Introduction

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CYP1A2 is one of the cytochrome P-450 (CYP or P-450)¹ enzymes in the human liver. This enzyme is constitutively expressed in the liver and catalyzes the metabolism of many drugs, such as theophylline, caffeine, phenacetin, and propranolol (Gonzalez, 1992). The enzyme also participates in the metabolic activation of chemical mutagens in cooked food, such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (Boobis et al., 1995). Of these mutagens, IQ has been identified as a carcinogen in rodents and monkeys (Sugimura, 1986; Wakabayashi et al., 1992). The activity of CYP1A2 is thus suspected to be one of the possible risk factors determining the carcinogenicity of heterocyclic amines in human beings.

It is well known that many species of P-450 are inducible after exposure to chemical compounds. Aryl hydrocarbons, 2,3,7,8-tetrachlorodibenzo-p-dioxin, isosafrole, and heterocyclic amines are known to induce the CYP1A gene family (Degawa et al., 1987; Gonzalez et al., 1993; Denison and Whitlock, 1995; Whitlock et al., 1996). Phenobarbital is one of the classical P-450 inducers; it induces forms of P-450 belonging to the CYP2A, CYP2B, CYP2C, and CYP3A gene families (Waxman and Azaroff, 1992; Gonzalez et al., 1993; Denison and Whitlock, 1995). Some studies have indicated that phenobarbital also induces CYP1A1 (McManus et al., 1986; Turner et al., 1988; Kärenlampi et al., 1989; Morris and Davila, 1996; Sadar et al., 1996). However, it had not been confirmed as to whether or not phenobarbital is capable of inducing CYP1A2 until one of our previous studies (Nemoto et al., 1995), which demonstrated that CYP1A2 mRNA could be induced by phenobarbital in female C57BL/6Ncrj (C57BL/6) mice in in vivo as well as in vitro experiments. In the present study, to analyze the mechanism of CYP1A2 induction by phenobarbital, we examined whether there is any strain difference in this phenomenon and in particular, any correlation between the induction of CYP1A2 by phenobarbital and the induction of CYP1A1 by polycyclic aryl hydrocarbons. We also compared the magnitude of the increases of CYP1A2 mRNA, protein, and enzyme activities to examine the possibility of post-transcriptional regulation.

	Experimental Procedures

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Materials. Phenobarbital sodium was purchased from Wako Pure Chemicals (Tokyo, Japan); barbital sodium, cyclobarbital, secobarbital, and pentobarbital sodium were purchased from Tokyo Kasei Kogyo (Tokyo, Japan); 3-methylcholanthrene (3-MC) was obtained from Sigma Chemical Co. (St. Louis, MO) and the [-³²P]dCTP was obtained from ICN Biomedicals, Inc. (Costa Mesa, CA). The radiolabeling kit and nylon or nitrocellulose membranes were obtained from Amersham (Aylesbury, UK) and the Taq DNA polymerase was purchased from Pharmacia (Uppsala, Sweden). Materials for cultivating hepatocytes were purchased from ICN Biomedicals Inc., Collaborative Research Inc. (Bedford, MA), and Kyokuto Seiyaku (Tokyo, Japan). Percoll and collagenase were products of Pharmacia and Sigma Chemical Co., respectively. Full-length cDNA for mouse CYP1A1 and CYP1A2 were generous gifts from Dr. D. W. Nebert (University of Cincinnati, Cincinnati, OH). The partial cDNA clone for mouse CYP2B10 was a gift from Dr. M. Negishi (U. S. National Institute of Environmental Health Sciences, Research Triangle Park, NC). Antibodies against rat CYP1A1 or rat CYP2B1 were generous gifts from Dr. Y. Funae (Osaka City University, Osaka, Japan). Salmonella typhimurium TA1535/pSK1002 was a generous gift from Dr. Y. Oda (Osaka Prefectural Institute of Public Health, Osaka). Other chemicals were of the highest grade commercially available.

