NIH Shift Research Articles

Aromatic hydroxylation. Part 6. Oxidation of naphthalene by dioxygen in the presence of iron(II) salts

The major products from the oxidation of naphthalene with dioxygen in the presence of an iron(II) salt and an organic reducing agent are cis- and trans-1,2-dihydroxy-l,2-dihydronaphthalene, 2,3-epoxy- 3,4-dihydro-cis- and -trans-4-hydroxynaphthalen-1(2H)-one, cis-2-formylcinnamaldehyde, and 1- and 2-naphthol. A series of labelling experiments using 18O2, and deuterionaphthalenes reveal the origin of the oxygen in the dihydrodiols and quinone epoxides and the absence of a large value of the NIH shift for formation of 1- or 2-naphthol. The suggested mechanism involves a free radical co-oxidation of naphthalene and the organic reducing agent by dioxygen with iron–oxygen complexes, behaving as oxy-radicals. as the active oxidising species.

A highly sensitive radiometric assay for zoxazolamine hydroxylation by liver microsomal cytochrome P-450 and P-448: Properties of the membrane-bound and purified reconstituted system

A radiometric assay for the in vitro metabolism of zoxazolamine has been developed which combines high sensitivity and rapid determination of product. [4,6- 3H]zoxazolamine was metabolized to 6-hydroxyzoxazolamine, and the tritium released as 3H 2O was determined after treating the incubation mixture with activated charcoal. This treatment efficiently removes labeled substrate (99.98%), permitting enzymatically released tritium to be measured directly in the aqueous medium. Since the preponderant in vitro product of zoxazolamine metabolism by rat liver microsomes and the purified reconstituted mixed function oxidase system is 6-hydroxyzoxazolamine, and since this aryl hydroxylation occurs without significant NIH shift, the subsequent release of tritium from the 6-position accurately represents metabolism of the molecule. The use of [4,6- 3H]zoxazolamine for a tritium release assay of mixed function oxidase activity is ideal since this compound shows no significant isotope effect or NIH shift during metabolic conversion to 6-hydroxyzoxazolamine. 3-Methylcholanthrene treatment of rats resulted in a fourfold induction of zoxazolamine hydroxylation while phenobarbital or pregnenolone 16α-carbonitrile pretreatment caused only a 20–50% increase in zoxazolamine metabolism. The use of a purified reconstituted system revealed that cytochrome P-448 from 3-methylcholanthrene-treated rats was approximately 10- to 15-fold more efficient than cytochrome P-450 from phenobarbital-treated rats in catalyzing the hydroxylation of zoxazolamine.

The metabolism of higher chlorinated benzene isomers.

The metabolism of the isomeric tri- and tetrachlorobenzene isomers, penta- and hexachlorobenzene was investigated in the rabbit. The major urinary metabolites of the isomeric tri- and tetrachlorobenzenes were identified as the corresponding tri- and tetrachlorophenols whose structures were confirmed by chromatographic analyses. The genesis of the formation of metabolites is discussed in terms of their possible arene oxide intermediates in which the NIH shift of a chlorine atom is observed in the oxidation of many of the isomers. Pentachlorobenzene is metabolized to give both pentachlorophenol and a dechlorination-hydroxylation product which was identified as 2,3,4,5-tetrachlorophenol. The hexachlorobenzene substrate did not give any phenolic metabolites.

Studies on carcinogenic azo dyes. VI. Effect of factors influencing drug metabolism on the NIH shift during hydroxylations of 4-dimethylamino-3'-methylazobenzene and 3-methylacetanilide.

