Different Forms Of Cytochrome P-450 Research Articles

The effect of some benzimidazoles on the disposition of antipyrine and tolbutamide from the rat isolated perfused liver.

The anthelmintic benzimidazoles, mebendazole, albendazole and flubendazole have been screened for any propensity to alter the disposition of antipyrine and tolbutamide in the rat isolated perfused liver preparation. The benzimidazoles were added as a 2.5 mg bolus dose into the perfusate reservoir 5 min before the administration of either antipyrine or tolbutamide. Neither mebendazole or albendazole produced any significant effect on the pharmacokinetics of either of the substrate drugs. In contrast, flubendazole significantly decreased the clearance of antipyrine (by 40%) indicating inhibition of mixed function oxidase activity. However, flubendazole did not alter the disposition of tolbutamide. The results suggest that not all benzimidazoles inhibit hepatic drug metabolizing enzymes and that different forms of cytochrome P-450 are involved in the metabolism of antipyrine and tolbutamide.

My passion for extreme conditions to solve the cytochrome P450 and NO synthase reaction mechanisms

Cytochrome P450, and especially its reaction mechanism, has always been a major research interest of Professor I.C. Gunsalus. The reaction cycle of this enzyme is complex, containing many elementary steps and intermediates, and constitutes a challenge for the scientific community. During his repeated stays in our laboratories in Paris and in Montpellier, he contributed decisively to our study of the P450 reaction mechanism under extreme conditions, i.e., at subzero temperatures and at high pressure. From this initial impulse, we continued the work with different forms of cytochrome P450 and later on with nitric oxide synthase. This paper gives an overview of the insights into these enzymes gained by the use of extreme conditions. These exciting achievements were initiated by numerous discussions with Professor Gunsalus and also reflect a long-lasting collaboration and friendship.

Pharmacogenetics: an opportunity for a safer and more efficient pharmacotherapy.

Drug treatment is in many cases ineffective. Besides patients who do not respond to the treatment despite receiving expensive drugs, adverse drug reactions (ADRs) as a consequence of the treatment, is estimated to cost the US society 100 billion USD and over 100,000 deaths per year. Pharmacogenetics is the discipline which takes the patient's genetic information of drug transporters, drug metabolizing enzymes and drug receptors into account to allow for an individualized drug therapy leading to optimal choice and dose of the drugs in question. It is believed that much cost for the society can be saved in this manner. Many drug transporters are polymorphic. In addition, the majority of phase I and phase II dependent drug metabolism is carried out by polymorphic enzymes which can cause abolished, quantitatively or qualitatively altered or enhanced drug metabolism. Stable duplication, multiduplication or amplification of active genes, most likely in response to dietary components that have resulted in a selection of alleles with multiple noninducible genes, has been described. Several examples exist where subjects carrying certain alleles suffer from a lack of drug efficacy because of ultrarapid metabolism caused by multiple genes or by induction of gene expression, or, alternatively, adverse effects from the drug treatment as a result of the presence of defective alleles. The information about the role of polymorphic drug receptors for efficiency of drug therapy is more scarce, although promising examples are seen in drug treatment of asthma where the efficiency can be severely enhanced by predictive genotyping of the drug targets. In addition, certain polymorphic genes can be used as markers for optimization of the drug therapy. It is likely that predictive genotyping is of benefit in 10-20% of drug treatment and thereby allows for prevention of causalities as a cause of ADRs and thus improves the health for a significant fraction of the patients. In 15-40% of the cases, the penetrance of genetic polymorphism is of less importance because of the polygenic influence on the outcome of drug treatment and in 50% of the cases, pharmacogenetics would be without influence because of other more important physiological and environmental factors. In the present contribution an overview about our present knowledge how polymorphic genes can influence the drug efficacy is presented. Some emphasis will be given to different forms of cytochrome P450 which are of importance for drug metabolism.

Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase

We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium ( S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY1002/3A4, which express respective human P450 enzymes and NADPH-cytochrome P450 reductase (reductase) and bacterial O-acetyltransferase ( O-AT). These strains were established by introducing two plasmids into S. typhimurium TA1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double tac promoter and the other, pOA102, carrying O-AT and umuC′′ lacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 125 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5- f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 3-Amino-1,4-dimethyl-5 H-pyrido[4,3- b]-indole and 3-amino-1-methyl-5 H-pyrido[4,3- b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B 1 exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. β-Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrom P450 enzyme involved in bioactivation of HCAs.

