A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals
Introduction
Cell-based in vitro toxicity testing of environmental contaminants has become an essential technique to rapidly assay compounds of potential concern to human health. In recent years a number of techniques utilizing cellular impedance biosensors have been developed to provide real-time, label-free toxicity testing [1], [2], [3], [4], [5], [6], [7]. The results of these assays can then be used to generate in vitro cytotoxicity values (IC50) which can be used similarly to in vivo LD50 for toxicity ranking of chemicals. One of the most recently developed biosensor technologies is real-time cell electronic sensing (RT-CES) which has been demonstrated to provide sensitive monitoring of cellular responses in a real-time continuous manner [5], [6], [8]. This technique utilizes a series of microwells, whose bottoms are 80% covered with microelectrodes to provide an advance in sensitivity in cell sensing compared to previous techniques. RT-CES measures cell viability by monitoring cell proliferation and morphology [5], [6], [8] using a dimensionless unit called the cell index (CI) which is based on the impedance changes caused by cells interacting with the microelectrodes. The results obtained using RT-CES have been shown to be comparable to more traditional cytotoxicity assays such as the MTT, neutral red uptake (NRU), lactose dehydrogenase, and acid phosphatase tests [5], [6], [9] and has been used to screen a number of chemicals including drugs and environmental metal contaminants [5], [6], [8]. Cell electronic sensing techniques have not been demonstrated for profiling and differentiating compounds with similar chemical structures and properties or for environmental studies.
Disinfection by-products (DBPs) are an unintended consequence of drinking water disinfection and are formed when the disinfectant residual reacts with natural organic matter or organic compounds present in the source water. The presence of DBPs in treated drinking water is an important public health concern as many DBPs are associated with adverse health outcomes. Rapid toxicity testing of DBPs is desirable as the number of identified DBPs increases. Some DBP testing methods based on microplate assays using both bacterial [10], [11] and mammalian cells [11], [12], [13] have been reported. The N-nitrosamines are a group of emerging DBPs. N-Nitrosodimethylamine (NDMA) is the nitrosamine most commonly detected as a DBP and has been detected in treated drinking water in California, Ontario, and Alberta [14], [15], [16], [17]. More recently, N-nitrosopyrrolidine (NPyr), N-nitrosopiperidine (NPip), and N-nitrosodiphenylamine (NDPhA) were discovered in a drinking water distribution system in Alberta [17]. NDMA, NPyr, and NDPhA are classified as probable human carcinogens [18] and NPip is considered a possible human carcinogen [19]. These four nitrosamines (Table 1) were chosen as testing compounds for demonstrating the RT-CES toxicity profiling method because they have already been identified in treated drinking water and have similar chemical structures and properties.
The purpose of this study is to develop an RT-CES method for profiling the toxicity of chemicals with similar structures and properties. The continuous sensing and quantitative measurements of the RT-CES system combined with use of multiple cell lines will produce a panel of cytotoxicity profiles that will allow differentiation between similar chemicals such as nitrosamines.
Section snippets
Materials and cell culture conditions
Standards of N-nitrosodimethylamine (NDMA), N-nitrosodiphenylamine (NDPhA), N-nitrosopiperidine (NPip), and N-nitrosopyrrolidine (NPyr) were purchased from Sigma–Aldrich (Oakville, ON) (Table 1). Methanol and water (HPLC grade) were obtained from Fisher Scientific (Nepean, ON).
A549 (Human type II pneumocyte derived adenocarcinoma) cells were cultured in RMPI 1640 (Sigma–Aldrich). T24 (human bladder carcinoma) cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA)
Method design
The RT-CES system has been previously described in detail [5], [6], [8]. Briefly, the RT-CES 16× system used in this study utilizes electronic microchips with 16 microwells on each E-plate. The bottom of each microwell has an approximate area of 20 mm2 and approximately 80% of this area is covered by microelectrodes. The microelectrodes measure changes in impedance between the electrode and the solution. Cells attaching to the microelectrodes cause a change in electrical impedance compared to
Acknowledgements
Funding for this project was provided by the Canadian Water Network, the Natural Sciences and Engineering Research Council of Canada (NSERC), Alberta Health and Wellness, and an NSERC University Faculty Award (to XFL). We thank Dr. Tom Hobman for providing CHO-K1 cells. We thank Dr. Gian Jhangri for his advice with the statistical analysis.
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These authors have contributed equally to this study.