Abstract  The 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) family of proteins regulates the levels of the active 17 beta-hydroxy forms of sex steroids. The expression of 17 beta-HSD type IV is induced by peroxisome proliferator chemicals (PPC) in rat liver. In order to characterize more generally the impact of PPC on 17 beta-HSD expression, we determined (1) if expression of other members of the 17 beta-HSD family was coordinately induced by PPC exposure, (2) the tissues in which 17 beta-HSD was induced by PPC, and (3) whether the induction of 17 beta-HSD by PPC was dependent on the peroxisome proliferator-activated receptor alpha (PPAR alpha), the central mediator of PPC effects in the mouse liver. The mRNA levels of 17 beta-HSD I, II, and III were not altered in the liver, kidney, and testis or uterus of rats treated with PPC. The mRNA or 80 kDa a full-length protein levels of 17 beta-HSD IV were strongly induced in liver and kidney, but not induced in adrenals, brown fat, heart, testis, and uterus of rats treated with diverse PPC. In liver and kidneys from treated rats, additional proteins of 66 kDa, 56 kDa, and 32 kDa were also induced which reacted with the anti-17 beta-HSD IV antibodies and were most likely proteolytic fragments of 17 bega-HSD IV. Treatment of mice which lack a functional form of PPAR alpha with PPC, demonstrated that PPC-inducibility of 17 beta-HSD IV mRNA or the 80 kDa protein was dependent on PPAR alpha expression in liver and kidney. Our results demonstrate that 17 beta-HSD IV is induced by PPC through a PPAR alpha-dependent mechanism and support the hypothesis that exposure to PPC leads to alterations in sex steroid metabolism.

Abstract  Oral administration of di-(2-ethylhexyl) phthalate (I) [117-81-7] and dibutyl phthalate (II) [84-74-2] in the diet for 1-3 months at 5.0 and 0.5 g/kg/day to mice resulted in marked lesions mostly in the liver and kidney. In the higher dose group, the liver showed remarkable vacuolar degeneration and necrosis of single cells, while the kidney showed cysts and degeneration of tubule epithelial cells. In the low dose group, degeneration of liver and kidney parenchyma was obsd. The cytotoxicity of I was greater than that of II.

Abstract  Effects of estrogenic compds. (bisphenol A, alkyl phenols, phthalate esters, and genistein) on T lymphocyte apoptosis were investigated in vitro. The assays were performed in the absence or presence of low concns. of apoptosis-inducing agents etoposide or dexamethasone to detect apoptosis-inducing, -enhancing, and -suppressing activities of the test compds. When T lymphatic Jurkat cells were exposed to 10 ?M bisphenol A for 20 h, apoptosis was not induced, but apoptosis induced by 1 ?M etoposide was significantly enhanced. The 4-n-Nonylphenol (10 ?M) showed an apoptosis-inducing activity significantly. The 4-tert-Octylphenol (10 ?M) exhibited an apoptosis-enhancing activity. In contrast, phthalate esters including di-Bu phthalate and di-2-ethylhexyl phthalate showed neither activity at 10 ?M. Genistein (10 ?M) significantly exhibited apoptosis-inducing and enhancing activities. On the other hand, 17?-estradiol did not show any of these activities at 10 nM, the concn. exerting the estrogenic activity comparable to or much higher than that of 10 ?M of the test compds. Effects of these estrogenic compds. on apoptosis were also investigated using mouse primary thymocytes. Mouse thymocytes similarly exposed to the estrogenic compds. in the absence or the presence of dexamethasone for 6 h were characterized. In agreement with Jurkat cells, apoptosis-inducing or/and -enhancing activities were obsd. for the cells coincubated with bisphenol A, alkyl phenols, and genistein, but not those with di-2-ethylhexyl phthalate or 17?-estradiol. The apoptosis-inducing or/and -enhancing effects of the estrogenic compds. obsd. here appear to be due to their unidentified properties other than estrogenic activity.

