Chem. Res. Toxicol. 1993,6, 866-871
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Epoxidation of Acrylonitrile by Rat and Human Cytochromes P450 Gregory L. Kedderis,*pt Renu Batra,t and Dennis R. Koopl Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709, and Department of Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201 Received July 28, 1 9 9 9
The cytochromes P450 (P450) involved in the epoxidation of the rat carcinogen acrylonitrile (ACN) to the mutagen 2-cyanoethylene oxide (CEO) have been investigated in hepatic microsomes from F-344 rats and humans. Induction of P450 2E1 by acetone treatment increased the Vmru for rat microsomal CEO formation &fold, while ACN treatment had little effect. Treatment with 8-naphthoflavone, dexamethasone, and phenobarbital had little effect upon V,, but increased the K M 3- to 5-fold. The P450 ligand l-phenylimidazole and substrate ethanol were potent inhibitors of ACN epoxidation after all treatments. 2-(Diethy1amino)ethyl 2,2diphenylvalerate (SKF 525A; 0.1 mM) was not an effective inhibitor with microsomes from untreated or acetone-treated rats, but inhibited 50% followingdexamethasone or phenobarbital treatment. Antibodies to P450 2E1 inhibited >85% of the ACN epoxidation activity in microsomes from untreated or B-naphthoflavone- or acetone-treated rats, but only produced 40 % and 60 % inhibition following dexamethasone or phenobarbital treatments, respectively. These results indicate that P450 2E1 is the major catalyst of ACN epoxidation in untreated rats and that other forms of P450 can also epoxidize ACN. Diethyldithiocarbamate (0.1 mM) was a potent irreversible inhibitor of ACN epoxidation after all of the induction treatments, indicating that it is not specific for P450 2E1. Chlorzoxazone (2 mM) produced 7 5 9 0 % inhibition after all of the induction treatments, indicating that it interacts with several rodent P450 isoforms in addition to 2E1. Human hepatic microsomes (n = 6) epoxidized ACN with Vmru’sranging from 129 to 315 pmol of CEO formed/(min.mg of protein) and KM’Sfrom 12 to 18 pM. The fidelity between the inhibition of ACN epoxidation by anti-P450 2E1 and chlorzoxazone (-60 % inhibition) is consistent with a specificity of chlorzoxazone for human P450 2E1. In contrast, diethyldithiocarbamate produced >95 % irreversible inhibition of the epoxidation reaction. SKF 525A produced 20-35 % inhibition of the human microsomal epoxidation reaction. These results show that human P450 2E1 is a major catalyst of ACN epoxidation, but that other forms of human cytochromes P450 are involved in ACN epoxidation to a greater extent than in untreated rats.
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Introduction The widely used industrial chemicalacrylonitrile(ACN)’ is carcinogenic in rats, causing tumors of the brain, stomach, and Zymbal‘s gland (1,2).Cancer bioassays of ACN have not been carried out in other animal species, and the potential carcinogenic risk to humans from ACN exposure is uncertain. Biotransformation of ACN to 2-cyanoethyleneoxide (CEO) is believed to be involved in the carcinogenic effects of ACN in rats (3, 4). CEO is significantly more DNA-reactive than ACN (3-5)and is a potent, direct-acting mutagen in human lymphoblast cells in vitro (6). In contrast, ACN reacts much more readily with proteins than with DNA (3-5)and requires metabolic activation to exert weak mutagenic effects in vitro (6). ACN is metabolized in rat liver by two pathways, glutathioneconjugation (3,7)and cytochrome P450 (P450)
* Addrees all correspondenceto this author at CIIT, P.O. Box 12137, ResearchTriangle Park,NC 27709. Telephone: 919-541-2070;FAX 919541-9015. t Chemical Industry Institute of Toxicology. t Oregon Health Sciences Univereity. Abstract published in Aduance ACS Abstracts, October 15, 1993. 1 Abbreviations: ACN, acrylonitrile; CEO, 2-cyanoethylene oxide; DDTC, diethyldithiocarbamate; DEX, dexamethasone; PNF, 8-naphthoflavone;P450, cytochromes P450; PB, sodium phenobarbital; SKF 525A, 2-(diethylamino)ethyl2,2-diphenylvalerats. @
eDoxidation to CEO (3, 8. 9). CEO is also readilv cbnjugated with glutathione (3, 10). The glutathion; conjugates of both ACN and CEO are catabolized in viuo and excreted in the urine as mercapturic acids or further degradation products (12,221. While the kinetics of ACN epoxidation by rat hepatic microsomes have been determined (€9,the P450s catalyzing ACN epoxidation have not been identified. Recent studies with human hepatic microsomes have correlated P450 2E1 activity with the epoxidation of ACN, which was measured indirectly by the formation of lp-ethenoadenosine from reaction of CEO with adenosine added to the incubation mixtures (23). In the present study, a variety of inducers and inhibitors commonly used in studies of P450-dependent metabolism were used to investigate the rat P450s catalyzing ACN epoxidation. The kinetics and inhibition of ACN epoxidation catalyzed by human hepatic microsomal P450s were also determined. In addition to these data. inhibitory antibodies to P450 2E1 were used to determine the contribution of this P450 enzyme to CEO formation from ACN.
