Introduction

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4-t-Butylcatechol (TBC)¹ (Fig. 1) is used principally as an antioxidant, stabilizer, and polymerization inhibitor for styrene, butadiene, neoprene, and other olefins and reactive monomers. Annual production of TBC in the United States was estimated to be 1.5 million pounds in 1989, and total consumption was predicted to increase (Chemical Marketing Reporter, 1989). Occupational and consumer exposures to TBC by skin contact have been implicated in numerous cases of allergic and depigmentation reactions. In some cases the residual TBC present in products made from TBC-stabilized monomers was sufficient to cause these reactions. TBC has been found a common factor in the development of contact dermatitis and leukoderma (Gellin et al., 1979). TBC is biochemically active and has been reported to be a substrate for tyrosinase and to inhibit the second step of melanogenesis (Usami et al., 1980). TBC-related leukoderma may be caused by its melanocytotoxic effects (Usami et al., 1980). The oral LD₅₀ for TBC in rat is 2820 mg/kg, and that for i.v. administration in mouse is 32 mg/kg (Smyth et al., 1954; Sax and Lewis, 1989). Some studies (Hirose et al., 1989) have called attention to the tumor-promoting ability of TBC, but this has not been confirmed in a full 2-year carcinogenicity bioassay.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Structure of TBC and gas chromatography/mass spectrometry and 1H-NMR of deconjugated metabolite of TBC. Mass spectra were determined by gas chromatography/mass spectrometry analysis on a HP-5989A mass spectrometer using electron impact ionization. The 1H-NMR was obtained on a Bruker AMX-500 MHz instrument. The sample was dissolved in a mixture of d4-methanol and D2O but also contained some CH3OH remaining from the purification procedure. The resonances at  3.1 to 3.5 are from (nondeuterated) methanol.

The objective of the present study was to investigate the absorption, disposition, metabolism, and excretion of TBC in rats and mice to complement toxicology studies in those species planned by the National Toxicology Program.

	Materials and Methods

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Chemicals. TBC was supplied by Aldrich Chemical Co, Inc. (Milwaukee, WI). [¹⁴C]TBC (15 mCi/mmol), labeled on the methine carbon, was prepared by Wizard Laboratories (West Sacramento, CA). The radiochemical purity of TBC was determined by reversed phase HPLC to be 94%.

Animal Studies. Adult male Fischer 344 rats (87-100 days old, 240-280 g) and B6C3F₁ mice (57-67 days old, 21-27 g) were purchased from Charles River Laboratories, Inc. (Raleigh, NC) and were furnished Purina Rodent Chow (no. 5002) and water ad libitum.

Collection of Biological Samples and Determination of Radiochemical Content. After dosing, rats and mice were housed in glass metabolism cages that provided for the separate collection of excreta into round-bottom flasks cooled with dry ice. In pharmacokinetic studies, blood (0.3 ml) was collected from rats in heparinized tubes at 0 (predose), 0.25, 0.5, 1, 2, 4, 8, and 24 h postdosing. Plasma was prepared by centrifugation for 10 min at 2500g. At the end of the experiments, the animals were anesthetized and sacrificed by exsanguination. Adipose tissues, muscle, and skin (from three locations each), blood, as well as the liver, kidneys, adrenal glands, spleen, lungs, prostate, seminal vesicles, testes, and brain were removed and assayed for radiochemical content. Additionally, stomach, small intestine, cecum, and large intestine were removed and assayed with contents for radiochemical content in some cases. At the conclusion of the dermal studies, the dose-site skin was excised, the appliance was removed, and both were washed and assayed as described previously (Mathews et al., 1998). Samples were assayed for radioactivity content as described previously (Mathews et al., 1998).

Metabolite Isolation. An initial purification of metabolites in urine collected 0 to 24 h after dermal administration (63 mg/kg) of TBC was accomplished by solid-phase extraction (SPE) using a Bond Elut C₁₈ SPE cartridge containing 500 mg of packing material in a 6-cc syringe barrel (Varian). Salts were eluted with 2 ml of water followed by 2 ml of 5% aqueous methanol before the metabolites were eluted with 2 ml of methanol. The methanol was evaporated and the sample was reconstituted and incubated with 20 to 30 µl of -glucuronidase/sulfatase (Sigma, from Helix pomatia) at 37°C overnight. The deconjugated metabolite (M3) was isolated by HPLC. The eluant containing M3 was prepared for NMR analysis by concentration by SPE, eluting with d₄-methanol.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

Distribution and Excretion. The excretion of i.v. and oral doses of TBC in male rats and mice is shown in Table 1. Rats rapidly excreted radiolabel in urine after an i.v. or oral dose, with 70% of the dose recovered there in the first 24 h postdosing. High percentages (ca. 90%) of oral and i.v. doses administered to mice were excreted in urine and feces. The high recoveries in mouse feces were likely due to contamination of feces with urine-soaked food particles. Less than 1% of the dose remained in the tissues of both species sampled 72 h postdosing, and there was no evidence of marked accumulation in any particular tissue type (data not shown).

Metabolism. Only polar metabolites were excreted in urine after oral, i.v., or dermal administration of TBC to rats. TBC, which elutes at a retention time of ca. 13 min, was not excreted in urine. The HPLC radiochromatogram of rat urine collected 0 to 6 h after a dermal dose (63 mg/kg) is shown in Fig. 2a and is representative of profiles observed after each route of administration for both species. Radiochromatograms of urine after treatment with purified -glucuronidase or purified sulfatase are shown in Fig. 2, b and c, respectively, and indicate that conjugates of TBC and at least two other metabolites were excreted in urine, one of which (M3) is less polar than TBC. These chromatograms were also typical for all species and routes. A greater proportion of the polar metabolites was liberated by treatment with sulfatase, indicating that the majority of the polar metabolites excreted were sulfate esters. The early eluting metabolites (M1 and M2) that remained after treatment of urine with sulfatase were isolated by SPE and HPLC techniques (similar to those used for M3) and treated again with sulfatase. HPLC analysis after enzyme incubation indicated that the radioactivity associated with these peaks had mostly shifted to the retention time of TBC.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   HPLC radiochromatograms of rat urine collected 0 to 6 h after dermal administration of [14C]TBC. The chromatographic system consisted of a DuPont Zorbax Rx-C18 column (250 × 4.6 mm, 5 µm; Mac-Mod Analytical, Chadds Ford, PA) and a mobile phase of methanol/water, 55:45 (v/v); the flow rate was 1 ml/min. The column effluent was monitored by an Applied Biosystems 759a (Bodman, Aston, PA) absorbance detector at a wavelength of 254 nm and by a Ramona-5-LS radioactivity detector equipped with a 500-µl solid scintillate flow cell. a, untreated urine; b, -glucuronidase-treated urine. Urine (200-400 µl) was added to 1000 U -glucuronidase (prepared from Escherichia coli) and the mixture was incubated 4 to 18 h at 37°C before analysis. c, sulfatase-treated urine. Sulfatase solution (10 µl, prepared from Aerobacter aerogenes; containing 14.5 U/ml) was added to 100 µl of urine and 300 µl of tris(hydroxymethyl)aminomethane (TRIZMA) buffer (0.05 M, pH 7.6) and incubated 2 to 4 h at 37°C.