A Proteomic Analysis of the Effects of Yessotoxin

Abstract

Seafood contaminated with diarrhetic shellfish poisoning (DSP) toxins cause a shortterm gastrointestinal illness characterised by diarrhea, vomiting and nausea. Yessotoxin (YTX) is classified as a DSP toxin because it often accompanies okadaic acid and the dinophysistoxins, which are toxins responsible for DSP. YTX causes the death of mice used in the routine monitoring of DSP toxins. However, YTX exhibits toxicological effects dissimilar to most DSP toxins. Unlike okadaic acid and the dinophysistoxins, YTX neither causes diarrhetic symptoms in mice after oral administration nor in vitro inhibition of protein phosphatase 2A. Intraperitoneal injection of YTX in mice causes cardiotoxic, neurotoxic and possibly tumourigenic effects. Morphological, apoptotic and specific protein changes have been uncovered from cellular studies with YTX, but no obvious mode of action exists. An investigation into the effects of YTX on protein expression in mouse liver and heart and the human HepG2 hepatocarcinoma cell line was conducted using a proteomic approach. Two-dimensional gel electrophoresis was used to separate proteins from individual samples, with matrix-assisted light desorption/ionisation time-of-flight mass spectrometry utilised for protein identification. Identified proteins that display a statistically significant change between control and toxin treatments may provide an insight into the cellular effects of YTX and the mechanisms that mediate its toxicity. Liver and heart from mice intraperitoneally dosed with 300 µg/kg YTX were assessed for in vivo protein abundance differences that may contribute to the effects of YTX. Liver carbonic anhydrase II displayed a YTX-dependant decrease in abundance and was the only statistically significant YTX-affected hepatic protein identified. A heart protein that was matched to the hypothetical protein RIKEN cDNA 2700085E05 exhibited a significant decrease in abundance in gels from YTX-dosed mice. However, no conclusions could be established regarding the effects of YTX in the liver or heart due to concerns with mouse interindividual variability. HepG2 cells were exposed to YTX to examine changes in protein abundance and to explore the possibility of an YTX bioassay based on these responses. At 1.4 µM YTX, a rounded cell morphology was observable by twelve hours. Of the eightythree spot expression profiles that changed significantly at the 5% level, profiles from twenty-seven spots were affected within twenty-four hours of YTX exposure. Many affected proteins were from the heterogeneous nuclear ribonucleoprotein (hnRNP), lamin and cathepsin families. In particular, the abundance of a complete hnRNP H1 protein increased, whereas two truncated hnRNP H1 isoforms decreased expression because of YTX exposure. Other affected hnRNP proteins include the A2/B1, E1, K and L members. Further investigations are required to ascertain whether these protein changes are in vivo and/or delayed effects of the toxin. Based on the hnRNP H1 expression changes induced by YTX, bioassay development is discussed. The HepG2 protein changes reported in this thesis have revealed new insights into the cellular effects of YTX.