Substrate Specificity for Rat Cytochrome P450 (CYP) Isoforms: Screening

Biochemical Pharmacology 63 2002) 889±896

Substrate speci®city for rat cytochrome P450 CYP) isoforms: screening with cDNA-expressed systems of the rat

Kaoru Kobayashia,*, Kikuko Urashimaa, Noriaki Shimadab, Kan Chibaa

aLaboratory of Biochemical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Chiba University, Chiba, Japan bDaiichi Pure Chemicals Co. Ltd., Tokyo, Japan Received 26 March 2001; accepted 8 October 2001

Abstract

In this study, we performed a screening of the speci®cities of rat cytochrome P450 CYP) isoforms for metabolic reactions known as the speci®c probes of human CYP isoforms, using 13 rat CYP isoforms expressed in baculovirus-infected insect cells or B-lymphoblastoid cells. Among the metabolic reactions studied, diclofenac 4-hydroxylation DFH), dextromethorphan O-demethylation DMOD) and midazolam 4-hydroxylation were speci®cally catalyzed by CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. These results suggest that diclofenac 4-hydroxylation, dextromethorphan O-demethylation and midazolam 4-hydroxylation are useful as catalytic markers of CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. On the other hand, phenacetin O-deethylation and 7-ethoxyresoru®n O-deethylation were catalyzed both by CYP1A2 and by CYP2C6. Benzyloxyresoru®n O-dealkylation and pentoxyresoru®n O-dealkylation were also catalyzed by CYP1A2 in addition to CYP2B1. Bufuralol 10-hydroxylation was extensively catalyzed by CYP2D2 but also by CYP2C6 and CYP2C11. p-Nitrophenol 2-hydroxylation and chlorzoxazone 6-hydroxylation were extensively catalyzed by CYP2E1 but also by CYP1A2 and CYP3A1. Therefore, it is necessary to conduct further study to clarify whether these activities in rat liver microsomes are useful as probes of rat CYP isoforms. In contrast, coumarin 7-hydroxylation and S-andR-mephenytoin 40-hydroxylation did not show selectivity toward any isoforms of rat CYP studied. Therefore, activities of coumarin 7-hydroxylation and S-andR-mephenytoin 40-hydroxylation are not able to be used as catalytic probes of CYP isoforms in rat liver microsomes. These results may provide useful information regarding catalytic probes of rat CYPs for studies using rat liver microsomal samples. # 2002 Elsevier Science Inc. All rights reserved.

Keywords: CYP; Rat; Substrate speci®city; cDNA-expression system; Probe; Drug metabolism

1. Introduction Although CYP enzymes make xenobiotics less toxic, the reactions frequently involve the formation of reactive CYP enzymes comprise a large family of hemoprotein intermediates or allow the leakage of free radicals capable [1], and enzymes from three families CYP1, CYP2, and of causing toxicity [3,4]. Therefore, differences in the CYP3) are mainly involved in the biotransformation of levels of individual CYP isoforms and indeed the expres- xenobiotics in both humans and rats [2]. However, con- sion of distinct isoforms signi®cantly contribute to inter- siderable differences exist in the expression and catalytic species differences in the toxicity of chemicals and the activities between human and rat CYP orthologues [2]. susceptibility to chemically induced cancer [2]. Never- theless, drug safety assessment studies, including pharma- cological and toxicological studies, have been performed * Corresponding author. Tel.: 81-43-290-2920; fax: 81-43-290-2920. using rodents although they are not necessarily excellent E-mail address: [email protected] K. Kobayashi). surrogate models for man. To assess the effects and toxi- Abbreviations: CYP, cytochrome P450; POD, phenacetin O-deethylation; EROD, 7-ethoxyresorufin O-deethylation; BROD, 7-benzyloxyresor- cities of xenobiotics (i.e. developmental drugs and envir- ufin O-dealkylation; PROD, 7-pentoxyresorufin O-dealkylation; CRH, onmental chemicals), it is necessary to identify CYP coumarin 7-hydroxylation; DFH, diclofenac 4-hydroxylation; SMH, isoforms responsible for the metabolism of drugs in each S-mephenytoin 40-hydroxylation; RMH, R-mephenytoin 40-hydroxylation; species. 0 DMOD, dextromethorphan O-demethylation; BLH, bufuralol 1 -hydro- Currently, many different strategies are employed for the xylation; PNPH, p-nitrophenol 2-hydroxylation; CZH, chlorzoxazone 6-hydroxylation; MD1H, midazolam 10-hydroxylation; MD4H, midazolam unambiguous identi®cation of CYP isoforms responsible 4-hydroxylation. for the biotransformation of therapeutic agents in humans.

