RELATIONSHIPS BETWEEN CELL-DENSITY, GLUTATHIONE AND PROLIFERATION OF A549 HUMAN LUNG ADENOCARCINOMA CELLS TREATED WITH ACROLEIN

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde to which humans are exposed in various situations. Acrolein reacts rapidl y with and depletes cellular glutathione (GSH), and is toxic to variou s types of cells. In the current study, the ability of acrolein to alt er proliferation of A549 cells was found to be dependent on cell densi ty as well as total cell number. Thus, 'doses' must be expressed per c ell rather than as a concentration, and all related studies need to be performed by plating a constant number of cells. A549 cells were plat ed at various densities and treated with acrolein after 48 h. Acrolein doses up to 47 fmol/cell at the time of treatment did not cause cell lethality. However, growth of A549 cells (as shown by thymidine incorp oration, alamarBlue and total protein) was inhibited at acrolein level s > 34 fmol/cell in 6-well plates seeded at 5000 cells/cm(2) 48 h prio r to treatment. Cellular GSH levels were decreased 34% by 2 h at acrol ein levels of 6.7 fmol/cell and by 65% at 47 fmol/cell. Recovery of GS H was rapid at 6.7-47 fmol/cell acrolein, returning to control levels or above by 12 h post-treatment. These data show a strong correlation between cellular GSH and proliferation. The apparent conflict with a p revious study of Ramu et al., suggesting that sublethal concentrations (up to 10 mu M) of acrolein inhibited the proliferation of A549 cells without a decline in total cellular GSH, arose because, while the acr olein concentration was the same in cells used for proliferation and G SH assays, GSH measurements were done in cells plated at a higher dens ity, resulting in a much lower acrolein dose per cell. Interestingly, very low dose levels of acrolein with cells seeded at low densities st imulated cell growth despite an initial decline in GSH content. Prelim inary studies with the stress genes hsp70 and gadd153 suggest that acr olein at 35 fmol/cell does not stimulate formation of their mRNA beyon d the level stimulated by a 2 h incubation in serum-free medium but ma y actually delay or decrease the induced expression. The mechanism(s) of the inhibitory and mitogenic effects of acrolein remains to be dete rmined, but could be due to changes in gene expression induced by this electrophile, perhaps mediated by changes in GSH. (C) 1997 Elsevier S cience Ireland Ltd.