Establishment of a transgenic yeast screening system for estrogenicity and identification of the anti‐estrogenic activity of malachite green

Abstract

Endocrine disruptors refer to chemical compounds in the environment which interfere with the endocrine systems of organisms. Among them, environmental estrogens pose serious problems to aquatic organisms, in particular fish. It is therefore important and necessary to have a fast and low-cost system to screen the large number of different chemical compounds in the aquatic environment for their potential endocrine disrupting actions. In this study, a screening platform was developed to detect xenoestrogens in the aquatic environment using the fission yeast Schizosaccharomyces pombe, and applied for compound screening. The aim was to demonstrate any significant potential differences between the fish screening system and the human screening system. To this end, a yeast expression vector harboring a fish estrogen receptor alpha and a reporter vector containing the estrogen responsive element fused with the Escherichia coli LacZ gene were constructed. After transformation with these two vectors, the transformed yeast clones were confirmed by Western blotting and selected on the basis of the beta-galactosidase activity. In this transgenic yeast system, the natural estrogen (estradiol) and other known xenoestrogens such as diethylstilbestrol, bisphenol A, genistein and dichloro-diphenyl-trichloroethane exhibited dose-dependent activities. Using this system, more than 40 putative endocrine disruptors including phytoestrogens, pesticides, herbicides, industrial dyes and other industrial chemicals were screened. Ten of them were demonstrated to exhibit estrogenic actions. Industrial dyes such as malachite green (MG) that disrupt thyroid hormone synthesis are extensively used and are widely distributed in the aquatic environment. Using this system, MG did not show any estrogenic action, but was demonstrated to exhibit anti-estrogenic activity.

In VitroEffect of Malachite Green on Candida albicans Involves Multiple Pathways and Transcriptional RegulatorsUPC2andSTP2

“…It has been shown that MG at 1 ppm is able to reduce the growth of different fungal species, such as Aspergillus parasiticus, Aspergillus flavus, Penicillium verrucosum, and Fusarium oxysporum (5). MG also is used in the fish industry to treat fungal and ectoparasitic diseases in eggs (22,44). We show here that MG has an advantage over conventional antifungal drugs in that it effectively inhibits the growth of C. albicans and non-C. albicans species at lower concentrations than those of triazoles, imidazoles, and allylamine.…”

Analysis of Environmental Samples with Yeast-Based Bioluminescent Bioreporters

“…Further, after 3 -5 days of incubation, it was unknown if any oxidation reactions were occurring that may activate the target compound. Some newer colorimetric assays have dramatically shortened the time required for color development (4-6 h) through the use of alternative substrates but have the disadvantage of requiring cell lysis steps (Jaio et al, 2008). To overcome these limitations, bioluminescent version of the YES and YAS reporters were developed by modifying the plasmid constructs of Gupta et al (2003).…”

Flavouring Group Evaluation 32 (FGE.32): Flavonoids (Flavanones and dihydrochalcones) from chemical groups 25 and 30

“…Naringenin, the aglycone of the candidate substance naringin .058], has been tested for estrogenic effects in a number of in vitro screening test studies with different end-points (Almstrup et al, 2002;Bovee et al, 2008;Bovee et al, 2004;Branham et al, 2002;Breinholt & Larsen, 1998;Galluzzo et al, 2008;Garrett et al, 1999;Jiao et al, 2008;Kretzschmar et al, 2009;Kuiper et al, 1998;van Meeuwen et al, 2007;Miksicek, 1993;Rosenberg Zand et al, 2000;Safe et al, 2002;Zierau et al, 2005;Zierau et al, 2003;Zierau et al, 2002). Generally it is concluded that naringenin is a weak in vitro estrogen compared to isoflavone phytoestrogens like genistein.…”