Removal of O6-methylguanine from plant DNA in vivo is accelerated under conditions of clastogenic adaptation.

Abstract

Previously it was shown that the clastogenic efficiency of high doses of alkylating agents in plant root meristems can be reduced significantly by conditioning pretreatment with either a low dose of the same agents, a sublethal heat shock, or heavy metal salts. The molecular mechanisms responsible for these protective effects are still unclear. Here we report on the quantification of O6-methylguanine [O6-MeG] by immuno-slot-blot analysis in DNA of root tip meristems of field bean (Vicia faba) seedlings under conditions of clastogenic adaptation. When root tips were pretreated with a low, conditioning dose of N-methyl-N-nitrosourea (MNU, 10(-4) M, 1 hour) 2 hours before exposure to a high dose of the same clastogen (10(-3) M, 1 hour), the frequency of chromatid aberrations was reduced by more than 50% at a recovery time of 1 B hours, as compared to treatment with the high dose alone. The same was observed when conditioning pretreatment was by a sublethal heat shock [10 minutes, 40 degrees C] or a heavy metal salt (Cd(NO)3, 10(-7) M, 1 hour). The frequency of O6-MeG immediately after exposure to a conditioning and a subsequent challenge treatment was reduced by 43% as compared to treatment with only the high dose. At a recovery time of 18 hours the corresponding frequency of adducts was reduced by 68.3% (related to the initial level) after treatment with the high dose alone, and by 81.3% under adaptive conditions. Sublethal heat shock or heavy metal salt used as conditioning pretreatments also resulted in a decrease of adducts immediately after treatment with the challenge dose. From these data and from prevention of the effects by pretreatment with cycloheximide or O6-benzylguanine we conclude that under conditions of clastogenic adaptation O6-MeG is more efficiently removed from the DNA, presumably by induction of an alkyl acceptor protein such as O6-methylguanine-DNA methyltransferase [MGMT]. This could explain the observed protective effects (clastogenic adaptation.