrations were determined by the Bradford method (Bradford, 1976) using bovine serum albumin as the standard.
Protein Immunoblotting. Protein blots were prepared using a modification of procedures described previously (Forkert, 1995). Microsomal proteins were separated by SDS-polyacrylamide gel electrophoresis using an 8.5% gel and transferred to a polyvinylidene difluoride membrane. Low-range SDS-polyacrylamide gel electrophoresis standards (Bio-Rad) were included on each gel. Microsomal proteins from human ß-lymphoblastoid cells expressing CYP2F1 were included as a positive control for murine CYP2F2 protein. Membranes were stained with Ponceau S to confirm uniform protein loading and membrane transfer. Membranes were then incubated overnight in a blocking solution consisting of 10% nonfat milk powder in 20 mM Tris-HCl containing 500 mM NaCl, pH 7.5. After rinsing in Tween 20/Tris-buffered saline, the membrane was incubated for 2 h with a CYP2E1, CYP2F1, or CYP2B1 antibody. The CYP2F1 antibody was generated against a cyclic peptide of 23 amino acids (Nichols et al., 2003) and was affinity-purified using a SulfoLink Coupling gel kit (Pierce Chemical, Rockford, IL). The blots were incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h as follows: CYP2E1 with rabbit anti-goat IgG (1:5000), CYP2F1 with goat anti-rabbit-IgG (1:18000), and CYP2B1 with a goat anti-rabbit IgG (1:5000). The protein bands were visualized using chemiluminescence reagents (Immuno-Star HRP substrate kit; Bio-Rad). Densitometric analyses of the Ponceau S-stained protein bands and radiographs were performed using a Gel Logic 200 Imaging System and Kodak 1D Image Analysis Software (Eastman Kodak Co., Rochester, NY).
p-Nitrophenol Hydroxylase and Pentoxyresorufin O-Dealkylase Activities. PNP hydroxylation was used as a catalytic marker for CYP2E1 and CYP2F2, whereas pentoxyresorufin O-dealkylation was used as a marker for the CYP2B enzymes (Shultz et al., 1999; Simmonds et al., 2004a). For determination of PNP hydroxylase activity, reaction mixtures contained 0.5 mg of lung microsomal protein from TCE-treated mice and 1.5 mM NADPH. A total volume of 250 µl of 100 mM potassium phosphate buffer containing 100 mM ascorbic acid, pH 6.8, was used. After preincubation for 3 min at 37°C, 1 mM PNP in dimethyl sulfoxide was added and the incubations were continued for an additional 10 min. Microsomal proteins were precipitated with 20 µl of perchloric acid (70%) and removed by centrifugation. Quantification of 4-nitrocatechol in the supernatant fraction was determined by HPLC analysis as described previously (Duescher and Elfarra, 1993). In brief, 100-µl samples were analyzed using a Beckman System Gold Programmable Solvent Module 126 HPLC, a reversed-phase C18 column (5 µm, 4.6 x 250 mm; Beckman Ultrasphere ODS), and a Beckman System Gold Module 168 UV detector (Beckman Coulter, Inc., Fullerton, CA). The isocratic mobile phase was 25% acetonitrile/75% H2O/0.1% trifluoroacetic acid with a flow rate of 1.5 ml/min. The column effluent was monitored at 345 nm. The levels of PNP hydroxylase activity were assessed by the formation of 4-nitrocatechol, which eluted from the column at 5.5 min, and quantified by relating the peak area to a standard calibration curve of known amounts of 4-nitrocatechol.
Lung microsomes from TCE-treated mice were used for measurement of pentoxyresorufin O-dealkylase activity. Reaction mixtures in a total volume of 200 µl of potassium phosphate buffer, pH 7.4, contained 0.1 mg of microsomal protein, 1.0 mM NADPH, and 2.0 mM pentoxyresorufin and were incubated for 15 min at 37°C. The reaction was terminated by cooling on ice. A volume of 200 µl of each sample was loaded into a 96-well microplate and read on a fluorescence plate reader (SpectraMax Gemini XS fluorescent plate reader/Softmax PRO software; Molecular Devices, Sunnyvale, CA) using an excitation wavelength of 535 nm and an emission wavelength of 580 nm. Pentoxyresorufin O-dealkylase activity was determined by the formation of resorufin and measured by reference to a standard curve containing known amounts of resorufin.
