Induction of cytochrome P-450 activities by nicotine in the tobacco hornworm,Manduca sexta

The induction by dietary nicotine of a series of cytochrome P-450 enzyme activities was investigated in early fifth-instarManduca sexta larvae. At a low nicotine concentration in the diet (0.1 %), three of 12 midgut microsomal enzyme activities were significantly increased. At a higher concentration (0.75%) commonly found in plants of the genusNicotiana, nine of 12 activities were induced by 1.4- to 10.0-fold. Total cytochrome P-450, P-450 reductase activity, and midgut microsomal metabolism of nicotine were also increased by feeding 0.75% nicotine. Nicotine was metabolized by midgut microsomes to nicotine-1-N-oxide and cotinine-N-oxide. Fat body microsomal nicotine metabolism was low and unaffected by dietary nicotine. Isolated nerve cords were able to metabolize nicotine in vitro but this metabolism was not inducible by dietary nicotine. Nicotine-fed fifth-instarM. sexta larvae showed an increased tolerance to subsequent nicotine injection when compared to larvae fed a control diet. These results support the idea that induction of midgut cytochrome P-450-related metabolism is an adaptation ofManduca sexta to dietary nicotine.     

Benzo(a)pyrene metabolites formed by the action of yeast cytochrome P-450/P-448

Benzo(a)pyrene metabolites have been identified by high-pressure liquid chromatography after production by a cytochrome P-450 in brewer's yeast disruptates. The major metabolites are 7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene, 9-hydroxybenzo(a)-pyrene and 3-hydroxybenzo(a)pyrene. Pretreatment of the yeast with benzo(a)-pyrene, during its growth decreases the Michaelis constant (K_m) of the enzyme for benzo(a)pyrene, using either NADPH or cumene hydroperoxide as a cofactor.     

Structure-activity correlations in pentachlorobenzene oxidation by engineered cytochrome P450cam

We had reported engineering of the heme monooxygenase cytochrome P450cam from Pseudomonas putida with the F87W/Y96F/L244A/V247L mutations for the oxidation of pentachlorobenzene (PeCB), a recalcitrant environmental contaminant, to pentachlorophenol. In order to provide further insights into P450 structure, function and substrate recognition, we have determined the crystal structure of this 4-mutant without a substrate and its complex with PeCB. PeCB is bound face-on to the heme, with a weak Fe--Cl interaction. One PeCB chlorine is located in the cavity generated by the L244A mutation, in striking illustration of the role of this mutation in promoting PeCB binding. The structures also show that the P450(cam) oxygen-binding groove between G248 and T252 is flexible and can tolerate significant deviations from their conformations in the wild type without loss of enzyme activity. Analysis of the PeCB binding interactions led to introduction of the T101A mutation to enable the substrate to reorient during the catalytic cycle for more efficient oxidation. The resultant 5-mutant F87W/Y96F/T101A/L244A/V247L is 3-fold more active for PeCB oxidation than the 4-mutant. Polychlorinated benzene binding by the mutants and the partitioning between substrate oxidation and non-productive (uncoupling) side reactions are correlated with the structural data.     

Synthesis and characterization of cytochrome P-450 epoxygenase metabolites of eicosapentaenoic acid

