Mechanisms underlying the anti-androgenic effects of diethylhexyl phthalate in fetal rat testis.

Diethylhexyl phthalate (DEHP) is widely used as a plasticizer in consumer products and is known to disturb the development of the male reproductive system in rats. The mechanisms by which DEHP exerts these effects are not yet fully elucidated, though some of the effects are related to reduced fetal testosterone production. The present study investigated the effects of four different doses of DEHP on fetal testicular histopathology, testosterone production and expression of proteins and genes involved in steroid synthesis in fetal testes. Pregnant Wistar rats were gavaged from GD 7 to 21 with vehicle, 10, 30, 100 or 300 mg/kg bw/day of DEHP. In male fetuses examined at GD 21, testicular testosterone production ex vivo and testicular testosterone levels were reduced significantly at the highest dose. Histopathological effects on gonocytes were observed at 100 and 300 mg/kg bw/day, whereas Leydig cell effects were mainly seen at 300 mg/kg bw/day. Quantitative RT-PCR revealed reduced testicular mRNA expression of the steroidogenesis related factors SR-B1, StAR, PBR and P450scc. Additionally, we observed reduced mRNA expression of the nuclear receptor SF-1, which regulates certain steps in steroid synthesis, and reduced expression of the cryptorchidism-associated Insl-3. Immunohistochemistry showed clear reductions of StAR, PBR, P450scc and PPARgamma protein levels in fetal Leydig cells, indicating that DEHP affects regulation of certain steps in cholesterol transport and steroid synthesis. The suppression of testosterone levels observed in phthalate-exposed fetal rats was likely caused by the low expression of these receptors and enzymes involved in steroidogenesis. It is conceivable that the observed effects of DEHP on the expression of nuclear receptors SF-1 and PPARgamma are involved in the downregulation of steroidogenic factors and testosterone levels and thereby underlie the disturbed development of the male reproductive system.

Diisobutyl phthalate has comparable anti-androgenic effects to di-n-butyl phthalate in fetal rat testis.

UNLABELLED: Phthalates are widely used as plasticizers in various consumer products and building materials. Some of the phthalates are known to interfere with male reproductive development in rats, and di-n-butyl phthalate (DBP), diethylhexyl phthalate (DEHP) and butyl benzyl phthalate (BBP) were recently banned for use in toys in the EU mainly due to their reproductive toxicity. Diisobutyl phthalate (DiBP) has similar structural and application properties as DBP, and is being used as a substitute for DBP. However, knowledge on male reproductive effects of DiBP in experimental animals is lacking. METHODS: In the current study, four groups of pregnant Wistar rats were exposed to either 0mg/kg bw/day or 600 mg/kg bw/day of DiBP from gestation day (GD) 7 to either GD 19 or GD 20/21. Male offspring was examined at GD 19 or GD 20/21 for effects on testicular testosterone production and testicular histopathology. Changes in anogenital distance (AGD) were evaluated as an indication of feminisation of males. RESULTS: Anogenital distance was statistically significantly reduced at GD 20/21 together with reductions in testicular testosterone production and testicular testosterone content. Histopathological effects (Leydig cell hyperplasia, Sertoli cell vacuolisation, central location of gonocytes and presence of multinuclear gonocytes) known for DBP and DEHP were observed in testes of DiBP-exposed animals at GD 20/21. Additionally, immunohistochemical expression of P450scc and StAR proteins in Leydig cells was reduced by DiBP. At GD 19, these effects on anogenital distance, testosterone levels and histopathology were less prominent. CONCLUSION: In this study, GD 20/21 rather than GD 19 appears to be the optimal time for investigating changes in anogenital distance, testosterone levels, and testicular histopathology. DiBP has similar testicular and developmental effects as DBP and DEHP, and although more developmental and especially postnatal studies are needed to clearly identify the reproductive effects of DiBP, this study indicates a reason for concern about the use of DiBP as a substitute for DBP.

Mechanisms of action underlying the antiandrogenic effects of the fungicide prochloraz.

The fungicide prochloraz has got multiple mechanisms of action that may influence the demasculinizing and reproductive toxic effects of the compound. In the present study, Wistar rats were dosed perinatally with prochloraz (50 and 150 mg/kg/day) from gestational day (GD) 7 to postnatal day (PND) 16. Caesarian sections were performed on selected dams at GD 21, while others were allowed to give birth to pups that were followed until PND 16. Prochloraz caused mild dysgenesis of the male external genitalia as well as reduced anogenital distance and retention of nipples in male pups. An increased anogenital distance indicated virilization of female pups. Effects on steroidogenesis in male fetuses became evident as decreased testicular and plasma levels of testosterone and increased levels of progesterone. Ex vivo synthesis of both steroid hormones was qualitatively similarly affected by prochloraz. Immunohistochemistry of fetal testes showed increased expression of 17alpha-hydroxylase/17,20-lyase (P450c17) and a reduction in 17beta-hydroxysteroid dehydrogenase (type 10) expression, whereas no changes in expression of genes involved in testicular steroidogenesis were observed. Increased expression of P450c17 mRNA was observed in fetal male adrenals, and the androgen-regulated genes ornithine decarboxylase, prostatic binding protein C3 as well as insulin-like growth factor I mRNA were reduced in ventral prostates PND 16. These results indicate that reduced activity of P450c17 may be a primary cause of the disrupted fetal steroidogenesis and that an altered androgen metabolism may play a role as well. In vitro studies on human adrenocortical carcinoma cells supported the findings in vivo as reduced testosterone and increased progesterone levels were observed. Overall, these results together indicate that prochloraz acts directly on the fetal testis to inhibit steroidogenesis and that this effect is exhibited at protein, and not at genomic, level.

