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A strain of Pseudomonas putida that can utilize naphthalene as its sole source of carbon and energy was isolated from soil. A mutant strain of this organism, P. putida 119, when grown on glucose in the presence of naphthalene, accumulates optically pure (+)-cis-1(R),2(S)-dihydroxy-1,2-dihydronaphthalene in the culture medium. The cis relative stereochemistry in this molecule was established by nuclear magnetic resonance spectrometry. Radiochemical trapping experiments established that this cis dihydrodiol is an intermediate in the metabolism of naphthalene by P. Fluorescens (formerly ATCC, 17483), P. putida (ATCC, 17484), and a Pseudomonas species (NCIB 9816), as well as the parent strain of P. putida described in this report. Formation of the cis dihydrodiol is catalyzed by a dioxygenase which requires either NADH or NADPH as an electron donor. A double label procedure is described for determining the origin of oxygen in the cis dihydrodiol under conditions where this metabolite would not normally accumulate. Several aromatic hydrocarbons are oxidized by cell extracts prepared from naphthalene-grown cells of P. putida. The cis dihydrodiol is converted to 1,2-dihydroxynaphthalene by an NAD+-dependent dehydrogenase. This enzyme is specific for the (+) isomer of the dihydrodiol and shows a primary isotope effect when the dihydrodiol is substituted at C-2 with deuterium.

The losses of CH$sub 3$ and CH$sub 6$ from the gaseous toluene cation (1) were studied using seven isotopic isomers of I and two of cycloheptatriene (2) utilizing high resolution (1: 500000) measurement of the abundance of the isobaric ionic products. Four distinct isomerization pathways, competitive with other I reactions, can be identified: (i) scrambling of the ring hydrogen atoms without involvement of the methyl group; (ii) isomerization I reversible 2 possibly through norcaradiene (7) as an intermediate; (iii) single exchange of an $alpha$-H and ring-H, possibly through methylenecyclohexadiene (8) as an intermediate; and (iv) complete scrambling of carbon as well as hydrogen atoms, possibly through opening of the aromatic ring. Reaction ii appears to involve the tightest activated complex, and i and ii the lowest activation energy; iii is intermediate, while iv has the loosest activated complex and highest activation energy. An additional pathway for the isomerization of 2, possibly through bis(methylene)cyclopentene, is also indicated. (auth)

The benzo[a]pyrene 4,5-, 7,8-, and 9,10-oxides and the six corresponding phenols (4-, 5-, 7-, 8-, 9-, and 10-hydroxybenzo[a]pyrene) have been tested for mutagenic and cytotoxic activity in bacteria and in a mammalian cell culture system. Benzo[a]pyrene 4,5-oxide (K-region) was highly mutagenic in two histidine-dependent strains (TA1537 and TA1538) of Salmonella typhimurium which detect frameshift mutagens. In contrast, benzo[a]pyrene 7,8- and 9,10-oxides were less than 1% as mutagenic as the 4,5-oxide. Benzo[a]pyrene 7,8- and 9,10-oxides were unstable in aqueous media, whereas the 4,5-oxide was stable for several hours. This difference in stability could not account for the different mutagenic activities of the three arene oxides. The benzo[a]pyrene oxides were inactive in a strain (TA1535) that is reverted by base pair mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine or in a strain (TA1536) that detects framshift mutagens similar to the acridine half-mustard ICR-191. Benzo-[a]-pyrene and the six phenols were all stable in aqueous media, but they had little or no mutagenic activity in any of the four Salmonella strains. Conversion of 8-azaguanine-sensitive Chinese hamster V79 cells to 8-azaguanine-resistant variants was increased by benzo[a]pyrene 4,5-oxide, whereas the 9,10-oxide was considerably less active. Benzo[a]pyrene and the other derivatives had little or no effect. Benzo[a]yrene 4,5-oxide was more cytotoxic to the Chinese hamster V79 cells than the 7,8- and 9,10-oxides, while 8-hydroxybenzo[a]pyrene was the most cytotoxic of the six phenols.

