Abstract  The hormone insulin-like factor 3 (INSL3) is produced by testicular Leydig cells. Production of INSL3 is dependent on the state of Leydig cell differentiation and is stimulated by the long-term trophic effects of luteinizing hormone. INSL3 is, along with the other major Leydig cell hormone testosterone, essential for testicular descent, which in humans should be completed before birth. The incidence of cryptorchidism (incomplete descent of the testis) may have increased in some developed countries during recent decades. Experimental studies have shown that maternal exposure to endocrine-disrupting chemicals (EDCs), such as phthalates, can result in cryptorchidism among male offspring and that INSL3 production, like steroidogenesis, is susceptible to phthalate exposure. Inhibition of these hormones may occur via a general phthalate-induced impairment of Leydig cell development and maturation. Recent studies have also addressed the sensitivity of human Leydig cells to EDCs, though with varied conclusions.

Abstract  Exposure to endocrine disrupting chemicals such as bisphenol A (BPA) and phthalates is prevalent among children and adolescents, but little is known regarding important sources of exposure at these sensitive life stages. In this study, we measured urinary concentrations of BPA and nine phthalate metabolites in 108 Mexican children aged 8-13years. Associations of age, time of day, and questionnaire items on external environment, water use, and food container use with specific gravity-corrected urinary concentrations were assessed, as were questionnaire items concerning the use of 17 personal care products in the past 48-h. As a secondary aim, third trimester urinary concentrations were measured in 99 mothers of these children, and the relationship between specific gravity-corrected urinary concentrations at these two time points was explored. After adjusting for potential confounding by other personal care product use in the past 48-h, there were statistically significant (p<0.05) positive associations in boys for cologne/perfume use and monoethyl phthalate (MEP), mono(3-carboxypropyl) phthalate (MCPP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), and in girls for colored cosmetics use and mono-n-butyl phthalate (MBP), mono(2-ethylhexyl) phthalate (MEHP), MEHHP, MEOHP, and mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), conditioner use and MEP, deodorant use and MEP, and other hair products use and MBP. There was a statistically significant positive trend for the number of personal care products used in the past 48-h and log-MEP in girls. However, there were no statistically significant associations between the analytes and the other questionnaire items and there were no strong correlations between the analytes measured during the third trimester and at 8-13years of age. We demonstrated that personal care product use is associated with exposure to multiple phthalates in children. Due to rapid development, children may be susceptible to impacts from exposure to endocrine disrupting chemicals; thus, reduced or delayed use of certain personal care products among children may be warranted.

Abstract  BACKGROUND: Insulin resistance (IR) is believed to be the underlying mechanism of metabolic syndrome and type 2 diabetes mellitus (DM). Recently, a few studies have demonstrated that phthalates could cause oxidative stress which would contribute to the development of IR. Therefore, we evaluated whether exposure to phthalates affects IR, and oxidative stress is involved in the phthalates-IR pathway.

METHODS: We recruited 560 elderly participants, and obtained blood and urine samples during repeated medical examinations. For the determination of phthalate exposure, we measured urinary levels of mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) as metabolites of diethylhexyl phthalates (DEHP), and mono-n-butyl phthalate (MnBP) as a metabolite of di-butyl phthalate (DBP). Malondialdehyde (MDA), an oxidative stress biomarker, was also measured in urine samples. We measured serum levels of fasting glucose and insulin, and derived the homeostatic model assessment (HOMA) index to assess IR. A mixed-effect model and penalized regression spline were used to estimate the associations among phthalate metabolites, MDA, and IR.

RESULTS: The molar sum of MEHHP and MEOHP (∑DEHP) were significantly associated with HOMA (β = 0.26, P = 0.040), and the association was apparent among participants with a history of DM (β = 0.88, P = 0.037) and among females (β = 0.30, P = 0.022). However, the relation between MnBP and HOMA was not found. When we evaluated whether oxidative stress is involved in increases of HOMA by ∑DEHP, MDA levels were significantly associated with increases of ∑DEHP (β = 0.11, P<0.001) and HOMA (β = 0.49, P = 0.049).

CONCLUSIONS: Our study results suggest that exposure to DEHP in the elderly population increases IR, which is related with oxidative stress, and that participants with a history of DM and females are more susceptible to DEHP exposure.

