Form 3a Research Articles

ChemInform Abstract: Sodium Dichloroisocyanurate Oxidation of a Sterically Hindered Tetrahydro‐9(10H)‐acridinone.

AbstractThe tetrahydroacridinone (I) is oxidized with the isocyanurate mentioned in the title to give the cis‐diol (II).

Reductive beta-scission of the hydroperoxides of fatty acids and xenobiotics: role of alcohol-inducible cytochrome P-450.

As shown previously in this laboratory, purified rabbit liver microsomal cytochrome P-450 form 2 (P-450 IIB4) catalyzes the reductive cleavage of hydroperoxides to yield hydrocarbons and either aldehydes or ketones. We have proposed that lipid hydroperoxides are the physiological substrates for the cleavage reaction and have shown that with 13-hydroperoxy-9,11-octadecadienoic acid the formation of pentane is roughly equimolar with respect to the NADPH consumed. In the present study, the other product was isolated and identified as 13-oxo-9,11-tridecadienoic acid. Of particular interest, the alcohol-inducible form of liver microsomal cytochrome P-450 form 3a (P-450 IIE1) is the most active of the isozymes examined in the reductive beta-scission of the 13-hydroperoxide derived from linoleic acid and the 15-hydroperoxide derived from arachidonic acid as well as the model compounds cumyl hydroperoxide (alpha, alpha-dimethylbenzyl hydroperoxide) and t-butyl hydroperoxide. In general, the forms of P-450 with lower activity, as judged by the rate of NADPH oxidation in the reconstituted system, give less of the cleavage products (hydrocarbon and oxo compound) and catalyze direct reduction of the hydroperoxides to the corresponding hydroxy compounds. The occurrence of the reductive cleavage reaction in liver microsomal membranes was demonstrated, and microsomes from animals treated with ethanol or acetone (P-450 IIE1 inducers) or phenobarbital (a P-450 IIB4 inducer) were more active than those from untreated animals. We suggest that the alcohol-inducible P-450, in addition to its known deleterious effects in chemical toxicity and chemical carcinogenesis, may enhance the reductive cleavage of lipid hydroperoxides with a resultant loss in membrane integrity.

Expression of Alcohol-Inducible Rabbit Liver Cytochrome P-450 3a (P-450IIE1) inSaccharomyces cerevisiaewith the Copper-Inducible CUP1 Promoter

The expression of the cDNA for alcohol-inducible rabbit liver microsomal cytochrome P-450 form 3a (P450IIE1) in Saccharomyces cerevisiae, with the use of the copper-inducible yeast metallothionein (CUP1) promoter and the ADH1 promoter, is described. Strains 50.L4 and PP1002 were compared for optimal levels of expressed protein. Immunoblot analysis showed that a much higher level of expression of cytochrome P-450 3a is obtained with strain 50.L4, and that the uninduced levels of expressed protein are similar with the two promoters. With the CUP1 promoter, transcription of the cDNA is strongly induced in the presence of cupric ions, and the amount of immunoreactive protein expressed in increased 20-fold in strain 50.L4, such that it constitutes 0.8% of the total cellular protein. The cytochrome P-450 holoenzyme content of these cells, calculated from the reduced CO difference spectrum, is about 0.02 nmole/mg of protein, or 0.1% of the total cellular protein. The holoenzyme content of microsomes prepared from these cells is up to 0.06 nmole/mg of protein, or 0.4% of the microsomal protein. Microsomal assays for ethylene formation from N-nitrosodiethylamine and for aniline p-hydroxylation, two reactions typical of purified rabbit cytochrome P-450 form 3a, showed that the cytochrome synthesized in yeast catalyzes both reactions. Furthermore, polyclonal anti-3a IgG completely inhibits the reactions with both substrates in yeast microsomes. A comparison of the product ratios from these substrates showed that the cytochrome P-450 3a expressed in yeast has catalytic activities similar to those of the authentic rabbit protein.

