PROTECTION OF A549 CELLS AGAINST THE TOXIC EFFECTS OF SULFUR MUSTARD BY HEXAMETHYLENETETRAMINE

The A549 cell line was used as a model of the deep lung to study the t oxicity and mechanism of action of sulphur mustard (HD), using the neu tral red (NR) dye retention and gentian violet (GV) assays as indices of cell viability. It was found that exposure to concentrations in exc ess of 40 mu M HD resulted in a rapid onset of toxicity. Exposure to 1 000 mu M HD reduced viability in A549 cell cultures to 61% after 2 h ( control cultures=100%), whereas exposure to 40 mu M HD did not result in deleterious effects until 26 h at which point viability fell to onl y 84% (NR assay). Agarose gel electrophoresis of cell cultures exposed to 40 and 1000 mu M HD and harvested at 4.5, 19 and 43 h after exposu re to HD, indicated that cell death was due to necrosis, despite the o bservation that at the higher concentration of HD cells displayed many of the features common to cells undergoing apoptotic, death. The abil ity of hexamethylenetetramine (HMT)to protect A549 cells against the e ffects of an LC(50) challenge dose of HD was assessed using the GV and NR assays. It was found that HMT (15 mM) could protect cells against the effects of HD though HMT had to be present at the time of HD chall enge. Cultures treated with HD only were 49% viable at 48 h after HD c hallenge, compared to 101% for protected cultures (NR assay) and 58% a nd 91% for unprotected and protected cultures respectively using the G V assay. Morphological observations of GV and NR stained cultures conf irmed these findings. HMT concentrations of 2.5 to 25 mM were used. Ma ximal protection against the toxic effects of HD (LC(50)) was found at 10 to 25 mM HMT, Over this concentration range, HMT did not exert any toxic effects on A549 cells. Pretreatment of A549 cultures with HMT f ollowed by its removal prior to HD challenge had no protective effect, Similarly, treating cultures with HD followed by addition of HMT did not increase the viability of the cultures, even if the HMT was added immediately after HD exposure. HMT was found to protect against the to xic effects of HD, though it must be present at the rime of HD challen ge. A549 cells were found to be a valuable experimental model for stud ying the toxicology of HD and other lung damaging agents, and for scre ening other compounds for potential therapeutic efficacy as a prelude to studies with nontransformed cell culture systems and in vivo models .