Eur. J Biochem. 200, 51 1-517 (1991) 0FEBS 1991 001429569100564H

Isolation and characterization of a cytochrome P450 of the IIA subfamily from human liver microsomes Manuelle MAURICE l , Stephane EMILIANI ', Isabelle DALET-BELUCHE I , Jean DERANCOURT' and Reinhard LANCE Institiit National dc la Santi. et dc la Recherchc Medicale U 128, Montpellicr, France

'Institut National de la Santk et de la Recherche Mkdicale U 249, Montpellier, France (Received Fcbruary 4/May 3, 1991) - EJB 910172

Antibodies raised against cytochrome P450, which is overexpressed in mouse hepatic tumors, (P450,J crossreact with two human liver microsomal proteins (49 kDa and 52 kDa). We have quantified these proteins in 60 human liver samples and found great interindividual variability in both of them. The concentration of the 49kDa protein varies up to 144 fold in the various samples and represents typically 10% of the total mincrosomal P450 content. Its immunologically determined concentration correlates well ( R = 0.78) with the microsomal coumarin-7-hydroylase (COH) activity. This activity is strongly and completely inhibited by anti-P450,, antibody (IC50 = 0.13 mg IgG/mg microsomal protein). The crossreacting 49-kDa protein shows an unusually high substrate specificity towards coumarin; it presents all human COH and part of 7-ethoxycoumarin 0-deethylase (ECOD). Besides these two activities, we did not find any activity with other typical P450 substrates. In primary cultures of human hepatocytes, it is inducible by phenobarbital and dexamethasone, but not by pyrazole and pnaphthoflavone. We isolated this protein from human liver microsomes and purified it to homogeneity by a com bination of aminooctyl-amino-Sepharose chromatography and immunoaffinity chromatography. The protein was identified as a cytochrome P450 of the IIA subfamily. Its N-terminal amino-acid sequence was identical with the first 20 residues deduced from the nucleotide sequence of P450IIA6.

Chemically induced hepatocarcinogenesis in rodents leads to modified cytochrome P450 expression; in general the P450 content decreases, whereas some individual isoenzyme contents increase [I]. One of these forms is P450,, which we have isolated from dimethyldibenzo(c,g)carbazole-induced hepatomas in female mice of the strain XVII nc/Z [2, 31. This form, which is overexpressed fourfold in mouse tumors [4], appears to belong to the IIA family; it has the same N-terminal aminoacid sequence as P450IIA4 (P45015a) and the closely related P450IIA5 (P450coh), which are characterized by strong 15ahydroxytestosterone and coumarin-7-hydroxylase activity [5 - 81. Due to the particular role of P450,, as a tumoral marker in mice, it is important to determine whether this form has a counterpart in humans. Studies of human forms are however difficult because of a great interindividual variation. Furthermore, the microsomal activities such as steroid hydroxylases, which are often used to discriminate between P450 isoenzymes in rodents [ 3 , 91 are much less informative for human P450 forms which produce primarily the 6p-hydroxy steroid derivative [lo]. No 15a-hydroxytestosterone hydroxylation can be detected with human liver microsomal fractions whereas coumarin 7-hydroxylation is relatively high in human liver. Human coumarin hydroxylase was found to crossreact with antiCorrespondence to R. Lange, INSERM U 128, Route de Mende (CNRS) BP 5051, F-34033 Montpellier, France Ahhreviutions. COH, coumarin 7-hydroxylase; ECOD, 7-ethoxycoumarin 0-deethylase; P450,,, cytochrome P450 form overexpressed in mouse hepatomas.

P450IIA5 antiserum, which indicates that it is related to P450,,. Recently, a human nucleotide sequence (P4501IA6) and later on several other closely related cDNAs, were identified which express coumarin hydroxylase in transfected cells [ l l , 121. However, until now the protein responsible of the human coumarin hydroxylase has not been isolated. Here we show that antibodies raised against mouse tumor P450,, crossreact with a human P450 form which is characterized by coumarin hydroxylation at position 7. We describe the isolation of this isoenzyme by immunoaffinity chromatography and compare it to other known P450 forms in humans and baboons. Furthermore we investigate its expression in human primary hepatocyte cultures. EXPERIMENTAL PROCEDURES

Chemicals Goat-peroxidase-conjugated anti-rabbit IgG, 4-chloro-lnaphthol, tergitol NP-10,7-ethoxycoumarin, benzphetamine, aniline, ethoxyresorufin, lubrol PX, nonidet NP-40 and 1,8diaminooctane were purchased from Sigma (USA). Coumarin and 7-hydroxycoumarin (umbelliferone) were from BDH chemicals (UK). [4-' 4C]progesterone was purchased from CEA, Saclay (France), [4-'4C]testosterone was obtained from Amersham (UK). 6p- and 7a-dihydroxytestosterone and 68hydroxyprogesterone were obtained from Steraloids (USA), 16a-hydroxytesterone, 16a-hydroxyprogesterone,dexamethasone, pyrazole and p-naphthoflavone were from Sigma (USA), 1Sa-hydroxytesterone and I 5a-hydroxyprogesterone

