Immunochemical detection and quantitation of microsomal cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase in the rat ventral prostate.

Treatment with beta-naphthoflavone (BNF) was found to induce 7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities about 500-fold in the microsomal fraction of the rat ventral prostate but had no effect on aminopyrine N-demethylase or reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activities. Phenobarbital (PB) treatment did not alter any of these enzyme activities. Antibodies raised in rabbits against rat liver cytochrome P-450 reductase (P-450 reductase) and against P-450 BNF-B2 and P-450 PB-B2, the major forms of P-450 isolated from liver microsomes of BNF- and PB-treated rats, respectively, were used to characterize the P-450-dependent monooxygenase system in the rat ventral prostate. Anti-P-450 reductase immunoglobulin G inhibited reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activity in prostatic microsomes, and anti-P-450 BNF-B2 but not anti-P-450 PB-B2 immunoglobulin G inhibited the BNF-induced prostatic microsomal 7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities. A highly sensitive immunoblotting method was used to quantitate P-450 BNF-B2, P-450 PB-B2, and P-450 reductase in prostatic microsomes. Using this technique, prostatic P-450 reductase with a molecular weight corresponding to that of purified liver P-450 reductase was detected at a level of 0.02 nmol/mg of microsomal protein. In the liver, the same enzyme amounts to 0.2 nmol/mg of microsomal protein. P-450 BNF-B2 was not detected in prostatic microsomes from control or PB-treated rats, whereas a protein band with a molecular weight corresponding to that of purified liver P-450 BNF-B2 was found in prostatic microsomes from BNF-treated rats at a level of 0.05 nmol P-450 per mg microsomal protein. P-450 PB-B2 was not detected in prostatic microsomes from either control, PB-treated, or BNF-treated animals.

Hybrid enzymes for structure-function analysis of cytochrome P-450 2B11.

Previous work has shown that P-450 2B11 is responsible for the unique ability of dogs to metabolize and eliminate certain highly-chlorinated biphenyls such as 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB), whereas the related P-450 2B forms in rat and rabbit are unable to metabolize the compound to any significant degree. To determine the structural basis for this functional diversity, hybrid enzymes were generated. Success with this approach required a careful choice of second enzyme and common substrate with which to assess the functional integrity of the hybrid proteins. The choices of P-450 2B5 from rabbit as the second enzyme and androstenedione as the substrate were based in part on the finding that P-450 2B11 and P-450 2B5 hydroxylate androstenedione with similar overall activities but distinct profiles. Enzymatic studies with eight hybrid enzymes provided evidence for two regions of P-450 2B11 and 2B5, between residues 95-239 and 240-370, that appear to be involved in defining substrate specificity for androstenedione, and three regions of P-450 2B11, between residues 95-239, 240-370, and 371-494, that contain amino acids necessary for metabolism of 245-HCB. This deliberate approach to the creation of hybrid cytochromes P-450 has generated a series of enzymes that will be central to further structure-function studies of the cytochromes P-450 2B.

Functional interaction between amino-acid residues 242 and 290 in cytochromes P-450 2B1 and 2B11.

Previous studies have revealed the functional importance of the negatively charged amino-acid residue Asp-290 of the phenobarbital-inducible dog liver cytochrome P-450 (P-450) 2B11 (Harlow, G.R. and Halpert J.R. (1996) Arch. Biochem. Biophys. 326, 85-92). A search for P-450 2B11 residues capable of forming a charge pair with Asp-290 suggested the positively charged residue Lys-242 as a likely candidate. Replacement of Lys-242 with Asp in a P-450 2B11 fusion protein with rat NADPH-cytochrome P-450 reductase (reductase) resulted in very low holoenzyme expression levels in Escherichia coli, as did replacement of Asp-290 with Lys. Remarkably, however, expression levels of the double mutant Lys-242 --> Asp/Asp-290 --> Lys were dramatically increased above either single replacement alone. Similarly, the pair-wise substitutions Lys-242 --> Leu/Asp-290 --> Ile in P-450 2B11 and Leu-242 --> Lys/Ile-290 --> Asp in P-450 2B1 showed greater holoenzyme expression levels than the constituent single mutants, providing further evidence for the close proximity of these residues within the three-dimensional structure of these two enzymes. These results support the hypothesis that a functional interaction exists between residues 242 and 290, which may help to coordinate the relative positions of proposed helices G and I. All of the mutant combinations, including the additional P-450 2B11 double mutants Tyr-242/Asn-290 and Tyr-242/Ser-290, displayed altered stereoselectivity of androstenedione hydroxylation.

