Microsomal Oxidation Research Articles

Induction of cytochrome P450 by Nrf2 activators: Implication for aldehyde metabolism

Acrolein is an environmental pollutant and product of lipid peroxidation in vivo. Acrolein mediates the activation of Nrf2, so we sought to determine whether Nrf2 regulates clearance of acrolein by inducing specific hepatic P450s. The routes of metabolism of acrolein and α,β-unsaturated aldehydes include carboxylic acid and alcohol formation. C57BL/6 mice were fed either 5 mg/kg of acrolein by gavage for 72 h or AIN76 diet containing 0.45 % BHA, a Nrf2 activator, for 7 days. Mouse liver was frozen at −80 ° and after extraction, the RNA was reverse transcribed to cDNA. Cyp2c29 mRNA levels were analyzed by QRT-PCR. Liver microsomal protein fractions were prepared from control or BHA-fed mouse liver. Our results indicate that hepatic Cyp2c29 mRNA levels were induced 5-fold in BHA-treated mice and 2-fold in acrolein-treated mice. Likewise, there was a two-fold higher activity for 9-anthracene aldehyde oxidation to 9-anthracene carboxylic acid and reduction to 9-anthracene methanol with BHA-treated mouse liver microsomes, reactions catalyzed by Cyp2c29 expressed in E. coli. These reactions were inhibited 90% by 0.5 mM miconazole, a general P450 inhibitor, but only slightly inhibited by troleandomycin, a Cyp3a11-specific inhibitor or cyanamide, an aldehyde dehydrogenase inhibitor. Cyp3A11 mRNA levels remained unchanged upon BHA feeding. This suggests that the aldehyde oxidation and reduction activity present in murine hepatic microsomes is predominantly mediated by Cyp2c29. Supported by USPHS P01 ES11860.

Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition

Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle and the arylation of intracellular nucleophiles. As the redox cycle is catalyzed by NADPH cytochrome P450 reductase, cytochrome P450 systems are expected to be related to the cytotoxicity induced by redox-cycling quinones. Thus, we investigated the relationship between cytochrome P450 systems and quinone toxicity for rat primary hepatocytes using an arylator, 1,4-benzoquinone (BQ), and a redox cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). The hepatocyte toxicity of both BQ and DMNQ increased in a time- and dose-dependent manner. Pretreatment with cytochrome P450 inhibitors, such as SKF-525A (SKF), ketoconazole and 2-methy-1,2-di-3-pyridyl-1-propanone, enhanced the hepatocyte toxicity induced by DMNQ but did not affect BQ-induced hepatocyte toxicity. The production of superoxide anion and the levels of glutathione disulfide and thiobarbituric-acid-reactive substances were increased by treatment with DMNQ, and SKF pretreatment further enhanced their increases. In addition, NADPH oxidation in microsomes was increased by treatment with DMNQ and further augmented by pretreatment with SKF, and a NADPH cytochrome P450 reductase inhibitor, diphenyleneiodonium chloride completely suppressed NADPH oxidations increased by treatment with either DMNQ- or DMNQ + SKF. Pretreatment with antioxidants, such as alpha-tocopherol, reduced glutathione, N-acetyl cysteine or an iron ion chelator deferoxamine, totally suppressed DMNQ- and DMNQ + SKF-induced hepatocyte toxicity. These results indicate that the hepatocyte toxicity of redox-cycling quinones is enhanced under cytochrome P450 inhibition, and that this enhancement is caused by the potentiation of oxidative stress.

Bioactivation of 4-Methylphenol (p-cresol) via CYP-mediated Aromatic Oxidation in Human Liver Microsomes

