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Chem. Res. Toxicol. 1998, 11, 778-785
Selective Inhibition of Cytochrome P450 2E1 in Vivo and in Vitro with trans-1,2-Dichloroethylene James M. Mathews,*,† Amy S. Etheridge,† James H. Raymer,† Sherry R. Black,† Donald W. Pulliam, Jr.,† and John R. Bucher‡ Research Triangle Institute, Research Triangle Park, North Carolina 27709, and National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Received December 22, 1997
The effect of trans-1,2-dichloroethylene (DCE), an inhibitor of cytochrome P450 (P450) 2E1, on the catalytic activities and total content of hepatic P450 was determined in vivo and in vitro. Hepatic microsomes were prepared from groups of rats prior to dosing and at 2, 5, 12, and 24 h postdosing, and total P450 content and the activities of P450 1A2, P450 2A1, P450 2B, P450 2C6, P450 2C11, P450 2D1, P450 2E1, and P450 3A were determined. The lowest dose of DCE that yielded maximal inactivation of P450 2E1 was found to be 100 mg/kg. Significant decreases in total content of P450 or the activities of P450 1A2, P450 2A1, P450 2B, P450 2C6, P450 2C11, P450 2D1, and P450 3A were not observed during the 24 h following administration of DCE (100 mg/kg ip), but P450 2E1 activity was diminished about 65% at 2 and 5 h after DCE treatment and returned to control levels at 24 h. Additionally, there was little or no significant effect on the activities of hepatic cytosolic alcohol dehydrogenase or mitochondrial or microsomal aldehyde dehydrogenases 5 h postdosing. DCE showed the same selectivity for P450 inactivation in vitro, and P450 2E1 activity was inhibited by >80% without affecting the other isozymes. However, DCE (5 mM) also proved to be a good competitive inhibitor of the probe activities of P450 1A2 and P450 2C6. The in vivo inhibition of P450 2E1 was accompanied by decreases in the levels of the immunoreactive protein, and an additional immunoreactive band appeared at ca. 30 kDa in the Western blot of microsomes from DCEtreated rats, possibly arising from proteolytic degradation of P450 2E1 protein after covalent modification by the inhibitor. DCE is an effective, relatively nontoxic inhibitor of P450 2E1 in vivo and in vitro that has greater selectivity than other agents currently used.
Introduction Cytochrome P450 (P450)1 2E1 catalyzes the oxidation of many lower-molecular-weight compounds, in addition to drugs such as chlorzoxazone and acetaminophen. Many of these compounds, including benzene, acetaminophen, and dimethylnitrosoamine, are activated to proximate carcinogens and other toxins by this biotransformation (1, 2). P450 2E1 activity varies with genetic factors and environmental exposures and is induced by ethanol (3-6). trans-1,2-Dichloroethylene (trans-1,2-dichloroethene, DCE) has been used as an industrial solvent for fats, oils, and waxes and in the manufacture of rubber. It is of relatively low toxicity, and the reported LD50 (ip) in rats and mice is g4000 mg/kg (7, 8). DCE has been shown to be a mechanism-based inhibitor of P450 2E1 in rats (911) and mice (12). In these studies rats and mice were exposed to DCE by inhalation, and P450 2E1, but not P450 2B, enzymes were inhibited in rats. A number of inhibitors of P450 2E1 are known, including many sulfur* Corresponding author: James M. Mathews, Ph.D., Research Triangle Institute, P.O. Box 12194, 3040 Cornwallis Rd, RTP, NC 27709. Tel: (919) 541-7461. Fax: (919) 541-6499. E-mail: mathews@ rti.org. † Research Triangle Institute. ‡ National Institute of Environmental Health Sciences. 1 Abbreviations: P450, cytochrome P450; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DCE, trans-1,2-dichloroethylene.
containing chemicals, pyrazoles, propylene glycol, and 3-amino-1,2,4-triazole, but the utility of each is limited by toxicity and/or lack of specificity in the inhibition of oxidative enzymes (Table 1). The present study was designed to determine if DCE can be used as an easily administered inactivator of P450 2E1 that has selectivity superior to agents currently employed. Such an agent would be a valuable tool in studying the involvement of P450 2E1 in processes of great pharmacological and toxicological importance.
