Persistent organic pollutants dysregulate energy homeostasis in human ovaries in vitro.

Exposure to persistent organic pollutants (POPs), such as dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCBs), has historically been linked to population collapses in wildlife. Despite international regulations, these legacy chemicals are still currently detected in women of reproductive age, and their levels correlate with reduced ovarian reserve, longer time-to-pregnancy, and higher risk of infertility. However, the specific modes of action underlying these associations remain unclear. Here, we examined the effects of five commonly occurring POPs - hexachlorobenzene (HCB), p,p'-dichlorodiphenyldichloroethylene (DDE), 2,3,3',4,4',5-hexachlorobiphenyl (PCB156), 2,2',3,4,4',5,5'-heptachlorobiphenyl (PCB180), perfluorooctane sulfonate (PFOS) - and their mixture on human ovaries in vitro. We exposed human ovarian cancer cell lines COV434, KGN, and PA1 as well as primary ovarian cells for 24 h, and ovarian tissue containing unilaminar follicles for 6 days. RNA-sequencing of samples exposed to concentrations covering epidemiologically relevant levels revealed significant gene expression changes related to central energy metabolism in the exposed cells, indicating glycolysis, oxidative phosphorylation, fatty acid metabolism, and reactive oxygen species as potential shared targets of POP exposures in ovarian cells. Alpha-enolase (ENO1), lactate dehydrogenase A (LDHA), cytochrome C oxidase subunit 4I1 (COX4I1), ATP synthase F1 subunit alpha (ATP5A), and glutathione peroxidase 4 (GPX4) were validated as targets through qPCR in additional cell culture experiments in KGN. In ovarian tissue cultures, we observed significant effects of exposure on follicle growth and atresia as well as protein expression. All POP exposures, except PCB180, decreased unilaminar follicle proportion and increased follicle atresia. Immunostaining confirmed altered expression of LDHA, ATP5A, and GPX4 in the exposed tissues. Moreover, POP exposures modified ATP production in KGN and tissue culture. In conclusion, our results demonstrate the disruption of cellular energy metabolism as a novel mode of action underlying POP-mediated interference of follicle growth in human ovaries.

Effects of DDT and DDE on placental cholinergic receptors.

The placental cholinergic system; known as an important factor in intracellular metabolic activities, regulation of placental vascular tone, placental development, and neurotransmission; can be affected by persistent organic pesticides, particularly organochlorine pesticides(OCPs), which can influence various epigenetic regulations and molecular pathways. Although OCPs are legally prohibited, trace amounts of the persistent dichlorodiphenyltrichloroethane(DDT) are still found in the environment, making prenatal exposure inevitable. In this study, the effects of 2,4'-DDT and 4,4'-DDT; and its breakdown product 4,4'-DDE in the environment on placental cholinergic system were evaluated with regards to cholinergic genes. 40 human placentas were screened, where 42,50% (17 samples) were found to be positive for the tested compounds. Average concentrations were 10.44 µg/kg; 15.07 µg/kg and 189,42 µg/kg for 4,4'-DDE; 2,4'-DDT and 4,4'-DDT respectively. RNA-Seq results revealed 2396 differentially expressed genes in positive samples; while an increase in CHRM1,CHRNA1,CHRNG and CHRNA2 genes at 1.28, 1.49, 1.59 and 0.4 fold change were found(p<0028). The increase for CHRM1 was also confirmed in tissue samples with immunohistochemistry. In vitro assays using HTR8/SVneo cells; revealed an increase in mRNA expression of CHRM1, CHRM3 and CHRN1 in DDT and DDE treated groups; which was also confirmed through western blot assays. An increase in the expression of CHRM1,CHRNA1, CHRNG(p<0001) and CHRNA2(p<0,05) were found from the OCPs exposed and non exposed groups.The present study reveals that intrauterine exposure to DDT affects the placental cholinergic system mainly through increased expression of muscarinic receptors. This increase in receptor expression is expected to enhance the sensitivity of the placental cholinergic system to acetylcholine.

The Association between Long-Term DDT or DDE Exposures and an Altered Sperm Epigenome-a Cross-Sectional Study of Greenlandic Inuit and South African VhaVenda Men.