Abstract  In a published controlled dosing experiment, a single individual consumed 5mg each of labeled di-n-butyl phthalate (DnBP) and diisobutyl phthalate (DiBP) on separate occasions and tracked metabolites in his blood and urine over 48h. Data from this study were used to structure and calibrate simple pharmacokinetic (PK) models for these two phthalates, which predict urine and blood metabolite concentrations with a given phthalate intake scenario (times and quantities). The calibrated models were applied to a second published experiment in which 5 individuals fasted over the course of a 48-h weekend (bottled water only), and their full urine voids were captured and measured for DnBP and DiBP metabolites. One goal of this model application was to confirm the validity of the calibrated models - their validity would be demonstrated if a profile of intakes could be found which adequately duplicated the metabolite concentrations measured in the urine. A second goal was to study patterns of exposure for this group. It was found that all metabolites could be duplicated very well with individual-specific "best-fit" intake scenarios, with one exception. It appears that the model predicted much lower concentrations of the metabolite, 3carboxy-mono-propylphthalate (MCPP), than were observed in all individuals. Modeled as a metabolite of DnBP, this suggests that DnBP was not the major source of MCPP in the urine. For all 5 individuals, the reconstructed dose profiles of the two phthalates were similar: about 6 small bolus doses per day and an intake of about 0.5μg/kg-day. The intakes did not appear to be associated with diary-reported activities (personal hygiene and medication) of the participants. The modeled frequent intakes suggested one (or both) of two possibilities: ongoing exposures such as an inhalation exposure, or no exposure but rather an ongoing release of body stores of the phthalate metabolites from past exposures.

Abstract  In utero exposure to the phthalate ester plasticizer di-n-hexyl phthalate (DnHP) is known to affect the development of the male reproductive system and induce alterations in androgen-dependent tissues of male rat offspring. Male reproductive malformations produced by several phthalates have been causally linked to decreased testosterone production during the gestational period. This study was designed to evaluate the dose-response relationship for the effects of DnHP on the synthesis and production of testosterone in the fetal rat testis. Pregnant Sprague-Dawley rats were administered the vehicle (olive oil) and either DnHP (5 to 625 mg kg(-1) per day) or diethylhexyl phthalate (DEHP) (50 or 625 mg kg(-1) per day), by gavage, from gestation day (GD) 12 to19. Fetal testes were assessed on GD 19. DnHP reduced ex vivo testosterone production and down-regulated the expression of several genes required for cholesterol transport and steroid synthesis (i.e. SR-B1, StAR, P450scc, 3βHSD and P450c17). These inhibitions were dose dependent. A no-effect level was established at 5 mg kg(-1) per day and a lowest-effect level at 20 mg kg(-1) per day. mRNA levels of SR-B1, StAR, P450scc and 3βHSD were not similarly decreased in the adrenals. In conclusion, DnHP shares the same mode of action as DEHP in disrupting fetal testicular androgen synthesis. Alterations in testosterone production and in key steroidogenic gene expressions were apparent at lower doses than those causing postnatal reproductive malformations after gestational exposure during the critical period of male sexual differentiation. This suggests that they can be considered early biomarkers of DnHP-induced fetal testicular effects in rats.

Abstract  Human exposure to chemicals commonly encountered in our environment, like phthalates, is routinely assessed through urinary measurement of their metabolites. A particular attention is given to the specific population groups, such as obese, for which the dietary intake of environmental chemicals is higher. To evaluate the exposure to phthalates, nine phthalate metabolites (PMs) were analyzed in urine collected from obese individuals and a control population. Obese individuals lost weight through either bariatric surgery or a conservative weight loss program with dietary and lifestyle counseling. Urine samples were also collected from the obese individuals after 3, 6 and 12months of weight loss. Individual daily intakes of the corresponding phthalate diesters were estimated based on the urinary PM concentrations. A high variability was recorded for the levels of each PM in both obese and control urine samples showing the exposure to high levels of PMs in specific subgroups. The most important PM metabolite as percentage contribution to the total PM levels was mono-ethyl phthalate followed by the metabolites of di-butyl phthalate and di 2-ethyl-hexyl phthalate (DEHP). No differences in the PM levels and profiles between obese entering the program and controls were observed. Although paralleled by a significant decrease of their weight, an increase in the urinary PM levels after 3 to 6months loss was seen. Constant figures for the estimated phthalates daily intake were observed over the studied period, suggesting that besides food consumption, other human exposure sources to phthalates (e.g. air, dust) might be also important. The weight loss treatment method followed by obese individuals influenced the correlations between PM levels, suggesting a change of the intake sources with time. Except for few gender differences recorded between the urinary DEHP metabolites correlations, no other differences were observed for the urinary PM levels as a function of age, body mass index or waist circumference. Linear regression analysis showed almost no significance of the relationship between measured urinary PMs and serum free thyroxine, thyroid-stimulating hormone (TSH) for all obese individuals participating to the study, while for the control samples, several PMs were significantly associated with the serum TSH levels.

