Regulation of cytochrome P-450-dependent catechol estrogen formation in rat liver microsomes. Evidence for involvement of estrogen receptors

Experiments were conducted to evaluate whether estrogen 2-hydroxylase activity in liver microsomes, the main pathway for oxidative metabolism of estrogens in the rat, is regulated by administration of synthetic estrogens. Ovariectomized rats were treated with ethinylestradiol (EE), 100 micrograms s.c. for 3 days. Liver microsomes from EE-treated animals showed a 2-fold increase over control in estrogen 2-hydroxylase activity measured over a substrate concentration range of 0.5 to 50 microM. Double-reciprocal plots of enzyme activity as a function of substrate concentration were linear; apparent Vmax values were 2-fold greater in microsomes from EE-treated animals while apparent Km values for control and EE preparations were not different. Administration of the triphenylethylene antiestrogen tamoxifen (TAM), 100 micrograms s.c. for 3 days, did not affect microsomal catechol estrogen formation activity, and apparent Km and Vmax values were comparable with controls. When EE and TAM were co-administered, no increase in microsomal estrogen 2-hydroxylase was observed, and apparent Km and Vmax values were not different from either control of TAM-treated preparations. Thus, acute administration of EE was associated with a specific increase in the apparent Vmax of estrogen 2-OHase activity, and this effect was not observed when TAM was co-administered with the estrogen.     

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) effects on hepatic microsomal steroid metabolism and serum estradiol of pregnant rats

Experiments were conducted to evaluate the effects of administration of low, but fetotoxic quantities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during pregnancy on steroid metabolism in liver microsomes. Oral administration of 1 microgram X kg-1 X day-1 of TCDD to pregnant rats on days 7-19 of gestation reduced maternal weight gain during pregnancy. Analysis of litters on day 20 showed that fetuses from TCDD-treated dams had a 66% incidence of visceral lesions characterized by intestinal hemorrhage. Liver microsomes prepared from TCDD-treated dams on day 20 of gestation exhibited a 2- to 3-fold increase in cytochrome P-450 content which was accompanied by a shift in the absorbance optimum of the dithionite reduced-CO spectrum to 448 nm. Catechol estrogen formation activity was decreased by 50-75% in hepatic microsomes from TCDD-treated dams. In contrast 7 alpha-hydroxylation of testosterone increased nearly 4-fold, while 16 alpha- and 6 beta-hydroxylase activities were unchanged in microsomes following exposure to TCDD. Thus, the inhibition of catechol estrogen formation associated with TCDD treatment did not reflect a general decrease in microsomal steroid hydroxylase activities. Insofar as catechol estrogen formation is physiologically a major pathway for estrogen metabolism, serum concentrations of 17 beta-estradiol were measured in a second group of pregnant rats treated with TCDD on days 4-15 of gestation. Serum estradiol levels were not different between control and treated dams at this stage of pregnancy. Thus, the present study does not support a link between TCDD-mediated inhibition of catechol estrogen formation measured in vitro in liver microsomes and altered circulating estradiol levels in vivo during pregnancy.     

Effects of aging on hepatic and pulmonary glutathione S-transferase activities in male and female Fischer 344 rats

The effects of aging on cytosolic glutathione S-transferase activities were evaluated with liver and lung cytosol from male and female Fischer 344 rats 3, 12, and 24 months of age. Age-related changes were tissue-, sex-, and substrate-specific. With liver and lung cytosol from both males and females, rates of metabolism of 1,2-epoxy-3-(p-nitrophenoxy)propane and p-nitrobenzyl chloride were lower in the old group than in the young group; however, patterns of decrease differed with tissue and sex. With 1,2-dichloro-4-nitrobenzene, metabolism was affected by aging only in liver and lung cytosol from males. Finally, with 1-chloro-2,4-dinitrobenzene, metabolic rates were altered during aging only with liver cytosol from females. However, the apparent Km was higher with liver cytosol from old males; those values from lung cytosol of males and liver or lung cytosol from females were unchanged. These data indicate that changes in the cytosolic glutathione S-transferase isozymes occurred during aging.     