Antiandrogenic effects in male rats perinatally exposed to a mixture of di(2-ethylhexyl) phthalate and di(2-ethylhexyl) adipate.

Di(2-ethylhexyl) phthalate (DEHP) is a well-known testicular toxicant inducing adverse effects in androgen responsive tissues. Therefore, di(2-ethylhexyl) adipate (DEHA) is currently being evaluated as a potential substitute for DEHP. Similarities in structure and metabolism of DEHP and DEHA have led to the hypothesis that DEHA can modulate the effects of DEHP. Wistar rats were gavaged with either vehicle, DEHP (300 or 750mg/kg bw/day) or DEHP (750mg/kg bw/day) in combination with DEHA (400mg/kg bw/day) from gestation day (GD) 7 to postnatal day (PND) 17. Decreased anogenital distance (AGD) and retention of nipples in male offspring were found in all three exposed groups. Dosed males exhibited decreased weights of ventral prostate and m. levator ani/bulbocavernosus. Histopathological investigations revealed alterations in testis morphology in both juvenile and adult animals. The litter size was decreased and postnatal mortality was increased in the combination group only, which is likely a combined effect of DEHP and DEHA. However, no combination effect was seen with respect to antiandrogenic effects, as males receiving DEHP in combination with DEHA did not exhibit more pronounced effects in the reproductive system than males receiving DEHP alone.

Early testicular effects in rats perinatally exposed to DEHP in combination with DEHA--apoptosis assessment and immunohistochemical studies.

This study aimed to characterize the effects of di(2-ethylhexyl) phthalate (DEHP) on the fetal rat testes and relate them to the effects seen in adults. Histopathological effects in fetal testes were examined with immunohistochemistry for anti-Mullerian hormone (AMH), 3beta-hydroxysteroid dehydrogenase, smooth muscle actin (SMA), proliferating cell nuclear antigen (PCNA), histone H3 and vimentin. Additionally, testicular apoptosis levels were assessed in fetal, prepubertal and adult rats. As the plasticizer di(2-ethylhexyl) adipate (DEHA) has similarities with DEHP in chemical structure and metabolism, we investigated if the testicular effects of DEHP were modulated by co-administration with DEHA. Wistar rats were gavaged during gestation and lactation with vehicle, DEHP (300 or 750 mg/kg/day), or DEHP (750 mg/kg/day) in combination with DEHA (400mg/kg/day), and male offspring were examined at gestation day (GD) 21, postnatal day (PND) 22, 26 and 190. In fetal testes, Leydig cells were found in large clusters containing AMH positive Sertoli cells. At GD 21, seminiferous chords appeared enlarged with an apparently increased number of gonocytes. However, proliferation of gonocytes did not appear increased. A few animals had a high number of TUNEL positive apoptotic cells in degenerating seminiferous tubules at PND 22 and 190, whereas most exposed animals had low levels of germ cell apoptosis at GD 21, PND 22 or PND 26, as evaluated by DNA laddering, TUNEL staining, Caspase-3 immunohistochemistry and Caspase-3 activity measurement. No differences between DEHP and DEHP+DEHA exposed groups were observed.

Steroidogenesis in fetal male rats is reduced by DEHP and DINP, but endocrine effects of DEHP are not modulated by DEHA in fetal, prepubertal and adult male rats.

The plasticizer di(2-ethylhexyl)phthalate (DEHP) exhibits antiandrogenic effects in perinatally exposed male rats. Di(2-ethylhexyl) adipate (DEHA) and diisononyl phthalate (DINP) are currently being evaluated as potential substitutes for DEHP, but similarities in structure and metabolism of DEHP with DEHA and DINP have led to the hypothesis that similarities in action may also exist. Pregnant Wistar rats were gavaged during gestation and lactation with vehicle, DEHP (300 or 750 mg/kg bodyweight per day), DINP (750 mg/kg bodyweight per day), DEHP (750 mg/kg bodyweight per day) in combination with DEHA (400 mg/kg bodyweight per day), or DEHP (300 mg/kg bodyweight per day) in combination with DINP (750 mg/kg bodyweight per day). DINP and DEHP were both shown to reduce testicular testosterone production ex vivo and testosterone levels in testes and plasma of male fetuses at gestation day 21, indicating a similar mechanism of action for DINP and DEHP. Additionally, plasma LH levels in male fetuses were elevated. Neonatal anogenital distance was reduced and the number of nipples at postnatal day 13 increased in DEHP-exposed male offspring. Serum inhibin B levels were significantly reduced in DEHP-exposed prepubertal male offspring, and in a few adult males. No modulating effects of DEHA on the endocrine effects of DEHP were detected, but a tendency towards an accumulating effect of DEHP and DINP in combination on suppression of testosterone synthesis was seen.