Specific Cytochrome P450 Enzymes Research Articles

Expression, structure-function, and molecular modeling of vitamin D P450s.

Although vitamin D(3) is a natural product of a sunlight-mediated process in the skin, the secosteroid's biological function is dependent upon specific cytochrome P450 enzymes that mediate the parent vitamin's bioactivation and inactivation. Cytochrome P450C1 (CYP27B1) is the regulatory rate-limiting enzyme that directs the bioactivation process through introduction of a C-1alpha hydroxyl group. The resultant 1,25-dihydroxyvitamin D(3) (1,25D) is the biologically active secosteroid hormone that directs the multitude of vitamin D-dependent actions involved with calcium homeostasis, cellular differentiation and growth, and the immune response. The circulating and cellular level of 1,25D is regulated through a coordinated process involving the hormone's synthesis and degradation. Central to the degradation and turnover of 1,25D is the regulatory multi-catalytic cytochrome P450C24 (CYP24) enzyme that directs the introduction of C-24R groups onto targeted 25-hydroxy substrates. Discussed in this article is the action of the rat CYP24 to catalyze the side-chain oxidation and cleavage of 25-hydroxylated vitamin D metabolites. Expression and characterization of purified recombinant rat CYP24 is discussed in light of mutations directed at the enzyme's active site.

Role of specific cytochrome P450 enzymes in the N-oxidation of the antiarrhythmic agent mexiletine

1. Mexiletine is extensively metabolized in man by C- and N-oxidation and the aim of the present study was to characterize major cytochrome P450 enzyme(s) involved in the formation of N -hydroxymexiletine. 2. Incubations with genetically engineered microsomes indicated that the formation rate of N -hydroxymexiletine was highest in the presence of microsomes expressing high levels of either CYP1A2 or CYP2E1 and the formation of N -hydroxymexiletine by human liver microsomes was inhibited about 40% by antibodies directed against CYP1A1/1A2 or CYP2E1. Additional incubations demonstrated that formation of N -hydroxymexiletine was decreased 47 and 51% by furafylline, 40 µm and 120 µm, respectively, and decreased 55 and 67% by α -naphthoflavone, 1 µm and 3 µm, respectively (all p < 0.05 versus control). 3. The formation rate of N -hydroxymexiletine in human liver microsomes was highly correlated with CYP2B6 (RS -mexiletine, r = 0.7827; R - (−) -enantiomer, r = 0.7034; S -(+)-enantiomer, r = 0.7495), CYP2E1 (S -(+)-enantiomer, r = 0.7057) and CYP1A2 (RS -mexiletine, r = 0.5334; S -(+)-enantiomer, r = 0.6035). 4. In conclusion, we have demonstrated that CYP1A2 is a major human cytochrome P450 enzyme involved in the formation of N -hydroxymexiletine. However, other cytochrome P450 enzymes (CYP2E1 and CYP2B6) also appear to play a role in the N-oxidation of this drug.

Identification of human hepatic cytochrome P450 sources of N-alkylprotoporphyrin IX after interaction with porphyrinogenic xenobiotics, implications for detection of xenobiotic-induced porphyria in humans.

Porphyrinogenicity of certain xenobiotics depends upon mechanism-based inactivation of specific cytochrome P450 (P450) enzymes, followed by formation of N-alkylprotoporphyrin IX (N-alkylPP). Examination of the porphyrinogenicity of xenobiotics in animals and extrapolation of the results to humans is associated with ambiguity due, in part, to differences between P450 enzymes. The goal of this study was to develop an in vitro test for the detection of N-alkylPPs, produced in human liver after administration of xenobiotics found to be porphyrinogenic in animals. This goal was achieved using fluorometry to detect N-alkylPP formation following mechanism-based inactivation by porphyrinogenic xenobiotics of single cDNA-expressed human P450 enzymes in microsomes prepared from baculovirus-infected insect cells (Supersomes) and in human liver microsomes. The following combinations of P450 enzymes were major sources of N-alkylPPs in Supersomes: CYP3A4 [3-[(arylthio)-ethyl]sydnone (TTMS)]; CYP1A2 and 2C9 [3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC)]; and CYP2C9, 2D6, and 3A4 [allylisopropylacetamide (AIA)]. Whereas similarities were found between results with human enzymes in Supersomes and their rat orthologs in rat liver microsomes, some differences were found. The results with TTMS and AIA, but not with 4-ethyl DDC, were the same in individual human enzymes expressed in Supersomes and human liver microsomes. We conclude that some differences exist between human liver P450 enzymes and their rat P450 orthologs in liver microsomes. It would therefore be prudent when dealing with xenobiotics in which porphyrinogenicity depends upon N-alkylPP formation to supplement animal data with studies using human P450 enzymes.

