Induction of the rat hepatic microsomal mixed-function oxidases by 3 imidazole-containing antifungal agents: selectivity for the cytochrome P-450IIB and P-450III families of cytochromes P-450

Administration of the imidazole antifungal agents ketoconazole, miconazole and clotrimazole gave rise to increases in the microsomal cytochrome P-450 levels and the NADPH-dependent reduction of cytochrome c. Clotrimazole, and to a much lesser extent miconazole and ketoconazole, stimulated the dealkylation of pentoxyresorufin. All 3 agents gave rise to small, but significant increases in the O-deethylations of ethoxycoumarin and ethoxyresorufin. The antifungal-induced O-deethylation of ethoxycoumarin was much more sensitive to inhibition by metyrapone rather than by -naphthoflavone. The binding of metyrapone to reduced microsomes was enhanced by treatment of animals with the 3 antifungal agents, clotrimazole being clearly the most potent. Immunoquantitation of cytochrome P-450 proteins using an ELISA procedure and employing anti-cytochrome P-450c (P-450IA1, P-448 low spin) and P-450b (P-450IIB1) antisera revealed that clotrimazole and miconazole, but not ketoconazole, induced the levels of phenobarbital-induced cytochromes P-450, while none of the antifungal agents increased the levels of cytochrome of P-448 proteins. Similar results were obtained using Western blots employing the above antibodies.

On SDS-polyacrylamide gel electrophoresis microsomes derived from animals pretreated with clotrimazole showed intensification of a band at 51 kDa which was identified by Western blotting as the PCN-inducible form of cytochrome P-450 (cytochrome P-450p, P-450III family). Similar, but less pronounced intensification was seen with microsomes from animals pretreated with miconazole and ketoconazole. Furthermore, microsomes from clotrimazole- and ketoconazole-treated animals interacted with erythromycin to yield type I spectra.

It is concluded that the imidazole-containing agents clotrimazole and miconazole, and to a much lesser extent ketoconazole, are potent inducers of the rat hepatic microsomal mixed-function oxidases, displaying selectivity towards the P-450IIB (phenobarbital-inducible) and P-450III (PCN-inducible) families of cytochrome P-450 proteins.     

Effects of polychlorinated terphenyls and paraffins on rat liver microsomal cytochrome P-450 and in vitro metabolic activities

The polychlorinated terphenyl Aroclor 5460 and the polychlorinated paraffins Witaclor 171 P and Witaclor 149 increased to different degrees the total microsomal concentration of cytochrome P-450 in the rat liver after intraperitoneal injection of 0.3, 1.0, and 1.0 g · kg^–1 body weight, respectively, each day for four days. The multiple forms of cytochrome P-450 were affected differently with an induction of RLvMc P-450₅₀ and RLvMc P-450₅₄ by all chemicals, and an additional induction of RLvMc P-450₅₅ by the polychlorinated terphenyl. The rat liver weights were extensively increased after treatment with the polychlorinated paraffins. Alterations were found in the in vitro metabolism of biphenyl, benzo(a)pyrene and the steroid hormones, 4-androstene-3,17-dione and 5-androstane-3,17-diol, after exposure to all chemicals. Changes in the in vitro formation of benzo(a)pyrene metabolites were found to correlate with changes in the multiple forms of cytochrome P-450. The present study demonstrate that only limited information can be obtained from alterations in the total concentration of cytochrome P-450 and show the importance of studying changes in the multiple forms and the metabolism of different substrates. Our results further indicate that exposure to any of the investigated polychlorinated chemicals may alter the biological effects of other environmental contaminants, drugs and endogenous substances which are metabolized by the cytochrome P-450 enzyme system.     

Differences in cytochrome P-450 of various strains of rats following chronic administration of pentobarbital.

Cytochrome P-450 levels were analyzed in rats of two pigmented (black Long-Evans and ACI) and two albino strains (Fischer 344 and Sprague-Dawley) following the administration of pentobarbital sodium and physiological saline. Differences between the albino vs pigmented strains were observed following injections of saline. The Fischer 344 albino strains responded similarly to the pigmented strains following a progressively increasing dose schedule of pentobarbital sodium.     