Abstract  Di(2-ethylhexyl)phthalate (DEHP) used as a common plasticizer additive in the manuf. of plastics, such as polyvinyl chloride (PVC). This study examd. the effect of DEHP on steroidogenesis or spermatogenesis in the testes of Sprague-Dawley male rats treated orally with 250, 500, 750 mg/kg over a 30-day period. The expression levels of the steroidogenic- or spermatogenic-related genes were analyzed in the testis using a reverse transcription-polymerase chain reaction (RT-PCR) and Western blot anal. High doses of DEHP (500 and 750 mg/kg) significantly decreased the testicular sperm counts and daily sperm prodn. (DSP). In addn., serum testosterone levels were significantly lower in the DEHP treatment groups than in the control. The mRNA levels of SR-B1, StAR, PBR and CYP17 increased in a dose-dependent manner. These increases were significant at 500 and 750 mg/kg. In the other hand, the mRNA levels of CYP19 decreased significantly in testes of rats exposed to DEHP 500 and 750 mg/kg. Dose-dependent decreases in Spag4 and LDHA mRNA in the testis were obsd. after DEHP exposure, while there was a significant decrease in thyroid hormone receptor (TR)?1 protein levels. After DEHP exposure, while there was a significant decrease in thyroid hormone receptor (TR)?1 protein levels. High doses of DEHP significantly increased the expression of peroxisome proliferator-activated receptor (PPAR)-? and retinoid X receptor (RXR)-? protein but markedly decreased the expression of RXR-?. These results suggest that DEHP exposure can later the expression of the spermatogenic- or steroidogenic-related genes resulting in a decrease in sperm prodn. in the testis. This study is expected to be helpful in research examg. the mechanisms for how DEHP reduces the expression pattern of the genes involved steroid hormone synthesis after chronic exposure to DEHP.

Abstract  Toxicologic effects of di-(2-ethylhexyl) phthalate, di-n-butyl phthalate, di-(n-octyl)tin S,S′-bis-(isooctyl mercaptoacetate), dibenzyltin S,S′-bis-(isooctyl mercaptoacetate), as well as their effect on reproduction and fetal development were examined in Wistar rats. In a 3-month toxicity study all the compounds produced a statistically significant increase in the mean liver weight of the animals. Rats fed dietary levels 0.35% of di-(2-ethylhexyl)phthalate and 0.02% of di-(n-octyl)tin S,S′-bis-(isooctyl mercaptoacetate) for periods up to 12 months showed a significant decrease in body weight and an increase in kidney weight; furthermore, di-(2-ethylhexyl) phthalate caused liver enlargement. Red and white blood cell counts, hemoglobin concentration and histopathology of liver, kidneys, and spleen were within normal limits. Reproduction studies showed an increased number of resorptions and decreased fetal body weights. Doses of 0.02 and 0.04 g/kg of di-(n-octyl)tin S,S′-bis-(isooctyl mercaptoacetate) also caused a 17% incidence in fetal deaths. No gross abnormalities were found in the fetuses.

Abstract  In order to study the biochemical changes involved in the testicular damage induced by phthalate ester plasticizers, dibutyl phthalate (DBP), a representative plasticizer, was administered orally to mature male rats for one week. The rats were sequently killed at 1, 7, 14, and 21 days after the final administration of DBP. One testis was examined histologically and the other was subjected to assay for the activities of the testicular enzymes lactate dehydrogenase-X (LDH-X) and ornithine decarboxylase (ODC). On day I, the testes were clearly atrophied. Histologically, the germ cells had almost disappeared in the seminiferous tubules. On day 21, the testicular damage had mostly recovered. Enzyme assay showed that LDH-X activity was depressed severely on day I, but recovered with time. Therefore, its activity showed good correlation with the histological changes in the testis. ODC activity was depressed on day I but elevated on day 14. This indicated that the recovery of ODC activity preceded the histological recovery. Because the pathological findings obtained in these damaged testes resembled to those of human male infertility, the changes in the activities of the two testicular enzymes after DBP treatment may provide a clue toward the understanding of the pathophysiological phenomena involved in clinical infertile testis.

