Drug Metab. Pharmacokinet. 23 (1): 45–53 (2008). Regular Article Comparison of Inducibility of Sulfotransferase and UDP-Glucuronosyltransferase mRNAs by Prototypical Microsomal Enzyme Inducers in Primary Cultures of Human and Cynomolgus Monkey Hepatocytes

Masuhiro NISHIMURA1,AkikoKOEDA2,TakefumiSHIMIZU2,MitsuoNAKAYAMA1, Tetsuo SATOH2,3,ShizuoNARIMATSU4,andShinsakuNAITO1,* 1Department of Drug , Division of Pharmacology, Drug Safety and Metabolism, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima, Japan 2Ina Research Inc., Nishiminowa, Ina, Nagano, Japan 3Non-Profit Organization Human & Animal Bridging Research Institute, Ichikawa General Hospital, Ichikawa, Chiba, Japan 4Laboratory of Health Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima-naka, Okayama, Japan

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

Summary: We investigated the change of the mRNA levels of sulfotransferase and UDP-glucuronosyltran- sferase isoforms by the prototypical microsomal enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) in primary cultures of cryopreserved human and cynomolgus monkey hepatocytes. Real- time RT-PCR analysis was performed using primers and TaqMan probes. Rif, Dex, and Ome increased SULT2A1 mRNA level in both human and cynomolgus monkey hepatocytes in dose-dependent manner, but not SULT1A1 mRNA level. Rif, Dex, and Ome increased the mRNA level of UGT1A1 in both human and cynomolgus monkey hepatocytes, Ome more potently in humans and Rif and Ome more potently in monkeys. They also increased the mRNA levels of UGT1A6 and UGT1A9 in cynomolgus monkey hepato- cytes, though the extent of elevation of UGT1A6 and UGT1A9 mRNA levels was smaller than that of UGT1A1 mRNA level. Furthermore, these inducers scarcely affected UGT1A6 and UGT1A9 in human hepatocytes. Rif, Dex, and Ome also showed no remarkable effect on the mRNA levels of UGT2Bs in human or cynomolgus monkey hepatocytes. We also studied in detail the time course of mRNA expression of these enzymes in primary cultures of hepatocytes. In conclusion, the results of the present study show that primary cultures of hepatocytes isolated from the cynomolgus monkey are as useful as human hepatocytes for evaluating the induction of drug-metabolizing enzymes in preclinical studies.

Keywords: induction; sulfotransferase; UDP-glucuronosyltransferase; human; cynomolgus monkey; hepatocytes

hepatocytes, Rif and Dex, a , are potent inducers Introduction of CYP3A in human,1–3) Dex is a potent inducer of CYP3A in Prototypical microsomal enzyme inducers such as rifampi- rats,2,3) and Ome is a potent inducer of CYP1A in humans.3,4) cin (Rif), dexamethasone (Dex), and omeprazole (Ome) are Phase II metabolizing enzymes such as sulfotransferases (STs) well known to induce a superfamily of phase I drug-metaboliz- and UDP-glucuronosyltransferases (UGTs) are also targets of ingenzymesincludingcytochromeP450(CYP)enzymes, microsomal enzyme inducers. Several studies have demonstrat- which are important for the bioactivation and biotransforma- ed that STs and UGTs are upregulated by microsomal enzyme tion of xenobiotics. For example, in primary cultures of inducers such as Rif, Dex, and Ome.5–7) We have previously

Received; August 30, 2007, Accepted; November 15, 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

45 46 Masuhiro NISHIMURA, et al.

