Hepatic Microsomal Cytochrome P-450 Content Research Articles

Polychlorinated biphenyls as inducers of hepatic microsomal enzymes: Effects of di- ortho substitution

All of the 13 possible polychlorinated biphenyl (PCB) isomers and congeners substituted at both para positions, at least two meta positions (but not necessarily on the same ring) and at two ortho positions have been synthesized and tested as rat hepatic microsomal enzyme inducers. The effects of these compounds were evaluated by measuring microsomal benzo[ a]pyrene (B[ a]P) hydroxylase, 4-chlorobiphenyl (4-CBP) hydroxylase, 4-dimethylaminoantipyrine (DMAP) N-demethylase and NADPH-cytochrome c reductase activities, the cytochrome b 5 content and the relative peak intensities and spectral shifts of the carbon monoxide(CO)- and ethylisocyanide(EIC)-difference spectra of ferrocytochrome P-450. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC) and PB plus MC (coadministered). At dose levels of 150 μmol · kg −1, all of the PCB congeners, except 2,3′,4,4′,5′,6-hexachlorobiphenyl, significantly enhanced hepatic microsomal cytochrome P-450 content, B[ a]P hydroxylase and/or DMAP N-demethylase activities compared to the control (corn oil-treated) animals. Only 5 of these compounds, namely 2,3,4,4′,5,6-hexa-, 2,2′,3,3′,4,4′-hexa-, 2,2′,3′,4,4′,5-hexa-, 2,3,3′,4,4′,6-hexa- and 2,2′,3,3′,4,4′,5-heptachlorobiphenyl, enhanced microsomal B[ a]P hydroxylase, 4-CBP hydroxylase, NADPH-cytochrome c reductase and DMAP N-demethylase activities in a manner consistent with a mixed pattern of induction. The results suggest that PCB isomers and congeners substituted at both para positions, at least two meta positions, at two ortho positions and containing a 2,3,4-trichloro substitution pattern on one ring are mixed-type inducers; in addition the effects of 2,3,4,4′,5,6-hexachlorobiphenyl were also consistent with a mixed pattern of induction.

Polychlorinated naphthalenes as hepatic microsomal enzyme inducers in the immature male rat.

1. The activities of several commercial polychlorinated naphthalenes including Halowaxes 100, 1001, 1099, 1013, 1014 and 1051 as hepatic microsomal enzyme inducers in the immature male rat were determined by measuring their effects on microsomal dimethylaminoantipyrine N-demethylase and benzo[a]pyrene (B[a]P) hydroxylase (or aryl hydrocarbon hydroxylase, AHH) activities, the cytochrome b5 content and the relative peak intensities and spectral shifts of the reduced microsomal cytochrome P-450:CO and ethylisocyanide binding difference spectra. 2. The results obtained for the Halowaxes were compared with the effects of animal pretreatment with phenobarbitone, 3-methylcholanthrene, phenobarbitone plus 3-methylcholanthrene (coadministered) and Aroclor 1254, a commercial polychlorinated biphenyl. 3. At the higher dose level (600 μmol.kg−1) all the Halowaxes enhanced microsomal benzo[a]pyrene hydroxylase activity (2- to 15-fold) and the hepatic microsomal cytochrome P-450 content (1.2- to 2.5-fold) whereas the dimethylaminoantipyrine N-demethylase activities were increased <40% compared to noninduced animals. 4. Based on the enzymic and spectral properties of the microsomal enzymes from the Halowax pretreated animals, Halowaxes 1000, 1001 and 1099 were phenobarbitone-type inducers and Halowaxes 1013, 1014 and 1051 were mixed-type inducers similar to Aroclor 1254 or phenobarbitone plus 3-methylcholanthrene (coadministered).

Effect of aflatoxin B1 on hepatic drug-metabolizing enzymes in female rats. Interaction with a contraceptive agent.