Enhanced expression of CYP1B1 in Escherichia coli

Conditions for the optimal expression of the human CYP1B1 hemoprotein in Escherichia coli have been investigated. CYP1B1 cDNA was prepared from a retinal cDNA template and used to generate cDNA fragments with modified 5′-sequences reported to allow enhanced expression in E. coli DH5α. Plasmids were constructed, using the pCWori+ expression vector and were used to examine necessity for thiamine, δ-aminolevulinic acid (ALA), and IPTG. The optimal shaking speed in an orbital incubator was 150 rpm at 30°C. Higher speeds resulted in increased cell death and lower speeds resulted in lower expression of cytochrome P450. IPTG was necessary for this expression system, which makes use of the lac repressor, but levels above 0.5 mM were without additional benefit. We were able to show thiamine to be unnecessary in this expression system, although included by others expressing CYP1B1. ALA has been reported to enhance expression of several different forms of cytochrome P450. We examined the dependence of CYP1B1 expression on ALA. The expression proved to be highly dependent upon this heme precursor, with levels of CYP1B1 increasing ∼20-fold, to 920 nmol/l in the presence of up to 2.5 mM ALA. The question of whether heme synthesis and apoprotein synthesis were coupled was then investigated. It could be shown that although heme synthesis was not limiting (CYP101 holoenzyme expression in the absence of ALA was four times higher than the ALA-supported CYP1B1 holoenzyme expression), it was necessary for optimal expression of CYP1B1. CYP1B1 protein synthesis appears to be coupled to heme precursor availability, as seen by SDS–PAGE, because in the absence of heme precursor apocytochrome P450 1B1 does not accumulate.

Cytochrome P450 monooxygenase system in echinoderms

The results of a limited number of studies on echinoderms provide evidence for the presence of a cytochrome P450 monooxygenase system in representatives of three classes of the phylum Echinodermata: the asteroids (sea stars), holothuroids (sea cucumbers) and echinoids (sea urchins). The monooxygenase system has been demonstrated to be involved in the metabolism of xenobiotic compounds, but is assumed to have its primary function in the metabolism of endogenous substrates, such as steroids. Available data on P450 cofactor requirement, P450-dependent metabolism of benzo[a]pyrene, studies with classical inhibitors of P450, specificity of P450 induction by planar compounds, and the changes in the benzo[a]pyrene metabolite profile in induced animals suggest similarities with the MO system present in vertebrates. However, the relatively high capacity of the monooxygenase system in sea stars to catalyse reactions with organic hydroperoxide as donor for activated oxygen, and the low induceability during exposure to xenobiotics indicate also important differences between the echinoderm cytochrome P450 monooxygenase system and that of vertebrates. Some evidence was found for the existence of different forms of cytochrome P450 in sea stars. Catalytic functions of the cytochrome P450 monooxygenase system of sea stars in the metabolism of steroids may be suppressed as a result of the induction of cytochrome P450 by xenobiotics.

Evidence for cytochrome P-450 catalysis and free radical involvement in the production of DNA strand breaks by benzo[a]pyrene and nitroaromatics in mussel ( Mytilus edulis L.) digestive gland cells