512 were kindly provided by Professor D. N. Kirk, Queen Mary College, London. Erythromycin was from Roussel-Uclaf (France) and resorufin from Aldrich (FKG). Hydroxyapatite (Hypatite C) was from Clarkson Chemical Company Inc. (USA). Sepharose 4B was from Pharmacia (Sweden). All other chemicals were of highest quality.

demethylase activities were quantified by the Nash method [I 81, aniline-4-hydroxylase activity was measured according to Mieyal and Blumer [19] an ethoxyresorufin-O-deethylase activity as previously described [20]. Irnmunoinhibition experiments were performed with antibodies at final concentrations from 0.16 to 10 mg IgG/mg microsonial protein.

Biochemicals

Sephurose-4B derivatization

a-NADPH/cytochrome-c reductase, sheep anti-(rabbit P4501A2) (LM4), P4501IB1 (LM2), P4501IC3 (LM3b) and goat anti-(rabbit P450IIIA4) (LM3c) polyclonal antibodies were previously prepared in this laboratory [13, 141. Anti(mouse P450,J antibodies were obtained from rabbit antiserum by precipitation with 40% (niassivol.) ammonium sulfate.

Aminooctyl-amino - Sepharose 4B was prepared by coupling 1,s-diaminooctane to cyanogen-bromide-activated Sepharose 4B [21] and finally stored at +4"C in 0.1 M potassium phosphate buffer, pH 7.25, containing 1 mM EDTA, 20% (by vol.) glycerol and 0.02% (massivol.) sodium azide. A color test with picrylsulfonic acid was performed according to the procedure of Cuatrecasas [22] to follow the course of the agarose derivatization. The antibodies directed against P450,, were covalently coupled to Sepharose 4B as follows: Sepharose 4B (I0 ml gel) in water was activated at pH 10.5 with 1 g cyanogen bromide in 2 ml acetonitrile. After successive washing with water, acetone and 0.1 M sodium bicarbonate buffer, pH 9.5, the activated gel was resuspended in 8 ml 0.1 M sodium bicarbonate buffer, pH 8.3, containing 0.5 M NaCl and then mixed with anti-P450,, antibodies (50 mg, 2 ml) previously dialyzed against 0.1 M sodium bicarbonate buffer, pH 8.3, containing 0.25 M NaC1. The solution was gently stirred for 2 h at room temperature and overnight at +4'C. Sepharose-4B-anti-P450,, was then treated for 3 h at 4°C with 0.1 M buffered ethanolamine at pH 8 and washed successively with water, 0.1 M potassium phosphate, buffer pH 7.4 and 20 mM potassium phosphate, buffer pH 7.5, containing 1 mM EDTA and 0.15 M NaCl, before immediate using.

Cell prcprations Primary cultures of human hepatocytes were prepared as previously described [15]. Pyrazole, dexamethasone and pnaphthoflavone were used at the final concentration of 50 pM, and phenobarbital at 1 mM. The cells were exposed to inducers for 72 h. Microsomes from hepatocyte cultures were prepared as described elsewhere [16]. Human liver microsomes

Human liver samples were obtained post-mortem from organ donors and from patients with secondary liver tumors (lobectomy fragment). Their use was approved by the French National Ethics Committee. For the isolation of P450 we started with 300-g liver from a 60-year-old man who had died in a road traffic accident. The liver was sliced and homogenized by Vortex in 10 mM Tris/HCl, pH 7.4, containing 150 mM KCl and 1 mM EDTA. The homogenate was filtered through a sterile compress, further homogenized in a glass/teflon potter (0.15-mm clearance) and centrifuged for 25 min at 15 300 x g at + 2' C. The supernatant was filtered and centrifuged for 1 h a t 105000 x g at 2°C. The pellet was resuspended in 10 mM Tris/HCl, pH 7.4, containing 1 mM EDTA and 20% (by vol.) glycerol and centrifuged again for I h at 105000 x g at +2"C. The microsomes (3 g microsomal protein, 1.6 pmol P450) were recovered in the same buffer and stored at -80°C.