Abstract  In this work, phthalic acid esters (PAEs): dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate, benzyl butyl phthalate, bis(2-ethylhexyl) phthalate, and di-n-octyl phthalate in indoor dust (used as passive sampler) were investigated. The settled dust samples were collected from thirteen indoor environments from Palermo city. A fast and simple method using Soxhlet and GC-MS analysis has been optimized to identify and quantify the phthalates. Total phthalates concentrations in indoor dusts ranged from 269 to 4,831 mg/kg d.w. (d.w. = dry weight). The data show a linear correlation between total PAEs concentration and a single compound content, with the exclusion of the two most volatile components (DMP and DEP) that are present in appreciable amounts only in two samples. These results suggest that most of the PAEs identified in the samples of settled dust originate from the same type of material. This evidence indicates that, in a specific indoor environment, generally is not present only one compound but a mixture having over time comparable percentages of PAEs. Consequently, for routine analyses of a specific indoor environment, only a smaller number of compounds could be determined to value the contamination of that environment. We also note differences in phthalate concentrations between buildings from different construction periods; the total concentration of PAEs was higher in ancient homes compared to those constructed later. This is due to a trend to reduce or remove certain hazardous compounds from building materials and consumer goods. A linear correlation between total PAEs concentration and age of the building was observed (R = 0.71).

Abstract  While risk assessment models attempt to predict human risk to toxicant exposure, in many cases these models cannot account for the wide variety of human responses. This review addresses several primary sources of heterogeneity that may affect individual responses to drug or toxicant exposure. Consideration was given to genetic polymorphisms, age-related factors during development and senescence, gender differences associated with hormonal function, and preexisting diseases influenced by toxicant exposure. These selected examples demonstrate the need for additional steps in risk assessment that are needed to more accurately predict human responses to toxicants and drugs.

Abstract  The effects of adult lifestyle--primarily smoking and diet in women, and sedentary habits generally--are important factors affecting the fertility of men and women, and can also impact the fertility of their children. This review summarizes the effects of season, modern lifestyles and environmental chemicals on human fertility, and discusses the implications of these effects for future generations.

Abstract  Male reproductive health has deteriorated in many countries during the last few decades. In the 1990s, declining semen quality has been reported from Belgium, Denmark, France, and Great Britain. The incidence of testicular cancer has increased during the same time incidences of hypospadias and cryptorchidism also appear to be increasing. Similar reproductive problems occur in many wildlife species. There are marked geographic differences in the prevalence of male reproductive disorders. While the reasons for these differences are currently unknown, both clinical and laboratory research suggest that the adverse changes may be inter-related and have a common origin in fetal life or childhood. Exposure of the male fetus to supranormal levels of estrogens, such as diethlylstilbestrol, can result in the above-mentioned reproductive defects. The growing number of reports demonstrating that common environmental contaminants and natural factors possess estrogenic activity presents the working hypothesis that the adverse trends in male reproductive health may be, at least in part, associated with exposure to estrogenic or other hormonally active (e.g., antiandrogenic) environmental chemicals during fetal and childhood development. An extensive research program is needed to understand the extent of the problem, its underlying etiology, and the development of a strategy for prevention and intervention.

