Induction of liver microsomal drug metabolism and inhibition of microsomal ribonuclease activity by phenobarbital

TOXICOLO‘iY

AND

APPLIED

PHARMACOLOGY

Induction of Liver of Microsomal

23,492-500 (1972)

Microsomal Ribonuclease

Drug Metabolism and Inhibition Activity by Phenobarbital1

R. T. LOUIS-FERDINAND~

AND G. C. FULLER

Department of Pharmacology and Toxicology, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881 Received March I,1972

Induction of Liver Microsomal Drug Metabolism and Inhibition Microsomal Ribonuclease Activity by Phenobarbital. LoursFERDINAND, R. T., and FULLER, G. C. (1972).ToxicoZ.Appl. Pharmacol. 23, 492-500.Microsomal ribonuclease(RNase)activity was determinedafter the administrationof phenobarbital(100mg/kg, ip) to rats. Eighty percent inhibition of RNaseactivity occurred 48 hr prior to maximal stimulation of p-chloro-N-methylaniline demethylation. Thereafter, RNase activity of

increased to control levels. Inhibition of RNase activity was also observed in a dose-dependent manner when phenobarbital(25 mg/kg or 50 mg/kg

twice daily) was administeredto intact, adrenalectomizedor hypophysectomizedrats. Theseresults suggestedthat RNase inhibition occurred during stimulation of drug metabolismin the absenceof the pituitary or adrenalglands.Recombinationexperimentsinvolving addition of 105,000g liver supernatantfractions to control microsomesresultedin inhibition of RNase activity of the latter fractions. The administration of 3-methylcholanthrene(40 mg/kg, ip for 2 days)did not producesignificant(p > 0.05) inhibition of microsomalRNase.The resultsof this study suggestedthat RNase inhibition did not require an intact pituitary-adrenal axis during induction of drug metabolismby phenobarbital.The inhibition of RNase wasprobably due to the presenceof a cytoplasmicinhibitor. The stimulation of hepatic microsomal drug metabolism by phenobarbital (PB) or 3-methylcholanthrene (MC) is reportedly mediated through enhanced de novo protein synthesisfollowing stimulation of DNA-dependent RNA polymerase activity. Support for such a mechanismrestson the observation of increaseduptake of precursor material into protein (Kato et al., 1966)and nuclear RNA (Gelboin et al., 1967)during induction, as well as the ability of inhibitors of transcription (Orrenius and Ernster, 1964) and translation (Conney and Gilman, 1963) to interfere with the stimulation of drug metabolism by PB and MC. However, several lines of evidence suggest that alternate mechanismsmay also contribute to the enhancement of drug metabolism. PB induction may be related to suppressionof degradative enzyme activity sincehepatic microsomal constituents are reported to accumulate following PB treatment through 1 Portions of this paper were presented at the 1970 Fall Meeting of the American Society for Pharmacology and Experimental Therapeutics, Palo Alto, California. 2 Present address: Department of Pharmacology and Toxicology, School of Pharmacy, University of Maryland, Baltimore, Maryland 21228. Copyright 0 1972 by Academic Press, Inc. 492 All rights of reproduction in any form reserved.

PHENOBARBITAL

lNHlBlTlON

OF RNASE

493

stabilization of preexisting protein (Jick and Shuster, 1966; Schenkman, 1970).Previous communications from this laboratory (Louis-Ferdinand and Fuller, 1970a, b) and others (Seifert and Vacha, 1970; Lechner and Pousada, 1971) have suggestedthat the enhancement of drug metabolism by PB is associatedwith an inhibition of microsomal ribonuclease (RNase) activity. This inhibition has been correlated with reduction of microsomal RNA breakdown (Mycek, 1971) and stabilization of hepatic ribosomal RNA (Cohen and Ruddon, 1970). RNase activity is also suppressedfollowing administration of growth hormone (Brewer et al., 1969), corticotropin (Imrie and Hutchinson, 1965)or cortisone (Barnabei and Ottolenghi, 1968).These findings suggestedthat inhibition of RNase by PB might be mediated through the pituitary or adrenal glands. The studies to be described were conducted to determine the importance of an intact pituitary-adrenal axis on the RNase inhibition produced by PB. The time course of RNase inhibition was also determined to provide a temporal correlation of RNase inhibition with stimulation of drug metabolism. In vitro experiments were conducted to examine factors which influence RNase and to detect the presenceof RNase inhibitors following PB treatment. METHODS

