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.     

Metabolic activation of Trp-P-2, a tryptophan-pyrolysis mutagen, by isolated rat hepatocytes

Metabolic activation of a tryptophan-pyrolysis product, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), by isolated rat hepatocytes was studied. The substrate (Trp-P-2) disappearance by hepatocytes from untreated rats was slow, but enhanced by 3-methylcholanthrene (MC) pretreatment of rats. The covalent binding of Trp-P-2 to cellular macromolecules was detected in hepatocytes from untreated rats. The amount of covalent binding of Trp-P-2 to protein and RNA was greater than that to DNA. The covalent binding to Trp-P-2 to DNA, RNA and protein in hepatocytes from untreated rats was about 5-10 times less than that in hepatocytes from MC-pretreated rats. 7,8-Benzoflavone strongly inhibited the substrate disappearance and the binding of Trp-P-2 to DNA in hepatocytes from MC-pretreated rats. These results indicate that Trp-P-2 is metabolically activated by the P-448 type of cytochrome P-450 which is induced by MC. Diethylmaleate enhanced by about 50% the binding of Trp-P-2 to DNA in hepatocytes from MC-pretreated rats. On the other hand, cysteine inhibited the binding of Trp-P-2 to DNA with a concomitant reduction in the accumulation of the active metabolite, N-hydroxy-Trp-P-2 (N-OH-Trp-P-2). Sulfhydryl compounds seemed to play important roles in the detoxification of Trp-P-2.     

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.     

Mestranol-induced hypertension: characterization of cytochrome P-450 dependent catechol estrogen formation in brain microsomes

An animal model of estrogen-induced hypertension was used to study the effects of chronic administration of the synthetic estrogen mestranol on cytochrome P-450 content and catechol estrogen formation in brain microsomes. Cytochrome P-450 content of brain microsomes from untreated female rats in estrus was 0.034 nmole/mg protein and the dithionite-reduced carbon monoxide absorbance peak (γ_max) was 452 nm. Catechol estrogen formation in brain microsomes was optimal in the presence of both NADPH and NADH cofactors with an apparent K_m value of 71 μM for 17β-estradiol substrate. Brain microsomes from animals in estrus and diestrus were compared, and no significant differences were observed in cytochrome P-450 content, or in the apparent K_m and V_max values of catechol estrogen formation. Administration of mestranol, 15 μg biweekly, for 3–4 weeks resulted in a significant increase in systolic blood pressure in unanesthetized female rats. Mestranol treatment was not associated with a change in microsomal cytochrome P-450 content or the spectral γ_max. At 10 μM substrate concentration, catechol estrogen formation in microsomes from mestranol-treated animals was increased 2- to 3-fold, with enzyme activity being expressed either per mg protein or per nmole cytochrome P-450. In contrast, no difference was observed between groups when enzyme activity was measured at 100 μM substrate concentration. These data suggest that chronic administration of a synthetic estrogen can regulate the enzyme system involved in formation of brain catechol estrogen metabolites, a mechanism which may alter the biological impact of the parent steroid.     

Activation of misonidazole by rat liver microsomes and purified NADPH-cytochrome c reductase

Rat liver microsomes and purified NADPH-cytochrome c reductase metabolized [14C]misonidazole anaerobically to a reactive intermediate that covalently binds to tissue macromolecules. Air strongly inhibited the binding whereas carbon monoxide had no effect, indicating that misonidazole is activated via reduction and not by cytochrome P-450-dependent oxidation. Both systems showed an absolute requirement for NADPH and were stimulated by flavine (FAD) and paraquat. The apparent Km for misonidazole binding to microsomal protein was 0.74 mM the apparent Vmax was 0.64 nmole 14C bound . mg-1 . min-1. At a single substrate concentration, nitrofurantoin, nitrofurazone and desmethylmisonidazole inhibited the covalent binding of misonidazole to microsomal protein by 47, 26, and 38% respectively. The effect of nitrofurantoin on the kinetics of misonidazole binding gave a complex interaction indicative of uncompetitive inhibition. Glutathione reduced the binding of misonidazole to microsomal protein below the level observed for boiled microsomes while ascorbic acid had no effect. Compared to nitrofurantoin and paraquat, misonidazole was a poor stimulator of superoxide production as measured by adrenochrome formation.     

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.     

