EVALUATION OF ESTROGENIC ACTIVITY OF PARABENS AND THEIR CHLORINATED DERIVATIVES BY USING THE YEAST TWO-HYBRID ASSAY AND THE ENZYME-LINKED IMMUNOSORBENT ASSAY

We assessed the estrogen agonist activities of 21 parabens and their chlorinated derivatives by using yeast two-hybrid assays incorporating either the human or medaka (Oryzias latipes) estrogen receptor alpha (hER alpha and medER alpha, respectively), and by using hER alpha competitive enzyme-linked immunosorbent assay (ER-ELISA). In the two-hybrid assay with hER alpha, five parabens and three chlorinated derivatives exhibited estrogenic activity, and their relative activity (17 beta-estradiol [E-2] = 1) ranged from 2.0 x 10(-5) to 2.0 x 10(-4), with the highest activity observed in i-butylparaben. In the medER alpha assay, six parabens and six chlorinated derivatives exhibited estrogenic activity and their relative activity ranged from 2.7 x 10(-5) to 3.5 x 10(-3), with the highest activity observed in benzylparaben, its monochlorinated derivative, i-butylparaben, and n-butylparaben. Although medER alpha demonstrated an activity to E-2 that was three times lower than that demonstrated by hER alpha, medER alpha has a higher sensitivity to parabens than hER alpha (1.3-8.9 times). Five parabens and two chlorinated derivatives exhibited a binding affinity to ER alpha in the ER-ELISA; of the parabens, i-butylparaben exhibited the strongest binding affinity. The yeast two-hybrid assay and the ER-ELISA also revealed that many of the assayed chlorinated parabens were much weaker than the parent compound. In addition, the results mainly showed that parabens with a bulk substituent (e. g., i-butyl and benzyl groups) had a higher activity than those with a sterically small substituent. It is considered that derivatization masks the apparent estrogenic activity of parabens, but the resulting chlorinated compounds may represent a potential hazard and therefore other toxicity tests should be performed to determine the toxicity of the chlorinated derivatives.