It has previously been proposed that 4-methylphenol (p-cresol) is metabolically activated by oxidation of the methyl group to form a reactive quinone methide. In the present study a new metabolism pathway is elucidated in human liver microsomes. Oxidation of the aromatic ring leads to formation of 4-methyl-ortho-hydroquinone, which is further oxidized to a reactive intermediate, 4-methyl-ortho-benzoquinone. This bioactivation pathway is fully supported by the following observations: 1) one major and two minor glutathione (GSH) adducts were detected in microsomal incubations of p-cresol in the presence of glutathione; 2) a major metabolite of p-cresol was identified as 4-methyl-ortho-hydroquinone in microsomal incubations; 3) the same GSH adducts were detected in microsomal incubations of 4-methyl-ortho-hydroquinone; and 4) the same GSH adducts were chemically synthesized by oxidizing 4-methyl-ortho-hydroquinone followed by the addition of GSH, and the major conjugate was identified by liquid chromatography-tandem mass spectrometry and NMR as 3-(glutathione-S-yl)-5-methyl-ortho-hydroquinone. In addition, it was found that 4-hydroxybenzylalcohol, a major metabolite derived from oxidation of the methyl group in liver microsomes, was further converted to 4-hydroxybenzaldehyde. In vitro studies also revealed that bioactivation of p-cresol was mediated by multiple cytochromes P450, but CYP2D6, 2E1, and 1A2 are the most active enzymes for formation of quinone methide, 4-methyl-ortho-benzoquinone, and 4-hydroxybenzaldehyde, respectively. Implications of the newly identified reactive metabolite in p-cresol-induced toxicity remain to be investigated in the future.

GLUTATHIONE TRAPPING TO MEASURE MICROSOMAL OXIDATION OF FURAN TOCIS-2-BUTENE-1,4-DIAL

Furan is a liver carcinogen and toxicant. Furan is oxidized to the reactive dialdehyde, cis-2-butene-1,4-dial, by microsomal enzymes. This reactive metabolite readily reacts with glutathione nonenzymatically to form conjugates. A high-performance liquid chromatography-electrochemical method for the detection of cis-2-butene-1,4-dial-glutathione (GSH) conjugates in microsomal preparations was developed to measure the extent of furan metabolism to cis-2-butene-1,4-dial in vitro. Previously unobserved mono-GSH reaction products of cis-2-butene-1,4-dial were detected in addition to the already characterized bis-GSH conjugates. Chemical characterization of these compounds indicated that the alpha-amino group of glutathione had reacted with cis-2-butene-1,4-dial to form a thiol-substituted pyrrole adduct. The analytical method was used to estimate the extent of furan oxidation in rat liver microsomes from untreated or acetone-pretreated F344 rats as well as in human P450 2E1 Supersomes. Our results confirm that cytochrome P450 2E1 can catalyze the oxidation of furan to cis-2-butene-1,4-dial. However, the data are also consistent with the involvement of other P450 enzymes in the oxidation of furan in untreated animals. This assay will be a valuable tool to explore tissue and species differences in rates of furan oxidation.

Effect of pH on Hemoglobin-Catalyzed Lipid Oxidation in Cod Muscle Membranes in Vitro and in Situ

The effect of pH and hemoglobin on oxidation of the microsomal lipids of cod was determined in isolated microsomes and in washed cod muscle. An increase of hemoglobin concentration from 0.5 to 15 microM accelerated lipid oxidation in both systems. In cod microsomes the rate of lipid oxidation increased in the order pH 6.8 >> pH 7.6 > pH 8.4 > pH 6.0 > pH 3.5. However, in washed cod muscle a decrease of pH from 7.8 to 6.8 greatly increased the lag phase and decreased the rate of lipid oxidation. A further decrease in pH to 3.5 decreased the lag phase and increased the rate of lipid oxidation further. A decrease of pH from 7.6 to 6.4 greatly reduced the affinity of hemoglobin for oxygen. Formation of methemoglobin due to autoxidation occurred more rapidly at pH 6.0 than at pH 7.5. Structural changes of the isolated microsomal membranes could be the reason for the unexpected slow lipid oxidation in microsomes at pH 6.0 and below.

