FAILURE TO OBSERVE A RELATIONSHIP BETWEEN BIS-(BETA-CHLOROETHYL)SULFIDE-INDUCED NAD DEPLETION AND CYTOTOXICITY IN THE RAT KERATINOCYTE CULTURE

It has been proposed that the activation of poly(ADP-ribose) polymeras e (Papirmeister et al., 1985), which results from the presence of stra nd breaks in bis-(beta-chloroethyl)sulfide (BCES) damaged DNA, causes depletion in the level of nicotinamide adenine dinucleotide (NAD) lead ing to cell death. This hypothesis has now been evaluated in the prima ry submerged culture of rat keratinocytes. The DNA content, the viable cell number, and the proliferative capability (measured by thymidine incorporation) of the culture were all reduced 48 h after exposure to 10 mu M BCES. However, the total NAD level, that is, NAD(+) plus NADH, was not changed at a dose of BCES lower than 50 mu M. This observatio n was the same in both proliferating and early differentiating culture s. To further test this hypothesis, the modifying effect of inhibiting poly(ADP-ribose) polymerase on cytotoxicity in BCES-exposed cells was investigated. After exposure to 250 mu M BCES, the NAD level was redu ced to approximately 26 pmol/mu g DNA. This value was increased to 34- 49 pmol/mu g DNA at both 24 and 48 h postexposure when the cultures we re incubated in medium supplemented with 1-10 mM nicotinamide. Neverth eless, the decrease in the DNA content of the culture was not reversed . These results suggest that in the rat keratinocyte culture exposed t o BCES, depletion of NAD is not a prerequisite for cell death.