Biomarkers Of Endocrine Disruption Research Articles

Biomarkers of Exposure and Reproduction-Related Effects in Mussels Exposed to Endocrine Disruptors

Biomarkers are useful tools to study the health of estuarine and marine ecosystems. Biomarkers can be measured in different organisms, but mussels have acquired a global importance as sentinels in marine pollution-monitoring programs. In the present work, we aimed to determine the effects of different endocrine disruptors in mussels by using peroxisome proliferation as a biomarker of exposure to organic pollutants and the levels of vitellogenin (Vtg)-like proteins as biomarker of endocrine disruption. In experiment 1, mussels Mytilus edulis were exposed for 3 weeks to North Sea crude oil (NSO 0.5 ppm) and a mixture of 0.5 ppm NSO, 0.1 ppm alkylphenol mix, and 0.1 ppm extra polycyclic aromatic hydrocarbons (PAHs) (MIX). In experiment 2, mussels were exposed for 3 weeks to diallylphthalate (DAP 50 ppb), bisphenol-A (BPA 50 ppb), and tetrabromodiphenylether (TBDE 5 ppb). Peroxisome proliferation was assessed by measuring acyl-CoA oxidase (AOX) activity and peroxisomal volume density (VVp) in digestive gland. Vtg-like protein levels were measured in gonads by the alkali-labile phosphate (ALP) method. Gonad was also analyzed histologically, and the gonad index (GI) calculated. Mussels exposed to NSO and MIX showed significantly increased AOX activities and VVP compared with control animals. Significantly higher VVP was also found in DAP- and TBDE-exposed mussels. Effects on ALP and GI depended significantly on sex and time of year. In female mussels, ALP levels and GI were lower in the NSO group. In male mussels, ALP levels were significantly increased in the MIX group. The volume density of athretic oocytes was higher in the NSO and MIX exposure groups than in controls, and gonad resorption was observed in the BPA exposure group. Our results confirm the usefulness of peroxisome proliferation as a biomarker of exposure to organic contaminants in mussels and indicate that changes in Vtg-like proteins could be used as potential indicator of pollutant effects on mussel reproduction.

Short-term bioaccumulation, circulation and metabolism of estradiol-17β in the oyster Crassostrea gigas

Steroids are active signal transmitters in Vertebrates. These roles have also been hypothesized in other Phyla and endocrine disrupting effects have been reported for different estrogen-like compounds in fishes and some marine invertebrates. As estradiol-17β has shown some physiological activities in the oyster and as estrogens or estrogen-like molecules can be present in water, we have investigated the bioaccumulation and metabolism of this estrogen in vivo in the oyster Crassostrea gigas. When dissolved in seawater, in less than 48 h estradiol-17β concentrated up to 31 times in the soft tissues of the suspension-feeder mollusc. Injected in the adductor muscle, estradiol-17β circulated from muscle to the gonad, the gills, the mantle, the labial palps, and to a lesser extent to the digestive gland. After 2 h, estradiol flow increased specifically towards this gland. Different hypotheses were raised concerning the circulation paths. However, in all cases estradiol metabolism primarily evidenced an in vivo transformation into estrone in the whole oyster and in its digestive gland. This strong 17β-hydroxysteroid-dehydrogenase activity confirms our previous in vitro results. In conclusion, it is proposed that oyster is able to take in charge estradiol as a potential contaminant in seawater. Therefore, its bioaccumulation and transformation into estrone could be studied as potential biomarkers of endocrine disruption. Furthermore, the experimental approach with dissolved steroids in the seawater combined to an anatomical screening appears as an interesting tool to investigate the bivalve endocrinology.

Larval Development and Vitellin-like Protein Expression in Palaemon elegans Larvae Following Xeno-oestrogen Exposure

