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6j mice by isosafrole: lack of correlation with the ah locus
Chem.-Biol.
Interactions,
Elsevier Scientific
58 (1986)
Publishers
Ireland
233
233-240
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Rapid Communication
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INDUCTION OF CYTOCHROME P-450 IN CONGENIC C57BL/6J MICE BY ISOSAFROLE: LACK OF CORRELATION WITH THE Ah LOCUS
JON C. COOK* Toxicology (U.S.A.)
and ERNEST
Program.
Box
HODGSON 7633,
North
Carolina
State
University,
Raleigh,
NC
27695
(Received October 10th. 1985) (Revision received February 2nd, 1986) (Accepted March 7th. 1986)
SUMMARY
Isosafrole induction of cytochrome P-450 was compared in congenic strains of C57BL/6J mice, one of which expresses normal levels of the Ah receptor [B6(Ahb)], and another that does not contain a measurable receptor concentration [ B6( Ahd)] . Using sucrose gradient analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding, an Ah receptor concentration of 69.1 + 3.8 fmol/mg protein was measured in the hepatic cytosol from B6 Ahb) mice, while no receptor could be detected in the cytosol from BG(Ah 6 ) mice. Isosafrole treatment (75 mg/kg X 3 days) increased the total hepatic microsomal cytochrome P-450 content to the same extent in the two congenic strains. The level of microsomal monooxygenase induction in the isosafrole-treated B6(Ahd) mice was greater than that of B6(Ahb) mice for ethylmorphine Ndemethylase and isosafrole metabolite-complex formation, the latter a measure of cytochrome P,-450. In the case of 7-ethoxycoumarin 0-deethylase only the isosafrole-treated B6(Ahd) mice had elevated microsomal activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) also revealed a similar induction pattern for the two congenic strains, following isosafrole treatment. Thus, the isosafrole treated B6 (Ahd) mice produced an
*Present address: Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, NC 27709, U.S.A. Abbreviations: B6(Ahb), C57BL/6J mice with measurable levels of the Ah receptor; BS(Ahd), C57BL/6J-Ahd’d [B6.D2-Ahd’d (NE8)] mice with no detectable Ah receptor; MDEG, buffer containing 25 mM MOPSpH 7.4, 1 mM dithiothreitol, 1.5 mM EDTA and 10% (v/v) glycerol; MDP, methylenedioxyphenyl; 3-MC, 3-methylcholanthrene; MOPS, 3(N-morpholino)propanesulfonic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. 0009-2797/86/$03.50
o 1986 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland
Ltd.
234 equivalent or slightly larger induction of cytochrome P-450 than the B6(Ah”) mice, suggesting that there is no direct role for the Ah receptor in the regulation of these cytochrome P-450 monooxygenase activities by isosafrole. Key words: Ah locus - Induction Methylenedioxyphenyl compounds
-
Cytochrome
P-450
-
Isosafrole
-
INTRODUCTION
Isosafrole is a naturally occurring methylenedioxyphenyl (MDP) compound that was originally reported to induce a novel form of cytochrome P-450 (P-450d) in rats [1,2]. Cytochrome P-450d was subsequently shown to be a second 3-methylcholanthrene inducible form [ 3,4]. In the mouse, isosafrole appears to induce cytochrome P2-450, as well as cytochrome P,-450, a form which corresponds to cytochrome P-450d in the rat [ 5,6]. Cytochrome P,450 in the mouse corresponds to cytochrome P-450~ of the rat [3,5]. Isosafrole induces cytochrome P-450~ in the rat [ 3,7], but conflicting data exists on cytochrome PI-450 induction in the mouse. Isosafrole treatment increases cytochrome P,-450-mRNA [S] yet monooxygenase activities associated with cytochrome PI-450 are not elevated [6,9,10]. The cytochrome P-450 induction pattern following isosafrole treatment has been described as either phenobarbital-like, 3-methylcholanthrene-like, or mixed in nature [ 1,9-121. MDP compounds have a biphasic effect on cytochrome P-450 levels and monooxygenase activity, namely, inhibition followed by induction [ 131. Inhibition is related to the formation of a stable MDP metabolite-cytochrome P-450 complex which is also responsible for the appearance of a Type III double-Soret optical difference spectrum [ 14,151. Bigelow and Nebert [ 161 reported that isosafrole produced 67% displacement of [3H] TCDD from the Ah receptor in C57BL/6N mice. From genetic evidence using C57BL/6N, DBA/BN, and (C57BL/6N X DBA/BN)F, mice, Ohyama et al. [6] concluded that cytochrome P,-450 induction by isosafrole was inherited as an autosomal additive trait and that the Ah locus, or a closely segregating gene, was involved. The involvement of the Ah receptor in the regulation of hepatic cytochrome P-450 levels by the 3-methylcholanthrene (3-MC) class of inducers has been the subject of numerous reviews [17,18]. Cook and Hodgson [19] examined the role of the Ah receptor in the induction of cytochrome P-450 by isosafrole, as well as two other MDP compounds, piperonyl butoxide and 5-t-butyl-1,3-benzodioxole. Using sucrose density gradient analysis, it was not possible to demonstrate the displacement of TCDD or 3-MC from the Ah receptor by these MDP compounds even though they were used at a lOOO-fold excess. In in vivo experiments on C57BL/6 mice, a single 500 mg/kg i.p. dose of isosafrole produced a twofold increase in the binding of 3-[3H] MC to the Ah receptor after 24 h. This increase may be due to induction of the receptor. If isosafrole does bind to the Ah receptor a decrease in 3-[3H] MC binding should have been observed
