In vivo and in vitro metabolism and organ distribution of nonylphenol in Atlantic salmon (Salmo salar)

Abstract

In the environment, nonylphenol (NP) occurs predominantly as a degradation product of nonylphenol ethoxylate (NPE). They can be found in many types of products including detergents, plastics, emulsifiers, pesticides, and industrial and consumer cleaning products. As a consequence of their use in a variety of products, they are quite common in rivers and other aquatic environments that receive sewage discharges. Because of its enhanced resistance towards biodegradation, toxicity, estrogenic effects, and ability to bioaccumulate in aquatic organisms NP has been regarded as the most critical metabolite of APEs. We have studied the in vivo and in vitro metabolism and organ distribution of NP in juvenile salmon. Fish were exposed in vivo to waterborne [³H]-4-n-NP for a period up to 72 h or were administered a single oral dose of [³H]-4-n-NP. In vitro biotransformation of NP was studied by exposure of cultured salmon hepatocytes to [³H]-4-n-NP in the presence or absence of a CYP1A-inducer, β-naphthoflavone (βNF). Our results show that 4-n-NP was mainly metabolized in vivo, to its corresponding glucuronide conjugates and hydroxylates. The major route of excretion was the bile. The half-life of residues in carcass and muscle was between 24 and 48 h in both waterborne and dietary exposure. In whole body autoradiography, intragastric administered [³H]-4-n-NP was mainly present in the gastrointestinal tract and bile. NP-derived radioactivity in fish exposed via water was more evenly distributed in the organs compared to intragastric exposure and were observed in the intestinal contents, liver, kidney, gills, skin, abdominal fat and brain. In vitro pretreatment of hepatocytes with βNF had no effect on rates or patterns of NP biotransformation. The in vitro metabolic rate of NP were 118 pmol NP metabolized/h/0.5×10⁶ cells without βNF, and 98 pmol NP metabolized/h/0.5×10⁶ cells when βNF was added to the culture medium.

Introduction

Alkylphenols (APs) are used as antioxidants and in the synthesis of alkylphenols polyethoxylates (APEs) detergents. APEs are also used as industrial detergents in the textile and paper industries, in toiletries and as spermicides (Talmage, 1994, Sonnenschein and Soto, 1998). A complex microbial degradation pattern, characterized by the shortening of the polyethoxylate chain to form several metabolic products (APs, mono- and diethoxylate) that are more toxic than the parent compound, has been established for APEs (Ekelund et al., 1990, Naylor et al., 1992). Recent studies have identified nonylphenol (NP) as the most critical metabolite of APEs because of its enhanced resistance towards biodegradation, toxicity, and ability to bioaccumulate in aquatic organisms (Ahel et al., 1994, Tyler et al., 1998). APE degradation products have been detected in drinking water (Clark et al., 1992). NP leaches from PVC tubing during milk processing (Junk et al., 1974) and from plastics used in food packaging (Gilbert et al., 1982). Recently, the estrogenic effects of NP have been documented in a number of in vivo and in vitro studies (Jobling and Sumpter, 1993, Jobling et al., 1996, Arukwe et al., 1997a, Arukwe et al., 1998, Celius and Walther, 1998).

Substantial amounts of alkylpenolic chemicals enter the aquatic environment. The major routes of entrance are through waste water discharges into rivers and the sea and from sewage sludge. Domestic sewage effluents can contain alkylphenolic compounds up to hundreds of μg/l (Ahel et al., 1994, Naylor, 1995). In contrast, certain types of industrial effluents, such as those originating from pulp mills and textile industries, can contain mg/l quantities (ibid.). In the UK, industrial effluents contain concentrations of NP that may exceed 100 μg/l (Tyler et al., 1998). In most of the rivers studied, concentrations are <10 μg/l (Blackburn and Waldock, 1995). However, the concentrations of estrogenic compounds in some UK rivers are high enough to stimulate egg yolk protein precursor (vitellogenin; Vtg) production in fish (Harries et al., 1997).

The initial demonstration of estrogenic properties of an NP, p-nonylphenol, followed the observation that estrogen-dependent growth of breast cancer cells occurred when this compound leached from plastic tubing in the laboratory (Soto et al., 1991). Other cell culture studies by Jobling and Sumpter (1993) and White et al. (1994) found that alkylphenolic compounds, including 4-NP, stimulated Vtg production in hepatocytes of male trout. Production of the eggshell zona radiata proteins (Zrp) was demonstrated in intact juvenile Salmon (Arukwe et al., 1997a, Arukwe et al., 1998). Vtg and Zrp are precursors of egg-yolk protein and the inner layer of eggshells, respectively, and are usually found only in female fish in which hepatic synthesis is estrogen dependent (Mommsen and Walsh, 1988, Oppen-Berntsen et al., 1992).

Because of their estrogenic properties, interest in APs is growing. For example, in vivo and in vitro studies have shown that NP is estrogenic in fish, birds, reptiles and mammals (for recent reviews see, Tyler et al., 1998, Sonnenschein and Soto, 1998, Arukwe and Goksøyr, 1998). In vivo studies with rainbow trout (Oncorhynchus mykiss) indicated that low μg/l concentrations of NP inhibited testicular growth (Jobling et al., 1996). To more completely understand the effects of NP on fish and the possible consequences to human and animal consumers, it is essential to elucidate the metabolic fate of nonylphenol.

Previous studies on metabolism and disposition of NP used radioactivity in the organs, urine and bile of rainbow trout after dietary (Talmage, 1994, Thibaut et al., 1998a; Thibaut et al., 1999) and water (Lewis and Lech, 1996) exposure. Both groups efforts were restricted to freshwater fish and radioactivity was shown to be mainly concentrated in the bile. As part of a larger effort to understand the fate and effects of APs in seawater fish, the main purpose of the present study was to investigate the capability of salmon to metabolize NP and to determine in vivo and in vitro metabolic pathways and organ distribution of this alkylphenol. The Atlantic salmon is an anadromic species that form the basis for economically important fisheries and aquaculture in Norway and other countries. In this regard, the effects of NP in seawater could be addressed. In addition, this study was designed to provide additional information on uptake and distribution following dietary and waterborne exposure under identical environmental conditions. Metabolism and excretion were analyzed using in vivo and in vitro studies and these were coupled to whole body autoradiographic localization studies.