BACKGROUND: The organochlorine dichlorodiphenyltrichloroethane (DDT) is banned worldwide owing to its negative health effects. It is exceptionally used as an insecticide for malaria control. Exposure occurs in regions where DDT is applied, as well as in the Arctic, where its endocrine disrupting metabolite, p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) accumulates in marine mammals and fish. DDT and p,p'-DDE exposures are linked to birth defects, infertility, cancer, and neurodevelopmental delays. Of particular concern is the potential of DDT use to impact the health of generations to come via the heritable sperm epigenome. OBJECTIVES: The objective of this study was to assess the sperm epigenome in relation to p,p'-DDE serum levels between geographically diverse populations. METHODS: In the Limpopo Province of South Africa, we recruited 247 VhaVenda South African men and selected 50 paired blood serum and semen samples, and 47 Greenlandic Inuit blood and semen paired samples were selected from a total of 193 samples from the biobank of the INUENDO cohort, an EU Fifth Framework Programme Research and Development project. Sample selection was based on obtaining a range of p,p'-DDE serum levels (mean=870.734±134.030 ng/mL). We assessed the sperm epigenome in relation to serum p,p'-DDE levels using MethylC-Capture-sequencing (MCC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq). We identified genomic regions with altered DNA methylation (DNAme) and differential enrichment of histone H3 lysine 4 trimethylation (H3K4me3) in sperm. RESULTS: Differences in DNAme and H3K4me3 enrichment were identified at transposable elements and regulatory regions involved in fertility, disease, development, and neurofunction. A subset of regions with sperm DNAme and H3K4me3 that differed between exposure groups was predicted to persist in the preimplantation embryo and to be associated with embryonic gene expression. DISCUSSION: These findings suggest that DDT and p,p'-DDE exposure impacts the sperm epigenome in a dose-response-like manner and may negatively impact the health of future generations through epigenetic mechanisms. Confounding factors, such as other environmental exposures, genetic diversity, and selection bias, cannot be ruled out. https://doi.org/10.1289/EHP12013.

Paternal p,p'-DDE exposure and pre-pubertal high-fat diet increases the susceptibility to fertility impairment and sperm Igf2 DMR2 hypo-methylation in male offspring.

The hypothesis of paternal origins of health and disease (POHaD) indicates that paternal exposure to adverse environment could alter the epigenetic modification in germ line, increasing the disease susceptibility in offspring or even in subsequent generations. p,p'-Dichlorodiphenyldichloroethylene (p,p'-DDE) is an anti-androgenic chemical and male reproductive toxicant. Gestational p,p'-DDE exposure could impair reproductive development and fertility in male offspring. However, the effect of paternal p,p'-DDE exposure on fertility in male offspring remains uncovered. From postnatal day (PND) 35 to 119, male rats (F0) were given 10 mg/body weight (b.w.) p,p'-DDE or corn oil by gavage. Male rats were then mated with the control females to generate male offspring. On PND35, the male offspring were divided into 4 groups according whether to be given the high-fat diet (HF): corn oil treatment with control diet (C-C), p,p'-DDE treatment with control diet (DDE-C), corn oil treatment with high-fat diet (C-HF) or p,p'-DDE treatment with high-fat diet (DDE-HF) for 35 days. Our results indicated that paternal p,p'-DDE exposure did not affect the male fertility of male offspring directly, but decreased sperm quality and induced testicular apoptosis after the high-fat diet treatment. Further analysis demonstrated that paternal exposure to p,p'-DDE and pre-pubertal high-fat diet decreased sperm Igf2 DMR2 methylation and gene expression in male offspring. Hence, paternal exposure to p,p'-DDE and pre-pubertal high-fat diet increases the susceptibility to male fertility impairment and sperm Igf2 DMR2 hypo-methylation in male offspring, posing a significant implication in the disease etiology.

