Phenobarbital, oxazepam and Wyeth 14,643 cause DNA damage as measured by the Comet assay

Although phenobarbital, oxazepam and Wyeth 14,643 are carcinogens that do n ot form DNA adducts, they induce mutations in the Big Blue(R) transgenic mo use model. The mutations produced by these compounds were predominantly G-- >T and G-->C transversions that we suspect arose from oxidative damage to D NA. To test this, we employed the single cell electrophoresis (Comet) assay that detects alkali-labile lesions in cells sustaining DNA damage. Human m yeloid leukemia K562 cells were treated with non-cytotoxic doses of the abo ve compounds for 3 h, then placed on slides containing low melting point ag arose. Cells were lysed, exposed to alkaline buffer, electrophoresed and an alyzed by microscopy for the existence of DNA damage. Extensive DNA damage, most likely due to the existence of single- and double-strand breaks and a purinic/ apyrimidinic (AP) sites, was observed in cells exposed to oxazepam (1 mM) and Wyeth 14,643 (0.5 mM). On the other hand, damage of this sort w as not observed in cells exposed to phenobarbital (1 mM). However, the addi tion of S9 liver extracts to cells exposed in the presence of phenobarbital resulted in significant amounts of DNA damage. We conclude from these stud ies that two of the three compounds evaluated in this study mediate their m utagenic. effects through oxidative stress, but that the mechanism of DNA d amage caused by phenobarbital differs from that elicited by oxazepam and Wy eth 14,643.