Chem. Res. Toxicol. 1993,6, 511-518
511
Cytochrome P450 Enzymes Involved in Acetaminophen Activation by Rat and Human Liver Microsomes and Their Kinetics Chris J. Patten,t Paul E. Thomas,t Robert L. Guy,$ Maojung Lee,t Frank J. Gonzalez,S F. Peter Guengerich,ll and Chung S. Yang'vt Laboratory for Cancer Research and Department of Toxicology and Pharmacology, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855, Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, and Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received October 23, 1992
ABSTRACT Acetaminophen (APAP), a commonly used analgesic, is catalyzed by cytochrome P450 (P450) enzymes to a toxic intermediate which can be trapped by glutathione. Using this approach, involvement of enzymes in the activation of APAP and their kinetics were studied. With human liver microsomes, there were three apparent K, values (approximately 10,474, and 13 000 p M ) for the oxidation of APAP to its glutathione conjugate. With rat liver microsomes (control and ethanol induced) the kinetic data were best fit to a two-Km model (approximately 30 and 1100 pM). Liver microsomes from dexamethasone (DEX)-treated female rats showed a single Kmof 56 pM and a V, of 7500 pmol of product formed/(min*mgof protein). Antibodies specific for rat P450s 2E1 and 1A2 each inhibited approximately 40% of the APAP metabolism in control male rat microsomes. Only slight inhibition was observed with the P450 3A1/2 antibodies in control male or female rat liver microsomes. Antibodies against rat P450s 3A1/2 inhibited the activity in DEX microsomes by 80 7%. Antibodies inhibitory to human P450 3A4 inhibited 38% of the activity in human liver microsome sample HL107 and 76% in human 2C8,2B6, microsome sample HL110. Human P450s (2A6,2El,lA2,3A4,3A5,3A3,2D6,2Fl, and 2C9) expressed in Hep G2 cells using a vaccinia virus expression system were each tested for APAP metabolism. Of these, P450 2E1, 1A2, and 3A4 showed substantial activity, with respective Km and Vm, values of 680 pM and 330 pmol/(min*mg) for P450 2E1 (with added cytochrome b b ) , 3430 pM and 74 pmol/(mimmg) for P450 1A2, and 280 pM and 130 pmol/ (mimmg) for P450 3A4. In the presence of a-naphthoflavone (a-NF), expressed P450 3A4 activity was increased 11-fold, expressed P450 2E1 activity was unaffected, and expressed 1A2 activity was inhibited 83% . With expressed P450 3A4, both the Km and V, for APAP oxidation were increased by a-NF. a-NF stimulated APAP oxidation 2- to 5-fold in human and control male rat liver microsomes. In the presence of a-NF, the P450 3A1/2 antibody became strongly inhibitory in control male rat liver microsomes, suggesting 3A2 activation by this flavone compound.
Introduction Acetaminophen (APAP)' is a widely used analgesic and antipyretic drug that is generally safe and nontoxic when administered at therapeutic doses. However, when taken in overdose quantities, severe liver and kidney necrosis occurs in man and laboratory animals (I). The major route of elimination of APAP is through conjugation of the parent compound with glucuronic acid and sulfate (2).It Address corresoondence to this author at the Laboratorv for Cancer +Laboratoryfor Cancer Research, College of Pharmacy, Rutgers University. t Department of Toxicology and Pharmacology,College of Pharmacy, Rutgers University. i National Institutes of Health. I Vanderbilt University School of Medicine. 1 Abbreviations: APAP,acetaminophen;DEX, dexamethasone;PCN, pregnenolone-16a-carbonitrile;SSD, s u m of squares deviation; bg, cytochrome bb; Hep G2-v2E1 (lA2,3A4), (e.g.) human P46Oe expressed m Hep G2 cella by the vaccinia virus expression system; TAO, troleandomycin; a-NF, a-naphthoflavone; MAb, monoclonal antibody; PAb, polyclonal antibody; NDMA, N-nitrosodimethylamine;EROD, ethoxyresorufim; P450, cytochrome P450.
can also be metabolized by a microsomal cytochrome P450 (P450)-dependent oxidation to N-acetyl-p-benzoquinone imine, a highly reactive and toxic intermediate (3). At low doses, the quinone imine is trapped by conjugation with GSH and subsequently excreted as the mercapturic acid and cysteine derivatives. At high doses of APAP, however, GSH is rapidly depleted and the quinone imine binds to cellular proteins, causing cell injury and death (4, 5).