Intact, hypophysectomized or adrenalectomized adult male Sprague-Dawley rats (200-300 g) obtained from Charles River Breeding Laboratories (Wilmington, Massachusetts) were used throughout the course of this investigation. These rats were maintained on commercial laboratory chow and water ( I “,,: saline for adrenalectomized rats) ad libitum in a room with controlled temperature and alternating I2 hr periods of light and dark. Analytical reagent grade chemicals or equivalent were used throughout these studies. Cofactors and substrates were obtained from Calbiochem, Los Angeles, California. Liver microsomal fractions were prepared aspreviously described(Louis-Ferdinand and Fuller, 1970a). Microsomal demethylation of aminopyrine was determined by assaying for formaldehyde formed (McMahon and Easton, 1962). Demethylation of p-chloro-N-methylaniline (PCMA) was determined according to the procedure of Kupfer and Bruggeman (1966) as modified by Fuller et al. (1969). p-Nitroanisole demethylase activity was determined by assayingforp-nitrophenol formed according to a modification of the procedure of Netter and Seidel ( 1964). Incubations were carried out at 37°C under air for 30 min in a medium containing 3 pmoles p-nitroanisole. 2 pmoles NADP, 20 pmoles glucose 6-phosphate, 80 pmoles nicotinamide, 120 pmoles MgC12, 5 IU glucose-6-phosphate dehydrogenase. microsomal protein (2-3 mg) and 1 ml 0.1 M (pH 7.9) phosphate buffer in a total volume of 3.9 ml. At the end of the incubation period 10 ml ice-cold acetone was added to the incubation flasks; I ml 0.5 M glycine-NaOH buffer (pH 9.5) was added. After centrifugation the resulting supernatant was read against the tissue blank at 410 nm on a Beckman DB-G spectrophotometer. The amount ofp-nitrophenol formed was determined by comparison with standard p-nitrophenol solutions. RNase activity was estimated by the method of Tsukada (1969) as modified by Louis-Ferdinand and Fuller (1970a). The incubation medium (1 ml) for RNase determinations contained 0.2 ml 0.2 M Tris*HCl buffer pH 7.6, 1.0 mg highly polymerized

494

LOUIS-FERDINAND

AND

FULLER

yeast RNA and IO-25 ~1 of microsomal suspension containing 18-39 mg protein/ml. RNA was added last, and the incubation was carried out in air at 37°C for 20 min in a Dubnoff incubator. Following the incubation, 1.0 ml 1 M HCl in 76 % ethanol was added as a precipitant, and the mixture was shaken thoroughly. The soluble fraction was separated by centrifugation at 9000 g at 0°C for 15 min. The absorbance of 1 ml of the clear supernatant after dilution with 2-5 ml of distilled water was read at 260 nm vs distilled water using a Beckman double-beam spectrophotometer. Readings obtained were corrected for absorbances of suitable tissue and RNA blanks. Protein content was determined by the procedure of Lowry et al. (1951). RESULTS

InIorder stimulation

to characterize the temporal relationship between RNase inhibition and of microsomal N-demethylation activity, one dose of PB (100 mg/kg, ip)

f \ \

HOURS

FOLLOWING

PHENOBARBITAL

(lOOmg/Kg)

TREATMENT

FIG. 1. Effect of phenobarbital on rat hepatic ribonuclease during stimulation of microsomal oxidative demethylation. Male rats (250-300 g) were given one dose of phenobarbital (100 mg/kg). Values represent mean * SE of at least 4 animals per point. RNase expressed as absorbance at 260 nm/20 min/mg microsomal protein. was given to male (250-300 g) rats. Groups of animals were killed at 24 hr intervals for

5 consecutive days. The PB treatment significantly (pc 0.05) enhanced PCMA demethylation

within

48 hr (Fig.