Attributions of causality for drinking behavior made by alcoholics and by normal drinkers

Alcoholic individuals often are assumed to deny personal responsibility for their alcoholism and to assign causation to external situational factors. To evaluate this assumption, 20 alcoholics and 14 nonalcoholics made causal attributions for a recent personal drinking episode and for the drinking behavior of three target individuals (an abstinent alcoholic, a nonabstinent alcoholic, and a nonalcoholic). Results showed that both alcoholic and nonalcoholic subjects tended to make external attributions for their own drinking behavior. Subjects' attributions for the target individuals depended on both the targets' and subjects' drinking histories. The results are discussed in terms of their relevance to models of alcoholism and to actor-observer differences in causal attribution processes.     

Neurotensin induces catalepsy in mice

Intracerebroventricular (i.c.v.) injection of neurotensin (NT) induced catalepsy in mice at doses greater than or equal to 0.02 microgram. The cataleptic effect progressively increased, reaching a maximum at approx. 2 hr after injection. In contrast, the hypothermic effect of neurotensin reached a maximum 1 hr after the injection, and was declining at 2 hr. Not all mice that showed hypothermia also showed catalepsy, and some mice showed catalepsy without hypothermia. Catalepsy induced by intracerebroventricular injection of neurotensin was not significantly correlated with the hypothermia. Furthermore, oxotremorine induced hypothermia without catalepsy. Thus, several lines of evidence indicate that the catalepsy induced by neurotensin is not the consequence of the neurotensin induced hypothermia. Thyrotropin releasing hormone (TRH), injected either intracerebroventricularly with neurotensin, or intraperitoneally before neurotensin abolished the hypothermia but only diminished the catalepsy scores. The cataleptic effect of neurotensin is consistent with its other neuroleptic-like activities.     

Purification and properties of cytochrome P-450 and NADPH-cytochrome c (P-450) reductase from human liver microsomes.

Cytochrome P-450 and NADPH-cytochrome c (P-450) reductase were purified to 10.6 nmoles per mg of protein and 19.9 units per mg of protein, respectively, from human liver microsomes. The purified cytochrome was assumed to be in a low spin state as judged by the absolute spectrum. n-Octylamine and aniline produced type II difference spectra and SKF 525-A and benzphetamine type I spectra when bound to the purified cytochrome P-450. The purified human cytochrome P-450 catalyzed laurate oxidation as determined by NADPH oxidation but not aniline hydroxylation, benzphetamine N-demethylation and 7-ethoxycoumarin O-deethylation when reconstituted with the reductases purified from human and rat liver microsomes. The human cytochrome P-450, however, catalyzed drug oxidations when cumene hydroperoxide was used as the oxygen source. The purified human NADPH-cytochrome c (P-450) reductase contained FAD and FMN at a ratio of 1:0.76. The reductase was capable of supporting 7-ethoxycoumarin O-deethylation activity of cytochrome P-448 purified from 3-methylcholanthrene-treated rat liver microsomes.     

Effect of cannabidiol on cytochrome P-450 and hexobarbital sleep time

The influences of acute and subacute cannabidiol (CBD) treatment and of subsequent drug withdrawal were investigated on hexobarbital-induced sleep time, on hepatic cytochrome P-450 concentration, on the in vitro formation of carbon monoxide (CO) associated with CBD metabolism, and on the kinetics of aminopyrine N-demethylase metabolism. In acutely treated mice, CBD prolonged sleep time, decreased cytochrome P-450 concentration, decreased the endogenous formation of CO, and increased an apparent K_m for aminopyrine N-demethylase activity. In subacutely treated animals, tolerance developed to the effect on sleep time but not to that on cytochrome P-450 concentration nor on the endogenous formation of CO in vitro nor on the K_m for the N-demethylase activity. Upon withdrawal from subacute treatment, tolerance to the sleep-time effect was still evident on day 14, but, by day 28, the sensitivity to CBD had returned to normal. In contrast, the cytochrome P-450 concentration returned to normal on day 14 of withdrawal, as did the K_m for the N-demethylase activity and the ability of CBD to induce CO synthesis in vitro. The comparative results lead us to conclude that the CBD effect on sleep time does not correlate with either the total amount of cytochrome P-450 or with the CBD depressant effect on the cytochrome.     

