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.

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.

Toxicity, dioxin-like activities, and endocrine effects of DDT metabolites--DDA, DDMU, DDMS, and DDCN.

BACKGROUND, AIM, AND SCOPE: 2,2-bis(chlorophenyl)-1,1,1-trichloroethane (DDT) metabolites, other than those routinely measured [i.e., 2,2-bis(chlorophenyl)-1,1-dichloroethylene (DDE) and 2,2-bis(chlorophenyl)-1,1-dichloroethane (DDD)], have recently been detected in elevated concentrations not only in the surface water of Teltow Canal, Berlin, but also in sediment samples from Elbe tributaries (e.g., Mulde and Havel/Spree). This was paralleled by recent reports that multiple other metabolites could emerge from the degradation of parent DDT by naturally occurring organisms or by interaction with some heavy metals. Nevertheless, only very few data on the biological activities of these metabolites are available to date. The objective of this communication is to evaluate, for the first time, the cytotoxicity, dioxin-like activity, and estrogenicity of the least-studied DDT metabolites. METHODS: Four DDT metabolites, p,p'-2,2-bis(chlorophenyl)-1-chloroethylene (DDMU), p,p'-2,2-bis(chlorophenyl)-1-chloroethane (DDMS), p,p'-2,2-bis(4-ch1oropheny1)acetonitrile (DDCN), and p,p'-2,2-bis(chlorophenyl)acetic acid (DDA), were selected based on their presence in environmental samples in Germany such as in sediments from the Mulde River and Teltow Canal. O,p'-DDT was used as reference in all assays. Cytotoxicity was measured by neutral red retention with the permanent cell line RTG-2 of rainbow trout (Oncorhynchus mykiss). Dioxin-like activity was determined using the 7-ethoxyresorufin-O-deetylase assay. The estrogenic potential was tested in a dot blot/RNAse protection-assay with primary hepatocytes from male rainbow trout (O. mykiss) and in a yeast estrogen screen (YES) assay. RESULTS: All DDT metabolites tested revealed a clear dose-response relationship for cytotoxicity in RTG-2 cells, but no dioxin-like activities with RTL-W1 cells. The dot blot/RNAse protection-assay demonstrated that the highest non-toxic concentrations of these DDT metabolites (50 µM) had vitellogenin-induction potentials comparable to the positive control (1 nM 17ß-estradiol). The estrogenic activities could be ranked as o,p'-DDT > p,p'-DDMS > p,p'-DDMU > p,p'-DDCN. In contrast, p,p'-DDA showed a moderate anti-estrogenic effect. In the YES assay, besides the reference o,p'-DDT, p,p'-DDMS and p,p'-DDMU displayed dose-dependent estrogenic potentials, whereas p,p'-DDCN and p,p'-DDA did not show any estrogenic potential. DISCUSSION: The reference toxicant o,p'-DDT displayed a similar spectrum of estrogenic activities similar to 17ß-estradiol, however, with a lower potency. Both p,p'-DDMS and p,p'-DDMU were also shown to have dose-dependent estrogenic potentials, which were much lower than the reference o,p'-DDT, in both the vitellogenin and YES bioassays. Interestingly, p,p'-DDA did not show estrogenic activity but rather displayed a tendency towards anti-estrogenic activity by inhibiting the estrogenic effect of 17ß-estradiol. The results also showed that the p,p'-metabolites DDMU, DDMS, DDCN, and DDA do not show any dioxin-like activities in RTL-W1 cells, thus resembling the major DDT metabolites DDD and DDE. CONCLUSIONS: All the DDT metabolites tested did not exhibit dioxin-like activities in RTL-W1 cells, but show cytotoxic and estrogenic activities. Based on the results of the in vitro assays used in our study and on the reported concentrations of DDT metabolites in contaminated sediments, such substances could, in the future, pose interference with the normal reproductive and endocrine functions in various organisms exposed to these chemicals. Consequently, there is an urgent need to examine more comprehensively the risk of environmental concentrations of the investigated DDT metabolites using in vivo studies. However, this should be paralleled also by periodic evaluation and monitoring of the current levels of the DDT metabolites in environmental matrices. RECOMMENDATIONS AND PERSPECTIVES: Our results clearly point out the need to integrate the potential ecotoxicological risks associated with the "neglected" p,p'-DDT metabolites. For instance, these DDT metabolites should be integrated into sediment risk assessment initiatives in contaminated areas. One major challenge would be the identification of baseline data for such risk assessment. Further studies are also warranted to determine possible additive, synergistic, or antagonistic effects that may interfere with the fundamental cytotoxicity and endocrine activities of these metabolites. For a more conclusive assessment of the spectrum of DDT metabolites, additional bioassays are needed to identify potential anti-estrogenic, androgenic, and/or anti-androgenic effects.