Table 1. Characteristics of hepatocytes and preparation of Isolation of cynomolgus monkey hepatocytes: Perfu- them sion of the cynomolgus monkey liver was performed according 13) 14) Age Sex Race/Strain Viability (%) to the Seglen method with some modifications. Briefly, the liver was perfused with Ca2＋-free Hanks solution containing Human 0.6 mM EGTA at 379C for 6–10 min and further perfused Donor #1 9 months male Caucasian 90 Donor #2 74 years female Caucasian 92 with Hanks solution containing 0.025% collagenase at 379C Donor #3 2 years female Caucasian 91 for 13–15 min. Hepatocytes were dispersed from the perfused Donor #4 6 days female Caucasian 91 liver in ice-cold HEPES solution. After filtration through gauze, Monkey the hepatocytes were separated from nonparenchymal cells by Monkey #1 3 years male Cynomolgus 80 centrifugation at 50×gfor1minat49C. The cells were Monkey #2 3 years male Cynomolgus 92 Monkey #3 2–3years male Cynomolgus 99 washed and resuspended in HEPES solution three times, and Monkey #4 2–3years female Cynomolgus 88 then washed with Williams' medium E containing 20% fetal Monkey #5 3 years female Cynomolgus 88 bovine serum, 2.5% polyvinylpyrrolidone, and 2% bovine se- 15) Viability was determined by Trypan blue dye exclusion. rum albumin (with the pH adjusted to approximately 7.4 with NaHCO3). Finally, the cells were resuspended in the same medium and cell viability was determined by the Trypan blue reported a novel method for evaluating the mRNA induction exclusion test. After addition of dimethyl sulfoxide (DMSO, fi- of phase II metabolizing enzymes by the enzyme inducers and nal concentration of 10%) to the cell suspension, cell density candidate drugs in primary cultures of human hepatocytes8–10) was approximately 1.5 to 2.5×107 viable cells/mL. The and in culture of HepG2 cells.11,12) However, there have been hepatocytes were cryopreserved at －309Cfor40minutes,at few studies of the comparison of inducibility of ST and UGT －809C for 15 minutes, and then in liquid nitrogen. Hepato- mRNAs in primary cultures of human and cynomolgus monkey cytes were stored in liquid nitrogen until use. hepatocytes following exposure to prototypical microsomal en- Monolayer culture of human and cynomolgus mon- zyme inducers (Rif, Dex, or Ome). key hepatocytes: Monolayer cultures of the cryopreserved In the present study, comparison of inducibility of ST and human and cynomolgus monkey hepatocytes were obtained ac- UGT mRNAs following exposure to Rif, Dex, or Ome was cording to the method of Nishimura et al.8) with some modifi- evaluated in primary cultures of cryopreserved hepatocytes cations.14) The cryopreserved human or cynomolgus monkey from humans and cynomolgus monkeys. Real-time RT-PCR hepatocytes were suspended in Hepatocyte Culture Medium. analysis was performed using an ABI PRISM 7700 Sequence The hepatocytes were centrifuged at 45×gfor3minat49C Detection System. and resuspended in the same medium. Numbers of cells were counted using a Coulter Counter (Beckman Coulter, Inc., Materials and Methods Fullerton, CA, USA). Human cell suspensions with viability Materials: Cryopreserved human hepatocytes (donor #1 rates of 90% to 92% as assessed by Trypan blue dye exclusion [Lot 079], donor #2 [Lot 100], donor #3 [Lot 130], and donor were used for the experiments, while cynomolgus monkey cell #4 [Lot RQO]) (Table 1) were purchased from In Vitro Tech- suspensions with viability rates of 80% to 99% as assessed by nologies, Inc. (Baltimore, MD, USA). Rif, Dex, and Ome were thesamemethodwereused(Table 1). The cell suspensions purchased from Wako Pure Chemical Industries, Ltd. (Osaka, were diluted to a final concentration of 2.5×105 viable Japan). Collagenase S-1 was purchased from Nitta Gelatin, Inc. cells/mL using Hepatocyte Culture Medium, and inocula of 1 (Osaka, Japan); bovine serum albumin (fraction V) from Sigma ×105 viable cells/0.4 mL/well were introduced into 24-well Aldrich (St. Louis, MO, USA): Trypan blue from Merck (Dar- plates that had been coated with type I collagen. The cells were mstadt, Germany) and Flow Laboratories, Ltd. (Irvine, UK); cultured for 3 hr after inoculation under 5% CO2 and 95% air Hepatocyte Culture Medium (CC-3198) from Cambrex Bio at 379C. The medium was then replaced with fresh medium, } Science Walkersville Inc. (Walkersville, MD, USA); RNeasy and the cells were cultured for 21 hr under 5% CO2 and 95% Mini Kit and QIAshredderTM from QIAGEN (Hilden, Germa- air at 379C. The medium was then replaced with fresh medi- ny); yeast tRNA from Life Technologies, Inc. (Rockville, MD, um without human epidermal growth factor (hEGF), hydrocor- USA); and TaqMan One-Step RT-PCR Master Mix Reagents tisone, gentamycin, or amphotericin B, and the cells were cul- from Applied Biosystems (Foster City, CA, USA). All other tured for 24 hr under 5% CO2 and 95% air at 379C. The cells chemicalsusedinthisstudywereofreagentgrade. were used for experiments at 48 hr after inoculation. Animals: Young adult male and female cynomolgus Treatment of primary cultures of human and monkeys were obtained from Ina Research Philippines, Inc. cynomolgus monkey hepatocytes with inducers: In the (Batangas, Philippines) (Table 1). These animals were allowed induction studies, Hepatocyte Culture Medium without hEGF, free access to food and water. The study was approved by the hydrocortisone, gentamycin, or amphotericin B was used. The Committee on the Care and Use of Laboratory Animals of Ina hepatocytes were exposed to the inducers Rif, Dex, and Ome Research Inc. and the Committee on the Care and Use of (2, 10 and 50 mM each) for 24 hr. All inducers were dissolved Laboratory Animals of Otsuka Pharmaceutical Factory, Inc. in DMSO at a final vehicle concentration of 0.1% (v/v). Con- Inducibility of ST and UGT in Humans and Cynomolgus Monkeys 47

