perinatal expression and inducibility of human cyp3a7 in c57bl/6n transgenic mice

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 228, 312–317 (1996)ARTICLE NO. 1658

Perinatal Expression and Inducibility of Human CYP3A7in C57BL/6N Transgenic Mice

Yong Li,* Tsuyoshi Yokoi,* Makoto Sasaki,* Kiyomi Hattori,*Motoya Katsuki,†,1 and Tetsuya Kamataki*,2

*Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan;and †Division of Molecular and Cellular Biology, Medical Institute of Bioregulation,

Kyushu University, Fukuoka 812, Japan

Received October 3, 1996

The expression and inducibility of CYP3A7 transgene in the fetus and suckling neonates from one ofthe transgenic lines (M10) were investigated by Northern and Western blot analyses. The mRNA expressioncould be detected as early as the 15th embryonic day and increased gradually with advancing gestation butthen remarkably so after birth. The protein expression was also detectable postnatally and increased.Inducibility was achieved in neonatal mice via maternal exposure to zinc sulfate. Midazolam hydroxylaseactivities could be detected in liver microsomes prepared from 14-day-old neonates. These activities weresignificantly higher in transgenic than nontransgenic lines of mice (põ0.001). q 1996 Academic Press, Inc.

The human fetus is susceptible to carcinogenesis and teratogenesis from maternal exposureto a wide range of harmful chemicals. Most of the chemicals undergo CYP-mediated biotrans-formations to exhibit their toxicological effects (1, 2). Exposure during gestation to manyxenobiotics such as drugs and environmental chemicals is believed to cause in utero death ofthe embryo or fetus, or bring about gross morphological defects in the surviving offspring.However, our knowledge about the underlying biochemical and molecular mechanism, or thedeterminants of susceptibility, is rather limited, especially in humans. It is well establishedthat human fetal tissues metabolize many foreign compounds (3) and endogenous substrates(4). Data from this and other laboratories have clearly shown that the human fetal liver differedfrom that of the experimental animals in terms of the expression of CYPs and capability ofbiotransforming chemicals (5-7). CYP3A7 was originally purified and cloned from humanfetal liver in our laboratory (8). It shares 95% nucleotide homology with CYP3A4 and isexpressed predominantly in human fetuses (9, 10). To get an insight into the physiologicalsignificance of the CYP3A7 expressed in human fetuses, we established several transgenicmouse lines carrying a CYP3A7 cDNA driven by a murine MT-1 promoter. In one of thetransgenic lines, M10, the CYP3A7 was expressed in the adult mouse liver with catalyticfunction towards aflatoxin B1 (11). We report here that the CYP3A7 transgene was expressedprenatally and postnatally in mice of the M10 transgenic line, and that CYP3A7 proteinexpressed in liver microsomes of suckling M10 neonates possessed detectable catalytic activityof midazolam 1*-hydroxylation.

1 Present address: Institute of Medical Sciences, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan.2 Corresponding author. Fax: /81-11-706-4978. E-mail: [email protected]: CYP, cytochrome P450, G-6-P, glucose 6-phosphate; HPLC, high-performance liquid chromatogra-

phy; MT-1, metallothionein 1; NaPi, sodium phosphate buffer; SDS-PAGE, sodium dodecyl sulfate-polyacrylamidegel electrophoresis.

0006-291X/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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AID BBRC 5635 / 6910$$$281 10-16-96 08:06:04 bbrca AP: BBRC

Vol. 228, No. 2, 1996 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS

Chemicals and reagents. Midazolam and its three metabolites (12) were provided by Hoffmann-La Roche Inc.(Nutley, NJ). Clonazepam was purchased from Sumitomo Chemical Inc. (Osaka, Japan). Other chemicals and reagentswere of the highest quality commercially available.

