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Effects of β-ionone on the expression of cytochrome P450s and NADPH-cytochrome P450 reductase in Sprague Dawley rats
Chemico-Biological Interactions 114 (1998) 97 – 107
Effects of b-ionone on the expression of cytochrome P450s and NADPH-cytochrome P450 reductase in Sprague Dawley rats Tae Cheon Jeong a,*, Hee Kyoung Gu a, Young Jin Chun b, Chul-Ho Yun c, Sang Seop Han a, Jung Koo Roh a a
Toxicology Research Center, Korea Research Institute of Chemical Technology, PO Box 107, Yusung, Taejon 305 -606, South Korea b College of Pharmacy, Chung-Ang Uni6ersity, Seoul, South Korea c Department of Biochemistry, Pai-Chai Uni6ersity, Taejon 302 -735, South Korea Received 14 November 1997; received in revised form 2 April 1998; accepted 15 April 1998
Abstract We have recently reported that b-ionone induces cytochrome P450 (P450) 2B1 in rats. Effects of b-ionone on the expression of other P450 isozymes and NADPH-P450 reductase were further investigated in Sprague Dawley rats. Administration of b-ionone subcutaneously 72 and 48 h before sacrificing the animals not only significantly induced the liver microsomal activities of P450-associated enzymes and NADPH-P450 reductase, but also clearly increased in the level of P450 1A1/2, P450 2C, and NADPH-P450 reductase proteins. The induction of P450 1A1/2 and 2C by b-ionone was much greater in male than in female as measured by western immunoblotting. Reverse transcriptase-polymerase chain reactions showed that, in addition to P450 2B1 and 2B2 mRNAs, P450 1A2, 2C6 and NADPH-P450 reductase mRNAs were increased when b-ionone was administered. Our previous and present results indicated that b-ionone may induce several P450s and NADPH-P450 reductase by the accumulation of their corresponding mRNAs. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: b-Ionone; Cytochrome P450; Induction; Sprague Dawley rats
* Corresponding author. Tel.: +82 42 8607470; fax: + 82 42 8607488. 0009-2797/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0009-2797(98)00044-1
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1. Introduction Many compounds in plants have been reported to modulate cytochrome P450 (P450) expression in experimental animals. For example, methoxsalen found in celery, parsnip and fig suppressed P450 1A1 induction in mouse hepatoma cells [1] and myristicin found in parsley, carrot, black pepper, nutmeg and flavoring agents induced liver P450 1A1/2, 2B1/2 and 2E1 in rats [2]. In addition, some phytochemicals like capsaicin in red peppers and diallyl sulfide in garlic have been characterized to possess chemopreventive effects against mutagenesis and tumorigenesis by inhibiting P450 2E1 which is responsible for bioactivating vinyl carbamate and dimethylnitrosamine [3]. Therefore, extensive efforts for evaluating the effects of compounds abundant in plants should be made, with a particular emphasis on P450 expression, because the modulation of P450 by certain compounds in dietary plants may interfere with drug action in the body and also play a role in chemical-induced carcinogenesis. b-Ionone (4-[2,6,6-trimethyl-1-cyclohexen-1-yl]-3-buten-2-one), which is a basic nucleus structure of retinoic acid, b-carotene and vitamin A, was first isolated from the volatile oil of Boronia megastigma Nees (Rutaceae) and also found in many plants including tea, tangelo and tomato [4]. In 1969, Parke and Rahman originally found that the pre-treatment with b-ionone induced the activity of biphenyl 4-hydroxylase and the content of P450, and that hexobarbital sleeping times were reduced by b-ionone [5]. Thereafter, b-ionone has been introduced as a model inducer of P450 in studying the role of metabolic activation mediated by P450 enzymes in cocaine-induced hepatotoxicity [6,7]. Recently, b-ionone was also shown to potentiate suppression of the antibody response induced by cocaine in female B6C3F1 mice, when the mice were pre-treated with b-ionone, suggesting that certain P450 isozyme(s) responsible for cocaine activation might be induced [8]. Most recently, we found that b-ionone clearly induced P450 2B1 through the accumulation of mRNA in Sprague Dawley rats, which are one of the most useful animal models in studying drug metabolism and chemical-induced toxicity [9]. In the same studies, the induction of P450 2B1 by b-ionone was much greater in male rats than in female rats, as measured by pentoxyresorufin O-dealkylase and western immunoblotting. The primary objective of the present study was to characterize the inductive effects of b-ionone on liver microsomal P450 enzymes and NADPH-P450 reductase in Sprague Dawley rats. Since the information on the effects of b-ionone on P450 induction is very limited, sex differences in P450 induction were also studied. b-Ionone was administered subcutaneously 72 and 48 h before sacrificing animals, as has been used in determining the role of metabolic activation in cocaine-induced hepatotoxicity and immunotoxicity in which b-ionone was a model inducer of P450 responsible for the activation of cocaine to its toxic metabolites [6,7,10].
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2. Materials and methods
2.1. Animals Specific pathogen-free male and female Sprague Dawley rats were obtained from the animal breeding laboratory at this institute. The animals received at 4–5 weeks old were acclimated for at least 1 week. Upon arrival, rats were radomized and housed four or five per cage. All animals were maintained on gamma-irradiated Jeil Lab Chow (Taejon, Korea) and UV-irradiated tap water ad libitum. Animals at 6–8 weeks old, ranging 200 – 250 g, were used in these studies. The animal quarters were strictly maintained at 23 9 3°C and 40–60% relative humidity. A 12-h light/dark cycle was used with an intensity of 150–300 Lux.
2.2. Materials b-Ionone, ethoxyresorufin, resorufin, aminopyrine, p-nitrophenol, erythromycin, NADPH, glucose 6-phosphate, cytochrome c, and nitrocellulose filters were purchased from Sigma (St. Louis, MO). Alkaline phosphatase-conjugated goat antirabbit IgG antibody, alkaline phosphatase substrate kit, and a kit for protein determination were purchased from Bio-Rad Laboratory (Richmond, CA). The kits for mRNA purification and first-strand cDNA synthesis were purchased from Pharmacia Biotech (Uppsala, Sweden). All other chemicals used were of reagent grade commercially available.
2.3. Animal treatment and microsome preparation Animals were treated subcutaneously with b-ionone in corn oil at 300, 600 and 1200 mg/kg 72 and 48 h before sacrificing animals by CO2 asphyxiation. The dose was selected from the previous studies in which b-ionone was used as a model inducer of P450 [6,7,10]. The control was given corn oil at 10 ml/kg. The microsomes were prepared from individual livers of four animals as described in our earlier studies [9] and were resuspended in 0.1 M potassium phosphate buffer, pH 7.4, containing 20% glycerol and 0.1 mM EDTA. Aliquots of liver microsomes were stored at − 70°C until use. The content of microsomal protein was determined according to the method of Bradford using bovine serum albumin as a standard [11]. For studying mRNA expression, male rats (two per groups) were treated with 600 mg/kg of b-ionone in corn oil subcutaneously once 6 h before sacrificing animals. The control was given corn oil at 10 ml/kg.
