Chem. Res. Toxicol. 1994, 7, 836-842
836
Lauric Acid as a Model Substrate for the Simultaneous Determination of Cytochrome P450 2E1 and 4A in Hepatic Microsomes Stephen E. Clarke,*’+Sandra J. Baldwin,? Jacqueline C. Bloomer,? Andrew D. Ayrton,$ Randall S. Sozio,$ and Richard J. Cheneryt Department of Drug Metabolism and Pharmacokinetics, SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Herts, AL6 9AR, U.K. and Department of Drug Metabolism and Pharmacokinetics, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, Pennsylvania 19406 Received July 29, 1994@
In vitro techniques have been utilized to investigate the microsomal enzymes involved in the metabolism of lauric acid and to establish conditions in which it can be used as a model substrate for both cytochrome P450 4A and cytochrome P450 2E1 in human liver microsomes. Studies of enzyme kinetics of lauric acid w-hydroxylation in human liver microsomes indicated the involvement of more than one enzyme in this pathway, a relatively low Km enzyme with a Km of 22 pM f 12 (n = 8) and a high K, enzyme with a Km a n order of magnitude higher (550 pM f 310 , n = 7). The apparent Vm, for this component correlated with the rate of cyclosporin metabolism and was highly sensitive to ketoconazole inhibition. These results indicated that this enzyme was a member of the 3A subfamily. The activity associated with the low Km enzyme (P450 4A) did not correlate with P450 1A2, 2A6, 2C9/8, 2C19, 2D6, 2E1, or 3A activities in a bank of human liver microsomes and was not appreciably inhibited by ketoconazole, furafylline, quinidine, sulfaphenazole, or diethyldithiocarbamate (DDC). Lauric acid 0-1hydroxylation demonstrated simple Michaelis-Menten kinetics in each of the human liver microsomal samples examined, with a Km of 130 pM It 42 (n = 8). This activity was highly correlated with chlorzoxazone 6-hydroxylation in human liver microsomes ( r = 0.98, n = 1 4 , p < 0.001) and was inhibited by both DDC and chlorzoxazone. Additionally, rats treated with the P450 2E1 inducer isoniazid demonstrated a 3-fold increase in lauric acid o-1 hydroxylation relative to the control group. Thus, the lauric acid hydroxylation assay, at a substrate concentration of 20 pM, appears to be a n effective and specific P450 model substrate capable of determining simultaneously P450 4A and P450 2E1 related activities in hepatic microsomal samples.
Introduction The role of the cytochrome P450 (P45O)l monooxygenases in the oxidation of a wide range of endogenous and exogenous compounds has been intensively studied. As a consequence, it is known that this system consists of a superfamily of related proteins which have been organized on the basis of sequence homology and divergent evolution (11. Many P450 enzymes can be characterized by specific substrates that they metabolize in a regio- and often stereospecific manner (2, 3). The metabolism of lauric acid has been extensively studied. In rat liver microsomes, lauric acid is regiospecifically metabolized to form both w-1 and w-hydroxylated products ( 4 ) . The ratio of these two products in rat liver microsomes varies significantly on drug administration (4, 5 ) and starvation (6). It is the effect of drug administration on lauric acid w-hydroxylation in rat liver microsomes that has been most intensively studied. The administration of some hypolipidemic drugs, phthalate esters, and other agents results in the proliferation of cellular organelles including peroxisomes and the smooth
* To whom all correspondence should be addressed. Telephone: (0438) 782707; Fax: (0438) 782814; e-mail address: CLARKE-WSB. COM. + SmithKline Beecham Pharmaceuticals, The Frythe. SmithKline Beecham Pharmaceuticals, King of Prussia. Abstract published in Advance ACS Abstracts, October 15, 1994. Abbreviations: P450, cytochrome P450; DDC, diethyldithiocarbamate; BNF, @-naphthoflavone.
*
@
endoplasmic reticulum (7). This inductive effect was associated with a n increase in lauric acid o-hydroxylation due to a n increase in the specific content of cytochrome P450 4A enzymes (8-10). This induction may be a n indicator of the non-genotoxic rodent hepatocarcinogenesis caused by this class of compound (5). There is also evidence that P450 4A enzymes are involved in the activation and detoxification of some carcinogenic nitrosamines ( I 1). Lauric acid hydroxylation activity has been reported in human liver and kidney microsomes a t high specific activities (12). The levels of the w - 1 and w-hydroxylase activities varied widely in human liver, but the whydroxylated lauric acid was the predominant product in kidney microsomes (12).More recently, a complete cDNA sequence for P450 4 A l l has been described that is believed to be the major lauric acid w-hydroxylase in the human kidney (13). Attempts to correlate lauric acid o-hydroxylase activity with immunoquantified (using rat antibodies) P450 4A in human liver microsomes have not been successful (14). However, it is likely that a P450 4A enzyme is present in human liver microsomes and is a t least partly responsible for the high levels of lauric acid w-hydroxylase observed. Lauric acid w-1 hydroxylation, which is typically determined simultaneously with the w-hydroxylation,has been the subject of fewer studies. Lauric acid hydroxy-
0 1994 American Chemical Society
Lauric Acid as a Substrate for P450 2E1 and 4A
lase has been used as a probe catalytic activity for P450 2E1 in site-directed mutagenicity studies (15,16).These studies demonstrated the importance of threonine-303 in modifylng the regioselectivity of rabbit P450 2E1 for lauric acid w-1 hydroxylation. More recently, we have reported that lauric acid w-1-hydroxylase activity is highly correlated with other markers of P450 2E1 in rat, dog, and human liver microsomes (17).Cytochrome P450 2E1 is of considerable relevance t o human health as it is inducible by agents such as ethanol and isoniazid and by physiological states such as diabetes and fasting (18). Additionally, it is involved in the activation of many toxic and carcinogenic chemicals (19). In the present study, microsomal kinetic, inhibitor, and induction techniques have been utilized to investigate the enzymes involved in the metabolism of lauric acid and to establish conditions in which it can be used as a n in vitro model substrate for both P450 4A and P450 2E1 in human liver microsomes.
