Whether cytochrome p450 induction accompanies glucuronosyl and glutathione transferase induction by isomers of dipyridyl appears unrelated to dose and iron chelation properties

Administration of 4, 4′dipyridyl to rats induces the activities of xenobiotic transferases (phase II drug metabolizing enzymes), UDP-glucuronosyl-tranferase and glutathione-S-transferase, and also the concentration and activity of cytochrome P450 (a phase I drug metabolizing enzyme). 2, 2′Dipyridyl, an isomer possessing iron chelation properties, only induces the phase II enzymes. Although the magnitude of the phase II induction by 2, 2′dipyridyl increases with increasing dosages, the selective induction of only phase II activities remains inviolate. Co-administration of 2, 2′dipyridyl does not prevent 4, 4′dipyridyl from inducing cytochrome P450, suggesting that the iron chelation property is not the factor that precludes 2, 2′dipyridyl from coordinately inducing cytochrome P450 with the transferases.     

Interleukin-1 (IL-1) depresses cytochrome P450 levels and activities in mice

Endotoxin depresses cytochrome P450 levels when injected into animals. The purpose of this study was to determine whether endotoxin itself, or monokine(s) released in response to endotoxin administration are responsible for this effect. Cytochrome P450 levels and drug metabolizing activities were measured in endotoxin resistant C3H/HeJ mice 24h after single intraperitoneal injections of either lipopolysaccharide (LPS), a semipurified murine monokine preparation containing interleukin-1 (IL-1), or murine recombinant IL-1. In endotoxin sensitive C3H/HeN mice, LPS (0.5 mg/Kg) decreased total cytochrome P450 levels, benzphetamine demethylase activities, and ethoxyresorufin-0-deethylase activities. This dose of LPS did not alter cytochrome P450 levels or activities in the C3H/HeJ mice. However, after injection of the semipurified monokine preparation or the recombinant IL-1, there were significant decreases in cytochrome P450 levels and activities similar to the decreases observed with LPS in the C3H/HeN mice. These findings suggest that the alterations in hepatic cytochrome P450 seen with endotoxin injection are mediated, at least in part, by IL-1.     

Defining the in Vivo Role for cytochrome b5 in cytochrome P450 function through the conditional hepatic deletion of microsomal cytochrome b5

In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50-90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner.     

The drug metabolism systems of liver and liver tumors: A comparison of activities and characteristics

Summary Transplantable rat liver tumors 5123 t.c., 7288 ct.c., 5123 t.c.(H) and the Novikoff hepatoma have active mixed function oxidase systems capable of metabolizing a variety of drug and polycyclic hydrocarbon substrates. The tumor drug metabolism systems are at best 20% as active as rat liver. The tumor drug metabolism activities are induced by pretreatment with phenobarbital or -naphthoflavone and can be inhibited with specific inhibitors such as carbon monoxide or 7,8-benzoflavone. Tumor drug metabolism systems appear to consist of cytochrome P-450 and cytochrome P-450 reductase. The properties of the two protein components from tumors are highly similar to the corresponding components of the liver drug metabolism system.Cytochrome P-450 reductase has been at least partially purified from the Novikoff hepatoma and hepatoma 5123 t.c.(H). The kinetic and physical properties of the tumor reductases are similar to those of the liver reductase except that the K_m of hepatoma 5123 t.c.(H)     

Microsomal omega-hydroxylated metabolites of N-arachidonoyl dopamine are active at recombinant human TRPV1 receptors

N-Arachidonoyl dopamine (NADA) is an endogenous lipid that modulates signal transduction in neuronal and immune pathways. NADA activates the non-selective cation channel, transient receptor potential vanilloid type 1 (TRPV(1)) and cannabinoid receptor 1. That NADA is comprised of an arachidonic acid (AA) backbone suggests that it may be metabolized through many of the enzymes that act upon AA such as the other AA-derived signaling lipids, the endogenous cannabinoids. To investigate the metabolism of NADA through the cytochrome P450 (CYP450) metabolic pathway, we studied the in vitro rat liver microsomal production of hydroxylated metabolites and their activity at recombinant human TRPV(1) receptors. We showed that following microsomal activation in the presence of NADA, omega and (omega-1) hydroxylated metabolites (19- and 20-HETE-DA) were formed. These metabolites were active at recombinant human TRPV(1) receptors, inducing a dose-dependent calcium influx. Both metabolites exhibited lower potency compared to NADA. We conclude that CYP450 enzymes are capable of metabolizing this signaling lipid forming a larger family of potential neuromodulators.     