Animals and treatment. All the transgenic mice established were maintained and bred within transgenic animalfacilities in the Faculty of Pharmaceutical Sciences, Hokkaido University. A sterilized and ventilated environmentwith constant temperature at 23 { 1 7C, a relative humidity of 55 { 5%, and 12 h light-dark cycle is automaticallycontrolled by a central system. Animals were housed in plastic stainless-steel cages with free access to a commercialrodent chow (Clea Inc. Japan) and tapwater, both of which were autoclaved at 121 7C for 20 min prior to feeding.

To test the perinatal expression of the transgene, we used adult mice (8 weeks old) of line M10 and their non-transgenic littermates. About 4 female and 2 male mice were allowed copulation overnight, and the next morningwas designated as day 1 of gestation if a vaginal plug was confirmed. When necessary, the pregnant mice were treatedwith 25 mM ZnSO4 in drinking water one week prior to the day of tissue collection of the fetuses or neonates asspecified in the figure legends.

RNA preparation and Northern blot analysis. Total RNA was prepared from the livers of fetuses or neonates, andNorthern blot analysis was carried out using an [a-32P]-labeled CYP3A7 cDNA as a probe as described previously(11). The hybridized signal on the membrane was first quantified with a FUJIX Bio-Imaging Analyzer BA 2000(Fujifilm, Tokyo, Japan), and the band density on an X-ray film was further confirmed with an ATTO Densitographsystem (version 4.0, ATTO Inc, Tokyo, Japan) after 10 days of autoradiography at 080 7C.

Preparation of liver microsomes and immunoblot analysis. Liver microsomes were prepared from the fetuses andneonates as detailed by Kitada et al. (4). Immunoblot analysis was performed using antibodies to a CYP3A7 peptideraised in a rabbit as described previously (11). The expression level was quantified by determining the band densityon the membrane with the ATTO Densitograph system.

Hydroxylation of midazolam. Midazolam 1*-hydroxylase activity in liver microsomes prepared from 14-day-oldsuckling neonates was measured according to the method described by Kronbach et al. (12) with minor modifications.Briefly, microsomes (80 mg) were incubated in NaPi (pH 7.4) at 37 7C for 10 min in a final volume of 200 ml, in thepresence of an NADPH-generating system (0.2 unit of G-6-P dehydrogenase, 0.5 mM NADP, 5 mM G-6-P, 8 mMMgCl2) and 8 mM substrate dissolved in acetonitrile as a 10-fold concentrated solution. The reaction was terminatedwith 200 ml of ice-cold methanol followed by addition of 25 ng of clonazepam as an internal standard. After centrifugingat 3,000 rpm for 5 min, a 100-ml aliquot of the supernatant was subjected to HPLC using a computerized HPLCsystem (HITACHI model L-7000 series, Hitachikouki, Tokyo, Japan) equipped with a TSKgel ODS-120T C18 reversed-phase column (5mm, 4.6 1 150mm, TOSOH, Japan). Elution was carried out with a linear gradient of 60% solventA (10 mM NaOAc:methanol:acetonitrile Å 9:1:1, v/v/v, pH 4.0) to 50% solvent B (methanol:acetonitrile Å 2:1, v/v)over 20 min, with a flow rate of 1 ml/min at 40 7C. Elution of midazolam and its metabolites was monitored at 240nm. The retention times for 1*-hydroxymidazolam and midazolam were about 11 and 15 min, respectively, under theabove-mentioned condition.

RESULTS AND DISCUSSION

The result of Northern blot analysis revealed that the mRNA of the CYP3A7 transgene wasexpressed in the livers of both fetuses and neonates without treatment of mice with ZnSO4 asan inducer. The transcript was detectable on the 15th day of gestation, and increased sharplyafter birth (Fig. 1). Previous studies in our laboratory indicated that a low level of mouseCyp3a16 expression was detectable only after the 20th day of gestation, and that a trace amountof Cyp3a11 expression was detected only postnatally (13). The [a-32P]-labeled CYP3A7 cDNAused as a probe in the present study did not cross-hybridize with the mouse endogenousorthologous (Fig.1, lane NC) despite the fact that a high expression level of Cyp3a16 on the14th day of age in the mouse was found to be rather high (13).

