Regular Article Comparison of Inducibility of CYP1A and CYP3A Mrnas by Prototypical Inducers in Primary Cultures of Human, Cynomolgus Monkey, and Rat Hepatocytes

Drug Metab. Pharmacokinet. 22 (3): 178–186 (2007).

Regular Article Comparison of Inducibility of CYP1A and CYP3A mRNAs by Prototypical Inducers in Primary Cultures of Human, Cynomolgus Monkey, and Rat Hepatocytes

Masuhiro NISHIMURA1, Akiko KOEDA2, Yasuyuki SUGANUMA2,EmakoSUZUKI2, Takefumi SHIMIZU2,MitsuoNAKAYAMA1,TetsuoSATOH2,3, Shizuo NARIMATSU4 and Shinsaku NAITO1,* 1Department of Drug Metabolism, Division of Pharmacology, Drug Safety and Metabolism, Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan 2Ina Research Inc., Nagano, Japan 3Non-Proˆt Organization Human & Animal Bridging Research Organization, Chiba, Japan 4Laboratory of Health Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: This study was conducted to investigate the eŠects of treatment with the prototypical inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on the mRNA levels of drug- metabolizing enzymes in primary cultures of cryopreserved human, cynomolgus monkey, and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. Treatment with Ome substantially increased the mRNA levels of both CYP1A1 and CYP1A2 in human hepatocytes, but increased only the mRNA level of CYP1A1 in monkey hepatocytes, whereas it had no marked eŠect on the mRNA levels of CYP1A1 or CYP1A2 in rat hepatocytes. Treatment with Rif or Dex did not markedly aŠect the mRNA level of CYP1A in any of the hepatocyte cultures under the conditions used. All three inducers increased the mRNA level of CYP3A8 in monkey hepatocytes (in the order RifÀDexÆOme), and a similar proˆle was observed for the mRNA level of CYP3A4 in human hepatocytes, but the potency of induction was markedly attenuated. In contrast, only Dex substantially increased the mRNA level of CYP3A1 in rat hepatocytes, with Rif and Ome showing no eŠects. These results indicate that the molecular mechanisms responsible for the regulation of CYP1A2 genes diŠer between humans and cynomolgus monkeys, although the regulatory mechanisms for CYP1A1 and CYP3A genes are similar.

Key words: induction, cytochrome P450, cynomolgus monkey, human, rat, hepatocytes

an experimental animal species because the monkey Introduction belongs to the order Primates, which also includes man. Drug discovery and development depend greatly on Various monkey species such as cynomolgus monkeys, the successful extrapolation of drug metabolism and rhesus monkeys, marmoset monkeys, and so on have toxicity data obtained from experimental animals to been used in a wide variety of experiments. Among humans. It is therefore important to understand the these species, cynomolgus monkeys have often been similarities and diŠerences between experimental employed in studies of drug metabolism and toxicity. animals and humans in terms of the ‰uctuation proˆles Recently, we have been studying the changes in the of the cytochrome P450 (CYP) subfamilies following mRNA levels of various drug-metabolizing enzymes in exposure to xenobiotics. From the viewpoint of data primary cultures of human and rat hepatocytes follow- extrapolation, the monkey is a promising candidate for ing treatment with prototypical inducers such as rifam-

Received; January 10, 2007, Accepted; May 2, 2007 *To whom correspondence should be addressed: Shinsaku NAITO,Ph.D.,Division of Pharmacology, Drug Safety and Metabolism, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan. Tel. ＋81-88-685-1151, Fax. ＋81-88-686-8176, E-mail: naitousn＠otsukakj. co.jp

