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

MORA SC; TAVALIERI YE; FRITZ M; GALOPPO GH; LUQUE EH; KASS L; MUÑOZ-DE-TORO M

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencias-LXIV Reunión de la Sociedad Argentina de Investigación Clínica; 2019

Sociedad Argentina de Investigación Clínica

Lack ofcontrol in the treatment of urban and industrial effluents, and the increasinguse of agrochemicals lead to an increased exposure to hormonal active chemicalsalso known as endocrine disruptors (EDC). EDC are hormonally active substancesthat mimic or antagonize endogenous hormones that can be found worldwide andcause adverse effects in humans and wildlife. The yeast estrogen screen (YES)and yeast androgen screen (YAS) assays are used to evaluate hormonal activityin complex samples. The assays are performed using two strains of Saccharomycescerevisiae stably transfected with human androgen receptor (AR) and estrogenreceptor alpha (ER) respectively, together with reporter plasmids containinghormone response elements upstream of reporter gene LacZ. Each assay is carriedout using serial dilutions of 17Estradiol (E2), tamoxifen (TMX),dihydrotestosterone (DTH) or flutamide (FLU) to build standard curves.Anti-hormonal activity is evaluated in the presence of TMX and FLU. Incubatedwith the yeasts and a chromogenic substrate, samples with hormonal activitydevelop a color changing reaction. Previously we reproduced the assay as describedby Sohoni and Sumpter (1998). Reliable results were obtained using that methodbut sensitivity was lower than expected. The aim of this study was to optimizethe methods of the YES and YAS assays. An alternative method was designed andcompared to the original one. Parameters changed include incubation times withthe chromogenic substrate, data analysis and incorporation of a lysis buffer.Solutions with different concentrations of BPA and water samples from theEcological Reserve of the Universidad Nacional del Litoral (UNL), Santa Fe wereevaluated to compare both methods. The new method presents advantages: reducedassay time, greater sensitivity to working dilutions, and reduced ranges ofabsorbance allowing better measurement discrimination and enhancing dataanalysis.