2.4. Monooxygenase assays Ethoxyresorufin O-deethylase (EROD) activity was determined as described by Blank et al. [12] with a slight modification. The reaction mixture (2 ml) consisted of 0.1 M potassium phosphate buffer, pH 7.4, containing 2 mg/ml of bovine serum albumin, 10 mM dicumorol, 5 mM glucose 6-phosphate, 1 U of glucose 6-phos-
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phate dehydrogenase, 5 mM NADPH and 2.5 mM 7-ethoxyresorufin. The formation of resorufin was monitored fluorometrically at an extinction maximum of 550 nm and an emission maximum of 585 nm. NADPH-cytochrome c reductase activity was determined as a surrogate for NADPH-P450 reductase activity by measuring the reduction rate of cytochrome c with an extinction coefficient of 21 mM − 1 cm − 1 at 550 nm following the addition of 0.12 mM NADPH [13]. p-Nitrophenol hydroxylase (PNPH) activity was determined as described by Koop [14]. The reaction mixture (1.0 ml) was composed of 0.1 M potassium phosphate buffer, pH 7.4, containing 100 mM p-nitrophenol, 1 mM NADPH and an enzyme source. The amount of 4-nitrocatechol formed was measured spectrophotometrically at 512 nm. Aminopyrine N-demethylase (APDM) and erythromycin N-demethylase (ERDM) activities were determined by measuring the amount of formaldehyde formed, as described previously [15,16]. Aminopyrine at 1.0 mM and erythromycin at 400 mM were used as substrates for assaying APDM and ERDM, respectively. The amount of formaldehyde formed was determined spectrophotometrically at a wavelength of 412 nm.
2.5. Western immunoblotting analyses Microsomal proteins (10 mg/well) were resolved by 10% SDS-PAGE and were transferred to nitrocellulose filters. The filters were incubated with 2.5% non-fat dry milk for 30 min to block the non-specific binding, and then were incubated with rabbit polyclonal antibodies against rat P450 1A1/2, 2C, and 3A, and NADPHP450 reductase, respectively, followed by an alkaline phosphatase-conjugated goat anti-rabbit IgG antibody. The primary antibody against each isozyme was prepared as described previously [17]. For immunostaining, the nitrocellulose filters were developed with a mixture of 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium and 0.1 M Tris buffer (1:1:10) under an instruction by the manufacturer.
2.6. mRNA extraction and re6erse transcriptase-polymerase chain reaction (RT-PCR) Cellular poly(A + ) mRNA from each animal was isolated and reverse transcribed using the commercially available kits under the manufacturer’s instruction. The reaction was stopped by incubating the mixture in boiling water bath for 5 min. A 5-ml aliquot of cDNA corresponding to approximately 20 ng of mRNA from each sample was amplified for 30 cycles by PCR in 50 ml of a mixture containing 50 pmol of specific primers, 1 mM dNTPs, 10x PCR buffer (100 mM Tris–HCl, 500 mM KCl, pH 8.3), and 0.5 ml of Taq polymerase (Pharmacia Biotech). The rat P450 1A2-, 2B1-, 2B2-, 2C6-, and NADPH-P450 reductase-specific primers producing 302-, 443-, 534-, 275-bp, and 407-bp PCR products, respectively, were synthesized at either Integrated DNA Technologies, Inc. (Coralville, IA) or Genosys Biotechnologies (Woodlands, TX). Glucose 3-phosphate dehydrogenase (G3PDH)specific primers producing 983-bp PCR product was also used as a control in this study. The sequence of each sense and anti-sense primer for P450 1A2, 2C6, and
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G3PDH has been described elsewhere [18–20]. The sequences for P450 2B1, 2B2, and NADPH-P450 reductase mRNAs were designed around the active centers: P450 2B1 sense primer, 5%-TTGTTTGGTGCTGGGACAGAG-3%; P450 2B1 antisense primer, 5%-GGCTAGGCCCTCTCCTGCACA-3%; P450 2B2 sense primer, 5%-CTCTTCTTTGCTGGCACTGAG-3%; P450 2B2 antisense primer, 5%-GGCAATGCCTTCGCCAAGACA-3%; NADPH-P450 reductase sense primer, 5%-GGCTCCCAGACGGGAACCGCT-3%; NADPH-P450 reductase antisense primer, 5%-CCTCCACGTGATGAAATCCTC-3%. RNA concentration and PCR cycle were titrated to obtain linearity of signal strength. The PCR products (10 ml/well) were visualized by UV illumination following an electrophoresis through a 1.5% agarose gel in Tris – borate – EDTA buffer containing 0.5 mg/ml of ethidium bromide.
2.7. Statistics Dunnett’s t-test was used to compare the significance of data obtained. The significant value at P B0.05 was represented as an asterisk.
3. Results Table 1 shows the effects of b-ionone on the activities of EROD, PNPH, APDM, ERDM and NADPH-P450 reductase in rat liver microsomes. The EROD activity was significantly induced by treatment with b-ionone from 300 mg/kg in both male and female rats. The activities of PNPH and APDM were induced at higher doses in both sexes. The ERDM activity was not changed by b-ionone in male rats, while the activity was significantly decreased in female rats at 300 and 600 mg/kg of b-ionone. The NADPH-P450 reductase activity was clearly induced from 300 mg/kg of b-ionone only in male rats. The constitutive enzyme activities were much greater in male rats than in female rats except NADPH-P450 reductase, as previously observed in PROD induction [9]. In Fig. 1, the liver microsomes prepared from b-ionone treated rats were electrophoresed and immunoblotted with polyclonal antibodies against either one of the following rat P450 1A1/2, 2C, 3A or NADPH-P450 reductase. Apparent changes in contents of P450 1A1/2, 2C and NADPH-P450 reductase proteins were observed in b-ionone-treated animals. The level of P450 1A1/2 and 2C induced by b-ionone was also higher in male rats than in female rats. Following the observation that the induction of P450 2B1 by b-ionone might be regulated by the accumulation of mRNAs in the previous work [9], the effects of b-ionone on P450 1A2, 2C6 and NADPH-P450 reductase mRNAs were further examined by using RT-PCR of mRNAs isolated from b-ionone-treated male rat livers in the present study. In addition, effects on the expression of P450 2B1 and 2B2 mRNAs were compared. b-Ionone was treated for 6 h, because our previous study implied that the P450 2B1 mRNA expression reached a maximum 6 hr after the treatment with b-ionone [9]. TCDD, a well-known inducer of P450 1A1/2, was used as a model inducer in the present study. As shown in Fig. 2, TCDD induced
Male
Female 0.76 90.04 1.69 90.13* 1.97 90.12* 2.81 90.39*
Male 1.20 9 0.18 2.09 90.19* 1.85 90.16* 2.34 90.25*
146 9 17 158 9 9 186 934 192 9 0.18*
PNPHb
ERODa
75 99 90 9 13 89 9 10 125 917*
Female 1.37 9 0.18 1.58 9 0.04 1.57 90.14 2.01 90.18*
Male
APDMc
0.9190.13 1.169 0.13 1.3090.06* 1.719 0.06*
Female
The rats were administered b-ionone in corn oil subcutaneously 72 and 48 h before sacrificing animals. Each value represents the mean activity 9 S.E. of four animals. An asterisk indicates the value significantly different from the vehicle control at PB0.05. a Ethoxyresorufin O-deethylase, nmol resorufin formed/min per mg protein. b p-Nitrophenol hydroxylase, pmol 4-nitrocatechol formed/min per mg protein. c Aminopyrine N-demethylase, nmol formaldehyde formed/min per mg protein. d Erythromycin N-demethylase, pmol formaldehyde formed/min per mg protein. e NADPH-P450 reductase, nmol/min per mg protein.