178 CYP1A and CYP3A Induction in Primary Cultures of Hepatocytes 179

Table 1. Characteristics of hepatocytes and preparations. reagent grade. Animals: Adult male and female cynomolgus Age Sex RaceWStrain Viability (z) monkeys were obtained from Ina Research Philippines, Human Inc. (Batangas, Philippines) and adult female rats were Donor #1 23 years female Caucasian 93.8 obtained from Charles River Japan, Inc. (Kanagawa, Donor #2 74 years female Caucasian 91.8 Donor #3 2 years female Caucasian 91.2 Japan) (Table 1). These animals were allowed free Monkey access to food and water. The study was approved by Monkey #1 3 years male Cynomolgus 80.3 the Committee on the Care and Use of Laboratory Monkey #2 2–3 years male Cynomolgus 99.3 Animals of Ina Research Inc. and the Committee on the Monkey #3 3 years female Cynomolgus 88.3 Care and Use of Laboratory Animals of Otsuka Phar- Rat Rat #1 7 weeks female Sprague-Dawley 91.8 maceutical Factory, Inc. Rat #2 7 weeks female Sprague-Dawley 89.3 Isolation of cynomolgus monkey hepatocytes: Per- Rat #3 7 weeks female Sprague-Dawley 88.1 fusion of the cynomolgus monkey liver was performed according to the Seglen method with some modiˆca- Viability was determined by the trypan blue exclusion test. tions.8) Brie‰y, the liver was perfused with Ca2＋-free Hanks solution containing 0.6 mM EGTA at 379Cfor picin (Rif), dexamethasone (Dex), and omeprazole 4 min and further perfused with Hanks solution con- (Ome). Rif induces CYP3A4 in primary cultures of taining 0.025z collagenase at 379C for 6 min. Hepato- human hepatocytes,1–3) while this drug is not an inducer cytes were dispersed from the perfused liver in ice-cold of CYP3A in rat hepatocytes,2–4) with the rat being the HEPES solution. After ˆltration through gauze, the most commonly used laboratory animal species for hepatocytes were separated from nonparenchymal cells toxicological studies. Dex is a potent inducer of CYP3A by centrifugation at 50×g for 2 min at 49C. The cells in rat hepatocytes, whereas it is a weak inducer of were washed and resuspended in HEPES solution three CYP3A in human hepatocytes.3) Ome is a potent in- times, then washed with Williams' medium E containing ducer of CYP1A in primary cultures of human hepato- 20z fetal bovine serum, 2.5z polyvinylpyrrolidone, cytes.5) and 2z bovine serum albumin7) (with the pH adjusted

Although various studies on the catalytic properties to approximately 7.4 with NaHCO3). Finally, the cells of CYP enzymes in cynomolgus monkey hepatocytes were resuspended in the same medium and cell viability have been reported,6,7) there is little information availa- was determined by the trypan blue exclusion test. After ble on the induction of CYP enzymes in cynomolgus the addition of dimethylsulfoxide (DMSO, ˆnal concen- monkey hepatocytes. In the present study, we conduct- tration of 10z) to the cell suspension, the cell density ed comparative experiments on the inducibility of was approximately 1.5 to 2.5×107 viable cellsWmL. The CYP1A and CYP3A mRNAs by the prototypical cynomolgus monkey hepatocytes were cryopreserved at inducers Rif, Dex, and Ome in primary cultures of －309C for 40 min, at －809C for 15 min, and then in cryopreserved human, cynomolgus monkey, and rat liquid nitrogen until use. hepatocytes. Isolation of rat hepatocytes: Perfusion of the rat liver was performed according to the Seglen method8) Materials and Methods with some modiˆcations.9,10) The rat hepatocytes were Materials: Rif, Dex and Ome were purchased from cryopreserved at －309C for 40 min, at －809Cfor15 Wako Pure Chemical Industries, Ltd. (Osaka, Japan). min, and then in liquid nitrogen until use. Collagenase S-1 was purchased from Nitta Gelatin, Inc. Monolayer culture of human and rat hepatocytes: (Osaka, Japan); bovine serum albumin (fraction V) Monolayer cultures of the cryopreserved human and rat from Sigma-Aldrich (St. Louis, MO); and trypan blue hepatocytes were obtained according to the method of from Merck (Darmstadt, Germany) and Flow Labora- Nishimura et al.9,10) Human cell suspensions with tories, Ltd. (Irvine, UK). Cryopreserved human hepato- viability rates of 91z to 94z and rat cell suspensions cytes (donor #1 [Lot 082], donor #2 [Lot 100], and with viability rates of 88z to 92z, as assessed by the donor #3[Lot130])(Table 1) were purchased from trypan blue exclusion test, were used for the experi- In Vitro Technologies, Inc. (Baltimore, MD). Hepato- ments (Table 1). The cells were used for experiments at cyte Culture Medium (CC-3198) was purchased from 48 hr after inoculation. BioWhittaker, Inc. (Walkersville, MD); the RNeasy} Monolayer culture of cynomolgus monkey hepato- Mini Kit and QIAshredderTM from QIAGEN (Hilden, cytes: Monolayer cultures of the cryopreserved Germany); yeast tRNA from Life Technologies, Inc. cynomolgus monkey hepatocytes were obtained accord- (Rockville, MD); and TaqMan One-Step RT-PCR ing to the method of Nishimura et al.11) with some Master Mix Reagents from Applied Biosystems (Foster modiˆcations. The cryopreserved hepatocytes were City, CA). All other chemicals used in this study were of suspended in Hepatocyte Culture Medium. The hepato- 180 Masuhiro NISHIMURA, et al. cytes were then centrifuged at 45×g for 3 min at 49C been reported previously. The primers and TaqMan and resuspended in the same medium. The number of probes were synthesized by Sigma-Aldrich Japan K.K. cells was counted using a Coulter Counter (Beckman Genosys Division (Ishikari, Japan). The TaqMan Coulter, Inc., Fullerton, CA). Cell suspensions with probes contained 6-carboxy‰uorescein (FAM) at the 5? viability rates of 80z to 99z were used for the end and 6-carboxytetramethylrhodamine (TAMRA) at experiments (Table 1). The cell suspensions were diluted the 3? end and were designed to hybridize to a sequence to a ˆnal concentration of 2.5×105 viable cellsWmL with located between the PCR primers. Hepatocyte Culture Medium, and inocula of 1×105 TaqMan RT-PCR conditions: Total RNA was viable cellsW0.4 mLWwell were introduced into 24-well diluted to about 4 mgWmL with 50 mgWmL yeast tRNA. platesthathadbeencoatedwithtypeIcollagen.The The RT-PCR assay was performed in 50 mLofTaqMan cells were cultured for 3 hr after inoculation under 5z One-Step RT-PCR Master Mix reagents containing 300

