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[33] Induction Protocols for C y t o c h r o m e s P 4 5 0 I I I A in Vivo and in Primary Cultures o f Animal and H u m a n H e p a t o c y t e s B y M A R T I N E D A U J A T , L Y D I A N E PICHARD, ISABELLE FABRE,
THIERRY PINEAU, G~RARD FABRE, CLAUDE BONFILS, and P A T R I C K M A U R E L Introduction Although induction of cytochromes P450IA and P450IIB by the prototypic inducers 3-methylcholanthrene and phenobarbital, respectively, was well established in the mid 1970s, the existence of a "third class" of inducers was only realized in the early 1980s. Pregnenolone 16a-carbonitrile and triacetyloleandomycin were independently shown to induce P450p and P450LM3C in rat and rabbit, respectively.~'2 These forms were later shown to be orthologs and members of the P450IIIA subfamily. 3,4 Since then, seven P450IIIA genes have been characterized in rat (P450IIIA1, P450IIIA2), human (P450IIIA3, IIIA4, IIIA5, and IIIA7), and rabbit P450IIIA6. 5-H In the last few years, expression of these genes was shown to be strongly inducible by a number of structurally unrelated chemicals. 12-23The protocols described here have been used successfully I N. A. Elshourbagy and P. S. Guzelian, J. Biol. Chem. 255, 1279 (1980). 2 C. Bonfils, I. Dalet-Beluche, and P. Maurel, Biochem. Biophys. Res, Commun. 104, 1101 (1982). 3 S. A. Wrighton, P. Maurel, E. G. Schuetz, P. B. Watkins, B. Young, and P. S. Guzelian, Biochemistry 2,4, 2171 (1985). 4 C. Bonfils, I. Dalet-Beluche, and P. Maurel, Biochem. Pharmacol. 34, 2445 (1985). 5 F. J. Gonzalez, D. W. Nebert, J. P. Harwick, and C. B. Kasper, J. Biol. Chem. 2,60, 7435 (1985). 6 F. J. Gonzalez, B. J. Song, and J. P. Hardwick, Mol. Cell. Biol. 6, 2969 (1986). 7 D. T. Molowa, E. G. Schuetz, S. A. Wrighton, P. B. Watkins, P. Kremers, G. MendezPicon, G. A. Parker, and P. S. Guzelian, Proc. Natl. Acad. Sci. U.S.A. 83, 5311 (1986). 8 R. W. Bork, T. Muto, P. H. Beaune, P. K. Srivastava, R. S. Lloyd, and F. P. Guengerich, J. Biol. Chem. 264, 910 (1989). 9 T. Aoyama, S. Yamano, D. J. Waxman, D. P. Lapenson, U. A. Meyer, V. Fischer, R. Tyndale, T. Inaba, W. Kalow, H. V. Gelboin, and F. J. Gonzalez, J. Biol. Chem. 264, 10388 (1989). 10 M. Komori, K. Nishio, T. Fujitani, H. Ohi, M. Kitada, S. Mima, K. Itahashi, and T. Kamataki, Arch. Biochem. Biophys. 272, 219 (1989). 11 C. DaleI, P. Clair, M. Daujat, P. Fort, J. M. Blanchard, and P. Maurel, DNA 7, 39 (1988). 12 p. B. Watkins, S. A. Wrighton, P. Maurel, E. G. Schuetz, G. Mendez-Picon, G. A. Parker, and P. S. Guzelian, Proc. Natl. Acad. Sci. U.S.A. 82, 6310 (1985).
METHODS IN ENZYMOLOGY,VOL. 206
Copyright © 1991by AcademicPress, Inc. All rightsof reproduction in any form reserved.
346
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in the authors' laboratory (as well as by others) to induce cytochromes P450IIIA both in vivo (rat and rabbit) and in primary cultures of animal and human hepatocytes, z4 Inducers
Chemicals characterized as inducers of class IIIA cytochromes P450 as well as the source from which they were purchased are as follows: triacetyloleandomycin (TAO) from Pfizer (Massy, France); erythromycin base (ER) from Roussel (Paris, France); diacetoxyerythralosamine (DAEM) and monobenzoyloleandomycin (MBO) from Dr. Daniel Mansuy (Facultd de Mddecine des Saint P6res, Paris, France); rifampicin (RIF) from Merrel Dow-Lepetit (Paris, France); dexamethasone (DEX) and carbamazepine (CAR) from Sigma (St. Louis, MO); pregnenolone 16t~-carbonitrile (PCN) from Upjohn (Kalamazoo, MI); phenobarbital (PB) from Specia (Paris, France); sulfadimidine (SUL) from Centre Pharmaceutique Europden Sanofi (Bordeaux, France); phenytoin (PHE) from Delalande
13 S. A. Wrighton, E. G. Schuetz, P. B. Watkins, P. Maurel, J. Barwick, B. S. Bailey, H. T. Hattie, B. Young, and P. S. Guzelian, Mol. Pharmacol. 28, 312 (1985). 14R. Lange, C. Larroque, C. Balny, and P. Maurel, Biochem. Biophys. Res. Commun. 126, 833 (1985). 15 E. G. Schuetz and P. S, Guzelian, J. Biol. Chem. 259, 2007 (1984). 16C. Dalet, J. M. Blanchard, P. S. Guzelian, J. Barwick, H. Hartle, and P. Maurel, Nucleic Acids Res. 14, 5999 (1986). 17M. Daujat, L. Pichard, C. Dalet, C. Larroque, C. Bonfils, D. Pompon, D. Li, P. S. Guzelian, and P. Maurel, Biochem. Pharmacol. 36, 3597 (1987). Is E. Sartori, M. Delaforge, D. Mansuy, and P. Beaune, Biochem. Biophys. Res. Commun. 128, 1434 (1985). 19 j. Combalbert, I. Fabre, G. Fabre, I. Dalet, J. Derancourt, J. P. Cano, and P. Maurel, Drug. Metab. Dispos. 17, 197 (1989). 20 C. Ged, J. M. Rouillon, L. Pichard, J. Combalbert, N. Bressot, P. Bories, H. Michel, P. Beaune, and P. Maurel, Br. J. Clin. Pharmacol.. 28, 373 (1989). 21 L. Pichard, I. Fabre, G. Fabre, J. Domergue, B. Saint Aubert, G. Mourad, and P. Maurel, Drug. Metab. Dispos. 18, 595 (1990). 22 K. A. Hostetler, S. A. Wrighton, D. T. Molowa, P. E. Thomas, W. Levin, and P. S. Guzelian, Mol. Pharmacol. 35, 279 (1989). 23 p. Bertault-Peres, C. Bontils, G. Fabre, S. Just, J. P. Cano, and P. Maurel, Drug Metab. Dispos. 15, 391 (1987). 24 The human P450IIIA family appears to have at least four genes coding for P450IIIA3, IIIA4, IIIA5, and IIIA7. 7-1°The genes encode proteins with at least 82% similarity in the primary sequence. Thus, polyclonal antibodies directed against any P450IIIA form are expected to cross-react with all other forms. We shall accordingly use the term P450IIIA to designate these proteins.
