Pathways of formation of 2-, 3- and 4-bromophenol from bromobenzene. Proposed mechanism for C-S lyase reactions of cysteine conjugates.

Bromobenzene is metabolized by the rat and guinea pig to 2-, 3- and 4-bromophenol. 3-Bromophenol is formed through the sulfur-series pathway to phenols. This route involves the enterohepatic circulation; the key intermediate is the S-(2-hydroxy-4-bromocyclohexa-3,5-dienyl)-L-cysteine derived from the 4-S-glutathione conjugate of the 3,4-oxide. A sulfonium ion C-S lyase reaction is proposed in order to account for the pyridoxal phosphate-dependent cleavage/aromatization step, and a C-S beta-lyase reaction sequence is also proposed for the formation of bromodihydrobenzene thiolols. This route of phenol formation may prove to be a general one for aromatic hydrocarbons and closely related compounds that show arene oxide conjugation with glutathione. 2-Bromophenol is formed predominately by spontaneous isomerization of the 2,3-oxide. 4-Bromophenol is formed by the sulfur-series route from the S-(2-hydroxy-5-bromocyclohexa-3,5-dienyl)-L-cysteine. Additional in vivo routes to 3- and 4-bromophenol involve dehydration/aromatization of the 3,4-dihydro-3,4-diol, possibly by way of conjugates; these routes have transient ketonic intermediates. The pathways from bromobenzene to phenols and to sulfur-containing metabolites derived from premercapturic acids show species and dosage variation.

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.

Analytical methods for the study of urinary thioether metabolites in the rat and guinea pig.

Methods are described for the isolation and identification of three classes of bivalent sulfur metabolites characterized as neutral methylthio ethers, ethyl acetate-soluble acidic thioethers and ethyl acetate-insoluble acidic thioethers from rat and guinea pig urine. After extraction of the metabolites by the ammonium carbonate-ethyl acetate procedure, the individual metabolites are separated by capillary gas chromatography and/or by high-performance liquid chromatography with both mu Bondapak C18 and Porasil columns. Identification of the metabolites is based on gas chromatography-mass spectrometry (electron impact) and on fast atom bombardment mass spectrometry. Interesting species differences in metabolism were observed. The major ethyl acetate-soluble acidic thioethers in rat urine are mercapturic acids. In contrast, in the guinea pig a new pathway involving mercaptopyruvic, mercaptolactic and mercaptoacetic acids is operative. The thioether metabolites of styrene oxide and phenanthrene are described, but the procedures have been applied in studies of several drugs and environmental chemicals in our laboratory.

Metabolism of bromobenzene. Analytical chemical and structural problems associated with studies of the metabolism of a model aromatic compound.

Our studies of bromobenzene metabolism have shown that the 3,4-oxide is metabolized to 3- and 4-bromophenol through an extended glutathione pathway. The mechanism of sulfur elimination from a dihydrobromobenzene metabolite is not known, although it is known that the aromatization reaction will occur in a 9000-g supernatant fraction of rat liver. The hepatotoxic and nephrotoxic metabolites of bromobenzene are most likely bromthiocatechols and a bromothiopyrogallol, respectively.

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.

Formation of methylthio metabolites of indene in the guinea pig and the rat.

The formation of methylthio metabolites of epoxides has been shown to be a significant route of metabolism in some species. Several aspects of this metabolic conversion for indene were examined. Two isomers of hydroxy(methylthio)indane were found in the urine of guinea pigs administered indene (14.3 and 100 mg/kg, ip). The major isomer, 2-hydroxy-1-methylthioindane (I) was present as 6-9% of the administered dose after 24 hr, while lower amounts (0-0.6%) of a minor isomer (II) were observed. A significant amount of isomer I was found as a urinary metabolite of indene oxide (14% of 12.5 mg/kg, ip). To further elucidate the route of formation of I, the glutathione (I-GLU) and mercapturic acid (I-MER) conjugates of indene oxide were synthesized and administered to the guinea pig. The methylthio metabolite I was present as a significant urinary metabolite of both conjugates of indene oxide, comprising 9.6% and 5.7% of the dose of I-GLU (5 mg, ip) and I-MER (4 mg, ip), respectively. These results show that the formation of a hydroxy(methylthio)indane is a significant route of metabolism for indene and indene oxide in the guinea pig, and that this metabolite arises via further metabolism of conjugates in the glutathione pathway. In the rat, isomer I is a minor metabolite. Mechanistic aspects of the formation of these thioether metabolites are discussed.