+

Enzymatic activities

COH and ECOD activities were determined by a direct fluorimetric method adapted from Creaven et al. [I71 on a spectrophosphorimeter Aminco-Keirs (USA). The microsonies were diluted to 0.5 mg proteinlml in 50 mM potassium phosphate buffer, pH 7.4 and were incubated at 3 7 T in a final volume of 0.5 in1 with 1 mM P-NADPH in the presence of 0.1 rnM coumarin or 0.2 mM 7-ethoxycoumarin. 7-Hydroxycournarin production was detected by the increase of the fluorescence intensity measured at 455 nm with excitation at 375 nm. For these two activities, a solution of 0.5 mM 7hydroxycoumarin in water was used as standard. [414C]progesterone and [4-'4C]testosterone metabolisms were detected according to a previously described method [2]. The TLC-separated metabolites were identified by comparison with the authentic standards 6p-, 7a-, 15a- and 16a-hydroxytestosterone and 6p-, 1 5 ~ -and 16a-hydroxyprogesterone. Erythromycin-demethylase and benzphetamine-N-

P450 isolation All steps were performed at +4"C under atmospheric pressure with Isco detectors set at 280 nm and 405 nm. Human liver microsomes (12.8 ml, 380 mg, total P450 210 nmol) were solubilized with 0.7% mass/vol.) sodium cholate and passed through a aminooctyl-amino - Sepharose-4B column in 0.1 M potassium phosphate buffer, pH 7.25, containing 1 mM EDTA, 20% (by vol.) glycerol, 0.42% (massivol.) sodium cholate. Elution was performed with 0.1 M potassium phosphate buffer, pH 7.25, containing 1 mM EDTA, 20% (by vol.) glycerol, 0.33% (mass/vol.) sodium cholate and 0.2% (by vol.) lubrol PX. The fraction containing cytochrome P450 (84 ml, 101 nrnol) was dialyzed against 20 mM potassium phosphate buffer, pH 7.5, containing 1 mM EDTA, 0.15 M NaCl and 0.33% (massivol.) sodium cholate (to decrease potassium phosphate concentration and remove glycerol) and mixed with 10 ml Sepharose-4B - anti-P450,,. The solution was gently stirred overnight at +4"C and centrifuged for 5 min at 3,000 x g. The supernatant was removed and the gel was poured into a glass column and washed successively with buffer A: 20 mM potassium phosphate, pH 7.5, containing 1 niM EDTA, 0.15 M NaCl and 0.5% (by vol.) tergitol NP-10; then with buffer B : 20 mM potassium phosphate, pH 7.5, containing 1 mM EDTA, 2 M EDTA, 2 M NaCl, 1 YO(by vol.) tergitol NP-10 and 0.5% (mass/vol.) sodium cholate to eliminate nonspecific interactions and again with buffer A. Elution was with 0.1 M glycerol/HCl, pH 3.0, containing 0.5% (by vol.) tergitol NP-10. The collected fractions (2 ml) were immediately neutralized with 0.2 ml K2HP04 and characterized with antiP450,, antiserum by Western blot analysis. The fractions containing the 49-kDa protein were pooled and the resulting

513 1

2 3 4

P450 5 6 tu 7 8

0.20

A

0

9 1 0 1 1 12 0.15 k Da 52

0.10

0.05

0.00 0.0

Fig. 1. Anti-PbSO,, crossreactirzgproteins in human liver samples. Western blot carried out with 25 pg microsomal protein and 2 pmol mouse P450,, (50 kDa) as standard. Lanes 1 - 12 represent various human liver samples

3.0

4. 0

coumarin 7-hydroxylation

.-Elw

N-terminal amino-acid sequence The N-terminal amino-acid sequence was determined with a model 470A Applied Biosystems (Foster City, CA) gasphase sequencer coupled to a model 120A phenylthiohydantoin analyzer as previously described [23]. A volume of 10 ml of the 49-kDa-protein-containing fraction was concentrated to 0.75 ml under vacuum and loaded (3 x 0.25 ml) on 8 % polyacrylamide electrophoresis gel with stained protein molecular mass standards (14.3-200 kDa, Bethesda Research Laboratories, USA). The migration was carried out at 60 mA and the transfer on Immobilon-P membrane (Millipore, USA) at 350 mA for 2 h. The proteins were colored on the membrane with Ponceau red and the band corresponding to the 49-kDa protein was cut out for direct sequencing. The N-terminal amino-acid sequence of the 49-kDa protein was determined on approximatively 20 pmol immobilized protein.

20,

RESULTS Identijkation of two anti-P450,, crossreacting proteins in human liver samples Antibodies directed against P450,, from mouse hepatoma crossreact with two human liver microsomal proteins. From a comparison with molecular mass standards and P450,, these crossreacting proteins have molecular masses of 49 kDa and 52 kDa (Fig. 1). We have compared liver microsomes from 50

2.0

(nmol productlminlmg microsomal protein)

fraction (13.5 ml) dialyzed against 20 mM potassium phosphate buffer, pH 7.5, containing EDTA 1 mM and stored at -8OOC.