Table 2. Primers and probes used for RT-PCR analysis of target mRNA of cynomolgus monkey

mRNA Sequence Position

SULT1A1 (PST) (GenBank accession number D85514) Forward primer 5?-ACTACACCACCATCCCCCA-3? 716–734 Reverse primer 5?-TCCCCAGTCATTCCTTTCCT-3? 788–769 Probe 5?-ATGGACCACAGCATCTCCCCCTTCAT-3? 742–767

SULT2A1 (HST) (GenBank accession number D85521) Forward primer 5?-GGGAGAGATCACCCTGGGTAG-3? 215–235 Reverse primer 5?-GGGAGGTGGGAGGAGAAGAG-3? 302–283 Probe 5?-TATAAATTACTCAGTGAAGAGGAGGGCCCACG-3? 250–281

UGT1A1 (GenBank accession number AF104339) Forward primer 5?-CCGCTCATTCAGATCACATGA-3? 593–613 Reverse primer 5?-AACCACGTCGCACAGAAAGTT-3? 678–658 Probe 5?-CTGCAGCGGGTGAAGAACATGCTCAT-3? 619–644

UGT1A6 (GenBank accession number AF104337) Forward primer 5?-CCTCAAGGAGAGCAAGTTCGA-3? 414–434 Reverse primer 5?-GTAGGCCCAAATACTCAGCCA-3? 496–476 Probe 5?-CTCTTTTCACAGACCCAGCCTTACCCTG-3? 437–464

UGT1A9 (GenBank accession number AF104336) Forward primer 5?-GATCTGGACCGGGAGTTCA-3? 253–271 Reverse primer 5?-TCAAAAATGTCATTGTAGGAACTCATTAT-3? 353–325 Probe 5?-TCGCCAGGCTCAATGGAAAGCACAAG-3? 279–304

UGT2B18 (GenBank accession number AF016310) Forward primer 5?-TGAGTTTGAGAATATCATCAGGCA-3? 252–275 Reverse primer 5?-TCCACATGATTTCTTGCATTTGT-3? 352–330 Probe 5?-CAAATTAAGAGATGGTCAGAACTTCCAAAAGATACATTT-3? 277–315