Experimental Procedures Chemicals. ACN (>99%pure) was obtained from Aldrich Chemical CO. (Milwaukee, WI) and contained 35-45 PPm hydroquinone monomethyl ether as a polymerization inhibitor.
0893-228x/93/2706-0866$04.00/00 1993 American Chemical Society
Chem. Res. Toxicol., Vol. 6, No. 6, 1993 867
Acrylonitrile Epoxidation by Cytochromes P450 CEO (>99% chemical purity) was purchased from Chemsyn Science Laboratories (Lenexa, KS). 2-(Diethylamino)ethy12,2diphenylvalerate (SKF 525A) was a gift from Smith, Kline and French (Philadelphia, PA). Chlorzoxazone [5-chloro-2(3H)benzoxazolone], dexamethasone (DEX), Folin and Ciocalteu’s phenol reagent, metyrapone (2-methyl-l,2-di-3-pyridyl-l-propanone), and j3-naphthoflavone(j3NF)were obtained from Sigma Chemical Co. (St.Louis, MO). Diethyldithiocarbamate (DDTC; sodium salt trihydrate) and 1-phenylimidazole were obtained from Aldrich. All other chemicals were of the highest quality available from commercial sources. Animals and Liver Microsome Preparation. Male Fischer344 rata [CDF(F-344)/CrlBR; 220-270 g] were obtained from Charles River Breeding Laboratories (Raleigh, NC). All animals were free from viral infection and maintained on a 12-h lightdark cycle at 22 f 2 OC and 55 5% relative humidity. Animals were acclimated for 2 weeks or more prior to use and were given NIH-07 rodent chow (Ziegler Bros., Gardners, PA) and purified water ad libitum. Groups of 3 rats were treated with acetone (1%v/v in drinking water for 7 days), ACN (10 mg/kg PO in water daily for 3 days), j3NF (40 mg/kg ip in corn oil 48 h prior to sacrifice), DEX (100 mg/kg ip in saline twice daily for 3 days), or sodium phenobarbital (PB; 0.1 % w/v in drinking water for 7 days) prior to sacrifice. The animals were exsanguinated under COz anesthesia, and their livers were removed,weighed,and rinsed with isotonic saline in Tris-HC1 (50 mM, pH 7.4). The livers were minced and homogenized in 3 volumes of the same buffer, and microsomes and cytosols were prepared by differential centrifugation (14,15). Protein was determined by the method of Lowry et al. (16) using bovine serum albumin as the standard. P450 content of the microsomal preparations was determined by the method of Omura and Sat0 (17). The specific contents of P450 in the rat hepatic microsomal preparations were (nmol of P450/mg of protein, mean f SD): untreated, 0.39 f 0.05; acetone, 0.42 f 0.09; ACN, 0.37 f 0.03; PNF, 0.57 0.04; DEX, 0.95 h 0.09; and PB, 1.14 f 0.04. Six human liver samples from kidney donors were obtained frozen from Dr. F. P. Guengerich (Vanderbilt University, Nashville, TN) through Tennessee Donor Services (Nashville, TN). The liver samples were thawed on ice, and microsomes were prepared by differential centrifugation (14,15).The P450 content of the human liver microsomal preparations ranged from 0.15 to 0.76 nmol of P450/mg of protein (9). All microsomal preparations were stored at -70 OC. Incubations and Analytical Procedures. All experiments were done under conditions where CEO formation was linear with respect to time and protein concentration. The reaction mixtures contained potassium phosphate buffer (0.1 M, pH 7.3), glucose 6-phosphate (10 mM), NADP+ (0.5 mM), magnesium chloride (3 mM), glucose-6-phosphate dehydrogenase (1 unit/ mL), and microsomal protein (0.1-1.0 mg/mL) in a final volume of 0.5mL. Following a 2-min preincubation at 37 OC, thereadions were initiated by addition of ACN in water (3-1200 wM) and incubated at 37 OC for 5-30 min. The reactions were terminated by addition of ice-coldmethylene chloride (1mL) to extract CEO. The quenched reaction mixtures were inverted approximately 50 times and allowed to stand at room temperature for 5 min to facilitate phase separation, and the methylene chloride layer was removed and stored at -20 OC until analysis. CEO formation was quantitated using the GC-MS procedure described by Roberts et al. ( 4 9 )by monitoring the fragment ion at m/z 41.0265 with a mass resolution of 4000 (M/AM). The interfering peak which was reported previously (8, 9) was not observed during GC-MS analysis. This previously observed interfering peak was most likely a contaminant in the methylene chloride. GC-MS analyses were carried out with a Carlo Erba/Mega Series GC equipped with a 30-m DB-5 capillary column (J & W Scientific, Folsom, CA) which was inserted directly into the ion source of a Kratos (Manchester, U.K.) MS89OMS mass spectrometer. CEO concentrations were determined from standard curves prepared in methylene chloride which were analyzed with each experiment. The extraction efficiency of CEO (50-80%) was determined in
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*
25001 20001
0
I
I
I
I
I
I
200
400
800
800
1000
1200
ACN (PM)
Figure 1. Concentration-dependent formation of CEO from ACN catalyzed by hepatic microsomes from untreated (A)and acetone-treated ( 0 )F-344 rats. The solid lines are the computer fit of the Michaelis-Menten equation to the data (mean SD of 4-6 determinations). The inset expands the region between 0 and 100 NM ACN. Table I. Effect of P450 Induction on the Hepatic Microsomal Epoxidation of ACN. treatment V, [pmol/(min.mg)] KM ( r M ) untreated 366 f 6 llfl acetone 2069 43 19f2 ACN 493 12 19f2 BNF 412 f 8 38h 2 DEX 610 32 51 f 9 PB 293 9 28f3 a The kinetic parameters were calculated by computer fit of the Michaelis-Menten equation to the data. The data are the mean h SD of 4-6 determinations.
* **
each experiment and accounted for in the quantitation. Immunological Procedures. Polyclonal goat anti-rabbit P450 2E1 and preimmune IgG used for immunoblot analysis were purchased from Oxford Biomedical Research (Oxford,MI). Immunoblots were prepared (18)using the Phastaystem (Pharmacia, Uppsala, Sweden) with detection by enhanced chemiluminescence (ECL, Amersham Corp., Arlington Heights, IL). Sheep anti-rabbit P450 2E1 and preimmune IgG used in the inhibition studies were prepared as described previously (19,20). The anti-rabbit P450 2E1 antibody recognizes the P450 2E1 orthologue in rat (21)and human liver (22) and has been shown to be a potent inhibitor of the P450 2El-linked activities aniline hydroxylase andN-nitrosodimethylamine demethylase in rat and human liver microsomes (21, 22). Data Analysis. The data presented are mean values SD. The kinetic data were analyzed by computer fit of the MichaelisMenten equation to the data.