Epoxidation of arachidonic acid as an active-site probe of cytochrome P-450 2B isoforms.

In the present study we determined the regioselectivity of arachidonic acid epoxidation by several members of the cytochrome P-450 2B subfamily, including rat P-450 2B1, 2B1-WM (an allelic variant of 2B1 expressed in Wistar-Munich rats), 2B2, and rabbit 2B4 and 2B5. The major products formed with all isoforms were the four regioisomeric epoxides, but each isoform produced a distinct distribution of the four epoxides. P-450 2B1 produced predominantly 14,15-epoxyeicosatrienoic acid (EET), while P-450 2B1-WM produced the 11,12-EET as the major product. P-450 2B2, 2B4, and 2B5 catalyzed the formation of all four epoxides in nearly equal amounts. The single Gly-478-->Ala substitution in the variant P-450 2B1-WM was sufficient to cause a dramatic change in the ratio of epoxides when compared with P-450 2B1. The Gly-478-->Ala mutation also changed the regioselective epoxidation of gamma-linolenic acid at the three double bonds. Four site-directed mutants of P-450 2B1 were also evaluated. The mutations included two single mutants where Ile-114 was changed to either Val or Ala and two double mutants where the Ala-478 mutation was coupled with either Val or Ala at position 114. When Ile-114 was mutated to Val, the degree of epoxidation of arachidonic acid at all four double bonds was nearly equal. However, substitution of Ile-114 with Ala, resulted in a significant reduction in the degree of epoxidation at the 14,15- and 11,12-double bonds, and the 8,9- and 5,6-EETs were the major products. When Ala was introduced at position 478 in conjunction with Val at position 114 the regioselective epoxidation of the mutant enzyme more closely resembled P-450 2B1-WM in that 11,12-EET was the major metabolite. The double mutation with Ala at both positions 114 and 478 produced a unique distribution of epoxide products with 5,6-EET as the major metabolite. The results of these studies indicate that residues 114 and 478 in the P-450 2B subfamily are important for the orientation of fatty acids in the active site.

cDNA and deduced amino acid sequences of a dog liver cytochrome P-450 of the IIIA gene subfamily.

A 1.96 kbp cDNA encoding a male Beagle dog liver cytochrome P-450 of 503 amino acid residues (Mr 57,636) has been isolated and sequenced. The deduced amino acid sequence is 79.8%, 69.3% and 74.1% identical to the P450IIIA forms human NF25, rat PCN1 and rabbit LM3c, respectively. The amino terminal sequence is identical to the first 28 residues of the dog P450IIIA form PBD-1. Southern blot analysis yields restriction patterns consistent with IIIA gene subfamily multiplicity.

The genetic determinants of the CYP3A5 polymorphism.

CYP3A proteins comprise a significant portion of the hepatic cytochrome P450 (CYP) protein and they metabolize around 50% of drugs currently in use. The dissection of the individual contributions of the four CYP3A genes identified in humans to overall hepatic CYP3A activity has been hampered by sequence and functional similarities. We have investigated the expression of CYP3A5 and its genetic determinants in a panel of 183 Caucasian liver samples. CYP3A5 expression is increased in 10% of livers in this ethnic group. Using a high density map of CYP3A5 variants, we searched for genetic markers of the increased CYP3A5 expression. In agreement with an independent, recent study, we report that a SNP within intron 3 (g.6986G>A) is the primary cause of the CYP3A5 protein polymorphism. The frequencies of the g.6986A variant which allow for normal splicing of CYP3A5 transcripts are 5% in Caucasians, 29% in Japanese, 27% in Chinese, 30% in Koreans and 73% in African-Americans. In the last ethnic group, the expression of CYP3A5 in some individuals who carry the g.6986A variant is affected adversely by a frame shift mutation (CYP3A5*7, D348., q = 0.10). In summary, these results should add to efforts to identify clinically relevant, CYP3A5-specific reactions and to further elucidate traits responsible for variable expression of the entire CYP3A family.

Identification and functional characterization of eight CYP3A4 protein variants.

