Premercapturic acid metabolites of bromobenzene derived via its 2,3- and 3,4-oxide metabolites

1. A new premercapturic acid metabolite of bromobenzene was isolated from the urine of β-naphthoflavone-induced rats; using ¹H-n.m.r., FAB mass spectrometry and chemical degradation it was identified as S-(2-hydroxy-3-bromocyclohexa-3,5-dienyl)-N-acetylcysteine.

2. Two regioisomeric premercapturic acids apparently derived from bromobenzene-3,4-oxide were isolated as an inseparable 1 :1 mixture from the urine of phenobarbital-induced rats and characterized by similar means.

3. Acid dehydration of bromobenzene 3,4- and 4,3-premercapturic acids (mixture) afforded only p-bromophenylmercapturic acid, whereas acid dehydration of 3,2-premer-capturic acid gave both o- and m-bromophenylmercapturic acids. This implies a shift of sulphur in acid dehydration of the 3,4- and 3,2- but not the 4,3-premercapturic acids.

4. Base dehydration of the 3,4- and 4,3-premercapturic acid mixture gave a mixture of p- and m-bromophenylmercapturic acids, whereas base dehydration of the 3,2-premercapturic acid gave only m-bromophenylmercapturic acid. This indicates these premercapturic acids dehydrate by direct elimination without rearrangement.

5. The 3,2-premercapturic acid was detected only in the urine of BNF-induced animals, whereas the 3,4- and 4,3-premercapturic acids were detected in the urines of untreated as well as PB- and BNF-induced animals.

6. Together with earlier reports of the isolation of the 2,3-dihydrodiol, the isolation of the 3,2-premercapturic acid as a urinary metabolite of bromobenzene implies that bromobenzene-2,3-oxide is a discrete metabolite of bromobenzene and not merely a hypothetical intermediate.     

Bromo(monohydroxy)phenyl mercapturic acids. A new class of mercapturic acids from bromobenzene-treated rats.

Alkaline permethylation and GC/MS analysis of urinary mercapturic acids from rats given bromobenzene yielded several quinone-derived bromodimethoxythioanisole isomers as expected. Unexpectedly, seven bromomonomethoxythioanisole isomers were also observed, suggesting the presence of bromomonohydroxyphenyl mercapturic acids in the urine. Alkaline permethylation of synthetic 4- and 5-bromo-2-hydroxyphenyl mercapturic acid gave 4- and 5-bromo-2-methoxythioanisole, respectively, which were also observed after alkaline permethylation of urine from bromobenzene-treated rats, as was 2-bromo-4-methoxythioanisole. To explore the biosynthetic origin of the bromonohydroxyphenyl mercapturic acids, rats were separately dosed intraperitoneally with synthetic racemic 2-, 3-, or 4-bromophenyl mercapturic acid, or biosynthetic L-(-)-4-bromophenyl mercapturic acid, or a biosynthetic mixture of the 3,4- and 4,3-premercapturic acids from bromobenzene, and their urine (0-24 hr) analyzed by alkaline permethylation and GC/MS. The administered mercapturic acids and premercapturic acids were partly excreted unchanged (60-80% and 24%, respectively), but both gave rise to bromomonohydroxyphenyl mercapturic acids (0.1-5.2% of dose). Results indicated that the latter could be formed by 1) dehydrogenation of premercapturic acids and 2) hydroxylation of mercapturic acids (or their cysteine equivalents).     