The NIH shift during aryl hydroxylations of 4-dimethylamino-3'-methylazobenzene (3'-Me-DAB) and 3-methylacetanilide under various conditions has been investigated. 4-Hydroxy-3-methylacetanilide was obtained from 3-methylacetanilide (4-2H, or 4-3H) by rat in vivo metabolism or by incubation with hepatic microsomal preparations of rat, mouse, hamster, or rabbit, and 3'-Me-4'-hydroxy-DAB by incubation of 3'-Me-DAB (4'-3H, or 4'-8H) with the liver homogenates. Retention of heavy hydrogen in the hydroxylated products was determined by mass spectrometry or liquid scintillation counting. The degree of retention of isotopic hydrogen during hydroxylation of the labeled 3-methylacetanilide was affected by the pH of incubation media, the species variation, the sex of animal used as the source of microsomal preparations, and the pretreatment of animal with inducing agents. Pretreatment of rat or mouse with phenobarbital caused an increase in the retention of tritium in 4-hydroxy-3-methylacetanilide produced from 3-methylacetanilide-4-3H, while pretreatment with 3-methylcholanthrene or 3, 4-benzpyrene caused a decrease in the retention. Pretreatment of rat with the carcinogenic azo dye, 3'-Me-DAB, did not affect the NIH shift during the in vivo hydroxylation of 3-methylacetanilide-4-2H. The effect of induction on the retention of tritium or deuterium is unique to 3-methylacetanilide (4-2H, or 4-3H), and is not observed during hydroxylation of 3'-Me-DAB (4'-2H, or 4'-3H) in which the tritium retention is also independent on the pH of incubation media and the species.

Metabolism of 4,4′-dihalogenobiphenyls

4,4′-Dichlorobiphenyl in rabbits gave three major urinary metabolites: 4,4′-dichlorobiphenyl-3-ol, 3,4′-dichlorobiphenyl-4-ol, and 4′-chlorobiphenyl-4-ol. 4,4′-Dibromobiphenyl gave the bromo analogues of the three 4,4′-dichlorobiphenyl metabolites. These results are consistent with 3,4-epoxidation of the biphenyl nucleus followed by epoxide ring opening accompanied by a 1,2-halogen shift (NIH shift). 4,4′-Di-iodobiphenyl gave only a single metabolite, 4,4′-di-iodobiphenyl-3-ol, and rearrangement products were not observed.

Metabolism of radioactive 17β-estradiol 3-methyl ether by humans

A mixture of 2- 3H and 4- 14C-17β-estradiol 3-methyl ether was administered orally to a man and to a woman. 34 And 35 percent of the 3h was liberated into the body water of the man and of the woman, respectively, reflecting reactions involving position 2. The metabolism of estradiol methyl ether was qualitatively similar to that observed previously for radioactive estradiol administered intravenously to the same subjects, as judged by the measurement of various urinary metabolites by reverse isotope dilution. Evidence was obtained for hydroxylation at position 2 without demethylation by the isolation of urinary 2-hydroxyestrone 3-methyl ether which retained 33% of the original 3H. This 3H was presumably at position 1, resulting from an NIH shift which does not occur during hydroxylation of estrone or estradiol. This was confirmed by subsequent administration of a mixture of 4- 14C and 3H-(methoxyl)-estradiol 3-methyl ether to the man. There was no evidence (by reverse isotope dilution) for 1-hydroxyestrone, 1-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether or 4-hydroxyestradiol 3-methyl ether as urinary metabolites of estradiol 3-methyl ether.

Studies on carcinogenic azo dyes. V. The NIH shift during the aryl hydroxylation of 3'-methyl-4-(dimethylamino)azobenzene and 3-methylacetanilide by the rat.

Studies on carcinogenic azo dyes. V. The NIH shift during the aryl hydroxylation of 3'-methyl-4-(dimethylamino)azobenzene and 3-methylacetanilide by the rat.

Mechanism of aromatic hydroxylation in fungi. Evidence for the formation of arene oxides.

Aromatic and aliphatic hydroxylations as well as O-demethylations occurred during metabolism of aromatic substrates by a range of fungi. para-Hydroxylation of monosubstituted aryl rings by fungi proceeded with migration and retention of deuterium label (NIH shift) and appeared to involve only arene 3,4-oxide intermediates, in common with plant and animal metabolism. The possibility of contributions from arene 1,2- or 2,3-oxide intermediates during ortho-hydroxylation is examined in the light of the results of oxygen-18 incorporation, the lack of deuterium–hydrogen primary kinetic isotope effects, and the wide range of deuterium migration and retention values. From these last experimental data no evidence for an arene oxide isomerase enzyme was found.