Sodium dichloroisocyanurate oxidation of a sterically hindered tetrahydro-9(10H)-acridinone

Treatment of 1,2,3,4-tetrahydro-9-(10H)-acridinone (1a) with sodium dichloroisocyanurate (2) yields cis-1,2-diol 3a or azepino[1,2-a]indole 4a. Here we present further developments which lead us to propose that a more plausible pathway to 4a form 3a involves a N-chloro 1,2-diol intermediate which undergoes ring contraction to the product

Identification of ethanol-inducible P450 isozyme 3a (P450IIE1) as a benzene and phenol hydroxylase

In this report, the identity of the cytochrome P450 isozyme(s) catalyzing the hydroxylation of benzene and the major hydroxylated metabolite of benzene, phenol, was investigated using rabbit hepatic microsomes and six purified isozymes of hepatic P450. Microsomes from acetone-treated rabbits showed about a 5-fold induction of benzene hydroxylation to phenol and hydroquinone. This increase correlated with the increase in form 3a determined immunochemically (about 7-fold). Antibody to isozyme 3a inhibited greater than 90% of the benzene and phenol hydroxylase activity of hepatic microsomes from acetone-treated rabbits. At high benzene concentrations (2 m m) in the presence of cytochrome b 5, form 3a was 1.3 times more active than form 2 and 7- to 10-fold more active than forms 3b, 3c, 4, and 6. At lower benzene concentrations (about 0.3 m m) form 3a was 5-fold more active than form 2. Furthermore, form 3a was the only isozyme to produce significant quantities of hydroquinone as did microsomes from acetone-treated rabbits. When phenol was used as the substrate, hydroquinone was the only product detected, and acetone treatment induced its formation 4- to 5-fold. Purified form 3a was 20- to 30-fold more active than the next most active isozyme, form 6, depending on the presence or absence of cytochrome b 5. These results suggest that isozyme 3a (P450IIE1) is a low- K m benzene hydroxylase and the principal phenol hydroxylase in rabbit hepatic microsomes. As a result, the induction of isozyme 3a could potentiate the toxicity of benzene by catalyzing an increase in the formation of both phenol and hydroquinone.

Comparison of the form(s) of cytochrome P-450 induced by ethanol and glutethimide in cultured chick hepatocytes

In this study, using a combination of immunological and enzymatic characterizations, we compared the forms of cytochrome P-450 induced by ethanol and glutethimide in primary cultures of chicken embryo hepatocytes. Recently we purified a cytochrome P-450 of 50K molecular weight from chicken embryo liver using glutethimide as a prototypic inducer. Antibodies to both this chicken cytochrome P-450 and to rabbit cytochrome P-450 form 3a from the IIE subfamily detected microsomal proteins of 50K induced by either ethanol or glutethimide in cultured chick embryo hepatocytes, indicating the antigenic homology of these subfamilies of cytochromes P-450 among different animal species. However, the antibody to glutethimide-induced chick cytochrome P-450 of 50K inhibited p-nitrophenol hydroxylase and benzphetamine demethylase activities 85–90% in microsomes from both ethanol- and glutethimide-treated cells, indicating similar epitopes whose integrity is required for catalytic activity. In contrast, antibodies to rabbit cytochrome P-450 form 3a had little to no effect on these same microsomal activities. Both ethanol and glutethimide induced microsomal p-nitrophenol and aniline hydroxylase activities in cultured chick embryo hepatocytes. In microsomes from ethanol-treated cells, the turnover of p-nitrophenol per cytochrome P-450 was 2-fold greater than that induced by glutethimide treatment, suggesting that ethanol is inducing a form of cytochrome P-450 that has greater catalytic activity with this substrate than glutethimide-induced forms. Thus, in cultured chick embryo hepatocytes, ethanol may induce cytochromes P-450 from both the IIB and IIE subfamilies.