The mechanism of formation of o-bromophenol from bromobenzene

Both o-bromophenol and p-bromophenol are formed from bromobenzene in rat liver microsomes. It has been established that p-bromophenol is formed via bromobenzene-3,4-oxide, but o-bromophenol could conceivably arise via either the 2,3-epoxide or the 1,2-epoxide or by direct insertion of oxygen. As described in the present article, we have isolated and identified bromobenzene 2,3-dihydrodiol as a microsomal metabolite of bromobenzene. Identification of the dihydrodiol therefore indicates the formation of its obligatory precursor, bromobenzene-2,3-oxide. Moreover, using bromo(2,4,6-2H3)benzene, we have clarified the mechanism of formation of o-bromophenol from bromobenzene. The rate of formation of o-bromophenol from bromobenzene and bromo(2,4,6-2H3)benzene in liver microsomes from 3-methylcholanthrene-treated rats was 0.72 +/- 0.02 and 0.74 +/- 0.06 nmol/mg/min (kH/kD = 0.99), respectively. The lack of a significant isotope effect indicates that the hydroxylation of bromobenzene to o-bromophenol is not by a direct insertion mechanism. Furthermore, the mass spectrum of o-bromophenol isolated from a microsomal incubation with bromo(2,4,6-2H3)benzene indicated that 70% of the product retained all three deuterium atoms. These results are consistent with the view that o-bromophenol is formed from the 2,3-epoxide intermediate but do not preclude formation by the addition of oxygen to the 2-position carbons followed by an NIH shift and rearrangement before an epoxide is formed.     

Hepatic microsomal epoxidation of bromobenzene to phenols and its toxicological implication

In vitro microsomal hepatic epoxidation of bromobenzene in rats and mice is presented in this study. Formation of o-bromophenol via bromobenzene-2,3-epoxide and p-bromophenol via bromobenzene-3,4-epoxide was assayed enzymatically and identified by a new, rapid and sensitive gas-liquid chromatography method using electron capture detection. Pretreatment of the animals with phenobarbital caused significant increases in both pathways whereas 3-methylcholanthrene or β-naphthoflavone caused a selective and marked increase of only the 2,3-epoxide pathway. Sodium dodecyl sulfate-gel electrophoresis of microsomal preparations resolved multiple forms of cytochrome P-450 and indicated that different forms of the heme protein were responsible for the formation of o-bromophenol and p-bromophenol. it is of interest that various inducers augment particular pathways for a common substrate especially since bromobenzene-3,4-epoxide and not the bromobenzene-2,3-epoxide has been proposed as the cytotoxic reactive metabolite of bromobenzene.     

Detection and half-life of bromobenzene-3,4-oxide in blood

Bromobenzene-3,4-oxide can be detected in venous blood of rats by trapping it as the corresponding 35[S]glutathione conjugates. More bromobenzene-3,4-oxide is detected in venous blood of rats treated with phenobarbital and diethyl maleate than in venous blood of rats treated with phenobarbital alone. The half-life of bromobenzene-3,4-oxide in venous blood was about 13.5 s. Bromobenzene-3,4-oxide may contribute to the extrahepatic covalent binding and presumably the toxicity observed after bromobenzene administration. The present technique may be used to determine in blood, the presence or absence of other reactive metabolites that form glutathione conjugates.     

Bromobenzene metabolism in vivo and in vitro. The mechanism of 4-bromocatechol formation

The metabolism of bromobenzene has been examined in isolated hepatocytes and liver microsomes from phenobarbital-induced rats and in phenobarbital-induced rats in vivo. The metabolite profile produced upon incubation of isolated rat hepatocytes with bromobenzene differed with the hepatocyte concentration. At a low hepatocyte concentration (0.5 x 10(6) cells/ml), 4-bromophenol was the major metabolite, while at higher hepatocyte concentrations (2.0 and 5.0 x 10(6) cells/ml) bromobenzene-3,4-dihydrodiol was the major metabolite. 4-Bromophenol was the primary metabolite in incubations with rat liver microsomes. In vivo, 3- and 4-bromophenol were more predominant, with very little dihydrodiol formed. 4-Bromocatechol, a potentially toxic metabolite of bromobenzene, was formed in vivo as well as in isolated hepatocytes and microsomes. However, the mechanism of catechol formation differed, as determined by the retention of a deuterium label at the para position of bromobenzene. In microsomes, 4-bromophenol was the predominant precursor metabolite of 4-bromocatechol. In isolated hepatocytes, although the relative contribution of 4-bromophenol as the bromocatechol precursor differed with hepatocyte concentration, bromobenzene-3,4-dihydrodiol was the predominant precursor at all concentrations. In vivo, as in isolated hepatocytes, 4-bromocatechol was formed primarily via bromobenzene-3,4-dihydrodiol.     

