Specimen pooling for efficient use of biospecimens in studies of time to a common event.

For case-control studies that rely on expensive assays for biomarkers, specimen pooling offers a cost-effective and efficient way to estimate individual-level odds ratios. Pooling helps to conserve irreplaceable biospecimens for the future, mitigates limit-of-detection problems, and enables inclusion of individuals who have limited available volumes of biospecimen. Pooling can also allow the study of a panel of biomarkers under a fixed assay budget. Here, we extend this method for application to discrete-time survival studies. Assuming a proportional odds logistic model for risk of a common outcome, we propose a design strategy that forms pooling sets within those experiencing the outcome at the same event time. We show that the proposed design enables a cost-effective analysis to assess the association of a biomarker with the outcome. Because the standard likelihood is slightly misspecified for the proposed pooling strategy under a nonnull biomarker effect, the proposed approach produces slightly biased estimates of exposure odds ratios. We explore the extent of this bias via simulations and illustrate the method by revisiting a data set relating polychlorinated biphenyls and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene to time to pregnancy.

Differential effect of DDT, DDE, and DDD on COX-2 expression in the human trophoblast derived HTR-8/SVneo cells.

The purpose of this study was to investigate the effect of 1,1,1-trichloro-2,2-bis-(chlorophenyl)ethane (DDT), 1,1-bis-(chlorophenyl)-2,2-dichloroethene (DDE), and 1,1-dichloro-2,2-bis(chlorophenyl)ethane (DDD) isomers on COX-2 expression in a human trophoblast-derived cell line. Cultured HTR-8/SVneo trophoblast cells were exposed to DDT isomers and its metabolites for 24 h, and COX-2 mRNA and protein expression were assessed by RT-PCR, Western blotting, and ELISA. Prostaglandin E2 production was also measured by ELISA. Both COX-2 mRNA and protein were detected under control (unexposed) conditions in the HTR-8/SVneo cell line. COX-2 protein expression and prostaglandin E2 production but not COX-2 mRNA levels increased only after DDE and DDD isomers exposure. It is concluded that DDE and DDD exposure induce the expression of COX-2 protein, leading to increased prostaglandin E2 production. Interestingly, the regulation of COX-2 by these organochlorines pesticides appears to be at the translational level.

Determinants of serum concentrations of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene and polychlorinated biphenyls among French women in the CECILE study.

BACKGROUND: Dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCBs) are persistent organochlorine compounds bioaccumulating in human tissues. Body burden of organochlorines may be influenced by individual characteristics such as age, weight variations, breastfeeding, dietary habits and place of residence. OBJECTIVES: To assess the current serum concentrations of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE), the main DDT breakdown product, and of PCBs in women from two French administrative areas (Ille-et-Vilaine and Côte d'Or). To identify determinants of the current serum levels among individual characteristics related to intake, metabolism, and excretion of organochlorines. METHODS: We measured serum p,p'-DDE and PCB levels in 1055 general population women who were recruited in 2005-2007 to serve as controls in a case-control study on breast cancer. Associations between organochlorine levels and age, current body mass index (BMI), BMI change during the last 10 years, dietary habits, breastfeeding history, residence area and education were assessed in multivariate analyses. RESULTS: Median concentrations of p,p'-DDE and total PCBs were 85 and 240ng/g lipid, respectively. Based on multivariate analyses, the main predictors of high p,p'-DDE levels included age and frequent consumption of saltwater fish in women below 50 years, and high BMI in older women. Total PCB levels increased markedly with age. Among older women, other important predictors of high PCB levels included frequent consumption of saltwater fish and low BMI. Our results are also suggestive of an inverse association between PCB levels and BMI gain during the last ten years. Women in Côte d'Or had significantly higher PCB levels than women in Ille-et-Vilaine. CONCLUSION: The patterns of associations between determinants and serum organochlorine concentrations suggest that human PCB contamination is still ongoing in France. The most important predictors of serum p,p'-DDE and PCB concentrations among French women include age, body mass index, dietary habits, and place of residence.

