Introduction

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-Methylstyrene (AMS;¹ 2-phenylpropylene) (CAS 83-98-9) is a volatile aromatic hydrocarbon (b.p. 165°C) used primarily in the production of specialty polymers and resins. The majority of the AMS used industrially is in the manufacture of acrylonitrile-butadiene-styrene copolymers, which are lightweight and have good heat-distortion properties at high temperatures (Lewis et al., 1983). AMS is less reactive than styrene and moderates the polymerization rate, resulting in coatings and resins with improved clarity. The National Occupational Exposure Survey released by NIOSH in 1976 indicated heavy construction contractors, paper and allied products, and other business services comprise industries with the largest number of workers potentially exposed to AMS.

AMS is not highly toxic, and lethalities probably arise mostly from oversedation. The oral LD₅₀ for AMS in rats is 4.9 g/kg (Wolf et al., 1956). The toxicity of AMS to B6C3F1 mice and Fischer 344 rats following exposure by inhalation of concentrations ranging from 125 to 1000 ppm for 6 h/day for 12 days has been recently reported (Morgan et al., 1999). Mortality was observed in female mice after the first exposure at concentrations greater than 600 ppm; no male mice died, but both male and female mice were sedated at these levels. Increased liver and decreased spleen weights were noted in both sexes of mice following 12 exposures at 600 ppm, although no microscopic treatment-related lesions were observed. Both single and repeat treatments at the higher doses resulted in significant decreases in hepatic glutathione levels. No mortality or sedation occurred in rats of either sex, but there were significant increases in liver weights. Additionally, there were no significant effects on serum chemistries. AMS is a weak inducer of sister-chromatid exchanges (Norppa and Vainio, 1983) and is not mutagenic in Salmonella typhimurium with or without activation (Zeiger et al., 1992).

The metabolism of AMS has not been comprehensively studied. 2-Phenyl-1,2-propanediol and atrolactic acid (2-phenyl-2-hydroxypropanoic acid) have been cited as major oxidative metabolites of AMS in humans, dogs, rats, and guinea pigs, and atrolactic acid has been proposed as an appropriate urinary marker of exposures in industrial workers (Bardodej and Bardodejova, 1970; Aizvert 1975, 1979). Humans are most likely to be exposed via the dermal route or by inhalation.

Citing a need to supplement data from its toxicity studies and limited information available in the literature detailing AMS disposition and metabolism, the National Toxicology Program nominated AMS for study. The goal of the present study was to conduct a definitive characterization of the metabolites of AMS produced in vivo in rat and to determine the tissue distribution and excretion of AMS after oral and i.v. administration to that species. The profile of metabolites produced by human liver slices was also determined and compared with those produced in vivo in rats.

	Materials and Methods

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Chemicals. Nonradiolabeled AMS (99%), 2-phenylpropionic acid (97%), and atrolactic acid (2-hydroxy-2-phenylpropionic acid; 98%) were purchased from the Aldrich Chemical Co., Inc. (Milwaukee, WI). A standard of 2-phenyl-1,2-propanediol was prepared at Research Triangle Institute (Triangle Park, North Carolina) by the reduction of atrolactic acid using the method of Shore and Yuen (1972). The identity of nonradiolabeled AMS was confirmed by proton NMR using CDCl₃ as a solvent. [¹⁴C]AMS, uniformly radiolabeled with carbon-14 in the phenyl ring, was obtained from Wizard Laboratories, Inc. (West Sacramento, CA) at a specific activity of 1.0 mCi/mmol. The radiochemical purity of [¹⁴C]AMS (98%) was established using a Microsorb-MV phenyl column (4.6 × 250 mm, 5-µm particle size; Varian, Palo Alto, CA) and an isocratic mobile phase of methanol/water (v/v); the flow rate was 1 ml/min. -Glucuronidase (prepared from Escherichia coli), sulfatase (prepared from Aerobacter aerogenes), and acylase (prepared from porcine kidney) were purchased from the Sigma Chemical Company (St. Louis, MO). Bis(trimethylsilyl)trifluoroacetamide was purchased from Supelco, Inc. (Bellefonte, PA). Emulphor EL-620 was obtained from the GAF Chemical Corporation (Wayne, NJ). Mass spectra were determined by GC/MS analysis on a Hewlett Packard (Palo Alto, CA) 5890 series 2 gas chromatograph and a HP-5989A mass spectrometer using electron impact. Metabolites and standards were dissolved in CD₃OD or CDCl₃ before obtaining NMR spectra on a Bruker (Newark, DE) AMX-500 MHz instrument.