Bromobenzene metabolism in isolated rat hepatocytes. 18O2 incorporation studies

Bromobenzene metabolites have been determined in incubations of hepatocytes isolated from untreated, phenobarbital-treated, and beta-naphthoflavone-treated rats. The total formation of bromobenzene metabolites was increased 9-fold in incubations with hepatocytes isolated from phenobarbital-treated rats, and the percentage of total metabolites recovered as bromobenzene-3,4-dihydrodiol and 4-bromocatechol was more than doubled, compared to incubations using hepatocytes from untreated rats. The formation of 2-bromophenol and bromobenzene-2,3-dihydrodiol was increased more than 10-fold in incubations of hepatocytes from beta-naphthoflavone-treated rats, as compared to those of hepatocytes from untreated rats, but recovery of 4-bromocatechol was unchanged. The mechanism of 4-bromocatechol formation from bromobenzene was investigated by examining the incorporation of 18O from 18O2 and H218O into 4-bromocatechol during incubations of bromobenzene with hepatocytes from untreated and phenobarbital-treated rats. Potential metabolic precursor molecules of 4-bromocatechol were also incubated individually with isolated hepatocytes, in order to clarify their roles in 4-bromocatechol formation. The results of these studies show that 4-bromocatechol is formed in intact cells almost exclusively from bromobenzene-3,4-dihydrodiol, rather than from the bromophenols. The bromophenols are, instead, mostly conjugated. The rapid and extensive conjugation of the bromophenols by intact cells may restrict their role as precursors of 4-bromocatechol, while bromobenzene 3,4-dihydrodiol is well converted into 4-bromocatechol by hepatocytes.     

Detection and half-life of bromobenzene-3,4-oxide in blood

1. Bromobenzene-3,4-oxide can be detected in venous blood of rats by trapping it as the corresponding ³⁵[S]glutathione conjugates.

2. More bromobenzene-3,4-oxide is detected in venous blood of rats treated with phenobarbital and diethyl maleate than in venous blood of rats treated with phenobarbital alone.

3. The half-life of bromobenzene-3,4-oxide in venous blood was about 13·5 s.

4. Bromobenzene-3,4-oxide may contribute to the extrahepatic covalent binding and presumably the toxicity observed after bromobenzene administration.

5. The present technique may be used to determine in blood, the presence or absence of other reactive metabolites that form glutathione conjugates.     

Conversion of bromobenzene to 3-bromophenol. A route to 3- and 4-bromophenol through sulfur-series intermediates derived from the 3,4-oxide

Premercapturic acids derived from bromobenzene 3,4-oxide were found to act as precursors of 3- and 4-bromophenol in the rat and guinea pig. The 4-S- and 3-S- positional isomers used in this study were rat urinary metabolites and were prepared in unlabeled, radioactive, and 2,4,6-d3-labeled forms. These are not guinea pig urinary metabolites; the guinea pig does not completely acetylate cysteine conjugates, and this effect leads to urinary products arising from deamination of the cysteine moiety rather than to urinary premercapturic acids. Conversion to phenols was found to be much greater in the guinea pig than in the rat. We interpret our results as indicating that cysteine adducts, rather than the N-acetylcysteine adducts which were administered, are required intermediates in this metabolic route to 3- and 4-bromophenol. This route to phenols may be the major mode of phenol formation for many aromatic compounds. Sulfur-series metabolic products from bromobenzene also include thiocatechols, and these metabolites may be responsible for the hepatotoxicity of bromobenzene in high dosage.     

6-Chloro-1,2,4-triazolo[4,3-b]pyrido[2,3-d]-and [3,2-d]pyridazines - Synthesis and Structure Determination

5,8-Dichloropyrido[2,3-d]pyridazine ( 2 ) gave with hydrazine hydrate in dioxane 5-chloro-8-hydrazino- and 8-chloro-5-hydrazinopyrido[2,3-d]pyridazines 3 and 4 . When 3 and 4 were allowed to react with formic acid they gave a mixture of the 6-chloro-1,2,4-triazolo[4,3-b]pyrido[2,3-d] - and [3,2-d]pyridazines ( 5 and 6 ).     

Dihydroxylated mercapturic acid metabolites of bromobenzene.

