Identification of novel metabolites of the xenoestrogen 4-tert-octylphenol in primary rat hepatocytes

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Abstract

A number of environmental pollutants, including 4-tert-alkylphenols, can mimic the actions of endogenous steroids and have the potential to disrupt the endocrine function in humans and animals. The biotransformation of a 4-tert-alkylphenol in isolated rat hepatocytes was studied in order to determine the possible fate and activity of these xenoestrogens in higher vertebrates. Hepatocytes were incubated with 30 μM 4-(1′,1′,3′,3′-tetramethylbutyl)[U-14C]phenol (4-tert-octylphenol; t-OP) for up to 60 min. Radiolabelled metabolites were detected by radio-HPLC and the structures determined by gas chromatography–mass spectrometry (GC–MS) analysis of the conjugated or aglycone products. After a 15 min incubation, over 97% of t-OP was metabolised to a complex mixture of metabolites. The initial metabolites formed were identified as products of hydroxylation of the aromatic ring to form catechols and methylated catechols, as well as glucuronide conjugates of the catechol metabolites or parent phenol. These products were further metabolised by hydroxylation of the alkyl chain followed by glucuronide conjugation of the alkoxy group. The conjugated metabolites of t-OP are unlikely to retain estrogen receptor activity, however t-OP is metabolised by some pathways that are similar to that of estrogen catabolism, namely by ortho-hydroxylation to form catechols, methylation by catechol O-methyltransferases and ring conjugation by uridine diphosphoglucuronosyl transferases. Further investigations are needed to determine whether 4-tert-alkylphenols can alter circulating sex steroid profiles by acting as substrates of enzymes determining estrogen metabolism and excretion.

Introduction

A variety of chemicals released into the environment are thought to act as endocrine disrupters and could potentially alter the reproductive physiology of both wildlife and humans [1]. Animals can be exposed to a complex mixture of chemicals some of which, although weakly estrogenic compared with estradiol, may bioaccumulate and act additively to cause effects on fertility, sexual differentiation, behaviour and cancer. It has been proposed that in utero exposure to estrogenic chemicals may be associated with the increased incidence of male reproductive disorders in human populations including the apparent decrease in human semen quality [2]. One such class of xenoestrogens are para substituted alkylphenols and humans can be exposed to these xenoestrogens from a variety of sources. For instance, 4-nonylphenol, a component of plastics, has been reported to leach from polyvinylchloride and polystyrene used in food-packaging [3], [4]. 4-alkylphenols are also formed from the environmental degradation of alkylphenol polyethoxylate surfactants which have been used as industrial and domestic detergents, and as additives in paint and pesticide formulations [5]. Short chain 4-nonyl- and 4-octylphenol polyethoxylates have been detected in drinking water [6] and, together with their parent alkylphenols, in groundwater [7]. Alkylphenol polyethoxylates are also the active ingredient in the majority of spermicides [8]. Although alkylphenol polyethoxylates with a high degree of ethoxylation are not estrogenically active per se [9], there is concern that alkylphenol polyethoxylates could be metabolised within animals by cleavage at the alkylphenol-ether bond, producing free alkylphenol and the corresponding polyethylene glycol [8].

4-tert-octylphenol (t-OP) is the most estrogenic of the 4-alkylphenols and in vitro studies have demonstrated that it binds to the estrogen receptor, activates estrogen-responsive genes, and stimulates mitogenesis of MCF breast cancer cells [10], [11]. In vivo studies have reported that t-OP is uterotrophic in immature rats [12], [13]. Exposure of pregnant rats to t-OP via the drinking water has been reported to reduce testicular size and sperm production in the offspring [14]. In addition, chronic administration of t-OP for two months to adult male rats has been shown to cause estrogen-like effects, including disrupted spermatogenesis, increased incidence of sperm deformities and lowered serum concentrations of testosterone and gonadotrophins [15], [16].

There is limited information on the metabolic pathways of alkylphenols in mammalian systems. It is not clear whether the effects of t-OP observed in vivo are a result of the activity of the parent compound, or whether t-OP is metabolised to more active structures. In addition, alkylphenols have been reported to be conjugated by estrogen sulfotransferases, which suggests that alkylphenols could alter the conjugation and elimination of endogenous estrogens [17], [18]. In the present study, we report the detailed structural analysis of t-OP metabolites formed by primary rat hepatocytes exposed to concentrations of alkylphenol which were below the estimated maximum oral intake level in humans [19].

Section snippets

Chemicals and enzymes

Radiolabelled 4-(1′,1′,3′,3′-tetramethylbutyl)[U-14C]phenol (4-tert-octylphenol; t-OP, specific activity 666 MBq/mmol, radiochemical and chemical purity>99%) was purchased from Amersham International, Little Chalfont, Buckinghamshire, UK. Collagenase (activity>125 U/mg) was supplied by Worthington Biochemical, Freehold, NJ, USA. Minimum essential medium (MEM) was purchased from Gibco BRL, Paisley, UK. Sodium pentobarbitone (Sagatal) was obtained from RMB Animal Health, Dagenham, UK. β

RP-HPLC analysis of metabolites

During incubations with 10–50 μM [14C] t-OP, hepatocyte viability was similar to control untreated cells and was typically >70% after 60 min. At this time >93% of radioactivity was recovered by methanol extraction of the hepatocytes and the remaining radioactivity was covalently bound within the precipitated protein pellet and was only extracted after alkaline hydrolysis with tissue solubiliser. In contrast, the recovery of methanol-soluble radioactivity from incubations with heat-inactivated

Discussion

There is little information on the fate of estrogenic alkylphenols in mammals and the study reported here represents the first detailed analysis of the metabolism of an estrogenic alkylphenol in mammalian hepatocytes. This study shows that 57% of the t-OP dose was metabolised during the first 5 min of incubation and after 15 min incubation, 94% of t-OP was transformed to a complex mixture of products. An earlier study has also reported that only 2–3% of the parent compound remained after a 48 h

Acknowledgements

We thank Drs. E.D. Ceppi, F.S. Smith and M.A. Titheridge (University of Sussex) for the provision of rat hepatocytes and Professor J.P. Sumpter, Brunel University, UK, for the kind gift of radiolabelled t-OP.

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