Abstract  The short-term test has been considered to be the most practical method for the rapid screening of carcinogens. Under a project of the Ministry of Health and Welfare, 182 compounds were examined by short-term tests over six years (1973-1978), using Salmonella typhimurium TAlOO and TA98 (mutations), Bacillus subtilis (rec assay), hamster lung fibroblast cells (chromosome aberrations, sister-3 23-324 KAWACHI ET AL. chromatid exchanges), human embryo fibroblasts (chromosome aberrations, sister chromatid exchanges), bone-marrow cells (chromosome aberrations in vivo) and silk worms (mutations). Compounds were selected by a research group organized by the Ministry and included carcinogens, noncarcinogens and compounds not previously tested for carcinogenicity. They were selected from the following categories: (1) structural analogues of known carcinogens; (2) food additives widely used in Japan; (3) drugs administered for long periods to patients; and (4) industrial intermediates with a high annual production. The object of the programme is not to screen possible carcinogens in our environment but to improve the tests and to investigate the validity of newly developed rapid screening techniques. Data from short- term assays for carcinogenicity are shown in Table 1: all data c ited as 'Salmonella test' are from the Salmonella/microsome assay system. Rec assays performed during 1973 did not include metabolic activation; however, from 1975-1978, a metabolic activation system was included. The chromosome aberration test in hamster fibroblasts in vitro included a metabolic ac tivation system between 1977 and 1978: among 65 compounds tested in this way, chlorpyrifos, cochineal and Eulan U-33 were the only ones t hat required metabolic activation. The chromosomal aberration test in human fibroblasts and the sister chromatid exchange assay in human fibroblasts and hamster fibroblasts were not activated metabolically. The validation of the short- term assays is summarized in Table 2. In the Salmonella/microsome test, of 49 compounds that showed mutagenic activity, 33 (67%) were found to be carcinogenic. On the other hand, of 66 nonmutagenic compounds, 48 (73%) were noncarcinogenic. Detailed data from the Salmonella/microsome tests are given in Table 3. Results from a combination of the Salmonella/microsome test and the chromosome aberration test (in vitro, in vivo) showed better validity than other combinations of short- term assays.

Abstract  Method was developed for the simultaneous determination of 17 classified and suspected endocrine-disrupting compounds (EDCs). Phenol, 2-nitrophenol, 4-nitrophenol, 4-dinitrophenol, 4-chlorophenol, 2,4-dimethylphenol, 2: methyl 4,6-dinitro-phenol, 2-chlorophenol, 2,4-dichlorophenol, 4-chloro-3-methylphenol, pentachlorophenol, dimethyl phthalate, diethyl phthalate, benzylbutyl phthalate, dioctyl phthalate, diethylhexylphthalate and dibutyl phthalate. Qualitative and quantitative analyses were performed by gas chromatography mass spectrometry (GC-MS) using)B-5MS column. These compounds were evaluated using solid-phase extraction for raw and treated wastewater from a municipal treatment plant. Phenols were derivatized with N-(t-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA) to form their respective t-butyldimethylsilyl derivatives. The extraction recovery range from 73.21 to 01.32% for 2,4,6-TCP and DEHP respectively. The occurrence pattern of phenol is in the order of PCP > 2CP > POH > 2NP > 2M- 4,6 DNP (old plant) and PCP > 2M- 4,6 DNP > POH 2CP 2NP for old and new plants respectively. Phthalates ranked as DEP > DBP > DEHP with corresponding values of 2473 991; 2000 : 236; and 192 = 127 mu gL(-1) in the new plant. The average percent removal of analytes range from 54.77 to 89.34% in the two plants investigated.

Abstract  Phthalate esters are found in a wide variety of consumer and food packing products. Hence there is widespread exposure of the human population to these chemicals. Some of the phthalate esters are known to be toxic to the developing male reproductive system. This paper derives a reference dose (RfD) for each of the phthalate esters (dibutyl phthalate, diisobutyl phthalate, butylbenzyl phthalate, diethylhexyl phthalate, dipentyl phthalate, and diisononyl phthalate) that cause these effects. As these phthalate esters cause similar adverse biological effects and have the same mechanism of action, it is appropriate in a risk assessment to consider the potential adverse effects from cumulative exposure to these chemicals using a dose addition model. This paper provides examples of a cumulative risk assessment using the hazard index and relative potency approaches from the RfDs derived from studies in laboratory animals and exposure information in people. The results of the cumulative risk assessments for both a US and a German population show that the hazard index is below one. Thus it is unlikely that humans are suffering adverse developmental effects from current environmental exposure to these phthalate esters.

