Determining water quality : development of a cell culture cytotoxicity assay

Abstract

Not only in South Africa, but also all over the world, pollution is threatening the quality of water resources. More than 100,000 chemicals are produced worldwide and some of these chemicals enter into the aquatic environment, posing a potential threat to humans, animals as well as other organisms. Therefore, determining the quality of water is of great importance. The testing of the toxicity of water samples are either based on a biological model or chemical model. In the biological model, organisms are utilized as indicators for toxicity of a particular sample, this sample may then be cytotoxic to the cells of the organism. In the chemical model the amount of a specific chemical present in water samples are predicted. Although, within the biological model there are many cytotoxicity screening assays (also called bioassays) that assess quality of water, there is a need for a fast, efficient and cost-effective cell culture system that may act as first screening procedure in a range of tests. Of particular importance in the context of this thesis, are bioassays that measure specific biochemical parameters, e.g. the Uridine uptake bioassay, MTT (3-(4,5¬dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay and the bioluminescent assay. An important advantage of these assays are their sensitivity and the fact that the tests are principally based on cell mortality as a consequence of membrane damage or influence on cell metabolism. Researchers at the Highveld Biological Laboratory in South Africa have the past few years paid particular attention to the MTT assay, originally developed by Mosmann, and have modified this assay. In this thesis the adjusted method is referred to as the Modified Highveld Biological assay. There were, however, still a few adjustments to be made in order to use this modified method to its full potential. The Bioluminescent assay, also were thought to have potential to be modified as water quality screening test. Therefore, the hypotheses investigated in the current thesis were: • The original Mosmann MTT method can be modified successfully to provide a sensitive, reproducible mass screening method for determining chemical cytotoxicity and water quality, by using the K-562 cell line. • Sensitivity of the assay can be increased by utilizing a 3 phase medium cycle (Medium type 1-3) instead of the previously used single culture medium. • Furthermore, this cytotoxicity assay can be successfully utilized to determine whether any chemical solution or water sample is cytotoxic. Thus information was needed to determine whether cytotoxicity correlates with real life toxicity. This requires time-consuming epidemiological surveys unless results can be correlated with previous surveys. The research in this thesis attempted to indicate that the obtained cytotoxicity can be used to predict toxicity of a water sample, and that the cytotoxicity findings of this dissertation may indeed be meaningful. The culturing conditions of the Modified Highveld Biological MTT assay were modified successfully and could thus be utilized as the first assay in a battery of tests to determine overall cytotoxicity by utilising K¬652 cells in culture. Due to successful modification of the assay, this MTT assay now reduces assay duration, thereby saving important resources. An attempt was also made to optimise the Bioluminescent assay. This method is based on the fact that adenosine triphosphate (ATP) is present in all living cells. The method utilizes an enzyme, firefly luciferase, which catalyses the formation of light from ATP and luciferin. The emitted light intensity is linearly dependent on the ATP . concentration and is measured using a luminometer. The following problems were, however, identified that renders this method unsatisfactory as cytotoxicity indicator for water samples. • The luciferase enzyme is too sensitive to too many different substances used in the cell culture medium and in the preparation procedures of the cells (ions, salts etc.) which makes it less suited for usage for fast, effective testing of water toxicity. • This method will probably function better when luciferase can be carried into the cells or form part of the cells' genetic material. • We therefore suggest that, the Bioluminescent assay might possibly be a method to determine cytotoxicity, if the sensitivity of the luciferase enzyme could be further investigated. Thus it could be concluded that luminescence is not suitable for assaying complex mixtures because it is possible for unknown non~toxic agents, present in test samples, to interfere with the process of light emission (quenching). Lastly, known chemical solutions as well as unknown water samples were screened using the adjustments to the Modified Highveld Biological Method proposed in this thesis. The cytotoxicity of unknown water samples and chemical solutions were successfully determined and different cytotoxic effects were obtained, e.g. synergism, antagonism, additive effects and neutral effects. It was also possible to reduce or remove the cytotoxicity of certain water samples by applying pre~treatment with either Na2CO3 (removing possible toxic divalent and polyvalent metals) or SepPak cartridge clean up (removing organic toxicants). Results indicated that the method is very sensitive and can detect even low traces of toxicants. Thus, it can be concluded that the method was successfully adjusted to be useful as a first screening assay for toxicity analysis of a series of environmental water samples.