In mammals, exposure to antiandrogenic chemicals during sexual differentiation can produce malformations of the reproductive tract. Perinatal administration of AR antagonists like vinclozolin and procymidone or chemicals like di(2-ethylhexyl) phthalate (DEHP) that inhibit fetal testicular testosterone production demasculinize the males such that they display reduced anogenital distance (AGD), retained nipples, cleft phallus with hypospadias, undescended testes, a vaginal pouch, epididymal agenesis, and small to absent sex accessory glands as adults. In addition to DEHP, di-n-butyl (DBP) also has been shown to display antiandrogenic activity and induce malformations in male rats. In the current investigation, we examined several phthalate esters to determine if they altered sexual differentiation in an antiandrogenic manner. We hypothesized that the phthalate esters that altered testis function in the pubertal male rat would also alter testis function in the fetal male and produce malformations of androgen-dependent tissues. In this regard, we expected that benzyl butyl (BBP) and diethylhexyl (DEHP) phthalate would alter sexual differentiation, while dioctyl tere- (DOTP or DEHT), diethyl (DEP), and dimethyl (DMP) phthalate would not. We expected that the phthalate mixture diisononyl phthalate (DINP) would be weakly active due to the presence of some phthalates with a 6-7 ester group. DEHP, BBP, DINP, DEP, DMP, or DOTP were administered orally to the dam at 0.75 g/kg from gestational day (GD) 14 to postnatal day (PND) 3. None of the treatments induced overt maternal toxicity or reduced litter sizes. While only DEHP treatment reduced maternal weight gain during the entire dosing period by about 15 g, both DEHP and DINP reduced pregnancy weight gain to GD 21 by 24 g and 14 g, respectively. DEHP and BBP treatments reduced pup weight at birth (15%). Male (but not female) pups from the DEHP and BBP groups displayed shortened AGDs (about 30%) and reduced testis weights (about 35%). As infants, males in the DEHP, BBP, and DINP groups displayed femalelike areolas/nipples (87, 70, and 22% (p < 0.01), respectively, versus 0% in other groups). All three of the phthalate treatments that induced areolas also induced a significant incidence of reproductive malformations. The percentages of males with malformations were 82% (p < 0.0001) for DEHP, 84% (p < 0.0001) for BBP, and 7.7% (p < 0.04) in the DINP group. In summary, DEHP, BBP, and DINP all altered sexual differentiation, whereas DOTP, DEP, and DMP were ineffective at this dose. Whereas DEHP and BBP were of equivalent potency, DINP was about an order of magnitude less active.

Phthalate esters (PE) such as DEHP are high production volume plasticizers used in vinyl floors, food wraps, cosmetics, medical products, and toys. In spite of their widespread and long-term use, most PE have not been adequately tested for transgenerational reproductive toxicity. This is cause for concern, because several recent investigations have shown that DEHP, BBP, DBP, and DINP disrupt reproductive tract development of the male rat in an antiandrogenic manner. The present study explored whether the antiandrogenic action of DEHP occurs by (1) inhibiting testosterone (T) production, or by (2) inhibiting androgen action by binding to the androgen receptor (AR). Maternal DEHP treatment at 750 mg/kg/day from gestational day (GD) 14 to postnatal day (PND) 3 caused a reduction in T production, and reduced testicular and whole-body T levels in fetal and neonatal male rats from GD 17 to PND 2. As a consequence, anogenital distance (AGD) on PND 2 was reduced by 36% in exposed male, but not female, offspring. By GD 20, DEHP treatment also reduced testis weight. Histopathological evaluations revealed that testes in the DEHP treatment group displayed enhanced 3ss-HSD staining and increased numbers of multifocal areas of Leydig cell hyperplasia as well as multinucleated gonocytes as compared to controls at GD 20 and PND 3. In contrast to the effects of DEHP on T levels in vivo, neither DEHP nor its metabolite MEHP displayed affinity for the human androgen receptor at concentrations up to 10 microM in vitro. These data indicate that DEHP disrupts male rat sexual differentiation by reducing T to female levels in the fetal male rat during a critical stage of reproductive tract differentiation.

Antiandrogenic chemicals alter sexual differentiation by a variety of mechanisms, and as a consequence, they induce different profiles of effects. For example, in utero treatment with the androgen receptor (AR) antagonist, flutamide, produces ventral prostate agenesis and testicular nondescent, while in contrast, finasteride, an inhibitor of 5 alpha-dihydrotestosterone (DHT) synthesis, rarely, if ever, induces such malformations. In this regard, it was recently proposed that dibutyl phthalate (DBP) alters reproductive development by a different mechanism of action than flutamide or vinclozolin (V), which are AR antagonists, because the male offsprings display an unusually high incidence of testicular and epididymal alterations--effects rarely seen after in utero flutamide or V treatment. In this study, we present original data describing the reproductive effects of 10 known or suspected anti-androgens, including a Leydig cell toxicant ethane dimethane sulphonate (EDS, 50 mg kg-1 day-1), linuron (L, 100 mg kg-1 day-1), p,p'-DDE (100 mg kg-1 day-1), ketoconazole (12-50 mg kg-1 day-1), procymidone (P, 100 mg kg-1 day-1), chlozolinate (100 mg kg-1 day-1), iprodione (100 mg kg-1 day-1), DBP (500 mg kg-1 day-1), diethylhexyl phthalate (DEHP, 750 mg kg-1 day-1), and polychlorinated biphenyl (PCB) congener no. 169 (single dose of 1.8 mg kg-1). Our analysis indicates that the chemicals discussed here can be clustered into three or four separate groups, based on the resulting profiles of reproductive effects. Vinclozolin, P, and DDE, known AR ligands, produce similar profiles of toxicity. However, p,p'-DDE is less potent in this regard. DBP and DEHP produce a profile distinct from the above AR ligands. Male offsprings display a higher incidence of epididymal and testicular lesions than generally seen with flutamide, P, or V even at high dosage levels. Linuron treatment induced a level of external effects consistent with its low affinity for AR [reduced anogenital distance (AGD), retained nipples, and a low incidence of hypospadias]. However, L treatment also induced an unanticipated degree of malformed epididymides and testis atrophy. In fact, the profile of effects induced by L was similar to that seen with DBP. These results suggest that L may display several mechanisms of endocrine toxicity, one of which involves AR binding. Chlozolinate and iprodione did not produce any signs of maternal or fetal endocrine toxicity at 100 mg kg-1 day-1. EDS produced severe maternal toxicity and a 45% reduction in size at birth, which resulted in the death of all neonates by 5 days of age. However, EDS only reduced AGD in male pups by 15%. Ketoconazole did not demasculinize or feminize males but rather displayed anti-hormonal activities, apparently by inhibiting ovarian hormone synthesis, which resulted in delayed delivery and whole litter loss. In summary, the above in vivo data suggest that the chemicals we studied alter male sexual differentiation via different mechanisms. The anti-androgens V, P, and p,p'-DDE produce flutamide-like ...

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