Nutritional factors affecting drug-metabolizing enzymes of the rat

Feeding diets rich in thiamin depresses aniline hydroxylase, cytochrome P-450 and cytochrome b₅ within 9–14 days. Pair-feeding experiments suggest that the depression of aniline hydroxylase, cytochrome c reductase and ethylmorphine demethylase is due to the thiamin; however, the depression of cytochrome P-450 and b₅ may be due primarily to the increased amount of carbohydrate ingested by rats fed the enriched diet. When starch was substituted for sucrose, cytochrome P-450 was not lowered by high thiamin ingestion, although cytochrome b₅ and NADPH cytochrome c reductase were depressed similarly to that of rats fed high thiamin levels in a sucrose-based diet. Although aniline hydroxylase and ethylmorphine demethylase activities were significantly depressed by both high thiamin diets, this effect was more pronounced in rats fed the sucrose-based diet.     

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

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 b₅ 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.     

Induction of xenobiotic metabolism in rats by short-term administration of brominated diphenyl ethers

Commercial preparations of fire retardant brominated diphenyl ethers were tested along with bis (-bromophenyl) ether and diphenyl ether for their ability to alter xenobiotic metabolism. The materials, 0.1 $\text{mmol}\text{kg}\text{day}$, were administered p.o. to male rats for 14 days. Pentabromodiphenyl and octa-bromodiphenyl ether preparations increased O-ethyl O-p-nitrophenyl phenyl-phosphonothioate (EPN) detoxification, p-nitroanisole demethylation, NADPH-cytochrome c reductase, cytochrome P-450, liver weight, UDP-glucuronyltrans-ferase and benzo[a] pyrene hydroxylase. Diphenyl ether increased only EPN detoxification and decabromodiphenyl ether only liver weight. Bis(p-bromo-phenyl) ether increased liver weight, cytochrome c reductase and cytochrome P-450. The data indicate that these materials are inducers of xenobiotic metabolism with activity dependent upon degree of bromination.     

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.     

Alterations in hepatic heme and cytochrome P-450 metabolism in murphy-sturm lymphosarcoma-bearing rats implications for drug metabolism

Previous studies have shown that tumor-bearing rats have significantly decreased hepatic microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity with, consequently, significantly decreased capacity for microsomal oxidative drug metabolism. Subsequent investigations have revealed that the rates of hepatic cytochrome P-450 apo-protein synthesis and degradation are decreased significantly and hepatic microsomal heme oxygenase activity is increased significantly in rats bearing an extra-hepatic tumor. Further studies have been done to attempt to clarify the pathogenesis and significance of these observations. Hepatic delta-aminolevulinic acid (ALA) synthetase activity in male Wistar rats declined to a nadir of 162 ± 34 (S.E.) pmoles ALA per mg protein per 30 min 6 days following i.m. transplantation of Murphy-Sturm lymphosarcoma (vs control = 218 ± 36 pmoles per mg per 30 min). Turnover of ³H-labeled heme in microsomal CO-binding particles (i.e. cytochrome P-450 heme) was increased significantly 8 days following i.m. transplantation of Murphy-Sturm lymphosarcoma with a T_{$\text{1}\text{2}$} of 5.5 hr for the fast phase of hepatic cytochrome P-450 heme disappearance in tumor-bearing rats as compared with a T_{$\text{1}\text{2}$} of 7 hr in control rats. Hepatic cytochrome P-450 apo-protein concentration was slightly, but not significantly, increased in Murphy-Sturm lymphosarcoma-bearing rats as compared with control rats up to 10 days following tumor transplantation. These results suggest that, in Murphy-Sturm lymphosarcoma-bearing rats, decreased microsomal cytochrome P-450 concentration is the result of both decreased cytochrome P-450 apo-protein synthesis and increased cytochrome P-450 heme turnover. Apo-cytochrome P-450 concentration was not appreciably altered because increased cytochrome P-450 heme turnover and decreased cytochrome P-450 apo-protein degradation were balanced by decreased cytochrome P-450 apo-protein synthesis. Because of their effects on cytochrome P-450 concentration and action, these alterations in heme and hemoprotein metabolism may be of importance in regulating oxidative drug metabolism in the tumor-bearing state.     

