Cellular NAD+ and ATP levels in alkylation-induced cytotoxicity enhanced by an inhibitor of poly(ADP-ribose) synthesis.

Alkylating agents cause a marked depletion of cellular NAD+ levels by activating nuclear ADP-ribosyl transferase (ADPRT), which utilizes NAD+ as a substrate in the synthesis of poly(ADP-ribose). As a consequence of NAD+ depletion, it is possible that cellular ATP pools could be depleted. Because of this, exogenously supplied NAD+ had been proposed as a way to counteract some of the effects of an alkylator. We found that exogenously supplied NAD+ significantly increased intracellular levels of NAD+ in MMS- and MNNG-treated V79 Chinese hamster cells. Cytotoxicity was not changed by the exogenously supplied NAD+, however. 3-Aminobenzamide (3-ABA), an ADPRT inhibitor, prevented the depletion of intracellular NAD+ by MMS or MNNG treatment and potentiated cytotoxicity. As was the case without 3-ABA, exogenously supplied NAD+ plus 3-ABA did not change the cytotoxicity, even though NAD+ levels were increased. Intracellular ATP levels were also measured and were found to be unaffected following MMS treatment, and only slightly depleted following MNNG treatment. Exogenously supplied NAD+ raised these levels above those for their respective controls. Because survival was unaffected by elevated levels of NAD+ and ATP, our results suggest that depletion of cellular NAD+ pools following MMS and MNNG treatment is not a critical factor in determining cytotoxicity for these V79 cells. The energy reserves of V79 cells, at doses of MMS or MNNG which kill 99% of the cells, are apparently adequate to maintain normal levels of ATP.