Materials and Methods Chemicals. DCE (>99% pure) was purchased from TCI America (Portland, OR). Erythromycin, tolbutamide, and dextromethorphan were purchased from Sigma Chemical Co. (St. Louis, MO). Animals and Treatments. Male Fischer 344 rats, 12-16 weeks old, were obtained from Charles River Laboratories (Raleigh, NC) and, upon arrival, were examined by a veterinarian for evidence of disease, quarantined for 1 week, and examined again before release to the study. Prior to experiments, rats were housed (maximum of 4/cage) in polycarbonate cages and provided tap water and NIH07 diet (Ziegler Brothers, Gardners, PA) ad libitum. Contact bedding for rats was AbSorb-Dri hardwood chips (Lab Products, Maywood, NJ). Three animals were used for each test group. In experiments designed to determine the effect of DCE on P450, the chemical was administered ip to rats at doses of 25, 50, 100, 400, and 800 mg/kg. In experiments designed to determine the effect of DCE
S0893-228x(97)00227-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 06/02/1998
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Table 1. Specificity and Toxicities of P450 2E1 Inhibitorsa inhibitor disulfiram
P450 isozymes affected AlcDH V 2E1 (27) v 2B1 (27)
V (31-33) v (27) V fructose-1,6-diphosphate dehydrogenase (34) V succinic dehydrogenase (34) V glyceraldehyde-3-phosphate dehydrogenase (34) V xanthine oxidase (34) V dopamine β-hydroxylase (34) V aldolases (34) V hexokinase (34) V (32, 33) V dopamine β-hydroxylase (33)
V 3A (27) v P450 reductase (27)
DASd
PEITCe
V 2E1 (1) V 2A6 (37) V 3A (37) V 1A (1) V 2C9 (1) V 2C19 (1) V 2D (1) V 2E1 (28) V 3A (39) v 2B (28, 39) v 1A2 (39) v 2C11 (39) V 2E1 (40-42) V 1A2 (42) V 3A (42)
V 2E1 (43) V 2E1 (45) V ethylmorphine N-demethylaseh (46)
toxicities neurotoxic (35, 36) covalently modifies proteins (35) hepatotoxic (34)
teratogenic (38) genotoxic (38) neurotoxic (38) covalently modifies proteins (35, 38)
v GST (39)
V (40)
v 2B (40-42) PEGf 3-ATg
other affected enzymes GSTb
V 1A (27) v 2A1 (27)
DDCc
AldDH
V (44)
v GST (42) v epoxide hydroxylase (41, 42) v NAD(P)H:quinone oxidoreductase (42) V sulfotransferase (42) Vv UDP glucuronosyltransferase (42)
V (44) V thyroid peroxidase (45) V lactoperoxidase (45)
pyrazole
vV 2E1 (48, 49) v 1A1/2 (50)
V (49)
4-MPi
vV 2E1 (48, 49) v 2B (49)
V (49)
V catalase (45) V δ-aminolevulinic acid dehydratase (45) V catalase (50)
chlormethiazole YH439 v 1A (53, 54) v 2B (55)
thyroid tumors (47) pituitary tumors (47)
hepatotoxic (49) hyperplasia of thyroid (51) hepatomegaly (51) atrophy of testis and accessory glands (51) anemia (51) depression of bone morrow (51) hepatotoxic (49) hepatotoxic (52)
a Literature references are in parentheses. b GST ) glutathione S-transferase. c DDC ) diethyldithiocarbamate. d DAS ) diallyl sulfide. PEITC ) phenethyl isothiocyanate. f PEG ) propylene glycol. g 3-AT ) 3-amino-1,2,4-triazole. h P450 3A, 2B1, 2C11, and 2C6. i 4-MP ) 4-methylpyrazole.