Abstract  Carcinogenesis in the upper aerodigestive tract is influenced by multiple factors. Besides tobacco and alcohol consumption, specific pollutants such as phthalates, nitrosamines, and polycyclic aromatic carbohydrates may be important in tumor initiation. Genetic factors related to mutagen sensitivity and DNA repair capacity also play a role. The aim of this study was to investigate whether human peripheral blood lymphocytes and mucosal epithelium of the upper aerodigestive tract, the target for volatile and liquid xenobiotics, are equally sensitive to genotoxic agents. The Comet assay was used to detect for DNA damage induced by genotoxic agents in mucosal epithelial cells and peripheral blood lymphocytes of 60 volunteers. Mucosa was harvested from larynx, oropharynx, and inferior nasal turbinates. Xenobiotics investigated were dibutylphthalate (DBP), diisobutylphthalate (DiBP), N'-nitrosodiethylamine (NDELA), benzo[a]pyrene (B[a]P), and N'-methyl-N'-nitro-N-nitrosoguanidine (MNNG). DBP, DiBP, B[a]P, NDELA and MNNG induced a significant increase in DNA migration in both cell populations. Peripheral blood lymphocytes were more sensitive than mucosal cells to DBP and DiBP, but not to NDELA and B[a]P. The correlation, in terms of DNA migration, between lymphocytes and mucosal cells among volunteers was relatively poor. Based on the poor correlation in response between the two cell types, the sensitivity of peripheral blood lymphocytes to genotoxic agents appears to be a poor predictor of sensitivity in the target cells of the upper aerodigestive tract. Further attention should be focused on intra-individual mutagen sensitivities and inter-individual genetic differences as regards susceptibility to upper aerodigestive tract cancer.

Abstract  BACKGROUND: Phthalates are widely used chemicals, and human exposure is extensive. Recent studies have indicated that phthalates may have thyroid-disrupting properties. OBJECTIVE: We aimed to assess concentrations of phthalate metabolites in urine samples from Danish children and to investigate the associations with thyroid function, insulin-like growth factor I (IGF-I), and growth. METHODS: In 845 children 4-9 years of age, we determined urinary concentrations of 12 phthalate metabolites and serum levels of thyroid-stimulating hormone, thyroid hormones, and IGF-I. RESULTS: Phthalate metabolites were detected in all urine samples, of which monobutyl phthalate was present in highest concentration. Phthalate metabolites were negatively associated with serum levels of free and total triiodothyronine, although statistically significant primarily in girls. Metabolites of di(2-ethylhexyl) phthalate and diisononyl phthalate were negatively associated with IGF-I in boys. Most phthalate metabolites were negatively associated with height, weight, body surface, and height gain in both sexes. CONCLUSIONS: Our study showed negative associations between urinary phthalate concentrations and thyroid hormones, IGF-I, and growth in children. Although our study was not designed to reveal the mechanism of action, the overall coherent negative associations between urine phthalate and thyroid and growth parameters may suggest causative negative roles of phthalate exposures for child health.

Abstract  Phthalate esters have been extensively used as a plasticizer of synthetic polymers. Previous studies have revealed that some phthalate esters including di(n-butyl) phthalate (DBP) induce spermatogenic cell apoptosis, although its mechanism is not yet clear. The present study describes that disruption of Sertoli cell vimentin filaments by DBP administration may relate to spermatogenic cell apoptosis. The present histopathological study revealed that a single oral administration of 500 mg/kg DBP caused progressive detachment and displacement of spermatogenic cells away from the seminiferous epithelium and sloughing of them into the lumen. Degenerative spermatogenic cells characterized by chromatin condensation were frequently observed in DBP-treated rats. Ultrastructurally, the degenerative spermatogenic cells were separated from their neighbours, and a collapse of Sertoli cell vimentin filaments was recognized in DBP-treated rats. Sertoli cell cultures showed the increased number and size of vacuoles in their cytoplasm. In agreement with the in vivo experiment, vimentin filaments clearly showed a gradual collapse in DBP-exposed Sertoli cells in vitro. These in vivo and in vitro experiments indicate that DBP-induced collapse of Sertoli cell vimentin filaments may lead to detachment of spermatogenic cells, and then detached cells may undergo apoptosis because of loss of the support and nurture provided by Sertoli cells.

Abstract  Human phthalate exposure occurs as mixtures of diesters with varying activity towards testosterone-dependent development. Dibutyl (DBP), diethylhexyl (DEHP) and butylbenzyl (BBP) phthalate disrupt sexual development in the fetal rat. Dimethyl (DMP) and diethyl (DEP) phthalate do not. These differences in potency may result from differential delivery of the monophthalates to the testes or from variation in the abilities of the compounds to alter steroidogenesis. We tested five phthalates in pregnant rats (500mg/kg-d, GD12-19) and analyzed the fetal testes for corresponding monoesters (MMP, MEP, MBP, MEHP, MBeP). Testes MMP and MEP levels were 2-40-fold higher than the active monoesters, MBP and MEHP. BBP exposure led to low concentrations of MBeP, but similar MBP levels to DBP. An in vitro MA-10 cell assay measured the direct effect of monophthalates on testosterone production. MEHP inhibited LH-stimulated testosterone production at 1microM. RT-PCR confirmed down-regulation of genes associated with cholesterol transport and steroid synthesis and metabolism by MEHP. Five additional phthalates were tested for testosterone inhibition. MBP and mono-n-octyl phthalate were similar to MEHP; MMP, MEP and MBeP were poor inhibitors of testosterone production. Based on these results, differences in the phthalates' ability to interfere with sexual development in vivo appears to be more associated with differential potency for testosterone inhibition than differences in tissue doses.