The mechanisms involved in the production of DNA strand breaks (SB) by model polycyclic aromatic hydrocarbon and nitroaromatic contaminants were investigated in isolated mussel ( Mytilus edulis L.) digestive gland cell mixtures using the model compounds benzo[a]pyrene (BaP), 1-nitropyrene (1-NP) and nitrofurantoin (NF). Isolated cells were exposed in vitro to sub-cytotoxic concentrations (50 μM) of BaP, 1-NP or NF for 1 h in the dark at 15°C in the absence or presence of various cytochrome P-450 inhibitors, antioxidant enzyme inhibitors, the free radical scavenger N- N- t-butyl- α-phenylnitrone (PBN), and other modulators. DNA strand breakage was measured using the comet assay (SB results presented as % tail DNA and was significant for each genotoxicant at least P<0.05). SB were seen for all three compounds and different metabolic pathways of genotoxicity were indicated for the three model compounds. BaP-induced strand breakage was indicated to be cytochrome P-450-catalysed and to occur via the production of BaP quinones because SB were inhibited 94% by 50 μM clotrimazole (inhibitor of digestive gland microsomal metabolism of BaP to quinones), stimulated 81% by 25 μM dicoumarol (inhibitor of DT-diaphorase, EC 1.6.99.2, which metabolises quinones to hydroquinones) and unaffected by 50 μM α-naphthoflavone (inhibitor of digestive gland microsomal metabolism of BaP to phenols and diols). Involvement of free radical(s) was indicated in BaP-induced strand breakage (75% SB inhibition by 50 mM PBN), consistent with either BaP cation radical formation (i.e. 1-electron oxidation) and/or reactive oxygen species (ROS) generation via BaP quinone formation and redox cycling. 1-NP-induced SB was indicated to occur via free radical mechanism(s) (84% SB inhibition by 50 mM PBN) and catalysis by different forms of cytochrome P-450 than for BaP (61% SB inhibition by 50 μM α-naphthoflavone but none by clotrimazole). In contrast to BaP and 1-NP, NF induced strand breakage was indicated not to involve cytochrome P-450(s) (no SB inhibition by clotrimazole or α-naphthoflavone), but to involve free radical(s) (88% SB inhibition by 50 mM PBN), consistent with redox cycling of NF and resultant DNA damage via superoxide anion radical (O 2 ·−) and other reactive oxygen species production. NF was more effective in producing SB compared to equimolar concentrations of BaP and 1-NP, possibly reflecting the greater direct redox cycling capacity of this compound.

Specificity of the cytochrome P-450 interaction with cytochrome b5

The specificity of the interaction of cytochrome b 5 with different forms of cytochrome P-450 was examined. Immunopurification of cytochromes P-450 1A1, 2B1 and 2E1 from rat liver microsomes resulted in co-purification of cytochrome b 5 with cytochrome P-450 forms 2B1 and 2EI but not 1A1. This specificity was evaluated in conjunction with multiple sequence alignment of the three cytochrome P-450s and a molecular model of the cytochrome P-450-cytochrome b 5 complex [(1989) Biochemistry 28, 8201–8205]. These analyses suggest two basic residues in the arginine cluster region of P-450, which are present in P-450s 2B1 and 2E1 but are absent in P-450 1A1, as potential binding sites for cytochrome b 5.

Isolation and Characterization of Four Cytochrome P450 Isozymes from Untreated and Phenobarbital-Treated Beagle Dogs.

Four different forms of cytochrome P450 (P450) were purified from liver microsomes of untreated or phenobarbital (PB)-treated male beagle dogs using HPLC techniques, and designated as DUT-1, DPB-1, DPB-2 and DPB-3, respectively. Specific contents of the purified DUT-1, DPB-1, DPB-2 and DPB-3 were 13.3, 9.6, 15.6 and 12.2 nmol/mg protein, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the monomeric molecular weights of DUT-1, DPB-1, DPB-2 and DPB-3 were estimated to be 57.5, 50.0, 47.0 and 50.0 kDa, respectively. The absolute spectra of the oxidized forms indicated that they exist in the low-spin state of heme in their oxidized forms. The NH2-terminal amino acid sequence of DUT-1 was unique and different from that of any other P450 so far reported. DUT-1 was active in the omega-hydroxylation of lauric acid. The amino-terminal sequences of DPB-1, DPB-2 and DPB-3 suggested that they belong to the P450 3A, 2C and 2B gene families, respectively. DPB-3 was a major form of P450 in PB-treated dog liver microsomes. Purified DPB-1 catalyzed nifedipine and (+)- and (-)-nilvadipine oxidations, as well as testosterone 6 beta-hydroxylation in the reconstituted system. These activities were enhanced 3- to 5-fold by the addition of cytochrome b5. DPB-2 and DPB-3 catalyzed aminopyrine N-demethylation, 7-ethoxycoumarin O-deethylation, biphenyl 4-hydroxylation and testosterone 16 alpha-hydroxylation. We believe that DUT-1 is a new form not purified previously.

Roles of different forms of cytochrome P450 in the activation of the promutagen 6-aminochrysene to genotoxic metabolites in human liver microsomes.

