2-Ethylhexyl Diphenyl Phosphate and Its Hydroxylated Metabolites are Anti-androgenic and Cause Adverse Reproductive Outcomes in Male Japanese Medaka (Oryzias latipes)

Abstract

Although high concentrations of 2-ethylhexyl diphenyl phosphate (EHDPP) have been detected in wild fish, its reproductive toxicity in fish remains unclear. In this study, we for the first time observed that EHDPP elicited androgen receptor (AR) antagonistic activity with a 50% inhibitory concentration of 37.5 μM. 2-Ethyl-5-hydroxyhexyl diphenyl phosphate was proved to be the dominant metabolite of EHDPP in Japanese medaka and elicited 3.1-fold stronger AR antagonistic activity than that of EHDPP. Medaka larvae (0-day post hatching) were exposed to EHDPP for 100 days, and intersex was observed in males from all exposure groups with significantly increased incidence (13.5−48.6%). 17β-E 2 was promoted at 104 ng/L, and androgens were suppressed at 434 ng/L, which account for the intersex incidence in the high-exposure groups but do not explain the significant incidence of intersex in the 29.9 ng/L exposure group. The AR antagonistic activity of EHDPP and its metabolites must therefore play a key role in intersex incidence. EHDPP also significantly (p < 0.05) repressed reproductive behaviors of males in the 434 ng/L group and decreased fertility in high-exposure groups compared with the control. All the adverse outcomes were observed under environmentally relevant concentrations, implying that EHDPP poses an ecological risk for wild fish populations.

Maternal Transfer of 2-Ethylhexyl Diphenyl Phosphate Leads to Developmental Toxicity Possibly by Blocking the Retinoic Acid Receptor and Retinoic X Receptor in Japanese Medaka (Oryzias latipes)

“…When observing the entire embryo development from each group, we only observed deformities in the eyes of the embryos. Such a phenomenon could be at least partly be due to the fact that the development of the eye is particularly regulated by RA signaling, , and the main development period of the eye is much earlier than those of other organs, such as blood vessels (3 dpf 10 h), the heart (6 dpf), and the vertebral column (after 9 dpf) . Eye deformities of various severity levels were observed.…”

“…Sixteen individuals were exposed in each treatment under a flow-through system that renewed the exposure water every 24 h. Water samples were collected daily during the exposure period. Aqueous EHDPP concentrations were measured according to a previous method . More information about the exposure conditions is provided in the Supporting Information.…”

“…The exposure water samples ( n = 5), tissue samples of female fish (each muscle sample contained ∼50 mg of muscle, each ovary sample contained ∼50 mg of ovary, and each liver or brain sample were pooled from three females, n = 3), and embryo samples (∼30 mg each, n = 3) from each group were pretreated according to our previous study with details provided in the Supporting Information. EHDPP and its metabolites were analyzed by UPLC-MS/MS, and the instrumental setup followed a previously reported method .…”

“…The RAR and RXR agonistic and antagonistic activities of EHDPP and its metabolites including 2-ethyl-5-hydroxyhexyl diphenyl phosphate (5-OH-EHDPP), 2-ethyl-3-hydroxyhexyl diphenyl phosphate (3-OH-EHDPP) were assessed using a yeast two-hybrid assay system with Japanese medaka RAR (mRAR) and RXR (mRXR). The assay was conducted according to our previous paper, 31 and the details are provided in the Supporting Information. The 50% effective concentration (EC 50 ) of atRA to agonize mRAR was 1.9 nM and that of 9cRA to agonize mRXR was 23.6 nM.…”

“…In this study, we assessed the agonistic and antagonistic activity of EHDPP to RAR and RXR in vitro to understand the potential adverse effects of EHDPP. EHDPP can be metabolized in fish, 31 and therefore, the RAR and RXR agonistic and antagonistic activities of its metabolites were also assessed. To investigate the maternal transfer and developmental toxicity of EHDPP in fish, adult female medaka were exposed to EHDPP at environmentally relevant concentrations.…”