Several P450 enzymes have been shown to carry out the activation of APAP to the quinone imine toxic intermediate. In reconstituted systems, substantial activity was demonstrated with purified rabbit P450s 2E1,1A2, and 1 A l (6), as well as by purified rat P450s 1A1, 1A2, and 2Cll(7). In human liver, P450s 2E1 and 1A2 have been suggested to be primary catalysts (8). Compounds that induce the levels of specific P450s, such as isoniazid and ethanol which induce 2E1 and 3-methylcholanthrane which induces 1A1/2, have been shown to increase the hepatotoxicity of APAP in severalspecies (SlI). Human alcoholics,with increased levels of P45O 2E1, show higher
0893-228x/9312706-0511$04.00/0 0 1993 American Chemical Society
512 Chem. Res. Toxicol., Vol. 6, No. 4, 1993 susceptibility t o t h e toxic effects of APAP (12). Pret r e a t m e n t of adult female r a t s with p r e g n e n o l o n e - 1 6 ~ ~ carbonitrile ( P C N ) , a steroidal c o m p o u n d which specifically induces P450 3A1 in these animals, has been shown to increase the rate of in vitro formation of APAP conjugate in microsomes, suggesting a role for P450 3A1 i n APAP metabolism (13). The reported stimulation of APAP oxidation in rat liver microsomes b y added flavone suggests a role for P450 3A2 (14). PCN has also been shown to increase the biliary excretion of APAP in rats, f u r t h e r suggesting t h e involvement of P450s 3A1/2 (15). A recent s t u d y has shown that p r e t r e a t m e n t of mice with dexamethasone ( D E X ) , also a P450 3A inducer, potentiates the hepatic toxicity of A P A P ; however, p r e t r e a t m e n t with PCN decreased APAP toxicity (16). It is also known that compounds which inhibit P450s, such as 4-methylpyrazole and ethanol, protect against t h e toxic affects of APAP when given concomitantly to the animals (10, 17). I n spite of the f a c t that several P450s have been shown t o oxidize APAP, the kinetic parameters and relative importance of different P450 forms is not well understood. Such information is very important for designing strategies for the prevention of APAP toxicity. The purpose of this s t u d y is to investigate t h e ability of different P450s i n h u m a n and r a t liver microsomes in the metabolism of APAP and to characterize the kinetic parameters involved. Human and rat liver microsomes, P450 enzymes expressed heterologously in Hep G2 cells, and specific inhibitory antibodies against different P450 forms were used in t h i s investigation. The results provide new evidence that human cytochrome P450 3A4 possesses high catalytic capacity for APAP activation, along with human P450s 2 E 1 and 1A2. In rat liver microsomes, the ability of P450s 3A1 and 3A2 to activate APAP is confirmed, and the role of P450 3A1 as a high-affinity (low-K,) APAP oxidase is established.
Materials and Methods Materials. APAP, GSH, DEX, and a-naphthoflavone (aNF) were purchased from Sigma Chemical Co. (St. Louis, MO). Troleandomycin (TAO) was a gift from Pfizer (Groton, CT). Tissue culture reagents were purchased from Gibco BRL (Gaithersburg,MD). The wild-typevacciniavirus and expression vector (pSC11) were obtained from Dr. B. Moss (National Institutes of Health, Bethesda, MD). Hep G2 cells were obtained from American Type Culture Collection (Rockville, MD). All other reagents were of the highest grade available commercially. Liver Microsome Preparation. Mature (250 g) and immature (100 g) Sprague-Dawley rata were obtained from Harlan Sprague Dawley (Altamont, NY) and Jackson Laboratories (Bar Harbor, MA). Mature female rata were administered DEX a t a dosage of 100 mg/kg body */day for 3 days by intraperitoneal injection. The animals were killed 24 h after receiving the last dose of inducer. Ethanol was administered to immature male rata as a 15% solution (v/v) in drinking water for 3 days. Rat liver microsomes were prepared by previously described methods (18). Human liver microsomes were prepared as previously described (19)and were labeled "HL" for human liver and a code number. Human liver samples were obtained from the Nashville Regionalorgan Procurement Agency, Nashville,T N (HLllOand HL107), and the National Disease Research Interchange, Philadelphia, PA (HL1 and HL2). Human microsome samples HL107, HL110, HL1, and HL2 were used throughout this study. Human P450 Expression. The characterization and the methods for construction of the 11recombinant vaccinia viruses containing the various P450 cDNAs (lA2,2A6,2B6,2C8,2C9, 2D6, 2E1, 2F1, 3A3, 3A4, and 3A5 ) have been fully described