[33]
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347
(Courbevoie, France); phenylbutazone (PBZ) and sulfinpyrazone (SULF) from Ciba-Geigy (Rueil-Malmaison, France); clotrimazole (CLO) from R. Bellon (Paris, France). Tween 20 and dimethyl sulfoxide (DMSO) used to prepare suspensions or solutions of inducers were from Sigma and Merck (Darmstadt, Germany), respectively. Induction Protocols in Whole Animals Male and female Wistar rats (150-200 g) and New Zealand rabbits (1.8-2.2 kg) are obtained from Ifa-Credo (Montlu~on, France) and INRAZootechnie (Montpellier, France), respectively. The animals are maintained on a 12 hr light-dark cycle (8 am to 8 pm), individually or in pairs in wire-bottomed cages with free access to food and water. The temperature of the room is controlled at 20° . In all cases treatment starts between 9 and 10 am, and animals are fasted overnight before sacrifice. All treatments described here have been standardized in terms of dosing and duration to give high level of induction of P450IIIA forms in rats and/or rabbits. Generally, 1 ml of suspension or solution of inducer is administered per kilogram body weight. Triacetyloleandomycin and Erythromycin
Triacetyloleandomycin and erythromycin are well tolerated at high dose (I mmol/kg/day, i.e., 814 or 734 mg/kg/day for TAO or ER, respectively) and can be administered either intraperitoneally or per os in both rats and rabbits. For intraperitoneal injection, the drug is resuspended either in corn oil or in 2% Tween 20 in water (v/v) and is injected once every day for 5 days. For per os administration, the drug is thoroughly mixed into ground chow (1 g/100 g of chow), and animals are fed this diet ad libitum for 7 days. Higher levels of induction are generally reached following per os treatment. These treatments work well in both the rat and the rabbit. Rifarnpicin
In the rabbit, including newborns (1 and 2 weeks) and adults of both sexes and pregnant females for transplacental fetus induction, rifampicin is a strong inducer when administered intraperitoneally as dissolved (50 mg/ml) in 40 mM NaOH in water at a dose of 50 mg/kg/day for 4 days. However, such treatment is inefficient in the rat, for which a "high dose" protocol has to be used. For this purpose RIF is thoroughly mixed into the ground chow (I g/100 g of chow), and animals are fed this diet ad
348
CELL CULTURE
SYSTEMS
[33]
A
-2,1kb UT
RIF
B
(f)
UT
RIF
UT
RIF (m)
FIG. 1. Induction of P450IIIAby rifampicin in male and female rats. Male and female rats were treated with RIF according to the "high dose" protocol described here. After an overnight fast, animals were sacrificed, and poly(A) RNA and mierosomes were prepared. (A). Northern blot of poly(A) RNA (5/zg per lane) from untreated and RIF-treated male animals (n = 3), developed with radiolabeled pDEX12 eDNA (P450IIIAI), kindly provided by Dr. P. S. Guzelian. (B) Western blot of liver rnicrosomes (2/zg per lane) from female (left) and male (right) rats (n = 2), developed with anti-P450IIIA6antibodies. In these microsomes the average ER demethylase activity was 0.92 and 3.83, and 2.11 and 4.02 nmol/mg/minin untreated and RIF-treated females and males, respectively. Average amounts of P450-TAO complex formed under standard conditions3'4 were 0.0 and 0.67, and 0.0 and 0.61 nmol/mg in untreated and RIF-treated females and males, respectively.
libitum for 3 weeks. By this time animals have received an average dose of 300 to 600 mg/kg/day (varying with individual food intake) and exhibit high levels of P450IIIA as reported in Fig. 1.
Pregnenolone 16a-Carbonitrile and Dexamethasone In rats and rabbits pregnenolone 16a-carbonitrile and dexamethasone are administered eitherper os (by gavage) or intraperitoneally as a suspension in 2% T w e e n 20 in water (v/v) and at a dose o f 150 mg/kg/day for 4 days.
Diacetoxyerythralosamine and Monobenzoyloleandomycin Diacetoxyerythralosamine and m o n o b e n z o y l o l e a n d o m y c i n , derivatives o f E R and TAO, are administered intraperitoneally after resuspension in corn oil at a dose o f I00 mg/kg/day for 4 days.