Levels of DDT and its metabolites in placenta, maternal and cord blood and their potential influence on neonatal anthropometric measures.

Previous studies of in utero exposure to dichlorodiphenyltrichloroethane (DDT) have shown mixed results for the harmful effects on fetal growth and development. This cross-sectional study was designed to: (1) examine the extent of DDT exposure in 1578 women, aged 28.5±6.0 who delivered between June 2005 and 2006 in the area of Al-Kharj, Saudi Arabia; and (2) assess its influence on neonatal anthropometric measurement of newly born babies. DDT and its metabolites, namely 1,1-dichloro-2,2-bis (p-chlorophenyl) ethylene (p,p'-DDE), 1,1-dichloro-2,2-bis (p-chlorophenyl) ethane (p,p'-DDD) and 1,1,1-trichloro-2,2' bis (p-chlorophenyl) ethane (p,p'-DDT) were measured in cord and maternal serum as well as placenta by Gas Chromatography coupled with an Electron Capture Detector (GC/ECD). p,p'-DDE was detected in 28.3% of cord and 54.4% of maternal serum, reflecting past exposure, whereas the p,p'-DDT was only found in 0.46% cord and 1.2% maternal samples. As expected the p,p'-DDE cord levels (0.197±0.961µg/L) were 2.8 times lower than the maternal levels (0.551±1.778µg/L), and both were significantly correlated (r=0.517) suggesting its transplacental transfer. The picture was different in placental tissues. p,p'-DDE and p,p'-DDT were detected in 84% and 99% of placental tissues, with the highest p,p'-DDT in placental tissues (29.62±158.282µg/kg dry wt.) compare to p,p'-DDE (10.167±18.851µg/kg dry wt.). In general, the presence of DDT metabolites in our participants indicates that these chemicals are still relevant despite the fact that they have been banned or restricted in the study area for many years. Our results support the view for an association between low in utero exposure to DDT and the anthropometric development of the fetus leading to a reduction in its head circumference, crown-heel length, birth weight and birth height. Since the reduction in these measures was independent of gestational age and/or preterm births, our findings suggest a detrimental effect of the DDT exposure on fetal growth. Neonatal anthropometric measures are considered as an important tool to detect newborns at higher risk of morbidity and impairment of growth. Efforts should be made to decrease exposure of women of reproductive age and to examine maternal DDT exposure in relation to long-term impact on health.

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.

Persistent halogenated compounds in aquaculture environments of South China: implications for global consumers' health risk via fish consumption.

This study examined the potential sources of persistent halogenated compounds (PHCs), including organochlorine pesticides, mainly DDXs (sum of o,p'- and p,p'-DDT, -DDD, and -DDE and p,p'-DDMU) and polybrominated diphenyl ethers, to typical aquaculture environments of South China, determined the relative importance of gill diffusion and fish feeding for exposure of fish to these contaminants and assessed potential health risk for global consumers via consumption of fish from South China. Fish feed is generally a direct and important source of PHCs in both freshwater and seawater aquaculture. In addition, gill diffusion is the predominant uptake route for PHCs (except p,p'-DDMU, o,p'-DDD and -DDT) in farmed freshwater fish, whereas accumulation from the diet is the major route for farmed marine fish. Risks to health of global consumers via consumption of fish from South China are minimal. However, increased risk can be foreseen due to continuous use of brominated fire retardants and electronic waste importation to China.

Input pathways of organochlorine pesticides to typical freshwater cultured fish ponds of South China: hints for pollution control.

Air, rain, pond water, bank soil, pond sediment, fish feed, and fish were sampled from four freshwater cultured fish ponds (FWCFPs) in rural areas within the Pearl River Delta (PRD) of South China. Compositional analyses indicated that historical residues were the main sources of DDXs (defined as the sum of dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethane (DDD), and dichlorodiphenyldichloroethylene (DDE) and 1-chloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDMU)), and hexachlorocyclohexanes (HCHs) in the FWCFPs. The input fluxes to the FWCFPs were estimated at 4.0, 1.6, 15, and -0.92 µg/m(2) ·year for DDXs and 3.8, 0.92, 2.9, and -1.4 µg/m(2) ·year for HCHs for dry deposition, wet deposition, feeding, and net air-water exchange in Dongguan, and 3.8, 1.2, 137, and -1.2 µg/m(2) ·year for DDXs and 3.6, 0.66, 5.0, and -1.0 µg/m(2) ·year for HCHs in Shunde, respectively. These results indicated that fish feed was the dominant input source of DDXs to the FWCFPs. As for HCHs, fluxes via dry deposition and feeding were similar and slightly higher than those via wet deposition. Biological effects due to the occurrence of DDXs in the FWCFPs were minimal, and consumption of freshwater fish from the PRD appeared to pose insignificant risk to human health based on some existing regulations and guidelines.