Animal Studies. Adult male Fischer 344 rats were purchased from Charles River Laboratories, Inc. (Raleigh, NC) and given Purina (St. Louis, MO) Rodent Chow (#5002) and water ad libitum. At dosing, rats were 79 to 85 days old and weighed 241 to 263 g. Intravenous dose formulations contained 21 to 24 µCi of [¹⁴C]AMS and an appropriate amount of Emulphor EL-620 and phosphate-buffered saline (1:20) in a single dose to deliver a volume of 1 ml/kg. An oral dose formulation was prepared for dosing one rat that contained 44 µCi of [¹⁴C]AMS, an appropriate amount of nonradiolabeled AMS, and sufficient corn oil for a single intragastric dose volume of 5 ml/kg.

Determination of Radiochemical Content. Radioactivity was determined using a Packard Tricarb 1500 Liquid Scintillation Analyzer (Packard Instrument Company, Meriden, CT). Aliquots of breath traps and urine were added directly to vials containing scintillation cocktail (Ultima Gold, Packard Instrument Company). Samples of tissue (0.1-0.5 g), feces (0.1-0.2 g), and blood (0.1-0.2 g) were digested in Soluene-350 overnight. After digestion, samples requiring bleaching were decolorized with 125 µl of 70% perchloric acid and 300 µl of 30% hydrogen peroxide before addition of scintillation cocktail.

Characterization of Metabolites in Urine. Samples from each urinary collection interval up to 48 h from one rat (11 mg/kg i.v.) were analyzed by HPLC. Radiochemical contents of the samples were determined by liquid scintillation spectrometry. Urinary metabolite profiles were obtained by HPLC using a Microsorb-MV phenyl analytical column. Urinary metabolites were eluted using a linear gradient, changing from 20 to 60% acetonitrile/aqueous 1% acetic acid (v/v) over 30 min; the flow rate was 1 ml/min. Metabolites eluting from the column were detected using both UV absorbance at 254 nm and a Ramona (Raytest, Pittsburgh, PA) 5-LS flow-through radioactivity detector equipped with a 500-µl solid scintillate flow cell.

Liver Slices. Human liver slices were received from the International Institute for the Advancement of Medicine (Exton, PA). The donor was a 45-year-old black male that died from a gunshot wound to the leg. He had renal cancer and used cocaine. The medications received during his hospital stay were DDAVP [1-(3-mercaptopropanoic acid)-8-D-arginine vasopressin], cephapirin sodium, and dopamine. Human liver slices were incubated with AMS (1 mM in medium) as previously described (Mathews et al., 1996).

	Results

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Distribution and Excretion. Intravenous doses of AMS (11 mg/kg) were mainly excreted in the urine with 76 ± 2% excreted in the first 24 h post dosing and 86 ± 1% excreted after 72 h (Table 1). Fecal elimination accounted for 2% of the dose. Exhalation as volatile organics and carbon dioxide accounted for only 2 and 0.02% of the dose, respectively. The profiles of metabolites present (see Metabolite Identification below for characterization) in each urinary collection interval up to 48 h post dosing for one rat are shown in Table 2, and a typical HPLC radiochromatogram is displayed in Fig. 1. Unfortunately, the diol glucuronide had partially hydrolyzed to the aglycone upon storage, but data from the analysis of urine freshly collected from one rat indicated that the majority of the diol metabolites were excreted as the glucuronide. The most abundant metabolite was 2-phenyl-1,2-propanediol glucuronide (41% of dose), followed by atrolactic acid (22%) and S-(2-hydroxy-2-phenylpropyl)-N-acetylcysteine (11%).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   HPLC radiochromatogram of urinary metabolites collected 6 to 12 h following an intravenous administration of [14C]AMS (11 mg/kg) to a male Fischer 344 rat. The HPLC chromatographic system consisted of a Microsorb-MV phenyl analytical column (4.6 × 250 mm, 5-µm particle size; Varian) and a linear gradient, changing from 20 to 60% acetonitrile/aqueous 1% acetic acid (v/v) over 30 min; the flow rate was 1 ml/min. Column effluent was monitored using UV absorbance at 254 nm on an Applied Biosystems 757 detector (Bodman, Aston, PA) and radioactivity on a Ramona 5-LS flow-through detector equipped with a 500-µl solid scintillate flow cell. See the Table 2 footnote for the identification of the metabolites.

