Evaluation of Xenoestrogenic Effects in Fish on Different Organization Levels

Schrag, Betting; Ensenbach, Uwe; Navas, José M.; Segner, Helmut

doi:10.1007/978-1-4899-0089-0_24

“…The rainbow trout primary hepatocyte vitellogenin or zona radiata protein assay includes these circumstances and therefore often is considered to be the most valid test to characterize the estrogenicity of a substance in vitro. However, high intra‐ [38] and interassay variability of the inducibility of vitellogenin or zona radiata proteins in hepatocytes is observed. Because immature female, immature mixed sex, or adult male rainbow trout were used for these studies, seasonal‐, age‐, and sex‐specific differences that result in different steroid levels and ER abundance may explain some of this variability.…”

Section: Discussionmentioning

confidence: 99%

“…Because immature female, immature mixed sex, or adult male rainbow trout were used for these studies, seasonal‐, age‐, and sex‐specific differences that result in different steroid levels and ER abundance may explain some of this variability. The concentration for half‐maximal vitellogenin induction for E 2 ranges from 1.8 nM [9] to 500 nM [39], whereas intermediate values of 46 nM [38] and 100 nM [40,41] have been measured.…”

Section: Discussionmentioning

confidence: 99%

“…Only very few studies with primary hepatocytes present complete dose‐response curves of environmental estrogens such as NP, OP, and Bis A. When testing these compounds, vitellogenin induction occurred between concentrations of 10 and 100 μM [38]. In contrast, White et al [8] detected OP‐and NP‐induced vitellogenin expression at 0.1 μM and 1 μM, respectively.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Development of a fish reporter gene system for the assessment of estrogenic compounds and sewage treatment plant effluents

Ackermann

¹

,

Brombacher

²

,

Fent

³

2002

Enviro Toxic and Chemistry

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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.

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“…Because immature female, immature mixed sex, or adult male rainbow trout were used for these studies, seasonal-, age-, and sex-specific differences that result in different steroid levels and ER abundance may explain some of this variability. The concentration for half-maximal vitellogenin induction for E 2 ranges from 1.8 nM [9] to 500 nM [39], whereas intermediate values of 46 nM [38] and 100 nM [40,41] have been measured.…”

Section: Comparison With Primary Fish Hepatocytesmentioning

confidence: 98%

“…Only very few studies with primary hepatocytes present complete dose-response curves of environmental estrogens such as NP, OP, and Bis A. When testing these compounds, vitellogenin induction occurred between concentrations of 10 and 100 M [38]. In contrast, White et al [8] detected OPand NP-induced vitellogenin expression at 0.1 M and 1 M, respectively.…”

Section: Comparison With Primary Fish Hepatocytesmentioning

confidence: 99%

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

Ackermann¹,

Brombacher²,

Fent³

2002

Environ Toxicol Chem

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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.

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