Materials and Methods Chemicals. All reagents were of analytical grade. Furafylline, sulfaphenazole, and 6-hydroxychlorzoxazone were obtained from Salford Ultrafine Chemicals and Research Ltd. (Manchester, U.K.). [lJ4C]Lauric acid and [Mebmt-j3-3H]cyclosporinA were purchased from Amersham Ltd (Bucks, U.K.). Chlorzoxazone, peanut oil, ketoconazole, j3-naphthoflavone(BNF),l phenobarbital, quinidine, isoniazid, and diethyldithiocarbamate (DDC)l were purchased from Sigma Chemical Co. (Dorset, U.K. or St Louis, MO). Phenobarbital was supplied by the department of Drug Substances and Products, SmithKline Beecham Pharmaceuticals (King of Prussia, PA). Animals. Male and female Sprague-Dawley rats (Cr1:CD [SD]BR) (225-250 g) were obtained from Charles River Breeding Laboratories (Raleigh, NC). Rats were group-housed in stainless steel wire cages in an environmentally controlled room (72 f 4 O F temperature; 50 & 10%relative humidity) with a 12-hour light-dark cycle. Rats were acclimatized for 5 days prior to the first day of dosing and provided food (Certified Rodent Chow 5002, Purina Mills, Inc., St. Louis, MO) and filtered tap water ad libitum. Following completion of the acclimatization period, all rats were housed individually; those rats receiving compound via the drinking water were placed in polycarbonate cages with hardwood bedding and bottled drinking water. All other rats remained in suspended stainless steel wire cages. BNF (40 mg/kg) or vehicle (peanut oil) was administered ip for 3 consecutive days to groups of three male and three female rats at 2.5 m u g . In addition, three groups of three male or three female rats received either phenobarbital (0.10% w/v) or isoniazid (0.10%w/v) in their drinking water for 10 consecutive days. A group of three male and three female rats receiving compound-free drinking water for 10 consecutive days served as controls. Following 10 days of exposure, those groups receiving the compound via the drinking water were allowed free access to compound-free drinking water 24 h prior to sacrifice. All other groups were sacrificed 24 h following final administration of their respective compound. Human Liver. Human liver tissue was obtained from either Vitron Inc. (Tuscon, AZ)or the International Institute for the Advancement of Medicine (Exton, PA). All samples were from otherwise healthy donors, and in all cases the cause of death was not believed to be due to any known biochemical deficiency in the liver. Preparation of Microsomes. Rat and human liver microsomal fractions were prepared by differential ultracentrifugation. After homogenization in 50 mM Tris-HC1 buffer (pH 7.4) containing 0.25 M sucrose, the microsomal fraction was isolated from the supernatant of a 20-min, lOOOOg spin by ultracentrifugation at lOOOOOg for 60 min. The microsomal pellet was resuspended in the above Tris-HC1 buffer and
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 837 recentrifuged a t lOOOOOg for 60 min. The final pellet was resuspended in 50 mM potassium phosphate buffer (pH 7.4) and stored at approximately -80 "C until required. P460 Activities. Lauric acid assays were carried out in 50 mM Tris-HC1buffer (pH 7.4);all other microsomal assays were carried out in 50 mM potassium phosphate buffer (pH 7.4). Reactions a t 37 "C were initiated by the addition of a NADPHgenerating system (0.2 mL/mL of incubation) consisting of 1.7 mg of NADP+, 7.8 mg of glucose 6-phosphate, and 6 units of glucose-6-phosphate dehydrogenase/mL of 2% (w/v) sodium hydrogen carbonate. Lauric acid hydroxylase was determined by HPLC using [l-l4C1lauric acid and radiometric assay for quantitation. Incubations (0.4 mL) containing 0.5 mg of microsomal proteid mL were terminated after 5 min by the addition of 0.2 mL of 3 M HC1. To determine the kinetic constants, lauric acid concentrations of 2, 8.8, 14.5, 27, 52, 127, 252, 502, and 1002 pM were used. Further experiments were performed at concentrations of 20,100, or 130 pM. The lauric acid and the metabolites were extracted by mixing with 7 mL of diethyl ether for 20 min. The organic layer was taken and evaporated to dryness under nitrogen and then resuspended in methanol (containing 0.1% v/v acetic acid). An equal volume of water (containing 0.1% v/v acetic acid) was added, and the resulting solution was filtered prior to HPLC analysis. A Supelcosil Cg, 3 pm, 15 cm x 4.6 mm column maintained a t 42.5 "C was eluted a t a flow rate of 1.0 mL min-l with an isocratic mixture of 50% solvent A (distilled water, 0.1%acetic acid)/50%solvent B (methanol, 0.1% acetic acid) from 0 to 20 min followed by a linear gradient to 100% B by 25 min and then isocratic 100% B for a further 5 min. Under these conditions, the order of elution was (u-1)hydroxy, o-hydroxy lauric acid, and then parent compound. The lowest limit of quantitation (LLQ) was equivalent to 50 pmoV mL of incubation. Chlorzoxazone 6-hydroxylation was determined by HPLC using a substrate concentration of 100 pM and 0.4 mg of microsomal proteidml. Incubations (0.25 mL) were terminated after 5 min by the addition of 50 pL of 6% (v/v) perchloric acid. After centrifugation to