235 following isosafrole treatment, in vivo [ 191. Phenobarbital, which does not interact with the Ah receptor [ 20,211, is the only other compound that has been shown to cause an increase in 3-[3H]MC binding to the Ah receptor [ 221. The data of Cook and Hodgson [ 191 suggest that the Ah receptor does not play a direct role in the regulation of cytochrome P-450 by isosafrole or by MDP compounds in general. In an attempt to resolve these conflicting findings, we have examined the ability of isosafrole to induce cytochrome P-450 in congenic C57BL/6J mice. These strains of mice differ in the gene locus which codes for the expression of the Ah receptor. The B6(Ahb) mice are C57BL/6J mice from Jackson Laboratories with normal levels of Ah receptor, while the B6(Ahd) mice are derived from C57BL/6J mice but do not express measurable levels of the Ah receptor. MATERIALS
AND
METHODS
We received B6(Ahd) homozygote breeding pairs from Dr. A. Poland, McArdle Laboratory for Cancer Research (University of Wisconsin, Madison). Offspring from these breeding pairs were used in the experiments reported. Dr. Poland derived the B6(Ahd) homozygotes from B6N.D2N-Ahd’d (NE13) mice by backcross-intercross breeding into C57BL/6J mice. Dr. D. Nebert, National Institute of Health, donated the NIH mice to Dr. Poland. At this time, 32 biochemical markers have been examined to confirm the congenicity of the B6(Ahd) homozygotes; no deviation from the normal C57BL/6J profile has been detected (Dr. Linda Birnbaum, NIEHS, pers. commun.). B6(Ahb) mice were obtained from Jackson Laboratories (Bar Harbor, ME). Seven-weekold male mice were dosed i.p. daily for 3 days with either corn oil or 75 mg/kg isosafrole. The animals, 4 per group, were killed on day 4 and liver microsomes and cytosol were prepared as previously described [10,19]. Cytochrome P-450 content, 7-ethoxycoumarin Odeethylase activity and ethylmorphine N-demethylase activity were also determined by previously described methods [ 10,19,23]. Isosafrole metabolite-complex formation was measured by the method of Fisher et al. [24]. Briefly, isosafrole was added to a final concentration of 0.15 mM to both sample and reference cuvets which contained 0.8 mg protein/ml of a microsomal suspension. Twenty five microliters of an NADPH regenerating system was added to the sample cuvet. After 6-min incubation sodium dithionite was added to both sample and reference cuvets. The amount of isosafrole-metabolite complex formed was calculated from AA455-490 nm using an extinction coefficient of 75 mM-’ cm-’ [ 251. SDS-PAGE was performed as previously described [ 231. The Ah receptor was measured using sucrose density gradient analysis [ 211 as modified by Cook and Hodgson [ 191. The source and purity of the chemicals used are as outlined in Cook and Hodgson [ 19,231. Statistical significance was determined using an analysis of variance to compute the least significant difference, with P < 0.05 as the criterion for significance [ 261.
236 RESULTS
AND DISCUSSION
Ah receptor concentration in hepatic cytosol from mice of the two congenie C57BL/6J strains was measured by sucrose gradient analysis using [3H] TCDD as the radioligand. B6(Ahb) mice had an Ah receptor concentration of 69.1 + 3.8 fmol/mg protein, while no Ah receptor could be detected in the hepatic cytosol from B6(Ahd) mice. Thus, if isosafrole does not require the Ah receptor to induce cytochrome P-450, as previously suggested [19], then similar levels of induction should be seen in these congenic strains. If isosafrole induction is dependent upon the Ah receptor, and if isosafrole behaves like ligands other than TCDD then, the B6(Ahb) strain should be inducible while the B6(Ahd) strain should not. Ohyama et al. [6] reported that isosafrole treatment at 150 mg/kg for 3 days induced cytochrome P2-450 equally well in C57BL/6N and DBA/BN mice even though DBA/BN mice do not contain measurable amounts of cytosolic Ah receptor. At lower doses of isosafrole, the induction of cytochrome PI-450 was greater in C57BL/6N mice than DBA/BN mice; a similar result is seen with TCDD for cytochrome PI-450 induction [ 271. We treated the congenic C57BL/6J mice with 75 mg/kg isosafrole for 3 days, since in the study of Ohyama et al. [ 61, this dose appears to produce a greater induction of C57BL/6N mice than DBA/BN mice even though it is at the high end of the dose-response curve.
