Molecular Markers of Endocrine Disruption in Aquatic Organisms

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Abstract
A wide range of organic contaminant compounds prevalent in the aquatic environment has been shown to exhibit hormone-disrupting activity. The actual potency of such compounds are low compared with endogenous hormones, such as 17 g -estradiol, but may still produce detrimental biological effects. Induced hormone levels are routinely measured using commercial testing kits, though these fail to relate to actual effects. Field and laboratory studies on the biological effects of environmental estrogens have, in the past, largely relied on assays of vitellogenin (vtg) induction in male fish, reduced growth in testes formation, and intersex incidence. Here, we critically review the current and potential application of molecular techniques in assessing the adverse biological reproductive effects of endocrine-disrupting chemicals in aquatic organisms. The role of fish (estrogen, androgen, and progestogen) hormone receptors and invertebrate (ecdysone) hormone receptor, egg production (vtg and chorion) proteins, steroid biosynthesis enzymes (aromatase, sulfotransferase, and hydroxysteroid dehydrogenase), DNA damage, apoptosis, and their potential development as biomarkers are discussed in turn. In each case, the sequences characterized are presented and homologies across species are highlighted. Molecular methods of gauging vtg and zona radiata (ZR) expression and protein concentrations have included immunoassay and reverse transcription polymerase chain reaction (RT-PCR). Suggestions for the isolation for key gene expression products (aromatase, ZR, and vtg, for instance), from a wider range of fish species using degenerate primers, are given. Endocrine disruption in invertebrates has received less attention compared with fish, partly because the knowledge regarding invertebrate endocrinology is limited. Here we review and suggest alternate isolation strategies for key players in the imposex induction process: vitellin (Vn), aromatase, and Ala-Pro-Gly-Trp (APGW) amide neurohormone. Current molecular-level techniques rely on ligand-binding assays, enzyme-linked immunosorbent assay (ELISA), and, more recently, gene expression. In the future, more reliance will be placed on the development of gene expression assays using reporter systems combined with cross-species PCR-based or polyclonal antibody-based assays. We discuss the use of recombinant receptors as a means of primary screening of environmental samples for estrogenicity and antiestrogenicity, which avoids species and seasonal variation in receptor response to ligand binding, a recognized problem of earlier bioassays. Most exciting is the potential that microarray and proteomics approaches have to offer. Such techniques are now used routinely in medical research to identify specific genes and proteins affected by treatment with endocrine disruptors, including estradiol. The technique has yet to be used to screen aquatic organisms, but it has the potential to implicate previously unsuspected estradiol-sensitive genes that may later become molecular markers of endocrine disruption.