Inhibition of DNA methylase activity by acrolein

Acrolein, a reactive metabolite of cyclophosphamide, may be responsible for bladder cancer induced by cyclophosphamide. DNA methylase was isolated from the liver and urothelium of rats by high salt extraction of purified nuclei. Acrolein at 10 microM inhibited liver and bladder DNA methylase activity by 30-50%. Kinetic studies with the liver enzyme showed a competitive type of inhibition with a Ki of 6.7 microM. Both dithiothreitol and glutathione afforded protection to the enzyme when added to the assay. At near equimolar concentrations of glutathione to acrolein, the methylase retained 80-90% activity. An increase in DNA had no effect on the inhibition by acrolein, whereas increased amounts of protein protected against acrolein inhibition, suggesting that acrolein reacted with the DNA methylase protein. On the other hand, DNA that had been reacted with acrolein was unable to serve as a substrate for DNA methylase. As the DNA adducts increased the methylation of the DNA decreased. Thus, acrolein has the ability to react with DNA and the DNA methylase protein, either of which results in inhibition of DNA methylation.