Other methods Microsomal proteins were determined with colorimetric bicinchoninic acid protein-assay reagent from Pierce (USA) and the P450 content was determined with a Cary 219 visible/ ultraviolet spectrophotometer using a value of E ~ = 91 ~ mM-’ . cm-’ [24]. Electrophoresis was carried out on a slab gel in 8% polyacrylamide in the presence of 0.1 % SDS. Western blots were carried out with anti-P450,, antiserum (1 mg IgG in 25 ml 3% (massjvol.) bovine serum albumin solution containing 10% (mass/vol.) fetal calf serum) and goatperoxydase-conjugated anti-rabbi t IgG with 4-chloro-I -naphthol and HzOz as substrates.

1.0

0.06

CI

0

I

B

L z 0 2

c-4

; 0.04

s;

0

.C

t e $

2

t;

-;E“ .

k E

3

0

W

d

E

-

0.02

D”

- & “anm

8

.-

h I

c

m

0.00 0.0

0

o”

9

I

I

I

1.0

2.0

3.0

4.0

coumar in 7- hydroxylation (nmol productlminlmg microsomal protein)

Fig. 2. COH activity related to the 49-kDa ( A ) or 52-kDa ( B ) protein levelin donor liver samples. Coumarin 7-hydroxylation was determined in niicrosomes from 50 donor liver samples by a fluorescence method; microsomal anti-P450,, crossreacting 49-kDa and 52-kDa proteins were immunoquantified o n a Western blot relative to mouse P450,,

donors and - 10 patients operated ~ ~ ~ ~ for secondary liver tumors and immunoquantified the crossreacting proteins relative to P450,, on Western blots with a Shimadzu Scanner CS930. The 49-kDa protein is characterized by a very high interindividual variability (144 X); in donors its level varies from 1.3 to 186.5 nmol/g microsomal protein (mean value = 45.3 38.3 SD). In extratumoral liver parenchyme its level varies between 3.3 and 79 nmol/g (mean value 36.0 & 24.0 SD), which is not significantly different from that observed in donor livers. The 52-kDa band shows a less pronounced variability (26 X) with a mean value of 19.0 A 12.1 SD nmol/g microsomal protein. Again, no significantly different values are obtained with extra tumoral parenchyme. Characterization of human liver samples f o r COH activity The 60 human liver microsomal samples were assayed individually for their P450-dependent COH activity. We observed again a strong interindividual variation (Fig. 2). The

514

60

50 40

30 20

10

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0

4.0

(pmol product/min/mg microsomal protein)

(nmol productlminlmg microsomal protein)

enzyme activities are comprised (except of one sample) between 0 and 2.6 nmol . min-' . mg microsomal protein-' (mean value = 0.88 0.61 SD). The distribution of this activity within the 60 samples tested appears to be homogeneous; we could not observe any distinct subpopulation (Fig. 3). As shown in Fig. 2A, coumarin 7-hydroxylation is well correlated with the immunoquantified concentration of the 49-kDa protein ( R = 0.78). Within experimental errors, the linear regression fit goes through zero, indicating that virtually all COH activity is due to the 49-kDa protein. No correlation was found however with the 52-kDa band (Fig. 2B). Inducibility of'the 49-kDa protein in hepntocyte cultures The 49-kDa protein which crossreacts with P450,, is expressed also in human hepatocytes in primary culture where it is induced by phenobarbital and dexamethasone two-tothreefold (Fig. 4). We did not observe however an induction by pyrazole, which has been reported to induce the coumarin hydroxylase (P450IIA5) in mice [25 - 271. Furthermore, flnaphthoflavone, a typical inducer of P450IA, did not induce human coumarin hydroxylase. The microsomal COH activity was again well correlated with the 49-kDa protein (R = 0.97) indicating that the induced cournarin hydroxylase is not different from the constitutive human P450t, analogue.

300

coumarin 7-hydroxylation

coumarin 7-hydroxylation

Fig. 3. Frequency distribution of coumarin 7-hydroxylase among 60 individual human liver samples

200

100

Fig. 4. Expression of human coumarin hydroxylase in primary human hepatotyte cultures. Correlation between COH activity and the 49kDa P450,, analogue in untreated (UT), pyrazole (PYR), phenobarbital (PB), P-naphthoflavone (BNF) and dexamethasone (DEX) treated cells

100

80

60 40

20 mg IgGlmg microsornal protein

0 0

I

I

2

4

.