*
Results Induction of Rat Hepatic Microsomal ACN Epoxidation. T h e initial rate of the microsomal epoxidation of ACN to CEO was markedly increased following acetone treatment of rats (Figure 1)to induce P450 2E1 (23).The V,, for the microsomal epoxidation of ACN following acetone treatment was more than 5 times greater than t h a t of microsomes from untreated rats (Table I). These data suggest that P450 2E1 is an effective catalyst of ACN epoxidation. Treatment with ACN itself slightly increased t h e V,, for rat liver microsomal ACN epoxidation while t h e K M value was similar t o those in microsomes from untreated or acetone-treated animals (Table I). These results suggest t h a t ACN treatment does not induce its
Kedderis et al.
868 Chem. Res. Toxicol., Vol. 6, No. 6, 1993 Table 11. Inhibition of the Rat Hepatic Microsomal Epoxidation of ACN. % control activity inhibitor (mM) untreated acetone ACN BNF DEX 71 f 3 45 4 88 9 86 6 90f5 SKF 525A (0.1) 120 f 8 101f 4 89 f 3 58 f 3 metyrapone (0.1) 87 f 7 6*2 If1 1-phenylimidazole(0.1) 4 f l 1fO o+o 22f3 22 f 1 22* 1 14f3 chlorzoxazone (2.0) 27 f 1 42f5 41 f 4 49 f 3 45 + 7 33 f 4 ethanol (25.0) 29f 1 811 14f3 9f2 17 f 2 DDTC (0.1)
PB 47 4 97 8
*
*
* *
l*O
910 33f3 13f2
a The reaction mixtures contained ACN (1.2 mM), the indicated inhibitor (in aqueous solution),and microsomal protein from the indicated treatments (0.2-0.4 mg/mL) in a final volume of 0.5 mL. Mixtures containing DDTC m e preincubated with microsomes for 7 min prior to ACN addition. The mixtures were incubated at 37 "Cfor 10 min, extracted with methylene chloride, and analyzed for CEO by GC-MS. The data are the mean f SD from 3 determinations.
own oxidative metabolism under the conditions investigated. Western blot analysis showed no change in P450 2E1 levels following ACN treatment compared to controls, while induction was evident following acetone treatment (data not shown). The kinetic parameters for ACN epoxidationby hepatic microsomes from rata treated with agents to induce various P450 enzymes are shown in Table I. All of the treatments employed changed the kinetic parameters for the microsomal epoxidation of ACN. Treatment with PNF to induce P45Os 1 A l and 1A2 (24) and with DEX to induce the P450 3A enzymes (25)increased both the V, and K M for ACN epoxidation catalyzed by microsomes from rats (Table I). Treatment with PB to induce P450s 2B1 and 2B2 (26) decreased the V, in microsomes from rata but increased the KM.The changes in the kinetic parameters for the microsomal epoxidationof ACN suggests that these treatmenta induce rat P45Os that are capable of epoxidizing ACN. Inhibition of Rat Hepatic Microsomal ACN Epoxidation. The roles of various P450 enzymes in the microsomal epoxidation of ACN were investigated by determining the effects of a number of P450 inhibitors on the reaction after the different induction treatments (Table 11). In microsomes from untreated rata, neither SKF 525A nor metyrapone were effective inhibitors of ACN epoxidation. The P450 ligand 1-phenylimidazole (27) was a potent inhibitorof ACN epoxidation,as were the substrates chlorzoxazone, ethanol, and 2-propanol, and the suicide substrate (13)DDTC. Treatment of rats with acetone or ACN did not alter the inhibition pattern (Table 11). After treatment of rats with DEX or PB, SKF 525A was a more effective inhibitor of ACN oxidation (Table 11). Metyrapone was an effective inhibitor of rat liver microsomal ACN epoxidation after DEX treatment. 1-Phenylimidazole, chlorzoxazone, ethanol, and DDTC were potent inhibitorsof ACN epoxidationfollowing all of the induction treatments employed. Experiments where microsomes from treated rats were incubated with DDTC, reisolated by ultracentrifugation, and analyzed for ACN oxidation demonstrated that the inhibition observed by DDTC was irreversible in all cases (data not shown). The changes in the degree of inhibition of the rat microsomal epoxidation of ACN following DEX and PB treatments are consistent with the interpretation that multiple P450 enzymes are capable of effectively epoxidizing ACN. The role of P450 2E1 in the rat liver microsomal epoxidation of ACN was further probed using polyclonal antibodies to P450 2E1(19,20). Anti-2El was an effective inhibitor of the microsomal epoxidation of ACN (Figure 2). Time course studies demonstrated that maximal inhibition was observed following a 4-min preincubation
0
,
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I
1
4
6
8
mg IgG I mg Microsomal Protein
Figure 2. Inhibition of the rat liver microsomal epoxidation of ACN by anti-P450 2E1. The indicated amounts of anti-P450 2E1 were incubated with microsomes (0.2mg/mL) from untreated (A),8-naphthoflavone-treated (01, phenobarbital-treated (W, acetone-treated (A),or dexamethasone-treated ( 0 )rata for 4 min at37 oCinpotassiumphosphatebuffer (0.1M,pH 7.3),containing glucose 6-phosphate (10 mM), NADP+ (0.5mM), and magnesium chloride (3 mM). The reactions were initiated by addition of glucose-6-phosphate dehydrogenase (1unit/mL) and ACN (1.2 mM), incubated for 10 min at 37 OC, extracted with methylene chloride, and analyzed for CEO by GC-MS. The data are the mean i SD from 3 determinations.
