Effect of UV screens and preservatives on vitellogenin and choriogenin production in male medaka (Oryzias latipes)
Introduction
Organic chemicals that absorb ultraviolet radiation, which are called UV screens, are added to sunscreen products in concentrations of up to 10% for skin protection. Some of these compounds are also included in other cosmetics such as beauty creams, lipsticks, skin lotions, hair sprays, hair dyes, shampoos, and bubble bath powders, for product stability and durability. The use of UV screens is increasing because of growing public concern about skin damage, due to sunlight. Thus far, in studies of humans, acute and subchronic systemic toxicity of these compounds has been considered to be rather low (Okereke et al., 1995, Hayden et al., 1997), although there have been some problems, such as photoallergic reactions (Schauder and Ippen, 1997). However, it has been reported recently that these UV screens, induce pS2 protein in MCF-7 breast cancer cells, and that 4-methyl-benzylidene camphor (4-MBC) and uterotropic assay showed dose-dependent increases in uterine weight due to 4-MBC and octyl-methoxycinnamate (OMC) (Schlumpf et al., 2001).
Parabens are the most commonly used preservatives in cosmetics, toiletries, pharmaceuticals, and foods because of their relatively low toxicity in humans and their effective antimicrobial activity (Elder, 1984). However, it has been reported that the estrogenic activity of parabens and their main metabolites may be demonstrated by in vitro recombinant yeast assay, human estrogen receptor (ER) assay, and E-screen (Lemini et al., 1997, Routledge et al., 1998, Blaier et al., 2000, Pedersen et al., 2000, Satoh et al., 2000, Okubo et al., 2001). In particular, propyl paraben (n-propyl-p-hydroxy-benzoate; PP) has the highest estrogenic activity among paraben esters (Routledge et al., 1998, Blaier et al., 2000, Nishihara et al., 2000, Satoh et al., 2000, Okubo et al., 2001). It has also been reported that PP directly affects the male reproductive system (Song et al., 1989, Song et al., 1991, Oishi, 2002).
These previous reports revealed that 4-MBC and OMC in UV screens, and PP in preservatives have estrogenic activity especially in in vitro system. Recently, international and national estrogenic compound screening programs have established methods for the screening and testing of environmental chemicals under the Organization for Economic Co-operation and Development (OECD) and the US Environmental Protection Agency (EPA). Examples are use of sensitive fish species (e.g. fathead minnow Pimephales promelas, zebrafish Danio rerio, rainbow trout Oncorhynchus mykiss, and medaka Oryzias latipes) for specific tests (Hutchinson and Pickfold, 2002). In particular, medaka fish have been used in Japan as a model in systems for monitoring chemicals in water. Vitellogenin (VTG) is well known as an estrogen-responsive phosphoprotein, and can also be used as a biomarker of contaminant exposure in fish such as medaka. VTG is a complex egg yolk precursor protein in oviparous vertebrates and is produced in the liver (Ng and Idler, 1983). Recently, it has become possible to conveniently measure medaka plasma VTG concentration, since enzyme-linked immunosorbent assay (ELISA) has been developed for measurement of medaka VTG (Nishi et al., 2002). Moreover, it has been reported that choriogenin (CHG), known to be a liver-derived precursor protein for the inner layer subunits of the fish egg envelopes (Murata et al., 1997), responds to estrogenic compounds (Lee et al., 2002). These reports show that two proteins, VTG and CHG, are key molecules that are present in medaka livers, and are estrogen-responsive proteins.
In the present study, we analyzed the VTG plasma concentration of medaka exposed by various concentrations of 4-MBC, OMC, and PP as compared to exposure to 17β-estradiol (E2) using a VTG ELISA system (Nishi et al., 2002). We also measured mRNA expression levels of the VTG subtypes VTG-1 and VTG-2 (Shimizu et al., 2002), of the CHG subtypes CHG-L and CHG-H (Murata et al., 1997), and of medaka sex hormone receptors, such as ERα, ERβ, and androgen receptor (AR) in the liver. In this study, we found increased plasma VTG concentration and increased mRNA expression level of VTG-1, VTG-2, CHG-L, and CHG-H in medaka exposed to 4-MBC, OMC, and PP.
Section snippets
Chemicals
OMC (Eusolex 2292) and 4-MBC (Eusolex 6300) were purchased from Sigma–Aldrich (Tokyo, Japan), and PP and E2 were purchased from Wako Pure Chemical Industries (Osaka, Japan).
Fish and study design
Adult male Japanese medakas (O. latipes) (body length 2.5–3.5 cm) were purchased from a local commercial supplier. Experimental fish were maintained for 7 days in 2 l of pure water, and were fed daily with tropical fish flake food (TetraFin, Tetra GmH, Melle, Germany). The acclimated fish (each treatment group: n=5) were
Plasma VTG concentration in medaka
Plasma VTG levels increased dose-dependently after all chemical treatments. However, plasma VTG levels were only slightly higher than those of the non-treated control following treatment with large doses of OMC. Plasma VTG levels of medaka treated with 4-MBC and PP were similar to those obtained following treatment with 10−5 times the E2 concentration (Fig. 1).
Estimation of mRNA expression of VTG, CHG, and sex hormone receptors using real-time RT-PCR analysis
To analyze the mRNA expression of egg proteins VTG and CHG, as well as sex hormone receptors ERα, ERβ, and AR in chemical-treated and
Discussion
In the present study, we analyzed the VTG plasma concentration and liver mRNA expression level of VTG, CHG, and sex hormone receptors of medaka exposed by various concentrations of 4-MBC, OMC, and PP as compared to exposure to E2.
We found for the first time that increase of plasma VTG concentration was induced in male medaka exposed to OMC and 4-MBC. In UV screens, plasma VTG concentration of medaka exposed to 4-MBC was higher than that of medaka exposed to OMC (Fig. 1). It has been reported in
Acknowledgements
The authors would like to thank EnBioTec Laboratories, Co., Ltd. for their donation of the VTG ELISA system. This project was supported by grants from the following: the Ministry of the Environment (Government of Japan), the Ministry of Education, Culture, Sports, Science and Technology (Government of Japan). This study was carried out under the NIBB Cooperative Research Program (3-129).
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Authors who contributed equally to this study.