| Melamine, which has a high nitrogen content by mass, is a common ingredient in fertilizers and a raw material in the production of melamine resin. As a non-protein nitrogen source, it is also added to cattle feedstuffs. Recently, melamine-contaminated dairy products have caused great public concerns about the problem of food security. Although the toxicity of melamine has been studied in animal tests, little information is available about its toxic effects on cells and proteins.In the present study, the toxicity of melamine was assayed on the unicellular eukaryotic system of Tetrahymena pyriformis (T. pyriformis). The half maximal inhibitory concentration (IC50) of melamine, changes of T. pyriformis cellular morphology and alterations of T. pyriformis cellular proteins'expression levels were examined as main topics in this toxicity assessment of melamine on eukaryotic cells.Firstly, cell growth curves of T. pyriformis cells cultivated in liquid complex culture media containing 0, 0.05, 0.25, 0.5, 2.5, and 5 mg/mL of melamine were plotted by recording the number of cells present in the culture medium during the course of a 52-hour cell growth experiment, with a strictly controlled time interval of 4 hours. In this study, cell counting was performed by using an inverted optical microscope and a hemocytometer. Then, the IC50 value of melamine was calculated from six linear equations deduced from these cell growth curves.Secondly, morphologic changes of these T. pyriformis cells were observed via a transmission electron microscope (TEM). The effects of melamine on T. pyriformis intracellular structure and cellular organelles were examined in this study originally.Finally, a new set of T. pyriformis cell cultures cultivated in liquid complex culture media containing eleven different concentrations of melamine, i.e. 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 mg/mL were prepared. Alterations of T. pyriformis main cellular proteins'expression levels were examined by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Then, the relative contents of top four main cellular proteins in Tetrahymena pyriformis cells were exclusively examined in order to reveal the rules of their alteration. Support Vector Machine (SVM) was employed in the data analysis.Results showed an obvious inhibitory effect of melamine on the growth rate of eukaryotic cells. The degree of inhibition increased with the increasing concentration of melamine. Cell growth dynamics indicated that the IC50 value of melamine on T. pyriformis cells was 0.82 mg/mL. Cellular morphology in the presence of melamine was also affected in a concentration- dependent manner, with characteristics of cellular atrophy and/or damage. Relative contents of four main cellular proteins corresponding to MALDI-TOF-MS peaks m/z 4466, m/z 6455, m/z 6514, and m/z 7772 were all found to be close correlated with the concentration of melamine, although their up- and/or down-regulations showed different patterns when the concentration of melamine changed. Support vector regression leave-one-out method, with the optimized polynomial kernel function and a poly-degree of 3, was chose to evaluate the correlation between the alteration of Tetrahymena pyriformis main proteins'relative contents and the concentration of melamine in the culture medium. The average of the relative error of this SVM model was 7.238%.In conclusion, an exposure of eukaryotic cells to melamine could inhibit cell growth, cause changes of cellular morphology, intracellular structure and organelles, and disturb the expression levels of main cellular proteins in a concentration-dependent way. The described method of discovering and examining cellular proteins sensitive to the concentration of melamine in the culture medium was proposed to be a preliminary study of protein biomarkers in T. pyriformis cells. The results are also useful to future studies about the mechanism of the toxicity of melamine. |
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