Real-time concurrent monitoring of apoptosis, cytosolic calcium, and mitochondria permeability transition for hypermulticolor high-content screening of drug-induced mitochondrial dysfunction-mediated hepatotoxicity.

A quantitative high-content screening (HCS) was suggested for the real-time monitoring of drug-induced mitochondrial dysfunction-mediated hepatotoxicity. This HCS is very advantageous in that it allows simultaneous observation of drug-induced activations of hepatotoxic pathways using hypermulticolor cellular imaging. The mitochondrial permeability transition (MPT), cytosolic calcium, and caspase-3 were selected as functional markers to verify drug-induced hepatotoxicity and were concurrently monitored in HepG2 cells in a real-time manner. Nefazodone, tolcapone, and troglitazone caused mitochondrial dysfunction and subsequent apoptotic HepG2 cell death in addition to marked cytosolic calcium increase. On the other hand, extrinsic pathway-mediated apoptotic cell death was monitored when HepG2 cells were treated with piroxicam. It was found that piroxicam-treated HepG2 cells showed apoptotic cell death without the MPT formation, while a cytosolic calcium increase was clearly observed. This finding was confirmed by the caspase-8 inhibition assay. These results demonstrated the unique potential of real-time hypermulticolor HCS to screen hepatotoxic drugs at the in vitro stage rather than the later in vivo stage based on an animal model and to ultimately reduce the probability of drug failure.

Determination of UV-induced DNA damages to suppress protein expression using reporter gene assay-based single cell cotransfection imaging cytometry.

Effects of UVB or UVC-induced DNA damage were simultaneously monitored at single cellular level by analyzing the change of yellow fluorescent protein (YFP) and red fluorescent protein (RFP) expression in human embryonic kidney (HEK) 293 cells using multicolor single-cell imaging cytometry. The method is based on the idea that there exists a quantitative correlation between the degree of UV-induced DNA damage and protein expression. A cotransfection assay was performed using UVB irradiated YFP and UVC irradiated RFP genes to eliminate cell-to-cell variation in protein expression yield. Up to an UVB irradiation dose of 50kJ/m(2), YFP expression yield did not change compared to control. On the other hand, RFP expression yield decreased remarkably as the UVC dose increased from 79.5 to 159J/m(2). The results showed that a certain level of DNA damage is efficiently repaired by intracellular repair mechanism and does not influence protein mutation. In addition, it was found that the amount of DNA damage induced by UVB in sunlight would not interfere with normal protein expression in the human body. Single-cell imaging cytometry is a cell lysis-free approach to directly monitor the intracellular correlation between the degree of UV-induced DNA damage and protein expression.

Glycosylation of flavonoids with E. coli expressing glycosyltransferase from Xanthomonas campestris.

Glycosyltransferase family 1 (UGT) uses small chemicals including phenolics, antibiotics, and alkaloids as substrates to have an influence in biological activities. A glycosyltransferase (XcGT-2) from Xanthomonas campestris was cloned and consisted of a 1,257 bp open reading frame encoding a 45.5 kDa protein. In order to use this for the modification of phenolic compounds, XcGT-2 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. With the E. coli transformant expressing XcGT-2, biotransformation of flavonoids was carried out. Flavonoids having a double bond between carbons 2 and 3, and hydroxyl groups at both C-3' and C-4', were glycosylated and the glycosylation position was determined to be at the hydroxyl group of C-3', using nuclear magnetic resonance spectroscopy. These results showed that XcGT-2 regiospecifically transferred a glucose molecule to the 3'-hydroxyl group of flavonoids containing both 3' and 4'-hydroxyl groups.