1). Conversely,

microsomal

RNase

was-reduced

to 20%

of

PHENOBARBITAL

INHIBITION OF RNASE

495

control values 24 hr following PB administration. The RNase inhibition was less pronounced at later time periods as induction of oxidative demethylation progressed. Significant inhibition of RNase activity persisted for 4 days. These results suggested that a temporal relationship may exist between RNase inhibition and stimulation of drug metabolism by PB. When two dosesof PB (50 mg/kg or 25 mg/kg twice daily for 4 days) were administered to inact male rats, the increase in microsomal aminopyrine demethylase and decreasein RNase were inversely related in a dose-dependentmanner

(Table I). TABLE 1 DOSE-RELATED INHIBITION OF RIBONUCLEASE DURING INDUCTION OF DRUG METABOLISMBY PHENOBARBITAL

Treatment” (N) Control (6) Phenobarbital,25 mg/kg (5) Phenobarbital,50 mg/kg (6)

Aminopyrine demethylationb (meani SE) 59.8 i 2.9

91.2 & 4.7d 100.5& 2.9d

Ribonuclease” (mean+ SE) 0.230 i 0.027

0.105i 0.021d 0.068 z 0.008d

aMale rats (170-200g)weretreatedwith phenobarbitaltwicedaily for 4 days andsacrificedon day 5. bNanomoles formaldehyde/30 min/mgmicrosomal protein. ’ Absorbanceat 260nm/20min/mgmicrosomalprotein. ’ Significantlydifferentfrom control(p x 0.05). In order to determine whether inhibition of microsomal RNase by PB required an intact pituitary-adrenal axis, PB (50 mg/kg or 25 mg/kg, ip) wasadministered to adrenal-

ectomized rats twice daily (Table 2). In each group, phenobarbital treatment significantly (p x 0.05) increased aminopyrine demethylation as well as microsomal protein, while RNase activity was reduced. Similar results were obtained when hypophysectomized rats were used in a parallel experiment (Table 2). These results indicate that RNase inhibition by PB does not require an intact pituitary-adrenal axis. However, the magnitude of the PB-induced increase in demethylation and RNase inhibition was smaller in the hypophysectomized and adrenalectomized rats compared to intact rats. Microsomes and 105,000 g liver supernatant fractions prepared from control and PB treated rats were recombined to determine the presence of RNase inhibitors. Negligible RNase activity was found in untreated supernatant fractions obtained from either control or PB treated animals. The incubation of control supernatants with microsomes from PB treated rats resulted in a 17 y0 stimulation of RNase activity (Table 3). Conversely, incubation of control microsomes with supernatants from PB treated animals resulted in an 87 % inhibition of control RNase. These data suggest that RNase inhibition following PB treatment may be related to the presence of an inhibitor of microsomal RNase in the soluble fraction. It has been reported that an endogenous RNase inhibitor normally present in liver fractions can be inactivated by 1O-6 M p-chloromercuribenzoate (PCMB) (Shortman, 1962). Repetition of these experiments in the presence of PCMB ( 10m6M) did not result in a stimulation of RNase activity to control values.