Abstract  Testis function in fetal and peripubertal male rats is disrupted by subchronic exposure to phthalate esters (PEs). In contrast to the male rat, it is generally held that reproduction in female rats is much less sensitive to phthalate-induced disruption. However, the current study demonstrates that oral administration of dibutyl phthalate (DBP) to female Long Evans (LE) hooded rats from weaning, through puberty, mating, and gestation disrupts pregnancy maintenance at dose levels similar to those that affect testis function in male rats. Administration of 500 and 1000 mg DBP/kg/day, but not 250 mg DBP/kg/day, to female LE rats induced midpregnancy abortions. The percentage of females delivering live pups was reduced by more than 50% at 500 mg/kg/day and by 90% at 1000 mg/kg/day in the absence of overt toxicity, whereas the ages at vaginal opening and first estrus, estrous cyclicity, and mating indices (N mated/N paired or N pregnant/N mated) were not significantly affected. On gestational day 13, prior to the stage when litters were being aborted, ex vivo ovarian hormone production was significantly decreased by in vivo DBP treatment at 500 and 1000 mg/kg/day. These results should be considered when evaluating mechanisms of reproductive toxicity for the PE because it is likely that these reproductive alterations in the female rat arise via a mode of action similar to that operative in male rats.

Abstract  Di(n-butyl) phthalate (DBP) is commonly used in personal care products and as a plasticizer to soften consumer plastic products. Male rats exposed to DBP in utero have malformations of the male reproductive tract and testicular atrophy characterized by degeneration of seminiferous epithelium and decreased sperm production. In the fetal testis, in utero exposure to DBP reportedly resulted in reduced testosterone levels, Leydig cell aggregates, and multinucleated gonocytes (MNG). We investigated whether exposure in utero to DBP affects rat fetal Sertoli cells and compromises interactions between Sertoli and germ cells in the developing testis. Histological examination showed that MNG occurred at low frequency in the normal fetal rat testis. Exposure in utero at the dose level of DBP above estimated environmental or occupational human exposure levels significantly increased the number of these abnormal germ cells. Postnatally, MNG exhibited aberrant mitoses and were detected at the basal lamina. MNG were not apoptotic in the fetal and postnatal rat testes, as indicated by TUNEL. Sertoli cells in DBP-exposed fetal testis had retracted apical processes, altered organization of the vimentin cytoskeleton, and abnormal cell-cell contacts with gonocytes. The effect of DBP on Sertoli cell morphology at the level of light microscopy was reversed after birth and cessation of exposure. Our data indicate that fetal Sertoli cells are targeted by exposure in utero to DBP and suggest that abnormal interactions between Sertoli and germ cells during fetal life play a role in the development of MNG.

Abstract  Di-n-butyl phthalate (DBP) appears to be a ubiquitous environmental contaminant, as indicated by its presence in air, water, and soil worldwide (Giam et al., 1980; ATSDR, 2001; Peterson & Freeman, 1982) and the presence of its major metabolite, monobutyl phthalate (MBP), in random human urine samples (Blount et al., 2000). Studies indicate that exposure to a variety of endocrine-disrupting chemicals, such as DBP, may be partially responsible for reported global amphibian declines; if so, amphibians may serve as ecological harbingers for the future of human health. Therefore, this study was undertaken to investigate the effects of environmentally relevant concentrations of DBP on development in Xenopus laevis African clawed frogs. Developmental effects of DBP on Xenopus embryos were determined using the 96-h frog embryo teratogenesis assay-Xenopus (FETAX). Embryos (n = 300/group) were exposed from gastrulation (stage 8-11) through primary organogenesis (stage 46) to 0.1, 0.5, 1, 5, 10, or 15 ppm DBP dissolved in 0.01% dimethyl sulfoxide (DMSO), vehicle alone (0.01% DMSO; solvent control), or FETAX culture medium only (control; n = 600). At 96 h, mortalities for control, solvent control, and 0.1, 0.5, 1, 5, 10, and 15 ppm DBP were 5, 4, 6, 5, 5, 9, 18, and 52%, respectively; the incidence of developmental malformations in the surviving tadpoles was 7, 9, 15, 37, 51, 53, 90, and 100%. The average length of embryos was significantly lower in all DBP treatment groups. Thus, DBP significantly affected development of Xenopus embryos at low, environmentally relevant concentrations.