Effect of protein deficiency on the inducibility of the hepatic microsomal drug-metabolizing enzyme system—I: Effect on substrate interaction with cytochrome P-450

Male, weanling rats divided into three groups were maintained for 15 days on a semipurified diet containing either 5% casein fed ad lib. (group 1), 20% casein pair-fed to group 1 (group 2), or 20% casein fed ad lib. (group 3). After each group was further subdivided, animals were injected i.p. on days 11, 12, 13 and 14 with either 0.9% saline or phenobarbital (80 mg/kg) in 0.9% saline. Twenty-four hr after the last injection, animals were decapitated and liver microsomes were prepared. Contents of microsomal protein, phosphatidylcholine and cytochrome P-450 were measured and used as bases of expression for spectral dissociation constants (K_s) and maximal spectral changes (ΔA_max) associated with the binding of ethylmorphine and aniline to the cytochrome P-450 hemoprotein of microsomes. Phenobarbital administration increased microsomal protein, cytochrome P-450, and phosphatidylcholine in all three dietary groups; however, in all groups, the increase in P-450 was relatively greater than that for phosphatidylcholine. Protein deficiency (group 1 vs 2) decreased P-450 and microsomal protein, but had no effect on phosphatidylcholine contents. The effect of total food restriction (group 2 vs 3) on each of these parameters was not significant. These data suggest that a portion of the induced cytochrome P-450 binding sites may be dependent on an association with phosphatidylcholine. The fraction of such phosphatidylcholine-associated sites relative to the total sites was greater during protein deficiency and was in agreement with a greater ΔA_max per nanomole P-450 for ethylmorphine. Phenobarbital induction decreases the proposed fraction of phosphatidylcholine-associated P-450 sites relative to the total P-450 sites and results in a decrease in the ΔA_max per nanomole P-450 for ethylmorphine. Phenobarbital increased the ΔA_max per milligram of microsomal protein for aniline, which paralleled the increase in total P-450, thus indicating that the type II site may be independent of any association of cytochrome P-450 with phosphatidylcholine. These results indicate that phosphatidylcholine may play an important role in distinguishing the effects of dietary deficiency on type I substrate binding and the corresponding capacity for induction of the rat liver microsomal enzyme system.     

In vitro hepatic drug metabolism and microsomal enzyme induction in genetically obese rats

Genetically obese Zucker-strain rats were used to determine if obesity produced alterations in in vitro drug metabolism in hepatic microsomes or cytosol. Adult homozygous obese rats of both sexes had significantly less active drug-metabolizing enzymes in vitro than homozygous lean Zucker rats or Sprague-Dawley rats. Specific activities of aniline hydroxylase, aminopyrine demethylase and arylhydrocarbon hydroxylase (AHH) from male obese rats were only 25–30 percent of control activities, while glutathione S-aryltransferase, biphenyl 4-hydroxylase, and cytochrome P-450 activities were 50–70 percent of control activities. K_m and V_max values for aminopyrine demethylation were significantly less in obese rats than in lean rats. Enzyme activities in weanling obese rats were nearly equivalent to activities in weanling lean rats. Pretreatment of obese and lean rats with 3-methylcholanthrene produced expected increases in activities of biphenyl 4-hydroxylase, AHH and cytochrome P-450. Phenobarbital (PB), however killed half of the PB-treated obese rats at 60 mg/kg, but killed no lean rats. At lower doses, PB pretreatment produced only marginal increases in specific activities of aniline hydroxylase, aminopyrine demethylase and cytochrome P-450 in obese rats, while producing larger increases in activities in lean controls. When genetically obese rats were pair-fed to body weights that were equal to lean rats, the deficiency in in vitro drug metabolism between lean and fat rats was qualitatively as great as in ad lib. fed rats. Testosterone treatment of obese rats produced no significant increase in in vitro drug metabolism. Heterozygous lean Zucker rats responded to all treatments in a manner similar to that of homozygous lean Zucker rats. The explanation for these results is complex and they cannot be accounted for by a simple increase in body weight.     

Influence of norethindrone on drug-metabolizing enzymes of female rat liver in various B-vitamin deficiency states.