Biphasic hormonal responses to the adrenocorticolytic DDT metabolite 3-methylsulfonyl-DDE in human cells.

The DDT metabolite 3-methylsulfonyl-DDE (3-MeSO(2)-DDE) has been proposed as a lead compound for an improved adrenocortical carcinoma (ACC) treatment. ACC is a rare malignant disorder with poor prognosis, and the current pharmacological therapy o,p'-DDD (mitotane) has limited efficacy and causes severe adverse effects. 3-MeSO(2)-DDE is bioactivated by cytochrome P450 (CYP) 11B1 in mice and causes formation of irreversibly bound protein adducts, reduced glucocorticoid secretion, and cell death in the adrenal cortex of several animal species. The present study was carried out to assess similarities and differences between mice and humans concerning the adrenocorticolytic effects of 3-MeSO(2)-DDE. The results support previous indications that humans are sensitive to the adrenocorticolytic actions of 3-MeSO(2)-DDE by demonstrating protein adduct formation and cytotoxicity in the human adrenocortical cell line H295R. However, neither the irreversible binding nor the cytotoxicity of 3-MeSO(2)-DDE in H295R cells was inhibited by the CYP11B1 inhibitor etomidate. We also report biphasic responses to 3-MeSO(2)-DDE in cortisol and aldosterone secretion as well as in mRNA levels of the steroidogenic genes StAR, CYP11B1 and CYP11B2. Hormone levels and mRNA levels were increased at lower concentrations of 3-MeSO(2)-DDE, while higher concentrations decreased hormone levels. These biphasic responses were not observed with o,p'-DDD or with the precursor DDT metabolite p,p'-DDE. Based on these results, 3-MeSO(2)-DDE remains a viable lead compound for drug design, although the adrenocorticolytic effects of 3-MeSO(2)-DDE in human cells seem more complex than in murine cells.

Prenatal exposure to the major DDT metabolite 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) and growth in boys from Mexico.

Recent data suggest that prenatal exposure to p,p'-DDE may reduce height and increase body mass index (BMI) in childhood, thus potentially raising the risk of adult health problems. The association between prenatal DDE exposure and growth was evaluated in 788 boys from Chiapas, an area of Mexico where DDT was recently used. The median DDE levels in maternal serum at birth (2002-2003) were 2.7 microg/g lipid. 2633 measurements of height (cm) and weight (kg) were obtained in 2004-2005. The median age of the children during follow-up was 18 months (quartiles 14 and 22 months). Height and body mass index (kg/m(2)) were age-standardized and expressed as standard deviation scores (SDS). Multivariate random-effect models for longitudinal data were fitted and predicted height and BMI SDS were estimated from the adjusted models. Overall, associations between prenatal DDE level and height or BMI SDS at any given age were not observed. For example, the predicted values showed that children with the highest exposure (DDE: >9.00 microg/g) compared to those least exposed (DDE: <3.01 microg/g) grew similarly and they had a BMI SDS similar to the referent group. The results do not support the prior findings of an association of DDE exposure with childhood height or BMI.

Modeling continuous auxiliary covariate data in generalized linear mixed models using the kernel smoother.

Auxiliary covariate data are often collected in biomedical studies when the primary exposure variable is only assessed on a subset of the study subjects. In this study, we investigate a semiparametric-estimated likelihood estimation for the generalized linear mixed models (GLMM) in the presence of a continuous auxiliary variable. We use a kernel smoother to handle continuous auxiliary data. The method can be used to deal with missing or mismeasured covariate data problems in a variety of applications when an auxiliary variable is available and cluster sizes are not too small. Simulation study results show that the proposed method performs better than that which ignores the random effects in GLMM and that which only uses data in the validation data set. We illustrate the proposed method with a real data set from a recent environmental epidemiology study on the maternal serum 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene level in relationship to preterm births.

Reductive dechlorination of DDE to DDMU in marine sediment microcosms.