Synthesis and elimination reactions of methylsulfonium ions formed from styrene oxide and methylthio compounds related to methionine and cysteine.

Methylsulfonium compounds were prepared through the nucleophilic addition of a number of methylthio reagents to styrene oxide. In the absence of water, methylsulfonium ions from styrene oxide were converted to 2-hydroxy-1-methylthio-1-phenylethane (I) and 1-hydroxy-2-methylthio-1-phenylethane (II) by brief heating. In the presence of water, the glycol 1,2- dihydroxyphenylethane (III) was obtained along with I and II. The addition and elimination reactions were combined in sequences that were carried out in an organic solvent (acetone) or in aqueous solutions containing acetone or methanol. With L-methionine as the reagent, both reactions proceeded in sequence at 37 degrees C in water/methanol (80:20) to give I, II, and III as reaction products. These methods can be used to prepare the methylthio metabolites of styrene oxide.

Metabolism of styrene oxide in the rat and guinea pig.

The metabolism of styrene oxide has been studied in the rat and guinea pig, with emphasis upon bivalent sulfur metabolites. Methylthio analogs of phenylethylene glycol, with the methylthio group in both possible positions, were found as urinary metabolites in both species. These compounds were present in more than trace amounts. The excretion of 2-hydroxy-1-methylthio-1-phenylethane amounted to about 7% of the administered dose in the guinea pig, and about 2% in the rat, in o-24 hr urine samples. The positional isomer 1-hydroxy-2-methylthio-1-phenylethane was excreted in lesser amounts in both species. Acidic urinary metabolites derived from glutathione conjugates are species dependent. In this study, the only products observed in the rat were the mercapturic acids expected as a result of reaction of the oxide with glutathione. In the guinea pig, the major bivalent sulfur acids were the corresponding mercaptoacetic acids. Other related metabolites included a mercaptolactic and a mercaptopyruvic acid, together with one of the mercapturic acids. These metabolites result from partial acetylation or acetylation/deacetylation of cysteine or cysteinylglycine adducts. The hitherto unobserved dihydrodiol formed via an arene oxide was found as a minor metabolite for both styrene and styrene oxide.

Dose-dependent kinetics of quinidine in the perfused rat liver preparation. Kinetics of formation of active metabolites.

The disposition of quinidine and the kinetics of metabolite formation were studied in the once-through perfused rat liver preparation by the stepwise increase in quinidine concentration. Dose-dependent kinetics of quinidine were observed; the steady-state hepatic extraction ratio decreased from 0.97 to 0.37 when input quinidine concentration varied from 1.34 to 33.9 micrograms/ml. Moreover, the formation of 3-hydroxyquinidine (detected only in perfusate as unconjugated 3-hydroxyquinidine) remained relatively linear while the formation of O-desmethylquinidine (present in bile and perfusate mostly as conjugates) apparently approached saturation kinetics within the quinidine concentrations used. The dose-dependent character of quinidine elimination cannot be attributed to changes in drug binding, as the blood/plasma ratio (4.07) and the degree of drug unbound in plasma (66.5%) remained unaltered for the quinidine concentrations used.

Urinary bivalent sulfur metabolites of phenanthrene excreted by the rat and guinea pig.