Vehicle 300 600 1200
b-Ionone (mg/kg)
Table 1 Effects of b-ionone on liver microsomal monooxygenases in Sprague Dawley rats
3359 48 309921 3349 40 388942
Male
ERDMd
128 911 79 9 7* 91 96* 143 9 14
Female
439 8 1199 4* 1329 40* 1039 7*
Male
Reductasee
128 9 13 112927 1839 16 98 914
Female
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the accumulation of P450 1A2, 2B1, 2B2 and 2C6 mRNAs, although undesirable minor bands were also observed in our present experimental conditions. In addition to P450 2B1 and 2B2 mRNAs, the amount of P450 1A2, 2C6 and NADPH-P450 reductase mRNAs were clearly increased by b-ionone when compared to the
Fig. 1. Western immunoblotting analyses for P4501A1/2, 2C, 3A, and NADPH-P450 reductase in liver microsomes. The liver microsomal proteins (10 mg/well) prepared from rats administered b-ionone at 300, 600 and 1200 mg/kg were resolved on 10% SDS-PAGE. The results represent typical experiments conducted at least twice for individual isozymes with same microsomes used in Table 1. A, P4501A1/2; B, P4502C; C, P4503A; D, NADPH-P450 reductase. Lane 1 and 5, vehicle control; lane 2 and 6, b-ionone 300 mg/kg; land 3 and 7, b-ionone 600 mg/kg; lane 4 and 8, b-ionone 1200 mg/kg. Lane 1 – 4, male rats; lane 5–8, female rats. M, purified rat NADPH-P450 reductase. The lower bands seen in A, B and C were non-specific binding.
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Fig. 2. RT-PCR amplification of P450 1A2, 2B1, 2B2, 2C6, and NADPH-P450 reductase mRNAs. b-Ionone at 600 mg/kg were treated to rats subcutaneously for 6 h and poly(A + ) mRNAs were prepared. A, P450 1A2; B, P450 2B1; C, P450 2B2; D, P450 2C6; E, NADPH-P450 reductase; F, glucose 3-phosphate dehydrogenase. The results represent typical experiments conducted at least twice for individual isozymes. Lane 1, molecular weight marker showing 100-bp ladder; lane 2, untreated control; lane 3, b-ionone-treated; lane 4, TCDD-treated at 10 mg/kg intraperitoneally once for 3 days; lane 5 in E, molecular weight markers showing 2000-, 1500-, 1000-, 750-, 500-, 300-, 150-, and 50-bp ladders from top; lane 5 in F, 100-bp markers from 1000-bp.
untreated control liver. Meanwhile, no induction of G3PDH mRNA was observed by the treatment with b-ionone. Taken together, in addition to P450 2B1, it is suggested that b-ionone may induce P450 1A2, 2B2, 2C6 and NADPH-P450 reductase in Sprague Dawley rats, possibly by the accumulation of corresponding mRNAs.
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4. Dicussion In the present study, the effects of b-ionone on P450s other than P450 2B1 were further investigated in male and female Sprague Dawley rats. To determine the inductive effects, the P450-associated enzyme activities and western immunoblotting for P450 isozymes were used in liver microsomes from b-ionone-treated rats. In addition, the expression of mRNA corresponding individual isozyme of P450s was studied by RT-PCRs. b-Ionone clearly induced P450 1A1/2 and 2C proteins dose-dependently in rats. The sex difference was so clear as in P450 2B1 induction that the induction was much greater in male rats than in female rats, particularly P450 1A1/2 and P450 2C (Fig. 1). Although the exact amount of isozyme-specific mRNAs induced by b-ionone could not be estimated in the present results, the induction of rat P450 1A1/2, 2C and NADPH-P450 reductase might be closely related with the increase in transcription of P450 1A2, 2C6 and NADPH-P450 reductase mRNAs (Fig. 2), indicating that b-ionone may induce P450 enzymes through the accumulation of P450 mRNAs. Since P450 1A1 and P450 1A2 are detected at the same time with polyclonal antibodies in the western immunoblotting (Fig. 1), the effects of b-ionone on expression of P450 1A1 mRNA should be further investigated. The induction profile of P450 enzymes by b-ionone may be very important in elucidating the former discrepancy in potentiation of cocaine hepatotoxicity –i.e. the SGPT activities resulting from cocaine administration were increased dramatically by the pretreatment with b-ionone, and this potentiation was much greater than that by phenobarbital pre-treatment [6]. It is of interest to speculate that, in addition to P450 2B which is responsible for cocaine activation [21], b-ionone might induce certain P450 isozyme(s) which may be involved in cocaine activation, because we demonstrated that the induction of P450 2B by phenobarbital was much greater than b-ionone [10]. To date the isozyme selectivity for cocaine activation by P450 has not been well characterized. Apparently, P450 1A1 induction by 3-methylcholanthrene did neither potentiate cocaine-induced hepatotoxicity [7] nor increase in cocaine N-demethylase activity in liver microsomes [8]. Moreover, P450 2E1 was not directly involved in the catalytic activation of cocaine, although the induction of P450 2E1 greatly enhanced cocaine cytotoxicity in vitro [22]. P450 3A has been suggested to demethylate cocaine to norcocaine in human liver microsomes [23]. The authors found not only that preincubation of the human microsomes with triacetyloleandomycin, a selective inhibitor of P450 3A, reduced norcocaine formation, but also that microsomes prepared from lymphoblastoid cells expressing transfected human P450 3A4 gene metabolized cocaine to norcocaine. Based on these results, we have speculated that b-ionone may induce P450 3A in addition to P450 2B1. In the present study, however, P450 3A was not induced by b-ionone treatment (Fig. 1), indicating that P450 3A may not be involved in cocaine activation to toxic metabolites, particularly in rats. Instead, the role of P450 2C should be further characterized in the near future to study more potent effects of b-ionone in cocaine-induced hepatotoxicity, because we found that b-ionone clearly induced P450 2C proteins (Fig. 1). In addition, the possible role of other
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forms of P450 in the metabolic activation of cocaine should be clearly elucidated, particularly in rats, because to date P450 2B1 has only been implied to activate cocaine to hepatotoxic metabolites in rats [21]. With regard to the sex differences, our present results and the previous results that b-ionone induced P450 enzymes at a greater rate in male rats than in female rats [9] are very consistent with the report that b-ionone pre-treatment potentiated cocaine-induced hepatotoxicity much greater in male mice, as measured by the elevation of SGOT activity [7]. The authors also found that female mice were more sensitive to cocaine hepatotoxicity when phenobarbital was injected as a model P450 inducer, indicating a supportive evidence that b-ionone pre-treatment induced other uncharacterized P450 isozyme(s), at least partially, responsible for cocaine activation. In summary, our present results clearly showed that b-ionone may induce P450 1A1/2, 2C and NADPH-P450 reductase proteins in Sprague Dawley rats. Since it has been demonstrated that b-ionone itself was neither hepatotoxic nor immunotoxic at the dose showing P450 induction [6–8], b-ionone may be a useful model inducer of P450s in investigating the possible role of metabolism in chemicalinduced toxicity. In fact, we have recently found that the pre-treatment of animals with b-ionone greatly potentiated the hepatotoxicity induced by thioacetamide, a hepatotoxicant which requires metabolic activation by P450s (manuscript in preparation).