Flavin-containing monooxygenase activity in camel tissues: comparison with rat and human liver enzymes

We previously reported the occurrence of multiple forms of drug metabolizing enzymes in camel tissues. In this study, we demonstrated for the first time, flavin-containing monooxygenase (FMO)-dependent metabolism of two model substrates methimazole (MEM) and N,N'-dimethylaniline (DMA) by camel liver, kidney, brain and intestine. FMO-catalyzed metabolism in the microsomes of camel tissues was independent of cytochrome P450 (CYP) activity and exhibited a pH and temperature dependence characteristic of FMO enzymes. Use of inhibitors of CYP activities, SKF525A, octylamine or antibody against NADPH-P450 reductase, did not significantly alter the FMO-dependent substrate metabolism. Using MEM as a model substrate for FMO activity, we show that camel liver has an activity similar to that in rat and human livers. MEM metabolism in extrahepatic tissues in camels was significantly lower (60%-80%) than that in liver. Our results suggest occurrence of FMO in camel tissues, with catalytic properties similar to those in rat and human livers. These results may help in better understanding the effects of pharmacologically and toxicologically active compounds administered to camels.     

Chemopreventive and therapeutic modulation of green tea polyphenols on drug metabolizing enzymes in 4-Nitroquinoline 1-oxide induced oral cancer

Oral cancer is one of the most common cancers in the world. Drugs can modulate the expression of drug metabolizing enzymes and are useful in chemoprevention as well as therapy in cancer. 4-Nitroquinoline 1-oxide (4-NQO) is used to induce oral cancer in the present study. In the present investigation, the effect of green tea polyphenols (GTP) on the activities of cytochrome b5, cytochrome P450, cytochrome b5 reductase (cyt b5 R), cytochrome P450 reductase (cyt P450 R), arryl hydrocarbon hydroxylase (AHH), DT-diaphorase (DTD)(Phase I enzymes) and glutathione-S-transferase (GST) and UDP-glucuronyl transferase (UDP-GT) (Phase II enzymes) were assessed in tongue and oral cavity. In induced rats, there was a decrease in the activity of Phase II enzymes and an increase in the activity of Phase I enzymes. On supplementation of GTP by both simultaneous and post treatment mode (200mg/kg) there was a significant increase in the activity of GST and UDP-GT and a significant decrease in the activity of Phase I enzymes. There was a significant decline in the number of tumors, tumor volume and oral squamous cell carcinoma in both simultaneous and post GTP treated animals relative to 4-NQO induced animals; on comparing simultaneous and post GTP treated animals the number of tumors, tumor volume and oral squamous cell carcinoma was significantly reduced in post treated animals. Thus inhibition of Phase I enzymes could be attributed to the protective efficacy of GTP which deactivates carcinogen and GTP induced the expression of Phase II enzymes that detoxifies the 4-NQO. It can be proposed that GTP plays role as a detoxifying agent by which its modulating role prevented/inhibited the formation of tumor.     

Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.     

Metabolism of benzo(a)pyrene,dimethylbenzanthracene and aflatoxin B1 by camel liver microsomes

The ability of camel liver microsomes to metabolise a range of common environmental carcinogens including benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 has been investigated. The camel liver has shown the ability to metabolise benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 to a number of metabolites. The major metabolites of benzo(a)pyrene produced by camel liver enzymes were identified as its mono-hydroxy derivatives and suggest that the metabolic detoxification pathways of carcinogen metabolism are predominant in this species. Benzo(a)pyrene metabolising activity in camel liver required NADPH and was inhibited by CO and alpha-naphthoflavone suggesting the involvement of cytochrome P450 in the metabolism of this carcinogen by camel liver. The cytochrome P450-dependent metabolism of carcinogen and other specific substrates such as ethoxyresorufin and ethoxycoumarin, by camel liver enzymes, was about 50% higher than that of rat liver enzymes. The cytochrome P450-dependent metabolism of a variety of carcinogenic and other substrates by camel liver demonstrated that there are multiple forms of cytochrome P450 enzymes involved in the metabolism of a wide array of xenobiotics and pollutants.     

Inhibitory effect of propylthiouracil-induced hypothyroidism in rat on oxidative drug metabolism

The effect of propylthiouracil (PTU) pretreatment on in vivo and in vitro oxidative drug metabolism was determined in the rat. Whereas pentobarbital sleeping time (PBST) and zoxazolamine paralysis time (ZZPT) were used as indices of in vivo drug metabolizing activity, biotransformation of aminopyrine and aniline by hepatic microsomal preparations were used as indices of in vitro drug metabolizing enzymes activities. PTU pretreatment significantly prolonged both PBST and ZZPT. Whereas PTU did not affect microsomal protein concentration or cytochrome P-450 content, it significantly decreased microsomal cytochrome c reductase and aniline hydroxylase activities. These changes in enzymatic activities were observed in microsomal preparations from either non-fasted or 24-hr fasted rats. Our results suggest that PTU-induced hypothyroidism modifies the metabolism and effectiveness or toxicity of concomitantly administered drugs.     

Treatment with poly I.C. enhances lipid peroxidation and the activity of xanthine oxidase, and decreases hepatic P-450 content and activities in mice and rats

Treatment of mice and rats with polyriboinosinic acid-polyribocytidylic acid (poly I.C., 5 mg/kg i.p.), a potent interferon inducer, decreased hepatic cytochrome P-450 system content and activities without influencing P-450-independent xenobiotic metabolizing enzymes. Treatment with poly I.C. decreased the content of P-450 by 28% in mice (P less than 0.05) and 30% in rats (P less than 0.05) but did not alter the activity of cytochrome c reductase. With treatment of poly I.C., the activity of XO increased 87% in mice (P less than 0.01) and 30% in rats (P less than 0.01). Lipid peroxidation was enhanced by 82% in mice (P less than 0.01) and 95% in rats (P less than 0.05). These results raise the possibility that a part of the depression of P-450 system content and activities by poly I.C. might be caused by enhanced lipid peroxidation associated with increased activity of XO.     