Putten et ul. elsewhere (20,211. Cell lysates of Hep G2 cells infected with the desired recombinant viruses were prepared by washing and resuspending the cells in phosphate-buffered saline (6.7 mM sodium/potassium phosphate buffer (pH 7.4) containing 137 mM NaCl and 2.7 mM KCl) followed by brief sonication (3-5 pulses each lasting 5 s). Preparation of microsomes from infected Hep G2 cells, and incorporation of cytochrome ba ( b b ) into Hep G2 microsomal membranes, has been described in detail previously (22). Final microsome preparations were suspended in 0.35 M sucrose. Acetaminophen Activation. In assays of the activation of APAP to a GSH conjugate, the incubation mixture contained 50 mM potassium phosphate buffer (pH 7.4), an NADPH-generating system (0.4 mM NADP+, 10 mM glucose 6-phosphate, and 0.4 unit of glucose-6-phosphate dehydrogenase mL-l), 15 mM GSH, selected concentrations of APAP, and rat or human liver microsomes (0.02-0.06 mg of protein) in a final volume of 200 rL. The assay was carried out a t 37 "C for 15-25 min and terminated by the addition of 3 % sulfosalicylicacid (w/v). With expressed human P450 microsomes, 0.16 mg of protein was used, and the incubations were carried out for either 25 or 40 min. (Under these assay conditions,the amount of product formed was linearly proportional t o the amount of cell microsomes added and to the incubation time, except in the 40-min incubation in which a 5-10% deviation from linearity was observed. However, the kinetic parameters obtained in the 40-min incubation were not significantly different from those obtained in the 25-min incubation.) The precipitated protein was removed by centrifugation, and 100 pL of the supernatant was injected onto the HPLC column. The electrochemical HPLC method for detection of the acetaminophen-glutathione conjugate has been described (22). In brief, samples were eluted from a Supelco C-18 column (4.6 X 150 mm) using an isocratic mobile phase of 10% CHSOH (v/v) and 0.1 % CF&OzH (v/v) in HzO. The flow rate was 1.5 mL min-', and the GSH-APAP conjugate eluted at approximately 8 min. The electrochemical detector was set a t +0.7 V. Acetaminophen was used as a standard to quantitate the conjugate (23). The conjugate peak area was multiplied by a factor of 1.8to correct for differences in the response of these two compounds as previously described (22). For kinetic analysis, the following acetaminophen concentrations were used: 7, 14, 29,59, 117, 234,469,937, 1875,3750,7500, and 15 000 pM. Kinetic Analysis. For expressed human P450s and purified P450 2E1, the kinetic parameters for APAP activation (K,and V-) were determined by a computer program (KaleidaGraph program from Synergy Software, Reading, PA) designed for nonlinear regression analysis of a first-degree Michaelis-Menten equation. In the case of rat and human liver microsomes, for which there was more than one P450 form contributing to the measured velocities, a computer program was used that can perform nonlinear regression on higher order Michaelis-Menten equations. The program, implementing Nash's modification of Marquardt's algorithm (24),is able to constrain parameters such that the upper and lower bounds are incorporated ( 2 5 ) . 2 The s u m of squares deviation (SSD) and the distribution of residuals around a zero mean were used as criteria to determine how well the raw data fit to the computer-derived models. Other Methods. Immunoinhibition experiments were performed with monospecific polyclonal antibodies raised against rat P45Os 2E1,1A2 (P450 d-c), 3A1/2 (this antibody recognized both 3A1 and 3A2), and human P450 3A4 (P45Om). These antibodies were prepared and characterized as described previously (18,26-29) and were used a t amounts known to produce maximal inhibition (7 mg of IgG/mg of microsomal protein). The monoclonal antibody against rat P450 2E1(1-91-3)(30)was added to incubations at a concentration of 2 mg/mg of microsomal protein. The antibody, buffer, and microsomes were mixed and kept a t room temperature for 10 min before adding the other components of the acetaminophen metabolism assay. The
* The program is availablefor the IBM PC from Robert L. Guy at the above address.
Chem. Res. Toxicol., Vol. 6, No. 4, 1993 513
Activation of Acetaminophen by Rat and Human P450s
Table I. Kinetic Parameters of Acetaminophen Oxidation in Human and Rat Liver Microsomes As Determined by Computer Modeling. microsomes Kml K d Kms V-1 V d VSSD human(HL107) ethanol-induced male (IM)b control male (IM) control male (M)b control female (M) DEX-induced female (M)
0
0
0.001
0.003 0.005 Velocity/[APAP]
0.007
Figure 1. Eadie-Hofstee plots of the substrate dependence of APAP oxidation by human liver microsomes (HL107). The values for the K, and V, are presented in Table I. The data points represent the experimental data,while the line was determined by the computer program for analysis of multiple kinetic parameters as described in the Materials and Methods section. The experimental data were fit to a three-Kmmodel. (0)