Isolation and partial characterization of the gene for cytochrome P-450 3a (P-450 ALC) and a second closely related gene

Two genes that hybridize to the cDNA for alcohol-inducible cytochrome P-450 form 3a (P-450 ALC) have been isolated from a rabbit genomic library and characterized by restriction mapping, hybridization, and partial sequence analysis. The genes show extensive sequence similarity as judged by hybridization at high stringency to the coding region of P-450 3a cDNA. However, only gene 1 hybridizes under these conditions to the 3′ nontranslated segment of P-450 3a cDNA. The hybridizing fragments derived from both cloned genes were found to be present in the genome of all rabbits examined by Southern blot analysis, indicating that the genes represent separate loci and are not polymorphic alleles. Partial sequence analysis indicated that gene 1 encodes P-450 3a. Gene 2, if transcribed, would encode a protein with greater than 96% sequence identity with P-450 3a in the NH 2-terminal region.

A Stable Stannaethene

The stable stannanediyl 2 and the carbene-like species 1 react to give stannaefhene 3. The structure of 3 was established by NMR spectroscopy and by X-ray structure analysis. The very high-frequency 119Sn-NMR signal indicates that the C=Sn bond has strongly polar character (ylide form 3A). R1 = Si(CH3)3; R2 = C(CH3)3; R3 = CHR.

CDNA and derived amino acid sequence of ethanol-inducible rabbit liver cytochrome P-450 isozyme 3a (P-450ALC).

Administration of ethanol to rabbits is known to induce a unique liver microsomal cytochrome P-450, termed isozyme 3a or P-450ALC, which is responsible for the increased oxidation of ethanol and other alcohols and the activation of toxic or carcinogenic compounds such as acetaminophen and N-nitrosodimethylamine. To further characterize this cytochrome P-450 we have identified cDNA clones to isozyme 3a by immunoscreening, DNA hybridization, and hybridization-selection. The cDNA sequence determined from two overlapping clones contains an open reading frame of 1416 nucleotides, and the first 25 amino acids of this reading frame correspond to residues 21-45 of cytochrome P-450 3a. The complete polypeptide, including residues 1 to 20, contains 492 amino acids and has a molecular weight of 56,820. Cytochrome P-450 3a is approximately 55% identical in sequence to P-450 isozymes 1 and 3b and 48% identical to isozyme 2. Hybridization of clone p3a-2 to electrophoretically fractionated rabbit liver poly(A)+ RNA revealed multiple bands, but, with a probe derived from the 3' nontranslated portion of this cDNA, only a 1.9-kilobase band was observed. Treatment of rabbits with imidazole, which increases the content of isozyme 3a, resulted in a transient increase in form 3a mRNA, but this was judged to be insufficient to account for the known 4.5-fold increase in form 3a protein. Genomic DNA analysis indicated that the cytochrome P-450 3a gene does not belong to a large subfamily.

The health knowledge of ninth grade students in Oyo State, Nigeria.

A stratified random sample of 540 ninth grade (Class 3) students in Oyo State, Nigeria, were administered the 60-item Health Education Test Form 3A. The purpose of the study was to obtain baseline data concerning the health knowledge of the students for use in the development of Health Education curricula in Oyo State. Subjects from the metropolitan area, from medium-size towns, and from rural areas recorded composite mean scores of 29.02, 24.78, and 25.01, respectively; indicating a general deficiency of health knowledge among the subjects. Recommendations are offered concerning curriculum, teacher preparation, and international cooperation to assist in the development of quality Health Education programs for Nigerian students.

Immunochemical evidence for a role of cytochrome P-450 in liver microsomal ethanol oxidation