Interlaboratory comparison of total cytochrome P-450 and protein determinations in rat liver microsomes

Assay conditions in determining total cytochrome P-450 in four laboratories were compared. Although the determination was derived from the original Omura and Sato method in each laboratory, the four standard protocols differed slightly, resulting in considerable differences in the results. Since the cytochrome P-450 content is usually expressed per mg protein, the protein assay conditions were evaluated as well. Furthermore, we compared the cytochrome P-450 values obtained by the CO- and the dithionite (DT)-difference methods. The effect of a number of variables in the assay was investigated. The influence of the storage temperature of the microsomes was ascertained as well as effects of the gassing time with CO and the time between addition of dithionite, CO-gassing and the recording of the difference spectra. After evaluating these variables a standard operation procedure was established. Using this procedure the interlaboratory coefficient of variation for total cytochrome P-450 was 4.8%, a value which was comparable to the intralaboratory coefficients of variation. The final results also show that the millimolar extinction coefficient for the DT-difference method is higher than for the CO-difference method.     

Characterization of hepatic and pulmonary cytochromes P-450 in 3-methylcholanthrene-treated hamsters

Two major forms of hepatic cytochrome P-450 (hepatic P-450_MCI and P-450_MCII) were purified approximately 5-fold from liver microsomes in Syrian golden hamsters treated with 3-methylcholanthrene (MC). The purified preparations of hepatic P-450_MCI and P-450_MCII contained 9.6 and 8.3 nmol cytochrome P-450 (P-450) per mg protein, respectively, and were essentially free from NADPH-cytochrome c (P-450) reductase (fpT), NADH-cytochrome b₅ reductase and cytochrome b₅. By sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weights of hepatic P-450_MCI and P-450 _mMCII were estimated to be 56 000 and 53 500. Further, a major form of pulmonary P-450 (P-450_MC) were purified from lung microsomes of MC-treated hamster, and contained 14.2 nmol P-450 per mg protein, and estimated to be 56 000 in monomeric molecular weight, indicating the similar molecular weight to hepatic P-450_MCI in the hamster. From the absorption spectra the oxidized forms of hepatic P-450_MCI and P-450_MCII were high- and low-spin ferric hemoproteins, respectively, and pulmonary P-450_MC was similar to hepatic P-450_MCII in their hemoprotein spin state. No difference, however, was observed in the CO-reduced forms among hepatic P-450_MCI, P-450_MCII and pulmonary P-450_MC, all exhibiting 446.5 nm Soret bands. In a reconstituted system containing fpT and dilauroylphos-phatidylcholine (DLPC), pulmonary P-450_MC efficiently catalyzed benzo[a]pyrene (BP) hydroxylation at a rate of 11.4 mol formed per min per mol P-450, but hepatic P-450_MCI and P-450_MCII both exhibited lower levels, e. g., 0.49 and 0.54, respectively. These findings indicated a clear tissue difference in the activity of BP hydroxylation between lung and liver in MC-treated hamsters.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Metabolism of diamantane by rat liver microsomal cytochromes P-450

1. Diamantane binds to liver microsomes from phenobarbital-treated rats with an apparent Ks value of 5.2 x 10(-7) mol/l. This value being lower than that obtained for perhydrophenanthrene indicates that diamantane is very strongly bound to microsomal cytochrome P-450. 2. Metabolic studies show that liver microsomes from phenobarbital-treated rats readily metabolize diamantane to mono-, di- and possibly tri-hydroxy derivatives, whereas liver microsomes from beta-naphthoflavone-induced rats do not bind this hydrocarbon or metabolize it. 3. Reconstituted cytochromes P-450 b and e were more efficient in the hydroxylation of diamantane than liver microsomes; metabolites formed by the reconstituted system do not include all the products formed by microsomes, which indicates the involvement of forms of cytochrome P-450 other than the isozymes b and e.     