Animal Treatment. Eight-week old male and female C57BL/6 and DBA/2NCrj (DBA/2) mice were purchased from Charles River Japan (Yokohama, Japan) and C3H/HeJSlc (C3H), AKR/JSea (AKR), NZB/NSlc (NZB), and ddY mice were obtained from Japan SLC (Hamamatsu, Japan). Regarding aryl hydrocarbon responsiveness, C57BL/6, C3H, and ddY mice are responsive, whereas DBA/2, AKR, and NZB mice are nonresponsive. Mice received a once-daily i.p. injection of phenobarbital sodium dissolved in saline at a dose of 80 mg/kg b.wt. for 3 days and were sacrificed 24 h after the last injection. For the preparation of positive control microsomes highly expressing CYP1A1 and CYP1A2 proteins for an immunoblot analysis, a female ddY mouse received a once-daily i.p. injection of 3-MC for 3 days at 20 mg/kg b.wt./day and was sacrificed 24 h after the last injection. The livers of all mice were excised immediately after sacrifice and used for the preparation of total RNA and microsomes.

Preparation of Primary Hepatocyte Cultures. The livers of female C57BL/6 mice weighing 20 to 25 g were perfused with collagenase and then viable hepatocytes were isolated by Percoll isodensity centrifugation as previously described (Nemoto et al., 1989; Nemoto and Sakurai, 1993). The culture conditions were the same as previously described (Nemoto et al., 1995).

Northern Blot Analysis. Total RNA was prepared from the liver by the method of Chomczynski and Sacchi (1987) using TRIzol reagent (Life Technologies, Gaithersburg, MD) and used for hybridization. Northern blotting was performed after the denatured RNA (10 µg) was size-fractionated on a 1.3% agarose gel containing formaldehyde. Hybridization proceeded at 42°C overnight in a mixture containing 50% formamide, 5× Denhardt's solution, 5× SSPE, salmon sperm DNA at 0.1 mg/ml, and ³²P-labeled cDNA probes. The cDNA probe of mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified by reverse transcription-polymerase chain reaction from the liver total RNA of a female C57BL/6 mouse. The primer sequences used were as follows: sense primer, 5'-TCCACTCACGGCAAATTCAACG-3'; antisense primer, 5'-TAGACTCCACGACATACTCAGC-3'. The blots were washed twice for 15 min with 2× SSC, 0.1% SDS at 65°C, and then twice for 15 min with 0.2× SSC, 0.1% SDS at 60°C. The intensity of the hybridized bands was measured with a bioimage analyzer (BAS2000; Fujix, Tokyo, Japan) and normalized with the intensity of the hybridized band with mouse GAPDH probe. The membranes were exposed to Fuji X-ray film at 80°C using an intensifying screen. The hybridized cDNA probe was stripped from the membranes between each hybridization by soaking the membranes in boiling water. The experiments were repeated at least twice and the same results were observed in all cases.

Immunoblot Analysis. Liver microsomes were prepared as described by Kamataki and Kitagawa (1974). The protein concentration was determined by the method of Bradford (1976) using a protein assay (BioRad Laboratories Inc., Hercules, CA) with BSA as a standard. Three micrograms of microsomal protein were resolved by SDS-polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane. The localized P-450 species were detected using rabbit polyclonal antibodies against rat CYP1A1 protein (Imaoka et al., 1990), which cross-reacted with the CYP1A2 protein, or antibodies against rat CYP2B1 protein (Imaoka et al., 1990), by a biotinylated goat anti-rabbit IgG and a biotinylated horseradish H avidin complex, followed by visualization with 3,3'-diaminobenzidine and hydrogen peroxide. Each membrane was scanned with an image scanner (Cano Scan 600; Canon Inc., Tokyo), and the signal intensity was quantified using the U. S. National Institutes of Health image analysis software program. In this study, levels of CYP1A2, 2B9, and 2B10 proteins were in the linear range for densitometry readings (ranging from 0.25 to 4 µg of microsomal proteins of phenobarbital-treated livers).

Analyses of Enzyme Activities. Methoxyresorufin-O-demethylase (MROD) and propoxyresorufin-O-dealkylase (PROD) activities were determined by the method of Sinjari et al. (1993) with minor modifications. Briefly, the reaction mixture containing 0.1 M Tris-HCl (pH7.8), 250 µM NADPH, 0.025 mg of liver microsomes, and 5 mM methoxyresorufin or propoxyresorufin in a final volume of 0.5 ml was incubated at 37°C for 3 min. The reaction was terminated by the addition of 2 ml of ice-cold methanol. After centrifugation at 3000g for 15 min, the amounts of resorufin in the aqueous solution were measured by the fluorescence intensity (excitation, 574 nm; emission, 596 nm). The P-450-mediated activation of a heterocyclic amine was measured by determination of the expression of the umu gene in S. typhimurium TA1535/pSK1002 in accordance with the method of Shimada and Okuda (1988). The mixture for the mutation assay consisted of 15 mM K-phosphate buffer (pH7.25), 0.025 mg of liver microsomes, an NADPH-generating system (0.5 mM NADP⁺, 5 mM glucose 6-phosphate, 5 mM MgCl2, and 1 U of glucose 6-phosphate dehydrogenase), 0.01 mM IQ, and the bacteria, in a final volume of 1 ml. The metabolic activation of IQ was measured by the induction of umu gene expression in the bacteria, which contained an umu C'-lac Z-fused gene that produced a hybrid protein with -galactosidase. The activity of -galactosidase was measured spectrophotometrically at 410 nm using o-nitrophenyl--D-galactoside as a substrate.