Antibodies to cytochrome P-450 isozyme 3a, the ethanol-inducible isozyme in rabbit liver, were used to determine the role of this enzyme in the microsomal oxidation of alcohols and the p-hydroxylation of aniline. P-450 isozymes, 2, 3b, 3c, 4, and 6 did not crossreact with anti-3a IgG as judged by Ouchterlony double diffusion, and radioimmunoassays indicated a crossreactivity of less than 1%. Greater than 90% of the activity of purified form 3a toward aniline, ethanol, n-butanol, and n-pentanol was inhibited by the antibody in the reconstituted system. The catalytic activity of liver microsomes from control or ethanol-treated rabbits was unaffected by the addition of either desferrioxamine (up to 1.0 m m) or EDTA (0.1 m m), suggesting that reactions involving the production of hydroxyl radicals from H 2O 2 and any contaminating iron in the system did not make a significant contribution to the microsomal activity. The addition of anti-3a IgG to hepatic microsomes from ethanol-treated rabbits inhibited the metabolism of ethanol, n-butanol, n-pentanol, and aniline by about 75, 70, 80, and 60%, respectively, while the inhibition of the activity of microsomes from control animals was only about one-half as great. The rate of microsomal H 2O 2 formation was inhibited to a lesser extent than the formation of acetaldehyde, thus suggesting that the antibody was acting to prevent the direct oxidation of ethanol by form 3a. Under conditions where purified NADPH-cytochrome P-450 reductase-catalyzed substrate oxidations was minimal, the P-450 isozymes other than 3a had low but significant activity toward the four substrates examined. The residual activity at maximal concentrations of the antibody most likely represents the sum of the activities of P-450 isozymes other than 3a present in the microsomal preparations. The results thus indicate that the enhanced monooxygenase activity of liver microsomes from ethanol-treated animals represents catalysis by P-450 isozyme 3a.

Alcohol metabolism and toxicity: Role of cytochrome P-450

A new isozyme of cytochrome P-450, designated form 3a on the basis of its relative electrophoretic mobility, has been purified to homogeneity from liver microsomes of rabbits treated chronically with ethanol. This cytochrome has the highest activity of the known rabbit P-450 isozymes in the oxidation of ethanol to acetaldehyde. In view of the reports of others that the hepatotoxicity of acetaminophen is increased in ethanol-treated animals and the human alcoholic, we have determined the activity of the six available P-450 isozymes in the activation of the drug to give an intermediate which forms a conjugate with reduced glutathione. Isozymes 3a, 4, and 6, the three major forms of cytochrome P-450 present in liver microsomes from rabbits chronically treated with ethanol, exhibited the highest activities in the recostituted enzyme system, whereas isozymes 3b and 3c were 10- to 20-fold less effective and phenobarbital-inducible isozyme 2 was essentially inactive, even in the presence of cytochrome b 5. The results obtained thus indicate that induction by ethanol of P-450 isozyme 3a may contribute to the toxicity of acetaminophen but that other cytochromes also play a significant role.

Alcohol oxidation by isozyme 3a of liver microsomal cytochrome P-450

Liver microsomes from rabbits treated chronically with ethanol were solubilized and fractionated to yield a new isozyme of cytochrome P-450 in a homogeneous state. This cytochrome, designated as isozyme 3a on the basis of its relative electrophoretic mobility, is distinct from the known isozymes as judged by its spectral properties, minimal molecular weight, amino acid composition, and NH 2- and COOH-terminal amino acid sequences. In addition, peptide mapping by high performance liquid chromatography following trypsinolysis indicates that form 3a is a unique gene product. This cytochrome has unusually high activity in the oxidation of ethanol and other alcohols to aldehydes and in the p-hydroxylation of aniline as compared with the other isozymes of P-450. The ethanol-oxidizing activity of isozyme 3a, which requires the presence of NADPH and NADPH-cytochrome P-450 reductase and is stimulated by the presence of phosphatidylcholine, is not due to contamination by catalase or an NAD +-or NADP +-dependent alcohol dehydrogenase.

Catalytic activity of cytochrome P-450 isozyme 3a isolated from liver microsomes of ethanol-treated rabbits. Oxidation of alcohols.