Abstract  DESCRIPTION (provided by applicant): The competitive renewal is responsive to recent epidemiologic and experimental evidence indicating that certain phthalates modulate thyroid function and reduce circulating thyroid hormone levels. The phthalates implicated include those already being studied under the current RO1 (Prenatal Phthalates, Placental Function and Fetal Growth R01 ES013543, Robin Whyatt, PI): di(2-ethylhexyl) phthalate (DEHP), butylbenzyl phthalate (BBzP) and dibutyl phthalate (DBP). Exposures to these phthalates are substantial among cohort subjects. Our proposed renewal will determine whether these exposures are associated with perturbations in thyroid function in children followed from birth through age 10-11 years. The study will also determine whether the phthalates are associated with deficits in child neuropsychological function, including in domains that might be associated with phthalate-induced reductions in thyroid function (intelligence, attention, executive function and motor skills). In addition, given that the purported mechanism whereby phthalates affect thyroid levels is through modulation of the Sodium Iodide Symporter (NIS) mediated iodide uptake activity in the thyroid gland, we will also evaluate whether prenatal phthalate exposures modulate expression of the NIS in placental tissue. Such a finding could have important implications for fetal thyroid function, as the NIS is the transport gene responsible for the active transfer of iodide across the placenta. Thyroid hormones during pregnancy and early childhood are critical to brain development, and even modest reductions in hormone levels may have long-lasting effects on child mental, motor and neuropsychological function. However, while prior studies have assessed the relationship between phthalates and thyroid hormones in adults, no prior studies have assessed these relationships in children, or evaluated the effects of prenatal and early-life phthalates exposures on child neuropsychological function. In our preliminary analyses, we found a significant inverse association between maternal prenatal DEHP exposure and child mental development at age 3 years among cohort children. This proposed renewal will build on these preliminary findings to evaluate the role of prenatal and early childhood phthalate exposures on: (1) measures of thyroid function in children from birth through age 11 years;(2) child cognition (language, working memory, executive function, problem solving);(3) child behavior (attention and impulsivity );(4) child motor development (fine manual control, manual coordination, body coordination, strength and agility, motor speed and visual scanning) and (5) expression of the NIS in placental tissue. PUBLIC HEALTH RELEVANCE: The competitive renewal is responsive to recent epidemiologic and experimental evidence indicating that certain phthalates modulate thyroid function and affect child cognitive development. The proposal will determine whether prenatal and early postnatal exposure to the phthalates are associated with perturbations in thyroid function and with deficits in child europsychological function (intelligence, attention, executive function and motor skills) in a cohort of 400 children followed from birth through age 10-11 years.

Abstract  BACKGROUND: Mono-(2-ethylhexyl) phthalate (MEHP), the metabolite of di-(2-ethylhexyl) phthalate (DEHP), was suspected to be toxic to human embryos. This study contributes to investigating its toxic effects by an embryonic stem cell test (EST) based on two human embryonic stem cell (hESCs) lines.

METHODS: CH1 established in our own lab and H1, a federally registered cell line were two human embryonic stem cell lines used in this test. Four endpoint measurements were performed consisting of cell viability, proliferation ability, apoptosis as well as changes of gene expression patterns after spontaneous differentiation were determined. For measuring effects on the first three endpoints, the cells were treated with various concentrations of MEHP dissolved in dimethyl sulfoxide (DMSO) and only with DMSO which served as control and harvested after 5 days. For measuring effects during spontaneous differentiation, the RNA of embryoid bodies (EBs) formed after 8 days' MEHP exposure was collected and changes in differentiation specific gene expression patterns were analyzed by quantitative real time RT-PCR.

RESULTS: As a result the viability and proliferation ability of both cell lines decreased significantly at 1000 µmol/L MEHP, while there was no effect on apoptosis or cell morphology. In addition MEHP also changed the gene expression pattern in the EBs of both cell lines.

CONCLUSION: MEHP in a high dose was cytotoxic and affected the development of hESCs, which indicates its embryo toxicity in human embryos.

Abstract  DESCRIPTION (provided by applicant): The proposed study will determine whether early childhood exposures to phthalates are associated with the development of current asthma and proallergic immunoglobulin (Ig) E production at ages five to seven and whether current exposures to phthalates are associated with augmented airway inflammation and diminished lung function at ages five to seven. The research will confirm or refute recent epidemiologic findings of associations between phthalate exposures and childhood asthma, atopy and reduced lung function. This prior research is limited by incomplete exposure and outcome measures. However, results from a pilot undertaken for the proposed study also support the hypothesis that phthalate exposures are risk factors in the development of asthma. The proposed study will be conducted among a cohort of 400 children who reside in minority communities in New York City. These communities experience some of the highest childhood asthma rates in the world. Phthalate exposures are also widespread. The research will be performed within the ongoing longitudinal birth cohort study being conducted by the Columbia Center for Children's Environmental Health (CCCEH). The CCCEH is evaluating the contribution of prenatal and postnatal exposures in the development of asthma and other health outcomes. Children are followed from pregnancy through age seven years. The proposed research is cost effective in that many of the required exposure and outcome measurements are already being gathered within the CCCEH cohort. These include detailed history of respiratory and allergic symptoms, asthma treatment and emergency room visits; lung function testing at age six; allergen levels in house dust samples collected from pregnancy through age five; and measurement of allergic sensitization (total and specific IgE measured at ages two, three, and five years). The phthalates will be measured in stored urine samples collected from the mother during pregnancy and from children at ages three and five years and in newly collected indoor air and urine samples at ages five to seven. Also at age five to seven years, exhaled nitric oxide will be measured as an indicator of airway inflammation and total and allergen-specific IgE levels will be determined. The case ascertainment of current asthma at ages five to seven will be made by a designated board- certified pediatric pulmonologist at the Morgan Stanley Children's Hospital of New York Presbyterian Hospital. Hypotheses to be tested are: (1) that early childhood exposures to the phthalates, as measured by metabolite levels in urine samples collected during pregnancy and at ages three and five years, predict current asthma at ages five to seven and production of IgE antibodies at age seven; and (2) after correcting for early phthalate exposure, current exposure as measured by levels of the parent compounds in indoor air samples and metabolites in urine samples collected at ages five to seven years, will be associated inversely with lung function and positively with airway inflammation at ages five to seven years.