remove the precipitated protein, 200 pL of the supernatant was analyzed by HPLC. A Nova-Pak C18, 4 pm, 15 cm x 3.9 mm column maintained a t 40 "C, was eluted a t a flow rate of 1mL min-' with an isocratic mixture of 83% solvent A (20 mM NaC104, pH 2.5) and 17% solvent B (acetonitrile) from 0 t o 5 min followed by a linear gradient to 100%B by 7 min and then isocratic 100%B for a further 3 min. Under these conditions, the retention time of 6-hydroxy chlorzoxazone was approximately 3 min. The 6-hydroxy chlorzoxazone was quantified from the W peak area a t 287 nm with reference to an external standard curve of the authentic metabolite. The LLQ was equivalent to 0.6 nmoles/mL of incubation, a t which concentration the accuracy was within 20% with a coeffkient of variance of less than 5%. Cyclosporin oxidation was determined by HPLC using [Mebmtj3-3Hlcyclosporin A a t a substrate concentration of 1 pM and radiometric assay for quantitation. Incubations (0.25 mL) containing 0.4 mg of microsomal proteidml were terminated after 10 min by the addition of 0.25 mL of acetonitrile. After centrifugation to remove precipitated protein, HPLC analysis was performed as has been reported previously (20). The LLQ was equivalent to 50 pmoVmL of incubation. Inhibition experiments were carried out as described above except for those with diethyldithiocarbamate and furafylline, which were preincubated for 10 min with the microsomes and the NADPH-generating system prior to initiation of the reaction by the addition of substrate. Analysis of Data. Enzyme kinetic data was analyzed by nonlinear regression using weighted (l/y) untransformed data with Grafit V3.0 (21).A simple, one-enzyme, model was used to determine the Michaelis-Menten parameters except where the use of a two-enzyme model was justified by the data.
Results Kinetics of Lauric Acid Hydroxylation. In human liver microsomes, the ratio of the w to w-1 hydroxylated
Clarke et al.
838 Chem. Res. Toxicol., Vol. 7, No. 6,1994
Table 1. Determination of Michaelis-Menten Parameters for Lauric Acid * and w l Hydroxylation in Human Liver Microsomesa
human liver sample H27 H28 H30 H31 HlOO HlOl H102b H103 mean SD
Km
33 15 13 14 14 18 47 20 22 12
low K, enzyme SE Vm, 8 5 4 7 6 7 2 10
lauric w-hydroxylase high K, enzyme
160 38 41 39 44 170 220 80 99 73
SE 47 10 14
16 17 64 6 39
Km 490 1200 200 480 550 410
530 550 310
SE 930 820 37 340 410 190
SE 97 160 13 55 70 130
Vm,
170 390 320 210 240 910
1100
lauric acid w-1-hydroxylase Km SE Vmax 140 15 130 110 110 18 110 20 290 87 6 80 80 190 220 100 12 290 150 22 110 120 17 140 170 130 82 42
40
75 330 270
SE 11 11
38 4
54 21 12 13
a V , expressed as nmol*h-l.(mg of protein-'); K, as pM. SE, standard error for determination of kinetic constants. SD, standard deviation of mean. Only one set of kinetic constants apparent for w-hydroxylation.
300
1
250
4
h
200
h!! \
m
i \
-
i
0
0.5
800
h
I
1.5
2
2.5
3
V/[Sl (nmol.h-'.mg protein"/pM)
Figure 1. Eadie-Hofstee plots for determination of MichaelisMenten parameters for lauric acid w - 1 hydroxylation in human liver microsomes. Data shown for human liver HlOl (squares) and H28 (circles). The kinetic parameters determined by nonlinear regression from untransformed data were V, of 290 and 110 nmol.h-l.(mg of protein-l) and a K, of 100 and 110 pM for HlOl and H28, respectively.
lauric acid varied with substrate concentration, and the Michaelis-Menten parameters associated with each of these reactions were clearly different (Table 1). Lauric acid 0-1hydroxylation displayed simple enzyme kinetic properties with only one component clearly apparent from Eadie-Hofstee plots (Figure 1). From eight human liver samples, the K, was 130 pM f 42 (mean f standard deviation), and the V, exhibited about a 4-fold range in activity in these samples (Table 1). In contrast, lauric acid w-hydroxylation Eadie-Hofstee plots were curved with two components apparent in seven of the eight human livers (Figure 2). The data for liver H102 fitted the one-site model and the two-site model equally, and as such the results from the simpler model were used. The relatively low K, component had a K, of 22 pM f 12 (mean f standard deviation), and the V, exhibited about a 6-fold range in activity in these samples. The relatively high K, component had a K, a t least an order of magnitude higher (550 pM f 310). Lauric acid o-Hydroxylase. The nature of the enzyme kinetics suggested that more than one P450 enzyme was capable of metabolizing lauric acid a t the w
0
2
4
6
8
10
12
V/[Sl (nmol.h".mg protein"/pM)
Figure 2. Eadie-Hofstee plots for determination of MichaelisMenten parameters for lauric acid w-hydroxylation in human liver microsomes. Data shown for human liver HlOl (squares) and H28 (circles). The kinetic parameters were determined by nonlinear regression from untransformed data, and in each liver a high and low K, component were apparent. HlOl had Vmax's of 170 and 910 nmol-h-l.(mg of protein-') and Km's of 18 and 410 pM for the high and low K , site, respectively.H28 had V-'s of 38 and 390 nmol*h-l.(mgof proteinF1)and K,'s of 15 and 1200 pM for the high and low K, site, respectively.