CO2 and 95z air at 379C. The medium was then nM forward primer, 900 nM reverse primer, 200 nM replaced with fresh medium, and the cells were cultured TaqMan probe, and about 20 ng of total RNA accord- 14,15) for 21 hr under 5z CO2 and 95z air at 379C. The ing to the method described previously. The RT- medium was then replaced with fresh medium without PCR assay was performed using the ABI PRISM 7700 human epidermal growth factor (hEGF), hydrocorti- Sequence Detector system (Applied Biosystems) under sone, gentamicin, and amphotericin B, and the cells the same conditions as in our previous studies.14,15) were cultured for 24 hr under 5z CO2 and 95z air at Statistical analysis: Data analyses were performed 379C. The cells were used for experiments at 48 hr after with the ABI PRISM Sequence Detector software. The inoculation. relative expression of each mRNA was calculated as the Experiments using primary cultures of human and DCt (the value obtained by subtracting the Ct value of cynomolgus monkey hepatocytes: In the induction b-actin mRNA from the Ct value of the target mRNA), studies, Hepatocyte Culture Medium without hEGF, as described in our previous reports.14–17) In particular, hydrocortisone, gentamicin, and amphotericin B was the amount of target mRNA relative to b-actin mRNA used. The hepatocytes were exposed to the inducers Rif, was expressed as 2－(DCt). Data are expressed as the ratio Dex, and Ome (2, 10, and 50 mMeach)for24hr.All of the target mRNA to b-actin mRNA. The experiments inducers were dissolved in DMSO at a ˆnal concentra- with the hepatocyte cultures shown in Figs. 1A and 2 tion of 0.1z (vWv). Controls were also exposed to were performed in triplicate, and the mean values were DMSO at a ˆnal concentration of 0.1z (vWv). Total calculated. In particular, the data are shown as mean± RNA was extracted from the hepatocytes using the SD for three animals or donors. The experiments with QIAshredderTM and RNeasy} Mini Kit. the hepatocyte cultures shown in Figs. 1B and 3 also Experiments using primary cultures of rat hepato- were performed in triplicate, and the mean values were cytes: In the induction studies, Williams' medium E calculated. Furthermore, the values shown in Figs. 1B (pH 7.4) with 3z bovine serum albumin (fraction V) and 3 indicate the results relative to controls without and without 10z fetal bovine serum, 4 mgWmL bovine DMSO, and the results are shown as mean±SD for insulin, 10 nM dexamethasone, 100 IUWmL penicillin, three animals or donors. Statistical analysis was per- and 100 mgWmL streptomycin was used. The hepatocytes formed using the Dunnett's test with a signiˆcance level were exposed to the inducers Rif, Dex, and Ome (2, 10, of pº0.05. and 50 mM each) or to vehicle for 24 hr as described Results above. Oligonucleotides: The pairs of forward and reverse In the present study, the regulation of the mRNA primers and the TaqMan probes for cynomolgus expression of CYP1A and CYP3A in primary cultures monkey b-actin, b-2-microglobulin (B2M), glyceralde- of three lots each of cryopreserved human, cynomolgus hyde 3-phosphate dehydrogenase (GAPDH), hypoxan- monkey, and rat hepatocytes was investigated, and thine phosphoribosyltransferase (HPRT), phosphogly- the mRNA expression levels in human, cynomolgus cerate kinase (PGK), CYP1A1, CYP1A2, and CYP3A8 monkey, and rat hepatocytes were compared. The and for rat CYP1A1 used in the RT-PCR sequences mRNA levels of b-actin as well as of several housekeep- were designed using Primer Express software (Applied ing genes were measured to conˆrm stable expression in Biosystems) (Table 2). Each primer and probe was human and rat hepatocytes under the same experimental homology searched by an NCBI BLAST search to conditions.9) The ratios of B2M, GAPDH, HPRT, and ensure that it was speciˆc for the target mRNA tran- PGK mRNA levels to the b-actin mRNA level in script. The pairs of forward and reverse primers and cynomolgus monkey hepatocytes were relatively con- the TaqMan probes for human b-actin,12) CYP1A1, stant from 48 to 72 hr of culture (Fig. 1A). The ratios CYP1A2, and CYP3A411) and for rat b-actin, CYP1A2, were not markedly changed in primary cultures of and CYP3A113) used in the RT-PCR sequences have cynomolgus monkey hepatocytes following exposure to CYP1A and CYP3A Induction in Primary Cultures of Hepatocytes 181