Phenobarbital Phenobarbital is dissolved in water (i g/liter p H 7.0), and animals are allowed free access to this drinking solution for 1 week.
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P450IIIA INDUCTION PROTOCOLS
349
IliA 2 3 4 5 6 7 8 9 1 0 ......
.....
IA2
FIG. 2. P450IIIA and P450IA2 levels in microsomes from primary cultures of human hepatocytes treated with various drugs. Hepatocytes from the whole liver of an organ donor were plated and maintained in culture in HCD medium for 72 hr in the absence or presence of 50/zM of various chemicals (except phenobarbital, which was used at 2 mM). Microsomes were prepared, and 10 ~g of protein from each sample was analyzed in Western blots developed either with anti-P450IIIA6or anti-P450IA2 antibodies. Authentic human P450IIIA (P450CsA)19and microsomes from freshly isolated hepatocytes, before plating, were also loaded on the gel (lanes 1 and 2, respectively). Lanes 3 to 10 refer to untreated, RIF-, DEX-, TAO-, PB-, fl-naphthoflavone-, 3-methylcholanthrene-, and PHE-treated cultures, respectively.
Induction Protocol with Rifampicin in Patients In the course of a clinical study on cyclosporin A m e t a b o l i s m and the relevance of urinary excretion of 6/3-hydroxycortisol as a m a r k e r of P450IIIA, 19,2° 14 patients w e r e treated with rifampicin according to a protocol a p p r o v e d by the Saint Eloi Hospital Ethic C o m m i t t e e (Montpellier, France). T h e s e patients w e r e admitted in the hospital for abdominal surgery and received no medication during the w e e k preceding the operation, except for t r e a t m e n t with R I F . R I F was given orally at a dose of 600 mg per day for 5 days. During surgery, a wedge liver biopsy was obtained and used to prepare m i c r o s o m e s . Induction Protocols in P r i m a r y Cultures of Rabbit and H u m a n Hepatocytes Procedures for isolating h e p a t o c y t e s f r o m 2-month-old or adult rabbit and h u m a n liver are described in detail e l s e w h e r e ) v,21,25 In our current protocol, 3.5 or 5.0 x l06 freshly isolated h e p a t o c y t e s are inoculated in 60-ram plastic dishes (Falcon, Becton Dickinson, Lincoln Park, N J) p r e c o a t e d with 50/zg rat tail collagen (Type VII, Sigma) in a total volume of 3 ml of either W a y m o u t h ' s 75215 or H C D medium consisting of a 1 : 1 D. Diaz, I. Fabre, M. Danjat, G. Fabre, B. Saint Aubert, P. Bories, H. Michel, and P. Maurel, Gastroenterology 99, 737 (1990).
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TABLE I INCREASED ACCUMULATION OF P450IIIA AFTER TREATMENT OF HUMAN HEPATOCYTE CULTURES WITH VARIOUS CHEMICALSa Inducer
P450IIIA (relative level)
None Rifampicin Triacetyloleandomycin Dexamethasone Phenobarbital Carbamazepine Suifadimidine Phenytoin Phenylbutazone Sulfinpyrazone Clotrimazole
1.0 3.7-24 8.5-25 3.4- I 1.7 4.0-33 16 2.0-6.0 5.0-8.0 2.0-4.0 6.0 7.0-53
Human hepatocytes were maintained in primary cultures for 48 to 72 hr after plating, in the absence or presence of 5 0 / z M of the various inducers (except phenobarbital which was used at 2 mM). At the end of the treatment, hepatocytes were scraped and microsomes prepared. Relative levels of P450IIIA (normalized to 1.0 in untreated cultures) were determined by Western blots developed with anti-P450IIIA6 antibodies (see Fig. 2). These results are representative of at least two different preparations per inducer, and data from seven different preparations are presented. In all cases, when the data from different preparations were within 20% only the average value is presented; when larger differences occurred the extreme values are given.
ratio of Williams' E and Ham's F12 (Sigma) supplemented as described, 26 except that dexamethasone concentration is reduced here to 0.1/zM, in the presence of 5% fetal calf serum (GIBCO BRL, Paisley, Scotland). Four to six hours after plating, the medium is renewed and thereafter every 24 hr in the absence of serum. Both culture media are convenient for P450IIIA (as well as for other forms) induction in rabbit hepatocytesl7; only HCD medium was used with human cells in our laboratory: 1'25 In our standard protocol, cells are allowed a period of 24 to 72 hr after plating before the beginning of inducer treatments. Inducers are added to the culture medium as a 1000x solution in DMSO. Except for PB, which is 26 H. C. Isom and I. Georgoff, Proc. Natl. Acad. Sci. U.S.A. 81, 6378 (1984).
[33]
P450IIIA INDUCTION PROTOCOLS
A
351
UT 12 24
48
72
9611 RIF
B
ikb 0
12 48 72 96 4
UT
8
24 48 72 96h
RIF
Fro. 3. Effect of time on P450IIIA6 gene transcription and mRNA accumulation in untreated and RIF-treated rabbit hepatocyte cultures. Rabbit hepatocytes were plated and maintained in culture in HCD medium for 24 hr. At this time the medium was renewed in the absence (UT) or presence (RIF) of 50/zM RIF. At the indicated times, cells were scraped, and nuclei and poly(A) RNA were prepared. (A) P450IIIA6 gene transcription (from l07 nuclei) and (B) mRNA accumulation (5 p.g per lane) were determined a s d e s c r i b e d , 16'17 pLM3C eDNA 11:6 being used as a probe.