Abstract  This study sought to establish whether reduced androgen levels/action in the fetal rat testis induced by di(n-butyl) phthalate (DBP) contributes to dysgenetic features, namely reduced Sertoli cell number, occurrence of multinucleated gonocytes (MNG), and Leydig cell aggregation. Pregnant rats were administered treatments or cotreatments designed to manipulate testosterone levels [DBP, testosterone propionate (TP)] or action [flutamide, 7,12-dimethyl-benz[a]anthracene (DMBA)]. The aforementioned end points were analyzed and related to intratesticular testosterone (ITT) levels and peripheral androgen action (anogenital distance). Dysgenetic features were also evaluated in mice with inactivation of the androgen receptor (testicular feminized or ARKO mice). Exposure to DBP alone, or combined with flutamide, DMBA, or TP, resulted in reduced Sertoli cell number and ITT levels, as did exposure to TP alone; coadministration of DBP + TP caused the most severe reduction in both parameters. A positive correlation between ITT levels and Sertoli cell number was found (r = 0.791; P = 0.019). Similarly, exposure to DBP alone, or as a cotreatment, significantly increased occurrence of MNG and Leydig cell aggregation, and these were negatively correlated with ITT levels. Exposure to flutamide or DMBA alone had no significant effect on these dysgenetic end points. These findings suggest that reduced ITT decreases fetal Sertoli cell numbers and might be involved in Leydig cell aggregation and MNG. However, of these three end points, only Sertoli cell number was affected significantly in ARKO/testicular feminized mice with absent androgen action. Therefore, induction of MNG and Leydig cell aggregation might result from DBP-induced effects other than suppression of ITT levels.

Abstract  Reproductive disorders of newborn (cryptorchidism, hypospadias) and young adult males (low sperm counts, testicular germ cell cancer) are common and/or increasing in incidence. It has been hypothesized that these disorders may comprise a testicular dysgenesis syndrome (TDS) with a common origin in fetal life. This has been supported by findings in an animal model of TDS involving fetal exposure to n(dibutyl) phthalate, as well as by new clinical studies. Recent advances in understanding from such studies have led to refinement of the TDS hypothesis, highlighting the central role that deficient androgen production/action during fetal testis development, may play in the origin of downstream disorders.

Abstract  Reproductive effects have been observed in experimental animals treated with di(n-butyl)phthalate (DBP), one of phthalate esters used in soft plastics and a variety of consumer products. In this study, we investigated whether testicular toxicity of DBP is influenced by diminished renal function. To generate an experimental condition reflecting chronic renal disease in man, adult male F344 rats were given five consecutive weekly subcutaneous injections of folic acid at a dose of 300 mg/kg and then a diet containing 1200, 5000, and 20,000 ppm of DBP for 4 weeks. These concentrations roughly correspond to 60, 250, and 1000 mg/kg per day per rat, respectively. Folic acid clearly induced interstitial nephritis accompanied by impairment of renal function. Seminiferous degeneration, diminished spermatogenesis and increase in the number of morphologically abnormal sperm were more prominent in rats given folic acid and then 20,000 ppm DBP as compared to those exposed to DBP alone. These data suggest that DBP-induced male reproductive toxicity can be increased by folic acid-induced renal dysfunction.

Abstract  Phthalates are a group of industrial chemicals with many commercial uses, such as solvents, additives, and plasticizers. For example, di-(2-ethylhexyl) phthalate (DEHP) is added in varying amounts to certain plastics, such as polyvinyl chloride, to increase their flexibility. In humans, phthalates are metabolized to their respective monoesters, conjugated, and eliminated. However, despite the high production and use of DEHP, we have recently found that the urinary levels of the DEHP metabolite mono-(2-ethylhexyl) phthalate (MEHP) in 2,541 persons in the United States were lower than we anticipated, especially when compared with urinary metabolite levels of other commonly used phthalates. This finding raised questions about the sensitivity of this biomarker for assessing DEHP exposure. We explored the utility of two other DEHP metabolites, mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), as additional DEHP biomarkers. These metabolites are formed by oxidative metabolism of MEHP. In urine from 62 people, both the range and the mean urinary levels of MEOHP and MEHHP were on average 4-fold higher than those of MEHP; the mean of the individual ratios of MEHHP/MEOHP, MEHHP/MEHP, and MEOHP/MEHP were 1.4, 8.2, and 5.9, respectively. These data suggest that MEOHP and MEHHP are more sensitive biomarkers of exposure to DEHP than is MEHP. These findings also suggest a predominant human metabolic route for DEHP hydrolysis to MEHP followed by oxidation of MEHP; they also imply that a similar mechanism may be relevant for other high-molecular-weight phthalates, such as di-n-octyl, di-isononyl, and di-isodecyl phthalates.