These include the use of CYP isoform-selective inhibitors, b5. Microsomes prepared from human B-lymphoblastoid immunoinhibitory antibodies, studies with puri®ed and cells expressing CYP2A1 lot 7) and CYP2B1 lot 6) were heterogeneously expressed CYP protein, and correlation obtained from Gentest. Recombinant CYP2A1 and CYP2E1 analyses of metabolic rates of drugs with immunoquanti- were coexpressed with NADPH-CYP oxidoreductase. Con- ®ed CYP levels or metabolic rates of speci®c substrates for trol microsomes were from insect cells infected with wild- each isoform. The use of cDNA-expressed human CYPs type baculovirus and from human B-lymphoblastoid cells has greatly facilitated the evaluation of the metabolic containing the expression vector without cDNA. The ratios speci®city of probe substrates and the identi®cation of of cytochrome c reductase activity nmol/min/mg) to CYP individual CYP isoforms involved in metabolism of drugs. content pmol CYP/mg) in each recombinant CYP isoform On the other hand, criteria and approaches for unambig- used in this study were 6 for CYP1A2, 4 for CYP2A1, uous identi®cation of individual CYP isoforms responsible 42 for CYP2A2, 7 for CYP2B1, 10 for CYP2C6, 6 for for the metabolism of drugs in rats have not been estab- CYP2C11, 27 for CYP2C12, 2 for CYP2C13, 13 for lished. This is because the speci®cities of metabolic reac- CYP2D1, 22 for CYP2D2, 10 for CYP2E1, 22 for CYP3A1 tions used as probes of rat CYP isoforms have not been and 17 for CYP3A2, respectively. thoroughly evaluated. In the present study, therefore, the metabolism of model 2.3. Incubation conditions substrates for human CYP-isoforms was examined using 13 rat CYP isoforms expressed in baculovirus-infected Activities of phenacetin O-deethylation POD), 7-ethox- insect cells or human B-lymphoblastoid cells. yresoru®n O-deethylation EROD), coumarin 7-hydroxy- lationCRH),7-benzyloxyresoru®nO-dealkylationBROD), 7-pentoxyresoru®n O-dealkylation PROD), DFH, R-and 2. Materials and methods S-mephenytoin 40-hydroxylation RMH and SMH), DMOD, bufuralol 10-hydroxylation BLH), p-nitrophenol 2-hydroxy- 2.1. Chemicals lation PNPH), chlorzoxazone 6-hydroxylation CZH), midazolam 10- and 4-hydroxylation MD1H and MD4H), and Bufuralol hydrochloride, 10-hydroxybufuralol and testosterone 6b-, 7a-and16a-hydroxylation were deter- 4-hydroxydiclofenac were purchased from Gentest. Mid- mined. Atypical incubation mixture 0.25 mL total volume) azolam, 10- and 4-hydroxymidazolam were gifts from contained 0.1 mM EDTA, 100 mM potassium phosphate F. Hoffmann-La Roche. Zaltoprofen was a gift from Zeria buffer pH 7.4), an NADPH-generating system 0.5 mM Pharmaceutical. Dextrorphan, R- and S-mephenytoin, NADP, 2 mM glucose-6-phosphate, 1 IU/mL of glucose-