Abstract  DESCRIPTION (provided by applicant): An apparent increase in the prevalence of human male reproductive tract abnormalities during the past half century have led Skakkebaek to articulate "testicular dysgenesis syndrome" as an explanation for this phenomenon. According to Skakkebaek, the observed abnormalities-including malformed external genitalia (hypospadias), undesecended testis (cryptorchidism), spermatogenic defects, and testis germ cell cancer- result from the alterations in the in utero and perinatal hormonal milieu causing disruption of sensitive male reproductive tract development events. Attention is focused on environmental endocrine disrupting chemicals as possible causes of this developmental disruption. Phthalates are ubiquitous environmental contaminants and endocrine disrupting chemicals. Rats exposed to di-(n-butyl)phthalate) (DBP) during critical in utero window of male reproductive tract development have altered gene expression and a number of reproductive tract abnormalities associated with testicular dysgenesis syndrome, including underdeveloped or absent reproductive organs, hypospadias, cryptorchidism and decreased sperm production. Although testis germ cell cancer has not been observed in this rat model, the fetal testes of DBP-exposed male rats contain dysplastic, multinucleated gonocytes. In this project, a new mouse model of gestational DBP-induced testicular dysgenesis is established. After characterizing alterations in gene expression in this genetically tractable species, p53-deficient mice will be used to allow DBP-induced dysplastic gonocytes to persist in the postnatal testis rather than degenerate and die. We expect these persistent dysplastic gonocytes to become transformed in the p53-deficient environment, resulting in tumorigenesis and the development of testis germ cell cancer. These goals will be pursued with the following working hypothesis as a guide: Disruption of mammalian in utero hormonal environment by phthalates produces a common spectrum of abnormalities across species, including a predilection to testicular carcinogenesis unmasked in p53-deficient mice.

Abstract  DESCRIPTION (provided by applicant): The overall objective of this proposal is to determine the mechanisms by which phthalate esters downregulate testosterone production in the developing fetal rat testes, resulting in antiandrogenic effects on the developing male reproductive tract. Phthalate esters are a class of environmental chemicals to which humans are ubiquitously exposed and which cause antiandrogenic effects on the developing male reproductive tract in rats. The mechanisms by which phthalate esters cause their effects remain to be determined. In preliminary studies, global changes in gene expression in the developing rat testis following in utero exposure to di(n-butyl) phthalate (DBP) were examined. A significant finding was that DBP produced a reduction in key genes in pathways associated with either steroid production or the provision of substrates for this activity. It is therefore hypothesized that phthalates decrease testosterone in the developing testes because of a coordinate disruption in cholesterol transport and steroid biosynthesis. This hypothesis will be tested by investigating the following specific aims: 1) Identify the key steps involved in cholesterol transport and testosterone biosynthesis that are targets for disruption by in utero exposure to DBP in the developing fetal rat testes, 2) Determine the consequences of DBP-induced changes in target gene and protein expression identified in Specific Aim 1 on fetal testicular cell signaling pathways, cholesterol transport, and steroidogenesis, and 3) Establish the molecular mechanism by which DBP downregulates expression of genes involved in cholesterol transport and steroid biosynthesis. Quantitative RT-PCR and protein analysis will be used to identify gene targets in the developing fetal testis following in utero exposure to DBP. The promoter regions of selected gene target will be further examined in vitro to determine the mechanism by which phthalate esters act on cholesterol transport and steroidogenesis. The proposed studies will identify critical genes and pathways associated with cholesterol transport that are targets for DBP in the male rat in utero and will aid in determining potential human risks from exposure to this class of environmental chemicals.