DDT is reductively dechlorinated to DDD and dehydrochlorinated to DDE; it has been thought that DDE is not degraded further in the environment. Laboratory experiments with DDE-containing marine sediments showed that DDE is dechlorinated to DDMU in both methanogenic and sulfidogenic microcosms and that DDD is dehydrochlorinated to DDMU three orders of magnitude more slowly. Thus, DDD does not appear to be an important precursor of the DDMU found in these sediments. These results imply that remediation decisions and risk assessments based on the recalcitrance of DDE in marine and estuarine sediments should be reevaluated.

A simple and fast liquid-liquid extraction method for the determination of 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE) from human serum for epidemiological studies on type 2 diabetes.

A simple and fast method is presented to be used for example in studies on the relationship between serum levels of persistent organic pollutants and type 2 diabetes mellitus. Method is based on liquid-liquid extraction and gas chromatography coupled with high-resolution mass spectrometry. In the sample pre-treatment special attention was paid to minimize the number of sample manipulation steps and the amounts of organic solvents needed. Compounds analyzed were 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p'-DDE), the major metabolite of DDT. The method included extraction and cleanup of 0.2ml of serum in a single test tube and subsequent analysis of the extract from 0.2ml final volume. Validation was conducted to explore the performance of the method. The limits of detection for p,p'-DDE and PCB-153 based on the standard deviation of the blank samples were 4.3 and 3.1pg/ml, respectively. Repeatability was less than 2.5% at three concentration levels tested and recovery from Certified Reference Material SRM 1589a was 84% for p,p'-DDE and 87% for PCB-153 of the certified values, respectively. Serum samples from the AMAP intercalibration round 2008-2 were also analyzed, and results were 101-116% of the assigned values. The presented method was used for an epidemiological study with more than 700 serum samples from a type 2 diabetes cohort from Sweden.

Cytotoxicity and decreased corticosterone production in adrenocortical Y-1 cells by 3-methylsulfonyl-DDE and structurally related molecules.

The persistent environmental pollutant 3-methylsulfonyl-DDE (3-MeSO2-DDE) undergoes bioactivation by cytochrome P450 11B1 (CYP11B1) in the adrenal cortex of several animal species in vivo and causes decreased glucocorticoid production and cell death in the zona fasciculata. This study presents extended investigations of the cytotoxic and endocrine disrupting effects of 3-MeSO2-DDE and some structurally related molecules in the mouse adrenocortical cell line Y-1. Both 3-MeSO2-DDE and, to a lesser extent, 3,3'(bis)-MeSO2-DDE decreased corticosterone production and produced CYP11B1-dependent cytotoxicity in Y-1 cells. Neither 2-MeSO2-DDE nor p,p'-DDE had any significant effect on either cell viability or corticosterone production, indicating that the presence and position of the methylsulfonyl moiety of 3-MeSO2-DDE is crucial for its biological activity. The adrenocortical toxicant o,p'-DDD decreased corticosterone production but was not cytotoxic in this cell line. None of the compounds altered Cyp11b1 gene expression, indicating that 3-MeSO2-DDE inhibits CYP11B1 activity on the protein level.

Pharmacokinetics of the adrenocorticolytic compounds 3-methylsulphonyl-DDE and o,p'-DDD (mitotane) in Minipigs.

The pharmacokinetics of the adrenocorticolytic drug candidate 3-Methylsulphonyl-DDE (3-MeSO2-DDE) and the anticancer drug o,p'-DDD (mitotane) were studied in Göttingen minipigs. The animals were given 3-MeSO2-DDE or o,p'-DDD as single oral doses (30 mg/kg). Concentrations in plasma and subcutaneous fat were measured by gas chromatography at different time points during 180 days. Maximal plasma concentrations appeared within 24 h for both compounds, but were about 2 times higher for 3-MeSO2DDE. o,p'-DDD plasma concentrations declined rapidly to low levels during 4 days. 3-MeSO2-DDE also decreased rapidly, but remained at high concentrations throughout the study. In fat, 3-MeSO2-DDE reached about 25-fold higher levels than o,p'-DDD at 30 days, and both substances were eliminated slowly from this tissue. 3-MeSO2-DDE liver concentrations were about 18-fold higher than those in plasma at 180 days. In contrast, o,p'-DDD liver and plasma levels were about equal at 180 days. o,p'-DDD had roughly 45 times larger CL/F than 3-MeSO2-DDE, confirming that the elimination of this compound was more rapid. Both compounds were characterised by their localisation and retention in fat tissue, and the individual size of the fat stores clearly determined the plasma concentrations. It is concluded that although 3-MeSO2-DDE is an interesting candidate for therapeutic use due to its potential characteristics to specifically target adrenocortical tumour cells the slow elimination of the compound might make it challenging to design appropriate dosage regimes.