MOLECULAR CLONING, BACULOVIRUS EXPRESSION, AND TISSUE DISTRIBUTION OF THE ZEBRAFISH ALDEHYDE DEHYDROGENASE 2

Ethanol is metabolized to acetaldehyde mainly by the alcohol dehydrogenase pathway and, to a lesser extent, through microsomal oxidation (CYP2E1) and the catalase-H(2)O(2) system. Acetaldehyde, which is responsible for some of the deleterious effects of ethanol, is further oxidized to acetic acid by aldehyde dehydrogenases (ALDHs), of which mitochondrial ALDH2 is the most efficient. The aim of this study was to evaluate zebrafish (Danio rerio) as a model for ethanol metabolism by cloning, expressing, and characterizing the zebrafish ALDH2. The zebrafish ALDH2 cDNA was cloned and found to be 1892 bp in length and encoding a protein of 516 amino acids (M(r) = 56,562), approximately 75% identical to mammalian ALDH2 proteins. Recombinant zebrafish ALDH2 protein was expressed using the baculovirus expression system and purified to homogeneity by affinity chromatography. We found that zebrafish ALDH2 is catalytically active and efficiently oxidizes acetaldehyde (K(m) = 11.5 microM) and propionaldehyde (K(m) = 6.1 microM). Similar kinetic properties were observed with the recombinant human ALDH2 protein, which was expressed and purified using comparable experimental conditions. Western blot analysis revealed that ALDH2 is highly expressed in the heart, skeletal muscle, and brain with moderate expression in liver, eye, and swim bladder of the zebrafish. These results are the first reported on the cloning, expression, and characterization of a zebrafish ALDH, and indicate that zebrafish is a suitable model for studying ethanol metabolism and, therefore, toxicity.

Molecular cloning and functional analysis of cytochrome P450 1A2 from Japanese monkey liver: comparison with marmoset cytochrome P450 1A2

A cDNA encoding a novel cytochrome P450 1A2 (CYP1A2) was cloned from the liver of an adult female Japanese monkey. The CYP1A2 protein was expressed in yeast cells and its enzymatic properties were compared with those of marmoset CYP1A2 using ethoxyresorufin (ER) and phenacetin (PN) as substrates. The nucleotide sequence of Japanese monkey CYP1A2 revealed 94.7, 99.5 and 93.5% identities to those of human, cynomolgus monkey and marmoset monkey CYP1A2, respectively. Multiple amino acid sequence alignment of Japanese monkey CYP1A2 with CYP1A2 of humans, cynomolgus monkeys and marmosets showed that Japanese monkey CYP1A2 had 92.4, 99.0 and 91.9% identities to the human, cynomolgus monkey and marmoset enzymes, respectively. Kinetic studies demonstrated that the enzymatic properties as ER and PN O-deethylases were considerably different between the Japanese monkey and the marmoset CYP1A2. Furthermore, both of these reactions in liver microsomal fractions from the Japanese monkey and marmoset showed biphasic kinetics. On the basis of the kinetic parameters, it is suggested that Japanese monkey CYP1A2 is a high- K m enzyme in both ER and PN O-deethylations, whereas marmoset CYP1A2 is a high- K m and low- K m enzyme in ER and PN O-deethylations, respectively. α-Naphthoflavone, an inhibitor of human CYP1A1 and CYP1A2, did not completely inhibit the liver microsomal oxidations of ER and PN even at the highest concentration (50 μM), supporting the notion that CYP1A2 enzymes are not the sole ER or PN O-deethylase in Japanese monkey and marmoset liver microsomes. Inhibitory effects of furafylline, an inhibitor of human CYP1A2, on ER O-deethylation by recombinant CYP1A2 enzymes were much lower than those of α-naphthoflavone, but marmoset CYP1A2 was more sensitive to furafylline than Japanese monkey CYP1A2. These results indicate that the properties of Japanese monkey CYP1A2 are considerably different from those of marmoset CYP1A2.

Design, synthesis and in vitro evaluation of novel water-soluble prodrugs of buparvaquone.

Novel water-soluble phosphate prodrugs (2b-5b) of buparvaquone-oxime (1a) and buparvaquone-O-methyloxime (1b) were synthesized and evaluated in vitro as potential oral prodrugs against leishmaniasis. Buparvaquone-oxime (1a), and most probably also buparvaquone-O-methyloxime (1b), released the parent buparvaquone via a cytochrome P450-catalysed reaction. The prodrugs 2b-5b showed significantly higher aqueous solubilities (>4 mg/ml) than buparvaquone (< or = 0.03 microg/ml) over a pH range of 3.0-7.4. The prodrugs 2b, 3b and 5b rapidly released (t1/2 = 7 min) the corresponding oximes of buparvaquone (1a and 1b), and prodrug 4b at a moderate rate (t1/2 = 22.5 min) in alkaline phosphatase solution in vitro. Prodrug 3b was the most chemically stable in the aqueous solutions over a pH range of 3.0-7.4 (t1/2 > 8 days). Although buparvaquone-oxime (1a) has been shown to undergo a cytochrome P450-catalysed oxidation in liver microsomes to the parent buparvaquone and behave as a novel bioreversible prodrug, its usefulness is limited in oral drug delivery due to its poor aqueous solubility, like buparvaquone itself. Further phosphorylation of an oxime form of buparvaquone significantly increased water solubility, and this novel approach is therefore useful to improve physicochemical properties of drugs containing a ketone functional group.