After the administration of phenanthrene (50 mg/kg, ip) to young adult male rats and guinea pigs, a series of bivalent sulfur urinary metabolites were isolated and characterized by gas chromatography and gas chromatography-mass spectrometry. Seven methylthio metabolites were isolated from the neutral fraction of hydrolyzed rat urine, whereas only two were detected in guinea pig urine. The major methylthio metabolite excreted by each species was 9-hydroxy-10-methylthio-9, 10-dihydrophenanthrene. This was observed as a second-day metabolite in the rat, and its appearance was accompanied by 9-phenanthrol. In addition to the methylthio compounds, which were excreted predominantly as glucuronides, six acidic bivalent sulfur metabolites were isolated from hydrolyzed rat urine and identified by GC/MS; five were present in hydrolyzed guinea pig urine. The major acidic metabolite in hydrolyzed rat urine was the hydroxydihydromercapturic acid N-acetyl-S-(9-hydroxy-9, 10-dihydro-10-phenanthryl)-L-cysteine. The major acidic metabolite in guinea pig urine was the mercaptoacetic acid S-(9-hydroxy-9, 10-dihydro-10-phenanthryl)mercaptoacetic acid, but the hydroxydihydromercapturic acid was also present.

Identification of new sulfur-containing metabolites of naphthalene in mouse urine.

Seven acidic sulfur-containing metabolites were isolated from mouse urine following administration of naphthalene. The metabolites have been identified as (1-hydroxy-1,2-dihydro-2-naphthalenylthio)acetic acid (I), 2-hydroxy-3-(1-hydroxy-1,2-dihydro-2-naphthalenylthio)propanoic acid (II), (1,2,3-trihydroxy-1,2,3,4-tetrahydro-4-naphthalenylthio)acetic acid (III), and N-acetyl-S-(1-hydroxy-1,2-dihydro-2-naphthalenyl)-L-cysteine (IV). The dehydration products of I, II, and IV, namely 1-(naphthalenylthio)acetic acid (V), 2-hydroxy-3-(1-naphthalenylthio)propanoic acid (VI), and N-acetyl-S-(1-naphthalenyl)-L-cysteine (VII), respectively, were also present in several urinary extracts. Nine methylthio derivatives were identified in the neutral extract of urine. These metabolites were the following: 1-methylthionaphthalene, trans-1-hydroxy-2-methylthio-1,2-dihydronaphthalene, two stereoisomeric 1,2,3-trihydroxy-4-methylthio-1,2,3,4-tetrahydronaphthalenes, 1,3-di(methylthio)-2,4-dihydroxy-1,2,3,4-tetrahydronaphthalene, 1,4-di(methylthio)-2,3-dihydroxy-1,2,3,4-tetrahydronaphthalene, two methylthiohydroxy-naphthalenes, and a methylthiodihydroxydihydronaphthalene. Following intraperitoneal administration of N-acetyl-S-(1-hydroxy-1,2-dihydro-2-naphthalenyl)-L-cysteine to mice, the acidic metabolites I, II, and unchanged IV were found. The gas-chromatographic and gas chromatographic-mass spectral properties of the methyl ester-trimethylsilyl derivatives of the acidic sulfur metabolites of naphthalene are presented.

Metabolite kinetics: formation of acetaminophen from deuterated and nondeuterated phenacetin and acetanilide on acetaminophen sulfation kinetics in the perfused rat liver preparation.