Abstract  DESCRIPTION (Adapted from the Investigator's Abstract) Understanding the mechanism of nongenotoxic liver carcinogens including peroxisome proliferators is important due to the wide range of sensitivity of different species. One mechanism for controlling the expression of genes is DNA methylation, i.e., specific changes in 5-methylcytosine (5-MEC) content of DNA. The principal investigator (PI) proposes that the effect of peroxisome proliferators on methylation of genes would be useful in understanding their mechanism and as biomarkers for their species sensitivity. There are four aims to test this hypothesis. Aim 1 will determine in rat liver, a sensitive species, whether dibutyl phthalate, gemfibrozil, Wy-14,643, and 2,4-D decrease methylation of DNA, IGF2 and c-myc while increasing methylation of connexin 32. By evaluating genes that are expected to be hypomethylated and a gene that should be hypermethylated, we hope to demonstrate specificity for the effect of peroxisome proliferators on DNA methylation. Aim 2 will determine whether the alterations in methylation found in rat liver also occurs in mouse liver, another sensitive species, and to a lesser degree in a less sensitive species, Syrian hamsters. Aim 3 will determine dose-response relationships. Aim 4 will determine whether hypomethylation of IGF2 and c-myc are associated with an increased expression of their mRNA and protein and whether hypermethylation of the connexin 32 gene is associated with decreased expression. The species sensitivity of the effect of the four peroxisome proliferators on the methylation of DNA and genes and on the expression of their mRNA will be compared to known species sensitivity of their carcinogenic activity, enhancement of cell proliferation, induction of peroxisomes, and other biological and molecular activity. A correlation between the species sensitivity of the effect of peroxisome proliferators on DNA (gene) methylation and the expression of their mRNA and/or protein would indicate usefulness as biomarkers of exposure to these chemicals. Furthermore, a correlation with carcinogenic activity would indicate the involvement of the gene(s) in a nongenotoxic mechanism for peroxisome proliferators.

Abstract  A reproductive assessment by continuous breeding study of di(n-butyl)-phthalate (DBP) was conducted by the US National Toxicology Program (NTP). When this plasticizing agent was administered continuously to rats of both sexes, fertility and reproductive tract effects were detected in adult F1 offspring, not in the F0 parents. The present study investigated the potential adverse effects on reproduction of in utero and lactational exposure of rats to DBP at dose levels similar to the NTP study, but with a shorter dosing period and production of a single litter. Pregnant CD rats (sperm-positive = gestation day (GD) 0; 10 dams/dose) were given DBP by gavage at 0, 250, 500 or 750 mg/kg body weight/day from GD 3 throughout pregnancy and lactation until the offspring were at postnatal day (PND) 20. Maternal body weights throughout the dosing period were not significantly affected by DBP treatment, and no clinical signs of toxicity were observed. DBP treatment had no apparent effect on parturition. Litter size was decreased in the 750 mg/kg/day group (9.0 +/- 0.7 vs 12.7 +/- 0.8 pups/litter in the control group). The number of implantation sites (on PND 21), proportion of pups born alive, sex ratio of live pups, and weight at birth were comparable in all groups. Adverse effects on the male reproductive system were induced in a dose-dependent manner. Anogenital distance on PND 2 was significantly decreased in males from dams treated with 500 and 750 mg DBP/kg/day. Undescended testes on PND 40 were observed in 0, 13, 29, and 25% of litters at 0, 250, 500, and 750 mg/kg/day, respectively. Small malformed prepuces and penises occured in 86 and 50% of litters at 500 and 750 mg/kg/day, respectively. The age at onset of vaginal opening and preputial separation was comparable in all groups, including gross morphologically normal males at 500 and 750 mg/kg/day. Decreased testicular size and poorly developed or absent epididymis were observed in all DBP groups. Lack of patent vagina and malformed or absent uteri and ovaries occured at 500 and 750 mg DBP/kg/day. Thus, exposure to high doses of DBP in utero and during the entire lactational period induced profound reproductive tract malformations in rats similar to those observed in the NTP study.

Abstract  The presence of phthalate esters (PAEs) in the environment is not desirable and therefore, needs to be monitored. This study reports the first data on the concentration levels of PAEs in water and sediments of the Jukskei River catchment area, South Africa. The study was conducted during the summer and winter seasons of 2005. Liquid-liquid extraction (LLE) and Soxhlet extraction (SE) methods were optimized, evaluated and used to determine PAEs of interest in water (unfiltered and filtered) and sediments samples, respectively. Mean percentage recoveries in spiked doubly distilled water ranged from 100 ± 5.32 dimethyl phthalate (DMP) - 122 ± 0.46 di-2-ethylhexyl phthalate (DEHP) and 91.6 ± 1.93 diethyl phthalate (DEP) - 117 ± 4.80 dibutyl phthalate (DBP) in sediments. The concentration levels of PAEs studied in unfiltered environmental water samples were in the range of 0.04(± 0.00) (DMP) - 9.76(± 00.1) ng mL(-1)(DEHP) for PAEs and from 0.09 (± 0.01) (DMP) - 4.38 (± 0.06) ng mL(-1)(DEHP) for filtered environmental water samples. Concentration levels obtained in sediments were from 0.05 (0.00) (DMP) - 4910 (0.36) ng/gdw (DEHP). PAEs adsorbed on the sample bottle gave concentration levels of up to 0.10 (± 0.03) ng mL(-1)for some samples and no analyte was detected (ND) in some cases Generally, concentrations obtained were below the water quality guideline values of United States Environmental Protection Agency (USEPA).