Cytochrome P-450 isozyme 3a, isolated from hepatic microsomes of rabbits treated chronically with ethanol, was found to have a unique substrate specificity when compared with isozymes 2, 3b, 3c, and 4. Form 3a has unusually high activity in the p-hydroxylation of aniline and in the oxidation of alcohols to aldehydes. These properties are reflected in the increased activities of these substrates in microsomes from ethanol-treated rabbits as compared to microsomes from untreated animals or those administered phenobarbital or 5,6-benzoflavone. The ethanol-oxidizing activity of isozyme 3a, which requires the presence of NADPH and NADPH cytochrome P-450 reductase and is stimulated by the presence of phospholipid, was shown not to be due to contaminating catalase or an NAD- or NADP-dependent alcohol dehydrogenase. Isozyme 3a catalyzes the oxidation of methanol, 1-propanol, and 1-butanol as well as ethanol; the relationships between the apparent Km values for these alcohols and their octanol-water partition coefficients is in accord with the known hydrophobic nature of the P-450 binding site. Whereas typical substrates of isozyme 2 are known to be metabolized with the stoichiometry predicted of a monooxygenase reaction, with isozyme 3a the sum of acetaldehyde formed from ethanol and of hydrogen peroxide generated is inadequate to account for the NADPH and oxygen consumed. Free hydroxyl radicals appear to mediate the slow oxidation of ethanol in the presence of the reductase alone but not the faster rate catalyzed by P-450 isozyme 3a. The results obtained, however, do not rule out the involvement of hydroxyl radical equivalent generated and bound at the active site of the cytochrome.

Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes of ethanol-treated rabbits.

A new isozyme of cytochrome P-450 has been purified to electrophoretic homogeneity from hepatic microsomes of rabbits treated chronically with ethanol. Several criteria indicate that the ethanol-inducible cytochrome, which has a minimal molecular weight of 51,000 and is designated form 3a on the basis of its relative electrophoretic mobility, is distinct from the known isozymes of P-450. As judged spectrally, the new isozyme is high spin in the oxidized state, as is form 4, but differs in that the spin state is unperturbed by nonionic detergents. The absolute spectrum of the ferrous carbonyl complex of form 3a is red shifted as compared to that of forms 2, 3b, 3c, 4, and 6 and exhibits a maximum at 452 nm. The amino acid composition of form 3a is different from that of the other isozymes, and both the NH2- and COOH-terminal sequences are distinct; form 3a has an NH2-terminal alanine and a carboxyl-terminal leucine residue. Peptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following treatment with papain, chymotrypsin, or Staphylococcus aureus V8 protease and by high performance liquid chromatography following trypsinolysis indicates that form 3a is a unique gene product. This cytochrome displays the highest activity of all of the rabbit isozymes in the oxidation of ethanol to acetaldehyde and the p-hydroxylation of aniline when reconstituted with NADPH-cytochrome P-450 reductase and phospholipid in the presence of NADPH and oxygen.

Zum verhalten von mono- und diolefinen gegenüber thallium(III)trifluoracetat

Cis-butene-2 yields with Tl(III)trifluoroacetate by cis-addition of two trifluoroacetoxygroups the bis-2,3-trifluoroacetate 1a of the mesobutane diol, whereas the trans-butene-2 affords the bis-2,3-trifluoroacetate of the racemic threo-compound as expected 2a. Cyclohexene is transformed by Tl(III)trifluoroacetate to the cyclotrimeric form 3a of cyclopentylaldehyde. Cis-cyclooctene is oxidized to trifluoroacetate 4a of cyclooctene-1-ol-3. The homoallylic alcohol 5 (cyclohexene-1-ol-4) suffers a remarkable—but not unexpected—fragmentation to Z-trifluoroacetoxy-hexene-4-al-5 5a. By treating the conjugated dienes cyclohexadiene and cyclopentadiene with this reagent only cis-addition of two trifluoroacetoxygroups takes place under formation of the mixture of cis-1,4-bis-trifluoroacetoxy-cyclohexene-2( 8a) and cis-1,2-bis-trifluoroacetoxy-cyclohexene-3( 8b) on the one and cis-1,4-bis-trifluoroacetoxy-cyclopentene-2( 9a) plus cis-1,2-bis-trifluoroacetoxy-cyclopentene-3( 9b) on the other side. 1,3-Butadiene and 2,3-dimethyl-butadiene are transformed under solely 1,2-addition of the trifluoroacetoxygroups to the bis-trifluoroacetoxy compounds 6a and 7a.