Acknowledgements This work was supported by the grants from the Ministry of Science and Technology, Korea, and the Ministry of Health and Welfare, Korea.
References [1] H.G. Jeong, C.-H. Yun, Y.J. Jeon, S.S. Lee, K.-H. Yang, Suppression of cytochrome P450 (Cyp1a-1) induction in mouse hepatoma Hepa-1c1c7 cells by methoxsalen, Biochem. Biophys. Res. Commun. 208 (1995) 1124–1130. [2] H.G. Jeong, C.-H. Yun, Induction of rat hepatic cytochrome P450 enzymes by myristicin, Biochem. Biophys. Res. Commun. 217 (1995) 966 – 971. [3] Y.J. Surh, R.C.-J. Lee, K.K. Park, S.T. Mayne, A. Liem, J.A. Miller, Chemoprotective effects of capsaicin and diallyl sulfide against mutagenesis or tumorigenesis by vinyl carbamate and N-nitrosodimethylamine, Carcinogenesis 16 (1995) 1471 – 2467. [4] Y.R. Naves, Some developments in the chemistry of ionones and their derivatives – a subject review, J. Soc. Cosmet. Chem. 22 (1971) 439 – 456. [5] D.V. Parke, H. Rahman, The effects of some terpenoids and other dietary nutrients on hepatic drug-metabolizing enzymes, Biochem. J. 113 (1969) 12. [6] L. Roth, R.D. Harbison, R.C. James, T. Tobin, S.M. Roberts, Cocaine hepatotoxicity: influence of hepatic enzyme inducing and inhibiting agents on the site of necrosis, Hepatology 15 (1992) 934–940. [7] M.L. Thompson, L. Shuster, E. Casey, G.C. Kanel, Sex and strain differences in response to cocaine, Biochem. Pharmacol. 33 (1984) 1299 – 1307.
T.C. Jeong et al. / Chemico-Biological Interactions 114 (1998) 97–107
107
[8] T.C. Jeong, S.D. Jordan, R.A. Matulka, E.D. Stanulis, E.J. Kaminski, M.P. Holsapple, Role of metabolism by esterase and cytochrome P-450 in cocaine-induced suppression of the antibody response, J. Pharmacol. Exp. Ther. 272 (1995) 407 – 416. [9] T.C. Jeong, H.J. Kim, C.-H. Yun, et al., Induction of liver cytochrome P450 2B1 by b-ionone in Sprague Dawley rats, Biochem. Biophys. Res. Commun. 216 (1995) 198 – 202. [10] T.C. Jeong, S.D. Jordan, R.A. Matulka, E.D. Stanulis, S.S. Park, M.P. Holsapple, Immunosuppression by acute exposure to cocaine is dependent on metabolism by cytochrome P-450, J. Pharmacol. Exp. Ther. 276 (1996) 1257 – 1265. [11] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248 – 254. [12] J.A. Blank, A.N. Tucker, J. Sweatlock, T.A. Gasiewicz, M.I. Luster, Alpha-naphthoflavone antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin induced murine ethoxyresorufin O-deethylase activity and immunosuppression, Mol. Pharmacol. 32 (1987) 168 – 172. [13] J. Vermillion, M.J. Coon, Purified liver microsomal NADPH-cytochrome P-450 reductase, J. Biol. Chem. 253 (1978) 8812–8819. [14] D.R. Koop, Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450 isozyme 3a, Mol. Pharmacol. 29 (1986) 399– 404. [15] T. Nash, The colorimetrical estimation of formaldehyde by means of the Hantzsch reaction, Biochem. J. 55 (1953) 416–421. [16] K.W. Johnson, D.H. Kim, A.E. Munson, M.P. Holsapple, Dependence on intact cells for the in vitro activation of dimethylnitrosamine to an immunosuppressive form, Mutat Res. 182 (1987) 211–221. [17] L.S. Kaminsky, M.J. Fasco, F.P. Guengerich, Production and application of antibodies to rat liver cytochrome P-450, Methods Enzymol. 74 (1981) 262 – 272. [18] D.R. Germolec, E.C. Henry, R. Maronpot, et al., Induction of CYP1A1 and ALDH-3 in lymphoid tissues from Fisher 344 rats exposed to 2,3,7,8-tetrachlorodibenzodioxin (TCDD), Toxicol. Appl. Pharmacol. 137 (1996) 57–66. [19] T. Oinonen, S. Saarikoski, K. Husgafvel-Pursiainen, A. Hirvonen, K.O. Lindros, Pretranslational induction of cytochrome P4501A enzymes by b-naphthoflavone and 3-methylcholanthrene occurs in different liver zones, Biochem. Pharmacol. 48 (1994) 2189 – 2197. [20] P.G. Zaphiropoulos, T. Wood, Identification of the major cytochrome P450s of subfamily 2C that are expressed in brain of female rats and in olfactory lobes of ethanol treated male rats, Biochem. Biophys. Res. Commun. 193 (1993) 1006 – 1013. [21] U.A. Boelsterli, A. Lanzotti, C. Go¨ldlin, M. Oertle, Identification of cytochrome P-450IIB1 as a cocaine-bioactivating isoform in rat hepatic microsomes and in cultured rat hepatocytes, Drug Metabol. Dispos. 20 (1992) 96–101. [22] U.A. Boelsterli, S. Atanasoski, C. Go¨ldlin, Ethanol-induced enhancement of cocaine bioactivation and irreversible protein binding: evidence against a role of cytochrome P-450IIE1, Alcohol. Clin. Exp. Res. 15 (1991) 779–784. [23] B.W. LeDuc, P.R. Sinclair, L. Shuster, J.F. Sinclair, J.E. Evans, D.J. Greenblatt, Norcocaine and N-hydroxynorcocaine formation in human liver microsomes, Pharmacology 46 (1993) 294 – 300.