Bioavailability to grains of rice of aged and fresh DDD and DDE in soils.

DDT had been widely used around the world before 1980s and is still under production and use for non-agricultural purposes in China. Because of their special physicochemical properties, p,p'-DDT and its main metabolites, p,p'-DDD and p,p'-DDE, accumulated and persisted in the environment, presenting potential menace on biota. A green-house study was conducted to determine the bioavailability of p,p'-DDD and p,p'-DDE to grains of rice and the influences of traditional Chinese farming practices on their bioaccumulation. Paddy rice and dry rice were grown in submerged paddy soils and non-submerged upland soils, respectively. Two types of soil, Hydragric Anthrosols (An) and Hydragric Acrisols (Ac), were employed. Bioaccumulation factors (BAFs) of DDE ranged from 0.67 for rice grown in non-submerged An to 0.84 in submerged An in the control group, whilst BAFs were all below 0.04 in experimental groups. BAFs of DDD varied from 1.39 for submerged An to 2.26 for submerged Ac in original soils. In contrast, BAFs were between 0.05 for non-submerged Ac and 0.08 for submerged An in DDD-contaminated soils. Flooding seemed to have two contradictory effects on the DDE/DDD accumulation by rice: on one hand, it made the pollutants more mobile and bioavailable; while on the other hand, it enhanced the degradation and binding of POPs. Adding rice straw to the soils protected DDE from being taken up yet promoted DDD accumulation by rice. Furthermore, the distinct inorganic component of the soils might also play an important role in the environmental activities of POPs.

Temporal trend of bis(4-chlorophenyl) sulfone, methylsulfonyl-DDE and -PCBs in Baltic guillemot (Uria aalge) egg 1971-2001--a comparison to 4,4'-DDE and PCB trends.

The dynamics of organohalogen contaminants and their metabolites are best studied over time by analysis of biota at high trophic levels. In this study, time trends, 1971-2001, of bis(4-chlorophenyl) sulfone (BCPS) and of methylsulfonyl-substituted metabolites of PCBs and 4,4'-DDE, were investigated in eggs of guillemot (Uria aalge) hatching in the Baltic Proper. Temporal trends of PCBs, trans-nonachlor, beta-HCH, 4,4'-DDT, and 4,4'-DDE were also assessed. Tris(4-chlorophenyl) methane (TCPMe), a 4,4'-DDT by-product, was detected in the eggs. The concentration of BCPS ranged between 2.6-0.76 microg/g on a lipid weight basis over the three decades and showed a significant 1.6% annual decrease. Three metabolites of PCBs, i.e. 3'-MeSO2-CB101, 4'-MeSO2-CB101 and 4-MeSO2-CB149, were quantified in all samples over time and showed an annual decrease of approximately 3% compared to MeSO2-DDE with a decrease of 8.9%. The methylsulfonyl-PCB and -DDE metabolites are eliminated more slowly than the persistent PCB congeners and 4,4'-DDE. Trans-nonachlor decreases by 16% compared to 19% and 9% for 4,4'-DDT and beta-HCH, respectively. The concentration of TCPMe in guillemot decreased by 8.2% per year. A linear relationship was found between TCPMe and 4,4'-DDE concentrations which supports the theory that TCPMe has an origin as a contaminant in commercial 4,4'-DDT products. The very slow decrease in BCPS concentrations is notable and remains to be explained. BCPS is still present at rather high concentrations in the guillemot eggs. The enantiomeric fraction varied between 0.27 and 0.67 which indicates less of a specific retention of the chiral MeSO2-PCBs in guillemot eggs than in grey seal tissues, for example. Independent of meta- or para-substitution of the sulfone group, the most accumulative atropisomer of each of four MeSO2-PCB pairs has been assigned an absolute R structure.