4-hydroxymephenytoin, 6-hydroxychlorzoxazone, 6b-, 6-phosphate dehydrogenase, and 4 mM MgCl2), cDNA- 7a- and 16a-hydroxytestosterone were purchased from expressed rat CYPs and a substrate. The reaction was Ultra®ne Chemicals. 7-Benzyloxyresoru®n, chlorzoxa- initiated by the addition of the NADPH-generating system zone, dextromethorphan hydrobromide, 7-ethoxyresoru®n, following a 1-min pre-incubation at 378. All reactions were 7-pentoxyresoru®n and resoru®n were purchased from performed in the linear range with respect to CYP concen- Sigma. Cyclobarbital was purchased from Tokyo Kasei tration and incubation time. After the reaction was stopped Kogyo. NADP, glucose-6-phosphate and glucose-6-phos- by the addition of 100 mL of ice-cold acetonitrile, an phate dehydrogenase were purchased from Oriental Yeast. internal standard was added. The mixtures were centrifuged p-Nitrophenol was purchased from Nacalai Tesque Inc. at 13,000 g for 10 min, and the supernatants 100 mL) were Acetaminophen, caffeine, coumarin, diclofenac, 7-hydroxy- analyzed by HPLC as described below. The substrate coumarin, 7-hydroxy-4-methylcoumarin, p-nitrocatechol, concentration, incubation time, content of cDNA-expressed phenacetin, testosterone and HPLC-grade acetonitrile CYP and amount of internal standard used for each assay and methanol were purchased from Wako Pure Chemical are listed in Table 1. 7-Ethoxyresoru®n, 7-benzyloxyresor- Industries. u®n and 7-pentoxyresoru®n were dissolved in dimethylsulfoxide, and this mixture was added to the incubation mixture 2.2. Microsomes at a ®nal dimethylsulfoxide concentration of 1%. Testos- terone was dissolved in methanol and added to the incuba- Microsomes prepared from baculovirus-infected insect tion mixture at a ®nal methanol concentration of 1%. cells expressing CYP1A2 lot 1), CYP2A2 lot 1), CYP2B1 Samples for POD activity were evaporated by a vacuum lot 2), CYP2C6 lot 1), CYP2C11 lot 1), CYP2C12 lot 1), evaporator for 15 min after the centrifugation, and the CYP2C13 lot 1), CYP2D1 lot 2), CYP2D2 lot 1), remaining samples 100 mL) were analyzed. CYP3A1 lot 1) and CYP3A2 lot 1) were obtained from Gentest. All recombinant CYPs were coexpressed with 2.4. HPLC analysis NADPH-CYP oxidoreductase. Recombinant CYP2A2, CYP2B1, CYP2C6, CYP2C11, CYP2C12, CYP2C13, The determination of respective metabolites was carried CYP3A1 and CYP3A2 were coexpressed with cytochrome out using a Hitachi HPLC system consisting of an L-7100 K. Kobayashi et al. / Biochemical Pharmacology 63 (2002) 889±896 891

Table 1 Substrate concentrations, incubation times, contents of CYP, and the internal standards used in the present study