Bromobenzene is metabolized to electrophilic epoxides and quinones which covalently bind to protein sulfur nucleophiles, yet no quinone-derived mercapturic acid metabolites of bromobenzene have been reported. To search for them, phenobarbital-induced Sprague-Dawley rats were dosed (0.5-1.5 mmol/kg, ip) with either bromobenzene, 2-, 3-, or 4-bromophenol (BP), 3- or 4-bromocatechol (BC), or 2-bromohydroquinone (BHQ). Urine (0-24 h) was alkaline permethylated (2 N NaOH/CH3I/80 degrees C), and the resulting thioanisole derivatives were analyzed by GC/MS. Three dimethoxythioanisoles and eight bromodimethoxythioanisoles were formed by alkaline permethylation of urine of rats treated with bromobenzene or 3-BP; alkaline permethylation of urine from rats in other treatment groups afforded characteristic subsets of these derivatives. The major thioanisole from all groups except 3-BC or 4-BC was 2,5-dimethoxythioanisole, which arises from (2,5-dihydroxyphenyl)mercapturic acid. The latter was isolated from rat urine and is the first debrominated metabolite of bromobenzene reported to date. Its formation from both 4-bromophenol and BHQ requires two parallel mechanisms for bromine loss: (1) nucleophilic addition to 1,4-benzoquinone formed by hydroxylative debromination of 4-bromophenol and (2) nucleophilic displacement of bromine from 2-bromo-1,4-benzoquinone by sulfur. The yields of quinone-derived mercapturic acids formed from bromobenzene are quite low (less than 1% of dose) compared to the high yields of epoxide-derived mercapturic acids (40% of dose). Potential reasons for this are discussed.     

Bromobenzene metabolism in the rat and guinea pig

The metabolism of bromobenzene was studied in the rat and guinea pig with respect to three considerations: the dose and species dependence of 3-bromophenol excretion; the formation of methylthio analogs of dihydrodiols and catechols; and the identification of acidic bivalent sulfur metabolites. In the guinea pig, 3-bromophenol was the major monohydric phenolic metabolite under conditions of both relatively low and relatively high dosage. In the rat, 3-bromophenol and 4-bromophenol were formed in approximately equal amounts. 2-Bromophenol was a minor metabolite in both species. Methylthio analogs of dihydrodiols were found as guinea pig, but not rat, metabolites. Two di(methylthio)dihydroxytetrahydrobromobenzene metabolites were excreted by the rat but not by the guinea pig. These methylthio compounds have not been reported in earlier studies of bromobenzene metabolism. In the guinea pig, the acidic urinary metabolites were a mercaptoacetate, a mercaptolactate, and a mercapturate. In the rat, the acidic metabolites were a mercapturic acid and premercapturic acids. This species difference in urinary acids indicates a difference in acetylation/deacetylation processes for cysteine conjugates.     

Absorption, distribution and metabolic fate of 2,3-dihydro-9H-isoxazolo-[3,2-b]quinazolin-9-one (W2429).

After oral administration to rats, mice, beagle dogs and human volunteers, 2,3-dihydro-9H-isoxazolo [3,2-b]-quinazolin-9-one (W 2429) is readily absorbed. The initial half-lives for the elimination from the circulation of these four species are about 240, 20, 40 and 120 min, respectively. Studies in the rat, using 9-14C-W-2429 and 3a-14C-W-2429, showed that more than half of the radioactivity is excreted in the urine. The major urinary metabolite in the rat and dog is a conjugated form of W-2429. Metabolic cleavage of the pyrimidone ring of W-2429 yields 3-(o-carboxyphenylimino)isoxazolidine, which in turn is converted to anthranilic and malonic acids. Anthranilic acid is acetylated and hydroxylated, and these products are excreted partly in the form of their glycine and glucuronic acid conjugates. The isoxazole ring of W-2429 is also dehydrogenated during metabolism to yield 9H-isoxanzolo-[3,2-b]quinazolin-9-one.     