TABLE
I
INDUCTION OF CYTOCHROME MONOOXYGENASE ACTIVITIES C57BL/6 MICE Treatment/ strain
Oil/B6(Ahb) ksafrole BG(Ah Oil/BG(Ah ba ) Isosafrole/ B6(Ahd)
Cytochrome
P-450 AND CYTOCHROME P-450-DEPENDENT BY ISOSAFROLE IN CONGENIC STRAINS OF
(nmol/mg protein)
Isosafrole metabolitecomplex formationa (nmol/min mg protein)
7-Ethoxycoumarin 0-deethylasea (nmol/min mg protein
Ethylmorphine N-demethylasea (nmol/min mg protein)
0.92 1.24b
0.017 0.031b
1.65 1.85
5.42 7.17b
0.90 1.32b
0.015 0.040b”
1.30 2.4gb”
5.42 8.96b’C
P-450a
aMean values are shown. The average S.E.‘s were 0.09, 0.004, 0.17 and 0.51 for cytochrome P-450, isosafrole-complex formation, 7-ethoxycoumarin 0-deethylase and ethylmorphine N-demethylase, respectively. bf’ < 0.05 compared to the appropriate control. ‘P < 0.05 compared to isosafrole-treated B6 (Ahb) mice.
237
60
50 45
1
2
3
4
5
Fig. 1. SDS-PAGE of hepatic microsomes from congenic C57BL/6J mice treated with isosafrole. A mixture of molecular weight standards was applied to well 1. Microsomes (20 pg protein) from treated mice were applied as indicated: (2) oil control/B6(Ahb), (3) isosafrole/B6(Ahb), (4) isosafrole/B6(Ahd) and (5) oil control/B6(Ahd). Migration is from the top of the gel to the bottom. Figures on the left represent mol. wt. x 10’. The arrows point to the molecular band regions which are induced by isosafrole.
Isosafrole induces the following cytochrome P-450 monooxygenase parameters in mice: biphenyl 2- and 4-hydroxylase [9,10], ethylmorphine Ndemethylase [ 9,101, acetanilide 4-hydroxylase [ 61, and isosafrole metabolitecomplex formation, a measure of cytochrome P,-450 [6]. In this study, isosafrole metabolitecomplex formation, ethylmorphine N-demethylase, and ethoxycoumarin 0-deethylase activities were measured to further define the role of the Ah locus in isosafrole induction of cytochrome P-450-dependent monooxygenase activities. Isosafrole treatment increased total hepatic cytochrome P-450 content to the same extent in the two congenic strains (Table I). The isosafrole-treated congenic mice also had increased microsomal isosafrole metabolite-complex formation and ethylmorphine N-demethylase activ-
238 ities compared to their respective controls. In addition, the level of induction seen in the isosafrole-treated B6(Ahd) mice was greater than that of the B6(Ahb) mice for these two monooxygenase activities. In the case of 7ethoxycoumarin 0-deethylase activity only the isosafrole-treated B6( Ahd) mice had elevated microsomal activity. Thus, the isosafrole-treated B6(Ahd) mice produced an equivalent or greater induction of cytochrome P-450 than the B6(Ahb) mice. The gel in Fig. 1 confirms that the congenic mice treated with isosafrole produced similar induction patterns. Isosafrole treatment induced two bands at 53 000 and 54 000, and possibly a third band around 52 000. The increase in isosafrole metabolite-complex formation suggests that one of the induced bands is cytochrome P2-450. A similar induction pattern has been reported in isosafrole-treated Dub: ICR mice [lo]. Based upon the dose-response curve for the induction of cytochrome P,450 by isosafrole, Ohyama et al. [6] concluded that cytochrome P,-450 induction by isosafrole involved the Ah receptor or a closely segregating gene. These studies were performed using an anti-cytochrome P2-450 antibody and measured precipitable protein. This data [6] may be difficult to interpret since the anti-cytochrome P2-450 antibody used in these experiments has been shown to