Reaction Substrate Incubation time min) CYP pmol) Internal standards

POD Phenacetin 10,500 mM) 15 5 Caffeine 0.25 mg) EROD 7-Ethoxyresorufin 5 mM) 10 5 None CRH Coumarin 10 mM) 60 10 7-Hydroxy-4-methylcoumarin 25 pmol) BROD 7-Benzyloxyresorufin 5 mM) 5 5 None PROD 7-Pentoxyresorufin 5 mM) 10 5 None DFH Diclofenac 25 mM) 20 5 Zaltoprofene 0.1 ng) SMH S-Mephenytoin 100 mM) 120 5 Cyclobarbital 62.5 ng) RMH R-Mephenytoin 200 mM) 30 10 Cyclobarbital 62.5 ng) DMOD Dextromethorphan 10 mM) 10 5 None BLH Bufuralol 10 mM) 15 0.5 None PNPH p-Nitrophenol 200 mM) 60 5 Phenacetin 0.125 mg) CZH Chlorzoxazone 20 mM) 20 5 Bupranolol 2 mg) MD1H Midazolam 25 mM) 20 10 Nitrazepam 0.125 mg) MD4H Midazolam 25 mM) 20 10 Nitrazepam 0.125 mg) pump, an L-7400 UV detector, an L-7200 autosampler and tion: 270 nm; emission: 312 nm). The mobile phase for a D-7500 integrator, an 821-FP intelligent spectro¯uorom- PNPH activity consisted of water/acetonitrile 80/20, v/v) eter Jasco) and a CAPCELL PAK C18 UG120 column adjusted to pH 3.0 by phosphoric acid and was eluted with 4:6mmÂ 250 mm, 5 mm; Shiseido). The activities of a ¯ow rate of 0.8 mL/min. The eluent was monitored at POD, CRH, DFH, SMH, BLH and CZH were determined 242 nm. The mobile phase for MD1H and MD4H activities as described elsewhere [5]. RMH activity was determined consisted of 10 mM potassium phosphate buffer pH 7.4)/ by the same method as that used for determination of SMH acetonitrile/methanol 5/3/2, v/v/v) with a ¯ow rate of activity. The mobile phase for EROD, BROD and PROD 0.8 mL/min. The eluent was monitored at 220 nm. The activities consisted of 20 mM potassium phosphate buffer mobile phase for 6b-, 7a- and 16a-hydroxylation activities pH 7.4) and acetonitrile 45/55, v/v) containing 2.5 mM of testosterone was eluted with a linear gradient of water/ tetra-n-octylammonium bromide, with a ¯ow rate of methanol/acetonitrile 62/36/2 to 46/50/4) for 15 min, 1.0 mL/min. The eluent was monitored ¯uorometrically followed by a linear gradient to water/methanol/acetoni- excitation: 530 nm; emission: 580 nm). The mobile phase trile 30/64/6) at 25 min, and then with an isocratic elution for DMOD activity consisted of 10 mM citrate buffer pH for 5 min, followed by a linear gradient to water/methanol/ 3.4)/acetonitrile 80/20, v/v) with a ¯ow rate of 1.0 mL/ acetonitrile 62/36/2) at 40 min. The ¯ow rate was 0.8 mL/ min. The eluent was monitored ¯uorometrically excita- min. The eluent was monitored at 254 nm.

Fig. 1. Activities of DFH A), DMOD B), MD1H and MD4H C) in cDNA-expressed rat CYPs. Substrates diclofenac, dextromethorphan and midazolam) were incubated at 378 with cDNA-expressed rat CYPs. Substrate concentrations, incubation times and contents of CYP used are shown in Table 1. Each column represents the mean of duplicate experiments. ND < 0:3 pmol/min/pmol CYP for DFH activity, <0.005 pmol/min/pmol CYP for DMOD activity and <0.2 pmol/min/pmol CYP for MD1H and MD4H activities. 892 K. Kobayashi et al. / Biochemical Pharmacology 63 (2002) 889±896

3. Results [8], were predominantly catalyzed by CYP3A1/3A2. CYP3A2 showed higher MD4H activity than did CYP3A1 3.1. Metabolism of diclofenac, dextromethorphan 8.2 vs. 2.8 pmol/min/pmol CYP), although CYP3A1 and and midazolam CYP3A2 showed similar MD1H activities 1.6 and 1.8 pmol/min/pmol CYP, respectively). Fig. 1 shows the activities of DFH A), DMOD B), MD1H and MD4H C) in cDNA-expressed rat CYPs. DFH 3.2. Metabolism of phenacetin, alkoxyresorufins, activity, a catalytic marker of human CYP2C9 [6], was bufuralol, p-nitrophenol, chlorzoxazone selectively catalyzed by CYP2C6 15.4 pmol/min/pmol - CYP). DMOD activity, a catalytic marker of human Fig. 2 shows the activities of POD A), EROD B), CYP2D6 [7], was extensively mediated by CYP2D2 BROD and PROD C), BLH D), PNPH E) and CZH F) in 3.4 pmol/min/pmol CYP). MD1H and MD4H activities, cDNA-expressed rat CYPs. Since the kinetics of POD in which are known to be catalyzed by human CYP3A4/3A5 rat liver microsomes has been reported to be biphasic [9],

