Using Genomic Approach To Select Response Genes Of FL Cell Exposed To Microcystin-LR

The outbreak of the cyanobacerial (specifically Microcystins aerugiosa) blooms due to eutrophication has been a worldwide threat since the late 1970s;it exerts severe adverse health effects on human and livestock by virtue of their ability to produce the heptapeptide toxin, microcystin. The general structure of microcystin (MC) is cyclo-(D-Ala-L-X-erythro-β-methyl-D-isoAsp-L-Y-Adda-D-isoGlu-N-Methyldehydr o-Ala), where Adda stands for 3-amino-9-methoxy-2, 6, 8-trimethyl-10-phenyldeca -4,6-dienoic acid, and X,Y are two variable amino acids. Adda is essential for biological activity of MC. Till now, more than 60 structure variants have been recognized. The X, Y variable amino acids for Microcystin-LR are leucine(L), arginine(R) respectively, which is the most predominant and the most toxic microcystin.Using tritium-labled MC, it has been demonstrated that the liver is the prime target organ affected, but to some extent, MC also concentrated in the intestine and the kidneys. The toxic effects of MC on different types of mammalian cells including hepatocytes, embryo kidney cells, fibroblast, endothelial, epithelial cell, and lymphocytes, were observed.Previous studies of molecular mechanisms of MC involved in: inhibition of protein phosphatases 1 and 2A (PP1 and 2A), effects of MC on the cell cytoskeleton, induction oxidative stress, induction of apoptosis, MC caused cell proliferation andDNA damage. So far, the exact mechanisms of MC have not been fully elucidated, even remains some controversial: MC could cause cell apoptosis while it also could cause cell proliferation. As an extraneous toxin, the effects of microcystin on the cell function must be complicated. To further clarify the biological machinery followed exposure to toxins, new high throughput methods are necessaryFollowing the completion of human genome project (HGP), researchers can now detect the transcription of almost all human genes to expatiate the disciplinarian and mechanism of life. The invention of gene chips (or microarrays) provides useful tools in high throughput detection of gene transcription. By compare the transcription of genes in different cells and tissues, in different development phases, under different pathophysiologic condition or different stimulates, we could elucidate the biological mechanism of life and diseases.Therefore, the "Human Genome U133 Plus 2.0 Array" from Affymitrix company and "Micro Fluidic card" from ABI company were used to detect differentially expressed genes following MC-LR exposure from the transcription level, meanwhile the dose response and time course of the cellular response to microcystin was studied. The differentially transcripted genes were classified according to their function through bioinformatics analysis and the functional implications of these genes were discussed. Results:1. In the time course study, more than 2000 differentially transcripted genes were detected, the number in dose-response study was 12. The protein functions of these genes involved in: apoptosis, cell cycle, cell growth and differentiation, transcription regulation, signal transducer, biosynthesis and metabolism, structure molecule and transporter etc.2. 50 genes which were differentially transcripted in all groups of time course study showed a time-dependent manner.3. Among the differentially expressed genes in two types of chips, 13 genesshowed the same transcription manner, most of those genes have not been reported previously to be involved in cellular processes responded to microcystin. Conclusion:1. Many genes were differentially transcripted after MC-LR exposure. The protein functions of these genes involved in a variety of cellular processes.2. There was a little time-dependent manner could be observed in differentially transcripted genes in time course study. Therefore, the biological responses of the long time exposure are not the prolongation of the short time exposure. Microcystin may activate the independent pathway to regulate the cell responses machinery followed different times exposure.3. Those time-dependent genes may be used as bio-marker of microcystin exposure, more work is needed to evaluate the physiological relevance of those genes.