Ingestion of high levels of thiamin significantly decreased the activity of cytochrome P-450, NADPH cytochrome c reductase, and the metabolism of aniline and ethylmorphine. Apparent VmaxS for ethylmorphine N-demethylase and aniline hydroxylase were decreased by high levels of riboflavin even though NADPH cytochrome c reductase was elevated. High levels of dietary pyridoxine significantly decreased only the Vmax for aniline hydroxylase. Generally, norethindrone produces either no change or slight depression of cytochrome P-450 regardless or diet, whereas the administration of norethindrone produced no change or an increase in activity of c reductase and ethylmorphine N-demethylase. Norethindrone induces aniline hydroxylase in animals fed all diets except those deficient in thiamin and riboflavin. The activities of the four parameters of the drug metabolizing system measured in these studies as well as the effects of norethindrone are clearly affected by the dietary status of the animal.     

Changes in the rat hepatic mixed function oxidase system associated with chronic ethanol vapor inhalation

Chronic ethanol vapor inhalation by rats increased hepatic microsomal aniline hydroxylase activity, increasing the turnover number and decreasing the K_m. Activity of ethanol-induced microsomes toward other substrates was also examined. The increase in aniline hydroxylase activity as a result of ethanol treatment is attributed to an increase in a form of cytochrome P-450 with a high specific activity toward aniline. Since the ethanol effect on aniline hydroxylation had disappeared 24 hr after treatment was discontinued, a high rate of turnover of this enzyme was deduced. Dimethylsulfoxide (56 mM) produced a reverse type I spectral change in ethanol-induced, but not in control, microsomes. This was interpreted as being due to a change in the spin state of the cytochrome P-450 in these microsomes. Acetone added to the incubation produced an increased rate of aniline hydroxylation by microsomes from control and ethanol-induced rats. The difference between the rate of aniline hydroxylation by control microsomes and the rate by ethanol-induced microsomes was, however, abolished at higher acetone concentrations.     

Dose-dependent actions of thyroxine on hepatic drug metabolism in male and female rats

Studies were carried out to compare the effects of various doses of thyroxine (T₄) on hepatic drug metabolism in male and female rats and to evaluate the role of the pituitary gland in the modulation of T₄ action. Administration of small amounts of T₄ (2.5 to 5 μg/100g body wt/day) to hypophysectomized rats of either sex increased hepatic ethylmorphine demethylase, benzo(a)pyrene hydroxylase and aniline hydroxylase activities. Larger amounts of T₄ (12.5 to 50 μg) reversed the stimulatory effects of the smaller doses. T₄ treatment produced dose-dependent decreases in hepatic cytochrome P-450 content and increases in NADPH-cytochrome c reductase activity in hypophysectomized rats of both sexes. Qualitatively similar effects were produced by T₄ administration to thyroidectomized male and female rats. However, larger doses of T₄ were required for maximum stimulation of drug metabolism in thyroidectomized than in hypophysectomized animals. The results indicate that physiological amounts of T₄ Uniformly stimulate hepatic drug metabolism in both male and female rats. Supraphysiological amounts, however, inhibit metabolism of some substrates and produce sex differences in T₄ actions. The effects of T₄ are demonstrable in the absence of the pituitary gland but pituitary-dependent factors appear to modulate the magnitude of the response to T₄.     

Kinetic alterations in rat liver microsomal cholecalciferol 25-hydroxylase associated with phenobarbital administration

The kinetics of cholecalciferol 25-hydroxylase in vitamin D-depleted rat liver microsomes, before and after phenobarbital induction, were studied. Three days of pretreatment with phenobarbital altered significantly both the apparent K_m and the V_max of the hydroxylase. Untreated vitamin D-repleted rats had lower cytochrome P-450 content and aminopyrine demethylase activity than the vitamin D-depleted animals. Phenobarbital administration reversed this nutritional effect on aminopyrine demethylase but not on cytochrome P-450 content. Furthermore, vitamin D deficiency potentiated the phenobarbital inductive effect upon microsomal protein. No inhibition of aminopyrine demethylase could be elicited in the presence of cholecalciferol or 25-hydroxycholecalciferol either prior to or after phenobarbital treatment, suggesting that these two oxidases are different entities.     

