An Integrated Approach Combining Regulatory Tests in tg(cyp19a1b:GFP) Zebrafish Embryos to Assess Toxicity, Developmental Effects, and Estrogenic Activity of Chemicals: A Case Study with Bisphenol A Substitutes

The use of efficient
testing strategies for chemical hazard assessment
is a current challenge in supporting regulatory requirements. Herein,
we combined two zebrafish eleuthero-embryo assays, a refined Fish
Embryo Acute Toxicity assay (FET, OECD TG 236) and the EASZY assay
(OECD TG 250), both using transgenic tg(cyp19a1b:GFP) embryos, to jointly assess acute toxicity, developmental effects,
and estrogenic activity of bisphenol A (BPA) and ten substitutes.
Several bisphenols were more toxic than BPA, inducing developmental
effects on zebrafish embryos, with some showing potential teratogenicity.
Increased GFP intensity was also detected for ten bisphenols, suggesting
estrogenic activity. FET results guided the selection of sublethal
concentrations for the EASZY assay, which confirmed estrogenic activity
for all bisphenols except TCBPA, most being more estrogenic than BPA in vivo and requiring functional zfERs to induce brain aromatase.
All bisphenols also activated zfERβ2 in a zebrafish-specific in vitro reporter gene assay, except BPS-MAE and BPS-MPE,
which only induced brain aromatase in vivo. Overall,
the combined FET and EASZY assays efficiently generated relevant data
for hazard assessment of chemicals and provided further evidence that
bisphenols modulate ER-dependent cyp19a1b expression
during early zebrafish brain development, raising concerns about their
potential short- and long-term adverse effects.