Cell culture biomarkers for monitoring of wastewater pollutants

Abstract

Wastewater is normally composed of a mixture of pollutants. The type and composition of pollutants in a particular wastewater depend on the source of origin. The source and characteristics of a particular wastewater determine the ideal method of sewage treatment. Specific treatment techniques are effective in the removal of certain types of pollutants and may have no impact on the levels of other types of pollutants. Therefore, a combination of treatments and assessment of the quality of effluent before release into the environment is normally recommended. The assessment of effluent can be achieved by various techniques including chemical analysis and biological assays. Chemical analyses are commonly employed; however, they often pose detection problems and are considered to be uneconomical. As a result increased efforts are being made to develop biological assays for pollutants that are easy to perform, have a broader pollutant detection range and are that are relatively cheap. The aim of this study was to use the mouse macrophage RAW264.7 cell line as a cell culture bioassay model for monitoring aquatic pollutants and quality of wastewater effluent. The RAW264.7 cells are mouse ascites leukaemia induced cells, which once stimulated secrete inflammatory mediators and pro-inflammatory cytokines. In this study the effects of selected common endocrine distrupting chemical (EDC) pollutants and effluent samples were evaluated using cytotoxicity and secretion of inflammatory mediators (NO) and pro-inflammatory cytokines (IL-6) in RAW264.7 cell culture. Cytotoxicity was evaluated by determination of cell viability. Inflammatory responses were evaluated by determination concentrations of NO and IL-6 in supernatant of RAW264.7 cell culture as biomarkers of inflammation. NO and IL-6 were determined in culture supernatant using Griess reaction assay and double-antibody sandwich enzymelinked immunoassay (DAS ELISA), respectively. The first specific objective of this study was to evaluate effects of endocrine disrupting chemicals on biomarkers of inflammation produced by lipopolysaccharide stimulated RAW264.7 macrophages. Endocrine disrupting chemicals (EDCs) are common pollutants in the environment and can induce disruption of the endocrine and immune systems. The EDCs investigated were Estradiol (E2), 5α-dihydrotestosterone (DHT) and Bisphenol A (BPA). To evaluate if the effects caused by EDCs were modulated by steroid hormone receptors, antagonists of estrogen and androgen receptors were used. The steroid receptor antagonists used were Tamoxifen, an estrogen receptor antagonist, and Flutamide an androgen receptor antagonist. The lipopolysaccharide (LPS) stimulated RAW264.7 cells were exposed to DHT, E2 and BPA alone or in combination with flutamide and tamoxifen. Secretion of biomarkers of inflammation, namely nitric oxide (NO) and interleukin 6 (IL-6), were monitored. RAW264.7 cell culture supernatants were collected for NO and IL-6 assays. The cells were used for cell viability assays. The results show that DHT, E2 and BPA at concentration of 5μg/ml had no cytotoxic effects to RAW264.7 cell culture. However, the same treatments significantly (P <0.001) induced suppression of both NO and IL-6 secretion in stimulated RAW264.7 cells. The suppression of NO and IL-6 indicates that DHT, E2 and BPA can induce anti-inflammatory activities in stimulated RAW264.7 cells. The anti-inflammatory effects were induced via their respective steroid receptors, because it was reversed by their respective antagonist compounds. DHT combined with flutamide reversed the anti-inflammatory effects of DHT, while a combination of an estrogenic E2 or BPA with tamoxifen reversed the effects of E2 and BPA respectively. The results show that stimulated RAW264.7 cells culture can be a useful bioassay model for monitoring androgenic and estrogenic pollutants. The second specific objective of this study was to assess toxicity and inflammatory activity of municipal wastewater samples using RAW264.7 cell culture model. The wastewater samples analysed were influent, post bio-filtration, post activated sludge treatment and final effluent collected from a wastewater treatment plant. RAW264.7 cell cultures were exposed to sterile filtered water samples from the treatment plant. RAW264.7 cell culture supernatants were collected for NO and IL-6 assays. The cells were used for cell viability assays. The results show that none of wastewater samples tested induced cell toxicity when compared to negative control. The results also show that all wastewater samples significantly (P<0.001) induced NO and IL-6 production in RAW264.7 cells. The highest inflammatory activities were induced by post bio-filtration wastewater sample. Final effluent sample induced the lowest inflammatory response. The lower inflammatory activity in final effluent indicates effective removal of pollutants upon sewage treatment. The findings of this study show that sewage samples can induce inflammatory responses in RAW264.7 cells. The results also give evidence that RAW264.7 cells can be used as a model for monitoring the quality of treated municipal sewage. The third specific objective of this study was to evaluate cytotoxicity and inflammatory activity of wastewater collected from a textile factory before and after treatment by coagulation-flocculation methods, using RAW264.7 cell culture model. RAW264.7 cell cultures were exposed to sterile filtered water samples from raw effluent and effluent treated with various coagulation-flocculation methods. RAW264.7 cell culture supernatants were collected for NO and IL-6 assays. The cells were used for cell viability assays. The results show that raw effluent induced cytotoxicity by reducing cell viability significantly (P<0.001) compared to the negative control. The results on inflammatory activities show that the raw effluent and effluent treated with 1.6g/L of Fe-Mn oxide induced significantly (P<0.001) higher NO production than the negative control. The inflammatory results further show that the raw effluent induced significantly (P<0.001) higher production of IL-6 than the negative control. Among the coagulants/flocculants evaluated Al2(SO4)3.14H2O at a dosage of 1.6 g/L was the most effective to remove both toxic and inflammatory pollutants from the textile effluent used in this study. Therefore, the inflammatory responses in RAW264.7 cells can be used as sensitive biomarkers for monitoring the effectiveness of coagulation/flocculation processes used for textile effluent treatment. The last specific objective of this study was to evaluate toxicity and inflammatory activities of a textile effluent treated with electrohydraulic discharge and coagulant/flocculants. A combination of coagulant/flocculants and electrohydraulic discharge (EHD) are tested for treatment efficiency of textile wastewater. Pre- and posttreatment samples were used to evaluate process efficiencies. Process efficiencies were evaluated using physicochemical characteristics, and cytotoxicity and inflammatory activities induced in macrophage RAW264.7 cell culture model. RAW264.7 cell cultures were exposed to sterile filtered water samples from raw textile industrial effluent and effluent after treatment with EHD in absence and presence of coagulant/flocculants. RAW264.7 cell culture supernatants were collected for NO and IL-6 assays. The cells were used for cell viability assays. The results show that raw effluent was characterized by high physicochemical parameters and intense colour. Treatment of textile wastewater with EHD alone successfully reduced the chemical oxygen demand (COD) and total organic carbon (TOC) values by 95.3 and 96.23 % respectively. The results show further that a single treatment approach is not effective in removing all pollutants. However, a combined treatment approach effectively removed complex organic pollutants, toxic and inflammatory pollutants based on the results of NO and IL-6 in RAW264.7 cell cultures. The study confirms that induction of NO and IL-6 secretions in macrophage RAW264.7 cells is a very sensitive model system to monitor the efficiency of textile effluent treated with electrohydraulic discharge and /or combined with other processes.