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Effects of b-ionone on the expression of cytochrome P450s and NADPH-cytochrome P450 reductase in Sprague Dawley rats Tae Cheon Jeong a,*, Hee Kyoung Gu a, Young Jin Chun b, Chul-Ho Yun c, Sang Seop Han a, Jung Koo Roh a a
Toxicology Research Center, Korea Research Institute of Chemical Technology, PO Box 107, Yusung, Taejon 305 -606, South Korea b College of Pharmacy, Chung-Ang Uni6ersity, Seoul, South Korea c Department of Biochemistry, Pai-Chai Uni6ersity, Taejon 302 -735, South Korea Received 14 November 1997; received in revised form 2 April 1998; accepted 15 April 1998
Abstract We have recently reported that b-ionone induces cytochrome P450 (P450) 2B1 in rats. Effects of b-ionone on the expression of other P450 isozymes and NADPH-P450 reductase were further investigated in Sprague Dawley rats. Administration of b-ionone subcutaneously 72 and 48 h before sacrificing the animals not only significantly induced the liver microsomal activities of P450-associated enzymes and NADPH-P450 reductase, but also clearly increased in the level of P450 1A1/2, P450 2C, and NADPH-P450 reductase proteins. The induction of P450 1A1/2 and 2C by b-ionone was much greater in male than in female as measured by western immunoblotting. Reverse transcriptase-polymerase chain reactions showed that, in addition to P450 2B1 and 2B2 mRNAs, P450 1A2, 2C6 and NADPH-P450 reductase mRNAs were increased when b-ionone was administered. Our previous and present results indicated that b-ionone may induce several P450s and NADPH-P450 reductase by the accumulation of their corresponding mRNAs. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: b-Ionone; Cytochrome P450; Induction; Sprague Dawley rats
* Corresponding author. Tel.: +82 42 8607470; fax: + 82 42 8607488. 0009-2797/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0009-2797(98)00044-1
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1. Introduction Many compounds in plants have been reported to modulate cytochrome P450 (P450) expression in experimental animals. For example, methoxsalen found in celery, parsnip and fig suppressed P450 1A1 induction in mouse hepatoma cells [1] and myristicin found in parsley, carrot, black pepper, nutmeg and flavoring agents induced liver P450 1A1/2, 2B1/2 and 2E1 in rats [2]. In addition, some phytochemicals like capsaicin in red peppers and diallyl sulfide in garlic have been characterized to possess chemopreventive effects against mutagenesis and tumorigenesis by inhibiting P450 2E1 which is responsible for bioactivating vinyl carbamate and dimethylnitrosamine [3]. Therefore, extensive efforts for evaluating the effects of compounds abundant in plants should be made, with a particular emphasis on P450 expression, because the modulation of P450 by certain compounds in dietary plants may interfere with drug action in the body and also play a role in chemical-induced carcinogenesis. b-Ionone (4-[2,6,6-trimethyl-1-cyclohexen-1-yl]-3-buten-2-one), which is a basic nucleus structure of retinoic acid, b-carotene and vitamin A, was first isolated from the volatile oil of Boronia megastigma Nees (Rutaceae) and also found in many plants including tea, tangelo and tomato [4]. In 1969, Parke and Rahman originally found that the pre-treatment with b-ionone induced the activity of biphenyl 4-hydroxylase and the content of P450, and that hexobarbital sleeping times were reduced by b-ionone [5]. Thereafter, b-ionone has been introduced as a model inducer of P450 in studying the role of metabolic activation mediated by P450 enzymes in cocaine-induced hepatotoxicity [6,7]. Recently, b-ionone was also shown to potentiate suppression of the antibody response induced by cocaine in female B6C3F1 mice, when the mice were pre-treated with b-ionone, suggesting that certain P450 isozyme(s) responsible for cocaine activation might be induced [8]. Most recently, we found that b-ionone clearly induced P450 2B1 through the accumulation of mRNA in Sprague Dawley rats, which are one of the most useful animal models in studying drug metabolism and chemical-induced toxicity [9]. In the same studies, the induction of P450 2B1 by b-ionone was much greater in male rats than in female rats, as measured by pentoxyresorufin O-dealkylase and western immunoblotting. The primary objective of the present study was to characterize the inductive effects of b-ionone on liver microsomal P450 enzymes and NADPH-P450 reductase in Sprague Dawley rats. Since the information on the effects of b-ionone on P450 induction is very limited, sex differences in P450 induction were also studied. b-Ionone was administered subcutaneously 72 and 48 h before sacrificing animals, as has been used in determining the role of metabolic activation in cocaine-induced hepatotoxicity and immunotoxicity in which b-ionone was a model inducer of P450 responsible for the activation of cocaine to its toxic metabolites [6,7,10].
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2. Materials and methods
2.1. Animals Specific pathogen-free male and female Sprague Dawley rats were obtained from the animal breeding laboratory at this institute. The animals received at 4–5 weeks old were acclimated for at least 1 week. Upon arrival, rats were radomized and housed four or five per cage. All animals were maintained on gamma-irradiated Jeil Lab Chow (Taejon, Korea) and UV-irradiated tap water ad libitum. Animals at 6–8 weeks old, ranging 200 – 250 g, were used in these studies. The animal quarters were strictly maintained at 23 9 3°C and 40–60% relative humidity. A 12-h light/dark cycle was used with an intensity of 150–300 Lux.
2.2. Materials b-Ionone, ethoxyresorufin, resorufin, aminopyrine, p-nitrophenol, erythromycin, NADPH, glucose 6-phosphate, cytochrome c, and nitrocellulose filters were purchased from Sigma (St. Louis, MO). Alkaline phosphatase-conjugated goat antirabbit IgG antibody, alkaline phosphatase substrate kit, and a kit for protein determination were purchased from Bio-Rad Laboratory (Richmond, CA). The kits for mRNA purification and first-strand cDNA synthesis were purchased from Pharmacia Biotech (Uppsala, Sweden). All other chemicals used were of reagent grade commercially available.
2.3. Animal treatment and microsome preparation Animals were treated subcutaneously with b-ionone in corn oil at 300, 600 and 1200 mg/kg 72 and 48 h before sacrificing animals by CO2 asphyxiation. The dose was selected from the previous studies in which b-ionone was used as a model inducer of P450 [6,7,10]. The control was given corn oil at 10 ml/kg. The microsomes were prepared from individual livers of four animals as described in our earlier studies [9] and were resuspended in 0.1 M potassium phosphate buffer, pH 7.4, containing 20% glycerol and 0.1 mM EDTA. Aliquots of liver microsomes were stored at − 70°C until use. The content of microsomal protein was determined according to the method of Bradford using bovine serum albumin as a standard [11]. For studying mRNA expression, male rats (two per groups) were treated with 600 mg/kg of b-ionone in corn oil subcutaneously once 6 h before sacrificing animals. The control was given corn oil at 10 ml/kg.