1. The effect of a contraceptive on aflatoxin B1 toxicity has been studied in female rats treated for 15 consecutive days with repeated doses of aflatoxin (0.40 mg/kg/day), norethindrone (0.60 mg/kg/day) and ethynylestradiol (0.012 mg/kg/day). 2. Increases occurred in hepatic microsomal cytochrome P-450 content (31%), and in the activities of epoxide hydrase (77%), UDP-glucuronyltransferase (67%) and gamma-glutamyltransferase (78%). 3. Aflatoxin alone caused a 22% increase in GSH S-epoxide transferase activity, whereas the contraceptive given alone or combined with aflatoxin increased the hepatic reduced glutathione. 4. The effect of aflatoxin plus contraceptive was not additive. 5. The effects caused by aflatoxin and the contraceptive were similar, and the contraceptive (depending on its progestogen/estrogen ratio), may modify aflatoxin toxicity by increasing the drug-metabolizing enzyme activities and the concentration of hepatic glutathione.

Induction of cytochromes P450 and mixed-function oxidase activity by polychlorinated biphenyls and β-naphthoflavone in carp ( Cyprinus carpio)

1. 1. Hepatic microsomal ethoxyresorufin- O-deethylase and benzo(a)pyrene hydroxylase activities consistently were increased and cytochrome P450 content and ethoxycoumarin- O-deethykase activity sometimes were increased in carp injected with Aroclor 1254 or β-naphthoflavone as compared to corn oil vehicle alone. 2. 2. Hepatic microsomal deethylase activities and cytochrome P450 content were not significantly elevated by injection with 2,4,2',4'-tetrachlorobiphenyl as compared to corn oil above. 3. 3. Kidney microsomal deethylase activities as compared to corn oil injected were unaffected by these ag agents except that Aroclor 1254 significantly elevated ethoxyresorufin- O-deethylase activity. 4. 4. Control and induced hepatic microsomal benzo(a)pyrene hydroxylase activities were unaffected by metyrapone but were significantly inhibited by α-naphthoflavone added in vitro. 5. 5. Patterns of carp hepatic microsomal enzyme activities and responses to inhibitors resemble those seen in hepatic microsomes from 3-methylcholanthrene induced mammalian species, i.e. are cytochrome P448-like.

The effects of phenobarbitone on urinary 6β-hydroxycortisol excretion and hepatic enzyme activity in the marmoset monkey ( callithrix jacchus)

The effect of phenobarbitone administration on hepatic drug metabolising activity and urinary 6β-hydroxycortisol excretion was investigated in the marmoset monkey ( Callithrix jacchus). Phenobarbitone produced a significant increase in hepatic microsomal cytochrome P-450 content, aminopyrine N-demethylase activity, 7-ethoxycoumarin O-deethylase activity but no change in ethoxyresorufin O-deethylase activity. A significant increase in the urinary excretion of 6β-hydroxycortisol was observed after two days treatment with phenobarbitone.

Effect of pyrrolizidine alkaloids from tansy ragwort ( Senecio jacobaea) on hepatic drug-metabolizing enzymes in male rats

Ad lib. consumption of diets containing 5% tansy ragwort ( Senecio jacobaea) for 1–4 weeks produced a 5- to 6-fold increase in hepatic microsomal epoxide hydrase and significant increases in cytosolic glutathione S-transferase activities in male Long-Evans rats. An enhancement of these enzyme activities was also observed when a diet containing 1% tansy ragwort was fed for a period of 3 weeks. Feeding a diet containing 0.5% pyrrolizidine alkaloid (PA) mixture extracted from tansy ragwort for 1 week produced a 5-fold increase in hepatic epoxide hydrase and a 73 per cent increase in glutathione S-transferase activities. In contrast, hepatic microsomal aryl hydrocarbon hydroxylase activity (AHH) was reduced significantly by feeding diets containing 5% tansy ragwort or a 0.5% alkaloid mixture. Hepatic microsomal cytochrome P-450 content was lowered following consumption of the 0.5% alkaloid mixture but not by feeding a 5% tansy ragwort diet, the difference presumably being a result of the lowered PA intake by the latter animals. Exposure to the pyrrolizidine alkaloids, therefore, may influence significantly the capacity of animals to metabolize endogenous or foreign compounds and possibly also affect the subsequent biotransformation and toxicity of these plant constituents.

Effect of unilateral nephrectomy on in vitro hepatic microsomal oxidative drug metabolism in the rat.

