Toxicity removal from contaminated water by constructed wetlands assessed using multiple biomarkers in human stem cell assays.

Constructed wetlands (CWs) have been developed rapidly as a sustainable water treatment technique. However, the capability of CWs for remediating the contaminated water based on toxicity assessment remains largely unknown. Four surface flow CWs and two integrated surface-subsurface flow CWs, from five cities in central and eastern region of China were evaluated, concerning the adverse effects of effluents and the toxicity reduction efficiency. Human bone marrow mesenchymal stem cells (hBMSCs) were employed as a human relevant in vitro model. The influent extractions caused cytotoxicity in a dose-dependent manner. The non-cytotoxic dilutions of the influents enhanced the genotoxicity marker γ-H2AX and reactive oxygen species levels. In addition, the influent repressed the osteogenic and neurogenic differentiation, and stimulated the adipogenic differentiation. Cytotoxicity of the contaminated water was reduced by 54 %-86 % after treatment with CWs. CWs were effective to remove part of the sub-lethal effects, with lower reduction than cytotoxicity. The integrated biomarker response (IBR) value of the effluents from the six CWs is lower than that of four secondary and one tertiary wastewater treatment plants. The IBR of the six CWs influents were in the range of 8.6-10.6, with a reduction of 15-50 % after the pollution restoration in CWs. The two integrated surface-subsurface flow CWs achieved higher IBR removal than the four surface flow CWs, possibly due to improved treatment effects by the combined systems. Cytotoxic and genotoxic effects of polar fractions in the CW effluents were stronger than the medium-polar and the non-polar fractions. Besides, PPARγ agonists present in the effluents played crucial roles and ERα agonists may make modest contributions. The present study enhances understanding of the role of CWs in achieving safe wastewater reclamation and provides evidence for further improving toxicity reduction in CWs performance.

Adipogenic and osteogenic effects of OBS and synergistic action with PFOS via PPARγ-RXRα heterodimers.

Sodium p-perfluorous nonenoxybenzenesulfonate (OBS) is a novel alternative to perfluorooctane sulfonate (PFOS), with environmental health risks largely unknown. The present study aims to unravel the adipogenesis effects and underlying molecular initiating events of OBS, which are crucial for understanding and predicting its adverse outcome. In undifferentiated human mesenchymal stem cells (hMSCs), exposure to 1-100 nM of OBS for 7 days stimulated reactive oxygen species production. In the subsequent multipotent differentiation, hMSCs favored adipogenesis and repressed osteogenesis. The point of departure (PoD) for cellular responses of OBS was 38.85 nM, higher than PFOS (0.39 nM). Notably, OBS/PFOS co-exposure inhibited osteogenesis and synergistically promoted adipogenesis. Consistently, the expression of adipogenic marker genes was up-regulated, while that of osteogenic marker genes was down-regulated. The decreased adiponectin and elevated tumor necrosis factor α (TNFα) secretion were observed in differentiated cells exposed to the mixture of OBS and PFOS. The co-treatment of a peroxisome proliferator-activated receptor γ (PPARγ) antagonist alleviated the adipogenic effects of PFOS and its combination with OBS. Moreover, OBS/PFOS co-exposure induced peroxisome PPARγ activation in reporter gene assays, and increased formation of PPARγ - retinoid X receptor α (RXRα) heterodimers measured by co-immunoprecipitation assays. Molecular docking showed interaction energy of OBS (-20.7 kcal/mol) with intact PPARγ-RXRα complex was lower than that of PFOS (-25.9 kcal/mol). Overall, single OBS exhibited lower potency in inducing adipogenesis but is comparable to PFOS in repressing osteogenesis, whereas OBS/PFOS co-exposure increases interaction with PPARγ-RXRα heterodimers, resulting in the synergistic activation of PPARγ, ultimately enhancing adipogenesis at the expense of osteogenic differentiation. The results indicate the potential health risks of increased obesity and decreased bone density caused by OBS and its co-exposure with PFOS, as well as other perfluorinated alkylated substances mixtures.

Integrated genotoxicity of secondary and tertiary treatment effluents in North China.

Genotoxic effects on aquatic organisms caused by wastewater discharging have raised extensive concerns. However, the efficiency of various wastewater treatment processes to reduce effluent genotoxicity was not well known. Genotoxic effects of effluents from four secondary wastewater treatment plants (SWTPs) and a tertiary wastewater treatment plant (TTP) in north China on Chinese rare minnows (Gobiocypris rarus) were evaluated and the toxicity reduction efficiency of various treatment techniques was compared. SWTPs and TTP final effluents disturbed the antioxidant system and lipid peroxidation, with malondialdehyde (MDA) contents in the fish livers and gills increasing to 1.4-2.4 folds and 1.6-3.1 folds of control, respectively. Significant increases in erythrocytes micronucleus (MN) frequency were induced by effluent, and liver DNA damage caused by final SWTPs effluent was 29-54 % lower than TTP effluent. Further, DNA repair gene atm and growth arrest gene gadd45a were remarkably upregulated by SWTP and TTP final effluents to 1.8-12 folds and 4.1-15 folds, respectively, being consistent with the chromosomal aberration and DNA damage in liver tissue. Integrated biomarker response (IBR) of the tertiary effluent was 49 %-69 % lower than the secondary effluents. However, the final ozone disinfection at TTP caused an increase in the DNA damage, suggesting the generation of genotoxic by-products. UV disinfection at secondary treatment removed part of genotoxicity, with a reduction in IBR of 0 %-47 %. The total semi-volatile organic compounds (SVOCs) detected in the final effluent contained 5 %-56 % potential genotoxic substances, removal of which was 9 %-51 % lower than non-genotoxic compounds. Microfiltration and reverse osmosis process exhibited good performance in removing both the integrated genotoxicity and the potential genotoxic SVOCs. Our finding shows that TTP is superior than SWTP for wastewater treatment due to higher genotoxicity removal, but ozone disinfection needs improvement by optimizing performance parameters or adding post-treatment processes, to achieve better protection for aquatic organisms against genotoxic contaminants.