Certain anthropogenic chemicals, most notably xeno-oestrogens, are known to have the potential to disrupt vertebrate endocrine systems. For example, induction of the female-specific protein, vitellogenin, in male fish is a well-known effect of exposure to xeno-oestrogens and serves as a biomarker of such exposure. There have been few comparable studies of putative biomarkers of endocrine disruption in invertebrates. An exception is the upregulation of vitellin-like larval storage protein (LSP) expression in the barnacle cypris larva following exposure to oestrogenic chemicals. The current study aimed to establish whether larvae of the glass prawn, Palaemon elegans, are likewise susceptible to xeno-oestrogen exposure. Using a polyclonal antiserum to P. elegans apolipovitellin, an 86 kDa polypeptide was detected by western blotting in the larval and early postlarval stages of this species. An indirect ELISA applied to the soluble protein fraction of larval homogenates determined that the titre of this putative LSP ranged, depending on larval stage, from 0.48-0.67 ng μg(-1). Exposure of P. elegans larvae to the xeno-oestrogen 4-n-nonylphenol (4-NP), at 0.2-20 μg L(-1), resulted in a significant, concentration-independent increase in putative LSP levels of 5-18%. Conversely, exposure to the natural oestrogen, 17β-oestradiol (E(2)), at 0.2 and 20 μg L(-1), led to a significant concentration-independent decline (up to 11%) in LSP levels. Whether the effect of 4-NP results from endocrine disruption is not known, however, an oestrogen receptor-mediated effect is unlikely. Other than a slight increase in larval mortality when exposed to 4-NP at 2 μg L(-1), neither 4-NP nor E(2) significantly affected development, growth or survival of P. elegans larvae.

Use of vitellogenin mRNA as a biomarker for endocrine disruption in feral and cultured fish.

The presence of the female-specific yolk protein precursor vitellogenin in blood and liver from male fish is widely used as an indicator of endocrine disruption. We studied the induction of vitellogenin mRNA in liver from several species of fish, both maintained in fish tanks or captured in the wild. Our procedure requires minute amounts of liver samples (down to 50 mg), and can be applied to field samples if the appropriate RNA-stabilisation agent is used. We used reverse-transcriptase PCR and quantitative real-time PCR for detection and precise quantitation of vitellogenin mRNA levels. Male mummichog (Fundulus heteroclitus) exposed to 17 beta-estradiol contained levels of vitellogenin mRNA up to 30 times higher than in untreated females and treatment with nonylphenol resulted in a weak but consistent induction of this transcript. We also studied levels of vitellogenin mRNA in a population of common carp (Cyprinus carpio) from the Anoia river, a river known for its high levels of estrogenic alkylphenols. The results were consistent with recorded data for fish from this sampling site. Finally, we also detected vitellogenin mRNA in barbs (Barbus graellsi), a species for which no vitellogenin sequence was available. The use of mRNA quantitation techniques for analysis of feral and cultured fish of different species opens the possibility of more precise detection and further control of the noxious effects of contaminants on the local fauna exposed to them.

Sequence analysis of choriogenin H gene of medaka (Oryzias latipes) and mRNA expression

Zona radiata proteins of medaka (Oryzias latipes) consist of two major subunit groups, ZI-1,2 and ZI-3. The precursor of ZI-1,2, which is a glycoprotein with a molecular weight of 74,000 to 76,000 Da, is called choriogenin H, and the precursor of ZI-3, with a molecular weight of 49,000 Da, is called choriogenin L. The precursor proteins are synthesized in the liver in response to estrogen in sexually mature female medaka. However, they are also induced in the male medaka when fish are exposed to estrogenic chemicals. Therefore, choriogenin is known as a possible sensitive biomarker for endocrine disruption in fish. In this study, the choriogenin H cDNA sequence was reanalyzed and the genomic DNA sequence was newly analyzed. This was done for the selection of proper reverse transcription-polymerase chain reaction (RT-PCR) primers to measure the choriogenin mRNA induction by estrogenic chemicals. In the results, the full cDNA sequence was found to be 2,109 bp long and the size of the open reading frame (ORF) was found to be a total of 1,998 bp encoding 666 predicted amino acids, which was found to be different from previously reported cDNA sequence of medaka choriogenin. In the choriogenin H cDNA, a repetitive domain and a nonrepetitive domain were shown. Regarding the repetitive domain, seven complete repeats of the 45 bp of 5'-ccc cag tac cca tca aag cct cag ccc cct cag aat cct cag gtc-3' encoding PQYPSKPQPPQNPQV were found. Medaka choriogenin H gene was found to possess seven exons and six introns, and the total length was 2,643 bp long. The seven repetitive nucleotide residues described above existed in exon 1, which was found to be 1,033 bp long. Based on the full sequence information, proper primers were synthesized for RT-PCR to detect choriogenin H mRNA induction in male and juvenile medaka by 17alpha-ethinylestradiol, and this type of measurement system was found to be effective as a simple tool for the screening of endocrine-disrupting chemicals.

