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EC number: 202-705-0 | CAS number: 98-83-9
Intravenous Study for Urinary Metabolite Identification
Intravenous doses of alpha-methylstyrene (10 mg/kg) were mainly excreted in the urine with 76 +/- 2 % excreted in the first 24 hours postdosing and 86 +/- 1 % by 72 hours. Faecal elimination accounted for 2 % of the dose. Exhalation of volatile organics and carbon dioxide accounted for only 2 % and 0.02 % of the dose, respectively. Concentrations of alpha-methylstyrene equivalents were low, and only 0.3 % of the radioactivity was recovered in the tissues. The concentration of alpha-methylstyrene and/or its metabolites in blood was 16 ng Eq per gram.
The profiles of metabolites present following an intravenous dose of alpha-methylstyrene (10 mg/kg) were determined for each urinary collection interval up to 48 hours postdosing for one rat. The peak eluting at 10:25 (metabolite D) coelutes with atrolactic acid, and over 20 % of the administered dose was excreted as this metabolite. Metabolite E was most abundant in the early urine collection (0 to 6 hours), with considerably less detected in later collections, suggesting that it may be an early intermediate metabolite of alpha-methylstyrene.
Oral Study for Urinary Metabolite Identification
Alpha-methylstyrene was administered orally to one rat at a dose of 1000 mg/kg in an effort to obtain as much urinary metabolite as possible. Urine from this experiment was chromatographed and gave a similar profile as the urine from the intravenous study. Five of the metabolite peaks were identified by GC/MS. Metabolite B was treated with beta-glucuronidase, and the aglycone was analyzed by GC/MS. The spectrum of the bis-trimethylsilane derivative of a 2-phenyl-1,2 -propanediol standard had no M+ at 296 but had peaks at m/z 281 (loss of a methyl), 193 (loss of CH2O-trimethylsilane ), and 147. The spectra of metabolite C and the aglycone of metabolite B were virtually identical to that of the bis-trimethylsilane derivative of a 2-phenyl-1,2-propanediol standard. Therefore, it was concluded that metabolite B is a glucuronide of 2-phenyl-1,2-propanediol and that metabolite C is 2-phenyl-1,2-propanediol.
Metabolite B was analyzed by 13C-DEPT NMR in an effort to determine the position of attachment of the glucuronide group in the conjugate. Of the two oxygenated carbon atoms, only the terminal methylene group had protons with which the pulsed carbon nucleus could relax. Therefore, the position of the methylene resonance was determined in these characterization experiments using NMR. The spectra displayed a doublet for each carbon bearing a proton, possibly indicating that this metabolite was present as a pair of diasteriomers. The 13C-DEPT NMR spectrum of 2-phenyl-1,2-propanediol was determined in order to interpret the spectra for metabolite B, and the methylene resonance appeared at 72 ppm. If the glucuronide conjugate were attached to the carbon alpha to the phenyl ring, the chemical shift of the methylene carbon would have been approximately 3 ppm upfield (at 69 ppm) relative to that for the unconjugated 2-phenyl-1,2-propanediol; had it been at the beta carbon, the shift would have been approximately 10 ppm downfield (at 82 ppm). In the 13C-DEPT NMR spectrum of metabolite B, the methylene resonances were at 79 ppm, consistent with the attachment of the glucuronide moiety to the carbon beta to the phenyl ring.
A bis-trimethylsilane derivative of an atrolactic acid standard and metabolite D were analyzed by GC/MS, and the spectra were identical. A standard of 2 -phenylpropionic acid and metabolite F were analyzed by GC/MS, and both spectra contained a molecular ion at m/z 150 and signals at m/z 105 (loss of COOH) and 77. Metabolite E was treated with acylase, and the incubation mixture was chromatographed as described previously for isolation of the peak. The profile was compared with the HPLC profile of the untreated urine. Treatment with acylase converted metabolite E to a new component that eluted between metabolites A and B. The 1H NMR spectrum of metabolite E was consistent with a N-acetylcysteine conjugate resulting from reaction of glutathione with the epoxide of alpha-methylstyrene, followed by further metabolism to the mercapturate. The 13C-DEPT NMR also corroborated this finding. The 13C chemical shift of the methylene group beta to the ring was consistent with attachment of the sulphur atom to this carbon atom. Trimethylsilane derivatives of the mercapturate(s) were prepared for analysis by GC/MS. Two di-derivatized and two tri-derivatized products were present in roughly equal amounts. The fragmentation patterns (in particular the ion at 193 a.m.u.) in these spectra were also consistent with the formation of just one of two possible positional isomers for the mercapturate. The NMR and mass spectral data indicated that a diasteriomeric pair of mercapturates was formed as metabolites.
The presence of roughly equal amounts of the diasteriomeric mercapturates suggests that the initial epoxidation of alpha-methylstyrene is not sterioselective and proceeds with no marked preference for the antarafacial or suprafacial addition of active oxygen to yield enantiomeric epoxides. Both enzymatic hydrolysis and glutathione conjugation of epoxides are known to proceed by SN2 reactions. Therefore, enzymatic hydrolysis can yield enantiomeric diols. Further oxidation of the terminal hydroxy group of these diols to form atrolactic acid does not affect the chiral centre at the benzyl position, and the potential products are enantiomers. However, conjugation with a chiral molecule such as glutathione or glucuronic acid would produce diasteriomeric metabolites from the enantiomeric products, as is the case with the mercapturates and glucuronides characterized in these studies.
