CONTRIBUTION OF HYDRAZINES-DERIVED ALKYL RADICALS TO CYTOTOXICITY ANDTRANSFORMATION-INDUCED IN NORMAL C-MYC-OVEREXPRESSING MOUSE FIBROBLASTS

Several hydrazine derivatives (KD) tested so far have pharmacological activities, but many also have toxic side effects, including carcinoge nesis, Their toxicity has been ascribed to carbocations (via formation of azoxy intermediates), alkyl radicals or reactive oxygen species, C ytotoxicity and transformation by carbocations is widely accepted, but the role of alkyl radicals is still questioned, We have investigated the cytotoxicity of HD to mouse fibroblasts in three activation system s in which enhanced alkyl radical formation is demonstrated by electro n spin resonance/spin-trapping. Cytotoxicity was assayed by inhibition of [H-3-methyl]thymidine uptake into DNA of Balb/c 3T3 and/or Myc 9E fibroblasts (normal Balb/c 3T3 cells over-expressing the c-myc proto-o ncogene). Based on the results obtained in the cytotoxicity assays we also investigated the transforming potential of procarbazine (PCZ) and methylhydrazine (MeH) activated by horseradish peroxidase (HRP) using the Myc 9E cell line, which aims at the activation of a second cooper ating oncogene, Our results show that: (i) cytotoxicity of no to mouse fibroblasts is increased by HRP activation of MeH, phenelzine and PCZ , which displayed enhanced alkyl radical formation, but not of 1,2-dim ethylhydrazine (DMH), which did not produce increased alkyl radical fo rmation under these conditions; (ii) cytotoxicity of neutrophil-activa ted MeH (producing a 10-fold higher concentration of methyl radicals), is more pronounced than DMH; (iii) Men and DMH activated by prolonged auto-oxidation in 24-h incubations have comparable cytotoxicity and a lkyl radical formation; and (iv) PCZ and MeH activation by HRP to alky l radicals increased the transformation induced in Myc 9E cells, Taken together, our results strongly support a role for hgdrazine-derived a lkyl radicals in HD-induced cytotoxicity and cell transformation.