DNA-damaging and transcription-terminating lesions induced by AF64A in vitro

Although immediate cholinergic deficits produced by AF64A can be explained adequately by inhibition of enzymes involved in acetylcholine metabolism, the structural similarity of AF64A to a number of DNA-damaging antitumor agents suggested that the observed long-term cholinergic deficits may involve damage to the cell's informational molecules. This study was initiated to determine if AF64A can damage DNA and prematurely terminate RNA transcription in vitro, and to produce cytotoxic and DNA damaging effects in cells exposed to the drug in vivo. The ability of AF64A to produce N-7 guanine alkylations in DNA in vitro was assessed using a modified Maxam and Gilbert DNA sequencing technique, and the ability of AF64A to terminate RNA transcription was assessed by an in vitro RNA transcription system. AF64A was capable of producing extensive dose-dependent N-7 guanine alkylations in DNA fragments exposed to AF64A in vitro, although no sequence specificity of AF64A attack could be discerned. Furthermore, AF64A was able to produce RNA transcription-terminating lesions in vitro, also in a dose-dependent fashion. Transcription of AF64A-damaged DNA resulted in RNA molecules terminated not at every alkylated guanine, but at various discrete sites along the DNA template. AF64A was also found to be cytotoxic in a dose-dependent manner in cultured mouse leukemia L1210 cells. The induced cytotoxicity was accompanied by DNA lesions which were detected as DNA single strand breaks using the DNA alkaline elution technique. The results of these experiments support the hypothesis that AF64A may alter the structure and function of cellular DNA and may help explain the observed long-term cholinergic deficits.