Metabolite Identification. AMS was administered orally to one rat at a dose of 1000 mg/kg to obtain greater quantities of metabolites for structural characterization. The percentage of radioactivity excreted in urine and the urinary metabolite profile from this experiment (data not shown) was similar to that from the intravenous study. Five of the metabolites were isolated from the urine and identified by GC/MS.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Gradient enhanced HMBC-NMR spectra of isolated metabolite B.  NMR spectra were obtained on a Bruker AMX-500 MHz instrument.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Gradient enhanced HMBC-NMR spectra of isolated metabolite E.  NMR spectra were obtained on a Bruker AMX-500 MHz instrument.

Metabolism by Human Liver Slices. In investigations of the metabolism of AMS by human liver slices, the same metabolites were produced as those identified in rat urine. As observed in rats, the major metabolite was 2-phenyl-1,2-propanediol (25% of the radioactivity) after 5 h of incubation (data not shown); atrolactic acid and 2-phenylpropionic acid each accounted for approximately 1% of the radioactivity, and the remainder of the metabolites accounted for less than 0.3% of the radioactivity.

	Discussion

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AMS was readily metabolized by rats to form products that were chiefly excreted in the urine. There was little accumulation of radiolabeled equivalents in the tissues. In the present study, 96% of the urinary radioactivity was characterized, and all of the metabolites were products of oxidation of the vinyl group; no ring oxidized metabolites were found (Fig. 4). Metabolism of AMS to atrolactic acid has been reported in humans and other mammals, and this metabolite has been suggested as an appropriate marker of exposure to this hydrocarbon (Aizvert, 1975). Five metabolites were identified in the present study. The predominant metabolite, 2-phenyl-1,2-propanediol glucuronide (accounting for 50% of the urinary radioactivity), was present in roughly twice the amount of that of atrolactic acid in rat urine. Only 3% of the urinary radioactivity was present as 2-phenyl-1,2-propanediol. 2-Phenyl-1,2-propanediol was the predominant metabolite present in the media of human liver slices incubated with AMS, and it and its glucuronide may be the major human urinary metabolites. Accordingly, the diol, or more likely its glucuronide metabolite, may also deserve consideration as a biomarker for exposure of humans to AMS.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Proposed pathway for the metabolism of AMS.

The mercapturate metabolite (E), formed after the reaction of the epoxide with glutathione, was most abundant in the early urine collections. It was the next most abundant metabolite and ultimately composed 13% of the urinary radioactivity. Its formation is consistent with the marked depletion of liver glutathione observed in inhalation studies with AMS (Morgan et al., 1999). Small amounts of 2-phenylpropionic acid (1% of urinary radioactivity) were also formed. It is likely that this derives from 2-phenylpropionaldehyde, formed from a 1,2-hydride shift during the transfer of active oxygen to the vinyl group, as has been proposed for the cytochrome P450-mediated oxidation of styrene to form phenylacetaldehyde (Ortiz de Montellano, 1995).

The presence of both diastereomeric forms of mercapturates and glucuronides suggests that the initial epoxidation of AMS is not stereoselective and proceeds with the addition of active oxygen to yield enantiomeric epoxides. Both enzymatic hydrolysis and glutathione conjugation of epoxides is known to proceed by S_N2 reactions. Therefore, enzymatic hydrolysis can yield enantiomeric diols. Further oxidation of these diols to atrolactic acid does not affect the chiral center at the benzyl position, and the potential products are enantiomers. However, conjugation with a chiral molecule such as glutathione or glucuronic acid would produce diastereomeric metabolites from the enantiomeric products, as was the case with the mercapturates and glucuronides characterized in these studies. In contrast to the reaction with styrene oxide (Seutter-Berlage et al., 1978; Sumner and Fennell, 1994), conjugation of glutathione with the epoxide of AMS occurs only at the less hindered -carbon. Seutter-Berlage et al. (1978) demonstrated the intermediacy of an enantiomeric epoxide of styrene from the diastereomeric mercapturates formed at the -carbon, but apparently the isochronicity of the NMR resonances of the -carbon mercapturates did not allow assessment of the relative contributions of mercapturate diastereomers formed at this position. Similarly, the -carbon mercapturates formed from AMS epoxide lent no NMR evidence of the formation of diastereomers, presumably due also to isochronous resonances, while the GC/MS data did.

This work is the first comprehensive study of the metabolism of AMS in rats and includes the first report of sulfur-containing AMS metabolites. The data are currently being used in support of the toxicological risk assessment of AMS exposure by inhalation and other routes of administration and in the construction of physiologically based pharmacokinetic models for this hydrocarbon.