precipitate cytochrome P3-450 [ 281. Isosafrole also induces cytochrome P,-450 [6]. Congenic C57BL/6J mice appear to differ only in the expression of the gene for the Ah receptor. When these congenic mice were treated with isosafrole, the monooxygenase activities associated with isosafrole induction, as well as the cytochrome P-450 content and SDS-PAGE, were similar. These data derived from congenic strains coupled with the in vitro and in vivo data of Cook and Hodgson [19] suggest that there is no direct role for the Ah receptor in the regulation of these cytochrome P-450 monooxygenase activities by isosafrole. ACKNOWLEDGEMENTS
Paper No. 10159 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC. Supported, in part, by PHS Grants ES-07046 and ES-00044 from the National Institute of Environmental Health Sciences. We are indebted to Dr. A. Poland, of the McArdle Laboratory for Cancer Research, University of Wisconsin, for the generous gift of B6(Ahd) breeding pairs. REFERENCES 1 M. Dickens, J.W. Bridges, C.R. Elcombe and K.J. Netter, A novel haemoprotein induced by isosafrole pretreatment in the rat, Biochem. Biophys. Res. Commun., 80 (1978) 89. 2 D.E. Ryan, P.E. Thomas and W. Levin, Properties of liver microsomal cytochrome P450 from rats treated with isosafrole, in: M.J. Coon, A.H. Conney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette and P.J. O’Brien (Eds.), Microsomes and Drug Oxidations, Academic Press, New York, 1980, p. 167.
239 3 L.M. Reik, W. Levin, D.G. Ryan and P.E. Thomas, Immunochemical relatedness of rat hepatic microsomal P-450~ and P-450d, J. Biol. Chem., 257 (1982) 3950. 4 J.A. Goldstein, P. Linko, M.I. Luster and D.W. Sundheimer, Purification and characterization of a second form of hepatic cytochrome P-488 from rats treated with a pure polychlorinated biphenyl isomer, J. Biol. Chem., 257 (1982) 2702. 5 S. Kimura, F.J. Gonzalez and D.W. Nebert, The murine Ah locus. Comparison of the complete cytochrome P,-450 and P,-450 cDNA nucleotide and amino acid sequences, J. Biol. Chem., 259 (1984) 10705. 6 T. Ohyama, D.W. Nebert and M. Negishi, Isosafrole-induced cytochrome PI-450 in DBA/BN mouse liver. Characterization and genetic control of induction, J. Biol. Chem., 259 (1984) 2675. 7 P.E. Thomas, L.M. Reik, D.E. Ryan and W. Levin, Induction of two immunochemitally related rat liver cytochrome P-450 isozymes, cytochromes P-450~ and P-450d, by structurally diverse xenobiotics, J. Biol. Chem., 258 (1983) 4590. 8 R.H. Tukey, M. Negishi and D.W. Nebert, Quantitation of hepatic cytochrome P,450 mRNA with the use of a cloned DNA probe. Effects of various inducers in C57BL/6N and DBA/BN mice, Mol. Pharmacol., 22 (1982) 779. 9 T.R. Fennell, B.C. Sweatman and J.W. Bridges, The induction of hepatic cytochrome P-450 in C57BL/lO and DBAI2 mice by isosafrole and piperonyl butoxide. A comparative study with other inducing agents, Chem.-Biol. Interact., 31 (1980) 189. 10 J.C. Cook and E. Hodgson, Induction of cytochrome P-450 by methylenedioxyphenyl compounds: importance of the methylene carbon, Toxicol. Appl. Pharmacol., 68 (1983) 131. 11 P.D. Lotlikar and M.B. Wasserman, Effects of safrole and isosafrole pretreatment on N- and ring hydroxylation of 2-acetamidofluorene by the rat and hamster, Biochem. J., 129 (1972) 937. 12 B.G. Lake, R. Hopkins, J. Chakraborty, J.W. Bridges and D.V. Parke, The influence of some hepatic enzyme inducers and inhibitors on extrahepatic drug metabolism, Drug Metab. Dispos., 1 (1973) 342. 