currently used at 2 mM, the final concentration of inducers is generally 50 or 100 /.tM, m o s t of the drugs being toxic to the cells a b o v e these concentrations. Control cultures receive the s a m e a m o u n t of D M S O (0.1%). Maximal induction of P450IIIA at the level of protein and m o n o x y genase activities requires at least 48 hr, w h e r e a s high levels of m R N A are reached b y 24 hr in both rabbit and h u m a n cultures (Figs. 2 and 3). General C o m m e n t s Besides the battery of biochemical tests, including gene transcription rate and m R N A and protein accumulation f r o m N o r t h e r n and Western blot analysis, the extent of P450IIIA induction can be evaluated through specific m o n o o x y g e n a s e activities (erythromycin N - d e m e t h y l a s e , nifedipine oxidase, or cyclosporin oxidase 19) and detection of a P 4 5 0 - T A O c o m p l e x absorbing at 457 nm after incubation of m i c r o s o m e s with T A O and N A D P H 3,4 (Fig. 1). P450IIIA inducers exhibit a m a r k e d species specificity in rats and rabbits. 13 T A O and E R are strong inducers in both species. R I F is a m o r e potent inducer in rabbits and h u m a n s than in rats as shown here (Fig. 1). P C N and D E X are strong inducers in the rat but fail to induce (PCN) or
352
CELL CULTURE SYSTEMS
[33]
TABLE II EFFECT OF RIFAMFICIN ON P450111A6 d e N o o o SYNTHESIS IN RABBIT HEPATOCYTES PRIMARY CULTURES a
P450IIIA6 d e n o o o synthesis (% of total)
Age of cultures (days) 1 3 11" 18"
UT
RIF
Ratio RIF/UT
0.4 0.8 1.4 1.9
1.7 3.2 5.4 5.3
4.2 4.0 3.9 2.8
a Rabbit hepatocytes were maintained in primary cultures in HCD medium for various periods of time (from 1 to 18 days) before a 48 hr treatment with 50 /zM RIF or DMSO (UT). At this time, hepatocytes were radiolabeled for 2 hr with tritiated Leu, and d e n o v o synthesis of P450IIIA6 was determined from immunoprecipitations of cell lysates with anti-P450IIIA6 antibodies as indicated.17 These experiments were carried out with different cultures. In cultures marked *, the dexamethasone concentration in the HCD medium was 1/zM.
only moderately induce (DEX) in the rabbit, perhaps simply because their dose-response is higher in this species. DAEM but not MBO is an inducer in rabbits, whereas both are potent inducers in rat. 18,23 Finally, PB is a moderate inducer in rats and does not induce in rabbits. This species specificity appears to be modified in primary cultures of hepatocytes. TAO and ER do not induce in either rats or rabbits, whereas they are good inducers in human cultures (Fig. 2). At the highest dose tested (100 tzM) RIF does not induce in rat cultures, but it is one of the best inducers in rabbits and humans. DEX and PB induce P450IIIA in hepatocyte cultures from all three species, whereas PCN only induces in rat cells. Interestingly, strong inducers of P450IIIA in animals, especially RIF which is a good inducer in humans in vivo, are also strong inducers in human hepatocyte cultures as shown in Fig. 2, where it also appears that the specificity of induction is conserved: class IIIA inducers do not increase P450IA forms and vice versa. 2L25 Other inducers of P450IIIA in human hepatocyte cultures are presented in Table I. It clearly appears that large interindividual variability occurs in the extent of induction. Note also the strong induction by CLO, already shown to be a P450IIIA inducer in rats in vioo. 22
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P450IVA INDUCTION
353
In both rabbit and human untreated hepatocyte cultures, P450IIIA gene transcription, mRNA and protein accumulation, and related monooxygenase activities rapidly decline after plating to levels hardly detectable after 48 hr (in contrast to other P450s). Nevertheless, these genes retain their capacity to be activated by their specific inducers in young (1 to 3 days old) and up to 3-week-old cultures from rabbits, as reported in Fig. 3 and Table II, and 1-week-old cultures from humans (owing to limited availability of human tissue longer times were not tried). Our experience is that, if the kinetics of protein or mRNA induction are to be evaluated, better results will be obtained in 3-day-old cultures where the postplating decline process has vanished. Acknowledgments We wish to thank our colleagues Drs. I. Dalet-Beluche, M. Maurice, and J. Combalbert for help in parts of this work. The collaboration ofDrs. D. Diaz, B. Saint Aubert, G. Mourad, J. Domergue, and H. Michel for providing human liver samples is gratefully acknowledged. This work was supported in parts by INSERM (R6seau de Recherche Clinique, convention No. 487020, P.M.), INRA (AIP No. 4432), la Caisse Nationale d'Assurance Maladie des Travailleurs Salad, s, I'ARC (M.D.), la Fondation pour la Recherche M6dicale Frangaise, and le Minist~re de la Recherche et de la Technologie.
[34] I n d u c t i o n P r o t o c o l s for t h e C y t o c h r o m e P 4 5 0 I V A S u b f a m i l y in A n i m a l s a n d P r i m a r y H e p a t o c y t e C u l t u r e s
By G. GORDON GIBSON and BRIAN G. LAKE Introduction The cytochrome P450IVA subfamily consists of seven members (termed P450IVA1 to P450IVA7), and all seven of the corresponding genes have been sequenced and their amino acid sequences predicted.1 To date, only the P450IVA1 (rat liver) and P450IVA7 (rabbit lung) enzymes from this subfamily have been isolated, purified to electrophoretic homogeneity, and their protein biochemistry and substrate specificity studied in any
i D. W. Nebert, D. R. Nelson, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. Waterman, D N A 8, 1 (1989).