e
on alcohol and aldehyde dehydrogenases, DCE-treated rats were sacrificed 5 h after administration of DCE (100 mg/kg), and their livers were excised and placed in ice-cold isotonic saline. No vehicle was used in any experiments, and control animals were untreated. P450 Assays. Rats were sacrificed by asphyxiation with carbon dioxide at the designated time points, and hepatic microsomes were prepared from the excised livers and assayed for protein concentration, P450 content, benzphetamine Ndemethylation activity, acetanilide hydroxylation activity, and p-nitrophenol (PNP) hydroxylation activity as previously described (13). Erythromycin N-demethylation activity was determined by a modified method of Nash (14). The reaction mixtures contained 100 mM potassium phosphate buffer (pH 7.3), 1-2 mg of microsomal protein, 0.4 mM erythromycin, and 1 mM NADPH in a final volume of 1.0 mL. The reaction was initiated with NADPH, incubated for 10 min at 37 °C, and terminated by the addition of 0.6 mL of 10% trichloroacetic acid (TCA). Following centrifugation, an aliquot of supernatant (1 mL) was added to 0.5 mL of Nash reagent and allowed to stand at 50 °C for 15 min and then at room temperature for 5 min. The absorbance at 412 nm of each resulting solution was determined. A standard curve was prepared using authentic
standards of formaldehyde. Tolbutamide hydroxylation activity was measured using the method of Miners et al. (15). Dextromethorphan O-demethylation activity was measured according to the method of Zysset et al. (16), using the modification of the HPLC analysis method of Laurenzana et al. (17). 2R-, 7R-, and 16β-testosterone hydroxylation activities, markers of P450 2C11, P450 2A1, and P450 2B activities, respectively, were determined by the method of Wood et al. (18). Immunoblotting. Electrophoresis grade sodium dodecyl sulfate (SDS), glycine, TRIZMA base, glycerol, bromphenol blue, and 2-mercaptoethanol and reagent grade TRIZMA HCl and Tween 20 were obtained from Sigma (St. Louis, MO). Rat P450 2E1 acetone-induced microsomes and goat anti-rat P450 2E1 were manufactured by Daiichi (Tokyo) and distributed by GENTEST Corp. (Woburn, MA). Anti-goat IgG, alkaline phosphatase conjugate, and 5-bromo-4-chloro-3-indolylphosphate/ nitro blue tetrazolium (BCIP/NBT) tablets were supplied by Sigma. P450 2E1 microsomal standards and microsomes from control and treated rats were diluted 1:20 in 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES) buffer, treated with SDS reagent, and heated in boiling water according to the manufacturer’s instructions. Microsome samples were centrifuged for 2 min at 900g, then applied to a 10- × 10-cm 10%
780 Chem. Res. Toxicol., Vol. 11, No. 7, 1998 polyacrylamide gel (Owl Scientific, Woburn, MA), and electrophoresed at 40 mA for 70 min in a Hoefer SE 260 Mini-Vertical Unit (Pharmacia Biotech, Piscataway, NJ). Proteins were transferred to nitrocellulose (0.45 µm; Bio-Rad, Hercules, CA) at room temperature for 90 min in 25 mM Tris, 192 mM glycine, and 20% ethanol in a Hoefer TE 22 Transfer Unit. The nitrocellulose was incubated overnight at 2-4 °C in 5% nonfat milk in phosphate-buffered saline (PBS) and then incubated for 1 h at room temperature in goat anti-rat P450 2E1 diluted 1:500 in 0.5% nonfat milk in PBS. The nitrocellulose then received three 5-min washings with 0.1% Tween 20 in PBS (PBST). Antigoat IgG alkaline phosphatase conjugate diluted 1:5000 in 0.5% nonfat milk in PBS was then applied for 1 h at room temperature; then the nitrocellulose was again washed for 5 min three times with PBST. BCIP/NBT in water was then applied for 10 min to visualize protein bands. Densitometry and comparative P450 2E1 protein quantitation were performed by a DeskTop scanner and Quantity One software (PDI, Huntington Station, NY). Inhibition in Vitro. Untreated rats were sacrificed and their livers removed and homogenized as described above. The homogenates were centrifuged at 9000g for 20 min. The supernatant fractions were then centrifuged at 100000g for 50 min. The microsomal pellets were removed and resuspended in approximately 25 mL of buffer to a protein concentration of ca. 4 mg/mL. Two types of in vitro experiments were conducted. Inactivation of the isozymes was assessed in the first experiments, and steps were taken to remove DCE following incubation with microsomes prior to