The isolated perfused rat brain preparation in the study of monoamine oxidase and benzylamine oxidase: Lack of selective brain perfusion

Monoamine oxidase (MAO) is present in brain blood vessels, and a different amine oxidase, benzylamine oxidase (BzAO), is claimed to exist in porcine cerebral vessels. The object of the present investigation was to evaluate these deaminating activities in the structurally intact brain, utilizing the isolated perfused rat brain preparation (IPRB). [¹⁴C]Benzylamine (10 μM), a substrate for both BzAO and MAO, was perfused via the internal carotid arteries for 5 min, and deaminated metabolites were measured. BzAO and MAO activities were distinguished by the use of the selective inhibitors semi-carbazide and pargyline. Both BzAO and MAO deaminated benzylamine (10 μm), but over 90 per cent of the total deaminated products resulted from BzAO. This was due to the much lower K_m, value of benzylamine for BzAO (2.8μM) than for MAO (169 μM). In vitro assays, however, revealed that the brain contained no measurable BzAO activity, whereas all other head structures (skull, mandible, skin, skeletal muscle, eyes, periocular tissues and tongue) contained BzAO activity. MAO was present both within and outside the brain. These results suggest that the IPRB preparation is not specific for brain perfusion. Experiments with technetium-labeled microspheres showed that, although the brain is preferentially perfused on a per gram basis, 52 per cent of the total perfusate passed through the skull and 13 per cent through extracranial structures. Only 35 per cent was specific for the brain. Other experiments showed that perfusion of the intact rat head produced greater deamination of benzylamine than when the skin and 70 per cent of the muscle were removed. Additionally, perfusion via the pterygopalatine arteries, to bypass the brain, resulted in increased deamination. It is concluded that the IPRB preparation is not specific for brain perfusion and that BzAO activity is present in all head structures other than the brain. The presence of BzAO in bone, skin, and muscle is consistent with suggestions for a physiological function of the enzyme in connective tissue.     

Differential metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate by rat and chicken hepatic microsomes

The in vitro hepatic metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) was investigated in the hen (a species that is sensitive to EPN delayed neurotoxicity) and the rat (an insensitive species). EPN, which produced a Type I binding spectrum on incubation with cytochrome P-450, was converted by liver microsomes from both species to its oxygen analog, O-ethyl O-4-nitrophenyl phenylphosphonate (EPNO), and to p-nitrophenol (PNP). The formation of EPNO and PNP was dependent on the presence of NADPH in the reaction mixture and could be inhibited by either SKF-525A or by anaerobic conditions. The rates of EPNO and PNP formation by rat liver microsomes were, however, 3- and 20-fold higher, respectively, than the rates of formation by chicken liver microsomes. There was also a 4-fold difference in the cytochrome P-450 contents of the liver microsomes. The EPNO-hydrolyzing activity of rat liver microsomes was much greater than that of chicken liver microsomes. EPNO metabolism, in contrast to EPN metabolism, did not require NAPDH nor was it inhibited by SKF-525A or by anaerobic conditions. Prior exposure of rats to phenobarbital (PB) or Arochlor 1254 resulted in an increase in hepatic microsomal EPN metabolism and cytochrome P-450 content. On the other hand, 3-methylcholanthrene (3-MC) treatment elevated microsomal cytochrome P-450 but did not increase EPNO or PNP formation. Pretreatment with EPN did not alter either microsomal EPN metabolism or cytochrome P-450 levels. In chickens, prior exposure to PB, 3-MC or 100 mg/kg EPN increased EPNO and PNP formation by liver microsomes as well as cytochrome P-450 levels; prior exposure of chickens to 15 mg/kg EPN did not alter these variables. The λ_max Soret bands of the reduced hepatic cytochrome P-450 complexes from these animals differed as follows (rat then chicken): untreated, 450 vs 452 nm; PB-treated, 450 vs 451 nm; and 3-MC-treated, 448 vs 449 nm. None of the above treatments had an effect on EPNO metabolism by liver microsomes.     