Thermal oxygen atom transfer from benz[cd]indazole 1,2-dioxide to benzenoid derivatives

Thermolysis of benz[cd]indazole 1,2-dioxide at 105–110 °C in the presence of various benzenoid derivatives gave benz[cd]indazole 1-oxide and oxidation products of the benzenoid substrates. Toluene was oxidised to benzyl alcohol (40% yield based on the dioxide), benzaldehyde (19%), benzoic acid (0.5%), o-cresol (11%), m-cresol (3%), and p-cresol ([graphic omitted]4%); an experiment with [4-3H]toluene showed no appreciable migration and retention of tritium (NIH shift) during formation of p-cresol. [4-2H]Anisole was converted mainly into deuteriated phenol (14%) and 2-hydroxyanisole (13%) together with 4-hydroxyanisole (4%) containing no significant amounts of deuterium. Naphthalene gave α- and β-naphthol in low yield. In contrast, 4-methylcinnoline 1,2-dioxide was stable in hot anisole. The results are compared with those reported for other, related oxidising systems.

Kinetic and mechanistic studies on the reaction of melilotate hydroxylase with deuterated melilotate.

Melilotate has been synthesized from melilotate by iodiantion followed by reductive deiodination in the presence of deuterated hydrazine. The deuterated melilotate has been employed in investigations of the reaction mechanism of melilotate hydroxylase. Stopped-flow spectrophotometry has revealed no isotope effect in the formation or decay of the oxygenated intermediate which is observed when reduced melilotate hydroxylase reacts with molecular oxygen. Steady-state analysis has corroborated this result, and in addition shows that there is no isotope effect in the reductive cycle of the enzyme mechanism. This analysis does reveal a reproducible 8% decrease in Vmax for the enzyme when using deuterated melilotate. These observations are compatible with the thesis that the above intermediate is an oxygenated form of the reduced flavine prosthetic group and that the last step of the proposed mechanism is rapid and involves a primary isotope effect. The existence of the NIH shift mechanism has been studied using combined gas chromatography-mass spectrometry. No evidence could be obtained for intramolecular migration of deuterium during the hydroxylation reaction. However, the small amount of migration expected when phenols are hydroxylated precludes elimination of the NIH shift as a possibility.

1-Tertiary-butylamino-3-(2,3-dimethylphenoxy)-2-propanol(D-32), 新β-遮断剤の代謝(第2報)血中濃度, 分布, 胆汁中の代謝物の構造

The distribution, blood level, and biliary metabolites of D-32, a new adrenergic β-blocking agent, were investigated in rats, rabbits, and dogs using 3H-labeled D-32. When D-32-3H was administered intravenously to the rat, radioactivity was taken up by the lung in higher concentration than by other organs and then decreased gradually with time. In the lung and brain, the main radioactivity accumulated was due to D-32, whereas most part of it was present as metabolites in the liver and kidneys. After both intravenous and oral administration, the half-life in plasma was approximately 45 min in rats, but was of the order of 110-160 min in dogs and rabbits. After oral administration there was virtually a complete absorption and peak blood levels of D-32 and active metabolites, 4'-hydroxy-D-32 and 3'-hydroxymethyl-D-32, were seen at about 1.5 hr after the administration. Further these metabolites appeared in the blood in amounts equal to D-32. On the other hand, after intravenous administration, total amount of active metabolites was only 1-10% of D-32 in the blood. These findings demonstrate that D-32 is absorbed easily from the gastrointestinal tract and is rapidly metabolized in the liver. The rat excreted approximately 40% of the dose in the bile during 24 hr. The major metabolites obtained from the bile were identified as 4'-hydroxy-D-32 4-O-glucuronide and 3'-hydroxymethyl-D-32 3-O-glucuronide by chemical degradation, nuclear magnetic resonance and mass spectra. D-32 undergoes metabolism in the rabbit liver involving NIH shift migration of the deuterium on metabolic 4'-hydroxylation of 4-deuterated D-32.

Aromatic hydroxylation mediated by flavin autoxidation: lack of the NIH shift

Aromatic hydroxylations by two flavin mono-oxygenase model systems show low values of the NIH shift suggesting that these oxidations are free-radical processes.

Metabolism of acetanilide with hepatic microsomes and reconstituted cytochrome monoxygenase systems

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-[ 3H]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.

Rearrangement of a non-arene oxide via an NIH shift related mechanism

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTRearrangement of a non-arene oxide via an NIH shift related mechanismDale L. Whalen and Angela M. RossCite this: J. Am. Chem. Soc. 1974, 96, 11, 3678–3679Publication Date (Print):May 1, 1974Publication History Published online1 May 2002Published inissue 1 May 1974https://doi.org/10.1021/ja00818a067RIGHTS & PERMISSIONSArticle Views131Altmetric-Citations7LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (277 KB) Get e-Alerts Get e-Alerts

Proceedings: Evidence for the NIH shift during pyrene hydroxylation.