Eicosapentaenoic acid, a major component of fish oil extracts, was recently shown to be metabolized to vicinal diol regioisomers by renal and hepatic cytochrome P-450 epoxygenases. The diol products were also found in the urine of people ingesting fish oil. In the present report, we developed a chemical method of making milligram amounts of the epoxide intermediates and their diol products. Eicosapentaenoic acid was reacted with 0.1 eq.m-chloroperoxybenzoic acid for 15 min. After normal- and reverse-phase high performance liquid chromatography plus capillary gas chromatography and electron impact mass spectrometry, the products were identified as 17,18-cis-epoxyeicosa-5,8,11,14-tetraenoic, 14,15-cis-epoxy-eicosa-5,8,11,17-tetraenoic, 11,12-cis-epoxy-eicosa-5,8,14,17-tetraenoic, 8,9-cis-epoxy-eicosa-5,11,14,17-tetraenoic and 5,6-cis-epoxy-eicosa-8,11,14,17-tetraenoic acids. The total epoxide yield from eicosapentaenoate was 10% per incubation. By reincubating (cycling) the unused substrate 10–20 times, the total epoxide yield could be increased to 66–88%. Epoxide regioisomers were not produced in equal amounts; epoxygenation was facilitated as the distance between the target double bond and carbomethoxy group increased. Upon hydrolyzing the epoxides, the gas-chromatographic retention times and mass spectra of the diol products were found to match those of biological metabolites. In addition, each of these standards was rapidly and quantitatively synthesized in an amount (milligram) adequate for biological tests.     

Subcellular distribution of the cytochrome P-450 complex and glutathione peroxidase activity in vitamin E and essential fatty acid deficiency

Glutathione peroxidase (EC 1.11.1.9) (GSHPx) and P-450 activity were measured in hepatic mitochondrial and microsomal fractions from rats deficient in vitamin E and/or essential fatty acids (EFA). The data were compared to corresponding normal values. GSHPx was significantly decreased in the mitochondrial matrix from animals in all 3 deficiency states. In vitamin E deficiency, a nonsignificant decreased GSHPx activity was found in mitochondrial membranes. Opposite to these findings, GSHPx was significantly increased in mitochondrial membranes of EFA-deficient animals. In combined EFA and vitamin E deficiency, the mitochondrial membrane GSHPx activity was only insignificantly increased. The P-450 complex activity was not detectable in the mitochondrial matrix. In mitochondrial membranes and microsomes, the P-450 complex activity changed parallel to the GSHPx activity.     

Dietary fish oil and cytochrome P-450 monooxygenase activity in rat liver and kidney

Lauric acid hydroxylation and aminopyrineN-demethylation were studied in kidney and liver microsomes from rats treated with fish oil. Different doses of fish oil containing 20% eicosapentaenoic acid and 10% docosahexaenoic acid were provided daily to the rats for seven days. In all the groups studied, the lauric acid metabolism was higher in kidney microsomes and the aminopyrine metabolism in the liver microsomes. Although no effect on the renal cytochrome P-450 concentration was detectable, all four fish oil doses increased the hepatic concentration of cytochrome P-450 by a mean 27%. The higher fish oil doses used increased the renal and hepatic microsomal metabolism of aminopyrine. The lauric acid metabolism was increased by fish oil only in the liver. Fish oil, a known inducer of fatty acid peroxisomal β-oxidation, also induced microsomal activity. These results show that liver and kidney respond in different ways to dietary factors such as fish oil. In addition, our study would suggest that fish oil increased the activity of two different families of liver cytochrome P-450. The activity of kidney lauric acid 11- and 12-hydroxylation, however, was not modulated by fish oil.     

Hydroxylation of fatty acids and alcohols by hepatic microsomal cytochrome P-450 system from the Mongolian gerbil

The liver microsomes of the Mongolian gerbilMeriones unguiculatus catalyzed the hydroxylation of various saturated fatty acids (C₈−C₁₈), alcohols (C₁₂ and C₁₆) and hydrocarbon (C₁₂) to the corresponding ω- and (ω-1)-hydroxy derivatives. Lauric acid was hydroxylated most effectively among saturated fatty acids and the order of activity as hydroxylation substrates was C₁₂>C₁₄>C₁₃>C₁₆>C₁₀>C₁₈>C₈. The specific activity of laurate hydroxylation (5.99 nmol/mg microsomal protein/min) in gerbil liver microsomes was higher than that observed in other species. 1-Dodecanol was also hydroxylated very effectively (4.58 nmol/mg microsomal protein/min) by gerbil liver microsomes, but in general the hydroxylation rates for fatty alcohols were much lower than those for the corresponding acids. It was found from both inhibitor and cofactor studies that the enzyme catalyzing the hydroxylation of fatty acids and alcohols in the liver microsomes of the Mongolian gerbil was a typical cytochrome P-450-linked monooxygenase, and at least two different cytochrome P-450 species were involved in the hydroxylation. Presented in part at the AOCS annual meeting (a joint meeting with the Japan Oil Chemists' Society), Honolulu, Hawaii, May 1986.     