The generality of the stimulus-binding hypothesis across drinking behavior and task performance in alcoholics

This experiment investigated the validity of applying the stimulus-binding hypothesis of obesity to conceptualize drinking and task performance behaviors in alcoholics. Twenty alcoholics and 12 nonalcoholics participated in two counterbalanced experimental sessions. One session involved an assessment of subjects' voluntary consumption of preferred and nonpreferred nonalcoholic beverages. The other session involved their performance of four tasks that involved or manipulated the presence of salient external cues. The prediction of heightened externality in alcoholics was supported on the beverage consumption measures and was marginally supported on the task performance measures. The results are discussed in terms of their implications for models and treatments of alcohol problems.     

Induction of hepatic metallothionein in the rabbit fetus following maternal cadmium exposure

Timed-pregnant rabbits were administered a single, sc injection of cadmium chloride (0, 0.125, 0.25, or 0.50 mg cadmium/kg body wt) 48 hr prior to being killed at term. Although the doses chosen were below that shown to produce significant fetal lethality, the 0.50 mg/kg dose did produce a significant reduction in fetal body weight, liver weight, kidney weight, and crown-rump length while having no effect on placental weight. Fetal hepatic metallothionein levels were significantly increased at all three doses; however, a significant increase in metallothionein zinc content was seen only at the two higher doses. Metal distribution studies indicated that the increase in fetal metallothionein and the accompanying increase in cytosolic zinc coincided with an apparent redistribution of the metal from extrahepatic sites to the liver. The whole fetal content of zinc was not significantly altered by the maternal administration of cadmium.     

Stimulation of rat liver 3-hydroxy-3-methylglutaryl-coenzyme a reductase activity by o,p′-DDD

To investigate the mechanism by which o,p′-DDD (2,2-bis[2-chlorophenyl-4-chloro-phenyl]-1,1-dichloroethane; Mitotane) produces hypercholesterolemia in man, we studied the effect of the drug on hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity in reverse light-cycled rats. o,p′-DDD markedly stimulated reductase activity in vivo and in vitro in a dose-dependent manner. This effect was not associated with demonstrable adrenocortical toxicity or changes in plasma corticosterone concentrations. Thus, o,p′-DDD may elevate circulating cholesterol levels in man by increasing endogenous cholesterol synthesis. In addition, the o,p′-DDD-treated rat may serve as a useful model for testing other agents for the ability to suppress endogenous cholesterol synthesis and lower circulating cholesterol levels.     

Metabolism of 2-nitro-1-phenylpropane by rabbit liver microsomes

The in vitro metabolism of 2-nitro-1-phenylpropane by rabbit liver microsomes was examined. The metabolites, identified by gas chromatography-mass spectrometry, were benzyl alcohol, benzoic acid, phenylacetone, 1-phenyl-2-propanon-1-ol, and 1-phenyl-1,2-propanediol. The mass spectra of these compounds are discussed in terms of the major diagnostic peaks. the levels of the metabolites formed were determined by quantitative gas chromatography, and the major metabolites were phenylacetone and 1-phenyl-2-propanon-1-ol. the formation of these two compounds appears to be P-450 dependent since the reactions are sensitive to CO and 2,4-dichloro-6-phenylphenolxethylamine (DPEA) and inducible by phenobarbital pretreatment of the rabbits.     