Abiotic transformation of DDT in aqueous solutions.

Significant concentrations of chlorinated pesticides such as 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) and its two main transformation products, 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD) and 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) are still present in soil and sediment systems more than 30 years after DDT use was banned in the United States. DDT enters waterways via the runoff from industrial point sources, agricultural lands and atmospheric deposition. We evaluated zero-valent iron (Fe(0)), ferrous sulfide (FeS), as well as combining them with hydrogen peroxide (H(2)O(2)) as viable treatment technologies for degrading DDT in an aqueous solution. Treatment of DDT with Fe(0) and FeS resulted in approximately 88% and 56% transformation of DDT within 150h, respectively. DDE production was insignificant in all systems. The DDT removal was slower with FeS than with Fe(0), but the amounts of DDD and DDE produced did not exceed baseline. Treatment with a 1:1 mixture of Fe(0)-FeS removed about 95% of the added mass of DDT within 4days and generated significant amounts of DDD and minor amounts of DDMU. When small amounts of H(2)O(2) were introduced halfway through the Fe(0) and FeS treatment times, the mass of DDT decreased by 87% and 96%, respectively, within 2days. Our results demonstrate that mixtures of Fe(0)-FeS in combination with H(2)O(2) can be used for rapid and efficient removal of DDT from aqueous solutions.

The persistent DDT metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene, alters thyroid hormone-dependent genes, hepatic cytochrome P4503A, and pregnane X receptor gene expressions in Atlantic salmon (Salmo salar) Parr.

The present study investigated the effects of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) on the thyroid and steroid-metabolizing system in Atlantic salmon (Salmo salar) parr. Fish were exposed to waterborne DDE and thyroxine (T4), both singly and in combination, for 5 d. Thyroid-stimulating hormone (TSHbeta), T4 deiodinase (T4ORD), thyroid receptors (TRalpha and TRbeta), and insulin-like growth factor type 1 receptor (IGF-1R) were analyzed using quantitative (real-time) polymerase chain reaction in liver, brain, and kidney, whereas cytochrome P4503A (CYP3A) and pregnane X receptor (PXR) mRNA levels were analyzed only in the liver. Exposure to DDE and T4, both singly and in combination, inhibited TSHbeta expression in the brain. The DDE induced TSHbeta in the liver, and T4 inhibited TSHbeta in the liver and kidney, both singly and in combination with DDE. The DDT-metabolite DDE induced T4ORD expression in the kidney and liver, and combined exposure with T4 inhibited T4ORD expression in the brain, kidney, and liver. The IGF-1R and TRalpha expressions were induced by DDE and T4 singly in the brain, whereas combined exposure with both compounds did not affect IGF-1R and TRd transcript levels. Whereas T4 inhibited TRbeta expression in the liver, exposure to DDE, both singly and in combination with T4, induced TRbeta transcript levels in the liver. Exposure to T4 and DDE, both singly and in combination, resulted in a parallel pattern of CYP3A and PXR mRNA induction in the liver. These results indicate that DDE alters thyroid hormone-dependent genes and hepatic CYP3A and PXR levels. The hepatic modulation of CYP3A and PXR transcript levels by DDE represents a novel aspect of DDE toxicity that, to our knowledge, has not been demonstrated previously in fish. Therefore, the present study demonstrates some possible physiological and endocrine consequences from exposure to endocrine-disrupting chemicals for salmon parr during smoltification.

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene and polychlorinated biphenyls and breast cancer: combined analysis of five U.S. studies.