amount of inhibition caused by the antibodies was determined by comparison with incubations containing preimmune IgG. Inhibitionby TAO was carried out by preincubatingmicrosomes, buffer, and NADPH-generating system for 25 min at 37 "C in the presence or absence of 40 pM TAO. The reaction was then initiated with APAP and extra generating system and was incubated for an additional 15 min. a-NF was prepared as a stock solution of 4 mM in CHaOH. The final concentration of the flavone used in the incubations was either 13 or 40 pM, and the final CHSOHconcentration was either 0.66% or 1.0 % . A control containingthe CHaOH vehicle was always assayed along with the a-NF incubations for comparison. The components of the reconstituted system and the order of addition were as follows: 0.04 nmol of P450 2E1, 480 units of NADPH:P450 oxidoreductase, 0.09 nmol of ba, 20 pg of ~-a-1,2-dilauroyl-snglycero-3-phosphocholine, and the other components of the acetaminophenassay system in a final volume of 160pL. Glycerol, a competitive inhibitor of P450 2E1 (31),was removed from the P450 and NADPH:P450 oxidoreductase preparations by dialysis for 24 h at 4 O C against 2 L (2 volumes) of a buffer consisting of 50 mM potassium phosphate (pH 7.25) and 0.5 M sucrose. Results Kinetic Analysis of APAP Activation in Rat and Human Liver Microsomes. The kinetic parameters of the conversion of APAP to its GSH conjugate were determined for human and rat liver microsomes over a wide range of substrate concentrations. The demonstrated curvature of the Eadie-Hofstee plots of the substrate dependency of the reaction rates suggests multiple K m s (Figures 1and 2A),a result consistent with previous studies demonstrating the involvement of several P450 enzymes in APAP metabolism (6-8). The values for the apparent K , and V,, were determined by nonlinear regression using a modified Marquardt procedure which adjusts for the contributions of enzymes for one Km to the values of the other K,s. Attempts were made to fit the data from each microsomesample to one-, two-, and three-K, models. The models producing the lowest SSD are presented. Kinetic data from human liver microsomes (HL107) were best fit to a three-K, model (Table I and Figure 1). The data from mature control male and female, immature control male, and immature ethanol-induced rat liver microsomes were best fit to a two-K, model (Table I and Figure 2A). Treatment of female rats with DEX produced a marked increase in the metabolism of APAP by these microsomes,
10 37
474 13000 80 79C 2700 0.0060 915 600 9700 0.0057
36 1495 30 872 12 1217 56
300 3400 80 1400 80 1100 7500
0.039 0.00092 0.037 0.026
a Kinetic constants were determined by nonlinear regression using a computer program based on the Marquardt procedure as described in Materials and Methods. Apparent Km values are expressed as pM, and V-valuesare expressedas pmolof product formed/(min.mg of protein). (M) indicates mature rats, (IM) indicates immature rata.
a result consistant with previous studies (13). The kinetic data could only be fit to a simple Michaelis-: lenten equation exhibiting a low K m of 56 pM (Figure 2B and Table I). The induced P450 form apparently became the predominant form in the activation of APAP. A substantial increase in the V,, of a low-K, isozyme will mask the higher Kms, such that only the lOW-Km isozyme is observed (32). In female rata, DEX has been shown to increase the amount of P450 3A1 whereas P450 3;i2 remains at low levels (33). In view of this, the present results strongly suggest that P450 3A1 has a high affinity (low K,) for APAP metabolism. Treatment of rats with ethanol increased both K m 1 and Km2 for APAP oxidation; however, Km2 was induced to a greater extent (2-fold vs 3-fold, respectively). A similar pattern of induction was seen with microsomes from acetone-treated rats.3 The fact that a single low-Km form did not become a dominate enzyme form in ethanolinduced muxosomes suggests that P450 2E1 is not a lowK , APAP oxidase. The increase in the Vm, of both K m 1 and Km2could suggest that ethanol induces P450 enzymes other than P450 2E1. Microsomes from immature control rats had consistently higher activity than mature rat liver microsomes, which is in agreement with a previous study showing that older rats are less susceptible to APAP toxicity (34). The hepatic P450 2E1 levels have been shown to decrease with age and could account for the lower acLlvity in mature rat microsomes (26). APAP Activation by Heterologously Expressed Human P450s. Eleven human P450s expressed in Hep G2 cells were tested for their abilities to metabolize APAP (5 mM final APAP concentration) to its GSH conjugate. Of these, only P450s 2E1, 1A2, and 3A4 showed activity significantly higher than control cell lysates, which demonstrated an activity value of less than 1pmol of product formed/(min*mgof protein). P450s 2A6,3A5,3A3, 2D6, 2F1, 2C8 2B6, and 2C9 did not show any activity. The involvement of P450 enzymes 2E1 and 1A2 are in agreement with previous studies (8). Determination of the kinetic parameters for APAP metabolism by microsomes from cells expressing the active enzymes demP450 3A4 onstrated that P450 2E1 had the highest V, showed the lowest K,,, value, and P450 1A2 demonstrated avery highK, (Table 11). Because P450 enzymesproduced by the vaccinia virus expression system are inserted into a relatively natural microsomal membrane, it is believed that the observed K , values should reflect their intrinsic 3
Unpublished results.