carbon. The V, associated with the high K, component was highly correlated with cyclosporin oxidation rate in seven human livers with a correlation coefficient of 0.97, p < 0.001 (Figure 3). The effect of ketoconazole a t 1 pM on lauric acid w-hydroxylation in microsomes from three human livers was tested a t 20 or 100 pM substrate concentration. The livers chosen had a relatively high (H28), intermediate (H27), or low (H103) cyclosporin oxidation rate. At a 20 pM concentration of lauric acid, the w-hydroxylation was not inhibited by 1 pM ketoconazole (Figure 4). Under these conditions, this activity was relatively insensitive to ketoconazole inhibition even a t far higher inhibitor concentrations (results not shown). In contrast, a t a 100 ,uM substrate concentration a proportion of the lauric acid w-hydroxylation activity was highly sensitive to ketoconazole inhibition (Figure 4). The proportion of activity sensitive to ketoconazole inhibition was related to the cyclosporin oxidation status of the sample. Lauric acid wl-hydroxylase. Lauric acid w-1 hydroxylation was highly correlated, r = 0.98, p < 0.001,
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 839
Lauric Acid as a Substrate for P450 2E1 and 4A
120
1
4i
0
3.5
0
0
0 0 0 0
0
20
u-
---O li0 0
0.5
100 200
300 400 500 600 700
0
j
800 900 1000 0
0.5
1
Vmxof high K,,, o-hydroxylase
1.5
2
2.5
3
4
3.5
Lauric acid o-1 hydroxylation (nmol.min*'.mgprotein-')
(nmo1.h-'.mgprotein-')
Figure 3. Correlation of the high K, lauric acid o-hydroxylase with cyclosporin oxidation activity in human liver microsomes. Coefficient of correlation, r, was 0.97 for seven samples, p < 0.001. Cyclosporin oxidation rate was the combined formation of all the oxidative products apparent in microsomes. This activity is indicative of P450 3A enzymes.
Figure 5. Correlation of lauric acid 0-1 hydroxylation with chlorzoxazone 6-hydroxylation in human liver microsomes. Coefficient of correlation, r, was 0.98for 14 s a m p l e s , ~< 0.001. Chlorzoxazone 6-hydroxylation activity is indicative of P450 0 0
T T
100
0
0
"
0
=. W
80
0
I
60
4
f
A
0
Control(ip)
0
Control (drinking water)
0
P-napthoflavone
40
Isoniazid A
Phenobarbital
20 I
0
0
1 H28
H27
H103
Human Liver Sample
Figure 4. Differential effect of 1pM ketoconazole on lauric acid o-hydroxylation at 20 pM (hatched columns) and 100 pM (open columns) substrate concentrations in human liver microsomes. H28 was believed to have high levels of P450 3A as determined by cyclosporin oxidation. H27 and H103 had intermediate and low P450 3A levels, respectively. Columns represent mean values from three determinations; error bars indicate the standard deviation. The mean control activities at 20 pM were 61.5, 24.6, and 26.4 and at 100 pM were 125.5, 72.4,and 56.3 nmol*h-l.(mg of protein-l) for H27, H28, and H103, respectively.
with chlorzoxazone 6-hydroxylation in 14 human liver microsomal samples (Figure 5). Treatment of rats with isoniazid at 0.1% (wh) in the drinking water clearly elevated both lauric acid 0-1hydroxylation and chlorzoxazone 6-hydroxylation(Figure 6). Within all animals, except those treated with BNF, these two activities were highly correlated, r = 0.98,n = 24, p -= 0.001 (Figure 6). By contrast, rats treated with BNF had markedly elevated chlorzoxazone 6-hydroxylation activity in liver microsomes, but lauric acid cu-1 hydroxylation activity
I
I
1
I
1
2
3
4
Lauric acid 0-1 hydroxylation (nmoles.min-'.mgprotein-')
Figure 6. Correlation of lauric acid 0-1hydroxylation with chlorzoxazone 6-hydroxylation in rat liver microsomes. Rats were treated with either BNF, phenobarbital, or isoniazid as described in the Materials and Methods and were compared to relevant controls. Correlation coefficient, r, for all rats except those treated with BNF was 0.98 for 24 samples, p < 0.001.
was unchanged (Figure 6 ) . Treatment with phenobarbital caused a n increase in lauric acid cu-1 hydroxylation when expressed on a per milligram protein basis only (Figure 6). This effect was less marked than that caused by isoniazid. Lauric acid cu-hydroxylase was not significantly elevated by these treatments (results not shown). Lauric acid cu-1-hydroxylase was inhibited by DDC. Concentrations up to 100 pM (preincubated with the microsomes for 10 min with NADPH before initiation of the reaction with the substrate) caused progressively greater inhibition (Figure 7). Higher concentrations caused further inhibition of this activity, but at the expense of specificity, since significant inhibition of lauric acid cu-hydroxylation was observed at concentrations of DDC greater than 100 pM (results not shown). Addition-
840 Chem. Res. Toxicol., Vol. 7, No.6,1994
Clarke et al.