Liver estradiol metabolism in rhesus monkeys with and without endometriosis and dioxin exposure

Objective: Recently, Rier et al. (2000) presented data showing that serum estradiol (E2) levels significantly decreased (p < 0.02) with exposure to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) in rhesus monkeys with and without endometriosis that were ovariectomized and receiving the same dose of E2 and progesterone replacement therapy. The purpose of this study was to determine whether these results could be explained by increased liver E2 metabolism, since TCDD is known to induce specific liver cytochrome P450 (CYP) enzymes, notably CYP1A1, that metabolize E2.

Antiepileptic Drugs Increase Plasma Levels of 4β-Hydroxycholesterolin Humans: EVIDENCE FOR INVOLVEMENT OF CYTOCHROME P450 3A4

The major cholesterol oxidation products in the human circulation are 27-hydroxycholesterol, 24-hydroxycholesterol, and 7alpha-hydroxycholesterol. These oxysterols are formed from cholesterol by specific cytochrome P450 enzymes, CYP27, CYP46, and CYP7A, respectively. An additional oxysterol present in concentrations comparable with 7alpha- and 24-hydroxycholesterol is 4beta-hydroxycholesterol. We now report that patients treated with the antiepileptic drugs phenobarbital, carbamazepine, or phenytoin have highly elevated levels of plasma 4beta-hydroxycholesterol. When patients with uncomplicated cholesterol gallstone disease were treated with ursodeoxycholic acid, plasma 4beta-hydroxycholesterol increased by 45%. Ursodeoxycholic acid, as well as the antiepileptic drugs, are known to induce cytochrome P450 3A. Recombinant CYP3A4 was shown to convert cholesterol to 4beta-hydroxycholesterol, whereas no conversion was observed with CYP1A2, CYP2C9, or CYP2B6. The concentration of 4alpha-hydroxycholesterol in plasma was lower than the concentration of 4beta-hydroxycholesterol and not affected by treatment with the antiepileptic drugs or ursodeoxycholic acid. Together, these data suggest that 4beta-hydroxycholesterol in human circulation is formed by a cytochrome P450 enzyme.

Evolution of benzoxazinone biosynthesis and indole production in maize.

The synthesis of a diverse spectrum of secondary metabolites has allowed plants to develop sophisticated chemical defense mechanisms. Maize (Zea mays L.), for example, releases a cocktail of volatile compounds when attacked by a caterpillar. These compounds attract a parasitic wasp, which deposits its eggs in the larvae, thereby controlling the population size of the herbivore. Indole, which is part of the cocktail, is produced by an enzyme recruited from primary metabolism. Indole can either function as a volatile signal or be converted by specific cytochrome P450 enzymes into benzoxazinoids, which function as important defense chemicals.

