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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 α (hERα and medERα, respectively), and by using hERα competitive enzyme‐linked immunosorbent assay (ER‐ELISA). In the two‐hybrid assay with hERα, five parabens and three chlorinated derivatives exhibited estrogenic activity, and their relative activity (17β‐estradiol [E2] = 1) ranged from 2.0 × 10−5 to 2.0 × 10−4, with the highest activity observed in i‐butylparaben. In the medERα assay, six parabens and six chlorinated derivatives exhibited estrogenic activity and their relative activity ranged from 2.7 × 10−5 to 3.5 × 10−3, with the highest activity observed in benzylparaben, its monochlorinated derivative, i‐butylparaben, and n‐butylparaben. Although medERα demonstrated an activity to E2 that was three times lower than that demonstrated by hERα, medERα has a higher sensitivity to parabens than hERα (1.3–8.9 times). Five parabens and two chlorinated derivatives exhibited a binding affinity to ERα 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.