Sawaguchi T., Sawaguchi A.

Dept. of Legal Medicine, Tokyo Women's Medical College, 8-1 Kawadacho, Shinjuku, Tokyo, Japan

Topoisomerase I relaxes supercoiled DNA by introducing transient nicks in the DNA substrate. Detection of DNA nicks requires trapping the enzyme in a cleavage complex using protein denaturants. When using a supercoiled DNA substrate, the resultant covalent DNA/topoisomerase I complex is relatively short, the trapping of nicked intermediates is inefficient; however, a high concentration of inhibitors stabilized the intermediate and thus led to an increase in the nicked DNA product. At lower concentrations of inhibitors, inhibition of relaxation is clearly observed. This forms the basis for a mechanistic drug screen designed to allow detection of agents that affect topoisomerase I by stabilizing the cleaved intermediate complex.
In this report, the inhibition of topoisomerase I activity was tested using a topoisomerase I drug screening kit (TopoGEN, Ohio). Four kinds of concentration of methamphetamine, namely, 100 µM, 200 µM, 500 µM, and 1000 µM, prepared for testing. The procedure was carried out under the instruction manual.
The testing showed there was a more than 200 µM methamphetamine increase of open, circular DNA and that 100 µM methamphetamine inhibits relaxation activity. These results clearly indicate the possibility of inhibition of topoisomerase I activity induced by methamphetamine.