Effects of 17β-estradiol, nonylphenol, and bisphenol-A on developing Xenopus laevis embryos

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Abstract

Many chemicals released into the environment have the capacity to disrupt the normal development of aquatic animals. We investigated the influence of nonylphenol (NP), bisphenol-A (BPA), and 17β-estradiol (E2) on developing Xenopus laevis embryos, as a model animal in the aquatic environment. Embryos were exposed to eight different concentrations of NP, BPA or E2 between 3 and 96 h post-fertilization (p.f.). Short body length, microcephaly, flexure, edema, and abnormal gut coiling were induced by 20 μM NP, BPA or 10 μM E2 by 96 h p.f. To clarify sensitive stages to these compounds, embryos were exposed to chemicals for 45 or 48 h starting at different developmental stages and experiments were terminated 96 h p.f. BPA and NP induced abnormalities in developing X. laevis, though the sensitive stages of embryos to these chemicals are different, BPA affecting earlier stages and NP affecting at later stages. To analyze the functional mechanisms of BPA and NP in induction of morphological changes, we adapted a DNA array technology and identified 6 X. laevis genes, XIRG, α skeletal tropomyosin, cyclin G1, HGF, troponin C2, and ribosomal protein L9. These findings may provide important clues to elucidate common mechanisms underlying teratogenic effects of these chemicals.

Introduction

Approximately 100,000 chemicals have been produced on industrial scale and ca. 1000–2000 new chemicals have been introduced each year and released into the environment, without evaluating the safety of these chemicals using developing animals (Younes, 1999). Many of these environmental pollutants have estrogenic activity with the potential to disrupt endocrine mechanisms and normal development (Colborn and Clement, 1992) by mimicking or blocking endogenous steroid hormones through interaction with hormone receptors (McLachlan, 2001). As a corollary, estrogenic chemicals have potential to induce feminization in nearly all classes of vertebrates (Fry and Toone, 1981; Guillette and Crain, 1996; Guzelian, 1982; Munkittrick et al., 1991; Pelissero et al., 1993). In amphibians, feminization can be induced by exogenous oestradiol, given between stages 52 and 55, converting testicular to ovarian tissue in laboratory experiments (Chang and Witschi, 1956; Witschi, 1971). Recently, gonadal abnormalities such as hermaphroditism and developmental delay have been induced in leopard frogs (Rana pipiens) and X. laevis by exposure to atrazine during larval development (Hayes et al., 2002a, Hayes et al., 2002b). Nonylphenol (NP) is widely used in the manufacture of non-ionic surfactants, lubricant additives, polymer stabilizers, antioxidant, and agricultural chemicals, and is released into the aquatic environment (Blackburn and Waldock, 1995; Kolpin et al., 2002). NP has estrogenic activity by binding to estrogen receptors (ER) in both in vitro and in vivo assay systems (Knudsen and Pottinger, 1999; Soto et al., 1991). Furthermore, NP has induced sex reversal in developing male X. laevis (Kloas et al., 1999). Developmental toxicity of nonylphenol ethoxylate has been reported using developing X. laevis, Litoria adelaidensis, and Crinia insignifera (Mann and Bidwell, 2000).

Bisphenol-A (BPA), a monomer of epoxy resins and polycarbonates, is used as a stabilizer or antioxidant in industrial chemicals, and has been used in the lacquer coating of food cans, and in dental sealants (Olea et al., 1996). BPA was originally introduced as a synthetic estrogen in 1938 (Dodds and Lawson, 1936). BPA has uterotrophic activity in rodent uterus (Colerangle and Roy, 1997), and binds to ER-α and ER-β (Gaido et al., 1997). BPA also induced feminization of male X. laevis larvae (Kloas et al., 1999), and also induced scoliosis, malformation of the head region, suppression of organogenesis of digestive and nervous systems in developing X. laevis (Iwamuro et al., 2003).

In X. laevis, the fertilized egg repeats synchronized cleavage until blastula stage (ca. 5 h p.f.). Through gastrula stage (ca. 12 h p.f.), neural tube is formed at 22 h p.f. The gray eye cups and the tail bud appear for the first time at 34 h p.f., and choroids fissure is nearly closed at 50 h p.f. Torsion of the intestine is 90° at 80 h p.f., then shows one and a half revolution until 98 h p.f. (Nieuwkoop and Faber, 1967). Effects of E2 on morphogenesis, such as small head and crooked vertebrae through these developmental stages have been reported previously (Nishimura et al., 1997). However, with the exception of the ER effects of E2 on gene expression in developing embryos have not been studied. In this study, we aimed to investigate effects of estrogen and estrogenic compounds on gene expression in developing X. laevis embryos using DNA macroarray techniques.

Section snippets

Embryos

In vitro fertilization of X. laevis eggs was performed as described previously (Yamamoto et al., 2001). The fertilized embryos were dejellied using 3% cysteine hydrochloride and washed with water several times. Embryos were cultured in 0.1× Steinberg’s solution (1× Steinberg’s solution composition in mg/L was NaCl 3400, KCl 50, Ca(NO3)24H2O 80, MgSO4 · 7H2O 205, and buffered to pH 7.2 with Tris–HCl) at 23 ± 0.5 °C. X. laevis embryos were staged according to Nieuwkoop and Faber (1967).

Chemical exposure

To evaluate

Teratogenic effects of NP, BPA, and E2 on developing X. laevis

Rates of normally developed embryos cultured in medium alone, dilution medium control (DMC), or cultured in dilution medium with solvent (control) showed the similar rate, 88.9 and 87.5%, respectively (Fig. 1, Table 1). There was no increase in abnormality or mortality in embryos exposed to NP at 1–5 μM. At 10 μM NP there was an increase in abnormality (55.8%), while at >30 μM NP there was increased abnormality and mortality (Fig. 1, Table 1). There was no increase in abnormality or mortality in

Discussion

Teratogenic effects of estrogen or estrogenic chemicals on developing X. laevis from stages 6 to 44 were identified in the present study. NP and BPA induced short body length, microcephaly, flexure, edema, and abnormal gut coiling. E2 also induced microcephaly, flexure, edema, and abnormal gut coiling but not short body length. These results of E2 exposure are consistent with a previous report (Nishimura et al., 1997). E2 has no effect on the body length at any developmental stages examined,

Acknowledgments

We are grateful to all the members of Dr. Ueno’s laboratory for their kind support. We thank Dr. Daniel B. Pickford, Department of Biological Sciences, Brunel University, UK, for critical reading of the manuscript. This work was supported by grants from the Ministry of Environment, Japan and the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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