BACKGROUND: Environmental exposure to organochlorines has been examined as a potential risk factor for breast cancer. In 1993, five large U.S. studies of women located mainly in the northeastern United States were funded to evaluate the association of levels of 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (DDE) and polychlorinated biphenyls (PCBs) in blood plasma or serum with breast cancer risk. We present a combined analysis of these results to increase precision and to maximize statistical power to detect effect modification by other breast cancer risk factors. METHODS: We reanalyzed the data from these five studies, consisting of 1400 case patients with breast cancer and 1642 control subjects, by use of a standardized approach to control for confounding and assess effect modification. We calculated pooled odds ratios (ORs) and 95% confidence intervals (CIs) by use of the random-effects model. All statistical tests were two-sided. RESULTS: When we compared women in the fifth quintile of lipid-adjusted values with those in the first quintile, the multivariate pooled OR for breast cancer associated with PCBs was 0.94 (95% CI = 0.73 to 1.21), and that associated with DDE was 0.99 (95% CI = 0.77 to 1.27). Although in the original studies there were suggestions of elevated breast cancer risk associated with PCBs in certain groups of women stratified by parity and lactation, these observations were not evident in the pooled analysis. No statistically significant associations were observed in any other stratified analyses, except for an increased risk with higher levels of PCBs among women in the middle tertile of body mass index (25-29.9 kg/m(2)); however, the risk was statistically nonsignificantly decreased among heavier women. CONCLUSIONS: Combined evidence does not support an association of breast cancer risk with plasma/serum concentrations of PCBs or DDE. Exposure to these compounds, as measured in adult women, is unlikely to explain the high rates of breast cancer experienced in the northeastern United States.

Metabolism of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane, and 1-chloro-2,2-bis(p-chlorophenyl)ethene in the hamster.

The urinary metabolites of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), and 1-chloro-2,2-bis(p-chlorophenyl)ethene in female hamsters are reported. The principal metabolite of both DDT and DDD is 2,2-bis(p-chlorophenyl) acetic acid. DDT- and DDD-treated animals also excreted small amounts of DDD, 1-chloro-2,2-bis(p-chlorophenyl)ethene, 1,1-dichloro-2,2-bis-(p-chlorophenyl)ethene, 2-hydroxy-2,2-bis(p-chlorophenyl)acetic acid, and 2,2-bis(p-chlorophenyl)ethanol. 1-Chloro-2,2-bis(p-chlorophenyl)ethene is metabolized to afford significant amounts of 2,2-bis(p-chlorophenyl)acetic acid, 2,2-bis(p-chlorophenyl)-ethanol, 2-hydroxy-2,2-bis(p-chlorophenyl)acetic acid, 2,2-bis-(p-chlorophenyl)acetaldehyde, and 1,1-bis(p-chlorophenyl) ethan-1,2-diol. These results indicate that the metabolic disposition of DDT in the hamster, a species refractory to DDT tumorigenicity, is very similar to that observed previously in the mouse, a species sensitive to DDT tumorigenicity. The one exception is that the hamster is not nearly as efficient as the mouse in converting DDT to 1,1-dichloro-2,2-bis(p-chlorophenyl)ethene, a metabolite that is tumorigenic in both species.

Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE.

Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO2-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO2-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO2-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO2-DDE (10 µM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO2-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO2-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO2-DDE exposure, but also display small number of proteins shared between culture conditions, suggesting the action of 3-MeSO2-DDE on several targeted pathways, including mitochondrial dysfunction, oxidative phosphorylation, EIF2-signaling, and glutathione-mediated detoxification. Further identification and characterization of these proteins and pathways may build our understanding to the molecular basis of 3-MeSO2-DDE induced endocrine disruption in Leydig cells.

Effects of perinatal combined exposure to 1,4-dichlorobenzene and 1,1-dichloro-2, 2-bis (p-chlorophenyl) ethylene on rat male offspring.

