Biotransformation of 4-methoxyphenol in rainbow trout (Oncorhynchus mykiss) hepatic microsomes
Introduction
Biotransformation reactions are generally considered to be a means of chemical detoxification. Phase I oxidation reactions serve to increase chemical polarity and provide adequate substrate for Phase II conjugation reactions, thus facilitating chemical elimination from organisms (Gibson and Skett, 1986, Parkinson, 1996). However, it is also widely recognized that metabolic transformation may result in more toxic chemical forms. While bioactivation has long been associated with increased toxicity in mammalian systems (Mitchell and Horning, 1984, Anders, 1985, Guengerich and Liebler, 1985, Hinson et al., 1994) and some aquatic systems (Ahokas and Pelkonen, 1984, Varanasi and Stein, 1991, Bradbury et al., 1993), little detailed information is available regarding specific bioactivation pathways in aquatic organisms. There is also a general lack of information as to rates of metabolic conversion to potentially more toxic chemical forms, regardless of species.
The measured toxicity of 4-methoxyphenol (4-MP) in an aquatic species has been hypothesized to involve metabolic activation. The 96-h acute toxicity of 4-MP to fathead minnows (Geiger et al., 1985) was investigated as part of a larger effort by the EPA to relate structure of industrial chemicals to biological activity, in this instance acute toxicity. Once chemical structure is correlated with toxicity for chemicals operating through a common mechanism of action, toxicity may be estimated for untested compounds (Bradbury, 1994, Bradbury, 1995). As part of these studies, 4-MP was predicted, based on structure, to be lethal to fathead minnows through the relatively non-specific narcotic mechanism of action (Russom et al., 1997). The potency measured for 4-MP was not inconsistent with what would be expected from chemical narcosis typical of phenolic compounds at acute levels. However, an examination of the dose-response curve suggested that, at lower doses, signs of toxicity were more consistent with formation of reactive, i.e. electrophilic/proelectrophilic, chemical species. It was hypothesized that 4-MP may be undergoing metabolic O-dealkylation and/or hydroxylation to chemical forms which are not only more hydrophilic, but also more reactive such as hydroquinone (HQ), benzoquinone (BQ) and trihydroxybenzene.
Bioactivation of 4-MP (also known as 4-hydroxyanisole or 4-hydroxy-1-methoxybenzene) has been shown to occur in mammalian systems. The depigmenting activity of 4-MP has lead to interest in its use as an antimelanoma drug (Riley, 1985, Pavel et al., 1989). The compound's cytotoxicity toward melanocytes had initially been thought to depend on oxidation of 4-MP by the enzyme tyrosinase. However, recent studies have investigated the role of rat and mouse cytochrome P450-mediated hydroxylations and demethylation in the toxicity of 4-MP (Schiller et al., 1991, Anari et al., 1995).
Thus, a study was undertaken to identify major metabolic products and rates of metabolic conversion of 4-MP in the rainbow trout, an aquatic species commonly used for metabolism studies (Lech, 1974, Franklin et al., 1980, Melancon and Lech, 1984, Buhler, 1995). An objective of this study was to establish the presence of hepatic microsomal O-dealkylation and/or ring-hydroxylation of 4-MP in rainbow trout by identifying metabolites formed in microsomal incubations. Microsomes were exposed to a range of 4-MP concentrations in an attempt to also calculate Michaelis–Menton kinetic constants for the reactions under study. It was the intent to add this information to a growing aquatic database of biotransformation and kinetic information for use in modeling the toxic effects of chemicals. This information is key to a better understanding of xenobiotic metabolism and potential for bioactivation in fish.
Section snippets
Chemicals
Ammonium acetate, 4-MP, BQ, HQ, methoxyhydroquinone (MHQ), 1,2,4-trihydroxybenzene (THB), 3-methoxycatechol (3-MCAT) and 5-methoxyresorcinol (5-MRES) were obtained from Aldrich Chemical Company (Milwaukee, WI). Reducing equivalents, buffer components, G-6-P dehydrogenase, 7-ethoxyresorufin, and mushroom tyrosinase were purchased from Sigma (St. Louis, MO). Acetonitrile (ACN) and methanol from Burdick and Jackson (Muskegon, MI) were of analytical grade. Resorufin was obtained from Pierce
Results
Qualitative examination of HPLC-ECD chromatograms of microsomal reaction mixtures (Fig. 4) indicated that, in addition to anticipated peaks for reaction product HQ (4.5 min) and substrate 4-MP (19.5 min), a relatively large peak was present at 10.5 min. The compound associated with this retention time was subsequently determined to be 4-MCAT, as follows. Initially a hydrodynamic curve (Eo=+200–+350 mV) for the compound eluting at 10.5 min was constructed utilizing a typical microsomal
Discussion
A proposed metabolic map for 4-MP, based on assumptions of O-demethylation and ring hydroxylation reactions, is shown in Fig. 8. Because it was postulated that O-demethylation would be the predominant reaction, initial efforts focused on the detection of HQ. Hydroquinone was found to be the primary metabolite of 4-MP in trout microsomes. Subsequent investigation determined the significant secondary metabolite to be 4-MCAT. BQ and THB were also detected in microsomal incubations. Unlike previous
Conclusions
In this study, 4-MP was found to be metabolized in trout microsomes primarily to HQ and 4-MCAT, the results of O-demethylation and ring hydroxylation reactions, respectively. Additional metabolites, identified as BQ (and/or 4-MQ) as well as THB, were present in small but detectable quantities. A metabolic pathway for metabolism of 4-MP in rainbow trout was proposed following confirmation of metabolite identity (utilizing hydrodynamic voltammograms generated by HPLC with electrochemical
Acknowledgements
The authors would like to acknowledge the laboratory assistance of Kurt Keogh and the additional guidance of several researchers at US EPA, Duluth: James McKim, III guided our use of microdialysis and reviewed the manuscript; Douglas Kuehl assisted with LC-MS analysis and manuscript review; and Russell Erickson consulted on analysis of kinetic data. This work was supported by the US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects
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