Recent Studies on Toxic Effects of Phthalate Esters on Reproduction and Development: Focus on Di(2-ethylhexyl) phthalate and Di-n-butyl phthalate

Phthalate esters extensively used as plasticizers in the plastic industry, are suspected of being endocrine disruptors. In this article, data for toxicity and the possible mechanisms of phthalate ester action in rodents are summarized for each endpoint related to reproduction and development, based on the latest research. As it is considered that the hydrolyzed metabolite of phthalate esters, the phthalate monoester, is the active material, this has been employed in most in vitro studies. Many kinds of phthalate esters have been reported to have weak estrogenic activity in vitro but there is no evidence of uterotrophic activity by phthalate esters such as di(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP). In addition, phthalate esters do not have a structure likely to bind to estrogen receptors. Therefore, they are not considered to be estrogenic compounds in vivo. It has been clearly shown that phthalate esters can exert testicular toxicity such as testicular atrophy and degenerative spermatogenesis in rats, mice and guinea pigs. Recent in vitro studies showed mono(2-ethylhexyl) phthalate (MEHP) to inhibit FSH stimulated cAMP accumulation in cultured Sertoli cells and induce apoptosis of germ cells in coculture with Sertoli cells. The proposed mechanism is as follows. First, MEHP affects Sertoli cells and separates germ cells from Sertoli cells. Secondly, solubilized FasL (sFasL) is produced from Sertoli cells and Fas is expressed on surface of the separated germ cells. Finally, the binding of sFasL and Fas induces apoptosis of the germ cells. Another effect of phthalate esters in the testis is known to be reduction of the biosynthesis of testosterone in Leydig cells. Therefore, the direct target sites of phthalate esters are probably both Sertoli cells and Leydig cells rather than germ cells. Oral administration of DEHP has been found to induce prolonged estrous cycle, suppression of ovulation and decrease of 17β-estradiol level in blood. On the bases of histopathological examination, preovulatory follicle granulosa cells in the ovary has been speculated to be target sites of phthalate esters. In in vitro studies using cultured granulosa cells, MEHP inhibited FSH stimulated c-AMP accumulation, and reduced 17β-estradiol production and aromatase mRNA expression. This might be the major cause of infertility in females in reproductive toxicity studies. In reproductive toxicity studies using mice and rats, DEHP and DBP induced infertility and decreased in the numbers and body weights of live offspring. Furthermore, this reproductive toxicity also occurs with mating of either phthalate esters-treated males or females with untreated animals. In the male case, the cause of infertility is presumably reduction of sperm counts in testis and epididymis due to degeneration of seminiferous tubules. One of the major toxic effects of phthalate esters is on development. The administration of DBP in the early pregnancy period results in embryolethality, and the exposure in mid pregnancy is teratogenic such as cleft palates, malformation of cervical and thoracic vertebrae, ribs, and dilatation of renal pelvis, but no changes in reproductive organs. When DEHP, DBP or n-butyl benzyl phthalate was administered to dams at late pregnancy and lactation periods, decreased anogenital distance, hypospadias, cryptorchidism, testicular and accessory organ atrophy in male offspring appeared but there were no effects in female offspring. Although these toxic effects were also induced by administration with flutamide, a typical androgen receptor antagonist, the mechanism of action of phthalate esters is not considered to be via androgen receptors because of the lack of interaction with androgen receptor. Oral administration of DEHP to neonatal males produced reduction of Sertoli cell number and MEHP induced separation of gonocytes from Sertoli cells in coculture system prepared from neonatal males. However, the mechanism of MEHP action in neonatal males may be different from that in adults because FSH stimulated cAMP accumulation in Sertoli cells was not affected by MEHP. Based on the above information, although phthalate esters are not estrogenic compounds, some of them are considered as endocrine disruptors in rodents because of antiandrogenic effect in male rats and decrease of 17β-estradiol level in blood in female rats. Other than abnormalities of the reproductive organs, the mechanisms of teratogenesis such as cleft palate and skeletal malformation remain to be elucidated. Presently, more than ten different phthalate esters are employed in industry and their toxicity seems to depend on their side chains. We will discuss the structure toxicity relationship of phthalate esters in the next article. _______________________________________________________________________________________ Corresponding author: 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Division of Risk Assessment, National Institute of Health Sciences, Mutsuko Koizumi