isozyme assays. Each microsomal sample was divided into two portions, each containing NADPH (1 mM) and one portion with DCE (5 mM). The samples were incubated for 30 min at 37 °C; then ice-cold buffer was added. Samples were then centrifuged at 100000g for 30 min. The resulting microsomal pellets were resuspended in buffer, flashfrozen in liquid nitrogen, and stored at -70 °C prior to assay. Total inhibition was assessed in the second set of experiments, and the assays were conducted in the presence and absence of DCE (5 mM final concentration), but without preincubation with DCE. Preparation of Mitochondria. Mitochondria were prepared by minor modifications of the method of Brabec et al. (19). Livers were removed and homogenized in three volumes of icecold 2.5 mM HEPES buffer (pH 7.4) containing 0.15 M KCl using a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 450g for 10 min. The supernatant was removed and centrifuged at 8800g for 10 min. The supernatant was discarded, and pellets were resuspended in fresh HEPES buffer and centrifuged at 480g for 10 min. The supernatant was removed and centrifuged at 7200g for 10 min. The pellet was removed, resuspended in fresh HEPES buffer, and resedimented at 7200g for 10 min. The final mitochondrial pellet was resuspended in fresh HEPES buffer, flash-frozen in liquid nitrogen, and stored at -70 °C. Preparation of Cytosol. Animals were sacrificed and livers removed and homogenized as described above. Homogenates were centrifuged at 9000g for 20 min. The supernatants were removed and centrifuged at 100000g for 50 min. The resulting 100000g supernatants, containing the cytosolic fraction, were flash-frozen in liquid nitrogen and stored at -70 °C. Aldehyde Dehydrogenase Activity. Aldehyde dehydrogenase activity was determined using the method of Tottmar et al. (20). The reaction mixtures contained 50 mM sodium pyrophosphate buffer (pH 8.8), 0.1 mM pyrazole, 2 µM rotenone (in methanol), and 0.5 mM NAD in a final volume of 2.4 mL. Mitochondrial aldehyde dehydrogenase activities were assayed using 0.05 mM acetaldehyde as the substrate and 0.5-1 mg of mitochondrial protein. Microsomal incubations contained 5 mM acetaldehyde and 1-2 mg of microsomal protein. Samples were solubilized with sodium deoxycholate (0.25 mg/mg of protein) immediately prior to addition to incubation tubes. Reactions were initiated by the addition of acetaldehyde. Activities were determined spectrophotometrically, measuring the reduction of
Mathews et al. Table 2. Total Hepatic P450 Content and Activity of P450 2E1a 2 h following ip Administration of DCE to Fischer 344 Ratsb DCE dose (mg/kg) control 25 50 100 400 800
cytochrome P450 (nmol/mg of protein) 0.75 ( 0.07 0.71 ( 0.05 (8) 0.69 ( 0.08 (10) 0.76 ( 0.11 (1)
p-nitrophenol hydroxylation activityc 0.78 ( 0.15 0.59 ( 0.18 (24) 0.45 ( 0.04 (42)* 0.28 ( 0.09 (64)*d,e 0.23 ( 0.03 (71)*d 0.36 ( 0.07 (54)*
a Expressed as p-nitrophenol hydroxylation activity. b Values are means ( standard deviation. Percent decrease from control given in parentheses. c nmol of 4-nitrocatechol formed/mg of protein/min. d 100 and 400 mg/kg dose levels significantly different from 25 and 50 mg/kg dose levels but not significantly different from each other. e This value has been reported previously (25). *Significantly different from control (p e 0.05).
NAD by monitoring the increase of visible absorbance at 340 nm for approximately 3 min at room temperature. Background absorbance was determined in incubations from which the substrate was omitted. After correction for background absorbance, formation of NADH was calculated using an value of 6.22 × 103 M-1 cm-1 (21). Alcohol Dehydrogenase Activity. Alcohol dehydrogenase activities were determined using a modified method of Crow et al. (22). Reaction mixtures contained 1.0 M Tris (pH 7.2), 45 mM ethanol, 1.5-2 mg of cytosolic protein, and 2 mM NAD in a final volume of 2.2 mL. Reactions were initiated by addition of cytosol. Activities were determined spectrophotometrically, measuring the reduction of NAD as described above. Statistical Treatment of Data. The values for the isozyme activities and total content of cytochrome P450 were compared by ANOVA followed by Dunnett’s test. Alcohol and aldehyde dehydrogenase activities were compared by ANOVA. Statistically significant differences were determined at the R ) 0.05 level.