1
T
100
7-
100
90 80
80 70
h U
>
.r(
60
w
.r(
2
60
c (
41
c-)
sg
40
20
0 0
10
20
30
40
50
60
70
80
90
100
[Diethyldithiocarbamate]
QUIN 2D6
1
1
T
T SULF 2C9
DDC 2E1
CLZ 2E1
KET 3A
Figure 9. Effect of specific P450 probes on lauric acid 0-1
(PM) Figure 7. Effect of DDC on lauric acid 0-1 hydroxylation in human liver microsomes. DDC was preincubated, for 10 min, with the microsomes and NADPH prior to the initiation of the reaction. Each point represents the mean value from three human liver microsomal samples; error bars indicate standard deviation. The mean control activity was 30.0 nmol-h-l*(mg of protein-l).
I
T FUR 1A2
T T
hydroxylation in human liver microsomes. Furafylline (FUR) at 10 yM and diethyldithiocarbamate (DDC) at 100 yM were preincubated, for 10 min, with the microsomes and NADPH prior to the initiation of the reaction. Sulfaphenazole (SULF) was added at a concentration of 10 yM, quinidine (QUIN) at 1 pM, chlorzoxazone (CLZ) at 1mM, and ketoconazole (KET) at 1 yM. The assay was conducted at the Km for the o-lhydroxylase. Each column represents the mean value from three human liver microsomal samples; error bars indicate standard deviation. The mean control activities were 25.6,28.9,31.4,30.0, 32.4, and 32.9 nmol*h-l.(mg of protein-l) for the furafylline, sulfaphenazole, quinidine, DDC, chlorzoxazone, and ketoconazole experiments, respectively.
Discussion
Es?
ta
40
30
2o 10
i
1 o
10-1
100
101
102
103
io4
105
[Chlorzoxazone]
(PM) Figure 8. Effect of chlorzoxazone on lauric acid o-1hydroxylation in human liver microsomes. Each point represents the mean value from three human liver microsomal samples; error bars indicate standard deviation. The mean control activity was 32.4 nmol*h-l.(mg of protein-l). The fitted curve represents an IC50 determination (500 yM); the assay was conducted at the Kmfor the o-1-hydroxylase. The mean control activity was 30.0 nmol*h-l.(mg of protein-l).
ally, the P450 2E1 substrate chlorzoxazone inhibited lauric acid cu-1 hydroxylation in human liver microsomes, with an IC50 of approximately 500 pM at a lauric acid concentration of 130 pM (Figure 8). Of the P450 enzyme probes tested, only DDC and chlorzoxazone caused significant inhibition of lauric acid cu-1 hydroxylation. Furafylline (P450 1A2), sulfaphenazole (P450 2C9), quinidine (P450 2D6), and ketoconazole (P450 3A) showed no significant inhibitory potential against this activity in human liver microsomes (Figure 9).
Lauric acid has been used as a model substrate for rat cytochrome P450, in particular the cu-hydroxylation for P450 4A, for many years. By convention, the substrate concentration used has invariably been 100 pM. Although studies determined the Km for lauric acid cu-hydroxylase in rat kidney cortex microsomes to be 7 p M , the reaction exhibited simple enzyme kinetics, and the ratio of cu to cu-1hydroxylated products was independent of substrate concentration (22). Thus, these saturation concentrations should reflect the amount of enzyme present, and this approach has been highly successful in quantifying the effect of exogenous compounds and physiological conditions on the levels of P450 4A in the rat (4-10). From our studies, however, it is clear that these conditions are not suitable for investigating lauric acid cu-hydroxylase in human liver. In the majority of human livers investigated, the microsomes had a t least two enzymes capable of cu-hydroxylating lauric acid. At substrate concentrations greater than 50 pM,the contribution from the high Km enzyme was more apparent. The activity associated with this component was highly correlated with cyclosporin oxidation. This suggested that this component was P450 3A. Further evidence of P450 3A involvement in lauric acid cu-hydroxylation was obtained from inhibition studies with ketoconazole. Ketoconazole has inhibitory potential against a number of human P450 enzymes, but a t low concentrations is selective for P450 3A (23). At a lauric acid concentration of 100 p M , the extent of P450 3A involvement in the cu-hydroxylation, as determined by ketoconazole inhibition, demonstrated considerable interindividual variation. In human liver known to have high P450 3A activities, P450 3A contributed to a t least
Lauric Acid as a Substrate for P450 2E1 and 4A 40% of the lauric acid o-hydroxylation. By contrast, in low P450 3A livers little inhibition with ketoconazole was observed, and the majority of the w-hydroxylation was probably due to a P450 4A enzyme such as described previously (13, 14). By using a substrate concentration close to the K, of the low K,,, component, the selectivity of this assay was recovered. Ketoconazole was ineffective a t inhibiting lauric acid w-hydroxylation in human liver microsomes, indicating that a t this substrate concentration the contribution from P450 3A was much less significant. The lauric acid a-hydroxylase activity at a 100 pM substrate concentration would appear to be dependent on the relative levels of expression of these P450 enzymes in human liver microsomes. Thus, it is not surprising that the attempts to correlate lauric acid w-hydroxylase with P450 4A immunoblotted protein levels in human liver microsomes failed to show significance (14). It may be possible to demonstrate such a correlation by either using a low substrate concentration (e.g., 20 pM)or in a human liver bank where the P450 3A levels are consistent or low. In our human liver bank, lauric acid w-hydroxylase a t a 20 pM substrate concentration did not correlate with the activity at a 100 pM substrate concentration, nor were significant correlations observed with P450 activities related to P450 1A2, 2A6, 2C9 (/8), 2D6,2E1, and 3A. It is likely that at the lower substrate concentration, lauric acid w-hydroxylation is a measure of a P450 4A in human liver, possibly related to that characterized in human kidney (13). Lauric acid o-1-hydroxylase activity is determined simultaneously with the w-hydroxylase, but has been the subject of far fewer studies. In our experience, in human liver microsomes, substrates that have specific activities of the order of nanomole/[minute.