Immobilization of cytochrome P-450 on MCM-41 with different silicon/aluminum ratios

The catalytic activity of two cytochrome P-450 (CYP450) isoforms (CYP2C9 and CYP2B4, 1.14.14.1) immobilized on a mesoporous material “MCM-41” synthesized with Si/Al ratios of ∞, 80 and 100 was evaluated. The amount of CYP2C9 and CYP2B4 immobilized in each material was different, due to isoform size, the number of hydrogen bonds and the electrostatic interactions between the aminoacid residues and the Lewis acid sites of the material. The catalytic activity of immobilized CYP2C9 and CYP2B4 took place without using cytochrome P-450 reductase; consequently MCM-41 participates in electron transfer through the Lewis acid sites. In addition, the catalytic activity of immobilized CYP2B4 on MCM-41 was similar to its free form when a Si/Al ratio of 100 was used. Finally, although the catalytic activity of immobilized CYP2C9 was also better on MCM-41 with a Si/Al ratio of 100, it maintained around 30% of the catalytic activity in relation to its free form.     

Engineering cytochrome P-450cam to increase the stereospecificity and coupling of aliphatic hydroxylation

Site-directed mutants were constructed in cytochrome P-450_camto re-engineer the stereochemistry and coupling of ethylbenzenehydroxyiation. The reaction with wild-type (WT) enzyme producesone regioisomer 1-phenylethanol with 5% reduced nicotinamideadenine deoxyribonucleic acid to product conversion of and aratio of 73:27 for the R and S enantiomers respectively. Ethyibenzenewas modeled into the active site of WT P-450_cam in a rigid modeand oriented to optimize either pro-R or pro-S hydrogen abstraction.Residues T101, T185 and V247 make extensive contacts with thesubstrate in the static complexes and were therefore chosenfor site-directed mutagenesis. Single mutants T101M, V247A andV247M are more stereospedik producing 89,87 and 82% (R)-1-phenylethanolrespectively. The coupling of the reaction is doubled for thesingle mutants T185L, T185F and V247M. In an effort to engineerincreased stereospecificity and coupling into a single catalystthe T101M, T185F and V247M mutants were combined in a multiplemutant of P-450_cam.This protein hydroxylates ethyibenzene resultingin an R:S ratio of 87:13 for the 1-phenylethanols and 13% couplingof reducing equivalents to product. The catalytic stereospecificityand stoichiometry with T101M–T185F–V247M does notrepresent a summation of the changes observed for the singlemutants. A portion of the individual effects on substrate recognitionproduced by the single substitutions is either eliminated ordegenerate within the triple mutant.     

Oxidation of benzyl alcohols to the corresponding carbonyl compounds catalyzed by copper (II) meso-tetra phenyl porphyrin as cytochrome P-450 model reaction

The oxidation of benzyl alcohols has been studied in the presence of isobutyraldehyde as co-catalyst, molecular oxygen as oxidant and copper (II) meso-tetra phenyl porphyrin (CuTPP) as catalyst. The CuTPP catalyst exhibits good activity and high selectivity under mild conditions. The effects of temperature and solvents have also been investigated in this catalyst system.     

Addition of unactivated thiols to epoxides and oxetanes catalyzed by a rhenium-oxo complex

A method for synthesizing β- and γ-hydroxy thioethers via addition of unactivated thiols to epoxides and oxetanes has been developed. This reaction is proposed to proceed through an unconventional mode of activation of the heterocycles. The transformation is catalyzed by ReO₂I(PPh₃)₂ affording β- and γ-hydroxy thioethers in moderate to excellent yield with excellent regioselectivity.     

