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 (10μM) 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.

Micro-organic compounds in the Humber rivers

The concentrations of a range of micro-organic compounds in the Humber rivers have been measured at weekly intervals over a period of 1 year. The compounds include the triazine herbicides (simazine, atrazine, propazine, desmetryn and prometryn), selected organophosphorus insecticides (fenitrothion, malathion and parathion), phenylurea (chlorotoluron, diuron, isoproturon and linuron) and phenoxyacid herbicides (2,4-D, MCPA, MCPB and mecoprop), phenol derivatives (phenol, 2-ethylphenol, 2-chlorophenol, 4-nitrophenol, 2-methylphenol, 4-ethylphenol and 2,4-dichlorophenol), organochlorine insecticides (HEOD, DDT, TDE, DDE, HCB, alpha-BHC and lindane), PCB's and some synthetic pyrethroid insecticides (cis/trans-permethrin, fenvalerate and cypermethrin). The results indicate the high frequency of occurrence of many compounds in the southern Humber rivers Aire, Calder, Trent and Don compared with the more occasional concentrations found in the upland rivers with catchments dominated by low intensity agriculture. The more water soluble herbicides, atrazine, simazine, isoproturon and diuron are detected frequently in the southern rivers with the highest concentrations and abundance in the rivers Aire, Calder and Trent. The most abundant phenolic compound is 2,4-dichlorophenol usually occurring at concentrations <1 mu g/l. The organochlorine insecticides and PCB's are generally at concentrations <0.01 mu g/l, cis/trans-permethrin are the only synthetic pyrethroids detected and these are found in the rivers Aire and Calder at about equal concentrations of between 0.01 and 0.11 mu g/l. The results of the occurrence of simazine and atrazine in the rivers Trent, Don and Aire illustrate peaks in concentration in the spring and then later in the year during the early autumn coinciding with the first major storm after the summer. In the rivers Trent and Don, the annual exports (March 1994-95) of atrazine are lower than for simazine whereas in the R. Calder the yield of atrazine is higher than for simazine suggesting differences in use in these catchments. The maximum triazine concentration observed, i.e. 8 mu g/l of atrazine in the R. Calder, could have inhibiting effects on the phytoplankton and algal growth, although because of the transient nature of the peaks, recovery is expected to be rapid. The only other compounds measured at concentrations likely to produce detrimental ecotoxicological effects are cis/trans permethrin in the rivers Aire and Calder. (C) 1997 Elsevier Science B.V.

The frequency of Environmental Quality Standard (EQS) exceedance for chlorinated organic pollutants in rivers of the Humber catchments

The southern industrial rivers (Aire, Calder, Don and Trent) feeding the Humber estuary were routinely monitored for a range of chlorinated micro- organic contaminants at least once a week over a 1.5-year period. Environmental Quality Standards (EQSs) for inland waters were set under the European Economic Community for a limited number of problematic contaminants (18). The results of the monitoring program for seven classes of chlorinated pollutants on the EQS list are presented in this study. All compounds were detected frequently with the exception of hexachlorobutadiene (where only one detectable measurement out of 280 individual samples occurred). In general, the rivers fell into two classes with respect to their contamination patterns. The Aire and Calder carried higher concentrations of micro- pollutants than the Don and Trent, with the exception of hexachlorobenzene (HCB). For Σ hexachlorocyclohexane (HCH) isomers (α + γ) and for dieldrin, a number of samples (~ 5%) exceeded their EQS for both the Aire and Calder. Often, ΣHCH concentrations were just below the EQS level. Levels of p,p'- DDT on occasions approached the EQS for these two rivers, but only one sample (out of 140) exceeded the EQS. No compounds exceeded their EQS levels on the Don and Trent. Analysis of the ratio of γ HCH/αHCH indicated that the source of HCH for the Don and Trent catchments was primarily lindane (γHCH) and, to a lesser extent, technical HCH (mixture of HCH isomers, dominated by α HCH), while the source(s) for the Aire and Calder had a much higher contribution from technical HCH.

Do persistent organic pollutants interact with the stress response? Individual compounds, and their mixtures, interaction with the glucocorticoid receptor

Persistent organic pollutants (POPs) are toxic substances, highly resistant to environmental degradation, which can bio-accumulate and have long-range atmospheric transport potential (UNEP 2001). The majority of studies on endocrine disruption have focused on interferences on the sexual steroid hormones and so have overlooked disruption to glucocorticoid hormones. Here the endocrine disrupting potential of individual POPs and their mixtures has been investigated in vitro to identify any disruption to glucocorticoid nuclear receptor transcriptional activity. POP mixtures were screened for glucocorticoid receptor (GR) translocation using a GR redistribution assay (RA) on a CellInsight(TM) NXT High Content Screening (HCS) platform. A mammalian reporter gene assay (RGA) was then used to assess the individual POPs, and their mixtures, for effects on glucocorticoid nuclear receptor transactivation. POP mixtures did not induce GR translocation in the GR RA or produce an agonist response in the GR RGA. However, in the antagonist test, in the presence of cortisol, an individual POP, p,p'-dichlorodiphenyldichloroethylene (DDE), was found to decrease glucocorticoid nuclear receptor transcriptional activity to 72.5% (in comparison to the positive cortisol control). Enhanced nuclear transcriptional activity, in the presence of cortisol, was evident for the two lowest concentrations of perfluorodecanoic acid (PFOS) potassium salt (0.0147mg/ml and 0.0294mg/ml), the two highest concentrations of perfluorodecanoic acid (PFDA) (0.0025mg/ml and 0.005mg/ml) and the highest concentration of 2,2',4,4'-tetrabromodiphenyl ether (BDE 47) (0.0000858mg/ml). It is important to gain a better understanding of how POPs can interact with GRs as the disruption of glucocorticoid action is thought to contribute to complex diseases.