We reported previously that the potent mutagen 6-aminochrysene is catalyzed principally by rat liver microsomal P4501A and P4502B enzymes to reactive metabolites that induce umu gene expression in O-acetyltransferase-over-expressing strain Salmonella typhimurium NM2009; the proposal was made that there are different mechanisms in the formation of reactive N-hydroxylated and diolepoxide metabolites by P450 enzymes (Yamazaki, H. and Shimada, T., Biochem. Pharmacol., 44, 913-920, 1992). Here we further examined the roles of human liver P450 enzymes and the mechanism of activation of 6-aminochrysene by rat and human P450 enzymes in the Salmonella tester strains. Liver microsomes from 18 different human samples catalyzed activation of 6-aminochrysene more efficiently in S. typhimurium NM2009 than in the original strain of S. typhimurium TA1535/pSK1002. The rates of 6-aminochrysene activation in 18 human liver samples showed good correlation to the contents of P4502B6 as well as contents of P4503A4 and the respective mono-oxygenase activities catalyzed by P4503A4. Among purified P450 enzymes examined, P4501A2 as well as P4503A4 were highly active in transforming 6-amino-chrysene to reactive metabolites, suggesting the involvement of different human P450 enzymes in the reaction. Four human samples that contained relatively high levels of particular P450 enzymes in their microsomes were selected and used for further characterization. Liver microsomes from human samples HL-13 and HL-4 that contained the highest levels of P4502B6 and P4503A4 respectively, were sensitive to the respective antibodies raised against monkey P4502B and human P4503A4; the activity in sample HL-16 having the highest level of P4501A2 was inhibited by anti-P4501A2 IgG. alpha-Naphthoflavone enhanced the activation of 6-aminochrysene very significantly in human liver microsomes enriched in P4503A4 and P4502B6 enzymes. Pentachlorophenol, an inhibitor of acetyltransferase activity, suppressed the activation of 6-aminochrysene in liver microsomes from phenobarbital-treated rats and from human samples HL-4, HL-13 and HL-18 but not HL-16. In contrast, 1,1,1-trichloropropane-2,3-oxide, an inhibitor of epoxide hydrolase activity, enhanced the activation of 6-aminochrysene catalyzed by liver microsomes from beta-naphthoflavone-treated rats and from human samples HL-16 but not HL-4, HL-13 and HL-18. Inclusion of purified rat epoxide hydrolase to the reconstituted system containing rat and human P4501A enzymes caused a decrease in the rates of 6-aminochrysene activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Elevated 4-hydroxylation of estradiol by hamster kidney microsomes: a potential pathway of metabolic activation of estrogens.

Characterization of enzymes mediating the formation of catecholestrogens (CE) by hamster kidney is of importance because of the proposed role of CE in renal cancer induced in this species by estrogens. We have reexamined the potential of hamster kidney to convert estradiol (E2) to 2- and 4-hydroxylated CE because of recent evidence of the limitations of assays used in previous studies, in particular in measuring 4-hydroxylation of estrogens. Under conditions optimized for NADPH-dependent activity, hamster kidney microsomes exhibited high levels of both E2-2- and E2-4-hydroxylase activities. Evidence that the two activities depend on different forms of cytochrome P-450 was obtained by the demonstration that 2- and 4-hydroxylation of E2 were affected differentially 1) by chronic treatment of hamsters with E2 and 2) by fadrozole hydrochloride, a selective cytochrome P-450 inhibitor. NADPH-dependent 2-hydroxylation of E2 from control and E2-treated hamsters, measured by a direct product isolation assay, was 1 order of magnitude higher (apparent maximum velocity, 24-32 and 6-12.5 pmol/mg protein.min in control and E2-treated hamsters, respectively) than that reported previously using radioenzymatic assays. NADPH-dependent 4-hydroxylation of E2 in controls approached and in E2-treated hamsters exceeded 2-hydroxylation of E2 (apparent maximum velocity, 17-21 and 7.5-19 pmol/mg protein.min in control and E2-treated hamsters, respectively). Thus, estrogen treatment reversed the ratios of NADPH-dependent E2-2-/4-hydroxylase activities by causing a much greater decline in 2- than 4-hydroxylation of E2 (P less than 0.007, by analysis of variance). Fadrozole hydrochloride caused a marked dose-dependent decrease in 2-hydroxylation of E2, in contrast to a small nondose-dependent inhibition of 4-hydroxylation. Under conditions optimized for peroxidatic organic hydroperoxide-dependent activity, hamster kidney microsomes generated 2- and 4-hydroxylated CE in similar amounts. The amounts of the two CE and, consequently, the ratios remained unaffected by estrogen treatment (1:0.9 and 1:1.0 in control and E2-treated hamsters, respectively). Thus, this study establishes that CE can be generated in the same tissue by three different pathways, i.e. NADPH-dependent E2-2-hydroxylase, NADPH-dependent E2-4-hydroxylase, and organic hydroperoxide-dependent E2-2/4-hydroxylase activities. We also show that these three activities can be regulated differentially and are, thus, probably mediated by different forms of cytochrome P-450. In hamster kidney, the potential to generate 4-hydroxylated CE metabolites with distinct properties could be a factor in this tissue's vulnerability to estrogen-induced carcinogenesis.