614
Chem. Res. Toxicol., Vol. 6, No. 4, 1993
Patten et al. I
I
-
-
-
-
-
-
W
I
0
Km
Hep G2-vlA2 Hep G2-v3A4 Hep G2-v2E1 purified P450 2E1
-bs
+bs
WM 3440 3430 313 276 1260 677 WM 4230 607
v,
-bs +h pmol/(min.mg) 74 129 328 nmol/(min.nmol) 119 52 536
17
-
3 -
-
-
2 -
e ,
4
Table 11. Kinetic Parameters of Acetaminophen Oxidation by Exwessed Human P450s and Purified P450 2E1. P450
4 -
V d Km(+bs) 0.02 0.47 0.48
44
a K , and V,, values are the average of 2 separate determinations. The incubation times were 40 min with the expressed human P450s, and 15 min for purified P450 2E1.
values in liver microsomes. Previous results have shown this to be true for expressed P450 2E1which demonstrated a Kmfor the N-demethylation of N-nitrosodimethylamine (NDMA) comparable to that observed in liver microsomes (22). The values for the enzyme efficiency constants ( V d Km)indicate that P450s 2E1 and 3A4 are more efficient catalysts of APAP than P450 1A2. Previous studies have shown that b5 may be a limiting factor in Hep G2 cells and that purified b5 can be incorporated into cell microsomal membranes in its native conformation (22). Incorporation of exogenous b5 produced an enzyme-specific effect on the kinetics of APAP activation. The hemoprotein stimulated the 2El-dependent activity at low substrate concentrations and decreased the activity at high APAP concentrations, resulting in decreases in the K m and Vm, by 47 % and 39 % , respectively. A similar b5 effect was seen with human P450 2E1 expressed in TK- 143 cells which are devoid of any b5.3 With expressed 3A4, exogenous b5 stimulated the Vm, 2.5-fold without affecting the K m . P450 1A2 activity was inhibited approximately 30-40% by bs at all substrate concentrations. Purified P450 2E1, reconstituted with phospholipid and in the absence of glycerol,showed a much higher K, for APAP oxidation than did the expressed protein (Table 11). The observed high K m value is in agreement with the value obtained for purified rabbit P450 2E1 (6). With the addition of b5 to the reconstituted system, purified P450 2E1 demonstrated a similar K m as the expressed P450 2E1 (Table 11). However, in this case b5 was shown to increase the Vm, of the reaction. Roles of Different P450 Forms Suggested by Immunoinhibition Studies. The involvement of P450 3A4 in APAP activation in human liver microsomes was examined with four human microsomesamples (Table 111).
1
I
I
I
I
1
Table 111. Inhibition of APAP Oxidation in Human Liver Microsomes by P450 Antibodies and Troleandomucin. activity [pmoU % microsomes inhibitor (miwmg)] inhibition (1)HL1 3A4PAb 503*6 6* (2) HL2 3A4PAb 491f5 12c (3) HL107 3A4PAb 194i9 38' (4) HL107 (+aNF)d 3A4 PAb 56e 93 (5) HLllO 3A4PAb 172*3 76c (6) HLllO TAO 259 49
*
a Control activity values were as follows: (1)533 17; (2) 558 f 3; (3) 315 9; (4) 772; (5) 729 f 22; and (6) 510. The final APAP concentration was 500 pM. b Values are not significantly different than controls (P> 0.05, N = 3). Values are significantly different than controls (P< 0.05, N = 3) as determined by Student's t-test. a-NF was added at a final concentration of 13 pM as described in Materials and Methods. e Values are the average of 2 separate determinations.
*
An antibody against human P450 3A4 produced 76% inhibition in HLllO microsomes, indicating that this enzyme plays a major role in APAP activation in this human liver microsome sample. With HL107, 38% inhibition was obtained, and with HL1 and HL2 there was 6 95 and 12% inhibition, respectively. The inhibition values are in close agreement with the levels of P450 3A4, which were found to vary considerably between the microsome samples (HL110 had the highest levels and HL1 the lowest based on erythromycin activity and immunoblot analy~is).~ Correlation coefficients (r)of 0.97 or 0.98 were obtained for percent inhibition of APAP oxidation by anti-3A4 vs erythromycin N-demethylase activity and P450 3A4 content (determined by immunoblot analysis), respectively. The relative levels of P450 3A4 in microsome samples HLllO and HL107 have been previously described (35)and are in agreement with the current results. In the presence of a-NF, the 3A4 antibody produced 93% inhibition in HL107 microsomes (Table 111). At low APAP concentrations (15 p M ) the anti-3A4 antibody increased APAP activation by 2-fold with HL107 microsomes. The reason for this increase is presently unknown and under investigation. Further evidence for the involvement of P450 3A4 is suggested by inhibition following incubation of HLllO microsomes with TAO, a suicide inactivator of P450 3A4 (36). Preincubation of microsomes with TAO and an NADPH-generating system resulted in 49 % inhibition (Table 111, whereas 26% inhibition was obtained without the preincubation step.
Chem. Res. Toxicol., Vol. 6, No. 4, 1993 515
Activation of Acetaminophen by Rat and Human P45Os
Table V. Effect of a-NFon APAP Oxidation by Liver Microsomes and Expressed Human P450s.
Table IV. Inhibition of APAP Oxidation i n Rat Liver Microsomes by P450 Antibodies.