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.

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.

Occurrence and fate of 1-chloro-2,2-bis(4-chlorophenyl)ethene in the environment of the Pearl River Delta, South China.

1-Chloro-2,2-bis(4-chlorophenyl)ethene (p,p'-DDMU), a metabolite of either 1,1-dichloro-22-bis(4-chlorophenyl)ethane (p,p'-DDD) or 1,1-dichloro-2,2-bis(4-chlorophenyl)ethene (p,p'-DDE), which is degraded from 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDT), was detected in a variety of environmental matrices of the Pearl River Delta (PRD), South China, including fish, fish feeds, semidigested fish stomach contents, marine surface sediment, surface soil, atmospheric gaseous phase, and particulates, rainwater, and coastal rainwater. Forfish species,the concentrations of p,p'-DDMU were significantly higher in farmed fish than in marine wild fish, with the highest value obtained in seawater farmed fish species (mear/median values of 262/173 ng/g lipid). The relative abundance of p,p'-DDMU to total DDTs (sum of o,p'- and p,p'-DDT, DDD, and DDE) was higher in samples of marine origin (approximately 5%) than in those of terrestrial origin (approximately 2%). Because p,p'-DDD was considerably abundant in all samples and a negative linear correlation was found between p,p'-DDD/(p,p'-DDD + p,p'-DDE) and p,p'-DDMU/DDTs in the marine sediments (r2 = 0.73) and seawater farmed fish (r2 = 0.29) under investigation, it is possible that DDMU was partially dehydrochlorinated from DDD in the environment of the PRD. A fugacity-based assessment suggested that p,p'-DDMU is likely to transport from sediment to seawater and then to air and from soilto air. The ubiquity of p,p'-DDMU in the PRD indicates that it may also be widespread worldwide particularly in countries with large amounts of DDT used currently and/or historically. Given the potential risk of p,p'-DDMU to human health, more efforts should be directed toward to this previously overlooked contaminant

Effects of maternal exposure to di-isononylphthalate (DINP) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) on steroidogenesis in the fetal rat testis and adrenal gland.

Exposure to antiandrogens during the critical developmental window (i.e. sexual differentiation) can permanently demasculinize the male phenotype. Here we have investigated the effects of developmental exposure to di-isononylphthalate (DINP) (250 and 750 mg/kg) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) (50 and 100mg/kg) on 19.5-day-old fetal Sprague-Dawley rat testicular and adrenal steroidogenesis. Maternal exposure to DINP or p,p'-DDE on embryonic days (EDs) 13.5-17.5 did not down-regulate the activity of steroidogenesis in ED 19.5 male rat fetus. Protein expression levels of testicular and adrenal StAR, P450scc, 3beta-HSD and androgen receptor (AR) did not show any changes. However, p,p'-DDE caused clear abnormalities in the ultrastructure of steroidogenic cells in ED 19.5 rat testis and adrenal. These structural alterations can disturb the development and function of fetal testis and adrenal that may become evident later in life.

Synthesis of 2-(4-chlorophenyl)-2-(4-chloro-3-thiophenol)-1,1-dichloroethene (3-SH-DDE) via Newman-Kwart rearrangement - a precursor for synthesis of radiolabeled and unlabeled alkylsulfonyl-DDEs.