(milligram of microsomal protein)] are typically substrates for P450 3A or P450 2E enzymes. This probably reflects the level of expression of these enzymes in human liver, but could conceivably be a result of these enzymes being significantly better catalysts. Both hydroxylations of lauric acid could be defined as being high rate reactions. Since the ratio of w to w-1 hydroxylated products varies widely between individuals, these pathways are not likely to be dependent on the same enzyme. On these considerations alone, the most likely candidates for a lauric acid w-1-hydroxylase in human liver microsomes are P450 3A or P450 2E. A highly significant correlation, in human liver microsomes, between the 6-hydroxylation of chlorzoxazone and the 0-1 hydroxylation of lauric acid was demonstrated in this study. These considerations and the previously reported correlation between this activity and P450 2E markers in other species (17) suggests that lauric acid w-1 hydroxylation is primarily catalyzed by P450 2E1. In this study, rats treated with isoniazid, known to induce P450 2E1 levels, had highly significantly elevated levels of lauric acid o-1-hydroxylase activity compared to controls. This suggests that in the rat P450 2E1 plays a major role in lauric acid w-1 hydroxylation. Additionally, this demonstrates that lauric acid, via the w-1 hydroxylation, is a suitable model substrate for detecting P450 2E1 inducers in rats. There are other wellestablished model substrates for investigating induction of P45O 2E1 in various species. p-Nitrophenol oxidation to 4-nitrocatechol has been shown to have utility in a number of species (24) and has been validated as a substrate probe for human liver P450 2E1 (25). p Nitrophenol oxidation is clearly increased in rats treated with P450 2E1 inducers, but does demonstrate a lack of
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 841
specificity. Treatment with phenobarbital causes increased p-nitrophenol oxidation (261, but does not cause a n increase in levels of P450 2E1 protein (27). Lauric acid w-1-hydroxylase activity, in rats, is not consistently increased by phenobarbital treatment, typically a small increase is apparent on a per milligram of protein basis, but no change on a per nanomol of P450 basis (28). In this study, phenobarbital caused a small increase in lauric acid w-1 hydroxylation and chlorzoxazone 6-hydroxylation when expressed on a per milligram of protein basis. In a recent review (291, the catalytic properties of a number of purified rat cytochrome P450s suggested that several of the enzymes were capable of lauric acid w-1 hydroxylation. Of the enzymes that were clearly identified, most noteworthy were P450 1A2, P450 2A1, P450 2C6, P450 2Cl1, and P450 2E1. The involvement of P450 1A2 and P450 2A1 in lauric acid w-1 hydroxylation is not borne out by our results. If these enzymes were significantly involved in this reaction, then treatment with the P450 1A inducer, such as BNF, would have significantly elevated lauric acid w-1-hydroxylase activity in an analogous manner to that seen for chlorzoxazone 6-hydroxylation. In fact BNF treatment caused no such increase in this study, and 3-methylcholanthrene has been reported to cause a decrease (28). The expression in rats of P450 2C6 is not effected by age or sex, and P450 2 C l l is a major male-specific form (29). The inductive effect of phenobarbital on the level of expression of P450 2C6 may be the cause of the slight elevation of lauric acid 0-1 hydroxylation seen on treatment of rats with this agent. Thus, lauric acid w-1 hydroxylation as a rat induction probe for P450 2E1 may suffer some limitations in specificity similar to p-nitrophenol, but lesser in degree. Chlozoxazone 6-hydroxylation has been demonstrated to be a specific model substrate for P450 2E1 in human liver microsomes (30). More recently, it has been shown that P450 1Al is also capable of this reaction and that in the rat P450 1Al inducers cause as great if not greater elevation of this activity than P450 2E1 inducers (31). This suggests that chlorzoxazone 6-hydroxylation is an inappropriate model substrate for rat induction studies and will also limit its use as an in vivo probe for P450 2E1 in man. In the current study, we confirm the marked inductive effect of P450 1Al inducers on chlorzoxazone 6-hydroxylation and demonstrate that lauric acid 0-1 hydroxylation does not have this particular specificity problem. It has been suggested that Nnitrosodimethylamine demethylation is the most specific probe of P450 2E1 in rat liver microsomes for induction studies (26). Lauric acid w-1 hydroxylation, however, may be equivalent to or better thanp-nitrophenol and is clearly superior to chlorzoxazone for rat induction studies. The most significant advantage in using this assay is the convenience of being able to detect induction of two P450 enzymes in one assay (P450 2E1 and P450 4A). In human liver microsomes, lauric acid w-1-hydroxylase activity was inhibited by DDC, which is a mechanismbased inhibitor of P450 2ElU9). At the highest concentration of DDC that retained specificity (100 pM), there was approximately a 50% inhibition of lauric acid w-1 hydroxylation. This inhibition is comparable to that reported under similar conditions for the P450 2E1 catalyzed defluorination of sevoflurane, isoflurane, and methoxyflurane (32), somewhat less than that reported for enflurane (33), and significantly less than that reported for chlorzoxazone (33). However, in the current
842 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
study, chlorzoxazone did specifically inhibit lauric acid 0-1 hydroxylation in human liver microsomes. In the rabbit, P450 2C enzymes as well as 2E1 have been described as laurate w-1hydroxylating P450s (16). From the inhibition studies, there was no significant inhibition with furafylline, a selective inhibitor of P450 1A2 (34); sulfaphenazole, a selective inhibitor of P450 2C9 (35); quinidine, a selective inhibitor of P450 2D6 (36); or ketoconazole, a selective inhibitor of P450 3A (20). This indicates that these P450s do not contribute significantly to lauric acid w-1 hydroxylation in man. Taken together, all of these results demonstrate that P450 2E1 is the predominant, if not singular, human P450 enzyme responsible for the w-1 hydroxylation of lauric acid. Thus, the simultaneous determination of the major metabolites of lauric acid in microsomes constitutes a convenient probe for two P450 enzymes in both rat and man. Lauric acid, in human liver microsomes a t a concentration of 20 p M , is a useful in vitro model substrate that can specifically and sensitively measure P450 4A and P450 2E1 levels and be used to determine the effect of various agents on these enzymes.