Although prenatal expression of a few forms of CYP in the 2B and 1A subfamilies havebeen reported to be inducible in the mouse (2, 14), no firm information is currently availableregarding the constitutive expression of mouse CYPs including Cyp3a isozymes to an apprecia-ble level in fetal mice or in the fetal tissues of other animal species (2, 15). In the presentstudy, the protein expression was not detectable in the mouse fetal livers prenatally withimmunoblot assay, but was detected right after birth (Fig. 2). The expression of CYP3A7mRNA sharply increased after birth, decreased gradually afterwards to the bottom on day 10postpartum and elevated again on day 14 postpartum (Fig. 3). The protein expression of the

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FIG. 1. Perinatal expression of CYP3A7 mRNA in the fetal and neonatal livers of M10 mice. Total RNAs wereprepared from livers pooled from fetuses and neonates at different embryonic or neonatal ages as specified in the figure.An aliquot (20 mg) of RNA from each of the samples was formaldehyde-denatured and separated electrophoretically ina 1.0% agarose gel. The RNAs were blotted onto a nylon membrane, hybridized with a [a-32P]-labeled CYP3A7cDNA and visualized by autoradiography at 0807C for 2 weeks (upper). The same membrane was rehybridized witha [a-32P]-labeled mouse b-actin probe after the removal of the CYP3A7 probe (bottom). NC, negative control, RNAfrom nontransgenic neonates at 14 days old was applied. PC indicates positive control in which RNA from humanfetal livers was used.

transgene also increased shortly after birth, even though the increase was somewhat delayedwhen compared with that of mRNA expression (Fig. 3). Such lag behind of protein expressionwas also observed in fetal and neonatal rats by Borlakoglu et al. (16). In our transgenic mice,the CYP3A7 transgene was under the control of the murine MT-1 promoter (11). The expressionpattern of the CYP3A7 transgene at both mRNA and protein levels resembles that of the nativemurine MT-1 perinatal life of the mouse (17) and of the rat (18). In the mouse, the MT-1mRNA expression was increased gradually in the late gestational period, rose sharply to amaximum level around birth, and diminished afterwards (17). The decreasing level of MT-1expression postpartum coincides with a decrease of zinc level in the neonatal mice (17, 19).The changes of MT-1 mRNA level during fetal and neonatal life mirrors the alterations ofmaternal MT-1 and zinc levels during the same period (20). It is well established that themurine MT-1 promoter is inducible by many factors, such as heavy metal ions, glucocorticoids,interferons, and interleukins, to name but a few (21, 22). The elevated CYP3A7 mRNA

FIG. 2. Western blot analysis of CYP3A7 protein expressed in fetal and neonatal M10 mice. Microsomal proteins(20 mg) from fetal and neonatal livers were electrophoretically separated in a 7.5% SDS-polyacrylamide gel andsubsequently transferred onto an Immobilon membrane. The protein blots were detected by antibodies to a CYP3A7peptide raised in a rabbit and visualized by 3,3*-diaminobenzidine staining. Lane NC contained 20 mg of livermicrosomal protein from nontransgenic pups of 14 days old as a negative control. Lane PC contained microsomesfrom a human fetal liver as the positive control.

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FIG. 3. Relative perinatal expression levels of CYP3A7 mRNA and protein in M10 mice. The densities ofhybridized bands on the X-ray film in Fig. 1 as well as the immunodetectable bands on the membrane in Fig. 2 werequantified with an ATTO Densitograph system. The relative abundance of the transcripts was corrected with the b-actin signals. Zero indicates the day of birth, and 05 and 5 indicate 5 days before and after birth, respectively.

expression on day 14 postpartum may result from induction by factors present in maternalmilk. A similar increase of maternal MT-1 mRNA level on about day 15 postpartum wasobserved by Andersen et al. (20). The parallel expressions of CYP3A7 mRNA and protein inthis study render individual variations unlikely (Fig. 3).

