Elsevier

Aquatic Toxicology

Volume 45, Issue 1, March 1999, Pages 47-61
Aquatic Toxicology

Biotransformation of 4-methoxyphenol in rainbow trout (Oncorhynchus mykiss) hepatic microsomes

https://doi.org/10.1016/S0166-445X(98)00088-5Get rights and content

Abstract

Rainbow trout liver microsomes were used to study the O-demethylation and ring hydroxylation of 4-methoxyphenol (4-MP) (4-hydroxyanisole) at 11 and 25°C by directly measuring the production of the primary metabolite hydroquinone (HQ), 4-methoxycatechol (4-MCAT), and additional metabolites. An HPLC method with integrated ultraviolet (UV) and electrochemical detection (ECD) was developed for metabolite identification and quantification at low concentrations. Sample handling with appropriate buffers, solvents, low temperature and light prevented loss of extremely labile metabolites. Saturation kinetics for the production of HQ via O-demethylation of 4-MP (0.66–40 mM) was never achieved, with substrate solubility being the limiting factor. The linear rate of HQ formation at 11°C was 22.0±2.2 (coefficient±S.E., r2=0.91) pmol min−1 per mg protein per mM substrate, and at 25°C was 34.0±1.3 (r2=0.99) pmol min−1 per mg protein per mM substrate. The second major microsomal metabolite 4-MCAT was also identified, with linear rates of ring hydroxylation determined to be 19.0±1.6 (r2=0.94) and 17.2±0.5 (r2=0.99) pmol min−1 per mg protein per mM substrate at 11 and 25°C, respectively. Unlike HQ production, the rate of 4-MCAT production was found to be similar at the two temperatures when linear formation rates were corrected for the effect of temperature on substrate and product solubility at 11°C. Measurement of `freely dissolved' fraction was essential to the accurate determination of ring hydroxylation and O-demethylation reaction rates in rainbow trout microsomes incubated at physiological temperature. Experimental conditions were shown to affect dissolved 4-MP and HQ at 11°C (verified using microdialysis) while not altering substrate and product levels at 25°C. Small but detectable levels of 1,4-benzoquinone were observed in 4-MP microsomal incubations. 1,2,4-Trihydroxybenzene was also detected, with possible routes of production through hydroxylation of HQ or O-demethylation of 4-MCAT. A metabolic scheme for bioactivation of 4-MP is proposed and the significance of observed metabolic conversions in rainbow trout microsomes discussed in relation to aquatic toxicity of 4-MP.

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

References (43)

  • J.M. McKim et al.

    In vivo microdialysis sampling of phenol and phenyl glucuronide in the blood of unanesthetized rainbow trout: implications for toxicokinetic studies

    Fundam. Appl. Toxicol.

    (1993)
  • M.J. Melancon et al.

    Metabolism of [14C]2-methylnaphthalene by rainbow trout (Salmo gairdneri) in vivo

    Comp. Biochem. Physiol.

    (1984)
  • T.J. Monks et al.

    Contemporary issues in toxicology: quinone chemistry and toxicity

    Toxicol. Appl. Pharmacol.

    (1992)
  • P.J. O'Brien

    Molecular mechanisms of quinone cytotoxicity-review

    Chem.-Biol. Interact.

    (1991)
  • A.H. Phillips et al.

    Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization, and kinetic studies

    J. Biol. Chem.

    (1962)
  • R.J. Pohl et al.

    A rapid method for assaying the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions

    Anal. Biochem.

    (1980)
  • C.D. Schiller et al.

    Mechanism of toxicity of the antimelanoma drug 4-hydroxyanisole in mouse hepatocytes

    Eur. J. Cancer

    (1991)
  • D. Schlenk et al.

    Influence of B-naphthoflavone and methoxychlor pretreatment on the biotransformation and estrogenic activity of methoxychlor in channel catfish (Ictalurus punctatus)

    Toxicol. Appl. Pharmacol.

    (1997)
  • J. Snegaroff et al.

    The effects of temperature on the basal activity of cytochrome P450 in rainbow trout (Salmo gairdneri)

    Comp. Biochem. Physiol.

    (1990)
  • M.R. Anari et al.

    Cytochrome P-450 peroxidase/peroxygenase mediated xenobiotic metabolic activation and cytotoxicity in isolated hepatocytes

    Chem. Res. Toxicol.

    (1995)
  • Anders, M.W. (Ed.), 1985. Bioactivation of Foreign Compounds. Academic Press, Orlando,...
  • Cited by (16)

    • MetaPath: An electronic knowledge base for collating, exchanging and analyzing case studies of xenobiotic metabolism

      2012, Regulatory Toxicology and Pharmacology
      Citation Excerpt :

      Knowledge of the occurrence, amounts and stability of activated metabolites is vital to chemical toxicity assessments, with activation processes often resulting in similar products across species, e.g., mammalian (Sahawata and Neal, 1983; Schlosser et al., 1993; Kenyon et al., 1995) and aquatic species (Kolanczyk and Schmeider, 2002; Kolanczyk et al., 1999).

    View all citing articles on Scopus
    View full text