	Results

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Induction of CYP1A2 mRNA, Protein, and Enzyme Activity by Treatment with Phenobarbital. The effects of phenobarbital treatment on the CYP1A and CYP2B gene subfamilies were assessed by the expressions of mRNA, protein, and microsomal enzyme activities. Figure 1 shows the Northern blot hybridization of total RNAs from the livers of both sexes of C57BL/6, C3H, DBA/2, AKR, and NZB mice. Both sexes of all five of the mouse strains examined showed a constitutive expression of CYP1A2 mRNA. Phenobarbital treatment increased the amounts of CYP1A2 mRNA in the livers of all mouse strains in both sexes, and the magnitude of the induction ranged from 1.5- to 2.8-fold. In contrast, CYP1A1 mRNA was not detected in liver RNA from the nontreated mice or phenobarbital-treated mice. A constitutive level of CYP2B mRNA in the livers of all strains of mice was sexually dimorphic, with higher levels in females than in males. This is in accordance with our previous observation (Nemoto and Sakurai, 1995). The induction of CYP2B mRNA was detected in all strains. In the C3H and DBA/2 mice, the level of CYP2B mRNA induced was higher in the males than in the females. Significant sex differences in the amounts of induced CYP2B mRNA were not observed in other strains.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Induction of CYP1A2 and CYP2B9/10 mRNA by treatment with phenobarbital in the liver of various strains of mice. A, total RNA was extracted from hepatic tissues and Northern blotted using mouse CYP1A1, mouse CYP1A2, mouse CYP2B10, and mouse GAPDH cDNA probes. Each lane contains 10 µg total RNA from the liver of a nontreated (N) mouse and a phenobarbital-treated (P) mouse. The lane "Pc" contains 10 µg of total RNA from benzanthracene-treated C57BL/6 mouse hepatocytes as a positive control for hybridization with CYP1A1 and CYP1A2 cDNAs. The same membrane was used for all hybridizations. We were able to identify separate signals for CYP1A1 and CYP1A2 mRNA based on their migration in a 1.3% denatured-agarose gel. CYP2B10 cDNA probe does not distinguish mRNA of CYP2B10 from that of CYP2B9. Thus the signal obtained with this probe indicates the total level of CYP2B mRNA in mice. B, signal intensity was quantified by a bioimage analyzer (BAS2000). The signal obtained with each P-450 probe was normalized by that of GAPDH. The open and closed columns represent the mean ± S.D. of three nontreated and three phenobarbital-treated mice, respectively. Each column shows the value relative to that of the nontreated female group in the respective strain. The sample number of each group was three in all groups except the phenobarbital-treated female NZB mice (n = 2). Significance was examined using Student's t test between phenobarbital-treated and nontreated mice. *P < .05; **P < .01; ***P < .001; between male and female; ##P < .01; ###P < .001. M, male; F, female.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Induction of CYP1A2, CYP2B9, and CYP2B10 protein in the liver of various strains of mice by treatment with phenobarbital. A, three micrograms of liver microsomes were separated by SDS-polyacrylamide gel electrophoresis, blotted onto a nitrocellulose membrane, and then probed with antibodies against rat CYP1A1 (top) or rat CYP2B1 (bottom). The results of one mouse of each group (n = 3) are shown. Liver microsomes prepared from a 3-MC-treated ddY mouse were used as a positive control in the blotting for CYP1A enzymes. It is highly probable that the smallest immunoreactive band indicated by an arrow in the bottom image is a member of the CYP2B subfamily in mouse. However, this has not yet been confirmed. B, a membrane blotted with all samples of one strain was scanned with an image scanner and then analyzed using National Institutes of Health image software. The open and closed columns represent mean ± S.D. (n = 3) of nontreated and phenobarbital-treated mice, respectively. Each column shows the value relative to that of the nontreated female group in the respective strain. Significance was examined using Student's t test between phenobarbital-treated and nontreated mice. *P < .05; **P < .01; ***P < .001.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Increase of CYP1A2 enzyme activity by treatment with phenobarbital in liver microsomes of mice. Activities of MROD and PROD were determined. The mutagenic activation of IQ by liver microsomes from phenobarbital-treated or nontreated mice was determined using umu mutagenicity testing. The open and closed columns represent mean ± S.D. (n = 3) of the nontreated and phenobarbital-treated mice, respectively. Significance was examined using Student's t test between phenobarbital-treated and nontreated mice. *P < .05; **P < .01; ***P < .001.

