Amphibians as a model to study endocrine disruptors: II. Estrogenic activity of environmental chemicals in vitro and in vivo

https://doi.org/10.1016/S0048-9697(99)80017-5Get rights and content

Abstract

Several environmental chemicals are known to have estrogenic activity by interacting with development and functions of endocrine systems in nearly all classes of vertebrates. In order to get a better insight of potential estrogenic effects on amphibians caused by environmental pollution this study aims to develop a model for investigating endocrine disruptors using the amphibian Xenopus laevis. In that model the potential estrogenic activity of endocrine disruptors is determined at several levels of investigation: (I) binding to liver estrogen receptor; (II) estrogenicity in vitro by inducing vitellogenin synthesis in primary cultured hepatocytes; and (III) in vivo effects on sexual development. Here we deal with establishing methods to assay estrogenic activity of environmental chemicals in vitro and in vivo. In vitro we used a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) technique to determine mRNA-induction of the estrogenic biomarker vitellogenin in primary cultured hepatocytes of male Xenopus laevis. Time courses of vitellogenin-mRNA in the presence and absence of 10−6 M 17β-estradiol (E2) resulted in a marked loss of mRNA from controls after 2 days while E2 treatment kept vitellogenin-mRNA at a relatively stable level. After 36 h of incubation estrogenic activities of E2, 4-nonylphenol (NP), and 2,2-bis-(4-hydroxyphenyl)-propan (bisphenol A) at concentrations ranging from 10−10 to 10−5 M were assayed by RT-PCR of vitellogenin-mRNA and showed the following ranking of dose-dependent potency: E2>NP>bisphenol A. These in vitro results were confirmed further by in vivo experiments determining sexual differentiation of Xenopus laevis after exposure to E2 and environmental chemicals during larval development. Concentrations of 10−7 and 10−8 M E2 as well as 10−7 M of NP or bisphenol A caused a significant higher number of female phenotypes compared to controls indicating a similar ranking of estrogenic potencies in vivo as in vitro. In addition, butylhydroxyanisol and octylphenol, both showed feminization at 10−7 M while octylphenol was also effective at 10−8 M. In summary these results demonstrate for the first time the use of a semiquantitative RT-PCR technique for screening estrogenicity by assaying mRNA induction of the estrogenic biomarker vitellogenin in vitro. The combination of this newly developed method with classical exposure experiments is necessary for determination of the biological significance of estrogenic chemicals.

Introduction

Several environmental chemicals are known to interact with development and functions of endocrine systems in wildlife and humans (Colborn and Clement, 1992). Many of these endocrine disruptors have estrogenic activity and can disrupt normal functions of sex steroids by causing feminization phenomena in all classes of vertebrates (Colborn et al., 1993; Sharpe and Skakkebaek, 1993). The possible impact of these endocrine disrupting chemicals on reproduction of animals needs to be considered because many of these compounds bioaccumulate due to their lipophility and persistence in the environment (Schäfer et al., 1996). Feminization of animals derived from areas polluted by estrogenic chemicals has been observed in nearly all classes of vertebrates including fish (Munkittrick et al., 1991), reptiles (Guilette and Crain, 1996), birds (Fry and Toone, 1981), and mammals (Beland, 1989). Unfortunately, information about a possible threat of endocrine disruptors for amphibian reproduction is scarce despite the dramatic declining of amphibian populations all over the world (Blaustein, 1994; Stebbins and Cohen, 1995). Although the biology of amphibians provides several features which make them an excellent model to study potential effects of endocrine disruptors, they have not been a well studied class of vertebrates.

In amphibians, like in all other vertebrates, estrogenic effects of compounds are mediated by binding to cytosolic estrogen receptors in target cells leading to dimerization of the ligand–receptor-complex. The homodimer binds to specific estrogen responsive elements of the DNA and promotes an estrogen specific genexpression pattern (Beato et al., 1995; Nimrod and Benson, 1996). One of the major target organs for the female sexual steroid 17β-estradiol (E2) is the liver. The induction of the yolk promoting protein vitellogenin is driven specifically by E2 in all egg laying vertebrates (Perlman et al., 1984; Tata, 1987). In addition, larval sexual development of amphibians is hormone-dependent meaning that treatment with E2 may shift sexes of a breed to phenotypic complete feminization (Witschi, 1971).