Formation of Chloral Hydrate in Murine and Human Lung Microsomal Incubations. Reaction mixtures in a total volume of 500 µl of 50 mM Tris-HCl buffer, pH 7.4, consisted of 0.25 mg of lung microsomal protein from untreated mice, 1.0 mM NADPH, and 1.0 mM TCE in 0.4% (v/v) acetonitrile. The incubations were performed at 37°C for 0, 20, 30, 45, and 60 min. Reactions were terminated by freezing in liquid nitrogen. Reaction mixtures in the human lung metabolism study contained 1.25 mg of microsomal protein, 1.0 mM NADPH, and 4.0 mM TCE in 0.4% (v/v) acetonitrile in a total volume of 250 µl of 50 mM Tris-HCl buffer, pH 7.4. Incubations were maintained for 20 min at 37°C. All of the reactions were terminated by the addition of 6.2 nmol of 1,3-dibromopropane in 250 µl of ethyl acetate.
Detection of Chloral Hydrate by Gas Chromatography. Quantitation of CH in murine and human lung microsomal incubations was performed using the methods described previously (Cummings et al., 2001). In brief, microsomal proteins from the murine (500 µl) and human (250 µl) lung microsomal incubations were thawed and extracted with 0.5 ml of ethyl acetate, after which 10 nmol of 1,3-dibromopropane was added as an internal standard. Analysis for TCE and CH was performed by gas chromatography with electron capture detection using a PerkinElmer PE-210 capillary column (30 m x 0.25 mm i.d. x 0.5 µm film thickness) and a PerkinElmer AutoSystem XL GC system (PerkinElmer Life and Analytical Sciences, Boston, MA). Injector temperature was 200°C, detector temperature was 300°C, and helium was the carrier gas at a flow rate of 24.8 ml/min at 150°C. The oven ramp method used for sample elution consisted of a temperature of 35°C for 11 min, a linear gradient from 35 to 20°C from 11 to 19 min, and a holding temperature of 120°C from 19 to 38 min. Retention times for TCE and CH were 3.90 and 6.15 min, respectively. Limits of detection were 0.2 and 0.05 pmol/mg protein for TCE and CH, respectively.
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Protein Immunoblotting. Immunoblots of lung microsomal proteins using the anti-CYP2E1 antibody yielded a single immunoreactive band with an apparent molecular mass of 51 kDa (Fig. 3A), similar to the molecular mass reported previously (Simmonds et al., 2004a). The amount of immunodetectable CYP2E1 was unchanged in lung microsomes from mice sacrificed from 5 to 60 min after TCE treatment. However, 70 and 35% decreases in immunoreactive CYP2E1 protein were observed at 2 and 4 h, respectively (Fig. 3, A and B). Protein blots probed with the CYP2F1 antibody demonstrated cross-reactivity with murine lung CYP2F2 as reported previously (Simmonds et al., 2004a). A protein band of approximately 56 kDa was detected and is similar to the molecular mass reported previously for this protein (Simmonds et al., 2004a).
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). Densitometric scanning revealed that, compared with controls, CYP2F2 protein content was decreased by approximately 75% at 15 min after TCE, abolished at 30 min, and partially recovered at 60 min when CYP2F2 levels amounted to approximately 60% of the control (Fig. 3D). The anti-CYP2B1 antibody detected a protein band of approximately 55 kDa (Fig. 3E). Densitometric scanning showed no changes in CYP2B1 protein at 5 to 15 min after TCE treatment (Fig. 3F). However, decreases of 37 to 57% in CYP2B1 protein contents were detected 1 to 4 h after TCE treatment. p-Nitrophenol Hydroxylase and Pentoxyresorufin O-Dealkylase Activities. Time course studies revealed marked decreases in lung PNP hydroxylase activity as a result of treatment of mice with TCE. A 30% loss of lung microsomal PNP hydroxylase activity was detected as early as 15 min after TCE treatment, and this was exacerbated to a 75% reduction by 30 min (Fig. 4A). The nadir was reached at 2 to 4 h when the levels of residual PNP hydroxylase activity comprised only approximately 7 to 10% of the control level. Thus, there is virtual elimination of hydroxylase activity observed at 2 to 4 h after TCE treatment. Treatment of mice with TCE also produced significant decreases in pentoxyresorufin O-dealkylase activity (Fig. 4B). A loss of 33% dealkylase activity was detected at 5 min after TCE treatment. Further decreases were observed at 15 min to 2 h after treatment, and at 4 h, the levels were reduced to 36% of the control level. Thus, TCE treatment caused decreases of lung microsomal PNP hydroxylase activity that were more pronounced than observed for pentoxyresorufin O-dealkylase activity.