Níveis séricos de DDT total de trabalhadores expostos no programa de controle de malária no Estado do Pará, Brasil / Serum levels of total DDT in exposed workers of malária control program in the state of Pará, Brazil

Foram avaliados os níveis de DDT total determinados através dos metabólitos pp-DDE e pp-DDT no soro de 67 agentes de saúde (borrifadores) que trabalharam no combate ao vetor transmissor da malária no Estado do Pará, aplicando o inseticida nas superfícies internas e externas das residências, e que ficaram expostos ao DDT durante anos. As análises do soro foram realizadas por Cromatografia Gasosa com detector de captura de elétrons. As concentrações médias encontradas foram de 48,27 mais ou menos 0,30mg por Litro de DDT total, variando de 2,32 a 231,73mg por Litro. O pp-DDE foi encontrado em 100 por cento das amostras em níveis considerados altos, sugerindo uma exposição pregressa ao DDT e demonstrando uma correlação positiva moderada entre o nível de concentração e tempo de exposição (r=0,57 e p=0,003). A concentração de DDT total do grupo constituído de pessoas que não sofreram nenhum tipo de contato conhecido com DDT foi de 0,96 mg por Litro. Não foi detectado nenhum outro inseticida organoclorado nas amostras analisadas.

Determinación de los niveles de DDT, DDE y DDE-MESO2 en leche materna y sangre de poblaciones contaminadas por DDT en México / DDT, DDE and DDE-MeSO2 levels in maternal milk and blood serum from DDT exposed populations in Mexico

Se determinaron los niveles de DDE-MeSO2, DDT y DDE en 51 muestras de leche materna y en 40 muestras de sangre venosa de mujeres que viven en zonas donde el DDT se aplicó para el control de la malaria. Las concentraciones de los tres compuestos en leche materna en las diferentes comunidades fueron: El Ramonal 2.8 ng/glípido, 911 ng/glípido, 3100 ng/glípido; La Cigüeña 1.2 ng/glípido,134 ng/glípido, 764 ng/glípido; Tapachula 0.5 ng/glípido, 17 ng/glípido, 683 ng/glípido; la Huasteca 1.4 ng/glípido, 46 ng/glípido,490 ng/glípido; y Faja de Oro 0.4 ng/glípido, 18 ng/glípido, 268 ng/glípido. Las concentraciones encontradas de DDE-MeSO2 enEl Ramonal son siete veces mayores a las reportadas en Suecia en 1992 (0.4 ng/glípido). Los niveles de exposición a los 3 compuestos en sangre venosa fueron: Lacanjá 64 ng/glípido, 1323 ng/glípido, 3738 ng/glípido; El Ramonal 20 ng/glípido, 748 ng/glípido, 1271 ng/glípido; La Cigüeña 66 ng/glípido, 424 ng/glípido, 1059 ng/glípido; y Ventanilla 43 ng/glípido, 335 ng/glípido,1362 ng/glípido; de DDE-MeSO2, DDT y DDE respectivamente. Las comunidades de Lacanjá y La Cigüeña presentaron los mayores niveles de exposición al DDE-MeSO2, que comparados con los reportes de Eslovaquia son nueve veces mayor (7.3 ng/glípido).

The levels of the DDE-MeSO2, DDT andDDE were determined in 51 breast milk samples and in 40 blood samples from women living in endemic areas of malaria. The concentrations of the three compounds in maternal milk were: El Ramonal 2.8 ng/glipid, 911 ng/glipid, 3100 ng/glipid; La Cigueña 1.2 ng/glipid, 134 ng/glipid, 764 ng/glipid; Tapachula 0.5 ng/glipid, 17 ng/glipid, 683 ng/glipid; Huasteca 1.4 ng/glipid,46 ng/glipid, 490 ng/glipid; and Faja de Oro 0.4 ng/glipid, 18 ng/glipid, 268 ng/glipid. The concentrations of DDE-MeSO2 found in El Ramonal were seven times higher than those reported in Sweden in 1992 (0.4 ng/glipid). In blood the levels of the threecompounds were: Lacanjá 64 ng/glipid, 1323 ng/glipid, 3738 ng/glipid; El Ramonal 20 ng/glipid, 748 ng/glipid, 1271 ng/glipid; La Cigueña 66 ng/glipid, 424 ng/glipid, 1059 ng/glipid; and Ventanilla 43 ng/glipid, 335 ng/glipid, 1362 ng/glipid. The concentrations of DDE-MeSO2 in Lacanjá and La Cigueña were nine times than those reported in Slovakia (7.3 ng/glipid).