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

Abstract

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.

Temporal profiles of cortisol accumulation and clearance support scale cortisol content as an indicator of chronic stress in fish

“…Scales develop by intramembranous ossification, are remodelled extensively during growth and can keep growing peripherally by appositional growth (Ohira et al , 2007; De Vrieze et al , 2011). Peripheral cells and blood supply enable dynamic properties of scales, such as functions in calcium homeostasis (Mugiya and Watabe, 1977), sensitivity of mineralization to estrogens (Pinto et al , 2017) and rapid regeneration abilities (Sire, 1989; Bereiter-Hahn and Zylberberg, 1993; Ohira et al , 2007; De Vrieze et al , 2011).…”

Genistein and estradiol have common and specific impacts on the sea bass (Dicentrarchus labrax) skin-scale barrier

“…Estrogens can have multiple modes of action that include regulation of 1) responsive-gene transcription, classically associated with nuclear estrogen receptors (Esrs), 2) rapid non-genomic effects mainly mediated by G-protein coupled estrogen receptors (Gpers) or, 3) indirect effects via other transcription factors and/or signalling cascades, that may cross-talk to achieve the final cellular response [3]. The expression of nuclear Esrs at both transcript and protein levels explains the responsiveness of fish scales to (E2 [3,[6][7][8]. Recently, duplicate Gper paralogues were found to be expressed in European sea bass (Dicentrarchus labrax) and Mozambique tilapia (Oreochromis mossambicus) scales [7][8][9] indicating that fish scales are estrogen targets through both classical genomic actions and rapid non-genomic actions [3].…”

“…The expression of nuclear Esrs at both transcript and protein levels explains the responsiveness of fish scales to (E2 [3,[6][7][8]. Recently, duplicate Gper paralogues were found to be expressed in European sea bass (Dicentrarchus labrax) and Mozambique tilapia (Oreochromis mossambicus) scales [7][8][9] indicating that fish scales are estrogen targets through both classical genomic actions and rapid non-genomic actions [3].…”

Fish as a model to assess chemical toxicity in bone

“…The (zebra)fish scales are of particular interest since they appear to recapitulate the process of intramembranous ossificationi.e. osteoblast differentiation, acellular matrix deposition, mineralization and resorption by osteoclastsand have been recently used as a non-invasive in vitro bioassay to demonstrate the estrogenic activity of different endocrine disruptors (Pinto et al, 2017). Scales are also technically advantageous since they can be easily harvested in large numbers with limited stress to the fish, can regenerate upon removal, and their matrix is translucent, thus facilitating imaging by microscopy (De Vrieze et al, 2014).…”