Frank E. Greene

Impairment of Drug Metabolism in Man by Allopurinol and Nortriptyline

Abstract Normal medical students received either allopurinol or nortriptyline. To quantitate changes in rates of drug metabolism produced by allopurinol or nortriptyline administration, the plasma half-lives of antipyrine or bishydroxycoumarin were determined both before and 24 hours after the last dose of allopurinol or nortriptyline. Allopurinol and nortriptyline each markedly prolonged the plasma half-lives of antipyrine and bishydroxycoumarin; large individual variations in prolongation times were observed. In rats, allopurinol and nortriptyline each reduced the activity of hepatic microsomal drug-metabolizing enzymes and slightly lowered the level of cytochrome P-450. These results in rats support the interpretation that in man prolongation of antipyrine and bishydroxycoumarin half-lives after allopurinol or nortriptyline pretreatment is produced by reduced rates of drug biotransformation in liver microsomes. Lowering of the usual dose and close monitoring of blood levels of drugs given simultaneousl...

Effect of partial hepatectomy on the responsiveness of microsomal enzymes and cytochrome P-450 to phenobarbital or 3-methylcholanthrene

Abstract The effects of phenobarbital and 3-methylcholanthrene (3-MC) pretreatment on liver weight, microsomal protein, cytochrome P-450 content, microsomal aniline hydroxylase, hexobarbital hydroxylase and P -nitroanisole O -demethylase activities have been studied in control, sham-operated and partially hepatectomized rats. In unoperated rats, phenobarbital pretreatment significantly increased liver weight, microsomal protein, P-450 content and enzyme activities toward all three substrates. 3-MC pretreatment significantly increased liver weight, P-450 content and p -nitroanisole O -demethylation without influencing the other parameters. When administered to shamoperated or partially hepatectomized rats, phenobarbital and 3-MC caused substantially the same effects, though the magnitude of the effects was less than in unoperated animals. Thus, significant increases were seen in the various parameters after injection of phenobarbital or 3-MC into hepatectomized rats. It is concluded that the regenerating liver, like the fetal or newborn liver and certain rodent hepatomas, although exhibiting low levels of microsomal enzymes, has the capacity to respond to the enzyme inducers, phenobarbital and 3-MC. Differential comparison of the enzyme changes expressed in terms of microsomal protein and also in terms of microsomal P-450 content revealed that the activity of the enzymes catalyzing p -nitroanisole and aniline oxidations parallel the microsomal P-450 content more closely than does the enzyme catalyzing hexobarbital hydroxylation.

Impairment of hepatic microsomal drug metabolism in the rat during daily disulfiram administration.

Abstract Male rats were given 0.1 or 0.4 g/kg of disulfiram (DS) daily by gavage for up to 12 days, in order to study the effects of chronic DS administration on hepatic microsoma] drug metabolism. Administration of 0.4 g/kg of DS resulted in significant impairment of aniline (ANL) hydroxylase after 1 day. but 2 days of DS treatment were required for significant inhibition of ethylmorphine (EtM) metabolism and depression of cytochrome P-450 levels. At this time, maximum impairment of ANL and EtM metabolism and maximum reduction of cytochrome P-450 levels were seen. Continued administration of DS for 10 additional days produced no further change in these parameters. ANL hydroxylase was also significantly reduced in treated animals throughout a 12-day period during which 0.1 g/kg of DS was given. EtM N -demethylase activity and cytochrome P-450 levels were also reduced in animals so treated, but not until DS had been given for at least 5 days. However, by the end of the 12-day experimental period. EtM metabolism and cytochrome P-450 levels had returned almost to control levels and only ANL hydroxylase was significantly different from control activity. Daily DS administration (0.4 g/kg produced small but significant increases in microsomal cytochrome b 5 levels and in NADPH-cytochrome c reductase activity, whereas NADPH oxidase and NADPH-cytochrome P-450 reductase activities were significantly lower in treated rats. In addition to these effects in vivo , DS competitively inhibited EtM N -demethylase in vitro and bound to cytochrome P-450, producing a type I difference spectrum, thus providing additional mechanisms to account for impairment in vivo of drug metabolism by DS.