APAP microsomes (1)control male (M)b (2) control male (M) (3) control male (IM)* (4) control male (IM) (5) control male (M) (6) control female (M) (7) DEX female (M) (8) DEX female (M)
(pM) lo00 lo00 1875 15 lo00 lo00 lo00 25
IgG 2ElPAb 1A2 PAb 2E1 MAb 2E1 MAb 3A1/2 PAb 3A1/2 PAb 3A1/2 PAb 3A1/2 PAb
activity bmoU % (minsmg)] inhibition 340 45 46c 369 91 4lC 134od 40 87d 5 567 53 1W 448 20 2O 674 78 81' 90 6 9lC
* * *
a Control activity values, containing preimmune IgG or ascites fluid, were as follows: (1,2 and 5) 630 9; (3) 2250; (4) 92; (6) 461 h 12; (7) 3508 79; and (8) 964 24. b (M) indicates mature rats, (IM) indicates immature rate. Significantlydifferent from controls (P< 0.05; N = 3) as determined by Student's t-test. d Activity values represent the average of 2 separate determinations. e Not significantly different from controls (P> 0.05; N = 3).
*
*
The levels of P450s 2E1 and 1A2 in the human samples, as determined by their NDMA N-demethylase and ethoxyresorufin (EROD) 0-deethylase activity, respectively, were found to be less variable, ranging from 0.6 to 1.1 nmol of product formed/(min*mgof protein) for NDMA metabolism, and from 25 to 30 pmol/(min-mg) for EROD metabolism. HL107 had the lowest NDMA demethylase activity; a reduced level of P450 2E1 could account for its lower APAP oxidase activity in comparison to the other human microsome samples (Table 111). Studies with inhibitory antibodies against P450s 2E1 and 1A2 demonstrated the involvement of these two enzymes in APAP metabolism in rat microsomes (Table IV). At an APAP concentration of 1 mM, these two antibodies inhibited 87% of the activity in control rat liver microsomes. At low substrate concentrations, antiP450 2E1 did not show significant inhibition, further suggestingthat P450 2E1is not a low-&, enzyme for APAP activation. An antibody that is inhibitory toward both P450s 3A1 and 3A2 in rats showed 510 % inhibition in adult control male rat microsomes. With control female microsomes,no inhibition was observed with this antibody. Anti-P450 3A1/2 caused extensive inhibition at high and low APAP concentrations in DEX female microsomes, further demonstrating that P450 3A1 is the major catalyst for APAP activation in these microsomes. Effect of a-Naphthoflavone on APAP Metabolism. Addition of a-NF to microsomal incubations was shown to increase the rate of APAP metabolism 2- to 5-fold in human microsomes (Table V). The extent of stimulation correlated with the amount of P450 3A4 in eachmicrosome sample. Microsomes isolated from control male rats were stimulated 3-fold, while microsomes isolated from control and DEX-treated female rats showed activation of less than 2-fold. These results are in agreement with previous studies showing the stimulation of APAP oxidation in control and DEX-treated rat liver microsomes by flavone (141, a compound similar in structure to a-NF. With all the liver microsome samples tested, the CH3OH vehicle showed some degree of inhibition. With expressed human P450s (Table V), a-NF was inhibitory toward 1A2 (83% inhibition) and had no effect on 2El-dependent metabolism. The CH30H vehicle inhibited 2E1 activity by 66 % , consistent with previous studies showing that CH30H is a competitive substrate for P450 2E1. However, doublereciprocal plots of activity vs substrate concentration in the presence and absence of CH30H indicated that CH30H
microsomes
control +CHsOH +a-NF a-NF/CHnOH ~
HL1 HL107 HLllO control male (M) control female (M) DEX female (M) Hep G2-vlA2 Hep G2-v2E1 Hep G2-v3A4
Experiment A 454 313 305 208 658 556 156 82 126 44 1470 1320
567 836 2760 245 80 1870
1.8 4.0 4.9 3.0 1.8 1.4
Experiment B 12 12 92 31 36 30
2 35 355
0.2 1.1 11.8
a Experiment A consisted of rat and human liver microsomes. In experiment B, microsomes from Hep G2 cella with expressed P450 were used. The final APAP concentration was 500 pM in each experiment. The concentration of a-NF was 13 pM, and the final CHaOH concentration was 0.66 % . Activity values are expressed an pmol of product formed/(min.mg of protein).
l/V
-0.002
0.002
0.004
0.008
0.006
0.010
l/Pl
Figure 3. Lineweaver-Burk plot of the substrate dependence of APAP oxidation by Hep G2-v3A4 microsomes in the presence and absence of a-NF. The incubations were carried out for 25 min at 37 O C . In the absence of a-NF (M) the K, and V,, values were 320 11pM and 43 7 pmol/(min.mg), respectively (N= 3). In the presence of 13 pM a - N F (*) the K, value was 486 f 50 pM,and the corresponding V, was 484 52 pmol/(min.mg). With 40 pM a-NF (v)the K, and V, values were 1178 f 134 r M and 606 23 pmol/(min.mg), respectively. The final CHgOH (vehicle) concentration in the incubations was 1%
*
*
*
*
.