The genetic component of the inter-individual variability in CYP3A4 activity has been estimated to be between 60% and 90%, but the underlying genetic factors remain largely unknown. A study of 213 Middle and Western European DNA samples resulted in the identification of 18 new CYP3A4 variants, including eight protein variants. A total of 7.5% of the population studied was found to be heterozygous for one of these variants. In a bacterial heterologous expression system, two mutants, R130Q and P416L, did not result in detectable P450 holoprotein. One mutant, T363M, expressed at significantly lower levels than wild-type CYP3A4. G56D, V170I, D174H and M445T were not significantly different when compared with wild-type CYP3A4 in expression or steroid hydroxylase activity. L373F displayed a significantly altered testosterone metabolite profile and a four-fold increase in the Km value for 1'-OH midazolam formation. The results suggest a limited contribution of CYP3A4 protein variants to the inter-individual variability of CYP3A4 activity in Caucasians. Some variants may, however, play a role in the atypical response to drugs or altered sensitivity to carcinogens.

Analysis of mammalian cytochrome P450 structure and function by site-directed mutagenesis.

Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and/or bioremediation.

Stoichiometry of 7-ethoxycoumarin metabolism by cytochrome P450 2B1 wild-type and five active-site mutants.

Recombinant P450 2B1 wild-type and the active-site mutants I114V, F206L, V363A, V363L, and G478S were purified and studied. The efficiency of coupling of reducing equivalents to 7-hydroxycoumarin formation was decreased for all the mutants except I114V. Uncoupling to H2O was increased for F206L, V363A, and G478S, decreased for V363L, and unchanged for I114V. Uncoupling to H2O2 was increased for V363L and decreased for I114V, F206L, and V363A. The findings from this study provide firm biochemical evidence that residues 206, 363, and 478 comprise part of the substrate binding site of P450 2B1.

Amino acid sequence of a postsynaptic neurotoxin from the venom of the Australian tiger snake Notechis scutatus scutatus.

Although 60 percent of the protein in tiger snake (Notechis scutatus scutatus) venom consists of the basic per-synaptically neurotoxic and myotoxic phospholipases notexin and Notechis II-5 and other phospholipase homologs such as Notechis II-1, several post-synaptic "curaremimetic" neurotoxins are present in small amounts. The major one of these is a typical "long" neurotoxin containing 73 amino acids in a single peptide chain cross-linked by five disulfide bridges. The formula weight calculated from the amino acid sequence is 8,051. The LD50 for intravenous injection into mice is 125 micrograms/kg.

Rat lung and liver microsomal cytochrome P-450 isozymes involved in the hydroxylation of n-hexane.

The primary metabolism of n-hexane in rat lung and liver microsomes was investigated. In liver microsomes from untreated animals the formation of each of the metabolites, 1-, 2- and 3-hexanol, was best described kinetically by a two-enzyme system, whereas for lung microsomes a one-enzyme system was indicated for each metabolite. Cytochrome P-450-PB-B, the major cytochrome P-450 isozyme induced in rat liver by phenobarbital, appeared to be responsible for the formation of 2- and 3-hexanol in lung microsomes from untreated rats as judged by antibody inhibition studies. The presence of this isozyme was confirmed by immunoblotting. In contrast, formation of 1-hexanol in rat lung was catalyzed by a cytochrome P-450 isozyme different from the major isozymes induced by either phenobarbital or beta-naphthoflavone. Similarly, formation of 2,5-hexanediol from 2-hexanol was catalyzed by a P-450 isozyme different from cytochrome P-450-PB-B and present in liver but not in lung microsomes. Furthermore, alcohol dehydrogenase activity with hexanols or hexanediol as the substrate was found exclusively in liver cytosol. These results suggest that inhaled n-hexane must be transported to the liver either intact or in the form of 2-hexanol before the neurotoxic metabolite 2,5-hexanedione can be formed.

Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450.

