| Abstract
| - Mitogen-activated protein kinases (MAPKs) play a central role in transmitting stress-inducedsignals stimulated by genotoxic agents. The present study is the first to investigate themechanisms by which genotoxic alkylating agents modulate MAPKs by directly measuringthe effects of methylating agents on MAPK activity, DNA methylation, and intracellularglutathione levels. The effects of acetoxymethylmethylnitrosamine (AMMN), N-nitroso-N-methylurethane (NMUR), and N-methyl-N-nitrosourea (MNU) on these parameters werecompared in a fetal rat lung cell line model (MP48). These compounds were chosen becausethey methylate DNA via a methanediazonium intermediate and, therefore, should inducesimilar cellular methylation patterns, although they produce different side products upondecomposition. All three compounds stimulated the activation of the stress-activated MAPKs,c-Jun N-terminal kinase, and p38. In contrast to what has been reported for other methylatingagents, these compounds also stimulated the activation of extracellular signal regulated kinase(ERK), a MAPK typically activated by mitogenic agents. O6-methylguanine (O6-mG) is widelyconsidered to be the critical toxic lesion induced by methylating agents, including AMMN,NMUR, and MNU, which form DNA adducts through SN1 reactions. O6-mG does not appearto be a key regulator of MAPK activity by these compounds, however. There is no directrelationship between the levels of O6-mG and the levels of MAPK activation, and formation ofO6-mG does not appear to be sufficient to stimulate MAPK activation. The present studiesalso indicate that depletion of glutathione is not required or sufficient to stimulate MAPKactivation by the methylating agents investigated here. The use of a pharmacological inhibitorindicates that these methylating agents activate ERK through a signaling pathway thatrequires the ERK kinase MEK. Altogether, these data indicate that genotoxic methylatingagents activate MAPKs through mechanisms that are likely to involve the alkylation of cellulartargets other than DNA.
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