Cholesterol dynamics in the foetal and neonatal brain as reflected by circulatory levels of 24S‐hydroxycholesterol

Oxysterols, particularly those hydroxylated in the steroid side‐chain, are formed from cholesterol by specific cytochrome P450 enzymes and may facilitate elimination of cholesterol from extrahepatic sources. In humans, the greatest portion of circulating 24S‐hydroxycholesterol (24S‐OH‐Chol) is derived from the brain and the absolute concentration depends on age. In the present study, concentrations of 24S‐OH‐Chol and for comparison 27‐OH‐Chol were determined by a highly sensitive isotope dilution method using gas chromatography‐mass spectrometry in serum samples from normal preterm and term neonates and those with Rhesus haemolytic disease, taken serially for diagnostic purposes. Serum concentrations of cholesterol, 24S‐OH‐Chol and 27‐OH‐Chol were similar in venous versus arterial cord blood of 6 term neonates. Serum concentrations of 24S‐OH‐Chol and 27‐OH‐Chol in 12 small for gestational age (SGA) preterm neonates were significantly lower than those in 12 appropriate for gestational age (AGA) preterm neonates (p &lt; 0.001), and also lower than those in 12 SGA (p &lt; 0.001) and 12 AGA term neonates (p &lt; 0.05). Serum cholesterol was significantly higher in preterm than in term neonates (p &lt; 0.001). 24S‐OH‐Chol serially determined in 8 infants with Rhesus haemolytic disease increased 5‐6‐fold during the first 3 mo after birth (from 42 ± 20 ng ml−1 to 227 ± 71 ng ml−1). 27‐OH‐Chol increased simultaneously from 30 ± 14 ng m ml−1 to 100 ± 39 ng m ml−1. Conclusion: Serum concentrations of 24S‐OH‐Chol increased 5‐6‐fold after birth. This could be an indication of normal cholesterol metabolism in the developing neonatal brain.

Metabolic activation of benzo[c]phenanthrene by cytochrome P450 enzymes in human liver and lung.

The environmentally occurring polycyclic aromatic hydrocarbon (PAH) benzo[c]phenanthrene (B[c]PH) is a weak carcinogen in rodents. In contrast, the dihydrodiol-epoxides of B[c]PH are among the most carcinogenic PAH metabolites tested so far. In rodents, B[c]PH is predominantly metabolized to B[c]PH-5,6-dihydrodiol (B[c]PH-5,6-DH) and only to a minor extent to B[c]PH-3,4-DH, the proximate precursor of the highly potent ultimate carcinogen, B[c]PH-3,4-DH-1,2-epoxide. This might explain why in rodents B[c]PH is a weak carcinogen. However, little is known about human metabolism of B[c]PH. Using microsomal preparations from human liver and lung, we investigated the metabolic activation of B[c]PH. In contrast to the findings in experimental animals, human liver microsomes predominantly generated B[c]PH-3,4-DH and only to a minor extent B[c]PH-5,6-DH. Only one lung tissue sample was found to be metabolically active, producing B[c]PH-5,6-DH together with small amounts of B[c]PH-3,4-DH. Catalytic activities known to be associated with specific cytochrome P450 (P450) enzyme activities were determined and correlated with the spectrum of B[c]PH metabolites. The results indicate that B[c]PH-DH formation in human liver is mainly mediated by P450 1A2. Studies with P450 enzyme selective inhibitors confirmed these findings. Further support was obtained using preparations of the respective human recombinant P450 enzymes expressed in Escherichia coli and yeast. In addition to P450 1A2, P450 1B1 effectively mediated B[c]PH-metabolism. The umu-assay for induction of SOS repair response in Salmonella typhimurium TA 1535 pSK 1002 containing a umuC-lacZ reporter gene was used to study metabolic generation of genotoxic metabolites from B[c]PH-DHs in human microsomal preparations. B[c]PH-3,4-DH was activated by human liver microsomes to a potent genotoxic agent. Taken together, the results clearly demonstrate that human liver microsomes can effectively catalyze the biotransformation of B[c]PH into highly genotoxic metabolites. The results provide evidence that B[c]PH should be considered a potentially potent carcinogen in humans, and that rodent models may underestimate the risk.

Bone marrow contributes to renal parenchymal turnover and regeneration.