On the other hand, significant induction was achieved in the transgenic newborns via lacta-tional transfer of zinc to the suckling pups (Fig. 4) although no measurable induction ofCYP3A7 transcript in fetal mice was observed (data not shown). The absence of inducibilityof CYP3A7 transcript in fetal mice was unlikely due to the inaccessibility of the inducingagent zinc in utero, since this metal ion is known to be readily transferred across the placentaand is intimately involved in the physiological processes of MT-1 synthesis (23). The bioavail-

FIG. 4. Inducibility of CYP3A7 mRNA in M10 neonates via maternal exposure to zinc sulfate. The M10 damswere either treated (closed column) or not treated (open column) with 25 mM ZnSO4 in drinking water for 1 weekprior to the sacrifice of their suckling pups at the ages indicated in the figure. Total RNAs were prepared from theneonatal livers and subjected to Northern blot analysis as detailed in the legend under figure 1. The relative expressionlevels were quantified by measuring the radioactivities of the hybridized bands on the membrane with a FUJIX Bio-Imaging Analyzer as described under Materials and Methods. The radioactivities of the hybridized band on day 3without zinc induction was arbitrarily assigned as 100%.

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FIG. 5. Midazolam 1*-hydroxylase activity in liver microsomes of M10 neonates. Liver microsomes were preparedfrom 5 specimens (each contained livers pooled from 2 suckling pups of 14 days old). The midazolam 1*-hydroxylaseactivity was measured as detailed under Materials and Methods. **Indicates significantly different from the non-transgenic control at põ0.001 (unpaired Student’s t test). NT, nontransgenic.

ability of zinc in utero can be evidenced by the fact that maternal gestational zinc deficiencyresults in a specific reduction of MT and zinc levels in fetuses (24). The low prenatal expressionlevel of the CYP3A7 transgene in M10 mice was similar to that of the native Cyp3a16 inmice (13) This, in addition to the poor prenatal inducibility of the CYP3A7 transgene, led usto speculate that some factors may exist in the maternal environment and affect the transcriptionand/or the stability of the transcript of the CYP3A7 transgene, as proposed by Borlakoglu etal. (16) and Wu et al. (25).

Midazolam 1*-hydroxylation is catalyzed exclusively by CYP3A isozymes (26, 27). CYP3A7was the predominant CYP expressed in the human embryonic liver (9, 28, 29). One of thedifficulties in the study of CYP3A7 is the scarcity of human fetal specimens. In addition,studies in our laboratory have shown that CYP3A7 is not easily expressed in E. coli (unpub-lished observation). To test the possible involvement of this fetal CYP3A isozyme in drugmetabolism, we used transgenic newborns as a model and midazolam as a probe substrate.As a result, we detected a significantly higher activity of the CYP3A7 enzyme in the M10newborns (Fig. 5). Gorski et al. suggested that CYP3A7 catalyzed the hydroxylation of midazo-lam in a regioselective manner, but this speculation was less conclusive due to the insufficiencyof human fetal liver samples used in that observation (30). In our study, no appreciable amountof 4-hydroxymidazolam was formed (data not shown). The present study is the first to assessthe in vivo catalytic property of a foreign P450 gene in transgenic mice. Further investigationis underway in this laboratory to delineate the catalytic function of CYP3A7 both in vitro andin vivo using adult transgenic mice.

ACKNOWLEDGMENTSThis work was supported in part by a Grant-in-Aid from The Ministry of Education, Science, Sports and Culture

of Japan. We thank the Department of Drug Metabolism and Pharmacokinetics, Hoffmann-La Roche, Nutley for theirassistance in the midazolam hydroxylase assay. Y. Li was supported by The Nagai Foundation Tokyo (October 1,1994 to March 31, 1995) and is currently a recipient of a fellowship from The Uehara Memorial Foundation, Japan.

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perinatal expression and inducibility of human cyp3a7 in c57bl/6n transgenic mice

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