2.4. Monooxygenase assays Ethoxyresorufin O-deethylase (EROD) activity was determined as described by Blank et al. [12] with a slight modification. The reaction mixture (2 ml) consisted of 0.1 M potassium phosphate buffer, pH 7.4, containing 2 mg/ml of bovine serum albumin, 10 mM dicumorol, 5 mM glucose 6-phosphate, 1 U of glucose 6-phos-
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phate dehydrogenase, 5 mM NADPH and 2.5 mM 7-ethoxyresorufin. The formation of resorufin was monitored fluorometrically at an extinction maximum of 550 nm and an emission maximum of 585 nm. NADPH-cytochrome c reductase activity was determined as a surrogate for NADPH-P450 reductase activity by measuring the reduction rate of cytochrome c with an extinction coefficient of 21 mM − 1 cm − 1 at 550 nm following the addition of 0.12 mM NADPH [13]. p-Nitrophenol hydroxylase (PNPH) activity was determined as described by Koop [14]. The reaction mixture (1.0 ml) was composed of 0.1 M potassium phosphate buffer, pH 7.4, containing 100 mM p-nitrophenol, 1 mM NADPH and an enzyme source. The amount of 4-nitrocatechol formed was measured spectrophotometrically at 512 nm. Aminopyrine N-demethylase (APDM) and erythromycin N-demethylase (ERDM) activities were determined by measuring the amount of formaldehyde formed, as described previously [15,16]. Aminopyrine at 1.0 mM and erythromycin at 400 mM were used as substrates for assaying APDM and ERDM, respectively. The amount of formaldehyde formed was determined spectrophotometrically at a wavelength of 412 nm.
2.5. Western immunoblotting analyses Microsomal proteins (10 mg/well) were resolved by 10% SDS-PAGE and were transferred to nitrocellulose filters. The filters were incubated with 2.5% non-fat dry milk for 30 min to block the non-specific binding, and then were incubated with rabbit polyclonal antibodies against rat P450 1A1/2, 2C, and 3A, and NADPHP450 reductase, respectively, followed by an alkaline phosphatase-conjugated goat anti-rabbit IgG antibody. The primary antibody against each isozyme was prepared as described previously [17]. For immunostaining, the nitrocellulose filters were developed with a mixture of 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium and 0.1 M Tris buffer (1:1:10) under an instruction by the manufacturer.
2.6. mRNA extraction and re6erse transcriptase-polymerase chain reaction (RT-PCR) Cellular poly(A + ) mRNA from each animal was isolated and reverse transcribed using the commercially available kits under the manufacturer’s instruction. The reaction was stopped by incubating the mixture in boiling water bath for 5 min. A 5-ml aliquot of cDNA corresponding to approximately 20 ng of mRNA from each sample was amplified for 30 cycles by PCR in 50 ml of a mixture containing 50 pmol of specific primers, 1 mM dNTPs, 10x PCR buffer (100 mM Tris–HCl, 500 mM KCl, pH 8.3), and 0.5 ml of Taq polymerase (Pharmacia Biotech). The rat P450 1A2-, 2B1-, 2B2-, 2C6-, and NADPH-P450 reductase-specific primers producing 302-, 443-, 534-, 275-bp, and 407-bp PCR products, respectively, were synthesized at either Integrated DNA Technologies, Inc. (Coralville, IA) or Genosys Biotechnologies (Woodlands, TX). Glucose 3-phosphate dehydrogenase (G3PDH)specific primers producing 983-bp PCR product was also used as a control in this study. The sequence of each sense and anti-sense primer for P450 1A2, 2C6, and
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G3PDH has been described elsewhere [18–20]. The sequences for P450 2B1, 2B2, and NADPH-P450 reductase mRNAs were designed around the active centers: P450 2B1 sense primer, 5%-TTGTTTGGTGCTGGGACAGAG-3%; P450 2B1 antisense primer, 5%-GGCTAGGCCCTCTCCTGCACA-3%; P450 2B2 sense primer, 5%-CTCTTCTTTGCTGGCACTGAG-3%; P450 2B2 antisense primer, 5%-GGCAATGCCTTCGCCAAGACA-3%; NADPH-P450 reductase sense primer, 5%-GGCTCCCAGACGGGAACCGCT-3%; NADPH-P450 reductase antisense primer, 5%-CCTCCACGTGATGAAATCCTC-3%. RNA concentration and PCR cycle were titrated to obtain linearity of signal strength. The PCR products (10 ml/well) were visualized by UV illumination following an electrophoresis through a 1.5% agarose gel in Tris – borate – EDTA buffer containing 0.5 mg/ml of ethidium bromide.
2.7. Statistics Dunnett’s t-test was used to compare the significance of data obtained. The significant value at P B0.05 was represented as an asterisk.
3. Results Table 1 shows the effects of b-ionone on the activities of EROD, PNPH, APDM, ERDM and NADPH-P450 reductase in rat liver microsomes. The EROD activity was significantly induced by treatment with b-ionone from 300 mg/kg in both male and female rats. The activities of PNPH and APDM were induced at higher doses in both sexes. The ERDM activity was not changed by b-ionone in male rats, while the activity was significantly decreased in female rats at 300 and 600 mg/kg of b-ionone. The NADPH-P450 reductase activity was clearly induced from 300 mg/kg of b-ionone only in male rats. The constitutive enzyme activities were much greater in male rats than in female rats except NADPH-P450 reductase, as previously observed in PROD induction [9]. In Fig. 1, the liver microsomes prepared from b-ionone treated rats were electrophoresed and immunoblotted with polyclonal antibodies against either one of the following rat P450 1A1/2, 2C, 3A or NADPH-P450 reductase. Apparent changes in contents of P450 1A1/2, 2C and NADPH-P450 reductase proteins were observed in b-ionone-treated animals. The level of P450 1A1/2 and 2C induced by b-ionone was also higher in male rats than in female rats. Following the observation that the induction of P450 2B1 by b-ionone might be regulated by the accumulation of mRNAs in the previous work [9], the effects of b-ionone on P450 1A2, 2C6 and NADPH-P450 reductase mRNAs were further examined by using RT-PCR of mRNAs isolated from b-ionone-treated male rat livers in the present study. In addition, effects on the expression of P450 2B1 and 2B2 mRNAs were compared. b-Ionone was treated for 6 h, because our previous study implied that the P450 2B1 mRNA expression reached a maximum 6 hr after the treatment with b-ionone [9]. TCDD, a well-known inducer of P450 1A1/2, was used as a model inducer in the present study. As shown in Fig. 2, TCDD induced
Male
Female 0.76 90.04 1.69 90.13* 1.97 90.12* 2.81 90.39*
Male 1.20 9 0.18 2.09 90.19* 1.85 90.16* 2.34 90.25*
146 9 17 158 9 9 186 934 192 9 0.18*
PNPHb
ERODa
75 99 90 9 13 89 9 10 125 917*
Female 1.37 9 0.18 1.58 9 0.04 1.57 90.14 2.01 90.18*
Male
APDMc
0.9190.13 1.169 0.13 1.3090.06* 1.719 0.06*
Female
The rats were administered b-ionone in corn oil subcutaneously 72 and 48 h before sacrificing animals. Each value represents the mean activity 9 S.E. of four animals. An asterisk indicates the value significantly different from the vehicle control at PB0.05. a Ethoxyresorufin O-deethylase, nmol resorufin formed/min per mg protein. b p-Nitrophenol hydroxylase, pmol 4-nitrocatechol formed/min per mg protein. c Aminopyrine N-demethylase, nmol formaldehyde formed/min per mg protein. d Erythromycin N-demethylase, pmol formaldehyde formed/min per mg protein. e NADPH-P450 reductase, nmol/min per mg protein.