The mobilities of 24 potential metabolites of benzo[ a ]pyrene were examined with high pressure liquid chromatography. Twelve phenols, five quinones, four dihydrodiols, and three oxides were studied. The chromatographic procedure employed allowed the separation and quantitation of benzopyrene metabolites into three major groups consisting of phenols, quinones, and dihydrodiols. Two of the benzopyrene oxides were unstable during chromatography, whereas the third oxide was more stable and chromatographed in the quinone fraction. Treatment of rats with phenobarbital or 3-methylcholanthrene enhanced the metabolism of benzopyrene by liver microsomes and altered the relative amounts of the various metabolites formed. In the absence of epoxide hydrase (EC 4.2.1.63), benzopyrene was metabolized primarily to phenols and quinones but was not appreciably metabolized to dihydrodiols by a solubilized, reconstituted cytochrome P -448 monooxygenase system. Addition of partially purified epoxide hydrase resulted in the formation of benzopyrene dihydrodiols with a concomitant decrease in the formation of phenolic metabolites, indicating that benzopyrene undergoes metabolism via arene oxides that are precursors for dihydrodiols and phenols.

Hepatic miscrosomes and reconstituted cytochrome monoxygenase systems from control rats and from rats that have been pretreated with phenobarbital or 3-methylcholanthrene convert radioactive acetanilide to ring-hydroxylated products, primarily 4-hydroxyacetanilide. 3-Methylcholanthrene pretreatment results in the greatest enhancement of activity: cytochrome fractions from 3-methylcholanthrene-pretreated rats have many-fold higher activity than cytochrome fractions from control or phenobarbital-treated rats. The percentage of migration and retention of tritium (NIH Shift) measured in 4-hydroxyacetanilide after enzymatic oxidation of 4-[ 3 H]acetanilide is nearly identical using microsomes or the corresponding reconstituted system, but in both cases the percentage of migration and retention of tritum is markedly lower for preparations from 3-methylcholanthrene-treated animals with values of 25%, as compared to the values of 40–60% for preparations from control or phenobarbital-treated animals. High-pressure liquid chromatography was employed for separation and quantitation of radioactive products.

Chlorobenzene is converted to mixture of o-, m-, and p-chlorophenols in perfused rat livers, and in the following cell-free hepatic preparations from rat: postmitochondrial supernatant, microsomes, and reconstituted soluble hemoprotein systems. The percentage of m-chlorophenol steadily decreases with increasing resolution of the hemoprotein-monoxygenase system. Prior treatment of rats with 3-methylcholanthrene results in a large increase in rate of formation of the ortho isomer in all hepatic systems. Prior treatment with phenobarbital results in moderate increases in rates of formation of all three chlorophenols. Formation of the three chlorophenols is inhibited to similar extents by carbon monoxide, metyrapone, and high concentrations of glutathione. SKF-525a and 7,8-benzoflavone inhibit formation of o- and p-chlorophenols to a greater extent than that of the meta isomer; with microsomal preparations NADH greatly potentiates NADPH-dependent formation of p-chlorophenol, moderately potentiates formation of m-chlorophenol and has little effect on formation of o-chlorophenol. Dihydrodiols are not significant metabolites of Chlorobenzene with the soluble hemoprotein systems. These results, in concert with changes in proportions of phenolic metabolites seen during resolution of hepatic systems from the intact cell of the perfused liver to the soluble hemoprotein, are at least consonant with the hypothesis that chlorophenol production from Chlorobenzene is catalyzed by three different enzymes, two of which form arene oxide intermediates and one of which catalyzes a direct formation of a phenol. Thus, o- and p-chlorophenols result, respectively, on isomerization of intermediate 3- and 4-chlorobenzene oxides, while formation of m-chlorophenol would appear to occur via a direct oxidative pathway. In vitro conjugation of the arene oxide with glutathione or hydration is not a significant pathway. Assay of chlorophenols and dihydrodiols of Chlorobenzene was by high-pressure liquid chromatography.