13 R.M. Philpot and E. Hodgson, The production and modification of cytochrome P-450 difference spectra by in vivo administration of methylenedioxyphenyl compounds, Chem.-Biol. Interact., 4 (1971/1972) 185. 14 R.M. Philpot and E. Hodgson, A cytochrome P-450-piperonyl butoxide spectrum similar to that produced by ethylisocyanide, Life Sci., 10 (1971) 503. 15 M.R. Franklin, The enzymatic formation of a methylene-dioxyphenyl derivative exhibiting an isocyanide-like spectrum with reduced cytochrome P-450 in hepatic microsomes, Xenobiotica, 1 (1971) 581. 16 S.W. Bigelow and D.W. Nebert, The Ah regulatory gene product. Survey of nineteen polycyclic aromatic compounds’ and fifteen benzo(a)pyrene metabolites’ capacity to bind to the cytosolic receptor, Toxicol. Lett., 10 (1982) 109. 17 H.J. Eisen, R.R. Hannah, C. Legraverend, A.B. Okey and D.W. Nebert, The Ah receptor: controlling factor in the induction of drug-metabolizing enzymes by certain chemical carcinogens and other environmental pollutants, in: G. Litwock (Ed.), Biochemical Actions of Hormones, Academic Press, New York, 1983, p. 227. 18 W.F. Greenlee and R.A. Neal, The Ah receptor: a biochemical and biological perspective, in: M. Conn (Ed.), The Receptors, V.11, Academic Press, New York, 1985, p. 89. 19 J.C. Cook and E. Hodgson, The induction of cytochrome P-450 by isosafrole and related methylenedioxyphenyl compounds, Chem.-Biol. Interact., 54 (1985) 299. 20 A. Poland, E. Glover and A.S. Kende, Stereospecific, high affinity binding of 2,3,7,8tetrachlorodibenzo-p-dioxin by hepatic cytosol: evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase, J. Biol. Chem., 251 (1976) 4936. 21 A.B. Okey, G.P. Bondy,M.E. Mason, G.F. Kahl, H.J. Eisen, T.M. Guenthner and D.W. Nebert, Regulatory gene product of the Ah locus. Characterization of the cytosolic
240
22
23 24
25
26 27
28
inducer-receptor complex and evidence for its nuclear translocation, J. Biol. Chem., 254 (1979) 11636. A.B. Okey and L.M. Vella, Elevated binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene to the Ah receptor in hepatic cytosols from phenobarbitaltreated rats and mice, Biochem. Pharmacol., 33 (1984) 531. J.C. Cook and E. Hodgson, 2,2-Dimethyl-5-t-butyl-1,3-benzodioxole: an unusual inducer of microsomal enzymes, Biochem. Pharmacol., 33 (1984) 3941. G.J. Fisher, H. Fukushima and J.L. Gaylor, Isolation, purification and properties of a unique form of cytochrome P-450 in microsomes of isosafrole-treated rats, J. Biol. Chem., 256 (1981) 4388. C.R. Elcombe, M. Dickins, B. Sweatman and J.W. Bridges, Substrate-elicited dissociation of the isosafrole metabolite-cytochrome P-450 complex and the consequent reactivation of monooxygenation, Hoppe-Seyler’s 2. Physiol. Chem., 357 (1976) 1026. R.G. Steel and J.H. Torrie, in: Principles and Procedures of Statistics. A Biometrical Approach, McGraw-Hill, New York, 1980. A. Poland and E. Glover, Genetic expression of aryl hydrocarbon hydroxylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin: evidence for a receptor mutation in genetically non-responsive mice, Mol. Pharmacol., 11 (1975) 389. R.H. Tukey and D.W. Nebert, Regulation of mouse cytochrome P,-450 by the Ah receptor. Studies with a P,-450 cDNA clone, Biochemistry, 23 (1984) 6003.
Interactions,
Elsevier Scientific
58 (1986)
Publishers
Ireland
233
233-240
Ltd.
Rapid Communication
-.
INDUCTION OF CYTOCHROME P-450 IN CONGENIC C57BL/6J MICE BY ISOSAFROLE: LACK OF CORRELATION WITH THE Ah LOCUS
JON C. COOK* Toxicology (U.S.A.)
and ERNEST
Program.
