In vitro assessment of potential endocrine disrupting activities of chlorinated paraffins of various chain lengths.

The production of chlorinated paraffins (CPs) has risen in the past two decades due to their versatile industrial applications. Consequently, CPs are now widely detected in human food sources, the environment, and in human matrices such as serum, the placenta and breast milk. This raises concern about prenatal and postnatal exposure. While some studies suggest that certain short-chained CPs (SCCPs) may have endocrine disrupting properties, knowledge about potential endocrine disrupting potential of medium- (MCCP) and long-chained CPs (LCCPs) remains relativity sparse. Here, we used a panel of in vitro assays to investigate seven pure CPs and two technical mixtures of CPs. These varied in chain length and, chlorination degree. The in vitro panel covered androgen, estrogen, and retinoic acid receptor activities, transthyretin displacement, and steroidogenesis. One of the SCCPs inhibited androgen receptor (AR) activity. All SCCPs induced estrogen receptor (ER) activity. Some SCCPs and MCCPs increased 17ß-estradiol levels in the steroidogenesis assay, though not consistently across all substances in these groups. SCCPs exhibited the most pronounced effects in multiple in vitro assays, while the tested LCCPs showed no effects. Based on our results, some CPs can have endocrine disrupting potential in vitro. These findings warrant further examinations to ensure that CPs do not cause issues in intact organisms, including humans.

Assessment of chemical mixtures using biomarkers of combined biological activity: A screening study in human placentas.

Humans are simultaneously exposed to complex mixtures of chemicals with limited knowledge on potential health effects, therefore improved tools for assessing these mixtures are needed. As part of the Human Biomonitoring for Europe (HBM4EU) Project, we aimed to examine the combined biological activity of chemical mixtures extracted from human placentas using one in vivo and four in vitro bioassays, also known as biomarkers of combined effect. Relevant endocrine activities (proliferative and/or reporter gene assays) and four endpoints were tested: the estrogen receptor (ER), androgen receptor (AR), and aryl hydrocarbon receptor (AhR) activities, as well as thyroid hormone (TH) signaling. Correlations among bioassays and their functional shapes were evaluated. Results showed that all placental extracts agonized or antagonized at least three of the abovementioned endpoints. Most placentas induced ER-mediated transactivation and ER-dependent cell proliferation, together with a strong inhibition of TH signaling and the AR transactivity; while the induction of the AhR was found in only one placental extract. The effects in the two estrogenic bioassays were positively and significantly correlated and the AR-antagonism activity showed a positive borderline-significant correlation with both estrogenic bioassay activities. However, the in vivo anti-thyroid activities of placental extracts were not correlated with any of the tested in vitro assays. Findings highlight the importance of comprehensively mapping the biological effects of "real-world" chemical mixtures present in human samples, through a battery of in vitro and in vivo bioassays. This approach should be a complementary tool for epidemiological studies to further elucidate the combined biological fingerprint triggered by chemical mixtures.

Assessment of source and treated water quality in seven drinking water treatment plants by in vitro bioassays - Oxidative stress and antiandrogenic effects after artificial infiltration.

Drinking water quality and treatment efficacy was investigated in seven drinking water treatment plants (DWTPs), using water from the river Göta Älv, which also is a recipient of treated sewage water. A panel of cell-based bioassays was used, including measurements of receptor activity of aryl hydrocarbon (AhR), estrogen (ER), androgen (AR), peroxisome proliferator-activated receptor alpha (PPARα) as well as induction of oxidative stress (Nrf2) and micronuclei formation. Grab water samples were concentrated by solid phase extraction (SPE) and water samples were analyzed at a relative enrichment factor of 50. High activities of AhR, ER and AR antagonism were present in WWTP outlets along the river. Inlet water from the river exhibited AhR and AR antagonistic activities. AhR activity was removed by DWTPs using granulated activated carbon (GAC) and artificial infiltration. AR antagonistic activity was removed by the treatment plants, except the artificial infiltration plant, which actually increased the activity. Furthermore, treated drinking water from the DWTP using artificial infiltration exhibited high Nrf2 activity, which was not found in any of the other water samples. Nrf2 activity was found in water from eight of the 13 abstraction wells, collecting water from the artificial infiltration. No genotoxic activity was detected at non-cytotoxic concentrations. No Nrf2 or AR antagonistic activities were detected in the inlet or outlet water after the DWTP had been replaced by a new plant, using membrane ultrafiltration and GAC. Neither target chemical analysis, nor chemical analysis according to the drinking water regulation, detected any presence of chemicals, which could be responsible of the prominent effects on oxidative stress and AR antagonistic activity in the drinking water samples. Thus, bioanalysis is a useful tool for detection of unknown hazards in drinking water and for assessment of drinking water treatments.

Effect-based assessment of recipient waters impacted by on-site, small scale, and large scale waste water treatment facilities - combining passive sampling with in vitro bioassays and chemical analysis.

Waste water treatment facilities are a major sources of organic micropollutants (MPs) in surface water. In this study, surface water samples were collected from seven sites along a river system in Uppsala, Sweden, during four seasons and evaluated based on the occurrence of MPs in the samples and bioactivity using in vitro bioassays. The sampling sites were differentially impacted by on-site sewage treatment facilities (OSSFs), small scale, and large scale waste water treatment plants (WWTPs). The bioassays used included activation of aryl hydrocarbon receptor (AhR), estrogen receptor (ER), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), nuclear factor erythroid 2-related factor 2 (Nrf2), and androgen receptor (AR). Occurrence of 80 MPs, were analyzed using liquid chromatography coupled to tandem mass spectrometry. Most water samples induced AhR activity, and all sampling sites showed a similar profile regarding this activity. With the exception of one water sample, we did not detect any NFkB, Nrf2 or AR activity of the water samples. The exception was a sample impacted by OSSFs, which showed an activity in multiple bioassays, but the activity could not be explained by the occurrence of target MPs. The occurrence of MPs showed a spatial trend, with the highest number and amount of MPs detected in the samples collected downstream of the WWTPs, where up to 47 MPs were detected in one single sample. A seasonal variation was observed with highest levels of MPs and highest AhR activities in samples collected in June and September 2015. However, neither the seasonal activity nor the on-site activity could be explained by the measured MPs, suggesting unknown contributory agents in the water.