METHODS IN ENZYMOLOGY,VOL. 206
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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[33] Induction Protocols for C y t o c h r o m e s P 4 5 0 I I I A in Vivo and in Primary Cultures o f Animal and H u m a n H e p a t o c y t e s B y M A R T I N E D A U J A T , L Y D I A N E PICHARD, ISABELLE FABRE,
THIERRY PINEAU, G~RARD FABRE, CLAUDE BONFILS, and P A T R I C K M A U R E L Introduction Although induction of cytochromes P450IA and P450IIB by the prototypic inducers 3-methylcholanthrene and phenobarbital, respectively, was well established in the mid 1970s, the existence of a "third class" of inducers was only realized in the early 1980s. Pregnenolone 16a-carbonitrile and triacetyloleandomycin were independently shown to induce P450p and P450LM3C in rat and rabbit, respectively.~'2 These forms were later shown to be orthologs and members of the P450IIIA subfamily. 3,4 Since then, seven P450IIIA genes have been characterized in rat (P450IIIA1, P450IIIA2), human (P450IIIA3, IIIA4, IIIA5, and IIIA7), and rabbit P450IIIA6. 5-H In the last few years, expression of these genes was shown to be strongly inducible by a number of structurally unrelated chemicals. 12-23The protocols described here have been used successfully I N. A. Elshourbagy and P. S. Guzelian, J. Biol. Chem. 255, 1279 (1980). 2 C. Bonfils, I. Dalet-Beluche, and P. Maurel, Biochem. Biophys. Res, Commun. 104, 1101 (1982). 3 S. A. Wrighton, P. Maurel, E. G. Schuetz, P. B. Watkins, B. Young, and P. S. Guzelian, Biochemistry 2,4, 2171 (1985). 4 C. Bonfils, I. Dalet-Beluche, and P. Maurel, Biochem. Pharmacol. 34, 2445 (1985). 5 F. J. Gonzalez, D. W. Nebert, J. P. Harwick, and C. B. Kasper, J. Biol. Chem. 2,60, 7435 (1985). 6 F. J. Gonzalez, B. J. Song, and J. P. Hardwick, Mol. Cell. Biol. 6, 2969 (1986). 7 D. T. Molowa, E. G. Schuetz, S. A. Wrighton, P. B. Watkins, P. Kremers, G. MendezPicon, G. A. Parker, and P. S. Guzelian, Proc. Natl. Acad. Sci. U.S.A. 83, 5311 (1986). 8 R. W. Bork, T. Muto, P. H. Beaune, P. K. Srivastava, R. S. Lloyd, and F. P. Guengerich, J. Biol. Chem. 264, 910 (1989). 9 T. Aoyama, S. Yamano, D. J. Waxman, D. P. Lapenson, U. A. Meyer, V. Fischer, R. Tyndale, T. Inaba, W. Kalow, H. V. Gelboin, and F. J. Gonzalez, J. Biol. Chem. 264, 10388 (1989). 10 M. Komori, K. Nishio, T. Fujitani, H. Ohi, M. Kitada, S. Mima, K. Itahashi, and T. Kamataki, Arch. Biochem. Biophys. 272, 219 (1989). 11 C. DaleI, P. Clair, M. Daujat, P. Fort, J. M. Blanchard, and P. Maurel, DNA 7, 39 (1988). 12 p. B. Watkins, S. A. Wrighton, P. Maurel, E. G. Schuetz, G. Mendez-Picon, G. A. Parker, and P. S. Guzelian, Proc. Natl. Acad. Sci. U.S.A. 82, 6310 (1985).
METHODS IN ENZYMOLOGY,VOL. 206
Copyright © 1991by AcademicPress, Inc. All rightsof reproduction in any form reserved.
346
CELL CULTURE SYSTEMS
[33]
in the authors' laboratory (as well as by others) to induce cytochromes P450IIIA both in vivo (rat and rabbit) and in primary cultures of animal and human hepatocytes, z4 Inducers
Chemicals characterized as inducers of class IIIA cytochromes P450 as well as the source from which they were purchased are as follows: triacetyloleandomycin (TAO) from Pfizer (Massy, France); erythromycin base (ER) from Roussel (Paris, France); diacetoxyerythralosamine (DAEM) and monobenzoyloleandomycin (MBO) from Dr. Daniel Mansuy (Facultd de Mddecine des Saint P6res, Paris, France); rifampicin (RIF) from Merrel Dow-Lepetit (Paris, France); dexamethasone (DEX) and carbamazepine (CAR) from Sigma (St. Louis, MO); pregnenolone 16t~-carbonitrile (PCN) from Upjohn (Kalamazoo, MI); phenobarbital (PB) from Specia (Paris, France); sulfadimidine (SUL) from Centre Pharmaceutique Europden Sanofi (Bordeaux, France); phenytoin (PHE) from Delalande
13 S. A. Wrighton, E. G. Schuetz, P. B. Watkins, P. Maurel, J. Barwick, B. S. Bailey, H. T. Hattie, B. Young, and P. S. Guzelian, Mol. Pharmacol. 28, 312 (1985). 14R. Lange, C. Larroque, C. Balny, and P. Maurel, Biochem. Biophys. Res. Commun. 126, 833 (1985). 15 E. G. Schuetz and P. S, Guzelian, J. Biol. Chem. 259, 2007 (1984). 16C. Dalet, J. M. Blanchard, P. S. Guzelian, J. Barwick, H. Hartle, and P. Maurel, Nucleic Acids Res. 14, 5999 (1986). 17M. Daujat, L. Pichard, C. Dalet, C. Larroque, C. Bonfils, D. Pompon, D. Li, P. S. Guzelian, and P. Maurel, Biochem. Pharmacol. 36, 3597 (1987). Is E. Sartori, M. Delaforge, D. Mansuy, and P. Beaune, Biochem. Biophys. Res. Commun. 128, 1434 (1985). 19 j. Combalbert, I. Fabre, G. Fabre, I. Dalet, J. Derancourt, J. P. Cano, and P. Maurel, Drug. Metab. Dispos. 17, 197 (1989). 20 C. Ged, J. M. Rouillon, L. Pichard, J. Combalbert, N. Bressot, P. Bories, H. Michel, P. Beaune, and P. Maurel, Br. J. Clin. Pharmacol.. 28, 373 (1989). 21 L. Pichard, I. Fabre, G. Fabre, J. Domergue, B. Saint Aubert, G. Mourad, and P. Maurel, Drug. Metab. Dispos. 18, 595 (1990). 22 K. A. Hostetler, S. A. Wrighton, D. T. Molowa, P. E. Thomas, W. Levin, and P. S. Guzelian, Mol. Pharmacol. 35, 279 (1989). 23 p. Bertault-Peres, C. Bontils, G. Fabre, S. Just, J. P. Cano, and P. Maurel, Drug Metab. Dispos. 15, 391 (1987). 24 The human P450IIIA family appears to have at least four genes coding for P450IIIA3, IIIA4, IIIA5, and IIIA7. 7-1°The genes encode proteins with at least 82% similarity in the primary sequence. Thus, polyclonal antibodies directed against any P450IIIA form are expected to cross-react with all other forms. We shall accordingly use the term P450IIIA to designate these proteins.