Effects of chlorinated paraffins on liver weight,cytochrome P-450 concentration and microsomal enzyme activities in chick embryos

Sublethal doses of three technical preparations of chlorinated paraffins (CPs) (Cereclor 42 (C_22–26, 42% Cl w/w), Cereclor 50LV (C_10–13, 49% Cl w/w) and Cereclor 70L (C_10–13, 70% Cl w/w)) were injected into the yolks of hens' eggs after 4 days of incubation. The liver weight, the cytochrome P-450 concentration in the liver and the liver microsomal activity of aminopyrine N-demethylase (APND), aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD) were determined in chick embryos incubated for 20 days. The degree of chlorination and probably also the carbon chain length of the CPs were of importance for their effects. Cereclor 70L was the most potent in causing increases in liver weight, cytochrome P-450 concentration and APND activity. Cereclor 42 was the least potent in these respects, even causing reduced APND activity. A decrease in AHH activity occurred in chick embryos treated with Cereclor 50LV, and a reduction in ECOD activity was noted as a result of treatment with Cereclor 42 and Cereclor 50LV.     

Effects of aminoglutethimide and its metabolite,N-acetylaminoglutethimide,on bovine adrenocortical and human placental cytochromes P-450scc

Summary The ability of aminoglutethimide to inhibit cholesterol conversion to pregnenolone was lost upon acetylation of the arylamine nitrogen. This appears to be due to failure of N-acetyl-d-aminoglutethimide to bind to cytochrome P-450scc, since it does not produce the altered low spin form of the enzyme formed upon binding ofd-aminoglutethimide. These findings provide further evidence for a role of the free arenamine function in aminoglutethimide and related inhibitors.     

Distribution and induction of cytochrome P-450 and two cytochrome P-450-dependent monooxygenase activities in rat liver parenchymal cell subpopulations separated by centrifugal elutriation

Liver parenchymal cells from the periportal and centrilobular zones differ in their morphological, biochemical and functional characteristics. In an effort to obtain fractions enriched in either periportal or centrilobular cells, isolated rat liver parenchymal cells were separated into five subpopulations by centrifugal elutriation. The mean diameters of the cells present in fractions I–V were 19.6, 21.1, 21.8, 22.7 and 23.5 m, respectively. The content of cytochrome P-450 as well as benzphetamine N-demethylase and 7-ethoxyresorufin O-deethylase activities were higher in the larger parenchymal cells than in the smaller ones. After administration of phenobarbital the content of cytochrome P-450 was approximately two-fold greater in the cells present in fractions 3–5, when compared to the same subpopulations isolated from untreated rats; the activity of benzphetamine N-demethylase was enhanced to a similar extent in all five fractions. 3-Methylcholanthrene treatment resulted in a significant increase of cytochrome P-450 content and 7-ethoxyresorufin O-deethylase activity in all five fractions: both parameters were slightly higher in fractions 4 and 5 than in fractions 1 and 2. In conclusion, the elutriated liver parenchymal cells seem to preserve the biochemical heterogeneity observed in the intact liver; the potential enrichment of periportal and centrilobular cells in the different fractions by centrifugal elutriation is discussed.     

Interactions of the histamine H2-receptor antagonist etintidine with rat liver cytochrome P-450: a comparison with cimetidine

Summary The two imidazole histamine H₂-receptor antagonists etintidine and cimetidine interact with the rat liver microsomal cytochrome P-450. From type II spectral changes follows that the affinity of rat liver microsomal preparations for etintidine is about 5 times as high as for cimetidine when comparing both high and low affinity binding sites. After pretreatment with phenobarbital etintidine inhibited benzphetamine N-demethylation competitively (app. K _i: 4.0 mmol/l). Cimetidine inhibited benzphetamine N-demethylation in the same range. After pretreatment with phenobarbital both drugs inhibited the oxidation of benzo(a)pyrene for which etintidine showed a higher inhibitory potency than cimetidine. However, this oxidation could not be inhibited when microsomes of 5,6-benzoflavone pretreated rats were used. After pretreatment with 5,6-benzoflavone only etintidine but not cimetidine inhibited the O-deethylation of ethoxyresorufin competitively (app. K _i: 0.2 mmol/l). Etintidine and cimetidine were metabolized by rat liver microsomes to their corresponding sulphoxides. In conclusion, etintidine may cause mainly the same drug interactions as cimetidine but seems to be a more potent inhibitor.Parts of the results were presented on the spring meeting of the German Pharmacological Society, Mainz 1986, Federal Republic of GermanyThis work forms part of the doctoral thesis of M. Schulz Send offprint requests to A. Schmoldt     