Vehicle 300 600 1200
b-Ionone (mg/kg)
Table 1 Effects of b-ionone on liver microsomal monooxygenases in Sprague Dawley rats
3359 48 309921 3349 40 388942
Male
ERDMd
128 911 79 9 7* 91 96* 143 9 14
Female
439 8 1199 4* 1329 40* 1039 7*
Male
Reductasee
128 9 13 112927 1839 16 98 914
Female
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the accumulation of P450 1A2, 2B1, 2B2 and 2C6 mRNAs, although undesirable minor bands were also observed in our present experimental conditions. In addition to P450 2B1 and 2B2 mRNAs, the amount of P450 1A2, 2C6 and NADPH-P450 reductase mRNAs were clearly increased by b-ionone when compared to the
Fig. 1. Western immunoblotting analyses for P4501A1/2, 2C, 3A, and NADPH-P450 reductase in liver microsomes. The liver microsomal proteins (10 mg/well) prepared from rats administered b-ionone at 300, 600 and 1200 mg/kg were resolved on 10% SDS-PAGE. The results represent typical experiments conducted at least twice for individual isozymes with same microsomes used in Table 1. A, P4501A1/2; B, P4502C; C, P4503A; D, NADPH-P450 reductase. Lane 1 and 5, vehicle control; lane 2 and 6, b-ionone 300 mg/kg; land 3 and 7, b-ionone 600 mg/kg; lane 4 and 8, b-ionone 1200 mg/kg. Lane 1 – 4, male rats; lane 5–8, female rats. M, purified rat NADPH-P450 reductase. The lower bands seen in A, B and C were non-specific binding.
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Fig. 2. RT-PCR amplification of P450 1A2, 2B1, 2B2, 2C6, and NADPH-P450 reductase mRNAs. b-Ionone at 600 mg/kg were treated to rats subcutaneously for 6 h and poly(A + ) mRNAs were prepared. A, P450 1A2; B, P450 2B1; C, P450 2B2; D, P450 2C6; E, NADPH-P450 reductase; F, glucose 3-phosphate dehydrogenase. The results represent typical experiments conducted at least twice for individual isozymes. Lane 1, molecular weight marker showing 100-bp ladder; lane 2, untreated control; lane 3, b-ionone-treated; lane 4, TCDD-treated at 10 mg/kg intraperitoneally once for 3 days; lane 5 in E, molecular weight markers showing 2000-, 1500-, 1000-, 750-, 500-, 300-, 150-, and 50-bp ladders from top; lane 5 in F, 100-bp markers from 1000-bp.
untreated control liver. Meanwhile, no induction of G3PDH mRNA was observed by the treatment with b-ionone. Taken together, in addition to P450 2B1, it is suggested that b-ionone may induce P450 1A2, 2B2, 2C6 and NADPH-P450 reductase in Sprague Dawley rats, possibly by the accumulation of corresponding mRNAs.
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4. Dicussion In the present study, the effects of b-ionone on P450s other than P450 2B1 were further investigated in male and female Sprague Dawley rats. To determine the inductive effects, the P450-associated enzyme activities and western immunoblotting for P450 isozymes were used in liver microsomes from b-ionone-treated rats. In addition, the expression of mRNA corresponding individual isozyme of P450s was studied by RT-PCRs. b-Ionone clearly induced P450 1A1/2 and 2C proteins dose-dependently in rats. The sex difference was so clear as in P450 2B1 induction that the induction was much greater in male rats than in female rats, particularly P450 1A1/2 and P450 2C (Fig. 1). Although the exact amount of isozyme-specific mRNAs induced by b-ionone could not be estimated in the present results, the induction of rat P450 1A1/2, 2C and NADPH-P450 reductase might be closely related with the increase in transcription of P450 1A2, 2C6 and NADPH-P450 reductase mRNAs (Fig. 2), indicating that b-ionone may induce P450 enzymes through the accumulation of P450 mRNAs. Since P450 1A1 and P450 1A2 are detected at the same time with polyclonal antibodies in the western immunoblotting (Fig. 1), the effects of b-ionone on expression of P450 1A1 mRNA should be further investigated. The induction profile of P450 enzymes by b-ionone may be very important in elucidating the former discrepancy in potentiation of cocaine hepatotoxicity –i.e. the SGPT activities resulting from cocaine administration were increased dramatically by the pretreatment with b-ionone, and this potentiation was much greater than that by phenobarbital pre-treatment [6]. It is of interest to speculate that, in addition to P450 2B which is responsible for cocaine activation [21], b-ionone might induce certain P450 isozyme(s) which may be involved in cocaine activation, because we demonstrated that the induction of P450 2B by phenobarbital was much greater than b-ionone [10]. To date the isozyme selectivity for cocaine activation by P450 has not been well characterized. Apparently, P450 1A1 induction by 3-methylcholanthrene did neither potentiate cocaine-induced hepatotoxicity [7] nor increase in cocaine N-demethylase activity in liver microsomes [8]. Moreover, P450 2E1 was not directly involved in the catalytic activation of cocaine, although the induction of P450 2E1 greatly enhanced cocaine cytotoxicity in vitro [22]. P450 3A has been suggested to demethylate cocaine to norcocaine in human liver microsomes [23]. The authors found not only that preincubation of the human microsomes with triacetyloleandomycin, a selective inhibitor of P450 3A, reduced norcocaine formation, but also that microsomes prepared from lymphoblastoid cells expressing transfected human P450 3A4 gene metabolized cocaine to norcocaine. Based on these results, we have speculated that b-ionone may induce P450 3A in addition to P450 2B1. In the present study, however, P450 3A was not induced by b-ionone treatment (Fig. 1), indicating that P450 3A may not be involved in cocaine activation to toxic metabolites, particularly in rats. Instead, the role of P450 2C should be further characterized in the near future to study more potent effects of b-ionone in cocaine-induced hepatotoxicity, because we found that b-ionone clearly induced P450 2C proteins (Fig. 1). In addition, the possible role of other
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forms of P450 in the metabolic activation of cocaine should be clearly elucidated, particularly in rats, because to date P450 2B1 has only been implied to activate cocaine to hepatotoxic metabolites in rats [21]. With regard to the sex differences, our present results and the previous results that b-ionone induced P450 enzymes at a greater rate in male rats than in female rats [9] are very consistent with the report that b-ionone pre-treatment potentiated cocaine-induced hepatotoxicity much greater in male mice, as measured by the elevation of SGOT activity [7]. The authors also found that female mice were more sensitive to cocaine hepatotoxicity when phenobarbital was injected as a model P450 inducer, indicating a supportive evidence that b-ionone pre-treatment induced other uncharacterized P450 isozyme(s), at least partially, responsible for cocaine activation. In summary, our present results clearly showed that b-ionone may induce P450 1A1/2, 2C and NADPH-P450 reductase proteins in Sprague Dawley rats. Since it has been demonstrated that b-ionone itself was neither hepatotoxic nor immunotoxic at the dose showing P450 induction [6–8], b-ionone may be a useful model inducer of P450s in investigating the possible role of metabolism in chemicalinduced toxicity. In fact, we have recently found that the pre-treatment of animals with b-ionone greatly potentiated the hepatotoxicity induced by thioacetamide, a hepatotoxicant which requires metabolic activation by P450s (manuscript in preparation).