UGT2B20 (GenBank accession number AF072223) Forward primer 5?-GAGACGTCATGAGGTGACTGTGT-3? 144–166 Reverse primer 5?-ATATCTAGCAGTTTCATAAGAGAATCTTCC-3? 287–258 Probe 5?-TACTTTTGTCAATGACAGTAAATCATCTGCTATTAAATTTGA-3? 183–224

UGT2B30 (GenBank accession number AF401657) Forward primer 5?-TCAAATGACTTTCATGGAGAGAGTAA-3? 609–634 Reverse primer 5?-TGTAAAACTGATCCCACTTCTTCGT-3? 709–685 Probe 5?-CCATGCTTGAAACCAAAAGTCAAAATAAACCA-3? 681–650

trols were also exposed to DMSO at the same final concentra- total RNA according to a method described previously.14) The tion. Total RNA was extracted from the hepatocytes using the RT-PCR assay was performed using the ABI PRISM 7700 Se- QIAshredderTM and RNeasy} Mini Kit. quence Detector system (Applied Biosystems) under the same Oligonucleotides: The pairs of forward and reverse conditions as in our previous studies.14) primers and the TaqMan probes for human and cynomolgus Statistical analysis: Data analyses were performed with monkeys used in the RT-PCR sequences were designed using the ABI PRISM sequence detection software. Relative expres- Primer Express software (Applied Biosystems). Each primer sion of each mRNA was calculated as the DCt (the value ob- and probe was homology-searched by an NCBI BLAST search tained by subtracting the Ct value of the b-actin mRNA from to ensure that it was specific for the target mRNA transcript. the Ct value of the target mRNA). The amount of target rela- The primer pairs and the TaqMan probes for human targets tive to b-actinmRNAwasthusexpressedas2－(DCt).Valuesare and cynomolgus monkey b-actin have been reported else- expressed as the ratio of target mRNA to b-actin mRNA. The where,8,11,14,16–18) and those for cynomolgus monkey targets are experiments with the hepatocyte cultures shown in Figure 1 listed in Table 2. The primers and TaqMan probes were syn- were performed in triplicate, and the mean values were calcu- thesized by Sigma-Aldrich Japan K.K. Genosys Division lated. In particular, the data are shown as mean±SD for four (Ishikari, Japan). The TaqMan probes contained 6-carbox- humans or five cynomolgus monkeys. The experiments with yfluorescein (FAM) at the 5? end and 6-carboxytetramethyl- the hepatocyte cultures shown in Figures 2–5 also were per- rhodamine (TAMRA) at the 3? end, and were designed to formed in triplicate, and the mean values were calculated. Fur- hybridize to sequences located between the PCR primers. thermore, the values shown in Figures 2–5 indicate the TaqMan RT-PCR conditions: Total RNA was diluted to results relative to controls without DMSO, and the results are about 4 mg/mL with 50-mg/mL yeast tRNA. The RT-PCR assay shown as mean±SDforfourhumansorfivecynomolgus was performed in 50 mLofTaqManOne-StepRT-PCRMaster monkeys. Statistical analysis was performed using the paired Mix reagents containing 300 nM forward primer, 900 nM Student's t-test (two-tailed) with a significance level of reverse primer, 200 nM TaqMan probe, and about 20 ng of pº0.05. 48 Masuhiro NISHIMURA, et al.

Fig. 1. Changes in STs and UGTs mRNA expression in primary cultures of human hepatocytes Values are ratios of target mRNA to b-actin mRNA. Experiments (culture of human and cynomolgus monkey hepatocytes) were performed in triplicate using four or five each. Values are means±SD for four humans and five cynomolgus monkeys.