The individual members of a homologous series of phenoxazone ethers related to ethoxyresorufin were O-dealkylated, and the parent compound phenoxazone was ring-hydroxylated, each at different rates with hepatic microsomes of untreated rats. A structure-activity relationship (SAR) was plotted, relating the rate of O-dealkylation to the length and type of the ether side-chain. Phenobarbitone (PB), 3-methylcholanthrene (MC), Aroclor 1254 (ARO), isosafrole (ISO) and SKF-525A each induced preferentially the O-dealkylation of different members of the homologous series, resulting in the appearance of 5 different SAR plots, which characterized and differentiated between the 5 different inducers. beta-Napthoflavone (BNF) had a similar effect to MC, whereas pregnenolone 16 alpha-carbonitrile treatment caused no large change in the metabolism of any of the substrates tested. For characterizing the effects of the different inducers it was largely sufficient to compare the O-dealkylations of just 4 of the ethers: methoxy-, ethoxy-, pentoxy- and benzyloxyphenoxazone. Very high degrees of induction were seen. MC and ARO each induced preferentially the O-dealkylation of ethoxyphenoxazone (51- and 61-fold respectively). PB and SKF-525A each induced preferentially the O-dealkylation of pentoxyphenoxazone (283- and 324-fold respectively). ISO induced preferentially the O-dealkylation of benzyloxyphenoxazone (43-fold). For any particular induced type of microsomes the substrate with the fastest metabolism was not necessarily the substrate whose metabolism was induced the most, so that in order to characterize each of the 5 different inducers (PB, MC/BNF, ARO, ISO, SKF) it was necessary to compare both the degrees of induction and the specific activities of the reactions. Experiments with purified cyt. P-450 isozymes showed that ethoxyphenoxazone and pentoxyphenoxazone were highly selective substrates for the major isozymes induced by MC and PB respectively, whilst benzyloxyphenoxazone was a good substrate for both isozymes. Experiments using the organic inhibitors metyrapone and alpha-naphthoflavone and inhibitory antibodies against individual cyt. P-450 isozymes indicated that similar substrate selectivities occurred with the monooxygenase system in the microsomal membrane. It is suggested that the use of some or all of these homologous phenoxazone ethers will provide both a simple routine test for the characterization of several types of inducing agents and a powerful tool for investigating the biochemical basis for cyt. P-450 isozyme substrate selectivity.

Effects of social support on medical students' performances.

BACKGROUND: Stress among medical students has been linked to poor academic performance, while supportive social relationships have been associated with the alleviation of psychological stress. This study examines social support as a potential buffer against stress and hence as a potential strengthener of students' academic performances. METHOD: A cohort of 153 third-year students at the University of Illinois College of Medicine at Chicago was asked in the fall of 1990 to complete a questionnaire assessing role stress (stress involving competing demands between school and social and/or family life), social support, and sources of support (outside or inside medical school). Grades for the five major clerkships through which all the students rotated during their third year were collected from student transcripts. Statistical analyses of the relationships among academic performance, stress, and social support included factor analysis, hierarchical multiple-regression analysis, and Pearson correlational analysis. RESULTS: Data from 120 students (78% of the cohort) were used for correlational analysis. Of these students, 79 (66%) were men and 41 (34%) were women. Because eight of the questionnaires contained incomplete data, 112 questionnaires (73%) were used for multiple-regression analysis. No buffering effect was found for social support. Rather, social support from outside the medical school explained significant variance in academic performances and in role stress. Higher levels of outside support were associated with poorer clerkship grades for women, but with lower levels of stress for men. Also, total support (outside and inside combined) was negatively related to clerkship grades for the entire sample. CONCLUSION: The results suggest that contrary to the study's hypotheses, social support in general is related to lower levels of academic performance for both men and women, and that the negative effects of support from outside the medical school context may be particularly salient for women. These results are understandable given the nature of medical training, which places great demands on students' time. Therefore, it may be more appropriate for medical schools to promote time-management strategies than support-building interventions, especially for women.

Rat lung and liver cytochrome P-450 isozymes involved in the hydroxylation of m-xylene.

The primary metabolism of m-xylene in rat lung and liver microsomes was investigated. The ratio of side chain to aromatic hydroxylation was found to be approximately 1:1 in lung microsomes from untreated rats and in a reconstituted system containing the major cytochrome P-450 isozyme induced in rat liver by phenobarbital, cytochrome P-450-PB-B2, as compared to 8:1 in liver microsomes. Antibody inhibition studies showed the major importance of cytochrome P-450-PB-B2 for the formation of both primary m-xylene metabolites (3-methylbenzylalcohol and 2,4-dimethylphenol) in lung microsomes. Antibodies to the major cytochrome P-450 isozyme induced in rat liver by beta-naphthoflavone, P-450-BNF-B2, did not inhibit m-xylene metabolism in either liver or lung microsomes from beta-naphthoflavone treated rats although this isozyme efficiently catalyzed m-xylene hydroxylation in a reconstituted system. m-Xylene metabolism by purified P-450-BNF-B2 appeared to cause rapid inactivation of the enzyme.

Use of homology modeling in conjunction with site-directed mutagenesis for analysis of structure-function relationships of mammalian cytochromes P450.