References (1) Nelson. D. R.. Tetsuva. " , K.. Waxman. D. J.. Guengerich. F. P.. Estabrook, R.' W., Feyereisen, R., Gonzalez: F. J.,"Coon,' M. J.: Gunsalus, I. C., Gotoh, O., Okuda, K., and Nebert, D. W. (1993) The P450 superfamily: Update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biol. 12, 1-51. Guengerich, F. P. (1992) Human cytochrome P450 enzymes. Life Sci. 50,1471-1478. Guengerich, F. P. (1992) Characterisation of human cytochrome P450 enzymes. FASEB J . 6, 745-748. Parker, G. L., and Orton, T. C. (1980) Induction by oxyisobutyrates of hepatic and kidney microsomal cytochrome P-450 with specificity towards hydroxylation of fatty acids. In Biochemistry, Biophysics and Regulation of Cytochrome P450 (Gustafsson, J. A., Duke, J. C., Mode, A., and Rafter, J., Eds.) pp 373-377, Elseviermorth Holland, Amsterdam. Hawkins, J . M., Jones, W. E., Bonner, F. W., and Gibson, G. G. (1987) The effect of peroxisome proliferators on microsomal, peroxisomal and mitochondrial enzyme activities in the liver and kidney. Drug Metab. Rev. 18, 441-515. Bjorkhem, I. (1973) w-oxidation of stearic acid in the normal, starved and diabetic rat liver. Eur. J . Biochem. 40, 415-422. Sharma, R., Lake, B. G., Foster, J., and Gibson, G. G. (1988) Microsomal cytochrome P452 induction and peroxisome proliferation by hypolipidaemic agents in rat liver: A mechanistic interrelationship. Biochem. Pharmacol. 37, 1193-1201. Hardwick, J. P., Song, B. J., Huberman, E., and Gonzalez, F. J. (1987) Isolation, complimentary DNA sequence and regulation of rat hepatic lauric acid w-hydroxylase (cytochrome P 4 5 0 ~ , ) : identification of a new cytochrome P450 gene family. J . Biol. Chem. 262,801-810. Kimura, S., Hardwick, J. P., Kozak, C. A., and Gonzalez, F. J. (1989) The rat clofibrate-inducible CYPM subfamily 11. cDNA sequence of IVA3, mapping of the Cypla locus to mouse chromosome 4 and coordinate and tissue specific regulation of the CYF'4A genes. DNA 8, 517-525. Aoyama, T., Hardwick, J . P., Imaoka, S., Funae, Y., Gelboin, H. V., and Gonzalez, F. J. (1990) Clofibrate-inducible rat hepatic P450s IVAl and IVA3 catalyze the w - and (w-1)-hydroxylation of fatty acids and the w-hydroxylation of prostaglandins E1 and Fzu. J . Lipid Res. 31, 1477-1482. Lawson, T. A. (1991) Involvement of lauric acid hydroxylase in the activation of P-substituted nitrosamines. Cancer Lett. 59,177182. Okita, R. T., Jakobsson, S. W., Prough, R. A., and Masters, B. S. (1979) Lauric acid hydroxylation in human liver and kidney cortex microsomes. Biochen. Pharmucol. 28, 3385-3390. Imaoka, S., Ogawa, H., Kimura, K., and Gonzalez, F. J. (1993) ComDlete cDNA seauence and cDNA-directed exmession of C Y P k l 1 , a fatty acid w-hydroxylase expressed in human kidney. DNA Cell Biol. 12, 893-899. Dirven, H. A. A. M.. Peters, J. G. P.. Gibson. G. G., Peters. W. H. M., and Jongeneelen, F. J. (1991)La'kic acid hydro'xylase activity I
Clarke et al. and cytochrome P450 IV family proteins in human liver microsomes. Biochem. Pharmacol. 42, 1841-1844. (15) Fukuda, T., Imai, Y., Komori, M., Nakamura, M., Kusunose, E., Satouchi, K., and Kusunose, M. (1993) Replacement of Thr-303 of P450 2E1 with serine modifies the regioselectivity of its fatty acid hydroxylase activity. J . Biochem. 113, 7-12. (16) Fukuda, T., Imai, Y., Komori, M., Nakamura, M., Kusunose, E., Satouchi, K., and Kusunose, M. (1994) Different mechanisms of regioselection of fatty acid hydroxylation by laurate (w-1)-hydroxylating P450s, P450 2C2 and P450 2E1. J . Biochem. 115, 338-344. (17) Clarke, S. E., Baldwin, S. J., Bloomer, J. C., Ayrton, A. D., and Chenery, R. J. (1994) Cytochrome P450 2E1 and lauric acid w-1 hydroxylation in rat, dog and human liver microsomes. Br. J. Clin. Pharmacol. 