I

I

6

8

10

antibodies (mg IgGlmg microsornal protein)

Inhibition of COH activity by anti-P450,, antibodies

Fig. 5. Immunoinhibition of COH activity in human microsomes. Anti(mouse P450,J antibodies (El) and anti-rabbit LM2 ( O ) ,LM3b (0), LM3c (m)and LM4 antibodies (+)were used a t a final concentration of 0.1 6 to 10 mg IgG/mg microsomal protein

Human liver microsomal COH activity is strongly inhibited by antibodies directed against P450,, (Fig. 5). Under our assay conditions, 50% inhibition is observed at ICs0 = 0.13 mg IgG/mg microsomal protein. However, antibodies directed against other P450 forms (rabbit liver LM2, LM3b, LM3c and LM4) do not inhibit this activity significantly. Although these antibodies were derived from another animal species, they are nevertheless inhibitory for other human liver microsomal activities. Antibodies against P450,, do not inhibit significantly the frequently investigated P450-dependent

activities erythromycin demethylase, benzphetamine N-demethylase, aniline 4-hydroxylase and ethoxyresorufin O-deethylase (Table 1). Similarly the progesterone and testosterone metabolite profiles, as measured by TLC separation and scintillation counting, are not modified by the presence of antiP450,, antibodies even at higher concentration (10 mg IgGJ mg microsomal protein). Apart from COH activity, the only other activity that we found to be significantly inhibited by anti-P450,, antibodies at high concentration was 7-ethoxy-

51 5 Table 1. Inhibition of some P450dependent activities in humun liver microsornes with unti-P-450,, and anti-rabbit L M 2 , LM3h. L M 3 c und L M 4 antibodies The different antibodies were used at a final concentration of 10 mg IgG/mg rnicrosomal protein. The experiments were performed in duplicate Inhibitor

Activity

Antibody anti-P450,,

Erythromycin demethylase Benzphetamine N-demethylase Aniline 4-hydroxylase Ethoxyresorufin U-deethylase COH ECOD

nmoI . min- . mg-

% inhibition

1.62 1. O l 0.71 0.013 1.ox 0.52

14 30 10 0 100 85

anti-LM2

anti-LM3b

anti-LM3c

anti-LM4

53 84

30 83

43 80

15 44

coumarin 0-deethylase. This activity was affected also by antiLM2, LM3b and LM3c antibodies (in decreasing order) in a similar extent as by anti-P450,, antibodies. Under the same conditions, the coumarin hydroxylase is however, much less inhibited by these antibodies. This indicates that ECOD activity is catalyzed, at least partly, by another P450 form than by the human P4501, analogue, which in turn appears to be the only form which catalyzes coumarin 7-hydroxylation and for which we have not found yet another substrate.

B

A 1

2

3

1

2

3

Purification of the 49-kDa protein

First attempts to isolate the 49-kDa protein by HPLC, using, as in the P45O1, purification, various ion-exchange chromatographic supports, were unsuccessful. Although the final fraction retained the COH activity and did not show any testosterone metabolism (as expected from the immunoinhibition experiments), it was impossible to raise the specific 49kDa content to more than 50% of total P450. The purity of the fraction was improved by the use of aminooctyl-amino Sepharose chromatography and elution with lubrol detergent. This step however, destroyed the enzyme activity. We therefore adopted an isolation procedure based on immunoaffinity chromatography. The anti- P450,, antibodies were covalently coupled to cyanogen-bromide-activated Sepharose 4B and the resulting gel SepharosedB - anti-P450,, was incubated with the aminooctyl-amino - Sepharose 4B prepurified fraction containing sodium-cholate solubilized cytochromes P450. Elution of the Sepharose-4B - anti-P450,, was performed in a glass column at pH 3 with glycine/HCl. A single elution peak with an increase in absorbance at 280 nm and 405 nm was obtained. The corresponding fraction showed a prominent band with a molecular mass of 49-kDa on Coomassie-blue-stained electrophoresis gel. A single 49-kDa protein band was recognized by Western blot with the anti-P450,, antibodies (Fig. 6). At each step of the purification the 49-kDa protein was immunoquantified relative to P450,, on Western blot. The initial microsomal49-kDa protein concentration was 12% of the total P450 content; its final purification yield was 1.8% (455 pmol, Table 2).

Fig. 6. Finalpurificution o f t h e fraction containing the 49-kDa protein. Electrophoresis (A) and Western blot (B) were carried out on a slab gel in 8% polyacrylamide, 0.1 % SDS with microsomal liver protein, (lane 1, 40 pg protein), mouse P450,, (lane 2, 2 pmol) and the final fraction (lane 3, 0.1 ml), obtained after elution of Sepharose-4Banti-P450,,

Table 2. Yield for 49-kDa protein at the dcferent steps of the purification The 49-kDa protein was immunoquantified relative to P450,, on a Western blot; total P450 in the final fraction was undetectable because of denaturation during the elution step Purification step

Total P450

49-kDa Protein

Yo

nmol Microsomal extract Solubilized P450 Aminooctyl-amineSepharose-4B fraction Final fraction