Fig. 2. Activities of POD A), EROD B), BROD and PROD C), BLH D), PNPH E) and CZH F) in cDNA-expressed rat CYPs. Substrates henacetin, 7-ethoxyresorufin, 7-benzyloxyresorufin, 7-pentoxyresorufin, bufuralol, p-nitrophenol and chlorzoxazone were incubated at 378 with cDNA-expressed rat CYPs. Substrate concentrations, incubation times and contents of CYP used are shown in Table 1. Each column represents the mean of duplicate experiments. ND < 2 pmol/min/pmol CYP for POD activity, <0.03 pmol/min/pmol CYP for EROD, BROD, PROD activities, <0.02 pmol/min/pmol CYP for BLH activity, <0.2 pmol/min/pmol CYP for PNPH activity and <1.0 pmol/min/pmol CYP for CZH activity. K. Kobayashi et al. / Biochemical Pharmacology 63 (2002) 889±896 893

POD activity was determined at low 10 mM) and high tively). The activities were also detected in CYP1A2, 500 mM) concentrations of the substrate. POD activity at a CYP2C11, CYP3A1 and CYP3A2. low substrate concentration, which is well established as a marker activity of human CYP1A2 [10], was observed in 3.3. Metabolism of coumarin, S-mephenytoin CYP1A2 and CYP2C6. CYP1A2 showed a higher POD and R-mephenytoin activity than CYP2C6 did 7.5 vs. 2.5 pmol/min/pmol - CYP). At 500 mM phenacetin, both CYP1A2 and CYP2C6 Fig. 3 shows the activities of CRH A) and RMH B) in showed high POD activity 15.1 and 18.0 pmol/min/pmol - cDNA-expressed rat CYPs. CRH activity, which is speci- CYP, respectively). CYP2A2, CYP2C11 CYP2C12, ®cally catalyzed by human CYP2A6 [16,17], was detected CYP2C13, CYP2D1, CYP2D2, CYP2E1, CYP3A1 and in multiple CYP isoforms, including CYP2A2, CYP2C6, CYP3A2 also showed POD activity >2.0 pmol/min/ CYP2C12, CYP2D2, CYP3A1 and CYP3A2 >1.5 fmol/ pmol CYP). Similarly, the activity of EROD, an alternative min/pmol CYP), although the activities were very low. marker of human CYP1A1 and CYP1A2 [11], was also Since SMH activity is speci®cally catalyzed by human catalyzed by CYP1A2 and CYP2C6 1.7 and 0.90 pmol/ CYP2C19 [18], SMH activity was determined using min/pmol CYP, respectively). As for the activity of BROD, cDNA-expressed rat CYPs. Negligible SMH activity which is used as a catalytic marker of human CYP2B6 <0.03 pmol/min/pmol CYP) was detected in all cDNA- [12], CYP2B1 showed the highest activity 0.47 pmol/min/ expressed rat CYPs studied. On the other hand, RMH pmol CYP), whereas CYP1A2 was also active 0.27 pmol/ activity was detected in CYP2A2, CYP2C6, CYP2C11, min/pmol CYP). Although PROD activity was catalyzed CYP2C12 and CYP3A2 >0.1 pmol/min/pmol CYP). by CYP1A2, CYP2B1 and CYP2C13 0.097, 0.053 and 0.036 pmol/min/pmol CYP, respectively), the activity was 3.4. Metabolism of testosterone much lower than BROD activity in both CYP1A2 and CYP2B1. BLH activity, which is used as a CYP2D6 probe The regioselective hydroxylation of testosterone has in human liver microsomes [13], was extensively metabo- been well used for the identi®cation of a speci®c isoform lized by CYP2D2 31.6 pmol/min/pmol CYP), whereas of CYP on the basis of results of studies using reconstituted low BLH activity was detected in CYP2C6 and CYP2C11 systems consisting of a puri®ed CYP isoform [19±21], 8.5 and 3.0 pmol/min/pmol CYP, respectively). PNPH although multiple CYP isoforms could catalyze a pathway activity and CZH activity, which are used as human of testosterone metabolism. In this study, the activities of CYP2E1 probes [14,15], were extensively catalyzed by 7a-, 16a- and 6b-hydroxylation of testosterone, which are CYP2E1 10.8 and 17.9 pmol/min/pmol CYP, respec- mainly catalyzed by CYP2A1, CYP2C11 and CYP3A,