In order to establish whether extra-renal cells contribute to the turnover and repair of renal tissues, this study examined kidneys of female mice that had received a male bone marrow transplant and kidney biopsies from male patients who had received kidney transplants from female donors. By using in situ hybridization to detect Y-chromosomes it could be demonstrated that circulating stem cells frequently engraft into the kidney and differentiate into renal parenchymal cells. In the human renal grafts it was confirmed that some of the recipient-derived cells within the kidney exhibited a tubular epithelial phenotype, by combining in situ hybridization with immunostaining for the epithelial markers CAM 5.2 and the lectin Ulex europaeus. Female mouse recipients of male bone marrow grafts showed co-localization of Y-chromosomes and tubular epithelial markers Ricinus communis and Lens culinaris, and a specific cytochrome P450 enzyme (CYP1A2) indicating an appropriate functional capability of clustered newly formed marrow-derived tubular epithelial cells. Y-chromosome-containing cells were observed within glomeruli, with morphology and location appropriate for podocytes. Within the murine kidney, these Y-chromosome-positive cells were negative for the mouse macrophage marker F4/80 antigen and leukocyte common antigen, but were vimentin-positive. The presence of bone marrow-derived cells was noted in both histologically normal mouse kidneys and in human transplanted kidneys suffering damage from a variety of causes. These data indicate that bone marrow cells contribute to both normal turnover of renal epithelia and regeneration after damage, and it is suggested that this could be exploited therapeutically.

Variable cytochrome P450 2D6 expression and metabolism of codeine and other opioid prodrugs: implications for the Australian anaesthetist.

Codeine is a popular opioid prodrug dependent on the activity of the specific cytochrome P450 enzyme 2D6 (CYP2D6). This enzyme catalyses the production of the potent analgesic metabolite morphine, but genetic studies have demonstrated that individuals from different ethnic groups exhibit considerable variability in the functional capacities of their expressed CYP2D6 enzymes, and pharmacological studies have shown many commonly prescribed drugs can reduce the action of CYP2D6 enzymes. These findings have significant clinical implications for the rational prescription of effective analgesia, especially in a multicultural country like Australia.

Cytochrome P4503A-Dependent Metabolism of Tocopherols and Inhibition by Sesamin

Carboxychroman metabolites of the major dietary tocopherols are excreted in human urine, but the mechanism of their synthesis is unknown. We employed well-characterized inhibitors of specific cytochrome P-450 (CYP) enzymes to determine which form was likely involved in tocopherol side chain oxidation. Ketoconozole (1.0 μM), a potent and selective inhibitor of CYP3A, substantially inhibited metabolism of γ- and α-tocopherol in rat primary hepatocytes, and metabolism of γ- and δ-tocopherol in HepG2/C3A cells. Sulphaphenazole and cyclosporin, inhibitors of CYP2C and CYP27, respectively, were without effect. Sesamin, a sesame lignan that causes elevation of tissue tocopherol concentration in rats, strongly inhibited tocopherol metabolism by HepG2/C3A cells at 1.0 μM. These results support a CYP3A-dependent mechanism of side chain metabolism of tocopherols to water-soluble carboxychromans, and provide the first evidence of a specific enzyme involved in vitamin E metabolism. The data further suggest that sesamin increases tissue tocopherol concentration by inhibiting tocopherol catabolism.

Using cytochrome P-450 gene knock-out mice to study chemical metabolism, toxicity, and carcinogenicity.