Effects of feeding in free-range conditions or in confinement with different dietary MUFA/PUFA ratios and α-tocopheryl acetate, on antioxidants accumulation and oxidative stability in Iberian pigs

The experiment was undertaken to provide information of the influence of feeding either free-range or in confinement with different dietary MUFA/PUFA ratios and α-tocopheryl acetate supplementation (40 vs. 200 mg/kg) on tocopherol content and susceptibility to lipid oxidation of muscle and microsomes in Iberian pigs. The grass provided to the pigs had a similar α-tocopherol concentration to that observed for diets supplemented with 200 mg/kg α-tocopheryl acetate, and acorns supplied fourfold higher content of γ-tocopherol than the experimental diets. The α- and γ-tocopherol contents of muscle reflected the tocopherol concentration of the diets. Mono and Medium diets produced a similar MUFA/PUFA ratio in neutral and polar lipids of pig muscle to those fed outdoors. The lowest TBARS numbers were found in muscle samples from pigs fed a MUFA-enriched diet in confinement. No significant influence of free-range feeding or dietary fat on drip loss was found. However, α-tocopheryl acetate supplementation reduced ( P < 0.05) drip loss. Dietary vitamin E supplementation decreased the membrane lipid oxidation by 18% after 120 min. However, free-range feeding decreased the extent of microsome oxidation by 20%, 56% and 82% after 120 min when compared with those groups fed in confinement with high, medium and low MUFA/PUFA ratios, respectively. The hexanal concentration of muscle showed a similar trend to that observed for microsome induced-oxidation, suggesting, that hexanal determination is a more accurate method to measure lipid oxidation in iberian pig muscle than the thiobarbituric acid test.

The Effects of Benzonal, Halonal, and Halodif on the Hepatocyte System of Microsomal Oxidation in Ontogeny

The Effects of Benzonal, Halonal, and Halodif on the Hepatocyte System of Microsomal Oxidation in Ontogeny

Oxidation of tertiary benzamides by 5,10,15,20-tetraphenylporphyrinatoironIIIchloride–tert-butylhydroperoxide

Tertiary benzamides are oxidized by the 5,10,15,20-tetraphenylporphyrinatoiron(III) chloride-Bu'(t)OOH system at the alpha-position of the N-alkyl groups. The major products are N-acylamides, although small amounts of secondary amides, the products of dealkylation, are also formed. Plots of initial rate versus initial substrate concentration for these reactions are curved, suggesting formation of an oxidant-substrate complex. The reaction rates are almost insensitive to the substituent in the benzamide moiety, but there is a kinetic deuterium isotope effect of 5.6 for the reaction of the N,N-(CH(3))(2) and N,N-(CD(3))(2) compounds. Comparison of the reaction products from N-alkyl-N-methylbenzamides reveals that, for all compounds studied except N-cyclopropyl-N-methylbenzamide, oxidation of the alkyl group is preferred, strongly so (by a factor of ca. 8) for N-allyl-N-methylbenzamide. In contrast to microsomal oxidation, there is no steric hindrance to oxidation of an isopropyl group. Thus, we propose that these reactions proceed via hydrogen atom abstraction to form an alpha-carbon-centred radical and we attribute the observed diminished reactivity of the N-cyclopropyl group to its known reluctance to form a cyclopropyl radical. Oxidation of N-methyl-N-(2,2,3,3-tetramethylcyclopropyl)methylbenzamide provides preliminary evidence for rearrangement of an intermediate radical. While it remains unclear how these reactions proceed directly to the N-acyl products, we have established that N-hydroxymethyl, N-alkoxymethyl and N-alkylperoxymethyl intermediates are not involved.