The main focus of our present research is to develop a comprehensive model using amphibians to detect estrogenic potencies of chemicals as well as of samples taken from the environment. Our modeling with Xenopus laevis for estrogenic effects of endocrine disruptors includes several levels of investigation: (I) binding to liver estrogen receptor; (II) biological significance in vitro by monitoring synthesis of the estrogenic biomarker vitellogenin in primary cultured hepatocytes; and (III) in vivo effects on sexual differentiation during larval development.

In the first phase of this research a radioreceptor assay using cytosolic liver homogenates to detect estrogen receptor binding of endocrine disruptors was established (Lutz and Kloas, 1999). Binding to estrogen receptor is the pre-requisite for estrogenic activity but it does not discriminate between activating or blocking effects on the receptor. Thus further experiments are needed to demonstrate the biological significance of xenoestrogens which bind to the estrogen receptor demonstrating their estrogenic effects. Effects on reproduction and development may also occur through other mechanisms. The present paper focuses attention on establishing experimental set-ups as evidence for estrogenicity of environmental chemicals in vitro by monitoring the estrogenic biomarker vitellogenin in primary cultured hepatocytes and in vivo by determining effects on sexual differentiation during larval development.

Section snippets

Animals

All experiments were carried out with Xenopus laevis tadpoles or adult male animals reared from the animal stock of the Department of Zoology II, University of Karlsruhe. Spawning of adult Xenopus laevis was induced as described elsewhere (Kloas et al., 1997) by injecting human chorionic gonadotropin (HCG, Sigma, Deisenhofen). Eggs and developing tadpoles were kept in 50-l tanks containing well-aerated tap water at 22°C under a 12:12-h light–dark cycle (lights on 06.00–18.00 h) up to

Semiquantitative RT-PCR of vitellogenin-mRNA in vitro

The applied procedures for RT-PCR of vitellogenin-mRNA as well as of elongation factor 1α were successful. Specific signals were obtained for amplified cDNAs encoding a vitellogenin A2 gene specific 529 bp sequence and an elongation factor 1α gene specific 285 bp sequence (Fig. 2). The analyses of these PCR products by automatic sequencing (Sequence Laboratories, Göttingen) revealed that both amplified cDNAs are identical with the predicted bp sequences for vitellogenin and elongation factor 1α

Discussion

The focus of the present paper aimed at establishing methods using amphibians as a model to investigate estrogenicity of environmental chemicals in vitro and in vivo. The techniques presented here were developed in order to get a comprehensive model using the amphibian Xenopus laevis to study endocrine disruptors including several levels of investigation: (I) binding to liver estrogen receptor; (II) estrogenicity in vitro by inducing vitellogenin synthesis in primary cultured hepatocytes; and

Conclusion

A novel semiquantitative RT-PCR technique for screening estrogenicity by assaying mRNA induction of the estrogenic biomarker vitellogenin in vitro was described. This newly developed in vitro method combined with classical exposure experiments in vivo demonstrates the biological significance of estrogenic endocrine disruptors on the amphibian Xenopus laevis. Both methods documented here in combination with the recently developed estrogen receptorassay (Lutz and Kloas, 1999) represents a

Acknowledgements

This work was supported by grants (PAÖ 9605.02 and 9702.02) for the project BW-PLUS (Baden-Württemberg-Programm Lebensgrundlage Umwelt und ihre Sicherung) from the Ministery of Environmental Affairs and Traffic Baden-Württemberg. The supporting interest of Dr Werner Scholz (LFU, Karlsruhe) on this research demonstrated by many discussions and helpful suggestions is also gratefully acknowledged.

References (24)

  • T Colborn et al.

    Developmental effects of endocrine-disrupting chemicals in wildlife and humans

    Environ Health Perspect

    (1993)
  • R Dosch et al.

    Bmp-4 act as a morphogen in dorsoventral mesoderm patterning in Xenopus

    Development

    (1997)
  • Cited by (0)

    View full text