21 0 179 101 -

Yield

25.2 21.5 9.5 0.455

85.3 37.7 1.8

phoresis on 8% polyacrylamide gel, cut out and sequenced on the membrane. 18 of the first 20 N-terminal amino-acid residues were determined. These amino-acid residues are identical to those of human cytochrome P450IIA6 (the CYP2A6 gene product), the primary sequence of which had been deduced from cDNA by Yamano et al. [27] (Fig. 7). The Determination of the N-terminal sequence amino-acid residues on positions 14 and 16 were not detected The isolated 49-kDa protein was then characterized by and not firmly identified. This is in agreement with the nature its N-terminal amino-acid sequence (first 20 residues) ; the of these two amino-acid residues in cytochrome P450IIA6; a denatured protein (0.3 nmol) was concentrated under Y ~ C - cysteine residue in position 14 which might have been chemiuum, transferred onto an Immobilon membrane after electro- cally derived before sequencing and a threonine residue in

516 5

1

isolated 49 kDa protein

15

10

20

ref

ML A S G ML L V A L L V X L X V M V L

baboon FI

L

A

(29)

mouse P450IIA4

T

L

A V A F

S

L

(577)

P45011A5

T

L

A V A F

S

L

(6,271

P450t,

T

L

A V A F

S

L

(2)

D T

L

rat P450IIA2 rat P450IIA3

L V

V A F

1

5

V I S V

10

A S

S

(30)

F

V L M S V W K Q R

15

(31)

20

Fig. I . N-terminal amino-acid sequence (first 20 amino acids) o j the isolated 49-kDa protein. Comparison with the cDNA-deduced N-terminal amino-acid sequence of human P450IIA6 and the sequences of the isolated proteins: baboon FI ; mouse P450IIA4, P450IIA5 and P450,,; rat P450IIA2 and P450IIA3. Only different amino acids are indicated

position 16 which gives a labile phenylthiohydantoin derivative during sequencing because of its hydroxyl group. The isolated human 49-kDa protein was consequently identified as a cytochrome P450 of the IIA family either identical to cytochrome P450IIA6 or closely related to it. The sequence of 20 amino acids at the N-terminus of human cytochrome P450IIA6 has 90% similarity with the P450IIA form, F1, recently isolated from baboon liver [29] which also displays COH activity (unpublished results); 60% similarity with P4501IA4 (P45015cr), P450IIA5 (P450coh) isolated from mouse liver, and P450,, isolated from mouse hepatomas, 55% similarity with P450IIA2 (15a-hydroxytestosterone hydroxylase) isolated from rat liver [30] and 20% similarity with P4501IA3 (P450a3) isolated from rat lung [31]. DISCUSSION In a previous study we have shown that mouse hepatic tumors are characterized by the overexpression of a particular P450 form, named P450,,. Our present study shows that antibodies against this new potential tumoral marker in mice crossreact with a 49-kDa protein present in microsomes from healthy human liver. These properties, as well as its activity, inducibility and N-terminal sequence which are discussed below, make it clear that this protein is a cytochrome P450. Its is characterized by one activity; the coumarin 7-hydroxylation. This activity is inhibited very strongly (ICso = 0.13 mg IgG/ mg microsomal protein) by anti-P450,, antibodies. We assayed without success a range of seven other typical monooxygenase substrates. The only other activity which could be inhibited significantly (ICs0 = 0.91 mg IgG/mg microsomal protein) was 7-ethoxycoumarin 0-deethylase, which appears to be carried out also by other P450 forms. The human liver coumarin hydroxylation on the contrary, appears to be catalyzed exclusively by the enzyme crossreacting with P450,". A correlation between a 49-kDa protein and human microsomal coumarin hydroxylase had been reported pre-

viously by Raunio et al. [32] and Miles et al. [12] who showed a crossreaction of anti-[mouse P450IIA5 (P450coh)l and anti(rat P450IIA1) antisera with human coumarin hydroxylase. Later on, on the basis of its cDNA nucleotide sequence, this form was named P450IIA6. The P450,, which served as a matrix for our antibodies probably belongs to the same subfamily as P450IIA4 and IIA5; all three forms have the same N-terminal amino-acid sequence. In view of these arguments, it appears likely that the P450,, crossreacting human coumarin 7-hydroxylase is identical to P450IIA6. This is further corroborated by its interindividual variability; Miles et al. [12] reported a 70-fold variability for human COH. We have confirmed these results by an investigation of 60 human liver samples and found a 144-fold interindividual variability of the crossreacting protein. In order to check further the identity of this P450 form, we decided to isolate it. For this purpose, we had to use a combination of aminooctyl-amino - Sepharose chromatography and immunoadsorption chromatography. It was not possible to obtain the enzyme in its active form, because, although its initial quantity was appreciable (12% of the total microsomal P450 content), this P450 form proved to be both very labile and difficult to separate from other copurified proteins. Its N-terminal amino-acid sequence proved to be identical to that determined by the nucleotide sequence of P450IIA6, confirming that the isolated P450 is the protein expression product of CYP2A6. It is the first time that this relatively abundant human liver microsomal protein has been isolated. The assignment of the isolated protein as the CYP2A6 gene product still needs however, some further analysis. It appears now that CYP2A6 encompasses several subforms; first, several cDNAs varying only a little (3 to 12 base pairs) from P4501IA6 have been isolated [33- 351; secondly, Miles et al. [12] showed by Northern blot analysis a hybridization of three different P4501IA mRNAs (1.9,2.7 and 3.8 kb). Each mRNA showed a wide interindividual variation. So the ques-