[33]
P450IIIA INDUCTIONPROTOCOLS
347
(Courbevoie, France); phenylbutazone (PBZ) and sulfinpyrazone (SULF) from Ciba-Geigy (Rueil-Malmaison, France); clotrimazole (CLO) from R. Bellon (Paris, France). Tween 20 and dimethyl sulfoxide (DMSO) used to prepare suspensions or solutions of inducers were from Sigma and Merck (Darmstadt, Germany), respectively. Induction Protocols in Whole Animals Male and female Wistar rats (150-200 g) and New Zealand rabbits (1.8-2.2 kg) are obtained from Ifa-Credo (Montlu~on, France) and INRAZootechnie (Montpellier, France), respectively. The animals are maintained on a 12 hr light-dark cycle (8 am to 8 pm), individually or in pairs in wire-bottomed cages with free access to food and water. The temperature of the room is controlled at 20° . In all cases treatment starts between 9 and 10 am, and animals are fasted overnight before sacrifice. All treatments described here have been standardized in terms of dosing and duration to give high level of induction of P450IIIA forms in rats and/or rabbits. Generally, 1 ml of suspension or solution of inducer is administered per kilogram body weight. Triacetyloleandomycin and Erythromycin
Triacetyloleandomycin and erythromycin are well tolerated at high dose (I mmol/kg/day, i.e., 814 or 734 mg/kg/day for TAO or ER, respectively) and can be administered either intraperitoneally or per os in both rats and rabbits. For intraperitoneal injection, the drug is resuspended either in corn oil or in 2% Tween 20 in water (v/v) and is injected once every day for 5 days. For per os administration, the drug is thoroughly mixed into ground chow (1 g/100 g of chow), and animals are fed this diet ad libitum for 7 days. Higher levels of induction are generally reached following per os treatment. These treatments work well in both the rat and the rabbit. Rifarnpicin
In the rabbit, including newborns (1 and 2 weeks) and adults of both sexes and pregnant females for transplacental fetus induction, rifampicin is a strong inducer when administered intraperitoneally as dissolved (50 mg/ml) in 40 mM NaOH in water at a dose of 50 mg/kg/day for 4 days. However, such treatment is inefficient in the rat, for which a "high dose" protocol has to be used. For this purpose RIF is thoroughly mixed into the ground chow (I g/100 g of chow), and animals are fed this diet ad
348
CELL CULTURE
SYSTEMS
[33]
A
-2,1kb UT
RIF
B
(f)
UT
RIF
UT
RIF (m)
FIG. 1. Induction of P450IIIAby rifampicin in male and female rats. Male and female rats were treated with RIF according to the "high dose" protocol described here. After an overnight fast, animals were sacrificed, and poly(A) RNA and mierosomes were prepared. (A). Northern blot of poly(A) RNA (5/zg per lane) from untreated and RIF-treated male animals (n = 3), developed with radiolabeled pDEX12 eDNA (P450IIIAI), kindly provided by Dr. P. S. Guzelian. (B) Western blot of liver rnicrosomes (2/zg per lane) from female (left) and male (right) rats (n = 2), developed with anti-P450IIIA6antibodies. In these microsomes the average ER demethylase activity was 0.92 and 3.83, and 2.11 and 4.02 nmol/mg/minin untreated and RIF-treated females and males, respectively. Average amounts of P450-TAO complex formed under standard conditions3'4 were 0.0 and 0.67, and 0.0 and 0.61 nmol/mg in untreated and RIF-treated females and males, respectively.
libitum for 3 weeks. By this time animals have received an average dose of 300 to 600 mg/kg/day (varying with individual food intake) and exhibit high levels of P450IIIA as reported in Fig. 1.
Pregnenolone 16a-Carbonitrile and Dexamethasone In rats and rabbits pregnenolone 16a-carbonitrile and dexamethasone are administered eitherper os (by gavage) or intraperitoneally as a suspension in 2% T w e e n 20 in water (v/v) and at a dose o f 150 mg/kg/day for 4 days.
Diacetoxyerythralosamine and Monobenzoyloleandomycin Diacetoxyerythralosamine and m o n o b e n z o y l o l e a n d o m y c i n , derivatives o f E R and TAO, are administered intraperitoneally after resuspension in corn oil at a dose o f I00 mg/kg/day for 4 days.