inhibition in human liver microsomes was of a noncompetitive nature? Noncompetitive inhibition has also been observed using C2H50H as an inhibitor of APAP metabolism (37). The activity of expressed P450 3A4 (not inhibited by CH30H) was increased 11-fold by a-NF. This result strongly suggests that the a-NF-dependent increase in metabolism by human liver microsomes is due primarily to the activation of P450 3A4. When anti-3A1/2 was included in an incubation of male control microsomes which contained a-NF,66-74 % inhibition was observed. In fact, the antibody inhibited the activity to a value equal to the activity observed with the CH30H vehicle. This result strongly suggests that the a-NF activation observed in control male microsomes is primarily due to an increase in the activity of P450 3A2. The effect of a-NF on the kinetic parameters of APAP oxidation by expressed P450 3A4 is demonstrated in Figure 3. The results show that the addition of a-NF at a final concentration of 13 p M produced a 1.5-fold increase in
616 Chem. Res. Toxicol., Vol. 6, No. 4, 1993
0
0.0016
0.0032 Velocity/[APAP]
Patten et al.
0.0048
0
0.002
0.006 Veloclty/[APAP]
0.01
Figure 4. Eadie-Hofstee plots of APAP oxidation by human liver microsomes (HL107). Both the kinetic parameters, and the lines were determined by the computer program designed for multiple K, fitting. The data points (*) represent the experimental data. (A) With CHaOH (vehicle) added to the incubation (0.66%). The data were best fit to a three-isozyme model (SSD= 2.6 x 10-2). The& values were 14,420,and 9OOO pM,and the corresponding V-values were 80,500,and 2300 pmol/(min.mg). (B)In the presence of a-NF(13pM). The data were fit best to a two-isozyme model (SSD= 1.3 X The K, values were 27 and 750 pM,and the corresponding V- values were 190 and 3500 pmol/(min.mg). the Km for APAP oxidation, while the Vm, of the reaction was increased to a greater extent (11-fold). Increasing the final a-NF concentration to 40 p M increased the Km and Vm, by 3.6- and 14-f0ld, respectively. Thus, the stimulation by the flavone was substrate concentrationdependent and greatest at high APAP concentrations. The effect of a-NF on the kinetic parameters of APAP metabolism by human liver microsomes is presented in Figure 4. The major effect of a-NF was that the high Km (Km3) could no longer be detected, and the Vm, values of Km2 and Kml were increased 7- and 2-fold, respectively. The loss of K m 3 could result from inhibition of P450 1A2 (Table V), which displayed a high Km in the expressed system (Table IV), and the activation of P450 3A4 could explain the increase in the V,, for Km2. The increase in the Km value of K m 2 (1.7-fold) by a-NF is consistent with the observed a-NF-dependent increase in the Km with expressed P450 3A4.
Discussion Previous studies have demonstrated that P450 3A2 is constitutively expressed in control male (mature and immature) and immature female rat liver microsomes. The levels of P450 3A2 in female rats decline with age but remain elevated in male rats (33). In control male rats, P450 3A2 makes up approximately 5 ?6 of the total hepatic P450 (28). Cytochrome P450 3A1, however, is not constitutively expressed in either sex. Treatment of adult female rats with DEX strongly induces P450 3A1, while P450 3A2 remains undetectable (33). Our studies show that a high-affinity (lOW-Km) APAP oxidase becomes predominate in liver microsomes of DEX-treated adult female rats, suggesting the involvement of P450 3A1. The antibody inhibition experiments further suggest that this activity is due to P450 3A1. The results from the immunoinhibition studies suggest that in control rats the major enzymes for the activation of APAP metabolism are constitutive P450s 2E1 and 1A2. P450 3A2 did not appear to be an effective catalyst unless the activator a-NF was present in the incubations. Using human P450 enzymes expressed in Hep G2 cells, we found that P450s 2E1,1A2, and 3A4 can all metabolize APAP to its GSH conjugate. The involvement of P450 3A4 in APAP activation in human microsomes is also established from immunoinhibition experiments. P450s
2E1,1A2, and 3A4 are some of the major P450s in human liver, but the relative amounts vary among individuals (35,381. Expressed human P450 2E1 showed the highest Vm, and P450 3A4 had the lowest Km. P450 1A2 showed a much higher Km and lower Vm,, which resulted in a lower value for its enzyme efficiency constant than the other two enzymes, and therefore may be less important than the other two P450s in the activation of APAP. This analysis is made on the basis of the assumption that all three P450s were expressed at about the same level in Hep G2 cells, because the same multiplicity of infection was used for each recombinant virus. In human liver microsomes we demonstrate a medium K m (Km2) which could be accounted for by P450s 2E1 and 3A4 as well as a high Km3corresponding to the Km of P450 1A2. The effects of a-NF on the kinetic parameters of APAP activation tend to support this idea. a-NF activated K m 2 as a result of stimulation of P450 3A4, but inhibited Km3 due to inhibition of P450 1A2. Purified b5 was shown to produce a different effect on the APAP oxidase activity of each of the expressed human P450s when