Gilbert J. Mannering

Depression of hepatic cytochrome P-450-dependent monooxygenase systems with administered interferon inducing agents

Abstract Cytochrome P-450-dependent monooxygenase activities and cytochrome P-450 levels were depressed in hepatic microsomes from rats treated with 12 interferon inducing agents of various types: small molecules (e.g. tilorone), an RNA virus (Mengo), a fungal mycophage (statolon), liver RNA, a synthetic double-stranded polynucleotide (poly rI · poly rC), a bacterial lipopolysaccharide ( E. coli endotoxin) and an attenuated bacteria ( B. pertussis vaccine). The results suggest that the depression of hepatic cytochrome P-450-dependent monooxygenase systems may be a general property of interferon inducing agents.

KINETICS OF DRUG METABOLISM BY HEPATIC MICROSOMES.

Abstract The metabolism of drugs by hepatic microsomes is thought to proceed through a coupled reaction involving NADPH oxidase and one or more ‘hydroxylases’ of limited specificity. If this concept is correct, it can be predicted that numerous drugs would inhibit competitively the metabolism of another drug employed as a substrate for the microsomal system. This proved to be the case. The N-demethylation of ethylmorphine was competitively inhibited by hexobarbital, chlorpromazine, zoxazolamine, phenylbutazone, and acetanilide; and ethylmorphine, and chlorpromazine were mutually inhibitory, each retarding the metabolism of the other. All these drugs are known to be oxidized by microsomal enzymes. Barbital and acetazoleamide, drugs which are not metabolized, failed to act as inhibitors. Evidence is presented to show that certain of the observed inhibitions may be explained on a basis of interaction of alternative substrates. Kinetic data were obtained on both phenobarbital-induced and noninduced microsomal systems. The close similarity of the Michaelis and inhibitor constants seen in the two systems, coupled with the increased maximal velocity values observed for the oxidation of a given drug after induction, argue strongly that a quantitative rather than a qualitative change is involved in the inductive process.

EFFECTS OF INTERFERON-INDUCING AGENTS ON HEPATIC CYTOCHROME P-450 DRUG METABOLIZING SYSTEMS*

Duration and intensity of drug action are greatly influenced by the rates of which drugs are biotransformed by the cytochrome P-450-linked monooxygenase systems of the hepatic endoplasmic reticulum. Several interferon-inducing agents (poly rI.rC, tilorone, vaccines, viruses, endotoxin) are shown to markedly depress hepatic P-450 systems when administered to rodents. The interferon (IF) inducers that depress hepatic drug metabolism also modulate certain immune responses; it is therefore not known whether the depression of P-450 is due to IF per se or to the action of IF-inducing agents on one or more components of the immune system. The loss of cytochrome P-450 elicited by IF-inducing agents is accompanied by a perturbation of heme metabolism associated with the dissociation of heme from cytochrome P-450. The agents also cause losses of hepatic catalase and tryptophan 2,3-dioxygenase. These studies predict that viral infections, vaccinations, and treatment with IF-inducing agents will be shown to seriously impair the metabolism of drugs in humans.

Kinetics of ethanol metabolism in the intact rat and monkey

Abstract Because ethanol distributes evenly throughout body water and because it can be given in saturating doses, it offers an opportunity for a comparison of kinetic studies in vivo with those performed in vitro . Ethanol was given intraperitoneally to rats and monkeys in a dose (1 g/kg) which not only saturated the enzyme system responsible for its oxidation, but which permitted sufficient time for its even distribution throughout body water before metabolism reduced its concentration to a level where its oxidation proceeded in accordance with first order kinetics. Blood alcohol concentrations were determined at intervals and these data were used to calculate apparent kinetic constants in vivo for the oxidation of ethanol. The apparent Michaelis constants in vivo obtained in this manner compared quite favorably with those for the reaction of ethanol with crude alcohol dehydrogenase preparations obtained from the livers of the same species. The calculated apparent maximum velocities in vivo were quite similar to the observed maximum rates in vivo of ethanol metabolism seen in each of the species. Kinetic data were more closely correlative in the rat than in the monkey.