Antioxidant defense system in the apple snail eggs, the role of ovorubin

A novel role of ovorubin as a protection system against oxidative damage in eggs from Pomacea canaliculata was investigated. Carotenoid composition, and their antioxidant capacity, as well as the carotenoid-apoprotein interaction, were studied for this lipoglycocarotenoprotein. Carotenoid extracts from ovorubin were analysed by TLC and spectrophotometry. The major carotenoid was astaxanthin in its free (40%), monoester (24%), and diester (35%) forms, mainly esterified with 16:0 fatty acid. The antioxidant capacity of ovorubin carotenoids was studied by the inhibition of microsomal oxidation in a non-enzymatic system, showing strong protection against oxidative damage (IC50=3.9 nmol/mg protein). The carotenoid–apoprotein interaction was studied by spectrophotometry and electrophoresis using reconstituted ovorubin. Astaxanthin does not seem to affect the structural characteristics of ovorubin, however the carotenoid–protein association significantly protected astaxanthin against oxidation. Ovorubin therefore, besides its role in providing energy and structural precursors during embryogenesis, would be an antioxidant carrier, protecting at the same time this pigment from oxidation in the perivitellin fluid environment of the egg.

Diallyl sulfide inhibits the oxidation and reduction reactions of stilbene estrogens catalyzed by microsomes, mitochondria and nuclei isolated from breast tissue of female ACI rats.

Previously, it has been demonstrated that microsomal, mitochondrial and nuclear enzymes isolated from the liver of male Sprague-Dawley rats catalyzed the oxidation of diethylstilbestrol (DES) to DES quinone. In the present study we have shown that diallyl sulfide (DAS) inhibits the oxidation of DES to DES quinone in all three subcellular fractions (microsomes, mitochondria and nuclei) isolated from breast tissue of female ACI rats. UV analysis of mitochondrial and microsomal fractions revealed that DAS decreased the rate of DES oxidation to DES quinone and DAS also decreased the rate in which DES quinone was reduced to DES. Lineweaver-Burk plots of the rate of DES quinone formation at various DES and DAS concentrations demonstrated that DAS inhibited the oxidation of DES and the reduction of DES quinone in a non-competitive fashion. In both microsomal and mitochondrial oxidation reactions the K(m) remained constant whereas the V(max) decreased with increasing DAS (0, 186 and 373 microM) concentrations (microsomes K(m) = 80 microM; V(max) = 5.56, 4.16 and 3.33 nmol/mg protein/min; mitochondria K(m) = 35.7 microM; V(max) = 3.45, 2.44 and 1.82 nmol/mg protein/min). Results were similar for reduction reactions. HPLC analysis revealed that a concentration of 186 microM DAS inhibited the mitochondrial, microsomal and nuclear oxidation by 27, 35 and 40%, respectively. A concentration of 373 microM DAS inhibited the mitochondrial, microsomal and nuclear oxidation by 50, 52 and 60% respectively. The data provide direct evidence that the breast tissue contain the metabolic machinery required to oxidize DES to reactive intermediates that may lead to genetic instability and cancer. This inhibition may play a role in the chemoprevention of stilbene estrogen-induced breast cancer.

Hepatic metabolism of diallyl disulphide in rat and man

1. The metabolism of diallyl disulphide was investigated in vitro with rat and human liver cell subfractions and ex vivo with an isolated perfused rat liver.2. Diallyl disulphide was oxidized to diallylthiosulphinate by rat liver microsomes with an apparent Km = 0.86 ± 0.1 mM and an apparent Vmax = 0.47 ± 0.12 nmol min−1 mg−1 protein (mean ± SE). Both cytochrome P450 (CYP) and flavin-containing monooxygenases were involved, with CYP2B1/2 and CYP2E1 being the most active CYP enzymes.3. In rat and man, microsomal oxidation of allylmethyl sulphide to allylmethyl sulphoxide and allylmethyl sulphone also occurred, although at a low rate. Diallyl disulphide was also metabolized to allylglutathione sulphide and allylmercaptan. In addition, diallylthiosulphinate reacted non-enzymatically with glutathione to form allylglutathione sulphide.4. When an isolated rat liver was perfused with diallyl disulphide, the metabolites allyl mercaptan, allylmethyl sulphide, allylmethyl sulphoxide, allylmethyl sulphone and allylglutathione sulphide were detected primarily within the liver tissue, with only small amounts of metabolites found in the bile and perfusion medium. The pharmacokinetic parameters for diallyl disulphide were t1/2 = 6.09 min; AUC0–∞ = 4.77 min mmol l−1; clearance = 34.22 ml min−1.5. A scheme for the metabolism of diallyl disulphide in rat and man is proposed.