38, 174P. (18) Koop, D., and Tierney, D. J. (1990) Multiple mechanisms in the regulation of ethanol-inducible cytochrome P450 IIE1. Bioessays 12, 429-435. (19) Guengerich, F. P., Kim, D.-H., and Iwasaki, I. (1991) Role of human cytochrome P450 IIEl in the oxidation of many low molecular weight cancer suspects. Chem. Res. Toxicol. 4, 168179. (20) Pichard, L., Fabre, I., Fabre, G., Domergue, J., Saint Aubert, B., Mourad, G., and Maurel, P. (1990) Cyclosporin A drug interactions: Screening for inducers and inhibitors of cytochrome P450 (cyclosporin A oxidase) in primary cultures of human hepatocytes and in liver microsomes. Drug Metab. Dispos. 18, 595-606. (21) Leatherbarrow, R. J. (1992) Grafit, Version 3.0, Erithacus Software Ltd., Staines, U.K. (22) Ellin, A., Orrenius, S., Pilotti, A,, and Swahn, C.-G. (1973) Cytochrome P450k of rat kidney cortex microsomes: Further studies on its interaction with fatty acids. Arch. Biochem. Biophys. 168, 597-604. (23) Maurice, M., Pichard, L., Daujat, M., Fabre, I., Joyeux, H., Domergue, J., and Maurel, P. (1992) Effects of imidazole derivatives on cytochromes P450 from human hepatocytes in primary culture. FASEB J . 6, 752-758. (24) Koop, D. R., Laethem, C. L., and Tierney, D. J. (1989) The utility ofp-nitrophenol hydroxylation in P450IIE1 analysis. Drug Metab. Rev. 20, 541-551. (25) Tassaneeyakui, W., Veronese, M. E., Birkett, D. J., Gonzalez, F. J., and Miners, J . 0. (1993) Validation of 4-nitrophenol as an in vitro substrate probe for human liver CYP2El using cDNA expression and microsomal kinetic techniques. Biochem. Phurmacol. 46, 1975-1981. (26) Lucas, D., Berthou, F., Dreano, Y., Floch, H. H., and Menez, J. F. (19901 Ethanol-inducible cytochrome P450: assessment of substrates' specific chemical probes in rat liver microsomes. Alcohol.: Clin. Exp. Res. 14, 590-594. (27) Thomas, P. E., Bandiera, S., Maines, S. L., Ryan, D. E., and Levine, W. (1987)Regulation of cytochrome P-450j, a high affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes. Biochemistry 26, 2280-2289. (28) Dirven, H. A. A. M., Van Den Broek, P. H. H., Peters, J. G. P., Noordhoek, J., and Jongeneelen, F. J. (1992) Biochem. Pharmacol. 43,2621-2629. (29) Funae, Y., and Imaoka, S. (1993) Cytochrome P450 in rodents. Handb. Exp. Pharmacol. 105, 221-238. (30) Peter, R., Bocker, R., Beaune, P. H., Iwasaki, M., Guengerich, F. P., and Yang, C. S. (1990) Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P450 IIE1. Chem. Res. Toxicol. 3, 566-573. (31) Caniere, V., Goasduff, T., Ratanasavanh, D., Morel, F., Gautier, J.-C., Guillouzo, A., Beaune, P., and Berthou, F. (1993) Both cytochromes P450 2E1 and 1Al are involved in the metabolism of chlorzoxazone. Chem Res. Toxicol. 6, 852-857. (32) Kharasch, E. D., and Thummel, K. E. (1993) Identification of cytochrome P450 2E1 as the predominant enzyme catalyzing human liver microsomal defluorination of sevoflurane, isoflurane and methoxyflurane. Anesthesiology 79, 795-807. (33) Thummel, K. E., Kharasch, E. D., Podoll, T., and Kunze, K. (1993) Human liver microsomal enflurane defluorination catalyzed by cytochrome P450 2E1. Drug Metab. Dispos. 21, 350-357. (34) Clarke, S. E., Ayrton, A. D., and Chenery, R. J. (1994) Characterization of the inhibition of P4501A2 by furafylline.Xenobiotica 24, 517-526. (35) Back, D. J., Tjia, J . F., Karbwang, J., and Colbert, J. (1988) In vitro inhibition studies of tolbutamide hydroxylase activity of human liver microsomes by azoles, sulphonamides and quinolines. Br. J. Clin. Pharmacol. 26, 23-29. (36) Otton, S. V., Crewe, H. K., Lennard, M. S., Tucker, G. T., and Woods, H. F. (1988)Use of quinidine inhibition to define the role of the sparteine/debrisquine cytochrome P450 in metoprolol oxidation by human liver microsomes. J. Pharmacol. Exp. Ther. 247, 242-247.