Cardiovascular and metabolic responses to thyroid hormones in animals after sympathectomy or treatment with nerve growth factor

The cardiovascular and metabolic effects of thyroxine (T₄) and triiodothyronine (T₃) were examined in rats and mice with decreased or permanently enhanced adrenergic innervations to the heart. 6-Hydroxydopamine (6-OHDA) and the antiserum to the nerve growth factor (AS) were administered to damage sympathetic fibers; the density of the cardiac adrenergic plexus was increased through the use of the nerve growth factor (NGF). Some animals were additionally adrenal demedullated or treated with N,N-diisopropyl- N′-isoamyl-N′-diethylaminoethylurea (P-286) to reduce circulating catecholamine levels. The results of these experiments showed that the rise in heart phosphorylase a activity produced by thyroid hormones was absent after adrenal demedullation and significantly inhibited after chemical sympathectomy with 6-OHDA. Interference with adrenal medullary function by P-286 in combination with chemical or immunological sympathectomy similarly prevented the hormone-induced activation of myocardial phosphorylase a. While an increase in the density of the peripheral sympathetic fibres to the heart accentuated the tachycardia characteristic of hyperthyroidism, the high metabolic rate which accompanies thyrotoxicosis was unaltered either by proliferation of adrenergic fibres or sympathectomy.     

Purification and properties of cytochrome P-450 from liver microsomes of phenobarbital-treated marmoset monkeys (Callithrix jacchus)

One form of cytochrome P-450 from phenobarbital-induced marmoset liver was purified to apparent electrophoretic homogeneity and compared with the major inducible form isolated from rat liver. Whereas spectral properties and molecular weights, as well as catalytic activities towards aminopyrine and ethylmorphine N-demethylation are quite similar, rates of O-dealkylation with enzymes from the two species are considerably different. While ethoxycoumarin deethylation for the marmoset cytochrome is about one-fortieth of that for the rat, ethoxyresorufin and even pentoxyresorufin dealkylations for the marmoset form are not detectable. By contrast, aldrin epoxidation as catalyzed by this cytochrome is about three times as high as that obtained from the rat.     

The relation between the sex-dependency of type I binding of ethylmorphine and the 1-butanol-induced spectral change in mouse liver microsomes

1. A sex difference in the spectral interaction of 1-butanol with liver microsomes from adult mice was observed. In males a profound reverse type I spectrum was elicited, whereas only a small spectral change of irregular shape was apparent in females. This sex difference is the opposite of that observed in the type I binding of ethylmorphine. In immature animals no sex difference was found. Testosterone pretreatment of female mice increased the size of the 1-butanol spectrum concomitantly with a decrease in ethylmorphine binding. 2. Microsomes from males and females did not contain different levels of endogenous substrates. Thus, the presence or displacement of such substrates does not explain the sex differences in type I and reverse type I binding respectively. 3. 1-Butanol was found to interfere with both type II and type I binding. It is concluded that the 1-butanol-induced spectral change consists of at least two components and that the sex difference is due to a sex-dependent type I component.     

Comparative studies on the oxidation and reduction of drug substrates in human placental versus rat hepatic microsomes.

Human placental microsomes prepared by conventional methods were compared with analogous preparations from adult, male rat livers with respect to biochemical components and systems which could affect rates of mixed-function oxidation and reduction of drugs and steroids in vitro. Each of the electron transport components required for the mixed-function oxidation of drugs in hepatic systems was present in lower concentrations in placental than hepatic microsomes. In contrast to hepatic microsomes, placental microsomes which exhibited unusually high aryl hydrocarbon hydroxylase activities did not contain increased concentrations of the electron transport components. Evidence was provided to indicate that rapid degradation of initial electron donors (reduced pyridine nucleotides) was not responsible for the observance of low or negligible drug metabolic activities observed in incubations with placental micro-somes. Cytochromes P-450 and b₅ and their corresponding reductases were shown to be present in human placental preparations. NADPH and NADH-dependent cytochrome c reductase activities in placental microsomes were somewhat lower but comparable to those determined in hepatic preparations. However, cytochromes b₅ and P-450 and contaminating hemoglobin and methemoglobin accounted for less than 56 per cent of the total heme present in placental microsomes. Rapid degradation of placental cytochrome P-450 was observed at 37° in the presence of sodium hydrosulfite, but conversion to cytochrome P-420 was minimal after incubation for 1 hr in the presence of NADPH at the same temperature. It was con- sidered probable that the low rates of drug biotransformation observed would be explicable in terms of high substrate specificities of the placental enzymic components.     

General pharmacological studies of fusarenon-X, a trichothecene mycotoxin from Fusarium species.