Induction of CYP1A2 by Other Barbiturates. The ability of barbiturate derivatives to induce mouse CYP1A2 was examined in hepatocytes of C57BL/6 mice in primary cultures. The induction of CYP1A2 mRNA, ranging from 6.5- to 13-fold increases, was achieved by treatment with all barbiturates except barbital (Fig. 4). All barbiturates except barbital also induced CYP2B mRNA, with 2.8- to 7.3-fold increases. These results indicate that all barbiturates examined except barbital have the ability to induce CYP1A2 in mice.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Induction of CYP1A2 and CYP2B9/10 mRNA by barbiturates in female C57BL/6N mouse hepatocytes in primary culture. A, total RNA was prepared from mouse hepatocytes in primary culture that were exposed to 1 mM phenobarbital sodium, barbital sodium, secobarbital, cyclobarbital, or pentobarbital sodium for 24 h from day 3 to day 4. All treatments were performed in duplicate, and total RNA samples were loaded separately. Northern blotting was done as described in the legend to Fig. 1. B, signal intensity was quantified with a bioimage analyzer (BAS2000). The signal obtained with each P-450 probe was normalized by that of GAPDH. The columns represent the mean fold-induction of two dishes.

	Discussion

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Phenobarbital is one of the typical P-450-inducing chemicals and induces CYP2A, 2B, 2C, and CYP3A gene subfamilies in rats, monkeys, and humans (Barry and Feely, 1990; Waxman and Azaroff, 1992; Gonzalez et al., 1993; Ohmori et al., 1993; Denison and Whitlock, 1995). Some reports indicate that phenobarbital also induces CYP1A1 (McManus et al., 1986; Turner et al., 1988; Kärenlampi et al., 1989; Morris and Davila, 1996; Sadar et al., 1996). However, it had not been confirmed whether phenobarbital is capable of inducing CYP1A2 until our previous study (Nemoto et al., 1995), which demonstrated that phenobarbital can induce CYP1A2 as well as CYP2B10 and CYP2B9 in C57BL/6 mice. These observations were of interest because CYP1A2 is not generally considered to be a phenobarbital-inducible P-450. Regarding the induction of CYP1A2, polycyclic aryl hydrocarbons are well known CYP1A2 inducers (Gonzalez et al., 1993; Denison and Whitlock, 1995), although the mechanism underlying this induction is less understood than the mechanism of CYP1A1 induction, which can also be achieved by treatment with polycyclic aryl hydrocarbons (Gonzalez et al., 1993). However, the aryl hydrocarbon receptor (AhR)-mediated pathway is a candidate for the pathway underlying the induction of CYP1A2 by aryl hydrocarbons, because the induction by these chemicals is completely abolished in AhR knockout mice (Fernandez-Salguero et al., 1995).

We examined whether there is a correlation between the induction of CYP1A2 by phenobarbital and the response to polycyclic aryl hydrocarbon using five strains of mice that are known as either aryl hydrocarbon-responsive or -nonresponsive (Poland and Glover, 1975). The mechanism underlying the difference in responsiveness has not been completely clarified. It has been reported, however, that the low responsiveness of DBA/2 mice to aryl hydrocarbon is due to a low binding affinity of AhR to ligands because of the amino acid substitution and elongation of the C-terminus in AhR (Ema et al., 1994). We did not observe any correlation in the present study between aryl hydrocarbon responsiveness and CYP1A2 induction by phenobarbital, suggesting that AhR does not directly participate in the induction by phenobarbital in mice. This speculation is also supported by the present finding that CYP1A1, which is induced by aryl hydrocarbon through the AhR-mediated pathway, is not induced by phenobarbital even in aryl hydrocarbon-responsive mouse strains. It was recently established by two groups using AhR-null mice that AhR does not directly participate in the CYP1A2 induction by phenobarbital (Zaher et al., 1998; Corcos et al., 1998).

