Urinary metabolites of 4-n-nonylphenol in rainbow trout (Oncorhynchus mykiss)

https://doi.org/10.1016/S0048-9697(99)00225-9Get rights and content

Abstract

Nonylphenol is present in surface water and aquatic sediments and because of its lipophilic characteristics shows a considerable potential to bioaccumulate in aquatic organisms. Nonylphenol inhibits testicular growth and induces vitellogenin synthesis in male rainbow trout. In order to better understand the effects of nonylphenol on fish and its impact in the aquatic environment, it is essential to elucidate the metabolic fate of this compound. A single oral dose (5 mg, 1850 KBq) of [3H]4-n-nonylphenol resulted in 1.1% and 3.0% of the ingested radioactivity eliminated in urine after 24 and 48 h, respectively. Four metabolites were separated by radio-HPLC and tentatively identified by mass spectrometry. Urinary metabolites likely resulted from the initial ω-oxidation of 4-n-nonylphenol to the putative 9-(4-hydroxyphenyl)-nonanoic acid which subsequent β-oxidation led to 4-hydroxybenzoic acid as major metabolite. Intermediary metabolites, namely 3-(4-hydroxyphenyl) propionic acid and 3-(4-hydroxyphenyl)-2-propenoic acid confirmed the occurrence of this β-oxidative pathway. Urinary metabolites identified in this study were quite different from biotransformation products previously described in bile of trout treated with 4-n-nonylphenol.

Introduction

Alkylphenols are the final products of the microbial biodegradation of a widely used class of non-ionic surfactants, i.e. alkylphenol-polyethoxylates during wastewater treatment (Giger et al., 1984, Ahel et al., 1994). Alkylphenols are also widely used in plastic industry as antioxidants and have been reported to leach from plastics used in food processing and packing (Soto et al., 1991). Nonylphenol (NP) is the predominant component of the alkylphenolic chemicals in the environment and in digested sewage sludges (Giger et al., 1984) and is a known estrogenic xenobiotic in fish, birds, and mammals (White et al. 1994).

In 1991, Soto et al. showed the estrogenicity of NP in mammals. Based on in vitro studies, they reported that NP induced both cell proliferation and the progesterone receptor in human MCF-7 breast tumor cells. They also reported in vivo data in which ovariectomised rats treated with NP exhibited increased uterine mitotic activity. This uterotrophic effect was recently confirmed by Lee and Lee (1996) in immature female rats. It is known that estrogenic chemicals increased the synthesis of vitellogenin in aquatic species (Wallace and Selman, 1981). Several in vitro studies demonstrated that NP stimulates the synthesis of vitellogenin in rainbow trout hepatocytes (Jobling and Sumpter, 1993, White et al., 1994, Petit et al., 1997). Lech et al. (1996) reported the in vivo induction of liver vitellogenin mRNA in both male and immature female rainbow trout exposed to NP under flow-through conditions. Jobling et al. (1996) confirmed that exposure of male rainbow trout to 4-NP caused synthesis of vitellogenin and a concomitant inhibition of testicular growth. Gray and Metcalfe (1997) recently observed an alteration of the gonadal development of Japanese medaka submitted, from hatch to 3 months of age, to 4-NP water exposure. At 50 μg/l NP dose, 50% of the male fish developed testis-ova, an intersex-condition, and at 100 μg/l NP dose, changes in sex ratios were observed, indicating a sex reversal of the male population.

In trout, the action of NP appears to be mediated by the estrogen receptor (Jobling and Sumpter, 1993, White et al., 1994, Petit et al., 1997), but little is known about the metabolic fate of this molecule. In previous works, dealing with the disposition and excretion route of [3H]4-n-NP in rainbow trout (Thibaut et al., 1998a), we observed that 48 h after dosing, the urinary excretion of radioactivity amounted to 3.0±2.2% of the ingested radiochemical. Recently some NP metabolites were identified in bile (Lewis and Lech, 1996, Coldham et al., 1998, Thibaut et al., 1998b). They mainly correspond to glucuronic acid conjugates and/or hydroxylated and carboxylated compounds. In order to complete our knowledge of the in vivo biotransformation pathways of 4-n-NP in rainbow trout, we undertook to identify urinary metabolites.

Section snippets

Chemicals

3-(4-Hydroxyphenyl)-propionic acid was obtained from Fluka (Buchs, Switzerland), 4-hydroxybenzoic acid from Merck (Darmstadt, Germany) and β-glucuronidase from bovine liver (type B-1), sulfatase from aerobacter aerogenes (type VI), MS 222 (tricaine methanesulfonate) and d-saccharic acid 1,4-lactone were from Sigma (Saint Quentin Fallavier, France). [R-2,6-3H]4-n-NP was synthesized according as previously described (Thibaut et al., 1998b).

Animals and treatment

Immature rainbow trout (Oncorhynchus mykiss, 180–200 g)

Results

Twenty-four hours after ingestion of a single dose of [3H]4-n-NP (25 mg/kg) rainbow trout excreted 1.1±1.1% of the radioactivity in urine. Forty-eight hours after dosing this excretion amounted 3.0±2.2% (data not shown). An HPLC method was developed to obtain an adequate baseline separation of the urinary metabolites of 4-n-NP. Radio-HPLC analysis of urine radioactivity (Fig. 1) showed the presence of four main metabolites (U1 -U4), more polar than the parent compound (4-n-NP RT=58 min). None

Discussion

Previous studies have shown that NP can be metabolized by rainbow trout. The Lech group (Lewis and Lech, 1996, Meldahl et al. 1996) investigated the metabolites of a mixture of some isomers of NP for which the phenol moiety was located at the two, three or four carbon position of the C9 alkyl chain. The products of in vivo metabolism isolated from the bile of waterborne exposed trout were found to be glucuronide of the parent isomers hydroxylated in the C-8 position of the nonane chain.

Acknowledgements

This work was supported in part by the EC programme Environment and Climate (contract ENV4-CT96-0223).

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