In vivo and in vitro effects of a new macrolide antibiotic roxithromycin on rat liver cytochrome P-450: comparison with troleandomycin and erythromycin

The effects of a new macrolide antibiotic (Roxithromycin) and one of its major metabolite (RU 39001) on rat hepatic drug metabolizing enzymes were compared to those of erythromycin, erythralosamine and troleandomycin (TAO) both in vitro and in vivo. In contrast to erythromycin, erythralosamine and TAO, roxithromycin and its metabolite RU 39001 exhibit: (i) a very poor affinity for rat liver cytochrome P-450, (ii) an unability to be metabolized into a stable inhibitory metabolite-cytochrome P-450 complex and (iii) a decreased ability to induce liver cytochrome P-450 PCNE, an isozyme implicated in drug associations involving some macrolide antibiotics.     

Modulatory Effect of Taurine on 7,12‐Dimethylbenz(a)Anthracene‐Induced Alterations in Detoxification Enzyme System,Membrane Bound Enzymes,Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue

The modulatory effect of taurine on 7,12‐dimethylbenz(a)anthracene (DMBA)‐induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na⁺/K⁺ATPase, Ca²⁺ATPase, and Mg²⁺ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione‐S‐transferase and UDP‐glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA‐induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA‐induced breast cancer.     

Influence of 3,5,3'-triiodo-L-thyronine on the hepatic enzyme activities responsible for the metabolism of xenobiotics during the development of the chick embryo

The influence of thyroid hormones on microsomal drug metabolizing enzymes was studied in hypothyroid newborn rats and chick embryos. Administration of 3,5,3'-triiodo-L-thyronine strongly decreased the microsomal cytochrome P 450 content in hypothyroid new-born rats and thus could render the rat pup more susceptible to hepatotoxicity from drugs. The drug metabolizing system in 20 days old chick embryos was less sensitive to the effects of thyroid hormone, but administration of phenobarbital was accompanied by a strongly induction effect on microsomal enzyme activities.     

Cytochrome P450 Enzymes and Drug Metabolism—Basic Concepts and Methods of Assessment

1. The cytochrome P450 enzyme family is one of the major drug metabolizing systems in man.2. Factors such as age, gender, race, environment, and drug treatment may have considerable influence on the activity of these enzymes.3. There are now well-established in vitro techniques for assessing the role of specific cytochrome P450 enzymes in the metabolism of drugs, as well as the inhibitory or inducing effects of drugs on enzyme activity. In vitro data have been utilized to predict clinical outcomes (i.e., pharmacokinetic interactions), with close correlations between in vitro and in vivo data.4. This information can be of considerable practical assistance to clinicians, to help with rational prescribing or to prevent or minimize the potential for drug interactions.     

细胞色素P450介导的果蝇抗药性研究进展

细胞色素P450 (cytochrome P450, CYP450)超基因家族是由一些数量多而功能复杂的血红蛋白酶基因所组成,该代谢酶系作为一种几乎地球上所有需氧生物都存在的重要生存策略,可以调控多种内源物质及外源化合物的代谢,参与了众多重要的生命过程,代谢解毒作用是该酶系重要功能之一。细胞色素P450的代谢解毒作用受药物影响,机体通过改变基因表达量,实现增强代谢解毒,加快机体对于有害物质的代谢,从而使得机体对有害环境产生一定的适应性,进而使得机体产生耐药性或抗药性。本研究说明果蝇细胞色素P450介导的杀虫剂类药物代谢机制及代谢抗性的特点等方面的研究,对明确果蝇的抗药性机制研究具有参考意义。     

Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC(50) values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z' factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format.     

Quantitative whole-cell cytochrome P450 measurement suitable for high-throughput application

The recombinant expression of cytochrome P450 enzymes involved in drug metabolism is of interest to the pharmaceutical and biotechnological industries due to the versatile catalytic properties of these enzymes. Accurate quantification of cytochrome P450 enzymes expressed in bacterial culture generally depends on disruption and fractionation of cells to prepare membranes for spectral analysis. Although whole-cell methods for spectral determination have been reported, problems with poor reproducibility and low signal-to-noise ratio confound the use of such techniques where P450 hemoprotein expression levels are relatively low, such as in cultures of certain mammalian forms. In particular, interference from bacterial hemoproteins often obscures the P450 peak. In the current study, the combination of culture concentration, incubation under microaerobic conditions, and a modified method of baseline correction enabled reproducible quantification of cytochrome P450s in whole cells. This whole-cell method is well suited to high-throughput application, as large sets or libraries of enzymes can be expressed in parallel and relative expression levels measured without downstream cell processing.