Solubilized cytochrome P-450 monooxygenase and epoxide hydrase activities from rat liver microsomes have been separated by column chromatography. The highly active epoxide hydrase fraction is still contaminated with cytochrome P-450, which has very low monooxygenase activity. The highly purified cytochrome P-450 fraction possesses high monooxygenase activity and is essentially devoid of epoxide hydrase activity. Purification factors for the epoxide hydrase through four purification steps are similar with [3H]styrene oxide, [3H]naphthalene oxide, [3H]cyclohexene oxide, and benzene oxide as substrates. Failure of benzene oxide to inhibit hydration of styrene or naphthalene oxide in the most purified preparations in indicative of the presence of at least two hydrases. These purified cytochrome monooxygenase and hydrase preparations represent valuable tools for the study of the intermediacy of arene oxides in drug metabolism. Thus, with naphthalene, only naphthol is formed with the monooxygenase, while both naphthol and the dihydrodiol are formed in the presence of monooxygenase and hydrase. A convenient radiochemical synthesis of [3H]naphthalene 1,2-oxide and assays for the measurement of the hydration of [3H]naphthalene oxide and benzene oxide, based on differential extractions and high-pressure liquid chromatography, respectively, are described.

The metabolism of [ 14 C]benzo[a]pyrene by liver microsomes from normal and 3-methylcholanthrene-treated mice and rats was quantitatively analyzed by high pressure liquid chromatography. The ratio of dihydrodiols of benzo[a]pyrene to total metabolites formed was greater with microsomes from Long-Evans rats when compared to the ratio produced by microsomes for DBA/2J or C57BL/6J mice. Treatment of rats or C57BL/6J mice with 3-methylcholanthrene resulted in marked increases in the metabolism of benzo[a]pyrene to phenols, dihydrodiols, and quinones; but this effect did not occur in DBA/2J mice. The profile of benzo[a]pyrene metabolites formed by rat and mouse liver microsomes was dependent on both substrate and protein concentration. The use of incubation conditions with limiting substrate concentrations resulted in massive secondary metabolism of benzo[a]pyrene metabolites and was accompanied by the production of substantial amounts of metabolites that were no longer extractable into the organic phase.

The in vivo and in vitro metabolisms of normal and deuterated aromatic substrates have been investigated in ratsmsignificant isotope effects (k(H)/k(d) = 1.3-1.75) were associated with in vivo formation of meta-hydroxylated metabolites from 1:1 mixtures of normal and perdeuterio-(aryl ring) nitrobenzine, methyl phenyl sulfide, and methyl phenyl sulfone. Since isotope effects of this magnitude are incompatible with arene oxides as intermediates in the formation of phenols, the results provide evidence that multiple pathways are responsible for the formation of phenols in mammals. Significant isotope effects were not associated with the formation of the other phenolic isomers of nitrobenzene, methyl phenyl sulfone, or methyl phenyl sulfide or with the formation of phenolic products from anisole, bromobenzene, chlorobenzene, fluorobenzene, benzonitrile, naphthalene, zoxazolamine, acetanilide, biphenyl, diphenylhydantoin, benzene, o- and p-xylene, toluene, and mesitylene. Significant isotope effects might not be observable with the latter substrates if the kinetic parameters for oxidation of substrate change or if the arene oxide pathway greatly predominates. Furthermore, extensive in vivo metabolism of any substrate would make isotope effects unobservable by the procedure employed, namely the analysis of isotope content in metabolites formed from 1:1 mixtures of normal and deuterated substrates.

