Although the only effect observed herein in PZ-treated female offspring was a transient increase in neonatal anogenital distance, it is important to note that our evaluation of the female reproductive system was limited to an examination of a few developmental landmarks and organ weights, as the female offspring were not the focus of the current study, and it is possible that the reproducible, high-dose effect of PZ on neonatal anogenital distance is associated with adverse developmental reproductive toxicity on fertility, estrous cyclicity, behavior, or histopathology (or a combination of these) of the female rat.
While in utero exposure to potent estrogenic drugs (personal communication) or steroids at high-dose levels does increase anogenital distance in female rats at birth, this effect is associated with malformations of the external genitalia (cleft phallus with hypospadias) in female offspring, an effect not observed in the PZ-exposed female offspring.
Although PZ also has been shown to display possible estrogenic, antiestrogenic, or aryl hydrocarbon receptor (AHR)-mediated effects in vitro, it is not certain whether PZ displays these endocrine activities in the whole animal. Furthermore, exposure to chemicals that disrupt female rat sexual differentiation by acting as estrogen or AHR agonists produce reproductive tract malformations that were not present in the PZ-treated female offspring.
Dystocia and pup retention during delivery likely accounted for most of the pup mortality in the current study. The ability to target multiple steroidogenic enzymes may allow PZ to simultaneously affect androgen- and estrogen-related physiology, and possible antiestrogenic activity of PZ has also been hypothesized to be mediated through P450 enzymes catalyzing estradiol hydroxylation.
The degree to which one form of endocrine toxicity predominates over the other (i.e., AR antagonism versus inhibition of aromatase) to disrupt reproductive function in the fetal male versus the pregnant dam depends to some degree on the duration of exposure. When PZ is administered at 250 mg/kg-BW per day or less for a brief period antiandrogenic effects are expressed along with delays in delivery that are not severe enough to reduce maternal or pup viability. However, had we expanded the dosing period to include the perinatal period it is likely that the effects on delivery would be more severe and occurred at lower dosage levels, which would preclude the survival of malformed pups.
For this reason, administration of PZ during sexual differentiation produces severe malformations of androgen-dependent tissues in male rat offspring but is without obvious effect on the female rat. In contrast, administration of androgens to the female fetus induces malformations of the bipotential undifferentiated tissue. Androgens activate AR-dependent male-like differentiation, but treated male rats differentiate normally.
With PZ, vinclozolin, and pro-cymidone exposures, male malformations were produced without the severe testicular lesions observed with phthalate exposure. Like PZ, procymidone and vinclo-zolin metabolites are AR antagonists, suggesting that AR antagonism may be a mechanism for malformation production through PZ exposure. Although further data are required before we can definitively determine what mechanisms of action are most responsible for the demasculin-ization of male pups, PZ antagonism of androgen in the Hershberger assay is consistent with in vivo AR antagonism by PZ.
The syndrome of male reproductive tract malformations produced by phthalates such as DEHP, DBP, and BBP includes high incidences of testicular, epididymal, and gubernacular agenesis, malformations that did not occur with PZ treatment in the current study.
The malformation profile of PZ-exposed males more closely resembles the profile observed with males exposed in utero to low doses of flutamide or intermediate doses of vinclozolin or procymidone.
As indicated above, the effects of PZ on sexual differentiation of androgen-dependent tissues could result from dual modes of action. PZ act as an AR antagonist and it inhibits testosterone production in the fetal testis. Ex vivo testosterone production is significantly reduced in testes at GD 18 from male fetuses exposed in utero from GD 14 to GD 18, whereas progesterone and hydroxyprogester-one production are increased by as much as 10-fold. Together, the results presented by Wilson et al. and the current study indicate that PZ effects on fetal testes differed from effects of antiandrogens linuron and diethylhexyl phthalate, which decreased testicular testosterone production and left levels of progesterone unaffected.
In the current study, we also demonstrated that in vitro PZ competed with a high-affinity synthetic androgen to bind to rat AR and that PZ also inhibited DHT-induced human AR-dependent gene expression at concentrations that did not induce cytotoxicity. Such results support the hypothesis that in vitro, PZ exhibits antiandrogenic activity by competing with androgens for receptor sites and support prior reports of PZ inhibition of AR transactivation. These results and findings from our laboratory demonstrating that PZ also inhibits fetal androgen production indicate that PZ could interfere with the androgen signaling pathway during sexual differentiation via two independent mechanisms (i.e., by acting as an AR antagonist and by inhibiting testosterone synthesis).