Induction of rat hepatic microsomal cytochrome P-450 by 2,3',4,4',5,5'-hexachlorobiphenyl

The following evidence suggests that 2,3',4,4',5,5'-hexachlorobiphenyl resembles isosafrole as an inducer of hepatic microsomal cytochrome P-450d in the immature male Wistar rat. First, the major hepatic microsomal polypeptide (Mr = 52,000), intensified after treatment of rats with 2,3',4,4',5,5'-hexachlorobiphenyl, comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with cytochrome P-450d (i.e. the major isosafrole-inducible polypeptide) but had an electrophoretic mobility intermediate between cytochrome P-450b (Mr approximately equal to 51,500) and cytochrome P-450c (Mr = 56,000) (i.e. the major phenobarbital- and 3-methylcholanthrene-inducible polypeptides respectively). Second, when pairs of various xenobiotics were coadministered to rats at doses effecting maximal induction of hepatic microsomal cytochrome P-450, the inductive effects of 2,3',4,4',5,5'-hexachlorobiphenyl were additive with those of phenobarbital, 3-methylcholanthrene and pregnenolone-16 alpha-carbonitrile but not with those of isosafrole. The inductive effects of phenobarbital, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile and isosafrole were all expressed additively with each other. Third, in contrast to phenobarbital and pregnenolone-16 alpha-carbonitrile treatment, treatment of rats with 2,3',4,4',5,5'-hexachlorobiphenyl, isosafrole or 3-methylcholanthrene failed to increase markedly the proportion of total cytochrome P-450 capable of forming a 446 nm-absorbing complex with metyrapone. Fourth, the in vitro metabolism of isosafrole, catalyzed by hepatic microsomes from rats treated with 2,3',4,4',5,5'-hexachlorobiphenyl, isosafrole or 3-methylcholanthrene, produced complexes between ferrous cytochrome P-450 and a methylenedioxyphenyl metabolite, the spectra of which were between 400 and 500 nm and were similar to each other but which were readily distinguishable from the spectra of the product adducts formed during the metabolism of isosafrole by hepatic microsomes from rats treated with corn oil (control), phenobarbital, or pregnenolone-16 alpha-carbonitrile.     

Morphine-mediated effects on rat hepatic heme and cytochrome P-450 in vivo: Antagonism by naloxone in the liver

Chronic morphine administration to adult male rats has long been known to lower hepatic cytochrome P-450 content and its dependent mixed function oxidase activity. More recently, we found that acute treatment of mature male rats with a dose of morphine higher than that used chronically also reduces their hepatic cytochrome P-450. In the present study, we demonstrate that this acute reduction of cytochrome P-450 in the rat liver is a result of morphine-mediated accelerated turnover (degradation) of its heme moiety and apparently is associated with hepatotoxicity of the drug. These morphine-mediated effects are largely prevented by concomitant administration of naloxone, a morphine antagonist.     

Partial purification and properties of cytochrome P450 from homogenates of human fetal livers.

Using ω-amino-n-octyl Sepharose 4B and hydroxylapatite columns, cytochrome P450 was purified to approx. 6.7 nmoles per mg of protein from the 105,000 g precipitate of homogenates of human fetal livers. The partially purified preparation of cytochrome P450 was free of detectable amounts of cytochrome b₅, NADPH-cytochrome P450 reductase and NADH-cytochrome b₅ reductase. The absolute spectrum of the preparation exhibited a peak at 417 nm in the Soret region, indicating that this cytochrome P450 is a low spin species. Binding of aniline and SKF 525-A to this partially purified preparation of cytochrome P450 produced a modified type II and a type I difference spectra, respectively. As judged by Ouchterlony double diffusion analysis, the cytochrome P450 preparation did not cross react with antibody against cytochrome P450 isolated from the livers of phenobarbital-pretreated rats. In reconstituted systems, the cytochrome exhibited considerable activity for aniline hydroxylation but only a low ethlymorphine N-demethylation activity compared to cytochrome P450 isolated from the livers of phenobarbital-pretreated rats.     