517 tion arises, whether we have isolated, and in more general terms whether human coumarin hydroxylase is, a single gene product, or whether we are in the presence of a mixture of very closely related proteins. Its pronounced substrate specificity, its relative abundance in man and the immunological relation with an enzyme overexpressed in mouse tumors, all suggest a peculiar physiological role for the isolated P450. Its inducibility by the steroid dexamethasone may reflect a possible endogenous regulation. It would be interesting of course, to determine whether this isoenzyme is overexpressed in human hepatomas. Preliminary experiments are however not conclusive, because of the advanced stage of available human tumors. However, the inducibility of this form by phenobarbital indicates that this enzyme is implicated in detoxication processes. Detoxication of coumarin may be important for cattle (it is present in sweet clover), but not for man, apart of its recent use as odour enhancer. We speculate therefore that coumarin is not the physiological substrate of human COH activity. It would be worthwhile to look for the true substrate of this monooxygenase by a search for P450IIA6 in certain human diseases. A point of departure may be that coumarin derivatives are used as vitamin K antagonists for blood coagulation, which in turn appears to be involved in metastases [36, 371. Furthermore, other coumarin derivatives such as aflatoxin (difuranocoumarin metabolites of Aspergillusfluvus) are suspected carcinogens [38]. This compound has recently be found to be metabolized by P45011A6 expressed in a human cell line [34]. Thus, in view of culminating arguments for a biological importance of this P450 form, one should look more systematically for other possible (perhaps endogenous) substrates. Probably the use of primary hepatocyte cultures will be helpful. The authors gratefully acknowledge Dr D. Diaz (CHR Montpellier) for providing the human liver samples and Dr P. Maurel (INSERM U 128) for his advice in human hepatocyte cultures. This study was supported in part by the Foundationpour la Formation par la Recherche b I’htrrface Chimie-Biologie FZRCB (Fondation de France, Paris).

REFERENCES 1. Buchmann, A,, Kuhlmann, W., Schwarz, M., Kanz, W., Wolf, C. R., Moll, E., Friedherg, T. & Oesch, F. (1985) Carcinogenesis (Lond.) 6, 513-521. 2. Lange, R., Pkrin, F., Larroque, C. & Zajdela, F. (1990) Biochim. Bioph-vs. Acta 1038, 130-135. 3. Lange, R., Ducloux, N., Ptrin, F., Larroque, C., Van Lier, J. E. & Zajdela, F. (1989) Carcinogenesis (Lond.) 6, 1871 -1877. 4. Maurice, M., Lange, R . & Pkrin, F. (1990) in Drug-metabolizing enzymes: genetics, regulation and toxicology (IngelmanSundberg, M., Gustafsson, J.-A. & Orrenius, S., eds) pp. 208, Karolinska lnstitutet, Stockholm. 5. Harada, N.&Negishi,M. (1984)J. Bid. Chem. 259,1265-1271. 6. Negishi, M., Lindberg, R., Burkhart, B., Ichikawa, T., Honkakovski, P. & Lang, M. (1989) Biochemistry 28, 41694172. 7. Lindberg, R., Burkhart, B. A,, Ichikawa, T. &Negishi, M. (1989) J . Biol. Chem. 264, 6465 - 6471. 8. Nebert, D. W., Nelson, D. R., Coon, M. J., Estabrook, R. W., Feyereisen, R., Fujii-Kuriyama, Y., Gonzales, F., Guengerich, F. P., Gunsalus, I. C., Johnson, E. F., Loper, J . C., Santos, R., Waterman, M. R. & Waxman, D. J. (1991) DNA Cell Biol. 10, 1-14.