Abstract  A large number of phthalate esters were screened for estrogenic activity using a recombinant yeast screen. a selection of these was also tested for mitogenic effect on estrogen-responsive human breast cancer cells. A small number of the commercially available phthalates tested showed extremely weak estrogenic activity. The relative potencies of these descended in the order butyl benzyl phthalate (BBP) > dibutyl phthalate (DBP) > diisobutyl phthalate (DIBP) > diethyl phthalate (DEP) > diisiononyl phthalate (DINP). Potencies ranged from approximately 1 x 10(6) to 5 x 10(7) times less than 17beta-estradiol. The phthalates that were estrogenic in the yeast screen were also mitogenic on the human breast cancer cells. Di(2-ethylhexyl) phthalate (DEHP) showed no estrogenic activity in these in vitro assays. A number of metabolites were tested, including mono-butyl phthalate, mono-benzyl phthalate, mono-ethylhexyl phthalate, mon-n-octyl phthalate; all were wound to be inactive. One of the phthalates, ditridecyl phthalate (DTDP), produced inconsistent results; one sample was weakly estrogenic, whereas another, obtained from a different source, was inactive. analysis by gel chromatography-mass spectometry showed that the preparation exhibiting estrogenic activity contained 0.5% of the ortho-isomer of bisphenol A. It is likely that the presence of this antioxidant in the phthalate standard was responsible for the generation of a dose-response curve--which was not observed with an alternative sample that had not been supplemented with o,p'-bisphenol A--in the yeast screen; hence, DTDP is probably not weakly estrogenic. The activities of simple mixtures of BBP, DBP, and 17beta-estradiol were assessed in the yeast screen. No synergism was observed, although the activities of the mixtures were approximately additive. In summary, a small number of phthalates are weakly estrogenic in vitro. No data has yet been published on whether these are also estrogenic in vitro. No data has yet been published on whether these are also estrogenic in vivo; this will require tests using different classes of vertebrates and different routes of exposure.

Abstract  We investigated the relationship between prenatal maternal urinary concentrations of phthalate metabolites and neonatal behavior in their 295 children enrolled in a multiethnic birth cohort between 1998 and 2002 at the Mount Sinai School of Medicine in New York City. Trained examiners administered the Brazelton Neonatal Behavioral Assessment Scale (BNBAS) to children within 5 days of delivery. We measured metabolites of 7 phthalate esters in maternal urine that was collected between 25 and 40 weeks' gestation. All but two phthalate metabolites were over 95% detectable. We summed metabolites on a molar basis into low and high molecular weight phthalates. We hypothesized the existence of sex-specific effects from phthalate exposure a priori given the hormonal activity of these chemicals. Overall we found few associations between individual phthalate metabolites or their molar sums and most of the BNBAS domains. However, we observed significant sex-phthalate metabolite interactions (p<0.10) for the Orientation and Motor domains and the overall Quality of Alertness score. Among girls, there was a significant linear decline in adjusted mean Orientation score with increasing urinary concentrations of high molecular weight phthalate metabolites (B=-0.37, p=0.02). Likewise, there was a strong linear decline in their adjusted mean Quality of Alertness score (B=-0.48, p<0.01). In addition, boys and girls demonstrated opposite patterns of association between low and high molecular weight phthalate metabolite concentrations and motor performance, with some indication of improved motor performance with increasing concentration of low molecular weight phthalate metabolites among boys. This is the first study to report an association between prenatal phthalate exposure and neurological effects in humans or animals, and as such requires replication.