Effects of legacy and emerging per- and polyfluoroalkyl substances on PPARα/ß/γ regulation and osteogenic/adipogenic differentiation.

As the primary molecular target, there is still a gap between the peroxisome proliferator-activated receptors (PPARs) regulation and the adverse health effects caused by per- and polyfluoroalkyl substances (PFASs). The effects of PFASs on cellular differentiation regulated by PPARs is likely significant given the association of PFASs exposure with obesity and decreased bone density. Human mesenchymal stem cells (hMSCs) were used as an in vitro model to assess the roles of PPAR subtypes in the multipotent differentiation of hMSCs affected by perfluorooctanesulfonate (PFOS), perfluorooctanoic acid (PFOA) and their replacement compounds. PFASs increased the expression of three PPAR subtypes in proliferating and differentiating hMSCs. Meanwhile, PFOS and PFOA decreased osteogenesis, enhanced adipogenesis, and increased bone turnover in hMSCs. Similarly, PFOA alternatives, hexafluoropropylene oxide dimer acid (HFPO-DA) and hexafluoropropylene oxide trimer acid (HFPO-TA), exhibited similar or even higher potency in affecting stem cell differentiation compared with PFOA. Perfluorohexanesulfonate (PFHxS) inhibited osteogenesis with comparable potency to PFOS. In contrast, 6:2 chlorinated poly-fluoroalkyl ether sulfonate (6:2Cl-PFESA) enhanced osteogenesis. PPARß expression is significantly positively correlated with osteogenesis and osteoprotegerin (OPG) secretion in 6:2Cl-PFESA treated cells. shRNA knockdown of PPARß remarkably reversed the osteogenic effects of 6:2Cl-PFESA and enhanced the adipogenic effects of the six chemicals. The results suggested that the adverse effects and relative potency of PFASs on the multipotent differentiation of hMSCs were dependent on the integrated action of the three PPAR subtypes, which facilitates a better understanding of the molecular initiating events of PFASs. The present study may well explain the mechanism of the decreased bone density and increased obesity incidence among those exposed to legacy PFASs, and indicates the necessity of further health risk assessment for the alternatives.

Persistent impairment of gonadal development in rare minnow (Gobiocypris rarus) after chronic exposure to chlorinated polyfluorinated ether sulfonate.

The delayed and persistent adverse effects caused by developmental exposure to per- and poly-fluorinated substances are of significant concern. Juvenile rare minnows (Gobiocypris rarus), were exposed to chlorinated polyfluoroalkyl ether sulfonate (Cl-PFESA) at measured medium concentrations of 86.5 µg/L, 162 µg/L and 329 µg/L, for 4 weeks followed by 12 weeks of depuration. After 4 weeks of exposure, the body weight and length of the juvenile fish were increased compared to controls. Gene expression of gnrh3, lhß, and cyp19a was decreased, and ar and erα were upregulated. Transcriptomic analysis revealed enrichment of multiple pathways related to gonadal development. After 12 weeks of depuration, the gonadosomatic indices were decreased in female fish in a concentration-dependent manner, with a significant decrease to 59% of control in 329 µg/L group. Histological analysis found increasing numbers of degenerating oocytes and perinucleolar oocytes, and decreasing numbers of mature vitellogenic oocytes in female fish treated by Cl-PFESA. Enlarged interstitial space of the testis was observed in the exposed male fish. Gene expression levels of gnrh3, lhß, ar, erα, and vtg were upregulated in the adult fish. Chronic developmental exposure to Cl-PFESA caused persistent effects on gonadal development of fish, highlighting the necessity of a comprehensive ecological risk assessment.

Interactive effects of diclofenac and copper on bioconcentration and multiple biomarkers in crucian carp (Carassius auratus).

Diclofenac (DCF), a non-steroidal anti-inflammatory drug, is widespread in aquatic environments and coexists with heavy metals to form combined pollution. However, the interactive effects of DCF and heavy metals on aquatic organisms remain unknown. This study aimed to investigate the interactive effects of DCF and copper (Cu) on the bioconcentration, oxidative stress status and detoxification-related gene expression in crucian carp (Carassius auratus). Fish were exposed to Cu (100 µg L-1) and DCF (1, 10, 100 and 1000 µg L-1) alone or in combination for 7 days. Results obtained showed that the treatment of Cu combined with high levels of DCF (100 and 1000 µg L-1) significantly decreased tissue concentrations of DCF and Cu compared to the correspondingly individual exposure. Concerning oxidative stress status, as reflected by the activities of antioxidant enzymes and malondialdehyde content, low exposure concentrations of DCF (1 and 10 µg L-1) seemed to mitigate the oxidative stress induced by Cu, whereas the co-exposure of Cu with the highest level of DCF (1000 µg L-1) led to stronger oxidative damage in fish liver than Cu exposure alone. With regarding to detoxification-related genes, in most cases, the expressions of cyp 1a, cyp 3a, gstα, gstπ, pxr and P-gp in crucian carp were significantly altered upon exposure to the compounds in combination compared to exposure to the compounds individually. Collectively, these findings indicate the capacity of each of these pollutants to alter bioconcentration potential, pro-oxidative effects and detoxification-related gene responses of the other when both co-occur at specific concentrations.