Biomonitoring hormonal activity in complex samples: Yeast estrogen and androgen screen assay optimization

MORA, S.C.; TAVALIERI, Y.E.; FRITZ, M.; GALOPPO, G.H.; LUQUE, E.H.; KASS, L.; MUÑOZ-DE-TORO, M.

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencias; 2019

Sociedad Argentina de Biología (SAB)

Lack of control in the treatment of urban and industrial effluents, and the increasing use of agrochemicals lead to an increased exposure to hormonal active chemicals also known as endocrine disruptors (EDC). EDC are hormonally active substances that mimic or antagonize endogenous hormones that can be found worldwide and cause adverse effects in humans and wildlife. The yeast estrogen screen (YES) and yeast androgen screen (YAS) assays are used to evaluate hormonal activity in complex samples. The assays are performed using two strains of Saccharomyces cerevisiae stably transfected with human androgen receptor (AR) and estrogen receptor alpha (ERα) respectively, together with reporter plasmids containing hormone response elements upstream of reporter gene LacZ. Each assay is carried out using serial dilutions of 17β-Estradiol (E2), tamoxifen (TMX), dihydrotestosterone (DTH) or flutamide (FLU) to build standard curves. Anti-hormonal activity is evaluated in the presence of TMX and FLU. Incubated with the yeasts and a chromogenic substrate, samples with hormonal activity develop a color changing reaction. Previously we reproduced the assay as described by Sohoni and Sumpter (1998). Reliable results were obtained using that method but sensitivity was lower than expected. The aim of this study was to optimize the methods of the YES and YAS assays. An alternative method was designed and compared to the original one. Parameters changed include incubation times with the chromogenic substrate, data analysis and incorporation of a lysis buffer. Solutions with different concentrations of BPA and water samples from the Ecological Reserve of the Universidad Nacional del Litoral (UNL), Santa Fe were evaluated to compare both methods. The new method presents advantages: reduced assay time, greater sensitivity to working dilutions, and reduced ranges of absorbance allowing better measurement discrimination and enhancing data analysis. Therefore is our choice.