of the antibody with the microsomes. Incubation of microsomes with preimmune IgG had no effect on ACN epoxidation (not shown). Anti-2El inhibited the ACN epoxidation reaction catalyzed by microsomes from untreated rats by >85% (Figure 2), indicating that P450 2E1 is the principal catalyst of ACN epoxidation. Anti2E1 also inhibited the ACN epoxidationreactionby >85 % inmicrosomes from acetone- and PNF-treated rata (Figure 21, indicating that P450 2E1 is the principal catalyst of ACN epoxidationin these microsomal preparations as well. In contrast, the ACN epoxidation reactions catalyzed by microsomes from PB- and DEX-treatedrata were inhibited by 60% and 40%, respectively, by anti-2El. These data are consistent with the participation of P450 enzymes other than 2E1 in ACN epoxidation following the induction treatments. Kinetics and Inhibition of Human Hepatic Microsomal ACN Epoxidation. The human hepatic microsomal epoxidation of ACN to CEO followed normal Michaelis-Menten kinetics (Figure 3). The kinetic parameters for the reaction catalyzed by hepatic microsomes from six different individuals are shown in Table 111.The V-values varied over a 3-fold range while the K M values were similar for the six liver samples investigated. As was observed with rodent liver microsomes, l-phenylimidazole (0.1 mM) was a potent inhibitor (1f 1%of control activity) of the epoxidation of ACN (1.2 mM)
Acrylonitrile Epoxidation by Cytochromes P450
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Chem. Res. Toxicol., Vol. 6, No. 6, 1993 869
rdoa
12w
Figure 3. Representative plot of the concentration-dependent formation of CEO from ACN catalyzed by human hepatic microsomes (HL 123).The solid lines are the computer fit of the Michaelis-Menten equation to the data (mean f SD of 3 determinations).The inset expands the region between 0 and 100 pM ACN.
V,,
[pmol/(min.mg)l
KM (FM)
129 f 3 264 4 315 i 7 274 6 218 6 183 3
12f 1 17k 1 16f 1 18* 1 12* 1 18* 1
* *
HL 100
HL 104 HL 115 HL 123 HL 126
4 The kinetic parameters were calculated by computer fit of the Michaelis-Menten equation to the data. The data are the mean SD of 3-6 determinations.