Table 2. Primers and probes used for RT-PCR analysis of target mRNAs.

mRNA Sequence Position Cynomolgus monkey b-Actin (GenBank accession number U20576) Forward primer 5?-TACAGCTTCACCACCACGG-3? unknown Reverse primer 5?-TGCTCGAAGTCTAGGGCGA-3? unknown Probe 5?-AATCGTGCGTGACATTAAGGAGAAGCTGTG-3? unknown B2M (GenBank accession number AF485817) Forward primer 5?-TGCTGGCGCTACTCTCTCTTT-3? 26–46 Reverse primer 5?-TGGTGGATGGCGTGAGTAAA-3? 105–86 Probe 5?-TGGAGGCTATCCAGCGTACTCCAAAGATTC-3? 53–82 GAPDH (GenBank accession number AB158631) Forward primer 5?-AGTTCCATGGCACCGTGAA-3? unknown Reverse primer 5?-GATTTTGGAGGGATCTCGCTC-3? unknown Probe 5?-AACGGGAAGCTCGTCATCAATGGAAGC-3? unknown HPRT (GenBank accession number S43335) Forward primer 5?-GCTTTCCTTGGTCAGGCAGTA-3? 438–458 Reverse primer 5?-TGGAGTCCTTTTCACCAGCA-3? 507–488 Probe 5?-AATCCAAAGATGGTCAAGGTCGCAAGC-3? 460–486 PGK (GenBank accession number AB125189) Forward primer 5?-GGCTGCATCACCATCATAGGT-3? 1096–1116 Reverse primer 5?-CAGTGCTCACATGGCTGACTT-3? 1183–1163 Probe 5?-TGTGCCAAATGGAACACGGAGGA-3? 1138–1160 CYP1A1 (GenBank accession number D17575) Forward primer 5?-ACCTGAACGGTTTATCACCCC-3? 1284–1304 Reverse primer 5?-ATGGTCTCACCGATGCACTTC-3? 1385–1365 Probe 5?-CTATCGACAAGGTGCTAAGTGAGAAGGTGATTCTC-3? 1313–1347 CYP1A2 (GenBank accession number D86474) Forward primer 5?-ATCCCCCAGGAGAAGATTGTC-3? 904–924 Reverse primer 5?-AGGAGATGGCTGTTGCAATTG-3? 982–962 Probe 5?-CCTTGTCAATGACATCTTCGGAGCAGGAT-3? 927–955 CYP3A8 (GenBank accession number S53047) Forward primer 5?-CCACACCTTTGCCTTTATTGG-3? 122–142 Reverse primer 5?-AAGCCCCACACTTTTCCATAC-3? 221–201 Probe 5?-TCCTACCGTAAGGGCTTTTGGACGTTTGATATG-3? 154–186 Rat CYP1A1 (GenBank accession number NM012540) Forward primer 5?-GTCATCTGTGCCATATGCTTTG-3? 601–622 Reverse primer 5?-GCTTAGATTGACTATGCTGAGCAG-3? 675–652 Probe 5?-CAGACGTTATGACCACGATGACCAAGA-3? 624–650