AbstractThe effect of ablation of renal mass on in vitro hepatic microsomal oxidative drug metabolism was studied in unilaterally nephrectomized rats which were not uremic. When compared to either sham-operated or unoperated control rats, hepatic microsomal cytochrome P-450 content and ethylmorphine N-demethylase activity were both decreased significantly at 4 days but not at 15 days after unilateral nephrectomy. Differences were not observed in hepatic microsomal cytochrome b5 content or NADPH-cytochrome c reductase activity at either 4 or 15 days following unilateral nephrectomy. These results demonstrate that the removal of renal mass can influence temporarily in vitro hepatic microsomal oxidative drug metabolism and that this effect is apparent in the absence of uremia.

Sulfonamide inhibition of rat hepatic uroporphyrinogen I synthetase activity and the biosynthesis of heme

Sulfonamides are known to provoke attacks of acute intermittent porphyria, which is characterized by a deficiency of uroporphyrinogen I synthetase (EC 4.3.1.8) activity. Various sulfonamides were examined for their potential as inhibitors of purified rat hepatic uroporphyrinogen I synthetase activity in vitro, and 10 were found to be inhibitory. Inhibition of uroporphyrinogen I synthetase activity by sulfonamides is reversible by dialysis and exhibits noncompetitive kinetics. Inhibition constants ( K i ) ranged from 100 μ m for sulfaguanidine to 270 μ m for sulfadiazine. These observations were extended to in vivo studies using sulfamerazine ( K i = 250 μm). Twenty-four hours after administration of sulfamerazine (orally; 1 g/kg) to male rats, hepatic microsomal heme and cytochrome P-450 contents were decreased approximately 33 and 42%, respectively. This was reflected by the inhibition of both aniline- p-hydroxylase and amino pyrine- N-demethylase activities, which are P-450 mediated. Heme oxygenase activity was not altered by sulfamerazine treatment. However, the relative rate of heme biosynthesis, as measured by the incorporation of δ-[4- 14C]aminolevulinic acid into heme, was 28% lower in sulfamerazine-treated rats. Thus, these results indicate that sulfamerazine acts to inhibit heme biosynthesis. Hepatic δ-aminolevulinic acid synthetase (EC 2.3.1.37) activity was increased 2.2-fold in the sulfamerazine-treated group, which is probably a compensatory increase caused by depression of the heme content. Consequently, it is suggested that sulfamerazine inhibits the synthesis of heme at the Uroporphyrinogen I synthetase step to impair hemoprotein formation and cytochrome P-450-mediated hepatic drug metabolism.

Effects of chlorinated hydrocarbons on plasma α-lipoprotein cholesterol in rats

Ten chlorinated hydrocarbons (tetradifon, Chlorobenzilate, kepone, lindane, Kelthane, toxaphene, dieldrin, hexachloro-benzene, dienochlor, and Aroclor 1254; see below) were evaluated in rats to assess agents which might selectively elevate high-density lipoprotein cholesterol (C-HDL). Single, nontoxic doses of Aroclor, dieldrin, and Kepone elevated C-HDL at day 7 (31%, 26%, and 24%, respectively, p < 0.05). The C-HDL elevations were maintained at days 21 and 60 (28% and 31%, p < 0.01) in the Aroclor group, while C-HDL returned to baseline in the dieldrin and Kepone groups. Liver function tests were unchanged from control, sugfesting that the changes in C-HDL were not the consequence of hepatotoxicity. The hepatic microsomal cytochrome P-450 content of animals receiving Aroclor, dieldrin, and Kepone was 67%, 56%, and 44% higher than control values ( p < 0.02), indicating probable hepatic enzyme induction. If HDL (an antiatherogenic lipoprotein) is also selectively elevated by chlorinated hydrocarbons in man, then analogues might potentially be developed as antiatherogenic pharmacologic agents. Alternatively, the implications of man's exposure to halogenated hydrocarbons may be better understood.

Interaction of pargyline with rat hepatic microsomes.

When administered by intraperitoneal injection daily for 3, 7 or 14 days, pargyline (75 mg/kg) significantly reduced rat hepatic microsomal ethylmorphine N-demethylase activity and cytochrome P-450 content. Injection of a lower dose of pargyline (15 mg/kg) failed to alter significantly either ethylmorphine N-demethylase activity or cytochrome P-450 content. Studies performed in vitro reveal that pargyline is metabolized to at least three compounds by rat hepatic microsomes. Thin-layer chromatography and other analyses suggest that one metabolite is an N-demethylated form, norpargyline.