Results In the present work, male Fischer 344 rats were administered trans-1,2-dichloroethylene, a mechanismbased inhibitor of P450 2E1 in rats (9, 11) and mice (12). Range-finding experiments were first conducted to determine the lowest dose that would give maximal inactivation of the hepatic microsomal p-nitrophenol hydroxylation activity, which is mediated by P450 2E1. Inhibition was measured at 2 h postdosing, since this time point showed maximal inhibition with varying dose levels. A dose of 100 mg/kg produced a 64% loss of P450 2E1 activity that was greater than the 25 and 50 mg/kg doses, but not statistically different from the 400 and 800 mg/kg doses (Table 2). In definitive experiments, rats were administered a 100 mg/kg intraperitoneal dose of DCE. DCE was selective in its action against total P450 and particular isozyme activities (Figure 1). The extent of 2E1 inhibition was maximal at 2-5 h, at which time ca. 65% of the activity was lost. This activity remained substantially inhibited out to 12 h but was not statistically different from control at 24 h. In contrast to the marked loss of P450 2E1 activity, there were no statistically significant changes in total content of P450 or the activities of P450 1A2, P450 2A1, P450 2B, P450 2C6, P450 2C11, P450 2D1, and P450 3A during the 24 h following administration of DCE, with the exception of a 25% increase in P450 2C6 activity at 24 h. P450 2E1 comprises less than 10% of the P450 in uninduced rat liver (23), and the minimal
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Figure 1. Total content and activities of hepatic microsomal cytochrome P450 isozymes following intraperitoneal administration of DCE (100 mg/kg) to F-344 rats. *Significantly different from control (p e 0.05). aExpressed as acetanilide hydroxylase; 100% value (control) ) 1.22 nmol/mg‚min. bExpressed as 7R-testosterone hydroxylase; 100% value (control) ) 0.12 nmol/mg‚min. cExpressed as erythromycin N-demethylase; 100% value (control) ) 0.34 nmol/mg‚min. dExpressed as benzphetamine N-demethylase; 100% value (control) ) 1.91 nmol/mg‚min. eExpressed as tolbutamide hydroxylase; 100% value (control) ) 1.22 nmol/mg‚h. fExpressed as 2Rtestosterone hydroxylase; 100% value (control) ) 1.20 nmol/mg‚h. gExpressed as dextromethorphan O-demethylase; 100% value (control) ) 43.1 nmol/mg‚h. hExpressed as p-nitrophenol hydroxylase; 100% value (control) ) 0.83 nmol/mg‚min.
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Mathews et al. Table 3. Effect of DCEa on the Activitiesb of Alcohol and Aldehyde Dehydrogenases alcohol dehydrogenase activity (cytosolic) aldehyde dehydrogenase activities mitochondrial microsomal
control
DCE-treated
6.5 ( 1.1
7.4 ( 1.5 (-15)
6.2 ( 1.4 16 ( 1.3
4.3 ( 0.76 (30) 13 ( 0.63 (18)*
a Rats (N ) 3-4) sacrificed 5 h following a 100 mg/kg ip dose of DCE. b Values are means ( standard deviation for the nmol of NADH formed/mg of protein/min. Percent decrease from control given in parentheses. *Significantly different from control (p e 0.05).
Figure 2. P450 2E1 immunoblot analysis of hepatic microsomes from DCE-treated rats. Lanes 1-5: microsomal standards; 1, 3, 5, 7, and 9 µg of microsomal protein standard containing 484 pmol of P450 2E1/mg, respectively. The optical density was linear with respect to the amount of protein (r2 ) 0.98). Lanes 6-10: 20.0 µg of hepatic microsomal protein from control and 100 mg/kg DCE-treated male rats sacrificed at 2, 5, 12, and 24 h. Molecular weight markers at 81.0, 53.5, 37.0, and 31.4 kDa. The average intensities (in arbitrary units) of the bands from duplicate determinations from pooled microsomes were 1.0, 0.73, 0.51, 0.41, and 0.68 for the control and 2-, 5-, 12-, and 24-h samples, respectively.
