Uptake, metabolism and excretion of bisphenol A in the rainbow trout (Oncorhynchus mykiss)
Introduction
For the past decades, an increasing effort has been made to elucidate the endocrine disrupting properties of natural as well as man-made compounds with hormonal activity (Toppari et al., 1996, Tyler et al., 1998). The endocrine disrupting chemicals (EDCs) can be found in a wide variety of matrices such as foodstuff, packaging materials and chemical formulations (Liehr et al., 1998). EDCs reach the aquatic environment mainly through sewage effluents thereby posing a potential threat to the organisms living in these ecosystems (Matthiessen and Sumpter, 1998). Field studies conducted on wild fish from English rivers show a high incidence of intersex among males living near sewage effluents (Jobling et al., 1998). Other experiments show that estrogenic responses can be detected in fish up to 5 km downstream from the site of sewage discharge (Harries et al., 1997). In addition to the estrogenic effects observed in natural aquatic environments, laboratory studies have revealed various endocrine disrupting properties of the compounds in question (Christiansen et al., 1998, Panter et al., 1998).
One of the compounds known to mimic the actions of natural oestrogen is bisphenol A (BPA) (Dodds and Lawson, 1936, Benjonathan and Steinmetz, 1998). In fish, an increased concentration of the yolk protein vitellogenin (VTG) has been observed following injection and water exposure of the compound (Christiansen et al., 2000, Lindholst et al., 2000). Apart from the endocrine disrupting effects observed for BPA and other hormonally active compounds, it is necessary to consider the uptake, metabolism and excretion in order to fully understand their modes of action. A rapid excretion or conversion of an oestrogenic compound into a less potent metabolite, might be a way in which an organism can reduce its oestrogenic load. Since EDCs often include phenols and other aromatic groups (e.g. alkyl phenols, phthalates, parabens and bisphenols) conjugation of polar moieties by phase II degradation enzymes is expected. The addition of a glucuronyl or sulfonyl group to the parent molecule is generally believed to increase the water solubility thereby facilitating the subsequent excretion (Giroud et al., 1998).
As regards the disposition of BPA, most of our present knowledge is based on mammalian studies. In 1966, Knaak and Sullivan examined the various routes of excretion of BPA in the rat, following oral exposure. They concluded that bisphenol A glucuronic acid (BPAGA) was a major degradation product subjected to renal excretion. Within recent years, the specific isoforms of glucuronosyl- and sulfonyl transferase, responsible for the enzymatic phase II conjugation of BPA have been characterised in the rat (Yokota et al., 1999, Suiko et al., 2000). In piscine systems, knowledge about metabolism and excretion of BPA, as well as most other EDCs, is scarce. Larsson et al. (1999) examined the presence of BPAGA in the bile from caged fish exposed to sewage effluent. Other studies measured the activity of phase II degradation enzymes in response to xenoestrogenic exposure (Andersson et al., 1985, Arukwe et al., 1997). However, the level of the BPAGA production as well as the excretion rate of BPA in fish has until now been unexplored.
The present study examines the toxicokinetic behaviour of BPA in the rainbow trout Oncorhynchus mykiss. The uptake, accumulation and excretion of nonmetabolised BPA was monitored in blood plasma and muscle- and liver tissue. Furthermore, the metabolism of BPA was estimated by direct measurements of BPAGA in the blood plasma. The quantification of degradation product was carried out by means of an enzymatically synthesised BPAGA standard. Finally the yolk protein vitellogenin (VTG) was measured in the plasma throughout the experimental period, in order to characterise the estrogenic response associated with BPA exposure.
Section snippets
Experimental set-up
Juvenile rainbow trout (O. mykiss: 90–130 g) were purchased from a local fish farm, and acclimated for 10 days in 400-l tanks supplied with running fresh groundwater at 15°C. Fish were held in a 12-h light, 12-h dark photoperiod and were not fed during the experiment.
The experiment was carried out in a flow-through system consisting of 400-l tanks, fitted with circulatory pumps (Eheim 1250, Germany). Administration of water was controlled through the use of a centrifugal pump (UPS 25-40
Characterisation of BPAGA
Following purification of BPAGA, a characterisation of the generated standard was carried out by 13C NMR and mass spectrometry (Fig. 1). The mass spectrum of BPAGA recorded at a fragmentor voltage of 150 V, shows the [M–H]− ion at m/z=403 and the [M-177]− ion at m/z=227 originating from BPA. The fragments observed at m/z=175 and 113 probably result from the glucuronic acid moiety.
Water, plasma and tissue levels of BPA and BPAGA
In one of the experimental groups, fish were exposed to a nominal concentration of 0.44 μM BPA (100 μg BPA/l). Every
Discussion
The aim of the present study was to examine the uptake, accumulation, degradation and excretion of a well established xenooestrogen in a teleostean system. Two separate experiments, one on the water uptake and another on the excretion following compound injection were performed using the rainbow trout (O. mykiss) as a test organism. Compound administration in the water-exposed group was controlled by the use of a continuous flow-through system, assuring good agreement between nominal and actual
Acknowledgements
The authors wish to thank Birthe Christensen for excellent technical assistance. Furthermore, Jakob Bunkenborg from the Department of Chemistry, Odense University, is thanked for his assistance in the use of NMR. This work was funded by a Danish Environmental Research Programme grant to Professor Poul Bjerregaard and Associate Professor Bodil Korsgaard.
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