Development of a Fish Reporter Gene System for the Assessment of Estrogenic Compounds and Sewage Treatment Plant Effluents

Abstract

This study reports on the development and application of a fish-specific estrogen-responsive reporter gene assay. The assay is based on the rainbow trout (Oncorhynchus mykiss) gonad cell line RTG-2 in which an acute estrogenic response is created by cotransfecting cultures with an expression vector containing rainbow trout estrogen receptor a complementary DNA (rtERalpha cDNA) in the presence of an estrogen-dependent reporter plasmid and an estrogen receptor (ER) agonist. In a further approach, RTG-2 cells were stably transfected with the rtERalpha cDNA expression vector, and clones responsive to 17beta-estradiol (E2) were selected. The estrogenic activity of E2, 17alpha-ethinylestradiol, 4-nonylphenol, nonylphenoxy acetic acid, 4-tert-octylphenol, bisphenol A, o,p'-DDT, p,p'-DDT, o,p'-2,2-bis(chlorophenyl)-1,1-dichloroethylene (o,p'-DDE), p,p'-DDE, o,p'-2,2-bis(chlorophenyl)-1,1-di-chloroethane (o,p'-DDD), p,p'-DDD, and p,p'-2,2-bis(chlorophenyl)acetic acid (p,p'-DDA) was assessed at increasing concentrations. All compounds except o,p'-DDT, p,p'-DDE, and p,p'-DDA showed logistic dose-response curves, which allowed the calculation of lowest-observed-effect concentrations and the concentrations at which half-maximal reporter gene activities were reached. To check whether estrogen-responsive RTG-2 cells may be used to detect the estrogenic activity of environmental samples, an extract from a sewage treatment plant (STP) effluent was assessed and found to have estrogenic activity corresponding to the transcriptional activity elicited by 0.05 nM of E2. Dose-response curves of nonylphenol, octylphenol, bisphenol A, and o,p'-DDD revealed that the RTG-2 reporter gene assay is more sensitive for these compounds when compared to transfection systems recombinant for mammalian ERs. These differences may have an effect on the calculation of E2 equivalents when estrogenic mixtures of known constitution, or environmental samples, such as STP effluents, are assessed.

Utility of in vitro test methods to assess the activity of xenoestrogens in fish

“…To our knowledge, however, the present study is the first to report the antagonistic behavior of ZM 189154 in rainbow trout primary hepatocytes. However, these results also agree with those of recent studies using rainbow trout ER␣-specific reporter gene systems in rainbow trout gonad or hepatoma cell lines [34,35] and with studies in rats demonstrating the antagonistic behavior of ZM 189154 in uterus, vagina, bone, and on luteinizing hormone secretion and growth rate [19].…”

“…The sensitivity of the reporter gene bioassay described in the present study was lower than that observed for the same reporter gene bioassay configuration using the chimeric human ER␣ [21,22] and with an ER reporter gene bioassay using the rainbow trout gonad cell line (RTG-2) [34]. The detection limit in the present study using the rainbow trout ER␣ construct was 10 nM E 2 , whereas the human ER construct has a detection limit of 0.01 nM and a maximum response at 1 nM [22].…”

Environmental contaminants and biomarker responses in fish from the Columbia River and its tributaries: Spatial and temporal trends

“…Largescale sucker from Station 506 also contained traces (0.01 μg/g) of o,p′-DDE and o,p′-DDD. Like many other pesticides and their metabolites, o,p′-DDD is weakly estrogenic even though the o,p′-homologs have historically been considered relatively benign (Guillette et al, 1996;Toppari et al, 1996;Tyler et al, 1998;Ackerman et al, 2002). The cumulative risk to fish and wildlife represented by concentrations of o,p′-DDT is unknown.…”

Assessment of estrogenic and thyrogenic activities in fish feeds

“…343 (Berthois et al, 1986). In 365 addition, serum deprivation during treatment resulted in weaker reporter gene induction when cells 366 were exposed to E2, again as previously reported (Ackermann et al, 2002). Moreover, the lack of 367 serum appears to affect cell fitness since they are not able to overcome the high concentrations of 368 fish feed extracts, resulting in cytotoxicity.…”