Activation of AP-1 through the MAP kinase pathway: A potential mechanism of the carcinogenic effect of arenediazonium ions

Arenediazonium ions such as those found in the common mushroom Agaricus bis porus have been convincingly demonstrated to be tumorigenic. The specific m echanism of their tumorigenicity remains unclear. It has been shown that ar enediazonium ions can be metabolized to aryl radicals, and that reaction of these aryl radicals with DNA produces aryl adducts. These metabolic proces ses also produce the reactive oxygen species superoxide and hydroxyl radica ls which have been implicated in AP-1 activation. To further investigate th e mechanism of tumorigenesis by arenediazonium ions, we studied the effect of a representative arenediazonium ion on AP-1 activation and phosphorylati on of the signal transduction proteins ERK1, ERK2, JNK, and p38 kinase, bot h in vitro and in vivo. We also identified the specific radicals produced b y spin trapping and ESR analysis. Here, it was found that p-methylbenzenedi azonium ion (2a) induced a 1G-fold increase in the extent of AP-1 activatio n at micromolar concentrations, and that this increase coincided with phosp horylation of the signaling kinases ERK1 and -2 and p38 kinase, but not JNK , in JB6 mouse epithelial cells. In vivo studies using AP-I luciferase repo rter-bearing transgenic mice supported the increase in the extent of AP-I a ctivation in Ba-treated mice over controls, and showed that this effect was different in different tissue types. The antioxidant N-acetylcysteine (NAC ), a general antioxidant, showed an inhibitory effect on aa-mediated AP-1 i nduction, while aspirin, a hydroxyl radical scavenger, had no effect. Spin trapping studies showed that while NAC suppressed radical formation from 2a , aspirin did not alter radical production from 2a. It appears that 3a, a c arbon-centered radical formed from 2a, is responsible for AP-1-induced acti vation, and therefore, radical species that are not oxygen-centered are als o capable of inducing AP-1. These results represent a step toward understan ding the mechanism of tumorigenicity of arenediazonium ions.