Fig. 3. Activities of CRH A) and RMH B) in cDNA-expressed rat CYPs. Substrates coumarin and R-mephenytoin) were incubated at 378 with cDNA- expressed rat CYPs. Substrate concentrations, incubation times and contents of CYP used are shown in Table 1. Each column represents the mean of duplicate experiments. ND < 1:5 fmol/min/pmol CYP for CRH activity and <0.03 pmol/min/pmol CYP for RMH activity. 894 K. Kobayashi et al. / Biochemical Pharmacology 63 (2002) 889±896 respectively, were reanalyzed using cDNA-expressed rat isoform to the reaction on the basis of the relative abun- CYPs. When testosterone 25 mM) was incubated with dance of CYPs in rat liver microsomes. However, the each 5 pmol of cDNA-expressed rat CYP for 30 min at turnover number of CYPs in rat liver microsomes is 378, CYP3A2 showed higher 6b-hydroxylase activity than affected by several factors such as NADPH-CYP reduc- did CYP3A1 8.8 vs. 1.8 pmol/min/pmol CYP). Testoster- tase, cytochrome b5, membrane lipid composition, and one 7a-hydroxylation activity was catalyzed by both ionic strength of the in vitro incubation matrix. Therefore, CYP2A1 and CYP2A2 1.7 and 0.48 pmol/min/pmol CYP, it is necessary to consider not only relative abundance of a respectively). Testosterone 16a-hydroxylation activity was particular CYP isoform in rat liver microsomes but also the catalyzed by CYP2C11 11.8 pmol/min/pmol CYP). differences in turnover number between the cDNA- expressed CYPs and liver microsomes to extrapolate the data obtained from cDNA-expressed CYPs to those of liver 4. Discussion microsomes. Additional studies are now underway in our laboratory to clarify the isoform-speci®city for activities of Since the 1990s, the use of cDNA-expressed human POD, EROD, BROD, PROD, BLH, PNPH and CZH in rat CYPs has greatly facilitated the evaluation of metabolic liver microsomes. These studies include the use of CYP speci®cities of probe substrates and the identi®cation of isoform-selective inhibitors, determination of catalytic individual CYPs involved in the metabolism of many drugs activities at different substrate concentrations, and com- in humans. On the other hand, the speci®cities of metabolic parison of catalytic activities in liver microsomes of rats reactions used as probes of CYP isoforms have not been treated with inducers of certain CYP isoforms with those of thoroughly evaluated by the study with cDNA-expressed control rats. rat CYP isoforms. Therefore, in the present study, the Unlike the speci®city to human CYPs, activities of CRH isoform-speci®cities of metabolic reactions known as and SMH did not show selectivity toward any isoforms of probes of human CYPs were examined by using 13 rat CYPs cDNA-expressed rat CYPs Fig. 3). Although CRH activity expressed in baculovirus-infected insect cells or human B- was catalyzed by multiple isoforms of cDNA-expressed rat lymphoblastoid cells. CYP CYP2A2, CYP2C6, CYP2C12, CYP2D2, CYP3A1 Among the metabolic reactions studied, activities of and CYP3A2), the activity was much lower than human DFH and DMOD were selectively catalyzed by CYP2C6 CYP2A6-mediated CRH activity [17]. This ®nding is in and CYP2D2, respectively Fig. 1Aand B). MD1H and agreement with previous results showing that CRH activity MD4H activities were predominantly catalyzed by in rat liver microsomes was much lower than that in human CYP3A1/3A2, whereas MD1H activity was lower than liver microsomes [23]. In addition, coumarin is extensively MD4H activity in both CYP3A1 and CYP3A2 Fig. 1C). metabolized to 7-hydroxycoumarin in human liver micro- These results suggest that activities of DFH, DMOD and somes, whereas only one metabolite, O-hydroxyphenyla- MD4H are useful as catalytic markers of CYP2C6, cetic acid, was detected in rat liver microsomes [24]. Our CYP2D2 and CYP3A1/3A2, respectively. results together with these observations suggest that CRH On the other hand, activities of POD, EROD, BROD, activity is unable to be used as a catalytic probe of a CYP PROD, BLH, PNPH and CZH were catalyzed by more than isoform in rat liver microsomes, unlike the speci®city of two isoforms in the present study using cDNA-expressed CRH activity to CYP2A6 in humans. Similarly, SMH rat CYPs Fig. 2). These results suggest that the activities activity was not substantially discernible in any of the of POD, EROD, BROD, PROD, BLH, PNPH and CZH, cDNA-expressed CYPs examined. Therefore, SMH activ- reactions known as catalytic markers of human CYPs, were ity is also unable to be used as a catalytic probe of a CYP not necessarily speci®c for rat CYPs. When multiple iso- isoform in rat liver microsomes. forms of CYP are involved in a metabolic reaction, iso- It has been reported that the kinetics of POD in rat liver form-selectivity occasionally differs depending on the microsomes is biphasic [9], which could indicate the substrate concentration [22], although the concentrations involvement of more than one isoform of CYP. Moreover, of probes used in this study were chosen on the basis of the Kahn and Ruben®eld [25] reported that 3-methylcholan-