Detection of adducts of bromobenzene 3,4-oxide with rat liver microsomal protein sulfhydryl groups using specific antibodies

The hepatotoxicity of bromobenzene (BB) has been attributed to covalent modification of cellular proteins by reactive metabolites generated during its oxidative biotransformation. Much of the net covalent binding which occurs originates via quinone metabolites, but bromobenzene 3,4-oxide (BBO), which is the reactive metabolite thought to be most significant toxicologically, also arylates protein side chains, although to a lesser extent. To facilitate the detection, isolation, and identification of rat liver proteins specifically adducted by BBO, we raised polyclonal antibodies capable of recognizing S-(p-bromophenyl)cysteine moieties (anti-BP) by immunizing rabbits with p-bromophenylmercapturic acid conjugated to keyhole limpet hemocyanin. The antiserum had a high titer, showed a high specificity for hapten in competitive ELISA with hapten analogues, and performed well in Western blot experiments using synthetically haptenized control proteins. When used for Western analysis of protein fractions from in vitro incubations of rat liver microsomes with [14C]BB, affinity-purified anti-BP recognized a limited number of bands, each of which also contained 14C. One of these bands corresponds to hydrolase B, a nonspecific esterase known to contain one free sulfhydryl group and previously shown to be a target protein for [14C]BB metabolites.     

Specific determination of bromobenzene metabolites by high-performance liquid chromatography

A chromatographic method for the quantitative and simultaneous determination of phenolic and mercapturic acid type metabolites of bromobenzene is presented. Acid hydrolysis (1.5 N HCl, 100 degrees C, 10 min) yielded unconjugated bromophenols. After extraction (ethyl acetate, pH 2), evaporation to dryness and dissolution in methanol, samples were analysed by high-performance liquid chromatography (HPLC) with a c18 reverse-phase column and a phosphate buffer (0.1 M,pH 9 + 5 mM tetrabutylammonium dihydrogen phosphate) - acetonitrile gradient with ultraviolet detection (225 nm). Benzylmercapturic acid and o-chlorophenol were used as internal standards. Using this method, it was possible to determine simultaneously o, m- and p-bromophenols together with o-, m- and p-bromophenylmercapturic acids. Additional validation data were obtained by analysis of urine samples from rats treated with bromobenzene. This new method was compared to existing procedures.     

Etodolac, a novel antiinflammatory agent. The syntheses and biological evaluation of its metabolites

The syntheses of five metabolites of the antiinflammatory drug etodolac (1,8-diethyl-1,3,4,9-tetrahydropyrano-[3,4-b]indole-1-acetic acid) are described, viz. 6-hydroxyetodolac, N-methyletodolac, 4-ureidoetodolac, 8-(1'-hydroxy)etodolac, and 4-oxoetodolac. These syntheses were used to confirm the identities of the metabolites. The metabolites themselves, as well as the previously reported metabolite 7-hydroxyetodolac, were tested in a rat adjuvant edema model and in vitro for their capacity to block prostaglandin production in chondrocyte cells. All either were inactive or possessed only marginal activity. The isolation of N-methyletodolac and 4-oxoetodolac from human and rat urine, respectively, is also described.     

4-Vinylphenol excretion suggestive of arene oxide formation in workers occupationally exposed to styrene

The toxicity of styrene has often been attributed to the formation of reactive epoxide intermediate, styrene-7,8-oxide. It has been suggested that in addition, an arene oxide, styrene-3,4-oxide, is a metabolite of styrene. Styrene-3,4-oxide is easily converted to corresponding phenols. In this study the presence of 4-vinylphenol in the urine is verified by gas chromatography/mass spectrometry and its quantity compared to mandelic acid excretion. Both 4-vinylphenol and mandelic acid were detected in the urine samples of workers occupationally exposed to styrene. No 4-vinylphenol was found in urine samples of unexposed individuals. The correlation between mandelic acid and 4-vinylphenol was fairly good (r = 0.93); increasing excretion of mandelic acid was also accompanied by increasing amounts of 4-vinylphenol in the urine. The interindividual variation of the 4-vinylphenol/mandelic acid excretion ratio was small, the mean ratio being about 0.3%. The presence of 4-vinylphenol in the urine of workers exposed to styrene suggests that, in man, styrene is also metabolized via arene oxidation. However, when the arene oxidation of styrene is compared to vinyl group oxidation the latter appears to be at least quantitatively by far the more important metabolic pathway.     