Modification of lipid composition and oxidation in porcine muscle and muscle microsomes as affected by dietary supplementation of n-3 with either n-9 or n-6 fatty acids and α-tocopheryl acetate

The effect of different oil combinations: olive oil (20 g/kg), sunflower oil (20 g/kg), linseed oil (5 g/kg) and either olive (15 g/kg) or sunflower oil (15 g/kg) and α-tocopheryl acetate supplementation (200 mg/kg feed) on fatty acid composition and lipid oxidation of pig muscle and muscle microsomes was investigated. The proportions of α-linolenic acid ( P<0.001), eicosapentaenoic acid ( P<0.001) and docosahexaenoic acid ( P<0.05) in neutral lipids were significantly greater when linseed oil was added to diets. However, the proportion of docosahexaenoic acid in polar lipids was not significantly affected by the addition of linseed oil to the diets. Adding n-3 fatty acids in the diet produced a higher ( P<0.05) linoleic acid proportion in muscle neutral lipids, particularly in those groups fed n-9 MUFA. Pigs fed n-9 and n-3 enriched diets had the closest n-6:n-3 ratio to human dietary recommendations. The addition of linseed oil produced a significantly ( P<0.05) lower proportion of oleic acid (C18:1n-9) in polar lipids of muscles from pigs fed olive and linseed oils (OL + LIN), but not in those fed sunflower and linseed when compared with groups fed diets not enriched with linseed oil. Supplementation with 200 mg α-tocopheryl acetate/kg feed also led to higher ( P<0.05) oleic acid concentrations in polar lipids. Muscle from the pigs fed sunflower and linseed oils had significantly ( P<0.05) higher Thiobarbituric acid Reactive Substances (TBARS) values throughout the study compared to those fed olive and linseed oils. α-Tocopheryl acetate supplementation consistently improved lipid stability. No interactions between α-tocopherol and oils were observed on lipid oxidation. Iron-induced peroxidation showed a similar trend to that observed for TBARS development. Iron-induced peroxidation of muscle microsomes showed a similar trend to oxidation in muscle. No interactions were detected between different oils in muscle microsomes. The partial replacement of n-9 MUFA (as olive oil) by n-3 PUFA in diet, results in a small increase in muscle and membrane lipid oxidation and could be an interesting alternative in pig diet formulation.

Dose-response studies on the induction of liver cytochromes P4501A1 and 1B1 by polycyclic aromatic hydrocarbons in arylhydrocarbon-responsive C57BL/6J mice

1. To determine the biological effects of 23 polycyclic aromatic hydrocarbons (PAHs) and 3,4,30,40-tetrachlorobiphenyl, the dose–response studies of the induction of CYP1- dependent xenobiotic oxidation activities by these chemicals in liver microsomes of C57BL/6J mice were studied. 2. In arylhydrocarbon-responsive C57BL/6J mice, the liver microsomal xenobiotic oxidation with substrates of 7-ethoxyresoru.n, 7-ethoxycoumarin, (±)-benzo[a]pyrene- 7,8-diol, dibenzo[a,l] pyrene-11,12-diol and 2-amino-3,5-dimethylimidazo[4,5-f]quinoline increased by increasing the doses of PAHs to mice, particularly when the PAHs that have been reported to be carcinogenic in experimental animals were used. In arylhydrocarbon receptor-knockout mice, there were no increases in liver microsomal 7-ethoxyresoru.n O-deethylation activities nor in liver mRNA levels of CYP1A1, 1A2 and 1B1 by these chemicals. 3. Of the chemicals examined, benzo[k].uoranthene, benzo[b].uoranthene, benzo [j] -. uoranthene, 3-methylcholanthrene, dibenz[a,h]anthracene, dibenz[a,c]anthracene and 3,4,30,40-tetrachlorobiphenyl were potent inducers of the induction of liver microsomal 7-ethoxyresoru.n O-deethylation in mice. 4. Other PAHs such as 5-methylchrysene, benzo[a]pyrene, dibenzo[a, l] pyrene, dibenz[a, j]acridine, benzo[a]anthracene and 7,12-dimethylbenz[a]anthracene moderately induced 7-ethoxyresoru.n O-deethylation activities in mice. PAHs reported to be weak or less carcinogenic in experimental animals did not induce the xenobiotic oxidation activities of CYP1A1 and 1B1 in the mice. 5. The results suggest that induction of liver microsomal CYP1-dependent xenobiotic oxidation activities is a good tool in determining the potencies of carcinogenic PAHs in arylhydrocarbon-responsive C57BL/6J mice.

