Embryoid body test with morphological and molecular endpoints implicates potential developmental toxicity of trans-resveratrol

Highlights: • Developmental toxicity of resveratrol is examined using morphogenetic embryoid body. • trans-resveratrol, but not cis isoform or metabolites, impairs morphogenesis. • Resveratrol alters gene expression patterns of gastrulation regulators. • Activation of the estrogen receptor or SIRT1 is not the major action of resveratrol. • Reduction in DNA replication rate accounts for the resveratrol action. Developmental toxicity of compounds, which women of reproductive age are exposed to, should be assessed to minimize the incidence of miscarriage and birth defects. The present study examined the potential developmental toxicity of resveratrol, a dietary supplement widely marketed with various health claims, using the P19C5 embryoid body (EB) morphogenesis assay, which evaluates adverse effects of chemical exposures on tissue growth and axial elongation. Resveratrol (trans isoform) impaired morphogenesis at 4 μM and higher, creating smaller and rounder EBs, whereas cis isoform, and glucuronated and sulfonated metabolites did not. Trans-resveratrol also altered expression levels of developmental regulator genes involved in embryonic patterning, such as Wnt3a, Tbx6, and Cyp26a1. To investigate the mechanisms of trans-resveratrol action, the roles of estrogen receptor, sirtuin 1 (SIRT1), and DNA replication in EB morphogenesis were examined. Neither activators of estrogen receptors (diethylstilbestrol [18 μM] and raloxifene [8 μM]) nor activator of SIRT1 (SRT1720 [2.4–3.2 μM]) caused morphological and molecular alterations that are comparable to trans-resveratrol (10 μM). By contrast, a reduction in the DNA replication rate with aphidicolin (0.4 μM) or hydroxyurea (40 μM) created smaller and rounder EBs and altered the expression levels of Wnt3a, Tbx6, and Cyp26a1 in a manner similar to trans-resveratrol. Consistently, trans-resveratrol significantly reduced the rate of EdU incorporation in P19C5 cells. These results suggest that a reduction in the DNA replication rate is one of the mechanisms by which trans-resveratrol impacts EB development. This study provides mechanistic insight for further investigations on the developmental toxicity of trans-resveratrol.