Rif, Dex, or Ome (Fig. 1B). b-Actin mRNA was there- of culture (Fig. 2A). The ratio of CYP1A2 mRNA to b- fore employed as the endogenous control for the meas- actin mRNA in monkey hepatocytes fell to 0.7z of the urement of CYP mRNAs in the present study. initial level during the ˆrst 24 hr and fell further to The ratio of CYP1A1 mRNA to b-actin mRNA in 0.05z at 72 hr of culture (Fig. 2B). In contrast, the human hepatocytes fell to 42z of the initial level during ratio of CYP1A1 mRNA to b-actin mRNA in rat the ˆrst 24 hr, and the ratio then remained constant hepatocytesat72hrofculturewas4700z of the initial from 24 to 72 hr of culture (Fig. 2A). The ratio of level (Fig. 2A). The ratio of CYP1A2 mRNA to b-actin CYP1A2 mRNA to b-actin mRNA in human hepato- mRNA in rat hepatocytes fell to 2z of the initial level cytes fell to 4z of the initial level during the ˆrst 24 hr, during the ˆrst 24 hr and fell further to 0.2z at 72 hr of and the ratio then remained constant from 24 to 72 hr of culture (Fig. 2B). The ratio of CYP3A4 mRNA to b- culture (Fig. 2B). The ratio of CYP1A1 mRNA to b- actin mRNA in human hepatocytes fell to 0.03z of the actin mRNA in monkey hepatocytes fell to 5z of the initial level at 72 hr of culture (Fig. 2C). A similar trend initial level during the ˆrst 24 hr, after which the ratio was observed for CYP3A mRNA levels in monkey and rose gradually, reaching 49z of the initial level at 72 h rathepatocytes.Thatis,theratioofCYP3A8mRNAto 182 Masuhiro NISHIMURA, et al.

Fig. 1. Changes in the mRNA levels of B2M, GAPDH, HPRT, and PGK during 72-hr primary culture of cynomolgus monkey hepatocytes (A) and eŠects of exposure to Rif, Dex, and Ome on the mRNA levels of housekeeping genes (B). Data are expressed as the ratio of the target mRNA to b-actin mRNA. Experiments (culture of cynomolgus monkey hepatocytes) were performed in triplicate using three monkey livers, and the values are shown as mean±SD (n＝3). In Fig. 1B, monkey hepatocytes were exposed to drugs at 48 hr after inoculation. The hepatocytes were treated with Rif, Dex, or Ome (2, 10, and 50 mM each) for 24 hr. The dotted line indicates the value for controls Fig. 2. Changes in the mRNA levels of CYP1A1 (A), CYP1A2 (B), without DMSO, which was assigned a value of 1. and CYP3As (C) during 72-hr primary culture of human, cynomolgus monkey, and rat hepatocytes. Data are expressed as the ratio of the target mRNA to b-actin mRNA. Experiments (culture of human, cynomolgus monkey, and rat hepatocytes) were performed in tripli- b-actin mRNA in monkey hepatocytes and the ratio of cate using three livers each, and the values are shown as mean±SD CYP3A1 mRNA to b-actin mRNA in rat hepatocytes (n＝3). fell to 0.04z and 0.05z of the initial levels, respec- tively,at72hrofculture(Fig. 2C). We previously examined the eŠect of DMSO on the middle panels). Only Ome substantially increased the levels of human CYP mRNAs in primary cultures of mRNA levels of CYP1A1 in a concentration-dependent cryopreserved human hepatocytes.18) Because 0.1z manner in both human and monkey hepatocytes DMSO did not aŠect the CYP mRNA level, the same (Fig. 3A, top and middle panels). Interestingly, Ome concentration of DMSO was employed in the present also signiˆcantly increased the mRNA level of CYP1A2 study. Treatment of human and cynomolgus monkey in human hepatocytes (Fig. 3B, top panel), whereas it hepatocytes with Rif and Dex did not have any marked did not aŠect the mRNA level of CYP1A2 in monkey eŠects on the mRNA levels of CYP1A1 or CYP1A2 hepatocytes (Fig. 3B, middle panel). Treatment with under the conditions employed (Fig. 3A and B, top and Dex and Ome also tended to increase the mRNA level of CYP1A and CYP3A Induction in Primary Cultures of Hepatocytes 183