Acetanilide-4- 2 H is converted to 4-hydroxyacetanilide-3- 2 H during incubation with liver microsomal preparations. The degree of migration and retention of deuterium in this reaction, previously shown to depend on the pH of the media, is also dependent upon the species, strain, and sex of the animal used as the source of microsomal preparations. Pretreatment of animals with phenobarbital causes an increase in the retention of deuterium in 4-hydroxyacetanilide produced from 4-deuteroacetanilide while pretreatment with benzpyrene or 3-methylcholanthrene causes a decrease in the retention. The effect of induction on the migration and retention of deuterium and of tritium is unique to acetanilide-4- 2 H, and is not observed during hydroxylation of anisole-4- 2 H, chlorobenzene-4- 2 H, or 4-fluorobiphenyl-4′- 2 H. It is proposed that two “acetanilide hydroxylases” exist in varying amounts in different species, and that one, characterized by exhibiting high retentions of deuterium in the hydroxylation of acetanilide-4- 2 H, is induced by phenobarbital, while the other, characterized by exhibiting low retentions of deuterium in the same reaction, is induced by 3-methylcholanthrene and benzpsyrene.

The hydroxylation of a variety of specifically deuterated aromatic substrates by liver microsomes has been investigated. Labeled substrates which cannot readily ionize by loss of a proton from the ring substituent display deuterium retentions varying from 40 to 64% upon hydroxylation at the ring position bearing the isotopic label. These substrates include compounds with either electron-donating or electron attracting substituents (anisole-4- 2 H, diphenyl ether-4- 2 H, biphenyl-4- 2 H, N -methyl- N -(phenyl-4- 2 H)-benzenesulfonamide, toluene-4- 2 H, fluorobenzene-4- 2 H, chlorobromobenzene-4- 2 H, benzonitrile-4- 2 H, benzamide-4- 2 H, and nitrobenzene-4- 2 H). Substrates that can, however, ionize by loss of a proton from the ring substituent exhibit lower retentions ranging from 0 to 30% at pH 8. This class of compounds includes phenols such as salicylic-5- 2 H acid, aniline-4- 2 H, N -(phenyl-4- 2 H)-benzenesulfonamide, and various N -acylanilines-4- 2 H. As the acidity of the amide hydrogen increases in the acylanilines, migration and retention of deuterium decreases. A mechanism is presented which accommodates the retention values observed for both classes of substrates. The degree of retention for acetanilide-4- 2 H is strongly dependent upon the pH of the incubating medium ranging from 21 (pH 10) to 44% (pH 6). N -(Phenyl-4- 2 H)-benzamide shows a similar pH dependence whereas nonionizable substrates (biphenyl-4- 2 H or anisole-4- 2 H) do not display this dependence. The relative reactivity of the substrates studied toward the microsomal hydroxylating system correlates well with their reactivity toward chemical reagents which cause electrophilic substitution: i.e., reactive or electron-rich rings act as the best substrates. In chemical terms, the microsomal hydroxylating system acts as though it is a weak, selective electrophile.

A range of ortho-disubstituted C-aryl aldimines has been synthesized. N.m.r. spectroscopic analysis revealed a significant proportion of the Z-isomer at equilibrium in solution. The E–Z-isomer distribution is critically examined in terms of electronic, steric, and solvent effects. The effect of trace amounts of carboxylic acid on imine stereomutation is discussed.

On incubation with rabbit liver microsomes, benzene oxide is converted enzymatically to trans-1,2-dihydro-l,2-dihydroxybenzene. The enzyme, “epoxide hydrase,” is found in both the microsomal and the soluble fractions and converts a variety of epoxides to 1,2-glycols. Hydration to glycols has been demonstrated for styrene oxide, indene oxide, and cyclohexene oxide. The trans-1,2-dihydro-1,2-dihydroxybenzene is dehydrogenated to catechol by the supernatant of microsomes. In the presence of acid, benzene oxide readily rearranges to phenol. This isomerization is also catalyzed by proteins, simple peptides, and acetamide, but not by N,N-dimethylacetamide. Consequently phenol is a major nonenzymatic product in all enzymatic studies with benzene oxide. An enzyme in the soluble fraction of rat liver catalyzes the addition of glutathione to benzene oxide forming the premercapturic acid, S-(1,2-dihydro-2-hydroxyphenyl)-glutathione.