Effect of dietary fats on pentobarbitone-induced sleeping times and hepatic microsomal cytochrome P-450 in rats

Female Wistar-derived rats were fed diets containing either sunflowerseed oil or tallow for 4 weeks. Rats fed the sunflowerseed oil diet had longer pentobarbitone-induced sleeping times and lower concentrations of hepatic microsomal cytochrome P-450 than rats fed the tallow diet. The addition of pentobarbitone to the drinking water of rats caused approximately 2-fold increases in the concentrations of hepatic microsomal cytochrome P-450 but did not alter the relative effect of the diets.     

Construction of a 3D model of cytochrome P450 2B4

A three-dimensional structural model of rabbit phenobarbital-inducible cytochrome P450 2B4 (LM2) was constructed by homology modeling techniques previously developed for building and evaluating a 3D model of the cytochrome P450choP isozyme. Four templates with known crystal structures including cytochrome P450cam, terp, BM-3 and eryF were used in multiple sequence alignments and construction of the cytochrome P450 2B4 coordinates. The model was evaluated for its overall quality using available protein analysis programs and found to be satisfactory. The model structure was stable at room temperature during a 140 ps unconstrained full protein molecular dynamics simulation. A putative substrate access channel and binding site were identified. Two different substrates, benzphetamine and androstenedione, that are metabolized by cytochrome P450 2B4 with pronounced product specificity were docked into the putative binding site. Two orientations were found for each substrate that could lead to the observed preferred products. Using a geometric fit method three regions on the surface of the model cytochrome P450 structure were identified as possible sites for interaction with cytochrome b5, a redox partner of P450 2B4. Residues that may interact with the substrates and with cytochrome b5 have been identified and mutagenesis studies are currently in progress.      

Composition of lipids bound to pure cytochrome P-450 of cholesterol side-chain cleavage enzyme from bovine adrenocortical mitochondria

Phospholipids bound to highly purified cytochrome P-450 from bovine adrenocortical mitochondria, part of the enzyme complex responsible for catalyzing the conversion of cholesterol to pregnenolone, have been examined for comparison with the bulk phospholipids of the mitochondria from the same tissue. In both cases, the major phospholipids are phosphatidylcholine (PC) (37%) and phosphatidylethanolamine (PE) (56%), as well as smaller amounts of sphingomyelin and diphosphatidylglycerol. The fatty acid compositions of the four classes of phospholipids and of the neutral lipids bound to the pure enzyme are indistinguishable from those of the respective mitochondrial lipids. They are also similar to those of mitochondria from other organs except for high levels of arachidonate and low levels of diphosphatidylglycerol.     

活性炭预处理对模型油吸附脱硫性能的影响

选取了两种商业活性炭(分别标记为AC_1和AC_2),以二苯并噻吩的正辛烷溶液为模型油,测定了温度、时间、硝酸预处理浓度、吸附剂用量对脱硫性能的影响。结果表明,AC_2的脱硫性能明显优于AC_1;在45℃,140 min,吸附剂用量为1.4 g(模型油10 mL),AC_2的脱硫率达96%。     

Surface modification of nanocast ordered mesoporous carbons through a wet oxidation method

An ordered mesoporous carbon was synthesized by chemical vapor deposition using SBA-15 silica as solid template and propylene as carbon precursor. It was submitted to several liquid oxidation treatments by means of HNO₃ and H₂O₂ under different oxidation conditions in order to modify its surface chemistry while keeping its structure and porous texture as unmodified as possible. Original and modified OMC samples were characterized using different techniques such as nitrogen adsorption at - 196 ºC , X-ray diffraction, scanning electron microscopy, elemental analysis, temperature programmed desorption and X-ray photoelectron spectroscopy. A higher amount of oxygen functional groups was introduced on the material surface by using HNO₃ than by means of H₂O₂, but the original surface texture and structural arrangement were kept unchanged in both cases.