The role of hepatic intrinsic clearance for metabolite formation from various precursors on subsequent metabolite elimination was was investigated in the once-through perfused rat liver preparation. Two pairs of acetaminophen precursors: [14C] phenacetin-d5 and [3H] phenacetin-do, [14C] acetanilide and [3H] phenacetin were delivered by constant flow (10 ml/min/liver) either by normal or retrograde perfusion to the rat liver preparations. The extents of acetaminophen sulfation were compared within the same preparation. The data showed that the higher the hepatocellular activity (intrinsic clearance) for acetaminophen formation, the greater the extent of subsequent acetaminophen sulfation. The findings were explained on the basis of blood transit time and metabolite "duration time." Because of blood having only a finite transit time in liver, the longer the drug requires for metabolite formation, the less time will remain for metabolite sulfation and the less will be the degree of subsequent sulfation. Conversely, when the drug forms the primary metabolite rapidly, a longer time will remain for the metabolite to be sulfated in liver to result in a greater degree of metabolite sulfation. Finally, the effects of hepatic intrinsic clearances for metabolite formation and zonal distribution of enzyme systems for metabolite formation and elimination in liver are discussed.

Metabolism of carbamazepine.

Thirty-two metabolites of carbamazepine, in addition to the 10,11-epoxide, have been isolated from enzymatically hydrolyzed urine by HPLC and identified by gas-chromatographic and mass-spectrometric techniques. Eight were sulfur-containing methylthio, methylsulfinyl, or methylsulfonyl derivatives. Eighteen new metabolites, including five iminostilbene derivatives that have not been described earlier, were found. All of the metabolites were formed by processes involving epoxidation and a peroxidation, the structures of the metabolites support the hypothesis that multiple epoxides and a cyclic peroxide are involved in the in vivo metabolism of carbamazepine by the rat and man.

Identification and synthesis of the isomeric tetrahydroxytetrahydronaphthalene metabolites excreted in rat urine.

The structures of three isomeric 1,2,3,4-tetrahydroxytetrahydronaphthalene metabolites of naphthalene have been confirmed by synthesis. Six isometric 1,2,3,4-tetrahydroxy-1,2,3,4-tetrahydronaphthalene structures occurring in for dl-pairs and two mesoforms have been synthesized. Five were synthesized by the action of osmium tetroxide on the cis- and trans-1,2-dihydrodiols and cis- and trans-1,4-dihydrodiols. The sixth isomeric tetrahydrotetrol was synthesized by hydrolysis of trans-1,2-dihydroxy-syn-3,4-epoxy-1,2,3,4-tetrahydronaphthalene (the syn-dihydrodiolepoxide of naphthalene). By comparing the gas-chromatographic and mass-spectrometric properties of the synthetic and urinary tetrahydrotetrols, two of the urinary metabolites were identified as 1 beta, 2 alpha, 3 alpha, 4 beta- and structure of the third tetrahydrotetrol metabolite, 1 beta, 2 alpha, 3 beta, 4 alpha-tetrahydroxy-1,2,3,4-tetrahydronaphthalene, identified in earlier studies, was confirmed.

The effect of 3-methylcholanthrene, Aroclor 1254, and phenobarbital induction on the metabolism of biphenyl by rat and mouse 9000g supernatant liver fractions.

The metabolism of biphenyl in vitro in 9000 g supernatant fractions from livers of noninduced rats and mice was compared with the metabolism in similar liver fractions of rats and mice induced with 3-methylcholanthrene (3-MC), Aroclor 1254 and phenobarbital (PB). Analyses were carried out by open-tubular capillary gas chromatography and by gas chromatography/mass spectrometry. The major metabolite of biphenyl in all instances was 4-hydroxybiphenyl, and only very small amounts of diols were observed before induction. After induction by 3-MC, an increase was observed in all monohydroxybiphenyls for rat liver 9000 g supernatant fractions, and the 2,5- and 3,4-diols were present in greater amount. The effect of Aroclor 1254 induction resembled that observed for 3 MC induction. Induction of PB showed very little effect. For the mouse, induction with 3-MC resulted in an increase in all monohydroxybiphenyls and an increase in 2,5- and 3,4-diols. Induction with Aroclor 1254 resulted in an increase in 2-hydroxybiphenyl formation, but not in 4-hydroxylation. Thus the effects of 3-MC and Aroclor 1254 induction on biphenyl metabolism are similar in the rat but not in the mouse. Very little change was observed after PB induction. The effect of 3-MC induction (rat and mouse) on hydroxylation of monohydroxybiphenyls was to increase ortho- and para-hydroxylation in the hydroxy-substituted ring. Single-stage oxidations can be studied in vitro, but in vivo experiments are more informative when two or more stages of oxidation are involved. Although 2-hydroxylation of biphenyl is not a specific effect of cytochrome P1-r50 induction, biphenyl can be used as a test substance in animals to recognize this type of induction.

