The highest concentrations from several tens
The highest concentrations (from several tens to often hundreds or thousands ng/L) are reported in animal farm flush water, and associated runoffs and lagoons in China and in the U.S. Lower concentrations are reported in WWTP influents and effluents. In WWTP influent waters DRO, LNG, NET and PRG have mainly been measured in concentrations ranging from a few to more than a hundred ng/L. In WWTP effluents, concentrations are generally lower with an order of magnitude. In surface waters, which are more relevant from an ecotoxicological point of view, the reported progestogen concentrations are approximately similar to WWTP effluent concentrations. In rivers, streams, lakes and ground water, DRO, LNG, NET and PRG are detected at concentrations of typically a few ng/L (Chang et al., 2011, Fent, 2015, Liu et al., 2012, Liu et al., 2014, Orlando and Ellestad, 2014, Vulliet et al., 2008, Yost et al., 2014). By analyzing freshwater samples from the catchment area of Lake Balaton and River Zala, varying progestogens concentration 0.26–4.30 (DRO), 0.85–3.40 (LNG) and 0.23–13.67 (PRG) ng/L were detected (Avar et al., 2016). There is increasingly high amount of evidence available about the toxic effects of individual progestogen. Fecundity or eggs production of fathead minnows (Pimephales promela) were dramatically decreased due to the 21 days long treatment by 22–596ng/L NET. Plasma concentration of the endogen 11-ketotestosterone in male and 17β-estradiol in female fathead minnows were significantly decreased after 21 days exposure to high concentration of NET (Paulos et al., 2010). Toxicity studies reported that DRO (6.5µg/L) reduces fertility in adult fathead minnows (Zeilinger et al., 2009). The disruption of the reproductive system occurred in male roach (Rutilus rutilus) after exposure to 31ng/L LNG (Kroupova et al., 2014). 6.5ng/L LNG can already disrupt the androgen-dependent reproductive SZL P1-41 pathway in male three-spined sticklebacks (Gasterosteus aculeatus) (Svensson et al., 2013, Svensson et al., 2014). Bioaccumulation of progestogenic chemicals in the rainbow trout (Oncorhynchus mykiss) has also been published (Liu et al., 2011). Being aware of the results of previously reported toxicity experiments with we aimed to investigate the combined impact of progestogens on the molecular and somatic level of a common fish (Rutilus rutilus). Our investigations include a 10ng/L treatment group, which represents a realistic level of contamination in freshwater. However, there are several reports about remarkable higher progestogen contamination (hundreds or thousands ng/L) in environment, so we aimed to also test the 50 and excessive 500ng/L concentrations in our experiment. According to literature data, the presence of protein deglycase DJ-1 chaperon molecule was also described in medeka (Oryzias latipes) (Ansai et al., 2013, Li et al., 2006), zebrafish (Danio rerio) (Bai et al., 2006, Bretaud et al., 2011), atlantic salmon (Salmo salar) (Shinbo et al., 2006) and pike (Esox lucius) (Leong et al., 2010). Fish DJ-1 protein (189 AA) is homologous with human DJ-1 which shows its evolutionary conserved appearance (Bai et al., 2006, Bandyopadhyay and Cookson, 2004). This protein deglycase chaperon protein possesses multiple functions in vertebrates. Besides antioxidant defense of DJ-1, it is also involved in cell survival and proliferation, cytoprotection and gene transcription. There are factors, such as nuclear E2-related factor 2 (NRF2) (Clements et al., 2006, Schultz et al., 2014), polypyrimidine tract-binding protein-associated splicing factor (PSF) (Dong et al., 2005, Xu et al., 2005) and low-density lipoprotein (LDL) receptor (Yamaguchi et al., 2012), which are common target molecules for steroids and DJ-1 (see Fig. S1). We hypothesized that steroids (progestogens) could elevate the level of DJ-1 and may also indirectly influence their targeted receptors through DJ-1 (Fig. S1), therefore, we examined the relationship between progestogens and DJ-1 protein in roach. Furthermore, using a cell stress array kit for observation our other aim was to follow the changes of 26 signal molecules of different biological function due to progestogen treatment.