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Table IV. Inhibition of the Human Hepatic Microsomal Epoxidation of ACN. % control activity
sample HL 98 HLl00 HL104 HL115 HL123 HL126 4
SKF 525A
DDTC
81 6 84*3 67*8 64t7 82*5 86*5
3*0 2*0 3*0 6*1 2*0
*
7fl
I
I
I
1
1.5
2
chlorzoxazone 40f 2 40 & 2 43 f 3 41fl 40 5 35 4
*
anti-2El 38* 1 39 3 33 5 42 2 30* 2 33 5
**
The reactions containing ACN (1.2 mM) were incubated under
the following conditions: SKF 525A (0.1 mM), 10-min incubation with 0.2 mg of microsomal protein/mL; DDTC (0.1 mM), 7-min preincubation with 0.4 mg of microsomal protein /mL prior to ACN addition, followed by a 20-min incubation; chlorzoxazone (2 mM), 20-min incubation with 0.4 mg of microsomal protein/mL; 7.5 mg of anti-2El/mg of microsomal protein, dmin preincubation with 0.2 mg of microsomal proteins/mL prior to ACN addition, followed by a 10-minincubation. The data are themean SD of 3 determinations. catalyzed by human liver microsomes, while metyrapone (0.1 mM) was not effective (91 f 3% of control activity). A number of solventa inhibited the reaction, such as 2-propanol, tetrahydrofuran, and N,N-dimethylformamide. Inhibition by SKF 525A ranged from 86% to 64% of control activity among the six human liver microsomal preparations (Table IV). DDTC was a potent irreversible inhibitor of the reaction (Table IV). Chlorzoxazone,which has been shown to be a specific substrate for human P450 2E1 (B), stimulated ACNepoxidation a t the submillimolar concentrations examined for other alternative substrate inhibitors (Figure 4). This kinetic behavior is not what would be expected for an alternative substrate inhibitor, given the low K M of chlorzoxazone and its specificity of metabolism by human P450 2E1 (28). The mechanism underlying the observed stimulation is not known. However, concentrations of chlorzoxazone >1mM significantly
*
Figure 4. Concentration-dependent effects of chlorzoxazone on ACN epoxidation by human hepatic microsomes (HL 126). The incubation conditionsare given in the footnoteto Table IV. The data are the mean SD from 3 determinations.
*
inhibited ACN epoxidation (Figure 4, Table IV). Antibodies to P450 2E1 produced 58-70% inhibition of the epoxidation of ACN catalyzed by the different human liver microsomal preparations (Table IV). Preimmune IgG had no effect on the reaction catalyzed by human liver microsomes (data not shown). These data indicate that while human P450 2E1 is a major catalyst of ACN epoxidation, other human P450 enzymes are also involved in the epoxidation of ACN.
Table 111. Kinetic Parameters for ACN Epoxidation by Human Hepatic Microaomesa sample
I
0.5
Chlorzoxazone (mM)
ACN (W)
HL 98
0
Discussion The results of these studies demonstrate that P450 2E1 is the major and essentially sole catalyst of ACN epoxidation in untreated F-344 rata. The induction and inhibition studies strongly suggest that other forms of P450 (such as 2B1, 2B2, and the 3A enzymes) can epoxidize ACN, but with specific activities that are apparently lower than that of P450 2E1. Immunochemical studies have suggested that P450 2E1 may account for approximately 10% of the total P450 detected in microsomes from untreated rata (29) and increases approximately 4-fold after induction by acetone treatment (30). This form of cytochrome P450 is thought to be involved in the metabolism of ketone bodies (23) and plays an important role in the oxidation and reduction of a wide variety of low molecular weight substrates, including many suspected carcinogens (13, 31). The data presented here indicate that human liver microsomal P450 2E1 is a major catalyst of ACN epoxidation, as has been proposed previously (13), but also show that other P450 enzymes participate in catalysis to a much greater extent than in rodent liver microsomes. The similarities between the kinetic parameters for the microsomal epoxidation of ACN by rata and humans (Tables I and 111) suggest that rata may be a good model species for the disposition of ACN in people. However, recent studies have shown that human liver microsomes actively catalyze the hydrolysis of CEO while this pathway is not significant in rat liver microsomes (32). Thus, the metabolic fate of CEO would be expected to be different in rata and people. The 6-hydroxylation of the muscle relaxant chlorzoxazone was proposed to be specifically catalyzed by human liver P450 2E1 on the basis of the results of kinetic, inhibition, and immunochemical studies (28). The data on inhibition of human liver microsomal ACN epoxidation presented here are consistent with this conclusion. There was an excellent correlation between the extent of inhi-