Phenobarbital Phenobarbital is dissolved in water (i g/liter p H 7.0), and animals are allowed free access to this drinking solution for 1 week.
[33]
P450IIIA INDUCTION PROTOCOLS
349
IliA 2 3 4 5 6 7 8 9 1 0 ......
.....
IA2
FIG. 2. P450IIIA and P450IA2 levels in microsomes from primary cultures of human hepatocytes treated with various drugs. Hepatocytes from the whole liver of an organ donor were plated and maintained in culture in HCD medium for 72 hr in the absence or presence of 50/zM of various chemicals (except phenobarbital, which was used at 2 mM). Microsomes were prepared, and 10 ~g of protein from each sample was analyzed in Western blots developed either with anti-P450IIIA6or anti-P450IA2 antibodies. Authentic human P450IIIA (P450CsA)19and microsomes from freshly isolated hepatocytes, before plating, were also loaded on the gel (lanes 1 and 2, respectively). Lanes 3 to 10 refer to untreated, RIF-, DEX-, TAO-, PB-, fl-naphthoflavone-, 3-methylcholanthrene-, and PHE-treated cultures, respectively.
Induction Protocol with Rifampicin in Patients In the course of a clinical study on cyclosporin A m e t a b o l i s m and the relevance of urinary excretion of 6/3-hydroxycortisol as a m a r k e r of P450IIIA, 19,2° 14 patients w e r e treated with rifampicin according to a protocol a p p r o v e d by the Saint Eloi Hospital Ethic C o m m i t t e e (Montpellier, France). T h e s e patients w e r e admitted in the hospital for abdominal surgery and received no medication during the w e e k preceding the operation, except for t r e a t m e n t with R I F . R I F was given orally at a dose of 600 mg per day for 5 days. During surgery, a wedge liver biopsy was obtained and used to prepare m i c r o s o m e s . Induction Protocols in P r i m a r y Cultures of Rabbit and H u m a n Hepatocytes Procedures for isolating h e p a t o c y t e s f r o m 2-month-old or adult rabbit and h u m a n liver are described in detail e l s e w h e r e ) v,21,25 In our current protocol, 3.5 or 5.0 x l06 freshly isolated h e p a t o c y t e s are inoculated in 60-ram plastic dishes (Falcon, Becton Dickinson, Lincoln Park, N J) p r e c o a t e d with 50/zg rat tail collagen (Type VII, Sigma) in a total volume of 3 ml of either W a y m o u t h ' s 75215 or H C D medium consisting of a 1 : 1 D. Diaz, I. Fabre, M. Danjat, G. Fabre, B. Saint Aubert, P. Bories, H. Michel, and P. Maurel, Gastroenterology 99, 737 (1990).
350
CELL CULTURE SYSTEMS
[33]
TABLE I INCREASED ACCUMULATION OF P450IIIA AFTER TREATMENT OF HUMAN HEPATOCYTE CULTURES WITH VARIOUS CHEMICALSa Inducer
P450IIIA (relative level)
None Rifampicin Triacetyloleandomycin Dexamethasone Phenobarbital Carbamazepine Suifadimidine Phenytoin Phenylbutazone Sulfinpyrazone Clotrimazole
1.0 3.7-24 8.5-25 3.4- I 1.7 4.0-33 16 2.0-6.0 5.0-8.0 2.0-4.0 6.0 7.0-53
Human hepatocytes were maintained in primary cultures for 48 to 72 hr after plating, in the absence or presence of 5 0 / z M of the various inducers (except phenobarbital which was used at 2 mM). At the end of the treatment, hepatocytes were scraped and microsomes prepared. Relative levels of P450IIIA (normalized to 1.0 in untreated cultures) were determined by Western blots developed with anti-P450IIIA6 antibodies (see Fig. 2). These results are representative of at least two different preparations per inducer, and data from seven different preparations are presented. In all cases, when the data from different preparations were within 20% only the average value is presented; when larger differences occurred the extreme values are given.
ratio of Williams' E and Ham's F12 (Sigma) supplemented as described, 26 except that dexamethasone concentration is reduced here to 0.1/zM, in the presence of 5% fetal calf serum (GIBCO BRL, Paisley, Scotland). Four to six hours after plating, the medium is renewed and thereafter every 24 hr in the absence of serum. Both culture media are convenient for P450IIIA (as well as for other forms) induction in rabbit hepatocytesl7; only HCD medium was used with human cells in our laboratory: 1'25 In our standard protocol, cells are allowed a period of 24 to 72 hr after plating before the beginning of inducer treatments. Inducers are added to the culture medium as a 1000x solution in DMSO. Except for PB, which is 26 H. C. Isom and I. Georgoff, Proc. Natl. Acad. Sci. U.S.A. 81, 6378 (1984).
[33]
P450IIIA INDUCTION PROTOCOLS
A
351
UT 12 24
48
72
9611 RIF
B
ikb 0
12 48 72 96 4
UT
8
24 48 72 96h
RIF
Fro. 3. Effect of time on P450IIIA6 gene transcription and mRNA accumulation in untreated and RIF-treated rabbit hepatocyte cultures. Rabbit hepatocytes were plated and maintained in culture in HCD medium for 24 hr. At this time the medium was renewed in the absence (UT) or presence (RIF) of 50/zM RIF. At the indicated times, cells were scraped, and nuclei and poly(A) RNA were prepared. (A) P450IIIA6 gene transcription (from l07 nuclei) and (B) mRNA accumulation (5 p.g per lane) were determined a s d e s c r i b e d , 16'17 pLM3C eDNA 11:6 being used as a probe.
