In vitro screening for estrogenic endocrine disrupting compounds using Mozambique tilapia and sea bass scales.

A wide range of estrogenic endocrine disruptors (EDCs) are accumulating in the environment and may disrupt the physiology of aquatic organisms. The effects of EDCs on fish have mainly been assessed using reproductive endpoints and in vivo animal experiments. We used a simple non-invasive assay to evaluate the impact of estrogens and EDCs on sea bass (Dicentrarchus labrax) and tilapia (Oreochromis mossambicus) scales. These were exposed to estradiol (E2), two phytoestrogens and six anthropogenic estrogenic/anti-estrogenic EDCs and activities of enzymes related to mineralized tissue turnover (TRAP, tartrate-resistant acid phosphatase and ALP, alkaline phosphatase) were measured. Semi-quantitative RT-PCR detected the expression of both membrane and nuclear estrogen receptors in the scales of both species, confirming scales as a target for E2 and EDCs through different mechanisms. Changes in TRAP or ALP activities after 30minute and 24h exposure were detected in sea bass and tilapia scales treated with E2 and three EDCs, although compound-, time- and dose-specific responses were observed for the two species. These results support again that the mineralized tissue turnover of fish is regulated by estrogens and reveals that the scales are a mineralized estrogen-responsive tissue that may be affected by some EDCs. The significance of these effects for whole animal physiology needs to be further explored. The in vitro fish scale bioassay is a promising non-invasive screening tool for E2 and EDCs effects, although the low sensitivity of TRAP/ALP quantification limits their utility and indicates that alternative endpoints are required.

Tissue responsiveness to estradiol and genistein in the sea bass liver and scale.

As in mammals, estrogens in fish are essential for reproduction but also important regulators of mineral homeostasis. Fish scales are a non-conventional target tissue responsive to estradiol and constitute a good model to study mineralized tissues effects and mechanisms of action of estrogenic compounds, including phytoestrogens. The responsiveness to estradiol and the phytoestrogen genistein, was compared between the scales and the liver, a classical estrogenic target, in sea bass (Dicentrarchus labrax). Injection with estradiol and genistein significantly increased circulating vitellogenin (for both compounds) and mineral levels (estradiol only) and genistein also significantly increased scale enzymatic activities suggesting it increased mineral turnover. The repertoire, abundance and estrogenic regulation of nuclear estrogen receptors (ESR1, 2a and 2b) and membrane G-protein receptors (GPER and GPER-like) were different between liver and scales, which presumably explains the tissue-specific changes detected in estrogen-responsive gene expression. In scales changes in gene expression mainly consisted of small rapid increases, while in liver strong, sustained increases/decreases in gene expression occurred. Similar but not overlapping gene expression changes were observed in response to both estradiol and genistein. This study demonstrates for the first time the expression of membrane estrogen receptors in scales and that estrogens and phytoestrogens, to which fish may be exposed in the wild or in aquaculture, both affect liver and mineralized tissues in a tissue-specific manner.

High- and low-frequency transcutaneous electrical nerve stimulation delay sciatic nerve regeneration after crush lesion in the mouse.

The stimulation of peripheral nerve regeneration has been studied in different ways, including the use of electrical fields. The capacity of this modality to enhance nerve regeneration is influenced by the parameters used, including current type, frequency, intensity, and means of administration. Transcutaneous electrical nerve stimulation (TENS) is a frequently used form of administering electrical current to the body, but its effects on peripheral nerve regeneration are not known. This study assessed the influence of TENS on sciatic nerve regeneration, using a model of crush lesion in the mouse. Mice were stimulated 30 min a day, 5 days a week, for 5 weeks with both high- (100 Hz) and low- (4 Hz) frequency TENS. Control animals had the sciatic nerve crushed but were not stimulated. Assessment was performed weekly by functional analysis using the Static Sciatic Index for the mouse and at the end of the experiment by light and electron microscopy. The results showed that although there were no differences between the groups regarding the Static Sciatic Index values, TENS led to nerves with morphological signs of impaired regeneration. At light microscopy level, TENS nerves presented more axons with dark axoplasm, signs of edema, and a less organized cytoarchitecture. Electronmicrographs showed fewer and thinner thick myelinated fibers and increased number of Schwann cell nuclei. Myelinated axon diameters and density and diameter of nonmyelinated fibers were not affected by TENS, leading to the conclusion that this regimen of electrical stimulation leads to a delayed regeneration after a crush lesion of the sciatic nerve in the mouse. All these effects were more pronounced on high-frequency TENS nerves.