Oxidative and reductive metabolism by cytochrome P450 2E1

We are constantly exposed to many potentially toxic chemicals. Most require metabolic activation to species responsible for cell injury. Although cytochrome P450 2E1 is only one of many different forms of cytochrome P450 that catalyze these reactions, it has an important role in human health as a result of being readily induced by acute and chronic alcohol ingestion. The enzyme efficiently catalyzes the low Km metabolism of compounds commonly used as solvents in industry and at home as well as components found in cigarette smoke, many of which are established carcinogens and hepatotoxins. As a result, there is the potential for increased risk to low level exposure to such chemicals while cytochrome P450 2E1 is induced. Many substrates have been identified for cytochrome P450 2E1. Of the 52 substrates for the enzyme identified in this review, the demethylation of N,N-dimethylnitrosamine and the hydroxylation of p-nitrophenol and chlorzoxazone are the most effective for monitoring the level of this enzyme. In addition to oxidative reactions, cytochrome P450 2E1 is also an efficient catalyst of reductive reactions. CCl4-induced hepatotoxicity is one of the best-documented cases for the participation of cytochrome P450 2E1 in a toxicologically important reductive reaction. The reduction of oxygen to superoxide and peroxide are also important reductive reactions of the enzyme and could be important in lipid peroxidation. However, the role of this reaction in vivo remains controversial.

Role of Cytochrome P450 in Tulip Bulb Probed by Substrate‐Binding Studies

AbstractMicrosomal fractions from dormant and wounded (sliced, aged) tulip bulbs showed a Soret peak at 448 nm in the carbon monoxide difference spectrum of dithionite‐treated membranes characteristic of cytochrome P450. Wounding and ageing induced a seven‐fold increase in P450 content. The enzyme‐substrate binding spectra and substrate affinity constants found under different conditions implied different forms of cytochrome P450. Of the several xenobiotics examined, two of them, benzo[a]pyrene and the herbicide 2,4‐dichlorophenoxyacetic acid, were inferred to be substrates from the production of P450 substrate‐binding spectra and varied substrate affinity constants were observed using the different microsomal preparations. In the wounded tissue, geraniol binding was also observed, which may reflect the endogenous function of the P450, possibly as part of a defence mechanism.

Immunohistochemical localization of cytochrome P-450

Distribution of different forms of cytochrome P-450 (forms C, D and B) in human placenta by immunohistochemical methods on light microscopy level with colloid gold was studied. Positive reaction with cytoplasm of endothelial cells, called the "epithelioid" plates of chorionic villi was found. Trophoblastic and stromal cells were not stained.

Multiple Forms of Plant Cytochromes P-450

Accumulating evidence indicates that there is a multiplicity of cytochrome P-450 enzymes in plants. These monooxygenases are implicated in the metabolism of sterols, terpenes, gibberellins, isoflavonoids, and xenobiotics. Evidence that cytochromes P-450 are involved in the detoxification of herbicides (chlorotoluron, primsulfuron, and diclofop) includes photoreversible CO inhibition of the reactions, and a requirement for O(2) and NADPH. Several cytochromes P-450, M(r) 45,000 to 65,000, have been isolated, including hydroxylases of cinnamic acid, 3,9-dihydroxypterocarpan, and digitoxin. In some cases the purified cytochrome P-450 has been successfully reconstituted with NADPH:cytochrome P-450 reductase (M(r) 72,000-84,000 protein). This reductase appears to be a nonspecific electron donor to different forms of cytochrome P-450. Immunological techniques and specific inhibitors (triazoles, imidazole derivatives) are being used to characterize plant cytochromes P-450 and the NADPH:cytochrome P-450 reductase. Specific cytochromes P-450 are induced by wounding or pathogens, others are expressed in specific cell types. Plant cytochromes P-450 are found in various subcellular locations, including endoplasmic reticulum, plasma membranes, glyoxysomes, and perhaps mitochondria. A cytochrome P-450 demethylase from avocado has recently been sequenced and found to have a hydrophobic N terminus similar to the membrane anchor of cytochromes P-450 from other organisms. The existence of cytochromes P-450 in different subcellular locations suggests that there are many genes for cytochromes P-450 in plants which have yet to be identified and classified.