Species of cytochrome P-450 in rat liver microsomes with different stereoselectivity for the binding and monooxygenation of (+)- and (−)-methylphenobarbital

Summary N-Demethylation to phenobarbital is the only detectable monooxygenation pathway for (+)- and (–)-methylphenobarbital in rat liver microsomes. In microsomes from control ratsV _max is about 3.5-fold higher for the (+)-isomer than for the (–)-form. This ratio increases significantly to 5 after pretreatment of rats with phenobarbital. Qualitatively similar but smaller ratios are found for the magnitude of the substrate binding spectra with corresponding values of 1.7 for control and 2.5 for phenobarbital treated rats.The spectral dissociation constants (K _s) and the distribution of both enantiomers between water and the microsomal membranes are not significantly different. Since only cytochrome P-450 is involved in the N-demethylation of both enantiomers the participation of two phenobarbital-inducible species of cytochrome P-450 with different Stereoselectivity is suggested. This is proved by inhibition studies with metyrapone which forms complexes more easily with the species responsible for the preferential binding and N-demethylation of the (+)-isomer of methyl phenobarbital.This work was supported by the Deutsche Forschungsgemeinschaft, Schwerpunktsprogramm: Struktur und Funktion biologischer Membranen. We thank Prof. Dr. J. Knabe for the generous gift of (+)- and (-)-methylphenobarbital and Prof. Dr. W. Rummel and Dr. H. Büch for helpful discussions.     

Stimulatory effects of benzene on rabbit liver and kidney microsomal cytochrome P-450 dependent drug metabolizing enzymes

Treatment of rabbits with benzene (880 mg/kg/day), s.c. for 3 consecutive days, caused 3.8- and 5.7-fold increases in aniline 4-hydroxylation rates of liver and kidney microsomes, respectively. Benzene treatment markedly enhanced hydroxylation rates ofp-nitrophenol by liver and kidney by 7.2- and 4.2-fold, respectively. Both of these enzymes are associated with cytochrome P-450 LM3a. In contrast, the activity of benzphetamine N-demethylase, associated with P-450 LM2, was not altered significantly in either liver or kidney microsomes. Although the total cytochrome P-450 contents of liver and kidney microsomes were not altered significantly by the benzene treatment, in the case of liver microsomes, formation of a new cytochrome P-450 with an apparent M_r of 51,400 was observed on SDS-PAGE. On the other hand, in the kidney microsomes, the intensity of the bands corresponding to approximate M_r of 50000 and 51400 was markedly increased. The results of the present work, in combination with those of the previous work (Arinç et al. 1988), indicate the existence of tissue specificity in the induction of rabbit P-450 isozyme by benzene.A preliminary account of this work has been presented at the Nato Advanced Study Institute on Molecular Aspects of Monooxygenases and Bioactivation of Toxic Compounds, August 27–September 7, 1989, Izmir, Turkey.     

The mechanism of the suicidal reductive inactivation of microsomal cytochrome P-450 by halothane

Anaerobic incubation of NADPH- or sodium dithionite-reduced rat liver microsomes with halothane resulted in a significant inactivation of cytochrome P-450 and parallel loss of the prosthetic group protohaem. When the loss of microsomal haem was measured in the same incubations by two different methods, the pyridine/haemochrome assay and the porphyrin fluorescence technique, halothane was responsible for a loss of haem in both assays, indicating that the tetrapyrrolic structure of haem has been modified by halothane metabolites. Cytochrome P-450 loss by halothane was found to be irreversible, saturable, inhibited by carbon monoxide and showed biphasic, pseudo first-order kinetics, thus fulfilling all the conditions of a typical suicide inactivation reaction. Pretreatment of rats with inducers of cytochrome P-450 isoenzymes modified the kinetics of cytochrome P-450 inactivation and the amount of total inactivable enzyme in microsomes. A partition ratio, between metabolic turnover of the substrate and enzyme inactivation, of about 121 was found with microsomes from phenobarbitaltreated rats, indicating that halothane is rather efficient as a suicide substrate of cytochrome P-450. A stable complex between reduced cytochrome P-450 and a halothane metabolite is responsible for the 470 nm peak observed in the difference spectrum of reduced liver microsomes obtained on addition of halothane. An extinction coefficient for this complex was calculated from the amount of enzyme involved.     