496

LOUIS-FERDINAND

AND FULLER

TABLE 2 INHIBITION OF RIBONUCLEASE BY PHENOBARBITAL DURING INDUCTION OF HEPATIC MICROWJMAL DRUG METABOLISM IN ADRENALECTOMIZED AND HYPOPHYSECTOMIZED RATS

Treatment’ (N) Adrenalectomized Control (6) Phenobarbital(11) Hypophysectomized Control (6) Phenobarbital(8)

Aminopyrine demethylationb (meanf SE)

Ribonuclease” (meani SE)

50.2 It 1.9 69.9i 3.0d

0.161rt 0.019 0.102f O.OIOd

48.0 ziz1.3 68.7 f 5.V

0.364f 0.050 0.1472z0.050d

aAt 2 wk after adrenalectomy or hypophysectomy. Male rats4.5daysold receivedphenobarbital(50 mg/kg ip) twice daily for 4 days and were sacrificedon day5. bNanomoles formaldehyde/30 min/mgmicrosomal protein. c Absorbanceat 260nm/20min/mgmicrosomal protein. dSignificantlydifferentfrom control (p < 0.05). TABLE 3 RIBONUCLEASE INHIBITION BY 105,000g SUPERNATANT FRACTION PREPARED FROM LIVERS OF PHENOBARBITAL TREATED RATS“

Microsomesb Control Phenobarbital Control Phenobarbital Control Phenobarbital

Supernatant Ribonuclease’ Control Phenobarbital Phenobarbital Control

0.151 0.057 0.231 0.123 0.031 0.159

4 Four male (375410 g) rats were treated with 50 mg/kg phenobarbitalfor 5 days. Controls were givendistilledwater.All animalswerekilled 24hr after the lasttreatment. bMicrosomesand supernatantsused were pooled from liver fractionspreparedfrom at least4 animals. c Mean of duplicatedeterminations(absorbance at 260nm/20min/mgmicrosomal protein). Since MC induction of hepatic drug metabolism differs in many respectsfrom that produced by PB treatment (Mannering et al., 1969), the following experiment was conducted to determine the effect of MC on hepatic microsomal RNase activity. 3-Methylcholanthrene (40 mg/kg, ip) was administered to male rats for 2 days, and the animals were killed on the third day. Hepatic microsomal fractions were assayedfor p-nitroanisole demethylase and RNase activity. Two day treatment with MC resulted in a 3-fold stimulation of microsomal O-demethylase activity (Table 4). However, stimulation of demethylase activity by MC was not associated with a significant (p > 0.05) inhibition of RNase.

PHENOBARBITAL

INHJBITION

497

OF RNASE

TABLE 4 EFFECT

0~ ~-METHYL~H~LAN~YI-~ENE DEMETHYLATION

Treatment Control (5) 3-Methylcholanthrene ’ Expressed

as nanomoles

0~ HEPA~IC MICROS~MALP-NITROANISOLE AND RIBONUCLEASE ACWITY p-Nitroanisole demethylation” (mean f

(N)

(40 mg/kg,

2 days)

p-nitrophenol

3.46 + 1.52 13.77 i 3.52"

(5)

formed/mg

Ribonucleaseb (mean ?r SE)

SE)

microsomal

protein/30

0.302 so.038 0.233 + 0.064" min

(male

rats

190.--

280 g). b Absorbance at 260 nm/mg microsomal ’ Significantly different from control (p a Not significantly different from control

protein/20

min.

< 0.05). (p > 0.05).

