Metabolite profiling and identification in human urine after single oral administration of DEHP.

Di(2-ethylhexyl)phthalate (DEHP) is known to be a reproductive toxicant in male rodents, and its primary or secondary metabolites seem to be causative agents. To identify and quantify urinary metabolites of DEHP in humans, urine samples collected from healthy male and female volunteers following oral administration of deuterium labeled DEHP (d(4)-DEHP) at 3mg/person were analyzed by a high performance liquid chromatograph/mass spectrometer (LC-MS), and the excretion behavior of orally taken DEHP was evaluated. Mono(2-ethylhexyl)phthalate (MEHP), OH-MEHP, oxo-MEHP, COOH-MEHP, and their glucuronides were identified as metabolites in the male and female urine. The ratios of the conjugate forms in the total urinary metabolites were remarkably high from immediately after administration (0 to 4-hr post-dose), which were approximately 69% to 86% (male) and 80% to 89% (female) until 36-hr post-dose. It was suggested that DEHP taken orally was promptly converted to MEHP and its oxidative metabolites such as OH-MEHP, oxo-MEHP, and COOH-MEHP, and most of these metabolites received the conjugation reaction and were excreted as glucuronides. Remarkable differences from rodents, in which most of these metabolites were excreted as free forms, were demonstrated.

Metabolism of di (2-ethylhexyl) phthalate (DEHP): comparative study in juvenile and fetal marmosets and rats.

We compared the metabolic profile of di (2-ethylhexyl) phthalate (DEHP) in juveniles and fetus between rats and marmosets. STUDY-I: (14)C-DEHP (100 and 2,500 mg/kg) was singly administered to juvenile and adult marmosets by gavage. C(max) of the radioactivity in juvenile marmosets was 6.45 and 31 µg eq./g, respectively. The radioactivity excreted mainly into feces; however, at least 10% of the radioactivity was absorbed even at 2,500 mg/kg. No abnormal accumulation was observed in the male reproductive organs. STUDY-II: (14)C-DEHP (100 mg/kg) was singly administered to juveniles of rat and marmoset. The plasma radioactivity in marmosets was about 5% to 9% of that in rats. Free forms of mono-2-ethylhexyl phthalate (MEHP) and its oxidized metabolites such as oxo-, OH-, and COOH-MEHP were detected as the main compositions in rat plasma. In marmosets, free form of MEHP was also detected as a major composition, but not for oxidized MEHP metabolites. In rats, oxidized MEHP metabolites were excreted into urine as unconjugated forms. MEHP and its oxidized metabolites were also detected in marmoset urine; however, they were mostly glucuronized. No specific accumulation of the radioactivity was noted in the testes of either species; however, the radioactivity concentration in the marmoset testes was much lower than that in rats. STUDY-III: (14)C-DEHP (100 mg/kg) was singly administered to dams on gestation day 130 for marmosets and day 20 for rats. In either species, no specific accumulation of radioactivity was noted in the testis of fetuses from the dams treated with (14)C-DEHP; however, the radioactivity in the rat testis was about 20-times higher than that in the marmoset. Major metabolite components in rat whole fetal tissue were free forms of MEHP, OH-MEHP, and oxo-MEHP. Free form of MEHP was also detected as only a peak in the marmoset fetal tissue.

Effect of di(2-ethylhexyl) phthalate (DEHP) on genital organs from juvenile common marmosets: I. Morphological and biochemical investigation in 65-week toxicity study.

Recent studies demonstrated that preadolescent male rats are more sensitive to testicular damage from exposure to DEHP than adults. Male and female marmosets were treated daily with 0, 100, 500, or 2500 mg/kg DEHP by oral gavage for 65 wk from weaning (3 mo of age) to sexual maturity (18 mo). No treatment-related changes were observed in male organ weights, and no microscopic changes were found in male gonads or secondary sex organs. Sperm head counts, zinc levels, glutathione levels, and testicular enzyme activities were comparable between groups. Electron microscopic examination revealed no treatment-related abnormalities in Leydig, Sertoli, or spermatogenic cells. Histochemical examination of the testis after 3beta-hydroxysteroid dehydrogenase (3beta-HSD) staining did not reveal any alterations in steroid synthesis in the Leydig cells. Thus, although marmoset monkeys were treated with 2500 mg/kg DEHP, throughout the pre- and periadolescent period, no histological changes were noted in the testes. For females, increased ovarian and uterine weights and elevated blood estradiol level were observed in higher dosage groups, 500 and 2500 mg/kg. These increased weights were associated with the presence of large corpus luteum, a common finding in older female marmosets. Although an effect on the female ovary cannot be completely ruled out, no abnormal histological changes were observed in the ovaries or uteri in comparison to controls. No increases in hepatic peroxisomal enzyme activities were noted in treated groups; isolated hepatic enzyme activities (P-450 contents, testosterone 6beta-hydroxylase, and lauric acid omega-1omega-hydroxylase activities) were increased in males and/or females of either the mid- or high-dose groups, but no consistent dose-related trend was observed.

Influence of di-(2-ethylhexyl)phthalate on fetal testicular development by oral administration to pregnant rats.

Influence of di-(2-ethylhexyl)phthalate (DEHP) on testicular development was studied by oral administration of DEHP at doses of 500 and 1000 mg/kg/day to pregnant rats on gestational days (G) 7 to 18. Ethinyl estradiol (EE) at dose levels of 0.25 and 0.5 mg/kg/day was used as a reference substance. Each 5-6 pregnant rats were sacrificed and their fetuses were examined on G12, 14, 16, 18 and 20. Fetal deaths averaging 20-36% were observed at every examination in the group receiving 1000 mg/kg of DEHP. Increases of fetal deaths over 50% were also observed in the reference group that received 0.5 mg/kg of EE. Microscopic examination of the fetal testis in groups treated with DEHP revealed degeneration of germ cells in G16 fetuses and localized proliferation or hyperplasia of interstitial cells in G18 and 20 fetuses. Germ cells having more than two nuclei were observed in a few cases including the control testes of G14 fetuses. These multinucleated cells were observed frequently in G20 fetuses treated with DEHP. Examination of testes of naturally delivered offspring of dams treated with 1000 mg/kg of DEHP at 7 weeks of age revealed scattered atrophy or dilatation of seminiferous tubules. Another experiment was carried out to confirm the dose of DEHP affecting testicular development and spermatogenesis. DEHP was given to pregnant rats at doses of 125, 250 and 500 mg/kg/day during G7-18. Similar histopathological changes were observed in fetal testes of the group exposed to 500 and 250 mg/kg of DEHP, but not in those exposed to 125 mg/kg. In postnatal examinations, however, no abnormality was found in the testes at 5 and 10 weeks after birth in any of the treated groups. Furthermore, no abnormal findings were observed in the function of sperm, sperm counts and sperm morphology in the offspring of the group treated with DEHP during the fetal period at 10 weeks of age. Thus, 125 mg/kg/day is considered the no-observed-effect-level of DEHP on testicular development of rats by exposure in utero during the period of organogenesis.