Cytochrome P-450 (CYP) enzymes are heme-containing proteins that carry out oxidative metabolism of a wide range of structurally diverse exogenous chemicals and therapeutic agents as well as endogenous compounds. For some of these xenobiotics, oxidative metabolism results in the formation of toxic, mutagenic, or carcinogenic metabolites. In the past, the role of CYP enzymes in metabolism and chemical-induced toxicity was studied indirectly through use of specific antibodies or inducers and inhibitors of these enzymes. Progress in molecular biology and the ability to bioengineer animal models that do not express CYP1A2, CYP1A1, CYP1B1, CYP2E1, or both CYP1A2 and CYP2E1 isozymes has allowed for direct investigations of the in vivo role of these enzymes in the metabolism, toxicity, and carcinogenicity of xenobiotics. This article reviews research conducted to date that utilizes these genetically bioengineered mice in metabolism, toxicity, or carcinogenicity studies of chemicals. Some studies showed a positive correlation between in vivo results and in vitro predictions for the role of a specific CYP in chemical-induced effects, whereas other studies did not support in vitro predictions. Work reviewed herein demonstrates the importance of using animal models for investigating the role of specific CYP enzymes in metabolism and chemical-induced toxicity or carcinogenicity rather than relying solely on in vitro techniques. Eventually, studies of this nature will facilitate a more accurate assessment of human risks with regard to chemicals by helping us to understand the relationships between chemical metabolism, carcinogenicity, and polymorphisms in CYP enzymes.

Tissue-specific synthesis and oxidative metabolism of estrogens.

Estrogen exposure represents the major known risk factor for development of breast cancer in women and is implicated in the development of prostate cancer in men. Human breast tissue has been shown to be a site of oxidative metabolism of estrogen due to the presence of specific cytochrome P450 enzymes. The oxidative metabolism of 17beta-estradiol (E2) to E2-3,4-quinone metabolites by an E2-4-hydroxylase in breast tissue provides a rational hypothesis to explain the mammary carcinogenic effects of estrogen in women because this metabolite is directly genotoxic and can undergo redox cycling to form genotoxic reactive oxygen species. In this chapter, evidence in support of this hypothesis and of the role of P4501B1 as the 4-hydroxylase expressed in human breast tissue is reviewed. However, the plausibility of this hypothesis has been questioned on the grounds that insufficient E2 is present in breast tissue to be converted to biologically significant amounts of metabolite. This critique is based on the assumption that plasma and tissue E2 levels are concordant. However, breast cancer tissue E2 levels are 10-fold to 50-fold higher in postmenopausal women than predicted from plasma levels. Consequently, factors must be present to alter breast tissue E2 levels independently of plasma concentrations. One such factor may be the local production of E2 in breast tissue through the enzyme aromatase, and the evidence supporting the expression of aromatase in breast tissue is also reviewed in this chapter. If correct, mutations or environmental factors enhancing aromatase activity might result in high tissue concentrations of E2 that would likely be sufficient to serve as substrates for CYP1B1, given its high affinity for E2. This concept, if verified experimentally, would provide plausibility to the hypothesis that sufficient E2 may be present in tissue for formation of catechol metabolites that are estrogenic and which, upon further oxidative metabolism, form genotoxic species at levels that may contribute to estrogen carcinogenesis.

Cytochromes P-450 catalyze the formation of marchantins A and C in Marchantia polymorpha

Two specific cytochrome P-450 enzymes were detected in cell suspension cultures of Marchantia polymorpha; the first catalyzes the coupling of two molecules of lunularic acid to form marchantin C and CO 2 and the second hydroxylates marchantin C to marchantin A. Cell free experiments using 3 H/ 14 C doubly-labeled substrates demonstrated that lunularic acid, and neither lunularine nor prelunularic acid, is the sole substrate for the coupling reaction. Both enzymes are dependent on the presence of oxygen and NADPH. Both reactions were inhibited in the presence of CO in the dark; this inhibition was partially reversed by white light. In addition, both reactions were inhibited by typical inhibitors of cytochrome P-450 enzymes such as cytochrome c and ancymidol.