Box
HODGSON 7633,
North
Carolina
State
University,
Raleigh,
NC
27695
(Received October 10th. 1985) (Revision received February 2nd, 1986) (Accepted March 7th. 1986)
SUMMARY
Isosafrole induction of cytochrome P-450 was compared in congenic strains of C57BL/6J mice, one of which expresses normal levels of the Ah receptor [B6(Ahb)], and another that does not contain a measurable receptor concentration [ B6( Ahd)] . Using sucrose gradient analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding, an Ah receptor concentration of 69.1 + 3.8 fmol/mg protein was measured in the hepatic cytosol from B6 Ahb) mice, while no receptor could be detected in the cytosol from BG(Ah 6 ) mice. Isosafrole treatment (75 mg/kg X 3 days) increased the total hepatic microsomal cytochrome P-450 content to the same extent in the two congenic strains. The level of microsomal monooxygenase induction in the isosafrole-treated B6(Ahd) mice was greater than that of B6(Ahb) mice for ethylmorphine Ndemethylase and isosafrole metabolite-complex formation, the latter a measure of cytochrome P,-450. In the case of 7-ethoxycoumarin 0-deethylase only the isosafrole-treated B6(Ahd) mice had elevated microsomal activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) also revealed a similar induction pattern for the two congenic strains, following isosafrole treatment. Thus, the isosafrole treated B6 (Ahd) mice produced an
*Present address: Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, NC 27709, U.S.A. Abbreviations: B6(Ahb), C57BL/6J mice with measurable levels of the Ah receptor; BS(Ahd), C57BL/6J-Ahd’d [B6.D2-Ahd’d (NE8)] mice with no detectable Ah receptor; MDEG, buffer containing 25 mM MOPSpH 7.4, 1 mM dithiothreitol, 1.5 mM EDTA and 10% (v/v) glycerol; MDP, methylenedioxyphenyl; 3-MC, 3-methylcholanthrene; MOPS, 3(N-morpholino)propanesulfonic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. 0009-2797/86/$03.50
o 1986 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland
Ltd.
234 equivalent or slightly larger induction of cytochrome P-450 than the B6(Ah”) mice, suggesting that there is no direct role for the Ah receptor in the regulation of these cytochrome P-450 monooxygenase activities by isosafrole. Key words: Ah locus - Induction Methylenedioxyphenyl compounds
-
Cytochrome
P-450
-
Isosafrole
-
INTRODUCTION
Isosafrole is a naturally occurring methylenedioxyphenyl (MDP) compound that was originally reported to induce a novel form of cytochrome P-450 (P-450d) in rats [1,2]. Cytochrome P-450d was subsequently shown to be a second 3-methylcholanthrene inducible form [ 3,4]. In the mouse, isosafrole appears to induce cytochrome P2-450, as well as cytochrome P,-450, a form which corresponds to cytochrome P-450d in the rat [ 5,6]. Cytochrome P,450 in the mouse corresponds to cytochrome P-450~ of the rat [3,5]. Isosafrole induces cytochrome P-450~ in the rat [ 3,7], but conflicting data exists on cytochrome PI-450 induction in the mouse. Isosafrole treatment increases cytochrome P,-450-mRNA [S] yet monooxygenase activities associated with cytochrome PI-450 are not elevated [6,9,10]. The cytochrome P-450 induction pattern following isosafrole treatment has been described as either phenobarbital-like, 3-methylcholanthrene-like, or mixed in nature [ 1,9-121. MDP compounds have a biphasic effect on cytochrome P-450 levels and monooxygenase activity, namely, inhibition followed by induction [ 131. Inhibition is related to the formation of a stable MDP metabolite-cytochrome P-450 complex which is also responsible for the appearance of a Type III double-Soret optical difference spectrum [ 14,151. Bigelow and Nebert [ 161 reported that isosafrole produced 67% displacement of [3H] TCDD from the Ah receptor in C57BL/6N mice. From genetic evidence using C57BL/6N, DBA/BN, and (C57BL/6N X DBA/BN)F, mice, Ohyama et al. [6] concluded that cytochrome P,-450 induction by isosafrole was inherited as an autosomal additive trait and that the Ah locus, or a closely segregating gene, was involved. The involvement of the Ah receptor in the regulation of hepatic cytochrome P-450 levels by the 3-methylcholanthrene (3-MC) class of inducers has been the subject of numerous reviews [17,18]. Cook and Hodgson [19] examined the role of the Ah receptor in the induction of cytochrome P-450 by isosafrole, as well as two other MDP compounds, piperonyl butoxide and 5-t-butyl-1,3-benzodioxole. Using sucrose density gradient analysis, it was not possible to demonstrate the displacement of TCDD or 3-MC from the Ah receptor by these MDP compounds even though they were used at a lOOO-fold excess. In in vivo experiments on C57BL/6 mice, a single 500 mg/kg i.p. dose of isosafrole produced a twofold increase in the binding of 3-[3H] MC to the Ah receptor after 24 h. This increase may be due to induction of the receptor. If isosafrole does bind to the Ah receptor a decrease in 3-[3H] MC binding should have been observed