DISCUSSION A temporal relationship may exist between the accumulation of nuclear RNA and inhibition of nuclear RNase since the administration of PB has been reported to enhance nuclear RNA synthesis (Nebert and Gelboin, 1969) and inhibit nuclear RNase (Seifert and Vacha, 1970) within 24 hr. A similar relationship between microsomal RNase inhibition and stimulation of protein synthesis remains to be established. Our results indicate that inhibition of microsomal RNase may occur prior to the enhancement of drug metabolism by PB. This inhibition may be responsible for a similar reduction in the breakdown of endogenous microsomal nucleic acid (Mycek, 197 1) and stabilization of liver RNA (Cohen and Ruddon, 1970; Steele, 1970) following PB treatment. It is unlikely that the suppression of RNase activity observed is attributable to dilution of RNase activity due to increased non-RNase liver microsomal protein since microsomal protein content is only increased 1.5-fold following 5 daily administrations of PB. Increases in microsomal protein greater than 5-fold are required for dilution to account for the magnitude of RNase inhibition observed 24 hr after PB treatment (Fig. 1). The inhibition of RNase during induction of drug metabolism in adrenalectomized or hypophysectomized rats indicates that an intact pituitary-adrenal axis is not required for RNase inhibition by PB. However, our results do not permit exclusion of a permissive effect of these glands during inhibition of RNase in livers of intact animals. In fact PB treatment of adrenalectomized rats did not result in the same degree of RNase inhibition or stimulation of demethylation as was observed in intact or hypophysectomized animals (Table 2). These results are consistent with previous findings which suggest that endogenous steroid hormones enhance the inducibility of drug metabolizing enzymes in vivo (Orrenius ef al., 1969). Induction of drug metabolism by PB may be mediated through the stimulation of nuclear RNA synthesis in intact or adrenalectomized animals; however, no enhancement of RNA synthesis is observed when PB is administered to hypophysectomized animals (Nerbert and Gelboin, 1969). Yet PB treatment produces increased microsomal amino acid incorporation (Jondorf et al., 1966) and induction of drug metabolism

498

LOUIS-FERDINAND

AND

FULLER

(Conney et al., 1961) following hypophysectomy. Thus, stimulation of nuclear RNA synthesis by PB may not be the only pathway contributing to the enhancement of drug metabolism in such animals. Inhibition of RNase may contribute to the induction by PB of drug metabolism in hypophysectomized rats. However, the relative importance of altered synthetic and/or degradative mechanisms in the intact animal during PB induction remains unclear. Ribonuclease inhibition is correlated with an RNase inhibitor in cell sap fractions obtained from livers of PB treated rats. This inhibition is not attributable to a direct effect of PB on RNase since direct addition of PB to incubation media does not influence RNase activity in vitro (Louis-Ferdinand and Fuller, 1970a). The addition of PCMB to the incubation media at concentrations (lO-‘j M) reported to inactivate endogenous RNase inhibitor material (Shortman, 1962) did not increase the microsomal RNase activity from PB treated rats to control values. Mycek (1971) has reported that breakdown of microsomal RNA of both control and PB treated rats is stimulated by PCMB (10V3 M); however, following PCMB addition, the microsomal RNase activity of PB treated rats was still less than that of control preparations. Thus, our results would not indicate that PB inhibition of RNase is attributable to increased levels of the previously described endogenous RNase inhibitors. The inhibition of microsomal RNase may not be an essential component of MC induction since 3-fold stimulation of microsomal demethylation by MC was not associated with significant (p > 0.05) inhibition of microsomal RNase. These findings support those of other workers (Sladek and Mannering, 1969) who have suggested that differences may exist between the mechanisms by which MC and PB induce drug metabolism. Since the data reported here suggest a partial role of microsomal degradative enzymes in the mechanism of PB induction, the results of toxicologic investigations in induced animals may be influenced by interactions on similar degradative systems. ACKNOWLEDGMENTS

This work was supported by an N.D.E.A. Predoctoral Fellowship and a grant from the U.R.I. Research Committee. REFERENCES BARNABIE, 0 and OTTOLENGHI, C. (1968). Glucocorticoid control of nuclear synthesis and of

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PHENOBARBITAL INHIBlTION OF RNA%

4YY

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in the mechanismsby which polycyclic hydrocarbons and phenobarbital product their inductive effectson microsomalN-demethylating systems.Mol. Pharmacol.5, 174-l 85. STEELE, W. J. (1970).Phenobarbital-inducedprolongation of the half-life of ribosomalRNA of rat liver. Fed.Proc. Fed. Amer. Sot. Exp. Biol. 29,737 (abstract). TSUKADA,K. (1969). Activity of serumribonucleaseafter partial hepatectomy and acute stressin the rat. Biochim.Biophys.Acta l&21-24.