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). A high-affinity enzyme present in liver microsomes from control and ethanol-treated rats catalyzed CH formation from TCE. An additional P450 isoform with low affinity for TCE metabolism was induced by phenobarbital, whereas a form with intermediate affinity was induced by 3-methylcholanthrene. Subsequent studies using inhibitory monoclonal antibodies and rat liver microsomes yielded findings implicating CYP2E1, CYP2B1/2, CYP2C11/6, and CYP1A1/2 in TCE metabolism to CH (Nakajima et al., 1992). However, the inhibitory effects of the antibodies were most pronounced for CYP2E1 and CYP2B1, suggesting relevant contributions of these P450 isoforms to TCE metabolism in rat liver. More recent studies found that the formation of chloral and TCE oxide was greater with liver microsomes from rats treated with phenobarbital to induce CYP2B1 than from rats treated with isoniazid to induce CYP2E1 (Cai and Guengerich, 2000). Findings from other studies confirmed that human liver CYP2E1 was also capable of metabolizing TCE, albeit at a diminished level (Guengerich et al., 1991). Although studies of TCE metabolism have been mainly conducted in the liver, similar studies have also been carried out in the lung. The results of studies in the lung showed that mice were more susceptible to TCE-induced toxicity than rats (Odum et al., 1992) and further that this effect was proposed to be due, in part, to higher rates of chloral formation in mice than in rats (Green et al., 1997). The enhanced rates of chloral formation were attributed to the higher CYP2E1 levels found in mice versus rats. Relevant in this context are recent findings showing that CYP2F is also present at higher levels in the lungs of mice than rats (Baldwin et al., 2004). However, the P450 enzymes responsible for lung metabolism of TCE have not been identified and characterized.
An objective of this study was to identify the P450 enzymes involved in TCE metabolism in murine and human lung. An initial approach was to select candidate lung P450 enzymes to determine their relative affinities for TCE metabolism. The enzymes CYP2E1, CYP2F2, and CYP2B1 are all present constitutively in lung tissue and are preferentially localized in the bronchiolar Clara cells (Buckpitt et al., 1995; Forkert, 1995), a cell type that is a selective target of TCE-induced lung cytotoxicity (Forkert et al., 1985). Incubations were performed with rat and human rCYP2E1, rCYP2F2, rCYP2F4, and rCYP2B1 to assess their relative capabilities for TCE metabolism to CH. These studies yielded data to demonstrate that all of the recombinant P450 enzymes catalyzed TCE metabolism to CH, with highly positive correlations between TCE concentrations and CH formation (Fig. 2). Kinetic analysis revealed that rat rCYP2E1 exhibited the lowest Km value and the highest Vmax/Km ratio for TCE metabolism to CH (Table 1), indicating that this P450 catalyzed TCE metabolism to CH with relatively high affinity and catalytic efficiency. Our results also showed that rCYP2F4 catalyzed TCE metabolism with greater affinity and catalytic efficiency than did rCYP2F2, rCYP2B1, and human rCYP2E1, which had the highest Km value and lowest Vmax/Km ratio. These findings demonstrated that all of the recombinant enzymes metabolized TCE to CH, albeit at differing catalytic affinities and efficiencies.
Although the present studies have clearly demonstrated the abilities of the recombinant enzymes CYP2E1, CYP2F4, CYP2F2, and CYP2B1 to metabolize TCE, the relative contributions of each isoform to in vivo lung metabolism of TCE remain unknown. To address this issue, immunoblotting studies were performed to determine the involvement of CYP2E1, CYP2F2, and CYP2B1 in TCE metabolism in murine lung. The rationale for this approach was based on findings from previous studies indicating that TCE metabolism produces reactive intermediates capable of inactivating P450 enzymes responsible for its bioactivation (Cai and Guengerich, 2001). Time course experiments were carried out with lung microsomes from mice treated with TCE (750 mg/kg, i.p.) for time periods ranging from 5 min to 4 h. The loss of immunoreactive CYP2E1, CYP2F2, and CYP2B1 proteins were all detected (Fig. 3). The protein content of CYP2F2 was decreased at 15 min after TCE treatment and was abolished by 30 min (Fig. 3, C and D). On the other hand, the loss of CYP2B1 was manifested from 1 to 4 h (Fig. 3, E and F), whereas decreases of the CYP2E1 protein were detected at a later time from 2 to 4 h (Fig. 3, A and B). The differing time points at which P450 proteins are lost and recovered preclude the possibility that the decreases are due to death and sloughing of Clara cells, leading to diminished recovery of lung microsomal proteins. These findings suggested that modification of the apoprotein moieties of lung CYP2F2, CYP2E1, and CYP2B1 takes place as a result of TCE treatment. The loss of immunoreactive CYP2E1, CYP2F2, and CYP2B1 proteins (Fig. 3) suggested that these P450 enzymes are inactivated by TCE metabolites at their site of formation. To verify that this inactivation occurs, PNP hydroxylation and pentoxyresorufin O-dealkylation were measured in lung microsomes from TCE-treated mice. Significant decreases in hydroxylase activity were observed from 15 min to 4 h (Fig. 4A). Residual hydroxylase activity detected at 2 to 4 h comprised only approximately 7 to 10% of the control level (Fig. 4A). Pentoxyresorufin O-dealkylase activity was reduced from 5 min to 4 h after TCE treatment, with the level remaining at 4 h comprising 36% of the control. The findings showed that the maximal loss of enzyme activity was higher for PNP hydroxylation (93% of control) than for pentoxyresorufin dealkylation (64% of control). These results are consistent with the greater affinity of rCYP2E1 and rCYP2F for TCE metabolism than rCYP2B1 (Table 1). The lack of concordance between the loss of enzyme catalytic activities and loss of immunoreactive proteins is in agreement with previous studies showing that catalytic deactivation in the case of CYP2E1 preceded a decrease in immunoreactive protein (Ronis et al., 1991). It should also be noted that previous studies have reported a loss of heme in incubations of TCE with rat liver microsomes (Miller and Guengerich, 1983). Whether the decreased catalytic activity in the lung may be ascribed also to the effect of TCE on P450 heme remains to be investigated. Taken together, the findings of this study supported the view that lung metabolism of TCE causes P450 inactivation, resulting in the loss of immunoreactive P450 proteins, including CYP2E1, CYP2F2, and CYP2B1 and associated catalytic activities.
In this study, the generation of CH in lung microsomal incubations was used as an index of TCE metabolism. The formation of CH was time-dependent, with rates that were incremental from 5 to 45 min and saturation at 60 min (Fig. 5). The maximal rates of CH produced by murine lung microsomes (7.9 ± 0.9 nmol/min/mg protein) were orders of magnitude greater than the rates found for human lung microsomes, which ranged from 0.44 to 0.58 pmol/min/mg protein from three subjects (Table 1). These data are consistent with the kinetic constants demonstrating the markedly lower Km and higher Vmax/Km ratios for rat rCYP2E1 versus human rCYP2E1 (Table 1). The low rate of CH production by human lung microsomes in the three subjects coupled with the lack of detectability in five other subjects suggested that TCE metabolism in human lung is not an important event. These findings are in agreement with those of previous studies that reported an absence of chloral formation in human lung microsomal incubations (Green et al., 1997). These data suggested that human lung is unlikely to generate a sufficient level of TCE metabolites to elicit a toxic response. However, further studies involving a larger population is required to fully establish the validity of this tentative conclusion.
It is of interest that, in previous studies with 1,1-dichloroethylene, a structural analog of TCE that also causes Clara cell toxicity, CYP2E1 and CYP2F2 mediated its metabolism to an epoxide and are both inactivated in the process (Simmonds et al., 2004a,b). However, it is not known whether CYP2B1 is involved also in the metabolism of 1,1-dichloroethylene, as was found with TCE in this study. Nevertheless, dichloroethylene and TCE may have common pathways of metabolism in lung because of their structural similarities. It is also of interest to compare the disposition of TCE in lung and liver; the findings indicated that the identities of the P450 enzymes responsible for TCE metabolism are not identical in the two tissues. Whereas CYP2E1 and CYP2B1 have important roles in TCE metabolism in the liver (Cai and Guengerich, 2001), as was also found for the lung in this study, CYP2F2, a P450 expressed mainly in the lung, is also involved and seems to have a more important role than CYP2B1, as suggested by the results from experiments with the recombinant P450 enzymes. In summary, the results of this investigation have provided evidence to support a pertinent role for CYP2E1, CYP2F2, and CYP2B1 in the metabolism of TCE in murine lung. Moreover, the findings suggested that CYP2E1 may have a more prominent role than either CYP2F2 or CYP2B1.
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Acknowledgments |
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Footnotes |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: TCE, trichloroethylene; CH, chloral hydrate; DCA, dichloroacetic acid; PNP, p-nitrophenol; rCYP2E1, recombinant CYP2E1; rCYP2F4, recombinant CYP2F4; rCYP2B1, recombinant CYP2B1; TCA, trichloroacetic acid; TCOH, trichloroethanol; HPLC, high-performance liquid chromatography.
Address correspondence to: Dr. Poh-Gek Forkert, Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6. E-mail: forkertp{at}post.queensu.ca
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