Fig. 3. EŠects of drug exposure on the mRNA levels of CYP1A1 (A), CYP1A2 (B), and CYP3As (C) in primary cultures of human, cynomolgus monkey, and rat hepatocytes. Data are expressed as the ratio of the target mRNA to b-actin mRNA. Experiments (culture of human, cynomolgus monkey, and rat hepatocytes) were performed in triplicate using three livers each, and the values are shown as mean±SD (n＝3). Hepatocytes were exposed to drugs at 48 hr after inoculation. The hepatocytes were treated with Rif, Dex, or Ome (2, 10, and 50 mMeach)for24hr.Thedottedline indicates the value for controls without DMSO, which was assigned a value of 1. *pº0.05, **pº0.01 and ***pº0.001 vs. controls at 0.1z DMSO.

CYP1A1 in rat hepatocytes, but in a concentration- cytes from the other sources examined. independent manner (Fig. 3A, bottom panel). As for In the human liver, CYP1A2 is constitutively CYP3A, Rif markedly increased the mRNA levels in a expressed, but CYP1A1 is infrequently expressed.19) We concentration-dependent manner in human and monkey also reported that the expression level of CYP1A1 hepatocytes (Fig. 3C, top and middle panels). Dex and mRNA was much lower than that of CYP1A2 mRNA20) OmealsoincreasedthemRNAlevelsofCYP3A4in in the human liver. Human CYP1A2 exhibits higher ac- human hepatocytes and of CYP3A8 in monkey tivity for methoxyresoruˆn O-demethylation (MROD) hepatocytes, but to lesser extent as compared with the than for ethoxyresoruˆn O-deethylation (EROD).21) In eŠects of Rif (Fig. 3C, top and middle panels). In rat contrast, Sakuma et al.22) reported that the expression hepatocytes, only Dex showed a tendency to increase level of CYP1A1 mRNA was higher than that of the mRNA level of CYP3A1, but in a concentration- CYP1A2 mRNA in the cynomolgus monkey liver. independent manner (Fig. 3C, bottom panel). Furthermore, hepatic microsomal MROD activity was reported to be lower than EROD activity in the Discussion cynomolgus monkey.23) In the present study, we compared the eŠects of treat- We also conˆrmed in the present study that the ment with Rif, Dex, and Ome on the levels of CYP1A expression level of CYP1A2 mRNA was much lower and CYP3A mRNAs in primary cultures of hepatocytes than that of CYP1A1 mRNA in hepatocytes from obtained from humans, cynomolgus monkeys, and rats. cynomolgus monkeys. Furthermore, we found that We found several interesting diŠerences with regard to CYP1A1 mRNA in cynomolgus monkey hepatocytes the changes in the mRNA levels of CYP1A and CYP3A was substantially induced by treatment with Ome, the between cynomolgus monkey hepatocytes and hepato- proˆle of which is similar to that of CYP1A1 and 184 Masuhiro NISHIMURA, et al.