Km values of human liver microsomes or cDNA-expressed threne selectively increased the high-af®nity component of human CYPs. Therefore, additional studies are necessary POD activity in rat liver microsomes, whereas phenobar- to clarify the speci®city of rat enzymes considering Km bital selectively increased the low-af®nity component. values of rat CYPs for the probes of human CYPs. Since 3-methylcholanthrene speci®cally induces both In addition, there is a large difference in the levels of CYP1A1 and CYP1A2 [26], the high-af®nity component individual CYP isoforms in rat liver microsomes [2]. of POD is thought to be catalyzed by CYP1A1/1A2. Moreover, levels of CYP isoforms in livers are very However, it is not clear which isoforms) catalyzes the different between control rats and rats treated with com- low-af®nity reaction of POD in rat liver microsomes pounds known to induce certain isoforms of CYP. There- because phenobarbital induces multiple CYP isoforms fore, when multiple CYPs are involved in certain metabolic such as CYP2B, CYP2C6 and CYP3A[27]. The present reaction, it is important to predict the contribution of each study using cDNA-expressed rat CYPs showed that POD K. Kobayashi et al. / Biochemical Pharmacology 63 (2002) 889±896 895 activity at a high phenacetin concentration 500 mM) was inhibited by a CYP1Ainhibitor. Therefore, CZH activity catalyzed by CYP1A2 and CYP2C6, but not by CYP2A1 in rat liver microsomes appears to be catalyzed by several and CYP2B1 Fig. 2A). Therefore, the low-af®nity reac- isoforms of CYP, such as CYP1A2, CYP2B1 and tion of POD in rat liver microsomes might be catalyzed by CYP3A1. CYP2C6. In humans, 40-hydroxylation of mephenytoin preferen- EROD activity and BROD activity in human liver micro- tially occurs with S-enantiomer [37]. The SMH activity is somes are widely used as probes for measuring CYP1A1/ mainly catalyzed by CYP2C19. However, negligible SMH 1A2 and CYP2B6, respectively. On the other hand, in vitro activity was detected in all cDNA-expressed rat CYPs study using rat liver microsomes showed that BROD activ- studied. Yasumori et al. [38] suggested that this is because ity was a selective probe for CYP1A1 in 3-methylcholan- rats showed a preferential RMH activity in a manner threne-induced microsomes and that both BROD activity opposite to that in humans. They reported that CYP2C11, and PROD activity were selective probes for CYP2B1 in CYP3A1 and CYP3A2 were able to ef®ciently catalyze phenobarbital-induced microsomes [28]. However, EROD RMH activity in a reconstituted system. Therefore, RMH activity was not a selective probe for CYP1A1 in non- activity was determined using cDNA-expressed rat CYPs. induced rat liver microsomes and was metabolized by The results showed that CYP2A2, CYP2C6 and CYP2C13 puri®ed CYP2C6 [28]. BROD and PROD activity were were also able to catalyze RMH activity as well as metabolized by several different CYP isoforms in non- CYP2C11 and CYP3A2, although CYP3A1 showed neg- induced microsomes but mainly by both CYP1A1 and ligible RMH activity Fig. 3B). These results