9. Waxman, D. J. (1988) Biochem. Pharmacol. 37, 71 - 84. 10. Waxman, D. J., Attisano, C., Guengerich, F. P. & Lapenson, D. P. (1988) Arch. Biochem. Biophys. 263,424-436. 11. Yamano, S., Tatsuno, J. & Gonzales, F. J. (1990) Biochemistry 29,1322 - 1329. 12. Miles, J. S., Mc Laren, A. W., Forrester, L. M., Glancey, M. J., Lang, M. A. &Wolf, C. R. (1990) Biochem. J . 267, 365-371. 13. Bonfils, C., Dalet-Beluche, I . & Maurel, P. (1982) Biochem. Biophys. Res. Commun. 104, 1011 - 1017. 14. Bonfils, C., Comhalbert, J., Pineau, T., Angevin, J., Larroque, C., Derancourt, J., Capony, J.-P. & Maurel, P. (1990) Eur. J . Biochem. 188, 187 - 194. 15. Pichard, L., Fabre, I., Domergue, J., Saint Aubert, B., Mourad, G. & Maurel, P. (1990) Drug Metab. Dispos. 18, 595 -606. 16. Daujat, M., Pichard, L., Dalet, C., Larroque, C., Bonfils, C., Pompon, D., Li, D., Guzelian, P. S. & Maurel, P. (1987) Biochem. Pharmacnl. 36, 3597 - 3606. 17. Creaven, P. J., Parke, D. V. & Williams, R. T. (1965) Biochem. J . 96,390 - 398. 18. Nash, T. (1953) Biochem. J . 55,416-421. 19. Mieyal, J. J. & Blumer, J. L. (1976) J . Biol. Chem. 251, 34423446. 20. Burke, M. D., Prough. R. A. & Mayer, R. T. (1975) Drug Metub. Dispos. 3, 245-253. 21. March, S. C., Parikh, I. & Cuatrecasas, P. (1974) Anal. Biochem. 60, 149-152. 22. Cuatrecasas, P. (1970) J . Bid. Chern. 245,3059-3065. 23. Hewick, R. M., Hunkapiller, M. W., Hood, L. E. & Dreyer, W. J. (1981) J . Biol. Chem. 256, 7990-7997. 24. Omura, R. & Sato, R. (1964) J . Bid. Chem. 239, 2370-2378. 25. Juvonen, R. O., Kaipainen, P. K. & Lang, M. A. (1985) Eur. J . Biochem. 152, 3 - 8. 26. Raunio, H., Kojo, A., Juvonen, R., Honkakovski, P., Jarvinen, P., Lang, M. A., Vahakangas, K., Gelboin, H. V., Park, S. & Pelkonen, 0. (1988) Biochem. Pharmacol. 37,4141 -4147. 27. Juvonen, R. O., Shkumatov, V. M. & Lang, M. A. (1988) Eur. J . Biochem. 171,205-211. 28. Yamano, S., Nagata, K., Yamazoe, Y., Kato, R., Gelboin, H. V. &Gonzales, J. (2989) Nucleic Acids Rex 17, 4888. 29. Dalet-Beluche, I., Boulenc, X., Combalbert, J., Maurel, P. & Bonfils, C. (1990) in Drug metabolizing enzymes: genetics, regulation arzd toxicology (Ingelman-Sundherg, M., Gustafsson, J.A. & Orrenius, S., eds) pp. 221, Karolinska Institutet, Stockholm. 30. Matsunaga, T., Nagata, K., Holsztynska, E. J., Lapenson, D. P., Smith, A., Kato, R., Gelboin, H. V., Waxman, D. J. & Gonzales, F. J. (1988) J . Biol. Chem. 263, 17995-18002. 31. Kimura, S., Kozak, C. A. & Gonzales, F. J. (1989) Biochemistry 28, 3798 - 3803. 32. Raunio, H., Syngelma, T., Pasanen, M., Juvonen, R., Honkakovski, P., Kairaluoma, M. A., Sotaniemi, E., Lang, M. A. & Pelkonen, 0.(1988) Biochem. Pharmacol. 37,3889 -389s. 33. Miles, J. S., Bickmore, W., Brook, J. D., Mc Laren, A. W., Meehan, R. &Wolf, C. R. (1989) Nucleic Acids Res. 17,2907 2911. 34. Crespi, C. L., Penman, B. W., Leakey, J. A. E., Arlotto, M. P., Stark, A., Turner, T., Steimel, D. T., Rudo, K. Davies, R. L. & Langenbach, R. (1990) Carcinogenesis (Lond.) 11,1293-1300. 35. Phillips, I. R., Shepard, E. A., Ashworth, A. & Rabin, B. R. (1 985) Proc. Nail Acad. Sci. U S A 82,983 -987. 36. Zacharski, L. R. (1982) in Interaction ofplatelets and tumor cells (Liss, A. R., ed.) pp. 113 - 129, New York, USA. 37. Liebman, H. A,, Furie, B. C., Tong, M. J., Blanchard, R. A., Lo, K. J., Lee, S. D., Coleman, M. S. & Furie, B. (1984) N. Engl. J . Med. 310,1427 - 1431. 38. Campbell, T., Chen, J., Liu, J. & Parpia, B. (1990) Cancer Res. 50,6882-6893.