target mRNAs in the present study. The ratios of SULT1A1 Results and SULT2A1 mRNA to b-actin mRNA in both human and Time course of mRNA expression of STs and UGTs in cynomolgus monkey hepatocytes decreased during the first 24 primary cultures of human and cynomolgus monkey hr of culture, and then remained constant from 24 to 72 hr of hepatocytes: In the present study, mRNA expression of STs culture (Fig. 1). The ratios of UGT1As and UGT2Bs mRNA to and UGTs in primary cultures of four lots of cryopreserved hu- b-actin mRNA in both human and cynomolgus monkey he- man hepatocytes and five lots of cryopreserved cynomolgus patocytes also decreased during the first 24 hr of culture, and monkey hepatocytes was measured, and the differences in then remained constant from 24 to 72 hr of culture (Fig. 1). mRNA expression levels between the human and cynomolgus Change of SULT1A1 and SULT2A1 mRNA by Rif, monkey hepatocytes were determined. b-Actin was confirmed Dex, and Ome in primary cultures of human and to exhibit stable expression of several housekeeping genes un- cynomolgus monkey hepatocytes: Due to its ability to der the same experimental conditions.19) b-Actin mRNA was dissolve a large number of organic chemicals, DMSO is usually therefore used as an endogenous control for measurement of the solvent of choice in studies of this type. We also examined Inducibility of ST and UGT in Humans and Cynomolgus Monkeys 49

Fig. 2. Effects of exposure to drugs on expression of SULT1A1 and SULT2A1 mRNA in primary cultures of human and cynomolgus monkey hepatocytes Hepatocytes were treated with Rif, Dex, or Ome for 24 hr. Concentrations of Rif, Dex, and Ome were 2, 10, and 50 mM each. Values are ra- tios of target mRNA to b-actin mRNA. Experiments (culture of human and cynomolgus monkey hepatocytes) were performed in triplicate us- ing four or five livers each. Values are means±SD for four humans and five cynomolgus monkeys. Dotted line is 1 (value of control without DMSO), which was assigned a value of 1. *pº0.05, **pº0.01 and ***pº0.001 vs. controls at 0.1% DMSO. the effects of DMSO on induction of human drug-metabolizing exposure to Dex (2 to 50 mM), and by 2.1- to 3.6-fold by ex- enzyme and transporter mRNAs in primary cultures of posure to Ome (2 to 50 mM), compared with control at 0.1% cryopreserved human hepatocytes, and found that 0.1% DMSO (Fig. 3). The mRNA levels of UGT1A6, UGT1A9, DMSO appeared to have no effects on induction.9) We further UGT2B7, and UGT2B10 mRNA were not increased by ex- confirmed that induction of the drug-metabolizing enzyme and posure to any of the inducers at 2, 10, or 50 mM(Figs. 3 and transporter mRNAs could be evaluated after 24 hr of exposure 4). The level of UGT2B15 mRNA was decreased by 0.75- to toinducerssuchasRifandOme.8,20) Therefore, 0.1% DMSO 0.90-fold by exposure to Rif (2 to 50 mM), by 0.43- to 0.60-fold was used and hepatocytes were exposed to inducers for 24 hr by exposure to Dex (2 to 50 mM), and by 0.74- to 0.92-fold by in the present study. In primary cultures of both human and exposure to Ome (2 to 50 mM), compared with control at 0.1% cynomolgus monkey hepatocytes, SULT1A1 mRNA expression DMSO (Fig. 4). The UGT1A1 mRNA level in primary cultures levels were not changed or only slightly changed by exposure of cynomolgus monkey hepatocytes was increased by 3.8- to to any of the inducers at 2, 10, or 50 mM(Fig. 2). The 8.7-fold by exposure to Rif (2 to 50 mM),by1.1-to2.2-foldby SULT2A1 mRNA level in primary cultures of human hepato- exposure to Dex (2 to 50 mM), and by 2.3- to 11.1-fold by ex- cytes were increased by 1.1- to 1.3-fold by exposure to Rif (2 to posure to Ome (2 to 50 mM), compared with control at 0.1% 50 mM), by 1.7- to 2.1-fold by exposure to Dex (2 to 50 mM) DMSO (Fig. 3). They also increased the UGT1A6 and and by 1.5- to 1.6-fold by exposure to Ome (2 to 50 mM), com- UGT1A9 mRNA levels as well as UGT1A1 mRNA level in pared with control at 0.1% DMSO (Fig. 2). SULT2A1 mRNA cynomolgus monkey hepatocytes, though the extent of the ele- levels in primary cultures of cynomolgus monkey hepatocytes vation of UGT1A6 and UGT1A9 mRNA levels was smaller were increased by 4.9- to 26.0-fold by exposure to Rif (2 to 50 than that of UGT1A1 (Fig. 3). Neither the mRNA level of mM), by 5.6- to 15.4-fold by exposure to Dex (2 to 50 mM), and UGT2B18 nor UGT2B20 was increased by exposure to any of by 1.3- to 9.4-fold by exposure to Ome (2 to 50 mM), com- the inducers at 2, 10, or 50 mM(Fig. 5). The level of pared with control at 0.1% DMSO (Fig. 2). These findings in- UGT2B30 mRNA was decreased by 0.85- to 0.92-fold by ex- dicate that SULT2A1 mRNA is increased by the inducers in posure to Rif (2 to 50 mM) and by 0.61- to 0.72-fold by ex- both human and cynomolgus monkey hepatocytes, and that the posure to Dex (2 to 50 mM), compared with control at 0.1% sensitivity to the inducers in the elevation of SULT2A1mRNA DMSO (Fig. 5). On the other hand, UGT2B30 mRNA levels level is higher in cynomolgus monkeys than in humans. were increased by 1.3- to 2.0-fold by exposure to Ome (2 to 50 Change of UGT1As and UGT2Bs mRNA by Rif, Dex, mM) compared with the control (Fig. 5). and Ome in primary cultures of human and cynomolgus Discussion monkey hepatocytes: The level of UGT1A1 mRNA in pri- mary cultures of human hepatocytes was increased by 1.3- to Several studies have demonstrated that STs and UGTs are 1.5-fold by exposure to Rif (2 to 50 mM), by 2.1- to 2.4-fold by upregulated by microsomal enzyme inducers in rat5,21) and hu- 50 Masuhiro NISHIMURA, et al.

