Bela Toth, D.V.M.

Professor, the Eppley Institute,
Department of Pathology and Microbiology

 

D.V.M. - University of Veterinary Science, Budapest, Hungary, 1956
A. Naturally occurring hydrazines and diazonium ion ingredients of edible mushrooms. Our pioneering investigations have shown that four chemical ingredients, the N’-acetyl derivative of 4-hydroxymethylphenylhydrazine, the tetrafluroborate and sulfate salts of  4-(hydroxymethyl)benzenediazonium ion, p-hydrazinobenzoic acid HCl and $-N-[(-L(+)glutamyl]-4-carboxyphenylhydrazine of the commonly eaten cultivated mushroom Agaricus bisporus, as well as the mushroom itself (raw, baked and lyophilized forms), and six hydrazines, acetaldehyde methylformylhydrazone, pentanal methylformylhydrazone, hexanal methylformylhydrazone, 3-methylbutanal methylformylhydrazone, N-methyl-N-formylhydrazine and methylhydrazine, of the edible false morel mushroom, Gyromitra esculenta, as well as the mushroom itself (raw form), are carcinogenic in laboratory animals. Because of the enormous exposure of the human population in North America and in Europe to Agaricus bisporus and since the world population is sporadically exposed to Gyromitra esculenta, the findings have practical environmental implications. Our work also revealed that the diazonum ion is the first chemical of this class shown to be carcinogenic. Subsequently, our research has proven the carcinogenicities of three additional diazonium salts: 4-methyl-, 4-hydroxy-, and the benzenediazonium sulfates.

B. Identification of synthetic new carcinogenic hydrazines of major environmental importance. A large number of experiments were conducted using mainly laboratory rodents (mice and hamsters) which were treated by various routes with hydrazines and related chemicals and observed for life. The results of these investigations demonstrated that most hydrazine derivatives are carcinogenic substances. We have discovered, for the first time, the carcinogenicity of 40 hydrazines, related chemicals, and hydrazine-containing natural products. The human population is exposed to approximately one-half of these carcinogenic agents in the form of drugs, agricultural herbicides and industrial chemicals. In these series of studies, the following compounds were found to be of major environmental importance: hydralazine, an antihypertensive drug; phenelzine, an antidepressant; alar 85, a herbicide; and methylhydrazine, an ingredient of rocket fuel, etc.
C. Identification of naturally occurring hydrazines. P-Hydrazinobenzoic acid (HBA) and N2-[(-L(+)-glutamyl]-4-carboxyphenylhydrazine (GCPH) were indentified in the cultivated mushroom Agaricus bisporus. This fungus was found to contain 10µg HBA/g and 42 µg GCPH/g. These chemicals were quantitated in the mushroom by high-performance liquid chromatography and their structure confirmed by mass spectrometry.
        In addition, we confirmed the presence of 4-(hydroxymethyl)benzenediazonium ion in Agaricus bisporus. This diazonium ion was found in the mushroom in a concentration of about 1µg/g.
D. Metabolism of hydrazines. We have demonstrated by in vivo and in vitro means the formation of methylhydrazine through an intermediate N-methyl-N-formylhydrazine from gyromitrin, an ingredient of Gytomitra esculenta. It was further shown that $-N-[(-L(+)-glutamyl]-4-hydroxymethylphenylhydrazine (agaritine), a component of Agaricus bisporus, is hydrolyzed to the 4-(hyrdroxymethyl)benzenediazonium ion by an enzyme system present in the mushroom. Finally, we have studied the metabolism of a variety of arylhydrazines, agaritine, N’-acetyl-4-(hydroxymethyl)phenylhydrazine, and 4-methylphenlhydrazine by cytochrome P-450 mediated mixed function oxidases from the mouse liver and by ram seminal vesicle protaglandin (H) synthase. Most of them were readily metabolized, pointing to the possibility that reactive carcinogenic metabolites are produced intracellularly. We also obtained adducts which resulted from reaction of adenine with diazonium ions. The reaction product appeared to be unique and was subsequently fully characterized. Later, we also found 8-arylguanine adducts from arenediazonium ions and DNA. In addition, the para-substituted benzenediazonium ions (-CH3,-CH2OH,-CH2OCH3 and -COOH) produced DNA-DNA crosslinks and DNA single strand breaks in V-79 cells in vitro. Furthermore, because Agaricus bisporus is mainly consumed in baked form, 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 activities of the baked and unbaked mushroom extracts. Finally, we have demonstrated the formation of possible metabolic intermediates derived from N-methyl-N-formylhydrazine (MFH) by chemical and biological oxidation that could act as the ultimate carcinogenic species: a diazenium ion or diazene and then fragmentation to formyl and methyl radicals. Lastly, we reacted 2'-deoxyguanosine, MFH and oxygen in the presence of Cu(II) or Fe(III), which yielded 8'-hydroxy-2'-deoxyguanosine. This adduct may be crucial in the carcinogenesis process.
Phone: (402) 559-4229
Fax: (402) 559-4651
E-Mail: Bela Toth
 
Last update: 01/04/2008