Effects of the interferon inducing agents tilorone and polyriboinosinic acid · polyribocytidylic acid (poly IC) on the hepatic monooxygenase systems of the developing neonatal rat

This paper describes the effects of the interferon inducing agents tilorone and polyriboinosinic acid · polyribocytidylic acid (poly IC) on the postnatal development of hepatic cytochrome P-450-linked monooxygenase systems of male rats from birth through early adolescence. The administration of tilorone to rats on days 1and 2 postpartum modified the changes in the activities of hepatic monooxygenase systems that occur normally during the first four days postpartum. Thus, aniline hydroxylase activity, which develops very rapidly during the first 2 days postpartum, was depressed markedly by tilorone, ethylmorphine N-demethylase activity was depressed moderately, and benzo[a]pyrene hydroxylase, normally the slowest of the three monooxygenase activities to develop, was induced. These changes in monooxygenase activities occurred without a significant change in the cytochrome P-450 content. These observations suggest that not all species of neonatal cytochrome P-450 are affected equally by tilorone administration. By day 7 postpartum, the cytochrome P-450 content and all three monooxygenase activities were depressed in rats that had received tilorone on days 1 and 2 postpartum. All three monooxygenase systems were depressed by the administration of a single dose of poly IC (10 mg/kg) in 1-, 2-, 21-, 28- and 56-day-old rats. The length of the period between maximal depression and complete recovery of cytochrome P-450 systems was shown to be a function of the age of the rat; it increased from about 6 hr in 1-day-old rats to 48 hr in 56-day-old rats. Protein is synthesized more rapidly and degraded more slowly in neonate than in adult animals; this may account for the more rapid recovery of poly IC-induced depression of monooxygenase systems in neonates.     

Hepatic drug metabolism after phenobarbital and diphenylhydantoin administration in the rat--influence of vitamin D3 status.

Measurements of aniline hydroxylation, aminopyrine N-demethylation and cytochrome P-450 content after a 3-week treatment with phenobarbital (PB), diphenylhydantoin (DPH) or a combination of the two drugs were undertaken during normal vitamin D status (D +) and vitamin D deficiency (D ?) with or without vitamin D₃(D₃) supplementation. Serum calcium concentrations were reduced after D deprivation but responded by a significant increase toward normal values to a single pharmacological dose of D₃. Serum phosphorus concentrations were also slightly raised by the supplementation. Even in the presence of higher cytochrome P-450 content in D ? rats, aniline hydroxylase and aminopyrine N-demethylase activities were lower in D ? than in D + animals. These two enzymatic parameters, as well as cytochrome P-450 content, were increased by anticonvulsant (ACV) drug treatment regardless of the D nutritional status. The in vivo hexobarbital sleeping time was shortened by ACV drugs but the sleeping time tended to be longer in D ? than in D + rats. Supplementation with 1000 I.U. of D₃, lowered aniline hydroxylase activity both in D + and D? animals; the supplementation had no effect on aminopyrine N-demethylase activity in D + animals but had an inhibitory effect after PB and a stimulatory effect after DPH treatment in D? animals. Cholecalciferol supplementation lowered cytochrome P-450 content toward normal values in D? rats while it had no effect in D + animals. These observations suggest that (1) PB and DPH pretreatment do not alter the normal response of serum calcium and phosphorus to a single pharmacological dose of D₃; (2) in a state of vitamin D deficiency accompanied by hypocalcemia, the inducing capacity of PB and DPH on the liver mixed function oxidase system is not lost; (3) under certain circumstances, vitamin D₃, can influence the catalytic activity of the mono-oxygenase complex; (4) cytochrome P-450 is influenced by vitamin D deficiency and/or changes in extracellular calcium but the forms induced by PB and DPH may not necessarily be the ones specifically involved in vitamin D metabolism.     

Effect of hexachlorobenzene on microsomal enzyme systems

Male rats were fed for 10 days on a diet containing 333 ppm hexachlorobenzene. Increased microsomal protein levels were noted compared to control rats. On a per g liver basis, the levels of aniline hydroxylase, biphenyl 4-hydroxylase, biphenyl 2-hydroxylase, 4-nitroanisole O-demethylase, esterase, cytochrome P-450 and cytochrome b₅ all increased compared with the control values. On a per mg microsomal protein basis, biphenyl 2-hydroxylase, 4-nitroanisole O-demethylase and cytochrome P-450 levels increased several-fold compared with the control values. It is suggested that, by inducing the 2-hydroxylation reaction, hexachlorobenzene might cause preferential ortho-hydroxylation, as do some carcinogenic polycyclic hydrocarbons, and that in some circumstances this could lead to the formation of carcinogens.     

Influence of drugs and chemical upon hepatic enzymes and proteins-I. Structure-activity relationship between various barbiturates and microsomal enzyme induction in rat liver.

