Development and validation of microcystin biomarkers for exposure studies

Microcystins (MCs) are hepatotoxic cyanotoxins produced mainly by the cyanobacteria Microcystis spp. They are distributed in waterbodies worldwide, and the toxicity on exposure to MCs was reported worldwide in fish, animals and in humans for over a century. There are about 70 known variants of MCs to date and of them the most toxic and widely distributed MC is Microcystin-LR (MCLR). MCLR is hepatotoxic and a potent tumor promoter. Epidemiological studies in China have linked exposure to MCs with high incidence of liver cancer. Although analytical tools have been reported to detect MCs in water and in food samples, and biomarkers for biochemical alterations like inhibition of protein phosphatases (PP) have been proposed, to date, validation of these analytical methods for simultaneous measurement of MCs in environmental samples and in body fluids of exposed individuals has not yet been done. In this study, we have developed new methods and validated existing methods to detect MCLR and its biomarkers in body fluids of animals and human hepatic cells treated with different concentrations of MCLR. The methods thus validated were used to monitor the seasonal fluctuations in MCLR concentrations in two lakes of western Texas. Studies were also conducted to explore molecular level targets of MCLR in normal (THLE-2) and cancerous (HepG2) human hepatic cell lines. Effect of MCLR on cell proliferation was explored, and validated methods were used to detect alteration in PP activity in these cell lines on exposure to MCLR. Alteration in expression of apoptosis regulatory proteins like Bax, Bcl2, Bad and PP2A on exposure to MCLR was also studied by immunoblotting in these cell lines. Studies were also conducted to explore molecular level targets of MCLR on acute exposure to a single dose, and on subacute exposure to repeated doses of MCLR in F-344 rats. We observed a dose dependent alteration in expression of PP2A, Bax, Bcl2 and Bad in both acute and subchronic exposures, as quantified by western blotting and immunohistochemistry. The study also focused on alteration in levels of sphingolipids in serum on exposure to MCLR. In another experiment, the combinative toxic effect of MCLR along with the tumor initiator aflatoxin-B1 was studied in normal and hepatocarcinoma cell lines, and the mechanism involved was explored.