DETECTION OF MNNG-INDUCED DNA LESIONS IN MAMMALIAN-CELLS - VALIDATIONOF COMET ASSAY AGAINST DNA UNWINDING TECHNIQUE, ALKALINE ELUTION OF DNA AND CHROMOSOMAL-ABERRATIONS

Human cells (VH10 or Hep G2) and hamster cells V79 were exposed to dif ferent concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) a nd the level of DNA lesions was evaluated by the DNA unwinding techniq ue, alkaline elution of DNA and the comet assay. All three methods wer e able to detect the effects of MNNG but with a clear difference in se nsitivity. At low concentrations of MNNG the most sensitive method app eared to be the comet assay. After the short-term treatment the comet assay was able to detect the lesions induced by MNNG at approx. 0.1 mu g/ml, alkaline elution of DNA at 1 mu g/ml and DNA unwinding at 1-2 m u g/ml. MNNG treated VH10 cells, human lymphocytes and V79 cells were also tested cytogenetically, confirming that MNNG induced chromosomal aberrations at concentrations > 1 mu g/ml in VH10 cells (short-term tr eatment); > 0.2 mu g/ml in V79 cells (long-term treatment) and > 8 mu g/ml in human lymphocytes (long-term treatment). In some experiments w e tried to increase the level of MNNG-induced DNA breaks with help of DNA repair inhibitors cytosine arabinoside (Ara C) and hydroxyurea (HU ) which were applied either after or during MNNG treatment. Our result s showed that the level of MNNG-induced lesions was increased by simul taneous treatment of cells with MNNG and Ara C and HU. 2 x 10(-5) M Ar a C and 2 x 10(-3) M HU were as effective as 10-times higher concentra tions of inhibitors. Ara C and HU increased the level of MNNG-induced DNA breaks mainly in combination with lower concentrations of MNNG (< 2 mu g/ml). Rejoining of DNA breaks was observed in human cells VH10 a nd Hep G2 as well as in Chinese hamster cells V79 damaged by both lowe r and higher MNNG-concentrations. All methods showed that MNNG-induced DNA breaks had been gradually rejoined.