1,4-Dichlorobenzene (DCB) is used as an air deodorant or a moth repellent and 1, 1-dichloro-2, 2-bis (p-chlorophenyl) ethylene (p,p'-DDE) is a persistent metabolite of 1, 1, 1-trichloro-2, 2-bis (p-chlorophenyl) ethane (DDT) which was used as a pesticide before. DCB concentrations of residential air and oral p,p'-DDE intake through marine products are demonstrated to be very high in Japan and consequently, foetuses and neonates may be exposed much more to DCB and/or p,p'-DDE via the maternal body. It has recently been reported that DCB is oestrogenic and that p,p'-DDE is antiandrogenic. Therefore, the combined effects of perinatal exposure to DCB and p,p'-DDE have been investigated in rat male offspring of dams ingesting these contaminants during the perinatal period from gestational day 1 to postpartum day 21 for 42 days. In this study, no obvious developmental effects on male offspring have been recognized until 6 weeks of age, following oral administration of 25 ppm DCB (approximately 2 mg/kg) and/or 125 ppm p,p'-DDE (approximately 10 mg/kg) to dams. In contrast to female offspring, the thymus weight in male offspring was not affected by DCB at 6 weeks of age, but there might be sexual differences concerning the effects of DCB on the thymus.

Cytochrome P450-catalyzed binding of 3-methylsulfonyl-DDE and o,p'-DDD in human adrenal zona fasciculata/reticularis.

3-Methylsulfonyl-2,2'-bis(4-chlorophenyl)-1,1'-dichloroethene (MeSO(2)-DDE) is a potent, tissue-specific toxicant that induces necrosis of the adrenal zona fasciculata following a local CYP11B1-catalyzed activation to a reactive intermediate in mice. Autoradiography was used to examine CYP11B1-catalyzed binding of MeSO(2)-[(14)C]DDE and the adrenocorticolytic drug 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichlorethane; (o,p'-[(14)C]DDD, Mitotane, Lysodren) in human adrenal tissue slice culture. Both compounds gave rise to a selective binding in the one sample of normal adrenal zona fasciculata/reticularis, leaving zona glomerulosa and the adrenal medulla devoid of binding. Addition of the CYP11B1 selective inhibitor metyrapone (50 microM) reduced MeSO(2)-[(14)C]DDE binding below the detection limit, whereas o,p'-[(14)C]DDD binding was reduced only by 42%. Selective binding of MeSO(2)-[(14)C]DDE and o,p'-[(14)C]DDD was also observed in an aldosterone-producing adrenocortical carcinoma and in a nonfunctional adrenocortical hyperplasia. Exposure of slices from the normal adrenal cortex to MeSO(2)-DDE (25 microM) resulted in an increased accumulation of 11-deoxycorticosterone, 11-deoxycortisol and androstenedione in the medium, and exposure to o,p'-DDD (25 microM) did not alter the steroid secretion pattern. No histological changes were found in either MeSO(2)-DDE- or o,p'-DDD-exposed slices, compared with nonexposed slices. We suggest that MeSO(2)-DDE might act as a potent adrenocorticolytic agent in humans. Further studies are needed to establish the usefulness of MeSO(2)-DDE as a possible alternative for the treatment of adrenocortical hypersecretion and tumor growth.

A mechanistic study of the metabolism of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD) to 2,2-bis(p-chlorophenyl)acetic acid (DDA).

The metabolism of [1-2H]-1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD-d) and [1-2H]-1-chloro-2,2-bis(p-chlorophenyl)ethene (DDMU-d) and their corresponding non-deuterated isomers DDD and DDMU was studied in female Swiss mice over a 96-hr period. The only detected urinary metabolite of DDD-d and DDD was 2,2-bis(p-chlorophenyl)acetic acid (DDA). Animals administered DDD excreted approximately 2.4-fold more DDA than those treated with DDD-d over the total collection period. The initial (0-36 hr) linear excretion rates of DDA for DDD and DDD-d were 17.1 and 5.5 micrograms/hr respectively. DDMU- and DDMU-d-treated mice excreted significant quantities of DDA, 2,2-bis(p-chlorophenyl)ethanol (DDOH) and 2,2-bis(p-chlorophenyl)ethanol (DDCHO). The only quantitative difference between DDMU and DDMU-d was that the non-deuterated isomer afforded approximately 1.8 times more DDA over the 96-hr collection. The initial (0-36 hr) linear excretion rates of DDMU and DDMU-d were 10.7 and 6.2 micrograms/hr respectively. The qualitative and quantitative results are consistent with DDD being metabolized to DDA via enzyme-mediated hydroxylation on the C-1 side chain carbon.