Antiandrogenic Effects of Phthalate Esters During Development

Recently, concerns about exposure of children to phthalates in toys and other products have resulted in a ban of phthalates in certain toys by the European Union. Although industry has repeatedly assured the safety of these chemicals [161], most of them, including di-2-ethylhexyl phthalate (DEHP), have never been rigorously examined by the manufacturers for multigenerational effects.

The phthalates represent a class of toxicants which alter reproductive development via a mechanism of action that does not appear to involve AR or ER binding. Although many of the same effects are seen in animals exposed in utero to AR antagonists, like vinclozolin, in vitro studies, conducted to determine the biochemical mechanism responsible for the adverse developmental effects of DEHP, found that neither DEHP nor the primary metabolite mono-2-ethylhexyl phthalate (MEHP) compete with androgens for binding to the androgen receptor [162]. While some have suggested that di-n-butyl phthalate (DBP) was estrogenic [163], this activity is only displayed in vitro. For example, we have found that DBP did not produce any signs of estrogenicity in the ovariectomized female rat [115]. DBP (by subcutaneous injection at 200 or 400 mg/kg/day or by gavage at 1000 mg/kg/day, administered for two days, followed on the third day by 0.5 mg progesterone subcutaneously) did not induce a uterotropic response or estrogen-dependent sex behavior (lordosis). In addition, phthalate-treatment did not increase uterine weight [164] in juvenile female rats, and oral DBP-treatment (250,500, or 1000 mg/kg/day from weaning through adulthood) failed to accelerate vaginal opening, or to induce constant estrus in intact female rats [115].

Recent publications demonstrate that perinatal exposure to a number of phthalate esters alters development of the male reproductive tract in an antian-drogenic manner, causing underdevelopment and agenesis of the epididymis at relative low dosage levels. Arcardi et al. [165] reported that administration of DEHP in the drinking water to the dam during pregnancy and lactation (estimated LOAEL of 3 mg/kg/day) produced testicular histopathological alterations in male rat offspring. Although DEHP is not an AR antagonist in vitro at concentrations up to 10 |imol/l, it inhibits fetal Leydig cell testosterone synthesis in vivo when orally administered to the dam at 0.75 g/kg/day starting at day 14 of pregnancy. As a consequence, fetal testosterone concentrations are reduced in males to female levels from day 17 of gestation to 2 days after birth. This reduction in testosterone levels results in a wide range of malformations of the androgen-dependent tissues in male rats including reduced AGD, retained nipples, hypospadias, cleft phallus, vaginal pouch, agenesis of the gubernacula cords and sex accessory tissues, underdevelopment of levator ani muscles, undescended testis, hemorrhagic testes, epididymal agenesis, and testicular atrophy. Mylchreest et al. [166] observed similar malformations in male rat progeny after prenatal oral exposure (GD 10-22) to DBP with effects occurring at dosage levels as low as 100 mg/kg/day. In our multigenerational assessment of the reproductive effects of DBP on the male and female parents and their progeny, daily oral administration of 500 mg/kg/day by gavage delayed puberty in male rats and reduced fertility in both male and female rats [115] while 250 mg/kg/day induced reproductive tract malformations and reduced fecundity in the offspring. In addition, when dams were dosed by gavage with 500 mg DBP/kg in oil or an equimolar dose of DEHP (750 mg/kg/day) during sexual differentiation (GD 14 - PND 4) the male offspring were profoundly malformed. More limited dosing in "pulses" during 4-day periods of gestation demonstrated that DBP at this dose was most effective on days 16-19 [115].

In a recent investigation [167] we examined several phthalate esters to determine if they also altered sexual differentiation in an antiandrogenic manner. We hypothesized that the phthalate esters that altered testes function in the puber-tal male rat [168] would also alter testis function in the fetal male and produce malformations of androgen-dependent tissues. In this regard, we expected that benzyl butyl phthalate (BBP) and DEHP would alter sexual differentiation, while dioctyl terephthalate (DOTP), diethyl phthalate (DEP), and dimethyl (DMP) phthalate would not. We also expected that the phthalate mixture, di-isononyl phthalate (DINP), would be weakly active due to the presence of some phthalates with a 4-7 carbon side chain. DEHP, BBP, DINP, DEP, DMP, or DOTP were administered orally to the dam at 0.75 g/kg from gestational day 14 to postnatal day 3. Male, but not female, pups from the DEHP and BBP groups displayed shortened AGDs (25%) and reduced testis weights (30%). As infants, males in the DEHP, BBP, and DINP groups displayed female-like areolas/nipples (86%, 70%, and 22%, respectively) vs 0% in other groups and they displayed reproductive malformations. The percentage of males with malformations was 91% for DEHP, 84% for BBP, and 7.7% (p <0.04) in the DINP group. These phthalate esters produced a wide range of malformations of the external genitalia, sex accessory glands, epididymides, and testes. In the DINP group, 2/52

males (from 2/12 litters) displayed nipples, another male from one of the above litters displayed bilateral testicular atrophy, and a fourth male in the DINP group, from a third litter, displayed unilateral epididymal agenesis with hy-pospermatogenesis and scrotal fluid-filled testis, devoid of spermatids. In summary, DEHP, BBP, DBP, and DINP all induce antiandrogenic effects on sexual differentiation. DEHP and BBP were of equivalent potency while DINP was about an order of magnitude less active.

It is evident that the phthalate esters are high production volume toxic substances that have not been adequately tested for transgenerational effects. It is also clear that standard developmental toxicity studies are inadequate in their assessment of the effects of antiandrogens like the phthalates on the reproductive system [169,170]. Developmental toxicity studies, conducted in several reputable laboratories using dosage levels well above those used herein, have failed to detect these malformations. There are many reasons for the insensitivity of the developmental toxicity test to detect reproductive tract malformations in the offspring. In studies conducted under regulatory test guidelines prior to 1998, exposure to the dams was on GD 6 to 15 in the rat, which is prior to the development of the fetal reproductive tract. In 1998, the period of exposure was extended to GD 19. However, this change did not resolve the major limitation of this protocol. Even when dosing is continued to GD 19, very few of the reproductive malformations cited above will be detected in a routine teratologic evaluation. Not only are some of these tissues very small and only detectable histo-logically, but also the process of sexual differentiation continues through perinatal life in the rat and some reproductive tissues are not fully differentiated until puberty is complete. For these reasons, the reproductive system cannot be fully evaluated during perinatal life.

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