For the first time, a pathway for synthesis of 2-(4-chlorophenyl)-2-(4-chloro-3-thiophenol)-1,1-dichloroethene (3-SH-DDE), is presented. The compound is of particular interest as a precursor for synthesis of alkylsulfonyl-DDE containing different alkyl groups to discover structural activity relationships, and to promote synthesis of radiolabeled methylsulfonyl-DDE. 2-Chloro-5-methylphenol was first methylated and further oxidized to the corresponding benzoic acid. The acid was reduced to the corresponding aldehyde (4-chloro-3-methoxy benzaldehyde) via 4-chloro-3-methoxy-benzene methanol. A lead/aluminium bimetal system was used to carry out the reductive addition of tetrachloromethane to 4-chloro-3-methoxy benzaldehyde to obtain 2,2,2-trichloro-1-(4-chloro-3-methoxyphenyl)ethanol, the desired starting material to synthesize the DDT-analogue (2-(4-chlorophenyl)-2-(4-chloro-3-methoxy-phenyl)-1,1,1-trichloroethane). Elimination of hydrochloric acid and removal of the methyl group led to the 3-OH-DDE. The Newman-Kwart rearrangement was applied to convert 3-OH-DDE to 3-SH-DDE via the dimethylcarbamothioate derivative. 3-SH-DDE is then used as a precursor for the radiolabel synthesis. The overall yield to acquire 3-SH-DDE after 11 steps was 3%. The step with the lowest yield was the DDT-analog synthesis with a yield of 30%. All other step had a yield of >50%. 3-SH-DDE was methylated with (14)C-labeled iodomethane and oxidized by hydrogen peroxide to obtain 3-[(14)C]MeSO(2)-DDE in an overall yield of 30%.

Detection of DDT and its metabolites in two estuaries of South China using a SPME-based device: first report of p,p'-DDMU in water column.

A solid-phase microextration-based sampling method was employed to determine the concentrations of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and its metabolites, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethene (DDE) and 1-chloro-2,2-bis(p-chlorophenyl)ethene (DDMU), in two estuarine bays, Daya Bay and Hailing Bay, of South China. Six DDT components including p,p'-DDT, o,p'-DDD, p,p'-DDD, o,p'-DDE, p,p'-DDE, and p,p'-DDMU were detected in Hailing Bay, while only p,p'-DDD was found in Daya Bay. p,p'-DDD was the most abundant DDT component in both bays, sharply different from the previous finding in the water column of the Palos Verdes Shelf, California, USA that p,p'-DDE was prevalent. In addition, the occurrence of p,p'-DDMU (with a range of 0.047-0.21 ng/L in Hailing Bay) has not been reported around the globe, and its presence in our study region appeared to stem from dehydrochlorination of p,p'-DDD, favored under aerobic conditions, but further investigations are clearly needed to confirm the mechanism for generation of DDMU in estuarine environments.

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.

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.

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

Prenatal or early postnatal exposure to some synthetic chemicals may affect the later reproductive system of the offspring. 1,4-Dichlorobenzene (DCB) is used as an air freshener and 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 that was used as a pesticide before. DCB concentrations of residential air and oral p,p'-DDE intake through marine products are demonstrated to be rather high in Japan. Such situations lead to high body burden of these pollutants in pregnant women. Consequently, foetuses and neonates will be exposed much more to DCB and p,p'-DDE via the mother. Therefore, the effects of the perinatal, combined exposure to DCB and p,p'-DDE on the female reproductive system have been investigated in mature rat female offspring of dams ingesting 25 p.p.m. DCB (approximately 2 mg/kg) and 125 p.p.m. p,p'-DDE (approximately 10 mg/kg) during the gestational and lactational period. Sexual maturation was fully developed in the rat female offspring perinatally exposed to DCB and/or p,p'-DDE through maternal exposure. The combined effect of DCB and p,p'-DDE was observed, and the ovarian weight was seen to decrease to approximately 80% of the control rat in matured female offspring following perinatal exposure to DCB and p,p'-DDE. This alteration might lead to reproductive dysfunction in matured female offspring. However, biological relevance of the alteration in the ovary remained uncertain in the present study. Further investigations concerning the reproductive function and mechanistic implication are required for elucidating the combined effects of perinatal exposure to DCB and p,p'-DDE on the later female reproductive system entirely.

Species differences in 3-methylsulphonyl-DDE bioactivation by adrenocortical tissue.

The CYP11B1-activated adrenocortical toxicant 3-methylsulphonyl-DDE (3-MeSO2-DDE) is proposed as a lead compound for an improved chemotherapy for adrenocortical carcinoma. We compared the binding of 3-MeSO2-[14C]DDE in the adrenal cortex of four rodent species; hamster, guinea pig, mouse and rat, using a precision-cut adrenal slice culture system ex vivo. Localization and quantification of the bound radioactivity were carried out using light microscopy autoradiography and radioluminography. The results revealed major species differences since 3-MeSO2-[14C]DDE was extensively bound to the hamster adrenal tissue while the guinea pig adrenals were devoid of binding. A high binding in mouse adrenal cortex was confirmed while binding in rat adrenal cortex was very weak. The results support previous observations that metabolic activation of 3-MeSO2-DDE is highly species dependent. Since CYP11B1 could be expressed in tissues other than the adrenal cortex, final toxicological characterization should be carried out in a species that can bioactivate this compound.

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.