currently used at 2 mM, the final concentration of inducers is generally 50 or 100 /.tM, m o s t of the drugs being toxic to the cells a b o v e these concentrations. Control cultures receive the s a m e a m o u n t of D M S O (0.1%). Maximal induction of P450IIIA at the level of protein and m o n o x y genase activities requires at least 48 hr, w h e r e a s high levels of m R N A are reached b y 24 hr in both rabbit and h u m a n cultures (Figs. 2 and 3). General C o m m e n t s Besides the battery of biochemical tests, including gene transcription rate and m R N A and protein accumulation f r o m N o r t h e r n and Western blot analysis, the extent of P450IIIA induction can be evaluated through specific m o n o o x y g e n a s e activities (erythromycin N - d e m e t h y l a s e , nifedipine oxidase, or cyclosporin oxidase 19) and detection of a P 4 5 0 - T A O c o m p l e x absorbing at 457 nm after incubation of m i c r o s o m e s with T A O and N A D P H 3,4 (Fig. 1). P450IIIA inducers exhibit a m a r k e d species specificity in rats and rabbits. 13 T A O and E R are strong inducers in both species. R I F is a m o r e potent inducer in rabbits and h u m a n s than in rats as shown here (Fig. 1). P C N and D E X are strong inducers in the rat but fail to induce (PCN) or
352
CELL CULTURE SYSTEMS
[33]
TABLE II EFFECT OF RIFAMFICIN ON P450111A6 d e N o o o SYNTHESIS IN RABBIT HEPATOCYTES PRIMARY CULTURES a
P450IIIA6 d e n o o o synthesis (% of total)
Age of cultures (days) 1 3 11" 18"
UT
RIF
Ratio RIF/UT
0.4 0.8 1.4 1.9
1.7 3.2 5.4 5.3
4.2 4.0 3.9 2.8
a Rabbit hepatocytes were maintained in primary cultures in HCD medium for various periods of time (from 1 to 18 days) before a 48 hr treatment with 50 /zM RIF or DMSO (UT). At this time, hepatocytes were radiolabeled for 2 hr with tritiated Leu, and d e n o v o synthesis of P450IIIA6 was determined from immunoprecipitations of cell lysates with anti-P450IIIA6 antibodies as indicated.17 These experiments were carried out with different cultures. In cultures marked *, the dexamethasone concentration in the HCD medium was 1/zM.
only moderately induce (DEX) in the rabbit, perhaps simply because their dose-response is higher in this species. DAEM but not MBO is an inducer in rabbits, whereas both are potent inducers in rat. 18,23 Finally, PB is a moderate inducer in rats and does not induce in rabbits. This species specificity appears to be modified in primary cultures of hepatocytes. TAO and ER do not induce in either rats or rabbits, whereas they are good inducers in human cultures (Fig. 2). At the highest dose tested (100 tzM) RIF does not induce in rat cultures, but it is one of the best inducers in rabbits and humans. DEX and PB induce P450IIIA in hepatocyte cultures from all three species, whereas PCN only induces in rat cells. Interestingly, strong inducers of P450IIIA in animals, especially RIF which is a good inducer in humans in vivo, are also strong inducers in human hepatocyte cultures as shown in Fig. 2, where it also appears that the specificity of induction is conserved: class IIIA inducers do not increase P450IA forms and vice versa. 2L25 Other inducers of P450IIIA in human hepatocyte cultures are presented in Table I. It clearly appears that large interindividual variability occurs in the extent of induction. Note also the strong induction by CLO, already shown to be a P450IIIA inducer in rats in vioo. 22
[34]
P450IVA INDUCTION
353
In both rabbit and human untreated hepatocyte cultures, P450IIIA gene transcription, mRNA and protein accumulation, and related monooxygenase activities rapidly decline after plating to levels hardly detectable after 48 hr (in contrast to other P450s). Nevertheless, these genes retain their capacity to be activated by their specific inducers in young (1 to 3 days old) and up to 3-week-old cultures from rabbits, as reported in Fig. 3 and Table II, and 1-week-old cultures from humans (owing to limited availability of human tissue longer times were not tried). Our experience is that, if the kinetics of protein or mRNA induction are to be evaluated, better results will be obtained in 3-day-old cultures where the postplating decline process has vanished. Acknowledgments We wish to thank our colleagues Drs. I. Dalet-Beluche, M. Maurice, and J. Combalbert for help in parts of this work. The collaboration ofDrs. D. Diaz, B. Saint Aubert, G. Mourad, J. Domergue, and H. Michel for providing human liver samples is gratefully acknowledged. This work was supported in parts by INSERM (R6seau de Recherche Clinique, convention No. 487020, P.M.), INRA (AIP No. 4432), la Caisse Nationale d'Assurance Maladie des Travailleurs Salad, s, I'ARC (M.D.), la Fondation pour la Recherche M6dicale Frangaise, and le Minist~re de la Recherche et de la Technologie.
[34] I n d u c t i o n P r o t o c o l s for t h e C y t o c h r o m e P 4 5 0 I V A S u b f a m i l y in A n i m a l s a n d P r i m a r y H e p a t o c y t e C u l t u r e s
By G. GORDON GIBSON and BRIAN G. LAKE Introduction The cytochrome P450IVA subfamily consists of seven members (termed P450IVA1 to P450IVA7), and all seven of the corresponding genes have been sequenced and their amino acid sequences predicted.1 To date, only the P450IVA1 (rat liver) and P450IVA7 (rabbit lung) enzymes from this subfamily have been isolated, purified to electrophoretic homogeneity, and their protein biochemistry and substrate specificity studied in any
i D. W. Nebert, D. R. Nelson, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. Waterman, D N A 8, 1 (1989).
METHODS IN ENZYMOLOGY,VOL. 206
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