Vacuolating cytotoxin purified from Helicobacter pylori causes mitochondrial damage in human gastric cells

We investigated the effects of vacuolating cytotoxin (VacA) prepared from Helicobacter pylori on the metabolism of gastric epithelial cells, AZ-521. VacA caused the ATP levels to decrease in a time-dependent manner; by approximately 20% in 6 h, 35% in 12 h and 50% in 24 h, at a concentration of 120 nM. This decrease was also dependent on the concentration of VacA. To evaluate the impairment of mitochondria by VacA, mitochondrial membrane potential was estimated by flow cytometric analysis using 3, 3'-dihexyloxacarbocyanine iodide as a substrate. VacA decreased membrane potential with the relative fluorescence intensity of AZ-521 cells in 6 h from 52+/-3 to 24+/-1. Treatment of the cells with bafilomycin A1, a specific inhibitor of vacuolar ATPase proton pump, showed no apparent effect on these changes in the levels of ATP and the mitochondrial membrane potential. Secondly, we estimated the effect of VacA on oxygen consumption. VacA inhibited oxygen consumption in AZ-521 cells: the levels of PO 2 in the medium of control cells decreased by 73% in 3 h and 37% in 6 h, whereas those in VacA-treated cells were 84% in 3 h and 59% in 6 h. Flow cytometric analysis showed the number of cells in the G0/G1 phase was increased by VacA. Taken together, VacA induced an inactivation of energy metabolism followed by mitochondrial damage, leading to impairment of the cell cycle in gastric epithelial cells.