Abstract  BACKGROUND: The ubiquitous use of phthalate esters in plastics, personal care products and food packaging materials results in widespread general population exposure. In this report, we extend our preliminary study on the relationship between urinary concentrations of phthalate metabolites and sperm DNA damage among a larger sample of men and include measurements of mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), two oxidative metabolites of di-(2-ethylhexyl) phthalate (DEHP). METHODS: Among 379 men from an infertility clinic, urinary concentrations of phthalate metabolites were measured using isotope-dilution high-performance liquid chromatography-tandem mass spectrometry. Sperm DNA damage measurements, assessed with the neutral comet assay, included comet extent (CE), percentage of DNA in tail (Tail%) and tail distributed moment (TDM). RESULTS: Monoethyl phthalate (MEP), a metabolite of diethyl phthalate, was associated with increased DNA damage, confirming our previous findings. Mono-(2-ethylhexyl) phthalate (MEHP), a metabolite of DEHP, was associated with DNA damage after adjustment for the oxidative DEHP metabolites. After adjustment for MEHHP, for an interquartile range increase in urinary MEHP, CE increased 17.3% [95% confidence interval (CI) = 8.7-25.7%], TDM increased 14.3% (95% CI = 6.8-21.7%) and Tail% increased 17.5% (95% CI = 3.5-31.5%). CONCLUSIONS: Sperm DNA damage was associated with MEP and with MEHP after adjusting for DEHP oxidative metabolites, which may serve as phenotypic markers of DEHP metabolism to 'less toxic' metabolites. The urinary levels of phthalate metabolites among these men were similar to those reported for the US general population, suggesting that exposure to some phthalates may affect the population distribution of sperm DNA damage.

Abstract  BACKGROUND: Phthalates may pose a risk for perinatal developmental effects. An important question relates to the choice of suitable biological matrices for assessing exposure during this period. OBJECTIVES: This study was designed to measure the concentrations of phthalate diesters or their metabolites in breast milk, blood or serum, and urine and to evaluate their suitability for assessing perinatal exposure to phthalates. METHODS: In 2001, 2-3 weeks after delivery, 42 Swedish primipara provided breast milk, blood, and urine samples at home. Special care was taken to minimize contamination with phthalates (e.g., use of a special breast milk pump, heat treatment of glassware and needles, addition of phosphoric acid). RESULTS: Phthalate diesters and metabolites in milk and blood or serum, if detected, were present at concentrations close to the limit of detection. By contrast, most phthalate metabolites were detectable in urine at concentrations comparable to those from the general population in the United States and in Germany. No correlations existed between urine concentrations and those found in milk or blood/serum for single phthalate metabolites. Our data are at odds with a previous study documenting frequent detection and comparatively high concentrations of phthalate metabolites in Finnish and Danish mothers' milk. CONCLUSIONS: Concentrations of phthalate metabolites in urine are more informative than those in milk or serum. Furthermore, collection of milk or blood may be associated with discomfort and potential technical problems such as contamination (unless oxidative metabolites are measured). Although urine is a suitable matrix for health-related phthalate monitoring, urinary concentrations in nursing mothers cannot be used to estimate exposure to phthalates through milk ingestion by breast-fed infants.

Abstract  Background: High exposure to phthalates, which are ubiquitous contaminants, has been shown in animal studies to produce detrimental effects on male reproductive functions. A recent study in humans reported dose–response relations between low phthalate levels in urine and human semen parameters, which raises the question whether humans are more sensitive to phthalate exposure than animals. Methods: Urine, serum, and semen samples were collected from 234 young Swedish men at the time of their medical conscript examination. Semen volume, sperm concentration, and motility were measured, together with sperm chromatin integrity (sperm chromatin structure assay) and biochemical markers of epididymal and prostatic function. We analyzed reproductive hormones in serum, and mono ethyl phthalate (MEP), mono ethylhexyl phthaltale (MEHP), mono benzyl phthalate (MBzP), mono butyl phthalate (MBP), and phthalic acid in urine. Results: For MBP, MBzP, and MEHP, no clear pattern of associations were observed with any of the reproductive biomarkers. Subjects within the highest quartile for MEP had fewer motile sperm (mean difference = 8.8%; 95% confidence interval = 0.8–17), more immotile sperms (8.9%; 0.3–18), and lower luteinizing hormone values (0.7 IU/L; 0.1–1.2), but there was no suggestion of harmful effects for most other endpoints. Phthalic acid actually was associated with improved function, as measured by several markers. Conclusions: The observed weak associations between 1 phthalate biomarker and impairment of a few aspects of reproductive function biomarkers were not consistent with results from a recent U.S. study. It is not yet possible to conclude whether phthalate exposure may reflect a hazard for human male reproduction.