Phenolic products obtained on isomerization of the three arene oxides of toluene, the nine arene oxides of ortho-, meta -, and para -xylene, the arene oxide of mesitylene, and 2-methyl and 1,2-dimethylnaphthalene-1,2-oxides are compared to the phenolic products obtained by hepatic metabolism of the parent aromatic hydrocarbons. The results are compatible with the intermediacy of certain of these arene oxides in the metabolic pathway from hydrocarbons to phenols. Migrations of methyl groups as well as a remarkable apparent migration of oxygen are observed. The isomerization of 1,4-dimethylbenzene oxide to 2,4-dimethylphenol is analogous to the methyl migration observed in the enzymatic conversion of 4-methylphenylalanine to 3-methyltyrosine with phenylalanine hydroxylase. There are multiple pathways of isomerization of alkylarene oxides to phenols and they are determined by environmental and structural factors. The proportion and nature of products vary greatly with pH, which suggests the presence of different mechanisms for acid-catalyzed or spontaneous isomerizations of arene oxides.

[7-3H]styrene oxide is converted to [7-3H]styrene glycol by a hepatic epoxide hydrase. Differential extraction of the incubation medium provides the basis for a rapid, simple method to assay epoxide hydrase activity. Unreacted substrate is first removed by extraction into petroleum ether followed by extraction of the glycol into ethyl acetate for assay by scintillation spectrometry. Enzyme activity is present in liver and kidneys and is localized exclusively in the microsomal fraction. The simplicity of the present assay permits the use of epoxide hydrase a a marker enzyme for microsomal membranes. The specific activity of this enzyme in hepatic microsomes increases during maturation of rats and following pretreatment of rats with phenobarbital or 3-methylcholanthrene.

A mutant strain of Beijerinckia, after growth with succinate plus biphenyl, contains an enzyme system that oxidizes benzo [a] pyrene and benzo [a] anthracene to mixtures of vicinal dihydrodiols. The major dihydrodiol formed from benzo [a] pyrene was identified as cis-9, 10-dihydroxy-9, 10-dihydrobenzo [a] pyrene by comparison with a synthetic sample. Benzo [a] anthracene was metabolized to four dihydrodiols, the major isomer being cis-1, 2-dihydroxy-1, 2-dihydroxy-1, 2-dihydrobenzo [a] anthracene.

Summary The NADPH-supported metabolism of chlorobenzene has been studied with a reconstituted hydroxylation system from rat liver microsomes. The maximal rate of metabolism required the presence of cytochrome P-450, NADPH-cytochrome c reductase, lipid, and cytochrome b 5 . In contrast, cytochrome b 5 was not required for the NADPH-supported cytochrome P-448-dependent hydroxylation of chlorobenzene. Thus, it appears that a conclusion as to whether cytochrome b 5 is or is not involved in NADPH-supported microsomal hydroxylation reactions is dependent not only on the specific reaction studied, but also on the particular hemeprotein catalyzing the reaction.

Die Epoxide der Nicht-K-Region wie z.B. (I) ergeben bei UV-Bestrahlung die beiden isomeren Phenanthrole wie z.B. Phenanthrol-(1) bzw.-(2).

The specific activities of glutathione- S -epoxide transferase present in the 100,00g supernatant from sheep liver and a highly purified preparation of this enzyme have been compared with a variety of epoxides as substrates. Purification factors of 30- to 60-fold were observed for nearly 50 simple epoxides and arene oxides. The similarity in purification factors suggests either that a single enzyme with activity toward simple epoxides and arene oxides has been purified or that several related enzymes have been copurified. Comparison of specific activities for benzene oxides, naphthalene oxide, phenanthrene oxides, and arene oxides of benzo[ a ]pyrene and dimethylbenzaanthracene has established that (i) benzene oxides are poor substrates unless strong electron withdrawing substituents are present, (ii) naphthalene oxide is a good substrate, and (iii) increasing the size of polycyclic arene oxides causes a steady decrease in activity. Reactivity toward simple epoxides was compared to that for epoxide hydrase. The transferase was not readily inhibited by any of the compounds which were used and showed a strict requirement for glutathione.