CYP1A2 in human hepatocytes. increased CYP3A mRNA levels in human and cynomol- The aromatic hydrocarbon receptor (AHR) is a gus monkey hepatocytes, with Rif being the more potent ligand-activated transcription factor that controls the inducer. In rat hepatocytes, however, among the three CYP1A1 and CYP1A2 genes.24) Ome was ˆrst reported compounds examined, only Dex increased the CYP3A1 to enhance the nuclear uptake of the human AHR and mRNA level, and the eŠect of Rif was negligible as to activate the transcription of a human CYP1A1- compared with that of Dex (Fig. 3). This may be due to luciferase chimeric gene in HepG2 cells.25) Ome is diŠerences in the sensitivity (ligand a‹nity) andWor thought to activate the AHR to translocate into the speciˆcity for these nuclear receptors between primates nucleus, where the AHR-AHR nuclear translocator and rodents, which is in accord with the previous (ARNT) complex interacts with the xenobiotic respon- ˆndings.34–36) sive element (XRE) on the CYP1A1 5?-‰anking gene to We observed in the present study that cynomolgus activate transcription.25,26) These ˆndings indicate that monkey CYP3A8 mRNA and human CYP3A4 mRNA Ome enhances CYP1A gene transcription via activation showed similar elevation proˆles in primary cultures of of the AHR. hepatocytes treated with the inducers. Ohmori et al.37) Quattrochi et al.27) and Fernandez-Salguero et al.28) estimated that CYP3A8 accounts for 20z of the total reported the involvement of the AHR in the regulation CYP in liver microsomes from untreated cynomolgus of CYP1A2 expression. In the present study, Ome monkeys, which is comparable to the levels of CYP3A4 increased only CYP1A1 mRNA but not CYP1A2 accounting for about 30z of the total CYP in human mRNA in monkey hepatocytes, while it induced both liver microsomes.38) We therefore consider that the CYP1A1 and CYP1A2 mRNAs in human hepatocytes. cynomolgus monkey is a good animal model for eval- In rat hepatocytes, neither CYP1A1 nor CYP1A2 uating the induction of CYP3A in preclinical studies. mRNA levels were substantially changed following Ome The results of the present study clearly demonstrated exposure. Backlund et al.29) reported that the induction that following treatment of hepatocytes with the of CYP1A1 in rat hepatoma H4IIE cells required high prototypical inducers Rif, Dex, and Ome, the response concentrations of Ome. This diŠerence in the response proˆles of CYP1A2 mRNA levels are diŠerent but those of CYP1A gene expression following treatment with of CYP1A1 and CYP3A mRNA levels are similar Ome may be due to diŠerences in sensitivity (ligand between cynomolgus monkeys and humans. These a‹nity) andWor speciˆcity for the AHR between the characteristics of the cynomolgus monkey should be animal species examined. However, this cannot explain taken into consideration when this animal species is the diŠerence in the response to Ome between CYP1A1 employed in studies on drug metabolism and toxicity. and CYP1A2 genes in the cynomolgus monkey. It seems In summary, we examined the eŠects of treatment likely that the mechanism for the transcriptional with Rif, Dex, and Ome on the mRNA levels of CYP1A activation of the CYP1A2 gene diŠers from that of the and CYP3A enzymes in primary cultures of human, CYP1A1 gene in the cynomolgus monkey. cynomolgus monkey, and rat hepatocytes. Of the three It has been proposed that the pregnane X receptor inducers examined, Ome substantially increased the (PXR) forms a heterodimer with the retinoid X receptor mRNA levels of both CYP1A1 and CYP1A2 in human a (RXRa), playing an important role in CYP3A4 gene hepatocytes but of only CYP1A1 in monkey hepato- regulation for several xenobiotics such as Rif in cytes. Treatment with Rif, Dex, and Ome increased the humans.30,31) Pascussi et al.32) observed that Dex in- mRNA levels of CYP3A4 in human hepatocytes and of creased both RXRa and PXR mRNA expression levels CYP3A8 in monkey hepatocytes, but not of CYP3A1 in through glucocorticoid receptor (GR)-mediated activa- rat hepatocytes. In contrast, treatment with Dex tion, whereas PXR activators such as Rif did not. markedly increased the mRNA level of CYP3A1 in rat Several lines of evidence suggest that the regulation of hepatocytes, but not of CYP3A4 in human hepatocytes CYP3A4 gene expression in human hepatocytes by or of CYP3A8 in monkey hepatocytes. These results glucocorticoids occurs through two distinct mechanisms indicate that the molecular mechanisms for the regula- involving the GR: ˆrst, by controlling the expression of tion of CYP1A2 genes diŠer between humans and the PXR and the constitutive androstane receptor cynomolgus monkeys, although the regulation mechan- (CAR) under physiological conditions through the isms of CYP1A1 and CYP3A genes are similar. classical GR pathway, and second, by activating the PXR under bolus or stress conditions.33) Acknowledgments: The authors would like to thank The molecular basis for species diŠerences in CYP3A David G. Spear, M.D., for reviewing this manuscript. induction by xenobiotics has already been reported.34,35) References It has also been reported that the ligand binding domains of monkey PXR show close similarity to those 1) Li, A. P., Rasmussen, A., Xu, L. and Kaminski, D. 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