Fig. 3. Effects of exposure to drugs on expression of UGT1As mRNA in primary cultures of human and cynomolgus monkey hepatocytes Hepatocytes were treated with Rif, Dex, or Ome for 24 hr. Concentrations of Rif, Dex, and Ome were 2, 10, and 50 mM each. Values are ra- tios of target mRNA to b-actin mRNA. Experiments (culture of human and cynomolgus monkey hepatocytes) were performed in triplicate us- ing four or five livers each. Values are means±SD for four humans and five cynomolgus monkeys. Dotted line is 1 (value of control without DMSO), which was assigned a value of 1. *pº0.05, **pº0.01 and ***pº0.001 vs. controls at 0.1% DMSO. man hepatocytes.6,7,22) However,therehavebeenfewstudies Dex.21) These findings suggest that inducibility of SULT2A1 on mRNA induction of these STs and UGTs in primary cul- mRNA is similar among human, cynomolgus monkey, and rat tures of human and cynomolgus monkey hepatocytes. There- hepatocytes, but that species differences exist in the inducibili- fore, in the present study, we have chosen to examine the STs ty of SULT1A1 mRNA. andUGTs,whichareexpressedathighmRNAlevelsinhu- The UGTs are a superfamily of typical membrane-bound en- man18) and cynomolgus monkey liver (Nishimura M., unpub- zymes that detoxify a large variety of xenobiotics and en- lished observations), and compared the inducibility of these dogenous substrates via the addition of UDP-glucuronate, and mRNAs in human and cynomolgus monkey hepatocytes. human UGTs have been grouped into the UGT1A and UGT2B Runge-Morris et al.21) found that Dex upregulates SULT1A1 families.23) However, there have been few studies on mRNA in- mRNA expression in primary cultured rat hepatocytes. On the ductionofUGTsinprimaryculturesofhumanandcynomol- other hand, the results of the present study indicated that the gus monkey hepatocytes. Therefore, in the present study, we level of SULT1A1 mRNA expression was only slightly changed directly compared the effects of prototypical microsomal en- by exposure to Dex in primary cultured human and cynomol- zyme inducers such as Rif, Dex, and Ome on the expression of gus monkey hepatocytes. Furthermore, the results of the UGTs mRNA in primary culture of hepatocytes derived from present study indicate that Rif, Dex, and Ome markedly up- humans and cynomolgus monkeys, and obtained several in- regulate SULT2A1 mRNA expression in human and cynomol- teresting findings regarding differences in the alteration of gus monkey hepatocytes. Rif22) and Dex6,22) have been identi- UGTs mRNA levels between humans and cynomolgus monkey. fied as SULT2A1 inducers in human hepatocytes. Expression Rif, Dex, and Ome elevated the mRNA level of UGT1A1 in of SULT2A1 mRNA in rat hepatocytes is also increased by both human and cynomolgus monkey hepatocytes, Ome more Inducibility of ST and UGT in Humans and Cynomolgus Monkeys 51