Choriogenin mRNA induction in male medaka, Oryzias latipes as a biomarker of endocrine disruption

The fish egg envelope has been known to consist of two distinct layers. The outer layer is thin and is formed around the oocytes during the later stage of previtellogenic development. The inner layer, called zona radiata, is thicker than outer layer and occupies most of the egg envelope. Zona radiata proteins of medaka ( Oryzias latipes) consist of two major subunit groups, ZI-1,2 and ZI-3. The precursors of ZI-1,2 and ZI-3 have been named choriogenin H and choriogenin L, respectively. They are synthesized in the liver in response to estrogen, and then released into the blood stream and incorporated into the zona radiata in sexually matured female medaka. However, choriogenin is also induced in male medaka when the fish are exposed to estrogenic chemicals. Choriogenin can be suggested as a possible biomarker of endocrine disruption in fish, however, relatively little has been known about chorogenin gene expression by estrogenic chemicals. In this study, full sequence of choriogenin L DNA was identified, and measurement of choriogenin mRNA induction was established in medaka by use of RT-PCR technique. Also estrogenic effects of industrial chemicals were evaluated by this tool. When mature male medaka was treated with bisphenol A, nonylphenol, and 17α-ethinylestradiol for 6 days, respectively, the expression levels of choriogenin L and H mRNA were elevated in treated groups. The induction of choriogenin subunits expression by these chemicals showed a dose-dependent pattern and choriogenin L was found to be more sensitive than choriogenin H.

Estrogenic responses of larval sunshine bass (Morone saxatilis × M. Chrysops) exposed to New York city sewage effluent

To determine the estrogenicity of effluents from sewage treatment plants (STPs) to larval fish, 2-day-old sunshine bass were exposed to effluents from three STPs serving New York City (NYC), varying in size and treatment level. Estrogenic response was evaluated by measuring vitellogenin (VTG) and estrogen receptor (ER) expression in cytosolic fractions of whole body homogenates. Concentrations of the presumptive endocrine disruptors in the effluents were also measured. VTG and ER levels in sewage-exposed fish were 3–5 times that observed in controls. Combined concentrations of estradiol and estrone ranged from 5 to 13 ng/l and nonylphenol-ethoxylate metabolites (NPEOs: 4-nonylphenol, and 1-, 2-, and 3-nonylphenol-ethoxylates) ranged from 180 to 470 μg/l in chlorinated effluent. Results indicate that both ER and VTG can be used as biomarkers for endocrine disruption in larval fish, and that 4-day exposure to sewage effluent is sufficient to elicit significant expression of these markers in sunshine bass larvae. The extremely higher concentrations of NPEOs found in effluent relative to hormones (approximately 40,000-fold) indicates that surfactant metabolites may be contributing significantly to the estrogenic effects observed.

Sensitivities of mRNA Expression of Vitellogenin, Choriogenin and Estrogen Receptor by Estrogenic Chemicals in Medaka, Oryzias latipes.

Gene expression levels of choriogenin, vitellogenin, and estrogen receptor were determined using a reverse transcription-polymerase chain reaction (RT-PCR) technique after exposure to estrogenic chemicals to compare the sensitivities of the biomarkers of endocrine disruption in medaka, Oryzias latipes. Mature male medaka were treated with a single dose of 100 μ/l of 17α-ethinylestradiol, nonylphenol, and bisphenol A for 6 days, then RNA was extracted from the livers of treated fish for RT-PCR. Primers of RT-PCR for choriogenin H and L, and estrogen receptor were synthesized based on previously known cDNA sequences, and primers for vitellogenin I and II were synthesized based on the partial cDNA which was sequenced in this study. When the five biomarker genes were amplified by RT-PCR under the same condition, the mRNA induction level of each gene was elevated with different sensitivities. Conclusively, choriogenin L, which is a precursor of zona radiata protein (ZI-3) with molecular weight of 49 kD, showed the most sensitive gene expression in all the treated groups.

Vitellogenin – a biomarker for endocrine disruptors

This article describes the use of vitellogenin as a biomarker for endocrine disruption in fish. The lowest effective concentrations of vitellogenin are reported for oestrogenic compounds like 17β-oestradiol, nonylphenol and bisphenol A. Comparative studies of oestrogens detected in sewage plants from Germany and the UK are discussed. The article points out future directions in the application of vitellogenin ELISA assays and how these assays can provide additional information for a better understanding of the environmental behaviour of pollutants.