Effect of cytochrome P-450 inhibitors on pharmacokinetics and safety of voriconazole

Our objective was to systematically assess the effect of cytochrome P-450 (CYP450) inhibitors on pharmacokinetics and safety of voriconazole (VORI). Cochrane Library, PubMed, Embase, CNKI, CBM and WanFang databases and Clinicaltrials. gov were searched up to Jan. 26th 2016. Two reviewers independently identified studies, extracted data and assessed quality of studies. Meta-analysis was performed with RevMan 5.3, and risk ratios (RRs) and mean differences (MDs) with 95% confidence intervals (CIs) were calculated. A total of 12 studies were included: three crossover randomized controlled trials, three single-arm before-and-after studies and six cohort studies. Compared with non-combination group, the group of VORI plus omeprazole had a significantly higher occurrence of hepatic dysfunction (RR = 4.11, 95% CI 1.12–15.08, P = 0.03). However, neurologic dysfunction and visual disturbance were not significantly different. Pantoprazole, rabeprazole, cimetidine and contraceptive significantly increased the peak concentration (Cmax) and area under the curve (AUC) of VORI, while indinavir had no significant effect on pharmacokinetics of VORI. The effects of esomeprazole, erythromycin and azithromycin on pharmacokinetic parameters of VORI presented inconsistent results. Co-administration of VORI and CYP450 inhibitors was safe except for omeprazole. Although Cmax and AUC of VORI were increased while co-administered with a couple of CYP450 inhibitors, no significant effect on clinical outcomes was observed.     

Alcohol-inducible cytochrome P-450 (P-450ALC)

Of the family of P-450 cytochromes occurring in rabbit liver microsomes, only isozyme 3 a (P-450_ALC) is induced by alcohol administration and is effective in catalyzing the reaction: ethanol+0₂+NADPH+H⁺ acetaldehyde +2H₂O+NADP⁺. As judged by immuno-chemical quantitation, P-450_ALC is also induced in the animals by other diverse agents, including imidazole, trichlorethylene, acetone, pyrazole, and isoniazid. Evidence has been obtained for the occurrence of a protein immuno-chemically related to P-450_ALC in human liver microsomes and of a similar alcohol-inducible protein in the rat and in the normal and alcohol dehydrogenase-deficient deer-mouse. P-450_ALC catalyzes the activation of foreign compounds such as acetaminophen, various nitrosamines, and carbon tetrachloride and is therefore believed to play an important role in the enhanced toxicity of these substances accompanying alcohol administrationDedicated to Professor Dr. Herbert Remmer on the occasion of his 65th birthday     

Effects of acetone administration on cytochrome P-450-dependent monooxygenases in hamster liver,kidney, and lung

The effects of acetone on liver, kidney, and lung monooxygenases were studied using hamsters administered 8% acetone in drinking water. Binding of aniline to liver microsomes induced a type II difference spectrum, and the spectral binding was enhanced in hamsters pretreated with acetone. Administration of acetone caused significant increases of cytochrome P-450 and cytochromeb ₅ contents in liver microsomes. The increases of the hemeproteins were associated with induction of monooxygenase activities toward test substrates, aniline, N-nitrosodimethylamine, benzphetamine, benzo(a)pyrene, and 7-ethoxycoumarin. In the kidneys, acetone administration increased microsomal contents of the hemeprotein and monooxygenase activities toward aniline, N-nitrosodimethylamine, and 7-ethoxycoumarin, but not benzphetamine or benzo(a)pyrene. In the lungs, acetone pretreatment increased aniline hydroxylase activity without affecting the levels of N-nitrosodimethylamine demethylase, cytochromes P-450 andb ₅. In marked contrast to the inductive effects in the liver, acetone administration markedly decreased lung microsomal benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase activities. Gel electrophoresis of liver and kidney microsomes from control and acetone-treated hamsters revealed that acetone treatment enhanced the intensity of a protein band(s) in the cytochrome P-450 molecular weight region. Immunoblotting of the microsomal proteins showed that the protein band induced by acetone in hamster liver, kidney and lung was cross-reactive with antibody raised against ethanol-inducible human liver cytochrome P-450. These results demonstrate that acetone has the ability to uniformly induce a specific form of cytochrome P-450, designated as IIE1, and to cause differential changes of monooxygenase activities in the hamster tissues. The complex effects of acetone on hepatic and extrahepatic monooxygenase systems may be important determinants of tissue-specific chemical toxicity.The nomenclature of P-450 used in this report follows the system recommended by Nebert et al. (1987, 1989). P-450 IIE1 has also been referred to as P-450ac by Patten et al. (1986), P-450j by Ryan et al. (1986), and as isozyme 3a and P-450_ALC by Coon and Koop (1987) in various species.     