Taurine conjugates as metabolites of arylacetic acids in the ferret.

1. The pattern of conjugation in the ferret of 8 arylacetic acids and, for comparison, benzoic acid and 4-nitrobenzoic acid was examined. 2. The arylacetic acids, phenylacetic, 4-chloro- and 4-nitro phenylacetic, alpha-methylphenylacetic (hydratropic), 1- and 2-naphthylacetic and indol-3-ylacetic acids, were excreted in the urine as taurine and glycine conjugates. Diphenylacetic acid did not form an amino acid conjugate and was excreted as a glucuronide. 3. The taurine conjugate was the major metabolite of 4-nitrophenylacetic, alpha-methylphenylacetic, 1- and 2-naphthylacetic and indol-3-ylacetic acids, whereas the glycine conjugate was the major metabolite of phenylacetic and 4-chlorophenylacetic acids. Taurine conjugation did not occur with benzoic and 4-nitrobenzoic acids which were excreted as glycine and glucuronic acid conjugates. 4. Phenacetylglutamine and 4-hydroxyphenylacetic acid were minor urinary metabolites of phenylacetic in the ferret. 5. A number of taurine conjugates of aliphatic and aromatic acids were synthesized and their characterization and properties were studied. The role of taurine as an alternative to glycine in the metabolic conjugation of arylacetic acids is discussed.     

Determination of 5-(3,4-Dihydroxy-1,5-cyclohexadien-1-yl)-5-phenylhydantoin (Dihydrodiol) and quantitative studies of phenytoin metabolism in man

A routine gas chromatographic assay for urinary 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), the major metabolite of phenytoin (PHT) in man, was adapted to allow quantitation of 5-(3,4-dihydroxy-1,5-cyclohexadien-1-yl)-5-phenylhydantoin (Dihydrodiol, DHD) is based on the observation that acid-catalyzed dehydration of DHD quantitatively yields a mixture of p-HPPH and m-HPPH in a reproducible molar ratio of 56:44p-HPPH: m-HPPH and on the assumption that all m-HPPH found in urine after heating with acid has been derived from DHD. The urinary DHD content was verified by a "specific" method in which urine was incubated with beta-glucuronidase and the released phenolic metabolites completely removed by extraction. Subsequent acid-catalyzed dehydration of the remaining DHD yielded p-HPPH and m-HPPH, from the sum of which the original DHD concentration in urine could be calculated. In all of the urine samples from PHT patients examined to date, there was close agreement between the DHD values obtained by the "specific" method and those calculated from m-HPPH, in the simple acid-hydrolysis method. It can be inferred that much the greater part (greater than 90%) of m-HPPH found in human urine after acid treatment has been derived from DHD. All samples of urine after acid treatment has been derived from DHD. All samples of urine from PHT patients examined have shown detectable quantities of DHD. The methods described here may be useful in a survey of PHT patients to reveal unusual patterns of PHT metabolism and to permit recognition of possible associations between such unusual patterns and the occurrence of adverse reactions.     

Metabolic hydroxylation of the thiophene ring: Isolation of 5-hydroxy-tienilic acid as the major urinary metabolite of tienilic acid in man and rat

The metabolism of tienilic acid, a drug containing a thiophene ring, was reinvestigated in man, rat and dog. The major urinary metabolite in man and rat was isolated and completely characterized by comparison with a synthetic compound. This metabolite derives from the hydroxylation of the thiophene ring of tienilic acid in position 5. Its isomers, 3- and 4-hydroxy-tienilic acids, were synthetized but could be detected neither in man nor in rat urine.Because of its particular behaviour toward electrophiles, 5-hydroxy-tienilic acid was found to react with diazomethane with the formation of a complex mixture of methylated products. This made difficult its measurement by a previously described GLC technique, after acidic extraction and methylation by diazomethane. A new very simple assay using HPLC and direct injection of urine is described in this paper. This assay led to a very precise and reproductible determination of tienilic acid and its hydroxylated metabolite in urine.Up to 50% of tienilic acid is excreted in man or rat urine as 5-hydroxy-tienilic acid whereas this metabolite does not appear in dog urine. These data describe the first example of metabolic hydroxylation of the thiophene ring.