Depletion of Adenosine Diphosphate-Ribosyl Transferase Activity in Rat Liver during Exposure to N-2-Acetylaminofluorene: Effect of Thiols

Abstract

The exposure of rats to a feeding regimen containing N-2-acetylaminofluorene (2AAF) causes an accumulation of lesions on liver DNA and a progressive impairment in DNA repair capacity. We used the in vivo experimental model of Teebor and Becker (Cancer Res., 31: 1–3, 1971) with the carcinogen given to rats during four consecutive cycles, each one composed of 3 weeks of treatment and 1 week of recovery. The extent of DNA damage and repair was determined during each cycle by the alkaline elution technique. The results obtained showed that the number of alkalilabile sites in DNA is significantly enhanced after the first cycle and remains increased during following cycles.

Since ADP-ribosyl transferase (ADPRT) is known to play a central role in the response to DNA damage, we investigated the effect of 2AAF on this enzyme during the carcinogenic process. The activity and the structure of ADPRT were analyzed using the activity gel and Western blot techniques. The catalytic band with a molecular weight of 116,000, clearly evident in liver extracts of control rats, was no longer detectable after one cycle of exposure to 2AAF returning progressively to an almost normal level within the last two cycles. When the aminothiol N-acetyl-l-cysteine (NAC) was added to the 2AAF diet, the extent of DNA damage was drastically reduced, and DNA repair activity preserved for a longer period. In addition, the loss of ADPRT was not observed after the first cycle, but delayed to the end of the second, indicating that NAC exerts a protective effect on DNA and on ADPRT. Such effect was not evident when NAC was substituted by glutathione.

The analysis of liver extracts on Western blot showed that the ADPRT immunoreactive band was almost undetectable after the first cycle suggesting that the loss in enzyme activity could be due to a block in de novo synthesis of the enzyme and not to an inhibition of its activity.