Pisatin demethylation by fungal pathogens and nonpathogens of pea: association with pisatin tolerance and virulence

Previous studies have indicated that detoxification of their hosts' phytoalexins is a tolerance mechanism for some true fungi, but not the fungus-like Oomycota, and may be involved in determining the virulence of a pathogen. In the present study, the associations between demethylation of the pea phytoalexin pisatin, tolerance to pisatin, and virulence on pea were examined for 50 fungal isolates which represent 17 species of pathogens and nonpathogens of pea. All isolates ofPythium coloratum and P. irregulare failed to metabolize and were sensitive to pisatin, consistent with previous observations that members of the Oomycota generally lack the ability to metabolize and are sensitive to their hosts' phytoalexins. Among true fungi tested, the ability to demethylate pisatin was common, regardless of whether the particular isolate was pathogenic on pea or not. However, when the rate of pisatin demethylation was compared to virulence, all but one of the moderate to highly virulent isolates rapidly demethylated pisatin. In addition, the more rapidly demethylating isolates were generally more tolerant of pisatin. These results suggest that a specialized enzyme system for quickly detoxifying pisatin might be present in most pea pathogens. In previous studies a specific cytochrome P450 enzyme for demethylating pisatin was identified in the pea pathogen Nectria haematococca mating population VI, and genes (PDA genes) encoding that enzyme have been cloned from this fungus. When DNA specific for these genes was used to probe genomic DNA from other fungi that demethylate pisatin, significant hybridization was detected with only one fungus, the pea pathogen Fusarium oxysporum f. sp. pisi. If the other pea pathogens possess a specific cytochrome P450 system for detoxification of pisatin, the genes encoding these enzymes apparently share limited nucleotide similarity with N. haematococca PDA genes.

Identification of the human cytochrome P450 enzymes involved in the in vitro metabolism of artemisinin.

The study aimed to identify the specific human cytochrome P450 (CYP450) enzymes involved in the metabolism of artemisinin. Microsomes from human B-lymphoblastoid cell lines transformed with individual CYP450 cDNAs were investigated for their capacity to metabolize artemisinin. The effect on artemisinin metabolism in human liver microsomes by chemical inhibitors selective for individual forms of CYP450 was investigated. The relative contribution of individual CYP450 isoenzymes to artemisinin metabolism in human liver microsomes was evaluated with a tree-based regression model of artemisinin disappearance rate and specific CYP450 activities. The involvement of CYP2B6 in artemisinin metabolism was demonstrated by metabolism of artemisinin by recombinant CYP2B6, inhibition of artemisinin disappearance in human liver microsomes by orphenadrine (76%) and primary inclusion of CYP2B6 in the tree-based regression model. Recombinant CYP3A4 was catalytically competent in metabolizing artemisinin, although the rate was 10% of that for recombinant CYP2B6. The tree-based regression model suggested CYP3A4 to be of importance in individuals with low CYP2B6 expression. Even though ketoconazole inhibited artemisinin metabolism in human liver microsomes by 46%, incubation with ketoconazole together with orphenadrine did not increase the inhibition of artemisinin metabolism compared to orphenadrine alone. Troleandomycin failed to inhibit artemisinin metabolism. The rate of artemisinin metabolism in recombinant CYP2A6 was 15% of that for recombinant CYP2B6. The inhibition of artemisinin metabolism in human liver microsomes by 8-methoxypsoralen (a CYP2A6 inhibitor) was 82% but CYP2A6 activity was not included in the regression tree. Artemisinin metabolism in human liver microsomes is mediated primarily by CYP2B6 with probable secondary contribution of CYP3A4 in individuals with low CYP2B6 expression. The contribution of CYP2A6 to artemisinin metabolism is likely of minor importance.

Cis-acting sequences from the rat cytochrome P450 2B1 gene confer pulmonary and phenobarbital-inducible expression in transgenic mice.