In the present study, when the magnitude of CYP1A2 induction was compared among three levels, i.e., mRNA, protein, and enzyme activities, the magnitude values were comparable. The differences in magnitude of the induction between mRNA and protein and between protein and enzyme activity within respective strains were less than 2-fold. Thus, it is highly probable that the CYP1A2 induction took place mainly at a pretranslational level. In accord with this possibility, Corcos et al. (1998) reported that CYP1A2 hnRNA in mouse liver was increased after treatment with phenobarbital. Based on these and the present findings, we speculate that the induction of CYP1A2 by phenobarbital is also a result of an increased rate of transcription, as has been suggested for the induction of rat CYP2B1 and CYP2B2 (Waxman and Azaroff, 1992).

In the present study, CYP1A1 mRNA was detected neither in liver RNA from the nontreated mice nor in those of phenobarbital-treated mice. This result is in accordance with our previous report (Nemoto et al., 1995) and the reports of Zaher et al. (1998) and Corcos et al. (1998), and is in contrast to earlier studies (McManus et al., 1986; Turner et al., 1988; Kärenlampi et al., 1989; Morris and Davila, 1996; Sadar et al., 1996). The reasons for the differences are not known at the present but may involve species-differences, difference between a liver and a hepatoma cell line, and specificities of antibodies and cDNA probes used for measurements of CYP1A1 expression.

To examine the ability of other barbiturates to induce CYP1A and CYP2B subfamilies, we exposed C57BL/6 mouse hepatocytes to five kinds of barbiturates. All barbiturates except barbital induced both subfamilies at comparable levels. This result indicates that barbiturates other than barbital have an intrinsic potential to induce CYP1A2 in mice. Of interest is the finding that barbital lacks the ability to induce CYP1A2 as well as CYP2B9 and CYP2B10. Because we observed the ability of barbital to induce CYP2B10 and CYP2B9 proteins in an in vivo experiment (not shown), the decomposition of the drug can be excluded as a possible reason for this finding. Another possibility is that the barbital concentration required for the induction is higher than those of other barbiturates.

The regulation mechanism of phenobarbital induction in mammals is mostly unknown. Possible cis-acting regulatory DNA elements for the induction of phenobarbital-inducible P-450s have been found. These are the so-called "Barbie Box" (He and Fulco, 1991) and "PBREM" (Honkakoski and Negishi, 1997). Thus, we compared nucleotide sequences between these elements and the 5'-flanking region of mouse Cypla2 gene (up to -890) (Gonzalez et al., 1985). However, no homologous sequence was found within the region reported.

Barbiturates, especially phenobarbital, have been widely used chronically. It is reasonable to speculate that even if the induction of CYP1A2 is slight, it has a large influence on the therapeutic efficacy of drugs, because the enzyme is one of the major constitutively expressed P-450s in humans. Therefore, it is necessary to clarify whether human CYP1A2 can be induced by barbiturate derivatives in further studies. There are at least two reports demonstrating a significant increase of theophylline clearance after chronic phenobarbital treatment in humans (Landay et al., 1978; Saccar et al., 1985). Additionally, Ratanasavanh et al. (1990) reported that phenobarbital treatment of a human hepatocyte coculture resulted in a slight increase of the N-3 demethylation of caffeine, which is catalyzed by CYP1A2 in human livers, as well as a slight increase in the overall caffeine metabolism. Therefore, it is possible that CYP1A2 is inducible by phenobarbital in humans as well as in mice.

CYP1A2 catalyzes the metabolic activation of a number of mutagenic or carcinogenic heterocyclic amines. Phenobarbital has pleiotropic effects on organisms, including the induction of other drug-metabolizing enzymes such as UDP-glucuronyltransferase and several glutathione S-transferases. These enzymes participate in detoxifying a number of active compounds that are activated by CYP1A2 via N-hydroxylation. Therefore, clarification of the effects of CYP1A2 induction after the administration of phenobarbital on drug metabolism and the carcinogenesis of heterocyclic amines in the whole body will require further study.