The effect of phenobarbital, three N-phenylbarbiturates and three N-cyclohexylbarbiturates on microsomal protein content, cytochrome P-450, cytochrome b₅, aniline hydroxylase, aminopyrine demethylase, p-nitrophenol-glucuronyltransferase and the ultrastructure of hepatic cells in rat liver were studied to elucidate the influence of barbiturate structure on enzyme-inducing activity. Smooth endoplasmic reticulum increased after administration of phenobarbital, phetharbital or bucolome. Phenobarbital and phetharbital, especially phenobarbital, induced cytochrome P-450 and glucuronyl-transferase. On the other hand, the other barbiturates showed little enzyme-inducing activity. From these results, the type and spatial position of barbiturate substituents are considered important for hepatic microsomal enzyme induction. Aniline hydroxylase and aminopyrine demethylase activities changed coincidentally with cytochrome P-450 content in almost all rat livers. However, quantitative agreement of the changes in cytochrome P-450 content and drug-metabolizing activity could not be obtained, suggesting the possibility of substrate inhibition or of intrinsic inhibitors in the microsomal fraction. Bucolome, one of the N-cyclohexylbarbiturates, has been reported to be effective in the reduction of serum unconjugated bilirubin level in Gilbert's syndrome. However, in. spite of the increased smooth endoplasmic reticulum, there was a reduction in the microsomal protein content, cytochrome P-450 and glucuronyltransferase after bucolome administration. This would seem to indicate that the serum bilirubin-reducing mechanism of bucolome is different from that of phenobarbital and phetharbital.     

Studies on the mechanism of cystamine prevention of several liver structural and biochemical alterations caused by carbon tetrachloride

Cystamine and cysteamine give type II spectral changes by interaction with microsomal suspensions while CCl₄ gives a type I spectral change. Neither cystamine nor cysteamine at saturating concentrations prevent the spectral binding of small amounts of CCl₄. Cystamine and cysteamine decelerate the reduction of cytochrome P-450 by cytochrome P-450 reductase. Cystamine administration partially prevents the irreversible binding of ¹⁴CCl₄ to microsomal lipids. This preventive effect does not significantly change when the ¹⁴CCl₄ dose is increased about 4-fold. In spite of the fact that this irreversible binding is decreased by about 60% at 6 hr by the previous cystamine administration, lipid peroxidation is only slightly prevented at 6 and 10 hr after CCl₄ and prevented to about 30% at 24 hr. Cystamine partially prevented the depression of glucose-6-phosphatase activity and the destruction of cytochrome P-450 caused by CCl₄. Cystamine pronouncedly lowers body temperature of the rats, and it is as effective in preventing CCl₄-induced necrosis and fatty liver at 24 hr as it is at 48 or 72 hr after CCl₄ administration. Cystamine stabilizes lysosomes in vitro but labilizes them when administered to rats. Cysteamine labilizes lysosomes in vitro. These results may suggest that cystamine administration partially prevents several structural and biochemical alterations caused by CCl₄ because it inhibits the CCl₄-activation step to ·CCl₃ free radical, which would take place during the reduction of the cytochrome $\text{P-450}\text{CCl}{}_4$ complex by cytochrome P-450 reductase.     

Effect of protein deficiency on the inducibility of the hepatic microsomal drug-metabolizing enzyme system—II: Effect on enzyme kinetics and electron transport system

Male, weanling rats divided into three groups were maintained for 15 days on a semipurified diet containing either 5% casein fed ad lib. (group 1), 20% casein pair-fed to group 1 (group 2), or 20% casein fed ad lib. (group 3). After each group was further subdivided, animals were injected i.p. on days 11–14 with either 0.9% saline or phenobarbital (80 mg/kg) in 0.9 % saline. Twenty-four hr after the last injection, animals were decapitated and liver microsomes were prepared. Apparent V_max and apparent K_m kinetic constants were determined for ethylmorphine and aniline. The V_max per milligram of microsomal protein was 64–66 per cent lower in the protein-deficient group. Equivalent reductions of the content of cytochrome P-450 and activities of cytochrome P-450 and c reductases were also observed. Phenobarbital induction increased specific enzyme activities (V_max per milligram of microsomal protein) in all groups with slightly greater percentage increases seen in the protein-deficient animals. Increases were also noted for the cytochrome P-450 content and cytochromes P-450 and c reductase activities. It was suggested that phosphatidylcholine and cytochrome P-450 both play important roles in the kinetics of metabolism determined after protein deficiency or phenobarbital induction, or both.     