The time weighted average alpha-methylstyrene vapour concentrations during exposure for the 300 and 900 ppm groups were 304 and 900 ppm. Animals exposed to 300 or 900 ppm received mean doses of 130.8 and 340.1 mg/kg, or 26.63 and 20.05 uCi, respectively.
For the 300 ppm group, urinary excretion was the main route of elimination, comprising 88.2 % of the absorbed dose, with volatile breath and faeces accounting for 3.1 and 2.2 %, respectively. These trends were consistent for all collection intervals. Over 90 % of the absorbed dose was eliminated within 48 hours post-initiation of exposure. The same pattern of elimination was observed in the 900 ppm group; 92.4 % was excreted in urine, with volatile breath and faeces accounting for 2.5 and 2.6 %, respectively.
Tissue distribution of radioactivity at 6, 24 (300 ppm only), and 72 hrs after initiation of inhalation: 2.6 – 10.1 % and 1.1 – 2.4 % of the inhaled radiolabeled alpha-methylstyrene was recovered in the residual carcass and tissues in the 300 and 900 ppm groups, respectively. The highest concentrations of radiolabeled alpha-methylstyrene (ug Eq/g tissue) were observed in adipose, bladder, liver, kidney, and skin at 6, 24 (300 ppm only), and 72 hours after initiation of exposure. This is consistent with the lipophilic nature of alpha-methylstyrene and the fact that most of the dose was eliminated in urine. Elevated levels of radiolabel were present in the small intestine compared to the stomach and large intestine, suggesting biliary excretion and re-absorption of [14C]alpha-methylstyrene-derived metabolite(s). Tissues for the 24-hour time point in the 300 ppm group were removed from animals used for serial blood sampling.
The metabolite profile observed in urine from 300 and 900 ppm male rats was qualitatively the same as that in the intravenous study. Much more atrolactic acid was observed in urine collected during the first 24 hrs in the inhalation studies compared to the intravenous study. Rats exposed to 900 ppm exhibited nearly a twofold increase in excretion of atrolactic acid, accompanied by a corresponding drop in excretion of 2-phenyl-1,2-propanediol glucuronide, in urine collected between 12 and 24 hrs compared to rats exposed to 300 ppm.
Characterization of the 14C Profile in Blood
In extraction method development experiments, recovery of carbon-14 from blood spiked with [14C]alpha-methylstyrene was 95 +/- 6 %. Recoveries of carbon-14 from blood samples obtained from the 300 and 900 ppm groups were 74 +/- 10 % and 82 +/- 9 %, respectively. These recoveries suggest sequestration of metabolites by red blood cells. Chromatographic analysis of red blood cell lysate showed that no radioactivity was associated with heme.
Alpha-methylstyrene concentrations in blood dropped precipitously in the 300 ppm rats upon cessation of exposure, from more than 6 ug/mL just prior to termination of exposure (5.5 hrs) to an average of 0.97 ug/mL at 7 hrs. From 7 – 24 hrs, alpha-methylstyrene concentrations in blood decreased at a much slower rate. Alpha-methylstyrene concentration in blood dropped from more than 24 ug/mL at 5.5 hrs into the exposure to ca. 10 ug/mL in the first hour after cessation of exposure in the 900 ppm rats.
Four metabolites were extracted from blood obtained in the inhalation study. The major component at all time points was 2-phenyl-1,2-propanediol. Atrolactic acid, 2 -phenylpropionic acid, and an additional radiolabeled component noted as blood metabolite 1 were also observed. Identities of atrolactic acid, 2-phenyl-1,2 -propanediol, 2-phenylpropionic acid, and alpha-methylstyrene peaks were established by co-elution with non-radiolabeled standards.
Male F344/N rats were exposed to alpha-methylstyrene via intravenous or nose-only inhalation exposure. In both studies, the substance was eliminated primarily in the urine (approximately 90 %) within 72 hours, with volatile breath and feces accounting for only a small amount (1 - 3 %) of elimination. In the inhalation study, the elimination half-life was calculated at 3 to 5 hours, with the highest concentrations of alpha-methylstyrene-derived radioactivity retained in the adipose tissue, urinary bladder, liver, kidney, and skin. Following intravenous dosing, the kidney, heart, lung, liver, urinary bladder, and spleen retained the highest concentrations of radioactivity. In both the intravenous study and the inhalation study, the major urinary metabolites of alpha-methylstyrene were the glucuronide conjugate of 2 -phenyl-1,2-propanediol and atrolactic acid. In the inhalation study, the major metabolites in the blood were 2-phenyl-1,2-propanediol and 2-phenylpropionic acid.
Based on these studies, the proposed metabolic pathway for alpha-methylstyrene involves an initial non-stereoselective epoxidation followed by hydrolysis to form 2-phenyl-1,2-propanediol followed by either oxidation to atrolactic acid or formation of the glucuronide conjugate, conjugation with glutathione and subsequent cleavage to the mercapturate, or rearrangement to form an aldehyde that is oxidized to yield 2-phenylpropionic acid. The dose-dependent pharmacokinetic parameters coupled with decreased excretion of 2-phenyl-1,2-propanediol glucuronide at 900 ppm indicate that glucuronide formation was saturated at this dose.
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