Impairment of hepatic microsomal and plasma esterases of the rat by disulfiram and diethyldithiocarbamate

Abstract Twenty-four hr after oral administration (0.2 to 2.0 g/kg) of disulfiram (DS) to male rats. significant impairment of hepatic microsomal carboxylesterase and plasma carboxyl- and cholinesterase was observed. Plasma esterase activities returned to control values between 48 and 72 hr after a single oral dose of DS (2.0 g/kg), but microsomal carboxylesterase activity was still significantly lower in treated animals at both times. Daily administration of DS (0.1 or 0.4 g/kg) resulted in decreased microsomal carboxylesterase activity after 2 days. However, continued administration of DS for a total of 12 days did not produce further depression of microsomal esterase activity. Microsomal and plasma carboxylesterase activities were also decreased 24 hr after oral administration (1.0 to 2.0 g/kg) of sodium diethyldithiocarbamate (DDTC), the reduced metabolite of DS. Hepatic microsomal esterases that migrated rapidly toward the anode during polyacrylamide disc gel elcctrophoresis were the most sensitive to impairment by DS or DDTC. Esterase activity in the lung was also impaired after DS or DDTC administration, whereas esterases of the heart, kidney and testis were essentially unaffected. Incubation in vitro of liver microsomes with DS decreased microsomal carboxylesterase activity, while incubation with DDTC had little effect. Plasma carboxylesterase was inhibited in vitro to a greater extent than microsomal esterases by both DS and DDTC. Diethylamine and CS2, the decomposition products of DDTC, were essentiallv inactive as esterase inhibitors in vitro.

Effect of adrenalectomy and cortisone administration on components of the liver microsomal mixed function oxygenase system of male rats which catalyzes ethylmorphine metabolism

Abstract The effects of adrenalectomy and cortisone administration on components of the mixed function oxygenase system of liver microsomes from adult male rats were studied. The N -demethylation of ethylmorphine (EM) as measured by formaldehyde production was used as an index of the overall activity of this system. EM demethylase activity in liver microsomes from adrenalectomized rats was 56 per cent of the value obtained with sham-operated controls, while cytochrome P-450 content fell by only 19 per cent. NADPH cytochrome c reductase and cytochrome P-450 reductase activities were decreased 68 and 69 per cent, respectively, in liver microsomes from adrenalectomized animals, while the apparent affinity constant ( K sp ) of EM for cytochrome P-450 spectral change was not altered. The decrease in maximal spectral change caused by adrenalectomy corresponded to the decrease in cytochrome P-450 content. Cortisone acetate administration (5 mg/kg/day for 8 days) to adrenalectomized rats prevented the decrease in EM demethylase activity. Accordingly, the steroid treatment increased the NADPH cytochrome c reductase and cytochrome P-450 reductase activities to 113 and 140 per cent, respectively, of the values obtained with the sham-operated controls, without significantly altering the cytochrome P-450 content or the kinetic constants for the spectral changes. These data suggest that the well known decrease in drug-metabolizing activity seen in liver microsomes of adrenalectomized animals is not related to changes in cytochrome P-450 content or to a decrease in the ability of cytochrome P-450 to bind drug substrates. However, there appears to be a relationship between the reductase activities and the adrenal function, because the changes in activities in NADPH cytochrome c reductase and cytochrome P-450 reductase in liver microsomes from adrenalectomized and cortisone-treated adrenalectomized rats paralleled the changes in EM demethylase activity.

Dieldrin-induced alterations in biogenic amine content of rat brain.

Dieldrin-Induced Alterations in Biogenic Amine Content of Rat Brain. Wagner, S. R. and Greene, F. E. (1978). Toxicol. Appl. Pharmacol. 43 , 45–55. Male and female Sprague-Dawley rats were exposed acutely and chronically to dieldrin. The effects of the cyclodiene insecticide on central biogenic amines were determined. Severe neurotoxic signs occurred in males after a single oral dose of dieldrin (50 mg/kg); females were less affected. Overt neurotoxicity was accompanied by decreased concentrations of norepinephrine (NE) in whole brains at 2 and 5 hr after dosing, but the decrease was statistically significant only in males. Reductions in whole brain NE were of limited duration; no decrease occurred after 48 hr, although neurotoxic signs were evident throughout this period. The serotonin concentrations decreased in both sexes at 2 hr, at which time hypothermia was greatest. Dopamine concentrations were unchanged by acute dieldrin exposure. Rats maintained on a diet containing 50 ppm of dieldrin for up to 8 weeks were also examined for effects on central biogenic amine content. In whole brain, this level of dietary exposure caused small, inconsistent decreases in biogenic amine concentrations. However, when brain regions were analyzed, NE concentrations in the medulla oblongata+pons, striatum, and hippocampus decreased during the initial weeks of the study. With continued dieldrin feeding, NE values returned to or exceeded control values, suggesting adaptations to the depleting effects of dieldrin. No consistent differences between sexes occurred during chronic exposure to dieldrin with regard to whole brain or regional amine concentrations, or to overt neurotoxicity.