The effect of phenobarbital and beta-naphthoflavone induction on the metabolism of biphenyl in the rat and mouse.

The effects of induction by beta-naphthoflavone (BNF) and by phenobarbital (PB) on the metabolism of biphenyl were studied in the rat (Sprague-Dawley) and the mouse (C57BL/6Tex and DBA/2Tex). Marked changes were observed after BNF induction. A major pathway of metabolism in C57BL/6 mice after induction was biphenyl leads to 2-hydroxybiphenyl leads to 2,5-dihydroxybiphenyl. This formerly minor pathways replaced biphenyl leads to 4-hydroxybiphenyl as the chief pathway of metabolism. No effect was observed in DBA/2 mice, in accord with observations that these animals lack the cytosolic receptor needed for cytochrome P1-450 induction. In Sprague-Dawley rats, the effect of BNF induction was to increase greatly the biphenyl leads to 4-hydroxybiphenyl leads to 3,4-dihydroxybiphenyl pathway and to decrease the 4-hydroxy leads to 4,4'-dihydroxy conversion. The effects of PB induction were less striking. Both genetic strains of mice showed a slight increase in 4,4'-dihydroxybiphenyl formation, and a slight decrease in 3,4-dihydroxybiphenyl formation. In the Sprague-Dawley rat, a slight increase in hydroxylation leading to 3-hydroxybiphenyl was observed. 2-Hydroxylation of biphenyl was not a specific feature of cytochrome P1-450 induction. The formation of the 2,5-diol as a major product was characteristic of cytochrome P1-450 induction in the C57BL/6 mouse, but not in the Sprague-Dawley rat. In both the mouse and rat, cytochrome P1-450 induction led to second-stage oxidation in the aromatic ring that was oxidized in the first stage, but the major pathways after induction were not in the same in both species.

Epoxide intermediates in the metabolism of naphthalene by the rat.

Twenty-one oxygenated metabolites of naphthalene have been isolated from rat urine and characterized by gas chromatography and gas chromatography and gas chromatography-mass spectrometry. Studied with naphthalene-1,4-d2 confirmed that the compounds characterized by GC/MS were derived from naphthalene. The structures of a number of metabolites were confirmed by synthesis. All but one of these metabolites were formed by processes involving epoxidation, and the structures of the metabolites support the hypothesis that multiple epoxides and a cyclic peroxide are involved in the in vivo metabolism of naphthalene by the rat. The evidence provided by this and our previous studied indicates that the anti-diepoxide of naphthalene is a mammalian metabolite. Ten methylthio metabolites were isolated in earlier studies. Excluding mercapturic acids, conjugates, and related compounds, the total number of isolated compounds is now 31, and several additional compounds must have been present as intermediates. The structures of epoxide intermediates are indicated by precursor-product relationships.

High-performance liquid chromatographic separation and isolation of quinidine and quinine metabolites in rat urine.

A procedure for the separation and isolation of the urinary metabolites of quinidine and quinine by reversed-phase high-performance liquid chromatography is described. Nine metabolites of quinidine and eight metabolites of quinine were detected in the urine of male Sprague-Dawley rats after a single dose of quinidine or quinine (50 mg kg-1). Following extraction from urine, the metabolites were separated on either an analytical or a semi-preparative reversed-phase column by gradient elution. After isolation and derivatization, the metabolites were analyzed by gas chromatography and gas chromatography--mass spectrometry.