In the current study we demonstrated for the first time that in utero PZ exposure induced permanent reproductive malformations such as retained nipples, hypospadias, cleft phallus, and vaginal pouch in male rat offspring. While the impact of these results on the risk assessment of PZ is uncertain, the determination that this fungicide is a teratogen can result in application of additional risk factors if PZ is deemed to be of special concern for the child. In this regard, reproductive malformations were not noted in the reproductive toxicity and teratology studies cited in PZ risk assessment documents.
In male rats born to dams treated with 125 mg/kg-BW per day, 15.6% retained nipples, 18.75% showed incomplete PPS, 12.5% showed cleft phallus and hypospadias, 3.1% (one animal) had an exposed os penis, and 6.2% (two animals) had vaginal pouches. Weights of ventral prostate (P = 0.048) and epididymis (P = 0.011) were reduced. Unadjusted testis weights were reduced compared with those of controls (P = 0.0325; Table 3) but not when body weight adjustment was used (P = 0.0907).
The mean number of nipples retained as adults were fewer than the number of areolas/ nipples noted on PCD 35 except in the highest-dosed group (Fig. 6). In all cases, inguinal areolas/nipples observed at PCD 35 were more likely to be transient, while thoracic and abdominal nipples were most likely to be retained in adults.
In the 250 mg/kg-BW per day group, 100% of males showed cleft phallus and hypospadias, 70% also had an exposed os penis (Fig. 5), and 40% had a vaginal pouch (Fig. 7).
Offspring Effects During Puberty and Adult Life
In the female offspring, pubertal landmarks, as well as age and body weight at vaginal opening, were not affected by prenatal PZ exposure. In the male offspring, 1 of 47 controls showed a persistent preputial thread (incomplete PPS) until PCD 82 (PND 60), but the prepuce was separated at the time of necropsy. Two of 46 (4.35%) 62.5 mg/ kg-BW per day-treated animals (from the same litter) showed incomplete PPS until PCD 82. A large preputial thread on one of the animals persisted to necropsy (PCD 169). In the 125 mg/kg-treated group, all males in one litter (four pups) and one male in another litter showed incomplete PPS until PCD 82, which persisted until necropsy (PCDs 147-213; Fig. 5).
On PCD 25 male offspring from dams treated with PZ 125 or 250 mg/kg-BW per day had shorter anogenital distances compared with male controls (P = 0.006 and 0.0004, respectively), whereas female offspring in these two groups had larger anogenital distances compared with offspring of controls (P = 0.0369 and 0.0002, respectively). When anogenital distance was analyzed with body weight as the co-variate, no treatment effects on anogenital distance were significant in the males, but females in the 250 mg/kg-BW per day group had longer anogenital distances compared with those of other groups (Fig. 4).
Dams at 125 mg/kg-BW per day or greater completed parturition more than 28 h after controls, all of whom delivered by time zero (0730 on PCD 23) (Table 2). Mean times to complete parturition were 2.5, 6.7, 22.2, and 28.4 h for 31.25, 62.5, 125, and 250 mg/kg-BW per day groups, respectively. Delivery times for dams dosed with 125 and 250 mg/kg-BW differed from times for dams dosed at 62.5 mg/kg-BW per day or lower (P < 0.0005) but were not statistically different from each other (P = 0.1847). Five of eight dams treated with 125 mg/kg-BW per day delivered at least one stillborn pup.
Males displayed areolas/ nipples on PCD 35 (PND 13) at frequencies of 40%, 71%, and 100% in the 62.5, 125, and 250 mg/kg-BW per day treatment groups, respectively. When necropsied as adults, the male offspring displayed reduced weight of the levator ani plus bulbocavernosus muscles in a dose-related fashion. At the time of necropsy, nipple retention in male offspring was observed at frequencies of 10%, 14%, and 100% in the 62.5, 125, and 250 mg/kg-BW per day groups, respectively.
In Vitro AR Transactivation and Binding
The ability of PZ to induce AR-mediated gene transcription was tested in MDA-kb2 cells containing endogenous human AR and stably transfected with an androgen-responsive luciferase reporter gene. Both hydroxyflutamide (1 |xM) and PZ at doses of 3 |xM or greater inhibited AR transcriptional activation induced by 0.1 nM DHT (Fig. 1A), with the effect of 1 |xM hydroxyflutamide being roughly equivalent to the effect of 10 |xM PZ. The IC50 for PZ effects on transactivation was between 3 and 10 |xM.
For in vivo data in the main study, although all measurements were recorded from individual animals (dams or pups), individual pup measurements were used to calculate litter means, and litter means were the unit of analysis. Thus the numbers per group are the number of litters (as opposed to number of pups). Significance for treatment effects on organ weights and anogenital distance data were determined using an analysis of covariance with body weight as the covariate.
Wet and dry weights were recorded for the reproductive tract (oviducts, uterus, cervix, and vagina), ovaries, adrenals, liver, and kidney. As the examination of control and high-dose animals revealed kidney weight and body weight differences among treatment groups, all remaining control, low-dose, and mid-dose females were examined (using the remainder of the randomized blocks) for body weight, liver weight, and kidney weight. Female necropsies were completed on PCD 255.