Abstract  Ultrasound-assisted dispersive liquid-liquid microextraction coupled with high performance liquid chromatography (UA-DLLME-HPLC) was developed for the determination of four typical phthalate esters (PAEs). The analyzed PAEs included dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP) and di-n-octyl phthalate (DnOP). The UA-DLLME parameters such as types/volumes of extraction/dispersion solvents, ultrasonic time, ionic strength and extraction time, were investigated. Enrichment factor (EF)was employed to evaluate the extraction efficiency. The conditions were finally chosen: CCl4 (40 microL)/ acetonitrile (1.0 mL) as extraction/dispersion solvents; 30 g/L NaCl; ultrasound/centrifugation of 5 min. Under the optimized extraction conditions, UA-DLLME exhibited strong enrichment ability for the four PAEs. The EFs for DMP, DEP, DBP and DnOP obtained were 71, 144, 169 and 159, respectively. The limits of detection were 3.78, 1.77, 3.07 and 3.30 microg/L for DMP, DEP, DBP and DnOP, respectively. The satisfactory recoveries for three water samples at three spiked levels ranged from 82.99%-114.47%, with the relative standard deviations of 1.93%-8.31%. It is a convenient, speedy, environmentally benign method for the routine analysis of PAEs in water samples.

Abstract  The pathway through which retinol (vitamin A) is converted to its active metabolite, all-trans-retinoic acid (atRA), and subsequent receptor-mediated regulation of gene transcription by atRA is essential for all mammal life stages. This pathway is required for normal embryonic development and maintenance of cellular phenotype in adult organisms; chemicals that cause even minor interference with its normal function are potential developmental and adult toxicants. A short-term (24 h) in vitro mode-of-action screen for detecting chemicals that disrupt this essential pathway is described. It uses the mouse pluripotent P19 stem cell in a 96-well format, RT-qPCR gene-expression assay that does not require RNA purification to detect chemicals that interfere with retinol-induced Hoxa1 gene expression, a target of retinol signaling in mammals. A total of 21 chemicals were screened at a single 45 μM concentration. Four chemicals known to disrupt the pathway in the rodent embryo (citral, disulfiram, and two rodent teratogens, nitrofen and bisdiamine) all significantly inhibited Hoxa1 upregulation by retinol. An additional four of seven chemicals with varying degrees of structural similarity to known disruptors or to the retinoid side chain, but not previously known to disrupt the pathway, were positive in the screen. The xenoestrogens, diethylstilbestrol, bisphenol A, 4-n-nonylphenol, and genistein and the phthalate esters, dibutyl phthalate and dipentyl phthalate, but not diethylhexyl phthalate, also significantly disrupted the pathway. Of the 21 chemicals tested, diethylstilbestrol was the only chemical that showed evidence in the MTT assay that cytotoxicity may have contributed to disruption of the pathway. Birth Defects Res (Part B) 98:276-282, 2013. © 2013 Wiley Periodicals, Inc.

Abstract  In the present study, we analyzed the oxidative stress related indices and immune related gene expression of zebrafish embryos after a short-term exposure to various concentrations of di-n-butyl phthalate (DBP), diethyl phthalate (DEP) and their mixture (DBP-DEP) from 4h post-fertilization (hpf) to 96hpf. Exposure to the chemicals was found to enhance the production of reactive oxygen species (ROS) and lipid peroxidation (LPO) in a concentration-dependent manner. Simultaneously, adaptive responses to DBP/DEP-induced oxidative stress were observed. The activity of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were all increased in a concentration-dependent manner. The transcription of innate immune related genes including interferon γ (IFNγ), interleukin-1β (IL1β), Myxovirus resistance (Mx), tumor necrosis factor α (TNFα), CC-chemokine, CXCL-clc, lysozyme (Lyz) and complement factor C3B (C3) were up-regulated upon DBP, DEP and their mixture exposure, suggesting the induction of immune response. In addition, co-exposure to DBP-DEP also induced antioxidant defense and immune response in zebrafish embryo. The results demonstrat that DBP/DEP exposure could induce the antioxidant and immune responses in zebrafish embryos.