suggest that CYP1A2 in 3-methylcholanthrene-induced microsomes. RMH activity is not selective toward any isoforms of The present results obtained from cDNA-expressed rat cDNA-expressed rat CYPs and that neither RMH activity CYPs showed that EROD activity was not only catalyzed nor SMH activity appears to be useful as catalytic probes of by CYP1A2 but also by CYP2C6 Fig. 2B) and that BROD CYP isoforms in rat liver microsomes. Therefore, it and PROD activity were catalyzed by CYP1A2 and appears that probes for CYP isoforms do not always exhibit CYP2B1 Fig. 2C). The present results obtained by using the same speci®city in human and rat. Caution should be cDNA-expressed rat CYPs are in agreement with the results exercised when extrapolating the metabolic data obtained of a previous study using puri®ed rat CYPs [28]. in rat to the human situation. In humans, only one CYP2D isoform, CYP2D6, has In the present study, 13 rat CYPs were investigated, been found [1]. On the other hand, ®ve CYP2D isoforms, however, they might be not all of the possible CYPs CYP2D1 through CYP2D5, have been found in the rat liver involved in the metabolism of the probes studied. There- [29±31]. The present results showed that BLH activity was fore, it should be noted that the other isoforms might extensively metabolized by CYP2D2, whereas CYP2D1 contribute to the metabolism of the probes used in the barely catalyzed the activity Fig. 2C). These results are in present study. agreement with previous results [32,33] showing that In conclusion, the present study suggested that activities heterogously expressed CYP2D2 had high af®nity for of DFH, DMOD and MD4H are useful as catalytic markers bufuralol compared with the other CYP2D isoforms. In of CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. In addition to CYP2D2, CYP2C6 and CYP2C11 also showed contrast, activities of CRH, SMH and RMH are not able to BLH activity. Therefore, BLH activity might not be a be used as catalytic probes of CYP isoforms in rat liver speci®c probe of CYP2D2 in liver microsomes from rats microsomes. However, it is necessary to conduct further treated with compounds known to induce CYP2Cs. study to clarify CYP isoform-speci®city for these activities The present study showed that activities of PNPH and in rat liver microsomes, since activities of POD, EROD, CZH are extensively catalyzed by CYP2E1 and that BROD, PROD, BLH, PNPH and CZH were found to be CYP1A2 and CYP3A1 are also capable of catalyzing those catalyzed by more than two isoforms in this study using activities Fig. 2E and F). These results are consistent with cDNA-expressed rat CYPs. These results may provide those of previous studies using rat liver microsomes. PNPH useful information regarding catalytic probes of rat CYP activity has been shown to be mainly catalyzed by CYP2E1 isoforms for studies using rat liver microsomal samples. in the rat [34]. However, CYP3Ainducer treatment increased PNPH activity in rat liver microsomes, and the activity was inhibited by a CYP3Ainhibitor [35]. 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