Acknowledgements This work was supported by the grants from the Ministry of Science and Technology, Korea, and the Ministry of Health and Welfare, Korea.
References [1] H.G. Jeong, C.-H. Yun, Y.J. Jeon, S.S. Lee, K.-H. Yang, Suppression of cytochrome P450 (Cyp1a-1) induction in mouse hepatoma Hepa-1c1c7 cells by methoxsalen, Biochem. Biophys. Res. Commun. 208 (1995) 1124–1130. [2] H.G. Jeong, C.-H. Yun, Induction of rat hepatic cytochrome P450 enzymes by myristicin, Biochem. Biophys. Res. Commun. 217 (1995) 966 – 971. [3] Y.J. Surh, R.C.-J. Lee, K.K. Park, S.T. Mayne, A. Liem, J.A. Miller, Chemoprotective effects of capsaicin and diallyl sulfide against mutagenesis or tumorigenesis by vinyl carbamate and N-nitrosodimethylamine, Carcinogenesis 16 (1995) 1471 – 2467. [4] Y.R. Naves, Some developments in the chemistry of ionones and their derivatives – a subject review, J. Soc. Cosmet. Chem. 22 (1971) 439 – 456. [5] D.V. Parke, H. Rahman, The effects of some terpenoids and other dietary nutrients on hepatic drug-metabolizing enzymes, Biochem. J. 113 (1969) 12. [6] L. Roth, R.D. Harbison, R.C. James, T. Tobin, S.M. Roberts, Cocaine hepatotoxicity: influence of hepatic enzyme inducing and inhibiting agents on the site of necrosis, Hepatology 15 (1992) 934–940. [7] M.L. Thompson, L. Shuster, E. Casey, G.C. Kanel, Sex and strain differences in response to cocaine, Biochem. Pharmacol. 33 (1984) 1299 – 1307.
T.C. Jeong et al. / Chemico-Biological Interactions 114 (1998) 97–107
107
[8] T.C. Jeong, S.D. Jordan, R.A. Matulka, E.D. Stanulis, E.J. Kaminski, M.P. Holsapple, Role of metabolism by esterase and cytochrome P-450 in cocaine-induced suppression of the antibody response, J. Pharmacol. Exp. Ther. 272 (1995) 407 – 416. [9] T.C. Jeong, H.J. Kim, C.-H. Yun, et al., Induction of liver cytochrome P450 2B1 by b-ionone in Sprague Dawley rats, Biochem. Biophys. Res. Commun. 216 (1995) 198 – 202. [10] T.C. Jeong, S.D. Jordan, R.A. Matulka, E.D. Stanulis, S.S. Park, M.P. Holsapple, Immunosuppression by acute exposure to cocaine is dependent on metabolism by cytochrome P-450, J. Pharmacol. Exp. Ther. 276 (1996) 1257 – 1265. [11] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248 – 254. [12] J.A. Blank, A.N. Tucker, J. Sweatlock, T.A. Gasiewicz, M.I. Luster, Alpha-naphthoflavone antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin induced murine ethoxyresorufin O-deethylase activity and immunosuppression, Mol. Pharmacol. 32 (1987) 168 – 172. [13] J. Vermillion, M.J. Coon, Purified liver microsomal NADPH-cytochrome P-450 reductase, J. Biol. Chem. 253 (1978) 8812–8819. [14] D.R. Koop, Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450 isozyme 3a, Mol. Pharmacol. 29 (1986) 399– 404. [15] T. Nash, The colorimetrical estimation of formaldehyde by means of the Hantzsch reaction, Biochem. J. 55 (1953) 416–421. [16] K.W. Johnson, D.H. Kim, A.E. Munson, M.P. Holsapple, Dependence on intact cells for the in vitro activation of dimethylnitrosamine to an immunosuppressive form, Mutat Res. 182 (1987) 211–221. [17] L.S. Kaminsky, M.J. Fasco, F.P. Guengerich, Production and application of antibodies to rat liver cytochrome P-450, Methods Enzymol. 74 (1981) 262 – 272. [18] D.R. Germolec, E.C. Henry, R. Maronpot, et al., Induction of CYP1A1 and ALDH-3 in lymphoid tissues from Fisher 344 rats exposed to 2,3,7,8-tetrachlorodibenzodioxin (TCDD), Toxicol. Appl. Pharmacol. 137 (1996) 57–66. [19] T. Oinonen, S. Saarikoski, K. Husgafvel-Pursiainen, A. Hirvonen, K.O. Lindros, Pretranslational induction of cytochrome P4501A enzymes by b-naphthoflavone and 3-methylcholanthrene occurs in different liver zones, Biochem. Pharmacol. 48 (1994) 2189 – 2197. [20] P.G. Zaphiropoulos, T. Wood, Identification of the major cytochrome P450s of subfamily 2C that are expressed in brain of female rats and in olfactory lobes of ethanol treated male rats, Biochem. Biophys. Res. Commun. 193 (1993) 1006 – 1013. [21] U.A. Boelsterli, A. Lanzotti, C. Go¨ldlin, M. Oertle, Identification of cytochrome P-450IIB1 as a cocaine-bioactivating isoform in rat hepatic microsomes and in cultured rat hepatocytes, Drug Metabol. Dispos. 20 (1992) 96–101. [22] U.A. Boelsterli, S. Atanasoski, C. Go¨ldlin, Ethanol-induced enhancement of cocaine bioactivation and irreversible protein binding: evidence against a role of cytochrome P-450IIE1, Alcohol. Clin. Exp. Res. 15 (1991) 779–784. [23] B.W. LeDuc, P.R. Sinclair, L. Shuster, J.F. Sinclair, J.E. Evans, D.J. Greenblatt, Norcocaine and N-hydroxynorcocaine formation in human liver microsomes, Pharmacology 46 (1993) 294 – 300.
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