235 following isosafrole treatment, in vivo [ 191. Phenobarbital, which does not interact with the Ah receptor [ 20,211, is the only other compound that has been shown to cause an increase in 3-[3H]MC binding to the Ah receptor [ 221. The data of Cook and Hodgson [ 191 suggest that the Ah receptor does not play a direct role in the regulation of cytochrome P-450 by isosafrole or by MDP compounds in general. In an attempt to resolve these conflicting findings, we have examined the ability of isosafrole to induce cytochrome P-450 in congenic C57BL/6J mice. These strains of mice differ in the gene locus which codes for the expression of the Ah receptor. The B6(Ahb) mice are C57BL/6J mice from Jackson Laboratories with normal levels of Ah receptor, while the B6(Ahd) mice are derived from C57BL/6J mice but do not express measurable levels of the Ah receptor. MATERIALS
AND
METHODS
We received B6(Ahd) homozygote breeding pairs from Dr. A. Poland, McArdle Laboratory for Cancer Research (University of Wisconsin, Madison). Offspring from these breeding pairs were used in the experiments reported. Dr. Poland derived the B6(Ahd) homozygotes from B6N.D2N-Ahd’d (NE13) mice by backcross-intercross breeding into C57BL/6J mice. Dr. D. Nebert, National Institute of Health, donated the NIH mice to Dr. Poland. At this time, 32 biochemical markers have been examined to confirm the congenicity of the B6(Ahd) homozygotes; no deviation from the normal C57BL/6J profile has been detected (Dr. Linda Birnbaum, NIEHS, pers. commun.). B6(Ahb) mice were obtained from Jackson Laboratories (Bar Harbor, ME). Seven-weekold male mice were dosed i.p. daily for 3 days with either corn oil or 75 mg/kg isosafrole. The animals, 4 per group, were killed on day 4 and liver microsomes and cytosol were prepared as previously described [10,19]. Cytochrome P-450 content, 7-ethoxycoumarin Odeethylase activity and ethylmorphine N-demethylase activity were also determined by previously described methods [ 10,19,23]. Isosafrole metabolite-complex formation was measured by the method of Fisher et al. [24]. Briefly, isosafrole was added to a final concentration of 0.15 mM to both sample and reference cuvets which contained 0.8 mg protein/ml of a microsomal suspension. Twenty five microliters of an NADPH regenerating system was added to the sample cuvet. After 6-min incubation sodium dithionite was added to both sample and reference cuvets. The amount of isosafrole-metabolite complex formed was calculated from AA455-490 nm using an extinction coefficient of 75 mM-’ cm-’ [ 251. SDS-PAGE was performed as previously described [ 231. The Ah receptor was measured using sucrose density gradient analysis [ 211 as modified by Cook and Hodgson [ 191. The source and purity of the chemicals used are as outlined in Cook and Hodgson [ 19,231. Statistical significance was determined using an analysis of variance to compute the least significant difference, with P < 0.05 as the criterion for significance [ 261.
236 RESULTS
AND DISCUSSION
Ah receptor concentration in hepatic cytosol from mice of the two congenie C57BL/6J strains was measured by sucrose gradient analysis using [3H] TCDD as the radioligand. B6(Ahb) mice had an Ah receptor concentration of 69.1 + 3.8 fmol/mg protein, while no Ah receptor could be detected in the hepatic cytosol from B6(Ahd) mice. Thus, if isosafrole does not require the Ah receptor to induce cytochrome P-450, as previously suggested [19], then similar levels of induction should be seen in these congenic strains. If isosafrole induction is dependent upon the Ah receptor, and if isosafrole behaves like ligands other than TCDD then, the B6(Ahb) strain should be inducible while the B6(Ahd) strain should not. Ohyama et al. [6] reported that isosafrole treatment at 150 mg/kg for 3 days induced cytochrome P2-450 equally well in C57BL/6N and DBA/BN mice even though DBA/BN mice do not contain measurable amounts of cytosolic Ah receptor. At lower doses of isosafrole, the induction of cytochrome PI-450 was greater in C57BL/6N mice than DBA/BN mice; a similar result is seen with TCDD for cytochrome PI-450 induction [ 271. We treated the congenic C57BL/6J mice with 75 mg/kg isosafrole for 3 days, since in the study of Ohyama et al. [ 61, this dose appears to produce a greater induction of C57BL/6N mice than DBA/BN mice even though it is at the high end of the dose-response curve.
TABLE
I
INDUCTION OF CYTOCHROME MONOOXYGENASE ACTIVITIES C57BL/6 MICE Treatment/ strain
Oil/B6(Ahb) ksafrole BG(Ah Oil/BG(Ah ba ) Isosafrole/ B6(Ahd)
Cytochrome
P-450 AND CYTOCHROME P-450-DEPENDENT BY ISOSAFROLE IN CONGENIC STRAINS OF
(nmol/mg protein)
Isosafrole metabolitecomplex formationa (nmol/min mg protein)
7-Ethoxycoumarin 0-deethylasea (nmol/min mg protein
Ethylmorphine N-demethylasea (nmol/min mg protein)
0.92 1.24b
0.017 0.031b
1.65 1.85
5.42 7.17b
0.90 1.32b
0.015 0.040b”
1.30 2.4gb”
5.42 8.96b’C
P-450a
aMean values are shown. The average S.E.‘s were 0.09, 0.004, 0.17 and 0.51 for cytochrome P-450, isosafrole-complex formation, 7-ethoxycoumarin 0-deethylase and ethylmorphine N-demethylase, respectively. bf’ < 0.05 compared to the appropriate control. ‘P < 0.05 compared to isosafrole-treated B6 (Ahb) mice.