Interaction of polyhalogenated compounds of appropriate configuration with mammalian or bacterial CYP enzymes: Increased bilirubin and uroporphyrinogen oxidation in vitro

Polyhalogenated compounds, such as 2,3,7,8-tetrachlorodibenzo- p-dioxin, are associated with toxic Uroporphyria and cause alleviation of jaundice in the Gunn rat. These effects have been attributed to a microsomal oxidation of uroporphyrinogen and bilirubin for which supportive evidence has been obtained in vitro. CYP1A1 required planar polyhalogenated biphenyls for these oxidative reactions, while CYP1A2 was capable of oxidation in their absence. We have now used rat CYP1A1 and confirmed with the pure enzyme that increased bilirubin oxidation was caused by the addition of 3,4,3′,4′-tetrachlorobiphenyl. CYP1A2 was more active than CYP1A1 at oxidizing bilirubin in presence of NADPH alone and reacted to addition of 3,4,3′,4′-tetrachlorobiphenyl with a depression rather than a stimulation of bilirubin oxidation. We have also tested a bacterial enzyme, CYP102. Dodecanoic acid and its polyhalogenated analogue (perfluorododecanoic acid) both stimulated NADPH oxidation by CYP102, but only the perfluoro analogue stimulated markedly bilirubin oxidation. The analogue exhibited much greater potency than the normal substrate in stimulating NADPH and bilirubin oxidation and also showed greater affinity for CYP102, as measured by the binding constant, Ks. The molar stoichiometry ratio between NADPH and O 2 consumption was 1 in the case of the substrate, but approximated 2 with the perfluoro analogue. We conclude that halogenated substrate analogues can interact with different CYPs to increase production of oxidative species, probably by an uncoupling mechanism. A role of the ferryl-oxygen intermediate is suggested in the oxidation of biologically important molecules, with possible implications for the therapy of jaundice and for toxic oxidative reactions, such as uroporphyria and cancer.

Role of ascorbate in oxidative protein folding.

Both in prokaryotic and eukaryotic cells, disulfide bond formation (oxidation and isomerization steps) are catalyzed exclusively in extracytoplasmic compartments. In eukaryotes, protein folding and disulfide bond formation are coupled processes that occur both co- and posttranslationally in the endoplasmic reticulum (ER), which is the main site of the synthesis and posttranslational modification of secretory and membrane proteins. The formation of a disulfide bond from the thiol groups of two cysteine residues requires the removal of two electrons, consequently, these bonds cannot form spontaneously; an oxidant is needed to accept the electrons. In aerobic conditions the ultimate electron acceptor is usually oxygen; however, oxygen itself is not effective in protein thiol oxidation. Therefore, a small molecular weight membrane permeable compound should be supposed for the transfer of electrons from the ER lumen. The aim of the present study was the investigation of the role of ascorbate/dehydroascorbate redox couple in oxidative folding of proteins. We demonstrated that ascorbate addition or its in situ synthesis from gulonolactone results in protein thiol (and/or glutathione; GSH) oxidation in rat liver microsomes. Since microsomal membrane is hardly permeable to ascorbate, the existence of a transport metabolon was hypothesized. Three components of the system have been described and partially characterized: (i) A microsomal metalloenzyme is responsible for ascorbate oxidation on the outer surface of the ER. Ascorbate oxidation results in ascorbate free radical and dehydroascorbate production. (ii) Facilitated diffusion of dehydroascorbate is present in microsomal vesicles. The transport is presumably mediated by a GLUT-type transporter. On the contrary, the previously hypothesized glutathione disulfide (GSSG) transport is practically absent, while GSH is transported with a moderate velocity. (iii) Protein disulfide isomerase catalyzes the reduction of dehydroascorbate in the ER lumen. Both GSH and protein thiols can be electron donors in the process. Intraluminal dehydroascorbate reduction and the consequent ascorbate accumulation strictly correlate with protein disulfide isomerase activity and protein thiol concentration. The concerted action of the three components of the system results in the intraluminal accumulation of ascorbate, protein disulfide and GSSG. In fact, intraluminal ascorbate and GSSG accumulation could be observed upon dehydroascorbate and GSH uptake. In conclusion, ascorbate is able to promote protein disulfide formation in an in vitro system. Further work is needed to justify its role in intact cellular and in vivo systems, as well as to explore the participation of other antioxidants (e.g. tocopherol, ubiquinone, and vitamin K) in the electron transfer chain responsible for oxidative protein folding in the ER.