In recent years, homology modeling has become an important tool to study cytochrome P450 function, especially in conjunction with experimental approaches such as site-directed mutagenesis. Molecular models of mammalian P450s can be constructed based on crystal structures of four bacterial enzymes, P450cam, P450 BM-3, P450terp and P450eryF, using molecular replacement or consensus methods. In a model built by molecular replacement, the coordinates are copied from those of a given template protein, while consensus methods utilize more then one protein as a template and are based on distance geometry calculations. The models can be used to identify or confirm key residues, evaluate enzyme-substrate interactions and explain changes in protein stability and/or regio- and stereospecificity of substrate oxidation upon residue substitution by site-directed mutagenesis. P450 models have also been utilized to analyze binding of inhibitors or activators, as well as alterations in inhibition and activation due to residue replacement.

Application of 3-dimensional homology modeling of cytochrome P450 2B1 for interpretation of site-directed mutagenesis results.

Three-dimensional structures of cytochrome P450 2B1 were modeled based on the crystallographic structure of P450cam. The effect of the alignment, loop choice, and minimization with or without water was assessed. Although final models were similar in overall structure, the identity of active site residues depended upon the alignment. An example is Phe-206, which may or may not form part of the active site. The choice of the loop conformation had a lesser effect, while including water in the final minimization step was essential for preserving the shape and size of the active site. The best model (model 2) was in good agreement with the data from site-directed mutagenesis studies, and correctly predicted the effect of substitutions at 9 out of 10 amino acid positions. Thus, residues important for P450 2B1 activity, such as Ile-114, Phe-206, Ile-290, Thr-302, Val-363, and Gly-478, constitute part of the active site and are able to interact with the substrate androstenedione through hydrophobic interactions. On the other hand, Ser-303, Ser-360 and Lys-473 are far from the active site and/or cannot interact with the substrate, in agreement with experimental data. The model indicates other residues likely to be important for enzyme function, such as Tyr-111, Leu-209, Ile-477, and Ile-480, which can be tested experimentally. The substrate may assume numerous binding orientations consistent with observed patterns of hydroxylation at C15 and C16. The replacement in the model of certain amino acid residues to mimic residue substitutions from site-directed mutagenesis studies and docking of the substrate into the modified active site allowed a plausible explanation for alterations in regio- and stereospecificities of some mutants of P450 2B1, such as Gly-478-->Ala or Val-363-->Ala.

Reduction of thyroid hormone may participate in the modulation of cytochromes P450 2C11 and 3A2 by retinol.

Both hypothyroidism and retinol supplementation in rats induce CYP 3A2 and suppress CYP 2C11. Therefore studies were performed to evaluate the role of thyroid hormones in the modulation of P450 expression by retinol. Adult male Sprague-Dawley rats were given retinol as a single oral dose of 75 mg/kg. Rats were killed and hepatic microsomes prepared at 24, 48, 72, and 96 hr following treatment. The catalytic activity of 2C11 was reduced maximally by retinol at 48 hr (by 30%) whereas 3A2 activity was elevated maximally at 24 hr (by 30%). The serum concentration of testosterone was not altered at any time point. However, retinol produced a decline in the concentration of thyroxine by 35% and 43% at 24 and 48 hr, respectively. These data suggest that administration of large doses of retinol may alter hepatic microsomal enzyme expression by perturbation of plasma thyroid hormone levels.

Covalent binding of benzo[a]pyrene to cytochrome P-450 beta NF-B2 and other proteins in reconstituted mixed-function oxidase systems.

A reconstituted mixed-function oxidase system, containing the major beta-naphthoflavone-induced isozyme of rat liver cytochrome P-450 bound benzo[a]pyrene covalently in the presence of NADPH. NADPH-cytochrome P-450 reductase was required for binding and a maximum rate of adduct formation was obtained at 8 units of reductase per nmol cytochrome P-450. Phosphatidylcholine inhibited this reaction. Benzo[a]pyrene was bound to the cytochrome, but not to the reductase, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Approximately 6 molecules of benzo[a]pyrene bound to each molecule cytochrome P-450 during prolonged incubations. No binding occurred when the beta-naphthoflavone-induced isozyme of cytochrome P-450 was replaced by the major isozyme induced by phenobarbital, but both cytochromes incorporated benzo[a]pyrene to approximately the same extent when they were incubated together in the presence of the reductase and NADPH. Metabolically activated benzo[a]pyrene also bound covalently to purified epoxide hydrolase, when this enzyme was added to the reconstituted mixed-function oxidase system.