effect of DCE on total P450 levels is consistent with specificity of action on that isozyme. Immunoblot analyses of the hepatic microsomes from control and DCE-treated animals are shown in Figure 2. The levels of P450 2E1 protein were more modestly decreased than were the corresponding P450 2E1 activities. A band of lower intensity is also visible at ca. 30 kDa only in the immunoblot from microsomes of DCEtreated rats. Experiments were conducted in vitro to assess (1) isozyme inactivation in microsomes (30-min incubation with DCE and NADPH, after which microsomes were washed and then assayed) and (2) total inhibition in the presence of DCE (the latter present at 5 mM during isozyme assays but not preincubated in the absence of assay substrates). In vitro inactivation experiments with microsomes and DCE gave similar results (Figure 3A) to those found in vivo. P450 2E1 activity was inactivated by >80%, while the activities of P450 1A2, P450 2A1, P450 2C6, P450 2C11, P450 2D1, and P450 3A showed no statistically significant changes. However, when assays were performed in the presence of DCE, there was marked inhibition of P450 1A2 (78%) and P450 2C6 (67%) (Figure 3B). There was no statistically significant change in the activities of P450 2A1, P450 2C11, P450 2D1, and P450 3A. As many of the agents used as inhibitors of P450 2E1 also inhibit alcohol and/or aldehyde dehydrogenases, the inactivation of those enzymes by DCE was also monitored. While alcohol dehydrogenase is predominately a cytosolic enzyme, the highest aldehyde dehydrogenase activities are localized in the mitochondrial and microsomal fractions of rat liver (20). The dehydrogenase activities of those fractions were determined after sacrifice of the rats 5 h following dosing with DCE (100 mg/ kg ip). The mean value for alcohol dehydrogenase activity was slightly higher (15%) in DCE-treated animals, but the difference was not statistically significant
(Table 3). Mean values for the aldehyde dehydrogenases were both decreased, but only the decrease (18%) in microsomal aldehyde dehydrogenase activity was statistically significant.
Discussion Previous investigations have shown that exposure of rats (9) or mice (12) to DCE by inhalation produced significant inhibition of P450 2E1. DCE also effectively inhibits P450 2E1 activity in rat liver microsomes under conditions in which P450 2B1/2 activity is unaffected (10). Inhibition of microsomal P450 by DCE has been shown to be a mechanism-based process which results in the destruction of the heme prosthetic group of the enzyme (24). In the present work it was shown that an easily administered 20-µL ip injection of DCE was effective in causing the inactivation of P450 2E1 without concomitant losses in total content of P450 or the activities of P450 1A2, P450 2A1, P450 2B, P450 2C6, P450 2C11, P450 2D1, and P450 3A and only modest effects on alcohol and aldehyde dehydrogenases. The dose was well-tolerated and caused only brief ataxia. The enzyme specificities and toxicities of other commonly used P450 2E1 inhibitors are summarized in Table 1. Almost all suffer from lack of specificity against P450 isozymes, inhibit either alcohol or aldehyde dehydrogenases, inhibit or induce other enzymes of metabolism, or have serious toxicities. Two relatively new agents, chlormethiazole and YH439, act by transcriptional inhibition of P450 2E1. While effective in inhibiting the induction of the isozyme, YH439 is relatively weak and slow in diminishing P450 2E1 levels in uninduced rats, and no report of the effect of chlormethiazole on P450 2E1 levels in uninduced animals could be found. The effects of these agents on other isozymes have been only partially evaluated. Additionally, chlormethiazole, and possibly YH439, are ineffective inhibitors in vitro (25). Recent work of this laboratory has demonstrated that the P450 2E1-mediated metabolism of endogenously generated volatile organic compounds measured in rat breath is inhibited by greater than 90% 5 h following a single 100 mg/kg ip dose of DCE and that lipid peroxidation is not stimulated by this treatment (26). Since mechanism-based inhibitors are substrates of the enzymes which they inhibit, they should also be competitive inhibitors of that enzyme. Therefore, it is likely that DCE also acts as a competitive inhibitor of the enzyme, and the 65% inactivation of the enzyme measured in the present studies is an underestimate of the total inhibition achieved. DCE also proved to be a very effective and selective inactivator of P450 2E1 in vitro. This selectivity was achieved under conditions in which DCE was washed