Alteration in Catalytic Properties of Human CYP2D6 Caused by Substitution of Glycine-42 with Arginine, Lysine and Glutamic Acid

The effects of the substitution of glycine at position 42 with various other amino acid residues on the functions of CYP2D6 were studied using debrisoquine (DB) and bunitrolol (BTL) 4-hydroxylations as indices of drug-metabolizing enzymes. The substitution with hydrophobic amino acid residues such as valine and phenylalanine did not affect the enzymatic properties such as reduced CO-difference spectra, microsomal CYP contents and oxidation activities towards DB and BTL. The substitution of glycine-42 with a polar but noncharged amino acid residue (serine) exhibited a similar reduced CO-different spectrum, but the substitution with a charged basic (lysine and arginine) or acidic (glutamic acid) amino acid residue commonly produced a peak at 420 nm in addition to a Soret peak at 450 nm. Cytochrome P450 contents and microsomal contents of G42S, G42K, G42R and G42E estimated spectrophotometrically and estimated by Western blot analysis, respectively, were lower than those of the wild-type. Kinetic analysis revealed that the substitution of glycine-42 with charged amino acid residues such as lysine, arginine and glutamic acid markedly increased the apparent K(m) values for DB and BTL oxidations without remarkable changes in the V(max) values. The subsitution with noncharged amino acid residues such as serine, valine and phenylalanine did not cause such a marked change in the K(m) values. Efficiencies (V(max)/K(m)) as DB and BTL 4-hydroxylases of CYP2D6 mutant proteins having charged amino acid residues were found to be decreased mainly by increasing their K(m) values. These results indicate that the properties of amino acid residues at position 42 affect the behavior of CYP2D6 proteins such as anchoring into ER membranes, conversion of P450 to P420 and incorporation of heme into apoproteins.

Roles of human liver cytochrome P450 3A4 and 1A2 enzymes in the oxidation of myristicin

The aim of this work was to identify the form(s) of human liver cytochrome P450 (CYP) involved in the hepatic transformation of myristicin to its major metabolite, 5-allyl-1-methoxy-2,3-dihydroxybenzene. When microsomes prepared from different human liver samples were compared, the activity of 5-allyl-1-methoxy-2,3-dihydroxybenzene formation was well correlated ( r 2=0.87) with nifedipine oxidation (a marker of CYP3A4). With a microsomal sample having high CYP3A4 activity, microsomal oxidation of myristicin to the major metabolite (5-allyl-1-methoxy-2,3-dihydroxybenzene) was markedly inhibited by gestodene and ketoconazole, selective inhibitors of CYP3A enzymes, but not by any of several other P450 inhibitors. Antibodies raised against CYPs 3A4 and 1A2 could also inhibit the oxidation of myristicin, but antibodies recognizing other CYPs had no effect. The oxidation of myristicin to 5-allyl-1-methoxy-2,3-dihydroxybenzene was catalyzed by purified bacterial recombinant CYPs 3A4 and 1A2. These results provide evidence that CYP3A4 (and possibly other CYP3A enzymes) and CYP1A2 play roles in the formation of the major metabolite, 5-allyl-1-methoxy-2,3-dihydroxybenzene.