Fusarenon-X (F-X) is one of the trichothecene mycotoxins. In the present work, pharmacological properties of F-X were examined. (1) F-X induced hypothermia but did not produce appreciable behavioral changes of mice. (2) In anesthetized rats, F-X caused a rise in the blood pressure and a decrease in the respiratory rate but induced no significant change in the heart rate. (3) In isolated tissues such as fundus muscle, vas deferens, tracheal muscle, ileum, and atrium, F-X had no detectable effects on spontaneous activity and the responsiveness to agonists. (4) Subplantar administration of F-X into rat hindpaw induced edema. F-X had little influence on the histamine release from mast cells and the membrane stability of erythrocytes. (5) F-X decreased the spontaneous peristalsis of intestine but increased the intestinal propulsion of charcoal meal. (6) F-X induced vomiting in dogs which was suppressed by preliminary administration of chlorpromazine and metoclopramide. F-X may induce vomiting by stimulating the chemoreceptor trigger zone in dogs.     

The use of competitive inhibition of substrate-binding to cytochrome P450 in the determination of spectral dissociation constants for substrates with multiple types of binding,as illustrated with 1-butanol

A method is described, which allows (a) the detection of competitive inhibition of binding to cytochrome P450 between two substrates which elicit the same type of spectral change, and (b) the estimation of dissociation constants for one type of spectral binding of a substrate which exhibits multiple interaction with cytochrome P450. This method was used to investigate the interaction of 1-butanol with the type I binding site of cytochrome P450 in liver microsomes from female mice. 1-Butanol was found to competitively inhibit the binding of ethylmorphine, and has an apparent dissociation constant for type I binding of 30 mM.     

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.     

Direct correlation between level of morphine and its biochemical effect on monoamine systems in mouse brain. Evidence for involvement of dopaminergic neurons in the pharmacological action of acute morphine

The pharmacokinetics (uptake and elimination) and pharmacodynamics (biochemical effects on monoamine systems) of morphine in the CNS were investigated concurrently. ICR mice, weighing about 25 g, were injected intravenously with several doses (2.5-80 mg/kg) of morphine. The animals were killed by microwave irradiation (5 kW, 0.6 sec) at 10 and 30 min, and 1, 2, 4, 8 and 24 hr after the injection. The intracerebral levels of morphine and metabolically related substances consisting of monoamines [noradrenaline, dopamine (DA), 5-hydroxytryptamine (5-HT), 3, 4-dihydroxyphenylacetic acid (DOPAC), 3-methoxy-4-hydroxyphenylacetic acid [homovanillic acid (HVA)], 5-hydroxyindoleacetic acid (5-HIAA), tyrosine and tryptophan] were determined in identical samples by a combination of organic extraction and high-performance liquid chromatography with electrochemical detection. The intracerebral level of morphine was found to depend on the dose injected, and the biological half-life of the drug was estimated to be about 1 hr. The morphine injection (2.5-80 mg/kg) caused significant increases in monoamine metabolites although only slight changes occurred in the concns of parent transmitters. The intracerebral level of morphine was significantly correlated with the ratios DOPAC/DA and HVA/DA (r = 0.7033, P less than 0.0001; and r = 0.6455, P less than 0.0001, respectively). On the other hand, the correlation between the morphine level and 5-HIAA/5-HT was lower than those for DOPAC/DA and HVA/DA. These results suggest that monoamine systems, especially DA, are closely involved in the biochemical effects of morphine. Furthermore, the proposed procedure is demonstrated to be useful as a new approach in biochemical pharmacology, where the direct correlation between the distribution of a drug (pharmacokinetics) and the biochemical effects of the drug (pharmacodynamics) can be measured.     

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.     

Induction of suppressor cells by intravenous administration of Bacillus Calmette-Guérin and its modulation by cyclophosphamide.

Intravenous administration of Bacillus Calmette-Guérin (BCG) activated cells in the bone marrow and induced cells in the spleen, which suppressed the development of cell-mediated immunity by T lymphocytes in vitro. The activity of these suppressor cells was dependent on both the dose and the schedule of BCG. A course of cyclophosphamide (60 mg/kg/day) given during the 4 days preceding or following the administration of BCG potentiated the induction of suppressor cells by BCG. If the 4-day course of cyclophosphamide was not initiated until 7 days after the administration of BCG, then the induction of suppressor cells by BCG was partially reversed.     

The effects of alcohol withdrawal and acute doses of alcohol on the acid-base balance in mice and rats

While respiratory alkalosis has been reported to occur in human alcoholics during the withdrawal period, and may causally contribute to the expression of the withdrawal syndrome, we found no evidence of this in either alcohol physically-dependent mice (DBA/2J) or rats (Sprague-Dawley) as reflected in blood pH, plasma pCO₂, or intracellular brain pH relative to pair-fed control (no alcohol) animals. The inhalation of atmospheres high in CO₂ (5% and 15%), which would be expected to reverse a respiratory alkalotic state, had no effect on the expression of the withdrawal syndrome in alcohol-dependent mice. These results indicate that a withdrawal syndrome of considerable intensity can develop in mice and rats in the absence of an alkalotic state.