870 Chem. Res. Toxicol., Vol. 6, No. 6, 1993
bition of the reaction by chlorzoxazone and antibodies to P450 2E1 (Table IV), implying that chlorzoxazone is only inhibiting that portion of ACN epoxidation catalyzed by human liver microsomal P450 2E1. However, the results with rat liver microsomes are not consistent with the suggested specificity of chlorzoxazone for rodent hepatic P450 2E1 (33). Chlorzoxazone was a potent inhibitor of microsomal ACN epoxidation regardless of induction treatment (Table 11),whereas antibodies to P450 2E1were less effective inhibitors of the reaction after PB or DEX treatments (Figure 2). The lack of specificity of chlorzoxazone for rat P450 2E1 suggests that other P450 enzymes interact with chlorzoxazone. While the isoforms may not metabolize chlorzoxazone to 6-hydroxychlorzoxazone,they may bind the compound at high concentrations. Similarly, while ethanol is most effectively metabolized by P450 2E1 (19), it can bind to most isoforms. This is reflected in inhibition of ACN epoxidation after all induction treatments (Table 11). DDTC has been suggested to be a selective mechanismbased inactivator of P450 2E1 at concentrations 95% of the epoxidation reaction catalyzed by human hepatic microsomes while chlorzoxazone and antibodies to P450 2E1 inhibited 60-70% (Table IV). These results are inconsistent with the suggestion that DDTC is a specific inhibitor of rat or human hepatic P450 2E1 (13). DDTC has also been found to potently inhibit the human P450 2A6-catalyzed 7-hydroxylation of coumarin (34). The data in Table I1suggest that DDTC may be a potent irreversible inhibitor of rat hepatic microsomal P450s induced by PB and DEX, in addition to P450 2E1. SKF 525A was not an effective inhibitor of ACN epoxidation catalyzed by microsomes from untreated rats (Table 11),as has been observed for several other P450 2El-catalyzed reactions (28,35). However, SKF 525A was an effective inhibitor of ACN epoxidation following treatment of rats with PNF, DEX, or PB (Table 111, consistent with the interpretation that these treatments induce other P450 enzymes that are capable of oxidizing ACN. The inhibition of human hepatic microsomal ACN epoxidation by SKF 525A was generally more pronounced than that with microsomes from rats, but the extent varied among the six samples (Table IV). The variation in the inhibitory potency of SKF 525A is probably due to interindividual variation in the participation of P450 enzymes other than P450 2E1 in the epoxidation of ACN. However, the specific contribution of other human hepatic P450s to the microsomal epoxidation of ACN was not investigated. Cytochrome P450 enzymes are known to be inducible in humans through exposure to exogenous and endogenous chemical agents (36,37). Induction of cytochromes P450 can be an important factor leading to interindividual differences in the disposition of toxic and potentially carcinogenic chemicals. Human P450 2E1 in particular has been shown to be induced by ethanol (38,391,isoniazid (381,and diabetes mellitus (40). In addition to diabetes, other physiologically-induced ketotic states, such as long distance running, may induce P450 2E1 in humans.
Kedderis et al.
Additionally, exogenous ketones such as methyl ethyl ketone have been shown to be potent inducers of P450 2E1 in rats (41), suggesting that human populations exposed to these solvents in the workplace may have elevated levels of P450 2E1. Individuals with elevated levels of P450 2E1 would be expected to produce CEO more rapidly after ACN exposure than uninduced individuals. Although the history of drug and alcohol use by the six human liver donors used in this study is not known, the cytochrome P450 2El-related activities among the six samples were similar (Table IV). Substantially greater variability in P450 2El-related activity has been reported in other studies (22,28,42-46). Considering that factors related to diet and lifestyle can readily modulate this enzyme, the range of hepatic P450 2E1 concentrations in the general population may be expected to vary widely. This potential variability needs to be taken into account when estimating the human disposition of cytochrome P450 2E1 substrates such as ACN. Acknowledgment. We thank Dr. F. P. Guengerich for supplying the human liver tissues used in this study. We also thank M. J. Turner, Jr., for his help with the mass spectral analysis and Dr. J. A. Bond for helpful discussions. This research was supported in part by a grant from BP America Inc., American Cyanamid, and Sterling Chemical Co., and by NIAAA Grant AA-08608 (to D.R.K.).
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