Specific cytochrome P450 enzymes show tissue-specific induction, and different regulatory units for expression of these enzymes have been identified. The regulation of the phenobarbital (PB)-inducible P450 genes has been relatively well characterized in terms of PB induction, but less so with regard to tissue-specific expression. CYP2B2 is not expressed in the rat lung, whereas cytochrome P450 2B1 (CYP2B1) is a dominating enzyme in the same tissue. The constitutive expression of CYP2B1 and CYP2B2 in liver is low, but inducible by PB, whereas the pulmonary expression of CYP2B1 is not induced by PB. This indicates utilization of different regulating mechanisms in the two organs. A gene construct consisting of the structural gene for LacZ coupled to a 1.3-kb 5' fragment of the rat CYP2B1 gene was used to generate transgenic mice in order to further elucidate the mechanism behind tissue-specific expression and PB induction of the CYP2B1 gene. Using reverse transcriptase-polymerase chain reaction on total RNA extracted from lung and liver tissue, a lung-specific transcription of the transgene was observed. Transcription of the construct was also observed in livers from PB-treated transgenic animals. By histochemical staining of lung sections with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal), we demonstrated expression at the protein level in bronchiolar cells. In conclusion, our results revealed that the region extending to -1. 3 kb in the 5' flanking region of the CYP2B1 gene included sequences that could partly account for the lung-specific transcription of CYP2B1 and the hepatic induction of CYP2B1 transcription by PB.

Dietary polyunsaturated fatty acids and hepatic gene expression.

Dietary polyunsaturated fatty acids (PUFA) have profound effects on hepatic gene transcription leading to significant changes in lipid metabolism. PUFA rapidly suppress transcription of genes encoding specific lipogenic and glycolytic enzymes and induce genes encoding specific peroxisomal and cytochrome P450 (CYP) enzymes. Using the peroxisome proliferator-activated receptor alpha (PPAR alpha)-null mouse, we showed that dietary PUFA induction of acyl CoA oxidase (AOX) and CYP4A2 require PPAR alpha. However, PPAR alpha is not required for the PUFA-mediated suppression of fatty acid synthase (FAS), S14, or L-pyruvate kinase (L-PK). Studies in primary rat hepatocytes and cultured 3T3-L1 adipocytes showed that metabolites of 20:4n-6, like prostaglandin E2 (PGE2), suppress mRNA encoding FAS, S14, and L-PK through a Gi/Go-coupled signal transduction cascade. In contrast to adipocytes, 20:4n-6-mediated suppression of lipogenic gene expression in hepatic parenchymal cells does not require cyclooxygenase. Transfection analysis of S14CAT fusion genes in primary hepatocytes shows that peroxisome proliferator-activated PPAR alpha acts on the thyroid hormone response elements (-2.8/-2.5 kb). In contrast, both PGE2 and 20:4n-6 regulate factors that act on the proximal promoter (-150/-80 bp) region, respectively. In conclusion, PUFA affects hepatic gene transcription through at least three distinct mechanisms: (i) a PPAR-dependent pathway, (ii) a prostanoid pathway, and (iii) a PPAR and prostanoid-independent pathway. PUFA regulation of hepatic lipid metabolism involves an integration of these multiple pathways.

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.

Production of Reactive Oxygen Species by Microsomes Enriched in Specific Human Cytochrome P450 Enzymes

Few studies have evaluated the production of reactive oxygen intermediates by human microsomes, especially the influence of the specific form of cytochrome P450. Experiments were carried out to evaluate the ability of CYP1A1, 1A2, 2B6, and 3A4 to consume NADPH, reduce iron, and catalyze production of reactive oxygen species. Microsomes enriched in each of these CYPs were obtained from commercial ± lymphoblast cells that had been transfected with cDNA encoding the specific human CYP. On a per nanomole cytochrome P450 basis, CYP3A4 was the most active P450 evaluated in catalyzing NADPH oxidation, production of superoxide anion radical, NADPH-dependent chemiluminescence, oxidation of dichlorofluorescein diacetate, and reduction of either ferric-EDTA or ferric-citrate. CYP1A1 was the next most reactive CYP, whereas CYP1A2 and 2B6 displayed a comparable, lower activity. Nitric oxide, which reacts with and inactivates hemoproteins, inhibited superoxide production by all the CYPs to a similar extent. Because CYP3A4 is present in high amounts in human liver microsomes and is active in catalyzing the formation of reactive oxygen species, this CYP may make an important contribution in the overall ability of human liver microsomes to generate active oxygen species.