237
60
50 45
1
2
3
4
5
Fig. 1. SDS-PAGE of hepatic microsomes from congenic C57BL/6J mice treated with isosafrole. A mixture of molecular weight standards was applied to well 1. Microsomes (20 pg protein) from treated mice were applied as indicated: (2) oil control/B6(Ahb), (3) isosafrole/B6(Ahb), (4) isosafrole/B6(Ahd) and (5) oil control/B6(Ahd). Migration is from the top of the gel to the bottom. Figures on the left represent mol. wt. x 10’. The arrows point to the molecular band regions which are induced by isosafrole.
Isosafrole induces the following cytochrome P-450 monooxygenase parameters in mice: biphenyl 2- and 4-hydroxylase [9,10], ethylmorphine Ndemethylase [ 9,101, acetanilide 4-hydroxylase [ 61, and isosafrole metabolitecomplex formation, a measure of cytochrome P,-450 [6]. In this study, isosafrole metabolitecomplex formation, ethylmorphine N-demethylase, and ethoxycoumarin 0-deethylase activities were measured to further define the role of the Ah locus in isosafrole induction of cytochrome P-450-dependent monooxygenase activities. Isosafrole treatment increased total hepatic cytochrome P-450 content to the same extent in the two congenic strains (Table I). The isosafrole-treated congenic mice also had increased microsomal isosafrole metabolite-complex formation and ethylmorphine N-demethylase activ-
238 ities compared to their respective controls. In addition, the level of induction seen in the isosafrole-treated B6(Ahd) mice was greater than that of the B6(Ahb) mice for these two monooxygenase activities. In the case of 7ethoxycoumarin 0-deethylase activity only the isosafrole-treated B6( Ahd) mice had elevated microsomal activity. Thus, the isosafrole-treated B6(Ahd) mice produced an equivalent or greater induction of cytochrome P-450 than the B6(Ahb) mice. The gel in Fig. 1 confirms that the congenic mice treated with isosafrole produced similar induction patterns. Isosafrole treatment induced two bands at 53 000 and 54 000, and possibly a third band around 52 000. The increase in isosafrole metabolite-complex formation suggests that one of the induced bands is cytochrome P2-450. A similar induction pattern has been reported in isosafrole-treated Dub: ICR mice [lo]. Based upon the dose-response curve for the induction of cytochrome P,450 by isosafrole, Ohyama et al. [6] concluded that cytochrome P,-450 induction by isosafrole involved the Ah receptor or a closely segregating gene. These studies were performed using an anti-cytochrome P2-450 antibody and measured precipitable protein. This data [6] may be difficult to interpret since the anti-cytochrome P2-450 antibody used in these experiments has been shown to precipitate cytochrome P3-450 [ 281. Isosafrole also induces cytochrome P,-450 [6]. Congenic C57BL/6J mice appear to differ only in the expression of the gene for the Ah receptor. When these congenic mice were treated with isosafrole, the monooxygenase activities associated with isosafrole induction, as well as the cytochrome P-450 content and SDS-PAGE, were similar. These data derived from congenic strains coupled with the in vitro and in vivo data of Cook and Hodgson [19] suggest that there is no direct role for the Ah receptor in the regulation of these cytochrome P-450 monooxygenase activities by isosafrole. ACKNOWLEDGEMENTS
Paper No. 10159 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC. Supported, in part, by PHS Grants ES-07046 and ES-00044 from the National Institute of Environmental Health Sciences. We are indebted to Dr. A. Poland, of the McArdle Laboratory for Cancer Research, University of Wisconsin, for the generous gift of B6(Ahd) breeding pairs. REFERENCES 1 M. Dickens, J.W. Bridges, C.R. Elcombe and K.J. Netter, A novel haemoprotein induced by isosafrole pretreatment in the rat, Biochem. Biophys. Res. Commun., 80 (1978) 89. 2 D.E. Ryan, P.E. Thomas and W. Levin, Properties of liver microsomal cytochrome P450 from rats treated with isosafrole, in: M.J. Coon, A.H. Conney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette and P.J. O’Brien (Eds.), Microsomes and Drug Oxidations, Academic Press, New York, 1980, p. 167.
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