The commonly eaten, cultivated mushroom, Agaricus bisporus (AB), contains several N-N-bond-containing chemicals. We demonstrated earlier the presence, carcinogenicity and mode of action of some of these mushroom chemicals. In the recent grant, we have shown the carcinogenicity of the uncooked mushroom and some of its ingredients in long-term experiments in mice. In addition, we have revealed the presence of two hydrazines: p-hydrazinobenzoic acid and beta-N-[gammaL(+)-glutamyl]-4- carboxyphenylhydrazine in the mushroom extracts. Further, we demonstrated in vitro the metabolism of several mushroom arylhydrazines using cytochrome P-450 mixed function oxidases and prostaglandin (H) synthase enzymes. Finally, using purine bases and nucleosides, we have modeled the reactions of 4-(hydroxymethyl)benzenediazonium ion (HMBD) with DNA and have observed that HMBD can react with DNA. Secondary reactions (e.g., cross-linking) may be important. Lastly, we determined the amount of hydrazines in the mushroom after baking: Only 25% was destroyed. Also, using the Ames assay we noted no significant difference between the mutagenic activities of the baked and unbaked mushroom extracts. In this new continuing proposal, we intend to extend our investigations to: (1) study the carcinogenicity of baked, lyophilized and raw AB by using a dose response protocol in lifelong experiments in mice; (2) reveal the tumorigenicity of 4-(hydroxy-benzenediazonium sulfate in mice; (3) analyze AB grown by us using radiolabeled compounds for the presence of diazonium ions and hydrazines; (4) determine the products of the reaction of HMBD, other diazonium ions and their reactive species with bases, nucleosides and DNA in vitro at physiological pH and in isolated cells; and (5) synthesize the above and other chemicals for analytical chemistry, biochemical and carcinogenicity studies. Positive findings from this project might have practical implications, because humans consume mainly baked mushrooms. Further, the mechanistic studies on mushroom hydrazines and related chemicals might help to reveal their mode of action on the molecular level. The annual estimated mushroom consumption in the United States was approximately 435 million kilograms in 1990-91. The corresponding figure in western Europe was more than 644 million kilograms.