Effect-directed analysis of contaminated sediment from the wastewater canal in Pancevo industrial area, Serbia.

Wastewater canal (WWC) in Pancevo industrial area in Serbia, whose main environmental receptor is the River Danube, is a well known hot-spot of contamination. WWC sediments have been assessed by UNEP based on chemical target analysis. However, integrative biological data on exposure to hazardous compounds are only provided by the present study which aims at evaluating whether the monitored compounds sufficiently reflect potential hazards and to suggest additional compounds to include in monitoring and hazard assessment by applying effect-directed analysis (EDA) based on arylhydrocarbon receptor-mediated activity and cytotoxicity. Multistep NP-HPLC fractionation provided 18 fractions co-eluting with polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), polycyclic aromatic hydrocarbons (PAHs) and more polar compounds. PAHs fractions exhibited great potencies to induce ethoxyresorufin-o-deethylase (EROD) in H4IIE rat hepatoma cell line expressed as 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQ) (0.1-34.6 x 10(3) pg g(-1)dry weight). Chemical analysis of the most active fractions revealed great concentrations of PAHs (up to 292 x 10(2)ngg(-1) sediment equivalents (SEQ)), methylated PAHs (up to 900 x 10(2) ng g(-1) SEQ), and other alkyl-substituted PAHs. Only minor portions of biologically derived TCDD-EQs could be attributed to monitored PAHs with known relative potencies (REPs). We hypothesize that a major part of the activity is due to non-monitored alkylated and heterocyclic PAHs. Results of the cell cytotoxicity/proliferation assay on H4IIE cell line suggest the presence of sediment pollutants with pronounced potency to disturb cell growth.

Atrazine oral exposure of peripubertal male rats downregulates steroidogenesis gene expression in Leydig cells.

In the present study, we investigated the effects of oral dosing of atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) to peripubertal male rats (50 and 200 mg/kg body weight daily from postnatal days 23-50) on ex vivo Leydig cell steroidogenesis. Leydig cells from treated rats were characterised by significant decline in mRNA transcripts of several genes responsible for steroidogenesis: luteinizing hormone receptor (LHR), scavenger receptor-B1, steroidogenic acute regulatory protein, translocator protein, steroidogenic factor-1, phosphodiesterase 4B, 3beta-hydroxysteroid dehydrogenase (HSD), CYP17A1, and 17betaHSD. In the presence of human chorion gonadotropin, the dose-dependent decrease in extracellular cAMP level and accordingly strong inhibition of androgenesis were obtained. The transcription of LHR gene in Leydig cells of atrazine-treated rats was downregulated in a dose-dependent manner, which could be the reason for reduction in cAMP level and expression of cAMP-dependent genes. To clarify the activity of the steroidogenic enzymes responsible for androgenesis, purified Leydig cells were challenged with different steroid substrates (22OH-cholesterol, pregnenolone, progesterone, and Delta(4)-androstenedione), and the obtained results indicated inhibition of androgen production in Leydig cells isolated from atrazine-treated animals in the presence of all those substrates. However, when Leydig cells were challenged with 22OH-cholesterol, the progesterone level in the incubation medium was unchanged, indicating that decrease in cholesterol transport and/or CYP17A1 and 17betaHSD activity are most probably responsible for inhibition of androgen production after the addition of different substrates. Our results demonstrated that in vivo exposure to atrazine affects Leydig cell steroidogenesis via the inhibition of steroidogenesis gene expression, which is accompanied by decreased androgenesis.