卤酚化合物LM49对大鼠肝微粒体细胞色素P450的影响

目的研究LM49对大鼠肝微粒体蛋白及细胞色素P450含量的影响。方法将大鼠分成空白对照组、溶剂对照组、阳性对照组(苯巴比妥组、地塞米松组、β-萘黄酮组)、LM49低、中、高剂量组。给药后采用超速离心法制备大鼠肝微粒体;BCA法测定大鼠肝微粒体蛋白浓度;Omura and Sato法测定大鼠肝微粒体细胞色素P450的含量。结果 给予不同剂量的LM49后,大鼠肝微粒体的蛋白及细胞色素P450含量均明显降低。低、中、高剂量组与对照组比较差异有统计学意义(P<0.05)。结论 LM49对大鼠肝微粒体细胞色素P450具有一定的抑制作用,可能引起肝药酶对某些药物代谢的改变。     

Cytochrome P-450 and Drug-induced Spectral Interactions in the Hepatic Microsomes of Trout,Salmo trutta lacustris*

Abstract The properties of various components of a drug-oxidizing mono-oxygenase system in the liver of trout (Salmo trutta lacustris) were studied. The concentration of the cytochrome P-450 was about 7 nmol/g liver wet weight when measured in the 12,000 × g supernatant and 0.2 nmol/mg protein when measured in the “microsomal” fraction. The activity of NADPH-cytochrome c reductase was about 2000 nmol cyt. c reduced/g liver/min. measured in the homogenate and 20 nmol/mg protein/min. when measured in the “microsomal” fraction. The distribution of the cytochrome P-450 into different centrifugal fractions indicates that it is localized in the endoplasmic reticulum. The cytochrome P-450 present in the fish liver microsomal fraction was capable of interacting with different substances resulting in type II (aniline, n-octylamine, cyanide) and a reversed type I (n-butanol) spectra. Substances producing type I spectra in the rat liver microsomes, did not give any spectra (hexobarbital, SKF 525A) or gave unclassified spectral changes with the fish liver cytochrome P-450 (bromobenzene, DDT, piperonyl butoxide). Spectral dissociation constants were calculated for aniline and n-octylamine. Further experiments indicated that the mono-oxygenase system in the trout liver microsomes can metabolize aminopyrine, aldrin and 3,4-benzpyrene.     

对氨基二苯醚类似物抑制细胞色素P－450的定量构效关系

测定了一组对氨基二苯醚类似物延长小鼠戊巴比妥睡眠时间和体外抑制未经处理的小鼠肝微粒体催化氧化对硝基茴香醚脱甲基的活性。用逐步多元回归分析法导出了这些类似物体内和体外抑制细胞色素Ｐ－４５０（Ｐ－４５０）的活性与量化指数的定量构效关系（ＱＳＡＲ）。结果表明：对氨基二苯醚类似物体内和体外抑制Ｐ－４５０的活性均与最低未占据分子轨道能级（ＥＬＵＭＯ）、氨基氮原子的亲核超离域度（ＳＮ（Ｎ））和醚氧原子的亲核超离域度（ＳＮ（Ｏ））相关。这些类似物的代谢中间体（ＭＩ）与Ｐ－４５０形成Ｐ－４５０代谢中间体络合物（Ｐ－４５０－ＭＩ）可能是它们能够抑制Ｐ－４５０的主要原因。