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Figure 3. Total content and activities of hepatic microsomal cytochrome P450 isozymes following in vitro incubation with DCE (5 mM). As described in the Materials and Methods section, in (A) microsomes were incubated with DCE and NADPH and then washed prior to assay, and (B) assays were performed in the presence of DCE. *Significantly different from control (p e 0.05). Panel A: aexpressed as acetanilide hydroxylase, 100% value (control) ) 0.97 nmol/mg‚min; bexpressed as 7R-testosterone hydroxylase, 100% value (control) ) 0.18 nmol/mg‚min; cexpressed as erythromycin N-demethylase, 100% value (control) ) 0.59 nmol/mg‚min; dexpressed as tolbutamide hydroxylase, 100% value (control) ) 1.25 nmol/mg‚h; eexpressed as 2R-testosterone hydroxylase, 100% value (control) ) 1.43 nmol/mg‚h; fexpressed as dextromethorphan O-demethylase, 100% value (control) ) 40.4 nmol/mg‚h; gexpressed as p-nitrophenol hydroxylase, 100% value (control) ) 1.41 nmol/mg‚min; hexpressed as nmol of P450/mg of protein, 100% value (control) ) 0.96 nmol/ mg. Panel B: aexpressed as acetanilide hydroxylase, 100% value (control) ) 1.39 nmol/mg‚min; bexpressed as 7R-testosterone hydroxylase, 100% value (control) ) 0.10 nmol/mg‚min; cexpressed as erythromycin N-demethylase, 100% value (control) ) 0.29 nmol/mg‚min; dexpressed as tolbutamide hydroxylase, 100% value (control) ) 1.29 nmol/mg‚h; eexpressed as 2R-testosterone hydroxylase, 100% value (control) ) 1.08 nmol/mg‚h; fexpressed as dextromethorphan O-demethylase, 100% value (control) ) 42.9 nmol/mg‚h; gexpressed as p-nitrophenol hydroxylase, 100% value (control) ) 0.65 nmol/mg‚min.
from the microsomes following incubation with the inhibitor and NADPH. However, in the presence of DCE the activities of P450 1A2 and P450 2C6 were also markedly depressed, presumably by competitive inhibi-
tion. Thus, DCE is useful as an inhibitor in vitro but 2 J. M. Mathews, N. Gaudette, and A. S. Etheridge, unpublished results.
784 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
requires application of methods tailored to the requirements of individual experiments. Immunoblot analyses of the hepatic microsomes from DCE-treated animals demonstrated that the levels of P450 2E1 protein were more modestly decreased than were the corresponding P450 2E1 activities. This phenomenon occurs also with diallyl sulfide and its sulfone and disulfiram (27, 28). Each of these mechanism-based inhibitors alkylates P450 2E1 protein, and the DCEinactivated protein may well be immunoreactive to the quantitating antibodies. Thus, the inhibition of this isozyme by DCE may be due in part to protein alkylation in addition to the heme alkylation mechanism reported earlier (24). The immunoreactive bands that appear consistently at ca. 30 kDa in the immunoblot from microsomes of DCE-treated rats may arise from partial proteolytic degradation of P450 2E1 protein covalently modified by the inhibitor, possibly by a proteolytic system that specifically degrades P450 2E1 having minor structural changes (29). If so, DCE may be a tool for probing the active site of P450 2E1. The relative ease of elimination of such a volatile chemical as DCE and the rapid turnover (half-life of about 7 h) of P450 2E1 in rat (30) probably contribute to the rebound of enzyme activity within 24 h. By using repeat doses of DCE in mouse (100 mg/kg ip every 4 h), we have shown that the P450 2E1-dependent metabolism of formamide to carbon dioxide can be suppressed by 90% over a 24-h period.2 The specificity and convenience of administration of DCE make it a useful tool for ascertaining the involvement of P450 2E1 in the in vivo and in vitro metabolism of xenobiotics.
Acknowledgment. The authors are grateful to Dr. John Lipscomb for helpful discussions and comments and to Ms. Sherry A. Tallent for her assistance in the preparation of this manuscript. This work was performed under National Institute of Environmental Health Sciences Contract No. NO1-ES-4-5380.
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