3R-Project 47-96

Development of an in vitro system for the detection of estrogenic compounds (xenoestrogens)

Karl Fent¹
Swiss Federal Institute for Environmental Science and Technology (EAWAG), 8600 Dubendorf, Switzerland
¹present address: Fachhochschule beider Basel, Institut für Umwelttechnik, 4132 Muttenz, Switzerland.
k.fent@fhbb.ch

Keywords: fish; toxicology; cell cultures: organ-specific; cell cultures: transgenic; reporter gene assay; reduction; toxicity testing: xenobiotics

Duration: 4 years Project Completion: 2000

Background and Aim
An increasing number of compounds are being recognized as possessing estrogenic activity by interfering with the sex hormone system. Many of them are widely used chemicals that enter the aquatic environment via effluents from sewage treatment plants. Estrogenic compounds (xenoestrogens) mimic the natural estrogen 17beta-estradiol and may negatively interact with the sex hormone system and reproduction. These compounds compete for binding to the estrogen receptor (ER) and influence gene expression. The aim of this project is the development of in vitro systems using permanent fish cell lines to detect estrogenic activity of environmental chemicals, chemical mixtures and environmental samples (wastewater). The production of ER-mediated gene products (ER, yolk precursor vitellogenin: VG) will be used to identify and assess estrogenic potential.

Method and Results
Fish cell lines used include a hepatocarcinoma cell line PLHC-1 from Poeciliopsis lucida and two cell lines from rainbow trout, gonadal RTG-2 cells and liver RTL-W1 cells. PLHC-1 and RTG-2 cells were adapted to serumfree culture conditions showing similar growth rates as when thriving in serum-containing medium (1). RTL-W1 cells need 1% serum in order to thrive. A palette of techniques to measure estrogenic activity have been developed using antibody, PCR and transfection techniques. A monoclonal antibody against conserved sequences of fish VG proteins has been produced, along with a monoclonal anti-VG antibody which crossreacts with trout-VG. A competitive RT-PCR technique has been developed. To quantify levels of ER and VG mRNA induction in vitro and in vivo. Transfection of PLHC-1 and RTG-2 cells is performed in a dual luciferase assay using estrogen responsive reporter genes. In RTG-2 cells, an expressed estrogen receptor mRNA fragment was detected by RT-PCR. Several plasmids carrying different fragments of the VG promoter and fused to the firefly luciferase encoding cDNA were compared for their induction potential when transfected into fish cell lines. The quantity of expressed ER seems too low in these cells. Further experiments are aimed at the detection of estradiol-induced reporter gene activity .

Conclusions and Relevance for 3R
Results of this ongoing study indicate the potential of these in vitro systems for detecting xenoestrogens. The transfection system is most promising and will be further developed and optimized. Once established, the systems will allow the identification and assessment of potentially estrogenic chemicals prior to production and release into the environment, hence contributing to the reduction of in vivo fish tests (2).

(see also 3R-Info-Bulletin 9)
Published updated Version 9/2007 (pdf)

References
1) Ackermann G. E., Fent K. (1998), The adaptation of the permanent fish cell lines PLHC-1 and RTG-2 to FCS-free media results in similar growth rates compared to FCS-containing conditions, Mar. Environ. Res. 46, 363-367.

2) Fent K. Ackermann G.E. Schwaiger J. (2000) Long-term effects of nonylphenol on vitellogenin and zona radiata protein expression in juvenile rainbow trout. Proceedings of the 6th International Conference on Reproduction Physiology of Fish, Bergen, Norway, p. 356-358.

3) Fent K., Ackermann, G.E., Schwaiger J. (2000) Analysis of vitellogenin mRNA by quantitative reverse transcription polymerase chain reaction (RT-PCR) in juvenile fish exposed of 12 months to nonylphenol. Marine Environmental Research 50: 193 (short communication).

4) Fent K. (2001) Fish cell lines as versatile tools in ecotoxicology: Asssessment of cytotoxicity, cytochrome P450 1A induction potential and estrogenic activity of chemicals and environmental samples.Toxicology In Vitro 15, 477-488.

5) Ackermann G.E., Brombacher E., Fent K. (2002) Development of a fish reporter gene system for the assessment of estrogenic compounds and sewage treatment plant effluents. Environ. Toxicol. Chem. 21/9, 1864-75.

6) Fent K. and Achermann, G.E. (2002) A fish cell line reporter gene assay for the assessment of estrogenic acivity of environmental chemicals and acquatic samples. Marine Environmental Research 54: 751-52 (short communication).

7) Ackermann G.E., Schwaiger J., Negele R.D, Fent K. (2002) Effects of long-term nonylphenol exposure on gonadal development and biomarkers of estrogen exposure in juvenile rainbow trout (Oncorhynchus mykiss). Aquat.Toxicol. 60: 203-221.