Foetal and neonatal development of cytochrome P-450 and cytochrome P-448 catalysed mixed function oxidases in the rat: induction by 3-methylcholanthrene

Benzphetamine N-demethylase (cytochrome P-450) and ethoxyresorufin O-deethylase activities (cytochrome P-448) were determined in the growing neonate and foetus of control and 3-methylcholanthrene-pretreated rats. Ethoxyresorufin O-deethylase activity was highest in the 1-2-week-old animals and then decreased with age. The inducibility of this activity by 3-methylcholanthrene was low in the young animals, but increased with age. In contrast, benzphetamine N-demethylase activity in the control animals was low at birth and increased with age, and was not induced by 3-methylcholanthrene. In the foetal liver, ethoxyresorufin O-deethylase was the only activity present at higher levels than in the maternal liver. Transplacental administration of 3-methylcholanthrene failed to induce the foetal activities while the maternal liver showed the expected response. These observations demonstrate that cytochrome P-448 may be a predominant hepatic form in the foetus and neonate but cytochrome P-450 becomes a major form as the animal grows. The implications of these findings in chemical toxicity are discussed.     

Role of hepatic microsomal cytochrome P-450 in the toxicity of fluorinated ether anesthetics

To evaluate the role of cytochrome P-450 in anesthetic toxicity, we investigated the effects of hepatic microsomal cytochrome P-450 inducers [phenobarbital (PB), 3-methylcholanthrene (3-MC) and pregnenolone-16 α-carbonitrile (PCN)] and inhibitors [SKF 525-A, metyrapone, and 2allyl2isopropylacetamide (ALA)] on the potentiation of lethal effects to rats of i.p. administered 2,2,2-trifluoroethyl vinyl ether (TFVE), ethyl 2,2,2-trifluoroethyl ether (TFEE), allyl 2,2,2-trifluoroethyl ether (TFAE) and 2,3-epoxypropyl 2,2,2-trifluoroethyl ether (EPTFE). The time courses of tail-vein blood anesthetic concentrations and quantities of exhaled anesthetics together with the in vitro metabolism of the anesthetics and their binding to microsomal cytochromes P-450 were also determined. The results indicate that (1) the majority of the administered anesthetics make a single pass through the liver prior to exhalation and apparently are metabolized to toxic products, (2) the epoxide (EPTFE) exerts its lethal effects independently of cytochrome P-450 catalyzed metabolism and does not lie on the major path of TFAE metabolism, (3) all the anesthetics yield 2,2,2-trinuoroethanol (TFE) on metabolism in vitro but lethality does not always correlate with the rates of TFE formation, (4) PB induced cytochromes P-450 potentiate lethal effects of TFVE and TFEE but not of TFAE, and inhibitors differentiate mechanisms of TFVE and TFEE lethality, (5) PCN induced cytochromes P-450 potentiate the toxicity of TFVE, TFAE, and TFEE in a similar manner, and (6) 3-MC induction potentiates TFEE and TFAE lethality apparently independently of cytochrome P-450 catalyzed metabolism.     

Mannich base derivatives of theophylline and 5-fluorouracil: syntheses,properties and topical delivery characteristics

Mannich base prodrugs of theophylline and 5-fluorouracil have been prepared and tested for their ability to deliver their parent drugs through hairless mouse skin. The Mannich base derivatives were more effective than the previously described N-acyloxyalkyl derivatives. In the case of theophylline the Mannich base derivative was also found to be as effective as the previously described N-hydroxymethyl derivative. All of the Mannich bases reverted to their parent compounds in water, but some were relatively stable in aprotic solvents such as isopropyl myristate which was therefore used as a vehicle for the diffusion experiments with the prodrugs.