Proceedings: Evidence for the NIH shift during pyrene hydroxylation.

Studies on drug metabolism by use of isotopes. X. Stability of the tritium attached to benzene ring (author's transl)

Basic studies were made on the applicability of a drug labeled with deuterium in the benzene ring for the metabolic study in man. After oral administration of l-ephedrine [arom. U-3H, α-14C] or l-ephedrine[p-3H, α-14C] to a rat or incubation with rabbit liver slices, the metabolites were separated and purified to a constant 3H/14C ratio. The percentage retention of tritium in each metabolite was calculated. Significant isotope effect of tritium was observed at the synthetic step where a propionyl group was introduced by the Friedel-Crafts reaction. Neither noticeable tritium loss nor isotope effect was observed in the formation of metabolites which were produced by the metabolic changes on the side-chain moiety of the substrate. This result supports the expected applicability. A near compete NIH shift of the label was observed during hydroxylation reactions. Percentage retention of tritium (over 90% in each case) was found with p-hydroxyephedrine. 3H/14C ratios of the glucuronide of p-hydroxyephedrine and p-hydroxyephedrine regenerated from the glucuronide were found to possess a significantly 1ower3H/14C ratio.

Monooxygenase activity in Cunninghamella bainieri: Evidence for a fungal system similar to liver microsomes

The fungus Cunninghamella bainieri effects the oxidative N-demethylation of aminopyrine, O-demethylation of 4-nitroanisole and anisole, the aryl hydroxylation of anisole, aniline, and naphthalene, and the reduction of nitro and azo groups. The hydroxylation of 4-[ 2H]-anisole and 2-[ 2H]-anisole proceeds with migration and retention of isotopic hydrogen (NIH shift). The above reactions and the formation of the trans-dihydrodiol of naphthalene and the incorporation of oxygen-18 from 18O 2 into the trans-dihydrodiol and hydroxylated anisole are characteristic of reactions catalyzed by the cytochrome P 450 monooxygenases of hepatic microsomes. The product ratios in these hydroxylations are very similar to those obtained using liver microsomes providing further evidence that the C. bainieri monooxygenase enzymes are similar to the liver monooxygenases. Furthermore, an epoxide hydrase enzyme similar to that present in hepatic microsomes must also be present in C. bainieri.

Oxygen walk as a complementary observation to the NIH shift

Oxygen walk as a complementary observation to the NIH shift

Formation of phenols from aromatic subtrates by plant and animal mono-oxygenases: The effect of adjacent deuteriums on the magnitude of the NIH shift of tritium

Hepatic metabolism of 4-[ 3H]acetanilide in vivo and in vitro yields 4-hydroxyacetanilide which retains, respectively, 40 and 62% of the tritium. When the 4-tritio-substrate contains adjacent deuteriums the retention of tritium is reduced to 26 and 40%. Hepatic metabolism of 4-[ 3H]anisole in vivo and in vitro yields 4-hydroxyanisole with 78% of the tritium. This retention is reduced to 62% in the corresponding 3,5-[ 2H 2]-4[ 3H]anisole. Similarly, the retention of tritium in trans-4-hydroxycinnamic acid derived by metabolism of trans-[4- 3H]cinnamic acid with chick pea microsomes is reduced from 91% to 68% by the presence of adjacent deuteriums in the substrate. Hydroxylation at the 4-position does not result in selective loss of tritium from the 3-position of acetanilide, anisole, or cinnamic acid. The above isotope effects indicate that isomerization of the probable arene oxide intermediates proceeds mainly via the keto-tautomer of the phenolic product.

Hydroxylation of phenylalanine by Pseudomonas sp.: measurement of an isotope effect following the NIH shift.

The conversion of [4-3H]- and [4-3H;3,5-2H2]phenylalanine into tyrosine by cultures of Pseudomonas sp. (ATCC 11 299a) has been studied. Comparison of the tritium retentions observed during hydroxylation of the two precursors showed an isotope effect, kD/kT= 2·8 ± 0·1 (kH/kD= 10 ± 1), for the final aromatisation reaction following the NIH shift. This value is compared with those of likely chemical models for the biochemical process.