Differential effects of cadmium on the hepatic microsomal cytochrome p-450 system in male and female rats

Cadmium (Cd) produced a marked sex-related difference with respect to inhibition of the hepatic microsomal monooxygenase enzyme system in the rat. Following in vivo cadmium (2 $\text{mg}\text{kg}$ i.p.) treatment, significant decreases in the levels of cytochrome P-450, significant reductions in the magnitudes of spectral binding (aniline or ethylmorphine), and significant inhibitions of microsomal metabolism (aniline and ethylmorphine) were observed with microsomes isolated from male but not female rats. Of these parameters only aniline metabolism was significantly altered in females. Following the in vitro addition of Cd (10^?6 M to 10^?3 M) to hepatic microsomes isolated from untreated male or female rats, sex-related changes were also observed in these parameters. Significant, concentration-dependent reductions were observed in cytochrome P-450 levels of both sexes but the males showed greater sensitivitiy to the cadmium effect. With respect to binding spectra, cadmium addition produced a concentration dependent inhibition of aniline only in the male rat. Ethylmorphine binding was inhibited only at the higher cadmium concentrations in both sexes. With respect to drug metabolism, cadmium addition inhibited both aniline and ethylmorphine metabolism in male rats and only aniline metabolism in female rats. These results showed that there are sex-related differences in the interaction of the hepatic microsomal monooxygenase enzyme system with cadmium both after in vitro addition as well as in vivo treatment with the metal.     

Induction and inhibition of hepatic drug metabolizing enzymes by rifampin.

The effects of the antibiotic rifampin on microsomal drug metabolizing enzymes were investigated. On acute administration, rifampin (100 mg/kg i.p.) doubled hexobarbital sleeping time and zoxazolamine paralysis time in mice. The in vitro metabolism of zoxazolamine (0.25 mM) and 17β-estradiol (10 μM) were inhibited 50% by rifampin in concentrations of 0.4 and 0.3 mM, respectively. The inhibition of ethylmorphine N-demethylase was competitive with an apparent K_i, of 52 μM. Repeated administration of rifampin to mice (50 mg/kg i.p. daily for 6 days) increased liver weight by 20%a cytochrome P-450 (50%), NADPH cytochrome c reductase (43%,). ethylmorphine N-demethylase (85%), zoxazolamine hydroxylase (77%), benzpyrene hydroxylase (174%). and 17β-estradiol metabolism (89%). Microsomal protein (mg/g). aniline hydroxylase and p-nitrophenol glucuronyl transferase activities were unaffected.Rat microsomal drug metabolizing enzyme activity was also inhibited after acute administration of rifampin as exemplified by an increase in hexobarbital sleeping time of 44% and a competitive inhibition of ethylmorphine N-demethylase by rifampin. The K_i, (33 μM) was close to that obtained with the mouse enzyme. The similarity in the in vivo and in vitro inhibition suggests that rifampin binds to microsomes in a similar manner in both species. Chronic administration of rifampin to rats (50 mg/kg i.p.) twice daily for six days did not lead to induction, indicating a species difference with respect to rifampin's inducing ability. Rifampin did not modify microsomal induction in rats by phenobarbital when both drugs were administered concomitantly. The mechanism responsible for the species difference and the clinical relevance of these results are discussed.     

Effect of stress by repeated immobilization on hepatic alcohol dehydrogenase activity and ethanol metabolism.

The hepatic activity of alcohol dehydrogenase was increased after 7, 14 and 42 days of stress induced by immobilization of rats for 2.5 $\text{hr}\text{day}$. A single immobilization had no effect on the activity of alcohol dehydrogenase. Immobilization for 14 days resulted in increases in the rates of ethanol metabolism. This was not associated with changes in the activity of the microsomal ethanol-oxidizing system, microsomal catalase, cytochrome P-450, or NADPH cytochrome c reductase. A decrease in the hepatic phosphorylation potential ([ATP]/[ADP][P_i]) was found to be due to a decrease in [ATP] and an increase in [P_i]; however, there were no changes in O₂ consumption by liver slices or in hepatic (Na⁺ + K⁺)-stimulated adenosine triphosphatase activity. The increased rate of ethanol metabolism after stress remains unexplained since alcohol dehydrogenase activity is not rate-limiting in ethanol oxidation and there were no increases in ethanol oxidation by microsomes or in mitochondrial oxidative rate.