Effect of different levels of corn oil in the diet upon the rate of hexobarbital, heptachlor and aniline metabolism in the liver of the male white rat

Abstract At low dietary intakes of corn oil there is a decreased ability of the liver, in an in vitro system, to metabolize drugs. Half of this decrease in rate of aniline, hexobarbital and heptachlor occurs at corn oil intakes equivalent to linoleate intakes of 0.1, 1 and 1% of calories, respectively. High corn oil intakes are equally deleterious (half decrease at intakes equivalent to 9% linoleate) in the case of hexobarbital. Biochemical results were confirmed by sleeping time measurements. Results are interpreted as confirming the linoleate requirement of the male rat as being about 2% of calories, and it is suggested that the optimal linoleate intake is about 3% of calories.

Mechanisms of diethyldithiocarbamate-induced loss of cytochrome P-450 from rat liver.

A single, intraperitoneal injection of diethyldithiocarbamate (DDTC) to adult, male Sprague-Dawley rats decreased hepatic cytochrome P-450 (P-450) concentrations. This effect was dose-dependent over a range of 250 to 750 mg/kg and most prominent 24-36 hr after dosing. Depletion of hepatic glutathione (GSH) by diethylmaleate (DEM) administration significantly decreased P-450 8 hr after concurrent treatment with DDTC at a dose which given alone had little effect on P-450 concentrations. When hepatic microsomes were incubated with DDTC in the presence of NADPH, P-450 was converted to cytochrome P-420 (P-420). Similar incubations employing [35S]DDTC demonstrated strict NADPH-dependent binding of labeled sulfur to microsomal membranes, suggesting that diminished P-450 concentrations are related to the metabolic activation of DDTC. Addition of reduced GSH to incubation mixtures blocked the binding of 35S to microsomal membranes, as well as conversion of P-450 to P-420. DDTC inhibited NADPH-ADP3+ mediated peroxidation of microsomal lipids in vitro, suggesting that the effect of DDTC on P-450 does not result from stimulation of lipid peroxidation, but may be influenced by the levels of hepatic GSH. DDTC treatment 1 hr after P-450 was pulse labeled by an intravenous injection of [3H]delta-aminolevulinic acid resulted in a 2-fold increase in the rate of loss of radioactivity associated with membrane-bound P-450 heme during the next 20 hr. Within this time interval, hepatic heme oxygenase (HO) activity increased and at 8 hr after dosing was 7-fold greater than control values in the livers, but was unchanged in the kidneys and spleens of DDTC-treated animals. An elevation of hepatic delta-aminolevulinic acid synthetase (delta-ALAS) activity occurred at 8 and 24 hr after DDTC treatment. Since this enzyme is rate limiting in the biosynthesis of heme, its increased activity may represent a compensatory response to offset the DDTC-mediated loss of P-450 heme.

Responsiveness of an SV40-immortalized hepatocyte cell line to growth hormone.

The response of an SV40-immortalized hepatocyte cell line (CWSV-1) derived from adult male rat hepatocytes to human growth hormone (hGH) was investigated. CWSV-1 cells, which have been characterized extensively, retain certain differentiated functions of normal liver (Woodworth and Isom, Mol Cell Biol 7: 3740-3748, 1987). This cell line consists of tightly associated polygonal, mononucleated cells that grow as monolayers. These cells showed no significant morphological changes with the addition of hGH. Northern blot analysis showed that continuous treatment of the CWSV-1 cells with hGH induced the expression of insulin-like growth factor I (IGF-I) and 5 alpha-reductase RNAs. In addition, continuous exposure to hGH resulted in the induction of expression of the growth hormone receptor/growth hormone binding protein (GHR/GHBP) genes. This study indicates that the CWSV-1 cells may serve as a valuable in vitro model system for studying the signaling pathway of GH.

Mouse Liver N-Demethylase Activity

Male-female differences in ethylmorphine N-demethylase activity and cytochrome P-450 content were studied in BALB/cJ, DBA/2J, C3H/HeJ, C57BL/10J and CRL:CD-1 mouse strains. Demethylase activity was gr