A short-term in vivo screen using fetal testosterone production, a key event in the phthalate adverse outcome pathway, to predict disruption of sexual differentiation

Abstract

This study was designed to develop and validate a short-term in vivo protocol termed the Fetal Phthalate Screen (FPS) to detect phthalate esters (PEs) and other chemicals that disrupt fetal testosterone synthesis and testis gene expression in rats. We propose that the FPS can be used to screen chemicals that produce adverse developmental outcomes via disruption of the androgen synthesis pathway more rapidly and efficiently, and with fewer animals than a postnatal one-generation study. Pregnant rats were dosed from gestational day (GD) 14 to 18 at one dose level with one of 27 chemicals including PEs, PE alternatives, pesticides known to inhibit steroidogenesis, an estrogen and a potent PPARα agonist and ex vivo testis testosterone production (T Prod) was measured on GD 18. We also included some chemicals with "unknown" activity including DMEP, DHeP, DHEH, DPHCH, DAP, TOTM, tetrabromo-diethyl hexyl phthalate (BrDEHP), and a relatively potent environmental estrogen BPAF. Dose-response studies also were conducted with this protocol with 11 of the above chemicals to determine their relative potencies. CD-1 mice also were exposed to varying dose levels of DPeP from GD 13 to 17 to determine if DPeP reduced T Prod in this species since there is a discrepancy among the results of in utero studies of PEs in mice. Compared to the known male reproductive effects of the PEs in rats the FPS correctly identified all known "positives" and "negatives" tested. Seven of eight "unknowns" tested were "negatives", they did not reduce T Prod, whereas DAP produced an "equivocal" response. Finally, a dose-response study with DPeP in CD-1 mice revealed that fetal T Prod can be inhibited by exposure to a PE in utero in this species, but at a higher dose level than required in rats.Key words. Phthalate Syndrome, Fetal endocrine biomarkers, Phthalate adverse outcome pathway, testosterone production, fetal rat testis.

Keywords: Fetal endocrine biomarkers; Phthalate Syndrome; Phthalate adverse outcome pathway; fetal rat testis; testosterone production.

FIG. 1.

Effects of the different in utero maternal treatments on fetal testis testosterone production, collected ex vivo for 3 h incubation (one testis for each of three males per litter, with 3–4 litters per dose group in most cases). Data are expressed as percentage of control from the respective block in which the PE was tested; T Prod data were log₁₀ transformed to correct for heterogeneity of variance. Phthalates are listed from left to right by increasing ester straight side chain length from C2 to C9. Several phthalates which do not have straight side chains from C4 to C6 disrupt fetal testis testosterone production including DIBP, DHeP, DINP, and DCHP. Gray histograms are not significantly different from control (p > 0.10), yellow were equivocal (p ≤ 0.05 to p > 0.01) and red differed significantly (p ≤ 0.01) from the concurrent control value.

FIG. 2.

Dose-related reductions in male rat testis testosterone production on gestational day 18, expressed as percentage of control values. The graph was generated using GraphPad Prism software using the nonlinear, four parameter logistic regression model, with the bottom constrained to 10% of control testosterone production from the same block as the treatment.

FIG. 3.

The ED₅₀ values from the logistic regression analyses of the dose-response data were ranked from left to right by decreasing potency to reduce testosterone production with the most potent chemical with the lowest ED₅₀ value on the left and the weakest chemical. Chemicals that did not significantly reduce testosterone production are not included in the figure.

FIG. 4.

Dipentyl, dibutyl, and diethylhexyl phthalate were run in several blocks in both Harlan SD and Charles Rivers SD (CR SD) rats in order to compare the sensitivity of these SD rats from different suppliers to phthalate-induced reduction of fetal testosterone production on GD 18. The results of the statistical comparison of the two logistic regression models with GraphPad Prism software indicate that the Harlan SD was slightly more sensitive than is the CR SD.

FIG. 5.

Comparison of the dose-related effects of dipentyl phthalate (DPeP) on testosterone production (T Prod) on gestational day 18 and fetal mortality as measured by postimplantation loss (PIL = 100 × (number of implantation sites – number of live fetuses)) in the fetal male rat and mouse. The results of the statistical comparison of the two logistic regression models with GraphPad Prism software indicate that T Prod was more sensitive to DPeP in the rat versus the mouse, whereas, fetal mortality was more affected in the mouse than the rat.