Hepatic cytochrome P-450-dependent monooxygenase systems of the trout, frog and snake—III. Induction☆

1. 3-Methylcholanthrene administration increased levels of cytochrome P-450, benzo[a]pyrene hydroxylase activity and p-nitrophenetole O-deethylase activity in hepatic microsomes from the brown trout (Salmo trutta), leopard frog (Rana pipiens) and the garter snake (Thamnophis). The level of aminopyrine N-demethylase activity was increased in trout microsomes, but not in those from the frog, snake or rat. 2. The shift in the Soret maximum of the reduced carbon monoxide difference spectrum of cytochrome P-450 from 450 to 448 nm, which is observed when 3-methylcholanthrene is administered to rats, was not seen in microsomes from the trout, frog or snake. 3. Benzo[a]pyrene hydroxylase activity induced by 3-methylcholanthrene in the trout, frog and snake was inhibited by relatively low concentrations of SKF 525-A, but that induced in the rat was not. alpha-Naphthoflavone inhibited 3-methylcholanthrene-induced benzo[a]pyrene hydroxylase activity in the trout, frog and rat, but not in the snake. 4. 3-Methylcholanthrene induced an increase in the 455/430 nm peak height ratio of the reduced ethylisocyanide spectrum of microsomes in the rat and trout, but not in the frog or snake. 5. These observations (items 1--4 of the abstract) show that different species of cytochrome P-450 are induced by 3-methylcholanthrene in the four vertebrates. 6. Phenobarbital did not alter the components or the activities of hepatic monooxygenase systems in the trout, frog or snake, even though it was shown to accumulate in the livers of these vertebrates as readily as it does in the liver of the rat.

Interactions of Δ9-tetrahydrocannabinol with the hepatic microsomal drug metabolizing system

Abstract Δ9-Tetrahydrocannabinol (Δ9-THC) was shown to combine with hepatic microsomes from both untreated and phenobarbital (Pb) treated male rats to give a typical type I difference spectrum. The affinity of the microsomes for Δ9-THC was very high, as reflected by the spectral dissociation constant (Ks) values of 18.5 and 9.1 μM for microsomes from untreated and Pb-treated animals, respectively. Δ9-THC inhibited competitively the microsomal metabolism of ethylmorphine, a typical type I substrate. The inhibitor constant (Ki) value obtained with untreated animals was very low, 15.4 μM. These studies support the view that Δ9-THC is metabolized by the hepatic mixed function oxidase system involving cytochrome P-450. The high reactivity of Δ9-THC with this system raises the possibility that Δ9-THC may interfere with the biotransformation of other drugs in vivo .

Induction of hepatic mono-oxygenase systems of pregnant rats with phenobarbital and 3-methylcholanthrene

Abstract When sodium phenobarbital was given to pregnant and non-pregnant female rats (40 mg/kg for 4 days), ethylmorphine N -demethylase, a cytochrome P-450-dependent system, was induced about 4-fold in non-pregnant females, but only 2-fold in pregnant females. The induction of microsomal cytochrome P-450 was also lower in pregnant animals. This impairment of phenobarbital induction occurred within 3 days of conception and disappeared after parturition within 5 days. 3-Methylcholan-threne induction of hepatic benzo[a]pyrene hydroxylase, a cytochrome P 1 -450-dependent mono-oxy-genase system not inducible by phenobarbital, was not impaired during pregnancy. The depressed response of the maternal liver to phenobarbital induction can be partially reversed by the coadministra-tion of 3-methylcholanthrene. The administration of a higher dose of sodium phenobarbital (80 mg/kg day for 4 days) overcame the pregnancy-related lowered response to phenobarbital induction observed with the smaller dose of the barbiturate. The similarity in responses of the maternal and fetal livers to inducing agents suggests that a common regulatory mechanism operates in both the fetus and the pregnant female.