Effects of chronic ethanol administration in the rat: relative dependency on dietary lipids. II. Paradoxical role of linoleate in the induction of hepatic drug-metabolizing enzymes in vitro

The effect of chronic ethanol administration on the hepatic microsomal cytochrome P-450 content and activities of NADPH - cytochrome P-450 reductase (EC 1.6.2.4), benzphetamine demethylase, aniline hydroxylase (EC 1.14.14.1), and of the microsomal ethanoloxidizing system were studied in various dietary models. When ethanol was given with linoleate as the only source of dietary lipid, the ethanol induction of these parameters was greater with diets containing 2 or 5% of total calories as linoleate than with diets containing 10% of total calories as linoleate. By contrast, when ethanol was given with high fat (35% of total calories) diets, the ethanol induction of these same parameters was slightly greater when linoleate provided 10% of total calories than when it provided 3% of calories. The apparent effect of dietary linoleate on the induction, by ethanol, of microsomal drug-metabolizing enzymes is markedly different when linoleate is given as the only source of dietary lipid as opposed to when it is given with other dietary lipids. Thus, conclusions on the effect of ethanol on hepatic microsomal drug-biotransformation enzymes, drawn from studies with dietary models in which linoleate provides the only source of dietary lipid, connot be extended to dietary modles of more complex lipid composition. When given as the only source of lipid, 2% of total calories in linoleate appears optimal for basal activity and inductibility, by ethanol, of mixed-function oxidases.

Androgen receptor dependent and independent activities of testosterone on hepatic microsomal drug metabolism.

Administration of testosterone for 6 days to intact female and castrate male BALB/cJ mice stimulated hepatic microsomal ethylmorphine N-demethylase activity and cytochrome P-450 content by 50-75%. Testosterone also stimulated hepatic microsomal NADPH-oxidase activity, but to a lesser degree. To probe the mechanism of this effect of androgens, two antiandrogens (cyproterone acetate and flutamide) were employed. Since cyproterone acetate was a potent stimulator of hepatic microsomal ethylmorphine N-demethylase activity and cytochrome P-450 content, no antiandrogenic activity of this steroid could be detected. By contrast, flutamide alone had little effect on either ethylmorphine N-demethylase activity or cytochrome P-450 content. However, this drug effectively blocked the stimulatory effects of testosterone on ethylmorphine N-demethylase activity and cytochrome P-450 content but not on NADPH-oxidase activity. This effect was not species specific, since flutamide also prevented androgen stimulation of ethylmorphine metabolism in adult castrate and prepubertal male Fisher rats. The testosterone-induced increase of hepatic weight and microsomal protein content was not affected by the administration of flutamide. The observations are consistent with the hypothesis that androgens have two distinct effects on the liver. First, testosterone may act as a general, nonspecific stimulant of liver weight and microsomal protein content which is independent of the androgen receptor. Secondly, testosterone action in the liver may be expressed via an androgen-specific or androgen receptor-dependent mechanism which controls, in part, the cytochrome P-450-dependent demethylase system.

The mechanism of the effect of acute stress on hexobarbital metabolism

The effect of acute stress (unilateral hind-leg ligation for 1 hr) on hepatic metabolism of hexobarbital (HB) was studied in the rat. A stress duration-response relationship was found for stress inhibition of HB metabolism. The hepatic microsomal protein content was not affected, but the hepatic microsomal cytochrome P-450 content was reduced approximately 45% by this stress treatment. Stress caused a twofold increase in plasma corticosterone level and had no effect on plasma corticosterone level of adrenalectomized rats. Hexobarbital metabolism was not affected by stress in adrenalectomized rats. Thus, the inhibition of hepatic HB metabolism by acute stress may be caused by the increased release of corticosterone induced by acute stress.