Isolation of four forms of acetone-induced cytochrome P-450 in chicken liver by h.p.l.c. and their enzymic characterization

The purpose of this study was to purify and characterize the forms of cytochrome P-450 induced in chicken liver by acetone or ethanol. Using high performance liquid ion-exchange chromatography, we were able to isolate at least four different forms of cytochrome P-450 which were induced by acetone in chicken liver. All four forms of cytochrome P-450 proved to be distinct proteins, as indicated by their N-terminal amino acid sequences and their reconstituted catalytic activities. Two of these forms, also induced by glutethimide in chicken embryo liver, appeared to be cytochromes P450IIH1 and P450IIH2. Both of these cytochromes P-450 have identical catalytic activities towards benzphetamine demethylation. However, they differ in their abilities to hydroxylate p-nitrophenol and to convert acetaminophen into a metabolite that forms a covalent adduct with glutathione at the 3-position. Another form of cytochrome P-450 induced by acetone is highly active in the hydroxylation of p-nitrophenol and in the conversion of acetaminophen to a reactive metabolite, similar to reactions catalysed by mammalian cytochrome P450IIE. Yet the N-terminal amino acid sequence of this form has only 30-33% similarity with cytochrome P450IIE purified from rat, rabbit and human livers. A fourth form of cytochrome P-450 was identified whose N-terminal amino acid sequence and enzymic activities do not correspond to any mammalian cytochromes P-450 reported to be induced by acetone or ethanol.

The effect of age on the oxidative metabolism of antipyrine by uninduced microsomal fractions of rat liver

The influence of ageing on the metabolism of antipyrine by different forms of cytochrome P-450 was studied in vitro, by measuring the formatin of antipyrine metabolites by microsomes isolated from untreated rats, which were grouped into 5 different ages. K m , V max and V max / K m values were determined for the metabolites: 3-hydroxymethylantipyrine (HMA), norantipyrine (NORA) and 4-hydroxyantipyrine (OHA). K m values for all metabolites ranged from 2.0 to 5.0 mM and V max values from 1.84 to 3.66 nmol/min per mg. The V max/ K m ratios varied from 0.65 to 1.73 μl/min per mg. With respect to the K m values, a decrease for HMA from 12 to 24 months, no change for OHA and an increase for NORA from 3 to 30 months, was observed. The V max values showed an increase for HMA from 3 to 24 months, a decrease for OHA from 12 to 30 months and no change for NORA. The absolute V max/ K m ratios for all metabolites showed no significant changes during ageing. However, regarding the relative V max/ K m ratios, an increase for HMA from 3 to 24 months, a decrease for OHA from 12 to 35 months and also a decrease for NORA from 3 to 24 months, was observed. It is concluded that ageing influenced the K m , V max and K m / V max ratio of different P-450 isoenzymes involved in antipyrine metabolism, in different ways. Howevers, the age-related changes are modest (10–100%), especially for the V max/ K m ratio.

Separation of the cytochromes P-450 in pig kidney mitochondria catalyzing 1α-, 24- and 26-hydroxylations of 25-hydroxyvitamin D 3

The cytochromes P-450 in pig kidney mitochondria catalyzing 1α-, 24- and 26-hydroxylations of 25-hydroxyvitamin D 3 have been separated. The cytochrome P-450 fractions required NADPH, mitochondrial ferredoxin and ferredoxin reductase for catalytic activity. The present report demonstrates that different forms of cytochrome P-450 are involved in 1α-, 24- and 26-hydroxylations of 25-hydroxyvitamin D 3 and provides a basis for further purification and characterization of these enzymes.