incorporated into Hep G2 microsomes. Such a result is consistent with previous studies showing that the b5 effect is dependent on both the substrate and the P450s employed (39). The increase in activity observed with expressed 3A4 is consistent with the previously demonstrated mechanism, whereby b5 functions to increase the rate of second electron transfer to the ferrous P450 dioxygen complex. The inhibition seen with expressed 1A2might involve an abortive mechanism resulting in the decomposition of the dioxygen complex to 0 2 and ferric P450 (40, 41). With expressed P450 2E1, bs decreased both the Kmand Vm, for APAP oxidation. This effect has been previously described for the N-demethylation of benzphetamine by P450 2B4 reconstituted with phospholipid (39). An effector role, in which b5 prompts a conformational change in the P450 structure, was suggested. Whether b5 also plays an effector role for the oxidation of APAP by P450 2E1remains to be determined. Purified rat P450 2E1, reconstituted with b5, showed a Kmvalue similar to that obtained with expressed human P450 2E1; however, in the reconstituted system the Vm, of the reaction was increased by b5, opposite to the effect seen with the expressed protein. In a previous study we observed a similar difference between expressed P450 2E1
Activation of Acetaminophen by Rat and Human P450s
and the reconstituted system in their response to b5 for the demethylation of NDMA and suggested that membrane structure is important in determining the b5 effect (22). It could affect the role of b6 in the specific step in the catalytic cycle or alter the rate-limiting steps of catalysis and thus result in a difference in the b5 effect. A similar difference exists between our present result that b5 inhibits APAP activation of membrane-bound human P450 1A2and the previous result that purified rabbit P450 1A2 (LM 4)-dependent APAP activation was stimulated by bs (6). At low APAP concentrations we detected a low-Km APAP oxidase in both human and rat liver microsomes. Preliminary studies show that metabolism of APAP at low concentrations is NADPH-dependent and sensitive to CO inhibition. Heating the microsomes to 37 "C for 25 min in the absence of NADPH, a condition which inactivates flavin-containing monooxygenase, did not significantly inhibit product formation. These results suggestthat the low-Kmactivity is due to a P450-dependent reaction. Another enzyme that can metabolize APAP to the GSH conjugate with alow Kmis prostaglandin synthase (42). However, this protein requires arachidonic acid as its cofactor and thus could not account for the low Km observed. In this study we demonstrate the ability of a-NF to stimulate APAP activation in human and male control rat liver microsomes. This increase is due to the activation of P450 3A2 in rats (3A2). In human microsomes, the stimulation is believed to be due to the activation of P450 3A4. The ability of flavanoids to activate rabbit P450 3c, the homolog of P450 3A2, has been studied in detail for benzo[a]pyrene metabolism (43). In a more recent study, it was demonstrated that a-NF stimulated the P450 3A4dependent formation of one AFBl metabolite while inhibiting that of another; it was suggested that a-NF might affect P450 3A4 activity by an allosteric mechanism (44). The results presented herein, showing that a-NF increases both the Km and Vm, for APAP oxidation, is consistent with the concept of an allosterically induced increase in transition-state complementarity (45). Alternatively, the increased Km could result from competitive inhibition by a-NF, suggesting the possibility that the flavone also binds to the active site of P450 3A4. Naturally occurring flavanoids with varied structures have been shown to have different effects on P450-dependent reactions (46,47). Because these flavanoids are present in substantial quantities in human diets, knowledge of their effects on the activation of APAP is of great interest. In conclusion, this study demonstrates the importance of human P450 3A4 in APAP bioactivation. It also demonstrates that P450 2E1 and 3A4 may be of greater significance than P450 1A2 on the basis of the high Km value of the 1A2 enzyme. In rat liver microsomes, P450 3A1 is shown to be a lOW-Km APAP oxidase whereas P450 3A2 is shown to require stimulation by a-NF for effective catalysis. The finding that P450 3A4 is involved in the activation of APAP is of potential clinical significance. The levels of this enzyme vary dramatically among individuals; its levels and activity may be influenced by a variety of drugs and dietary chemicals. These factors may impact on APAP toxicity. Acknowledgment. We thank Fang Xiao and Er Jia Wang for excellent technical assistance, Dr. S. S. Park for the 1-91-3 monoclonal antibody, Nakissa Sadrieh for
Chem. Res. Toxicol., Vol. 6, No. 4, 1993 517
preparation of the adult rat microsome samples, and Toshifumi Aoyama for advice on the vaccinia virus expression system. We also thank Dorothy Wang for computer assistance and Teresa Smith and Jennifer Hu for helpful discussions. This research was supported by U.S.Public Health Science Grants ES03938 and E505022 (C.S.Y.) and CA44353 and ES00267 (F.P.G.).
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