Abstract  It has been hypothesized that oils containing high levels of omega-3 polyunsaturated fatty acids, such as canola and fish oil, could counteract some of the adverse effects induced by phthalates. In the present study, the influence of different oily vehicles on di-butyl phthalate (DBP)-induced testicular toxicity and lipid profile was investigated. Pregnant Wistar rats were treated by oral gavage from gestation days 13 to 20 with DBP (500 mg/kg/day) diluted in three different vehicles: corn, canola or fish oil. Male fetuses were analyzed on gestation day 20. DBP exposure lowered intratesticular testosterone levels and anogenital distance, regardless of the vehicle used. The percentage of seminiferous cords containing multinucleated gonocytes and cord diameter was increased in DBP-exposed groups, compared with vehicle controls, with no difference between the three DBP-exposed groups. Clustering of Leydig cells was seen in all DBP groups. Lipid profile indicated that administration of canola and fish oil can increase the content of omega-3 fatty acids in rat testis. However, content of omega-3 was diminished in DBP-treated groups. Overall, our results indicate that different oily vehicles did not alter fetal rat testicular toxicity induced by a high DBP dose.

Abstract  OBJECTIVE: To observe the effects of dibutyl phthalate (DBP) andmonobutyl phthalate (MBP) on the mRNA and protein expression of insulin-like factor 3 (INSL3) in the Leydig tumor cells (MA-10) of mice and the level of testosterone secreted from MA-10 cells.

METHODS: The MA-10 cells of mice, used as a cellular model, were exposed to DBP and MBP. The content of testosterone in the supernatant medium was measured by enzyme-linked immunosorbent assay; the mRNA and protein expression levels of INSL3 in MA-10 cells were measured by quantitative PCR and Western Blot.

RESULTS: Compared with the control group, MA-10 cells showed increased synthesis of testosterone when exposed to low concentrations of DBP and MBP (10(-9) ∼ 10(-6) mol/L) and inhibited synthesis of testosterone when exposed to high concentrations of DBP and MBP (10(-3) mol/L), and the typical two-way effects became more significant as the time went one and the concentrations increased (P < 0.05). Compared with the control group, MA-10 cells showed significantly lower mRNA and protein expression levels of INSL3 when exposed to 10(-6) and 10(-4) mol/L DBP (P < 0.05); MA-10 cells showed increased protein expression of INSL3 when exposed to 10(-7) mol/L MBP, and the mRNA and protein expression levels of INSL3 decreased as the concentration of MBP increased.

CONCLUSION: DBP and MBP can inhibit the secretion of testosterone from MA-10 cells at high concentrations, but stimulate the secretion of testosterone at low concentrations. Both DBP and MBP have inhibitory effects on the mRNA and protein expression of INSL3 in MA-10 cells.

Abstract  Environmental compounds are known to promote epigenetic transgenerational inheritance of adult onset disease in subsequent generations (F1-F3) following ancestral exposure during fetal gonadal sex determination. The current study was designed to determine if a mixture of plastic derived endocrine disruptor compounds bisphenol-A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and dibutyl phthalate (DBP) at two different doses promoted epigenetic transgenerational inheritance of adult onset disease and associated DNA methylation epimutations in sperm. Gestating F0 generation females were exposed to either the "plastics" or "lower dose plastics" mixture during embryonic days 8 to 14 of gonadal sex determination and the incidence of adult onset disease was evaluated in F1 and F3 generation rats. There were significant increases in the incidence of total disease/abnormalities in F1 and F3 generation male and female animals from plastics lineages. Pubertal abnormalities, testis disease, obesity, and ovarian disease (primary ovarian insufficiency and polycystic ovaries) were increased in the F3 generation animals. Kidney and prostate disease were only observed in the direct fetally exposed F1 generation plastic lineage animals. Analysis of the plastics lineage F3 generation sperm epigenome previously identified 197 differential DNA methylation regions (DMR) in gene promoters, termed epimutations. A number of these transgenerational DMR form a unique direct connection gene network and have previously been shown to correlate with the pathologies identified. Observations demonstrate that a mixture of plastic derived compounds, BPA and phthalates, can promote epigenetic transgenerational inheritance of adult onset disease. The sperm DMR provide potential epigenetic biomarkers for transgenerational disease and/or ancestral environmental exposures.