Induction of cytochrome P-450 isozymes during long-term nifedipine administration

The wide substrate specificity of the microsomal monooxygenase system, catalyzing oxidation of many xenobiotics and endogenous compounds, is determined by the existence of multiple forms of cytochrome P-450 [13]. A modern approach to the characterization and identification of the individual cytochrome P-450 isozymes is to study specific reactions catalyzed by these hemoproteins. There are substrates whose metabolism is more specific for a particular cytochrome P-450 isozyme. For example, the O-de-ethylation of 7-ethoxyresorufin is catalyzed by cytochrome P-450 c [3] . The steroid hormones testosterone, androstenedione (AD), and progesterone are utilized as specific substrates by the microsomal monooxygenase of the liver, with well-marked regional and stereospecificity of metabolism [16]. The rate of formation of hydroxylated metabolites gives information on activity of different forms of cytochrome P-450. For example, the formation of 16a-OH-AD is catalyzed by cytochrome P-450h, which is characterized only of male rats. The formation of 16~-OH-AD is catalyzed by P-45%, that of 7a-OH-AD by cytochrome P-450a, and that of 6fl-OH-AD by cytochrome P-450p [15]. Thus the specificity of the reaction of metabolism of certain substrates can serve as an important criterion for the identification of many forms of cytochrome P-450. On the other hand, the immunochemical method using monospecific antibodies against individual cytochrome P-450 isozymes makes it quantitative and qualitative determination in microsomes possible. This combined approach can be used in pharmacologic investigations, especially those in which the drug influences the microsomal monooxygenase system. The calcium antagonist nifedipine, widely used in cardiologic practice, is metabolized by cytochrome P-450p in rat liver microsomes [6] or by P-450NF in human liver microsomes [14], which are induced by the synthetic steroid pregnenolone-16carbonitrile and steroids of the glucocorticoid class, such as dexamethasone and macrolide antibiotics [12]. Previously the writers described induction of a cytochrome P-450-dependent monooxygenase system after long-term administration of nifedipine to rats [7]. The aim of the present investigation was to discover what forms of cytochrome P-450 can be induced under these conditions.

ChemInform Abstract: Inhibitors of Sterol Biosynthesis and Growth in Plants and Fungi

Abstract Sterols and their derivatives promote and maintain growth and development in plants and fungi by acting as membrane constituents and probably also as hormones, engaged in control of metabolism. Inhibitors of sterol biosynthesis, operating at various stages in the pathway, are useful probes for investigating these functions. Some of the inhibitors have assumed considerable commercial importance as agricultural fungicides and antimycotic drugs in medicine. There is also potential for using similar compounds to regulate plant growth. The work reviewed here, much of it done within the last few years, points unmistakably to a fundamental role for sterols in promoting growth and development in both plants and fungi. A clear target enzyme in the biosynthetic pathway for growth inhibition in both plants and fungi is the 14α-methylsterol demethylase. Work with triazoles, particularly with closely related analogues and separated enantiomers, has provided an insight into the affinity of these inhibitors for the different forms of cytochrome P-450 which catalyse fungal sterol 14α-demethylation, plant sterol 14α-demethylation and plant ent -kaurene oxidation. It may become increasingly necessary to evaluate the relative contribution of these processes to optimise performance of fungicides, herbicides and PGRs, both with regard to their activity at the primary site of action and with a view to controlling side effects. There is clear scope here for extending and improving in vitro enzyme assay methods. The effectiveness of the morpholines as agricultural fungicides contrasts with the high tolerance of plants to cyclopropyl and Δ 8 -sterols which are readily induced by application of these compounds. Further study of the basic reason for morpholine toxicity is, therefore, an area well worthy of further investigation. Inhibition of squalene epoxidase by allylamines has proved successful in the development of medical antimycotics but no agricultural fungicides, based on this mode of action, have been forthcoming. Other enzymes of the pathway have yet to be commercially exploited but the C-24 alkylation process seems clearly linked to growth in both plants and fungi and hence would appear to be a particularly promising site for new inhibitors. The pre-squalene enzyme HMG-CoA reductase seems to offer potential for regulating or stopping plant growth. Above all there is a need to evaluate precisely how sterols and their derivatives control growth and developmental processes. The judicious use of inhibitors of the type described here may help in this undertaking which, in turn, could generate new ideas for the design and discovery of more active and specific compounds.