Divergence of growth hormone actions on hepatic drug metabolism in the presence and absence of the pituitary gland

Experiments were carried out to compare the effects of growth hormone on hepatic drug oxidation in normal and hypophysectomized rats. Administration of growth hormone to normal male rats lowered hepatic microsomal cytochrome P-450 content and decreased the rates of ethylmorphine n-demethylation and aniline hydroxylation. These effects were fully manifested in orchiectomized or adrenalectomized males, excluding a dependence upon endogenous steroids. Growth hormone was without effect on hepatic drug metabolism or cytochrome P-450 content in normal female rats. In contrast to its actions in animals with intact pituitary glands, administration of growth hormone to hypophysectomized rats of either sex increased the rate of ethylmorphine metabolism. Furthermore, in both males and females, aniline hydroxylation and microsomal cytochrome P-450 content were unaffected by growth hormone in the absence of the pituitary gland. Prolactin administration did not affect hypophysectomized or in normal rats of either sex. The results indicate that the nature of growth hormone actions on hepatic drug oxidation is pituitary-dependent and probably intertwined with the effects of other hormones. Furthermore, the direct physiological effects of growth hormone on hepatic mixed function oxidases seem to depend upon the substrate employed.

The metabolism of [UREYL- 14C] tolbutamide [formula omitted] and [formula omitted][formula omitted] in man

Hepatic microsomal kinetic constants and plasma half-lives and plasma clearances for the metabolism of tolbutamine were measured in the same patients. No significant correlations were found for the seven experiments performed. There was a trend to an inverse correlation between hepatic microsomal cytochrome P-450 content and the plasma clearance. The negative findings suggest a lack of variation in the hepatic drug-metabolizing activities of normal humans.

Relationship between plasma antipyrine half-lives and hepatic microsomal drug metabolism in dogs.

Plasma antipyrine half-lives exhibited an inverse relationship to hepatic microsomal aniline hydroxylase (r = –0.78) and ethylmorphine N-demethylase (r = –0.79) activities in healthy mongrel dogs. However, no close correlation occurred between plasma antipyrine half-life and hepatic microsomal cytochrome P-450 content (r = –0.07) cytochrome c reductase activity (r = –0.21) or NADPH oxidase activity (r = –0.30). In dogs retested at 21 days, plasma antipyrine half-life was a highly reproducible value in each animal. However, at shorter time intervals the dose of antipyrine used (75 mg/kg, i.v.) probably induced hepatic microsomal drug metabolism since retesting 10 days after the initial dose disclosed shortening of the plasma antipyrine half-life in each animal. These experiments suggest that under certain conditions plasma antipyrine half-lives may be useful indices of rates of hepatic metabolism of several compounds chemically unrelated to antipyrine.

Effect of reduced barometric pressure on drug action and metabolism in mice

Mice kept at a simulated altitude of 18,000 ft for 5 days had a shorter duration of loss of righting reflex with hexobarbital, zoxazolamine and mephenesin, but not with pentobarbital. Exposure to reduced pressure did not alter brain receptor sensitivity to these drugs. Decreased hexobarbital sleeping time was produced 3 days after initial simulated altitude exposure and was reversed within 3 days after return to normobaric conditions. The hepatic microsomal metabolism of aniline, hexobarbital, nitroanisole and dichlorophenolindophenol was increased in mice kept in a hypobaric chamber while that of methylaniline was not altered. Mice exposed to reduced pressure had a loss of total body weight, liver weight, and liver water content. Hepatic RNA-P and DNA content was increased, but there was no change in the hepatic microsomal protein or cytochrome P-450 content. A decreased hexobarbital sleeping time in different mice strains was observed at a simulated altitude of 18,000 ft but not at 8000 ft. The results suggest that the alterations in drug action observed may be due primarily to increased drug metabolism produced by hypobaric hypoxia.

The inhibitory effect of aureomycin (chlortetracycline) pretreatment on some rat liver m1crosomal enzyme activities

Rats were subjected to intraperitoneal injections of varying amounts of chlortetracycline to study the effects of this antibiotic on rat liver microsomal drug metabolism. The chlortetracycline-treated rats were then compared with sham-injected controls and phenobarbital-treated animals. Compared with the control animals, the chlortetracycline-treated animals gained weight more slowly, their livers were smaller, the microsomal protein per unit weight of liver was less, the hepatic microsomal cytochrome P-450 content and metabolism in vitro of aminopyrine and hexobarbital were decreased while the hexobarbital sleeping time was markedly increased. The phenobarbital-treated animals showed the usual increase in all of the hepatic microsomal components and a marked decrease in hexobarbital sleeping time. Our data would indicate a possible inhibition of microsomal protein synthesis by chlortetracycline treatment, supporting the suggested mechanism of action of the tetracycline antibiotics.