Fig. 4. Effects of exposure to drugs on expression of UGT2Bs Fig. 5. Effects of exposure to drugs on expression of UGT2Bs mRNA in primary cultures of human hepatocytes mRNA in primary cultures of cynomolgus monkey hepato- Hepatocytes were treated with Rif, Dex, or Ome for 24 hr. Concen- cytes trations of Rif, Dex, and Ome were 2, 10, and 50 mM each. Values Hepatocytes were treated with Rif, Dex, or Ome for 24 hr. Concen- are ratios of target mRNA to b-actin mRNA. Experiments (culture of trations of Rif, Dex, and Ome were 2, 10, and 50 mMeach.Values human hepatocytes) were performed in triplicate using four livers are ratios of target mRNA to b-actin mRNA. Experiments (culture of each. Values are means±SD for four humans. Dotted line is 1 cynomolgus monkey hepatocytes) were performed in triplicate us- (value of control without DMSO), which was assigned a value of 1. ing five livers each. Values are means±SD for five cynomolgus *pº0.05 and **pº0.01 vs. controls at 0.1% DMSO. monkeys. Dotted line is 1 (value of control without DMSO), which was assigned a value of 1. *pº0.05 vs. controls at 0.1% DMSO. potently in humans and Rif and Ome more potently in monkeys. They also increased the mRNA levels of UGT1A6 results of the present study indicate that Rif, Dex, and Ome and UGT1A9 in cynomolgus monkey hepatocytes, though the markedly upregulate SULT2A1 and UGT1A1 mRNA expres- extent of the elevation of UGT1A6 and UGT1A9 mRNA levels sion in cynomolgus monkey hepatocytes as well as in human was smaller than that of UGT1A1. Moreover, these inducers hepatocytes. The SULT2A1 and UGT1A1 genes may thus be scarcely affected the mRNA levels of UGT1A6 and UGT1A9 regulated by CAR, PXR, and/or Ah receptor in cynomolgus in human hepatocytes. Rif, Dex, and Ome also showed no monkeysaswellasinhumans. remarkable effect on the mRNA levels of UGT2Bs in human or In this study, we estimated only the levels of mRNA of STs cynomolgus monkey hepatocytes. These findings indicate that and UGTs. As described in our previous report,8) we think that the pattern of mRNA expression of UGTs in cynomolgus the alteration in the mRNA levels, rapidly determined by the monkey hepatocytes is similar to that in human hepatocytes, real time RT-PCR, may cause a change in the levels of proteins and that the sensitivity to the inducers in the elevation of or activities of target enzymes. The preset data on the altered mRNA levels of UGTs is higher in cynomolgus monkeys than mRNA levels should prompt future studies, in which a large in humans, which is similar to the case of SULT1A2 mRNA. amount of samples is required to evaluate possible changes in It has been reported that pretreatment of human hepatocyte the levels of the corresponding proteins and activities. cultures with Dex, a PXR activating agent, produced concen- In conclusion, the mRNA induction profiles of SULT2A1 tration-dependent increases in human SULT2A1 mRNA and and UGT1A1 in hepatocytes pretreated with Ome, Rif, and protein contents,6) indicating that the human SULT2A1 gene is Dex were similar in cynomolgus monkeys and humans. The regulated by PXR. 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