Role of the intracellular distribution of hepatic catalase in the peroxidative oxidation of methanol.

Previous to 1950, when Bonnichsenl isolated crystalline alcohol dehydrogenase (ADH) and showed that it did not react with methanol,

Effect of phenobarbital administration on the oxygen requirement for hexobarbital metabolism in the isolated, perfused rat liver and in the intact rat

it was believed that the ADH system was responsible for the oxidation of all primary aliphatic alcohols, including methanol (FIGURE 1 ) . This observation redirected attention to the peroxidative system as a means of oxidizing methanol. As early as 1936, Keilin and Hartree3 showed that catalase catalyzed the oxidation of alcohols to their aldehydes when hydrogen peroxide was supplied in low concentrations, as might be provided in living cells through the action of flavin and other peroxide-generating enzymes. They presented arguments to support their view that catalase is not present in the tissues to protect against peroxide intoxication, as was widely contended, but rather to carry out coupled (peroxidative) oxidations. Employing techniques that permitted very rapid spectral determinations, Chance4 identified the intermediate complexes and analyzed the kinetics of the components of the reaction (FIGURE 1 ) . Ideas were exchanged for several years as to whether or not the peroxidative system participated in the in vivo oxidation of methanol and other alcohols. A direct means of examining the question was provided when Heim and coworkers5 showed that the intraperitoneal injection of 3-amino-1,2,4-triazole (AT) caused a reduction in hepatic and renal catalase activities of 90% or more. Mannering and Parkss found that the AT-induced inhibition of hepatic catalase was accompanied by a 70% reduction of the methanol-oxidizing capacity of rat liver homogenates. The addition of crystalline beef liver catalase to these homogenates restored methanol oxidation to normal. While these in vitro studies pointed to a role of catalase in methanol oxidation, they did little to establish its participation in vivo. Because of the complexity of the system, involving as it does the rate of formation of hydrogen peroxide, which in turn depends upon the concentration of substrates available to the peroxide-generating enzymes as well as the availability of hepatic catalase to the hydrogen peroxide produced by these enzymes, it seemed unlikely that in vitro studies would provide much information as to what was occurring in the intact animal. This presentation is devoted largely to a review of the evidence that shows that the intact rat oxidizes methanol largely through the peroxidative system, but that ethanol oxidation proceeds differently, probably almost entirely via the ADH system. The evidence is based on in vitro studies that employed several approaches: 1 ) 14C-methanol and 1 -Wethano1 oxidation were studied

Induction of xanthine oxidase and depression of cytochrome P-450 by interferon inducers: Genetic difference in the responses of mice

Abstract Rates of hexobarbital metabolism at various oxygen tensions were studied in the isolated, perfused liver of the rat and in the intact animal. Both untreated and phenobarbital-treated rats were employed. The rate of hexobarbital metabolism remained constant in livers from both untreated and phenobarbital-treated animals when oxygen tensions of the perfusate were above 45 mm of Hg. At tensions below 45 mm of Hg the rate declined sharply. A similar decline in the rate of hexobarbital metabolism was observed in intact rats when the oxygen tension of the arterial blood was reduced below 45 mm of Hg. Regardless of oxygen tension, the rates of hexobarbital metabolism in livers from phenobarbital-treated rats were about twice those seen in livers from untreated rats. In the intact animal hexobarbital was metabolized more rapidly at any given arterial oxygen tension in the phenobarbital-treated animals than in untreated animals. The livers from the phenobarbital-treated rats contained 2·9 times more cytochrome P-450 than the livers from untreated animals. The finding that hexobarbital is metabolized more rapidly in phenobarbital-treated rats than in untreated rats when oxygen is rate limiting is interpreted to mean that the higher level of cytochrome P-450 in the liver of the treated rats enables this cytochrome to compete more successfully with other terminal oxidases and thus channel oxygen into the drug metabolizing system.