title: Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE.
abstract: 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.
authors.affiliation: Section of Experimental Biomedicine, Norwegian University of Life Sciences, Oslo, Norway; Mekelle University College of Veterinary Medicine, Mekelle, Ethiopia. Electronic address: shewit.kalayou.teklehaimanot@nmbu.no.
authors.forename: Shewit
authors.initials: S
authors.lastname: Kalayou
authors.affiliation: Section of Experimental Biomedicine, Norwegian University of Life Sciences, Oslo, Norway.
authors.forename: Cesilie
authors.initials: C
authors.lastname: Granum
authors.affiliation: Section of Experimental Biomedicine, Norwegian University of Life Sciences, Oslo, Norway.
authors.forename: Hanne Friis
authors.initials: HF
authors.lastname: Berntsen
authors.affiliation: Section of Experimental Biomedicine, Norwegian University of Life Sciences, Oslo, Norway.
authors.forename: Per Kristian
authors.initials: PK
authors.lastname: Groseth
authors.affiliation: Section of Experimental Biomedicine, Norwegian University of Life Sciences, Oslo, Norway.
authors.forename: Steven
authors.initials: S
authors.lastname: Verhaegen
authors.affiliation: Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Northern Ireland, UK.
authors.forename: Lisa
authors.initials: L
authors.lastname: Connolly
authors.affiliation: Department of Environmental Toxicology, Uppsala University, Norbyvägen 18 A, SE-752 36 Uppsala, Sweden.
authors.forename: Ingvar
authors.initials: I
authors.lastname: Brandt
authors.affiliation: Department of Immunology, Oslo University Hospital Rikshospitalet, University of Oslo, PO Box 4950, Nydalen, Oslo 0424, Norway.
authors.forename: Gustavo Antonio
authors.initials: GA
authors.lastname: de Souza
authors.affiliation: Section of Experimental Biomedicine, Norwegian University of Life Sciences, Oslo, Norway.
authors.forename: Erik
authors.initials: E
authors.lastname: Ropstad
mesh_terms: D000818:Animals; D003633:Dichlorodiphenyl Dichloroethylene / Q000009:adverse effects / Q000031:analogs & derivatives* / Q000494:pharmacology; D007306:Insecticides / Q000009:adverse effects* / Q000494:pharmacology; D007985:Leydig Cells / Q000378:metabolism* / Q000473:pathology; D008297:Male; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D013552:Swine
chemical_list: D007306:Insecticides; D020543:Proteome; D003633:Dichlorodiphenyl Dichloroethylene; C055238:1-chloro-4-(2,2-dichloro-1-(4-chlorophenyl)ethenyl)-3-(methylsulfonyl)benzene
keywords: 3-Methylsulfonyl-DDE; Endocrine disruption; IPA; Leydig cells; Persistent organic pollutants; Proteomics; Steroidogenesis; Sus scrofa