Use of HepG2 cell line for direct or indirect mutagens screening: comparative investigation between comet and micronucleus assays.
In the present study, DNA-damage and clastogenic or aneugenic effects of genotoxic compounds were examined in a metabolically competent human cell line (HepG2 cells) using the micronucleus and the comet assays. Compounds with various action mechanisms were tested: direct mutagens such as 4-nitroquinoline-N-oxide (4-NQO) and methyl methanesulfonate (MMS) and indirect mutagens requiring biotransformation to be active such as N-nitrosodimethylamine (NDMA), benzo[a]pyrene (B[a]P) and 2-acetylaminofluorene (2-AAF). The compounds were first tested for cytotoxicity by measuring their effects on RNA synthesis inhibition in HepG2 cells. 4-NQO, B[a]P and 2-AAF were the most potent compounds; their IC(50) values were, respectively, 1.9 micro M (4h contact), 3.4 and 112 micro M after 20 h. MMS was mildly cytotoxic (IC(50)=0.9 mM) and NDMA had a weak effect (IC(50)=110 mM) after 4h contact. In the micronucleus and comet assays, concentrations required to obtain a significant genotoxic effect in HepG2 cells varied over a broad range, NDMA being active only at very high concentrations. To compare the sensitivity of the two assays, we measured the so-called FIC(2)-the concentration necessary to induce a 2-fold increase of the measured genotoxicity parameter. The data show that genotoxic effects were consistently observed at lower concentrations in the micronucleus test, except in the case of MMS. The measured FIC(2) values were 0.12 micro M (4-NQO), 0.17 micro M (2-AAF), 0.26 micro M (B[a]P) and 6.4mM (NDMA). MMS had such a weak effect in the HepG2 cells that we could not calculate its FIC(2) value. In the comet assay, FIC(2) values were observed, respectively, at 1.48 micro M (4-NQO), 3.67 micro M (B[a]P), 13.42 micro M (MMS) and 27 mM (NDMA). 2-AAF failed to induce DNA-damage in this assay. The present study shows that HepG2 cells could be a suitable tool for assessing the genotoxicity of direct and indirect mutagens and for establishing the lowest genotoxic concentration.
Valentin-Severin
Isabelle
I
UMR (0938), Toxicologie Alimentaire, INRA/ENSBANA, 1, Esplanade Erasme, 21000, Dijon, France.
Le Hegarat
Ludovic
L
Lhuguenot
Jean-Claude
JC
Le Bon
Anne-Marie
AM
Chagnon
Marie-Christine
MC
D015073:2-Acetylaminofluorene / Q000633:toxicity; D015112:4-Nitroquinoline-1-oxide / Q000633:toxicity; D001564:Benzo(a)pyrene / Q000633:toxicity; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000235:genetics*; D020552:Comet Assay / Q000379:methods*; D004128:Dimethylnitrosamine; D006801:Humans; D020128:Inhibitory Concentration 50; D008113:Liver Neoplasms / Q000188:drug therapy / Q000235:genetics*; D008741:Methyl Methanesulfonate / Q000633:toxicity; D015162:Micronucleus Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D009602:Nitrosamines / Q000633:toxicity; D015363:Quinolones / Q000633:toxicity; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
C120195:4-nitroquinolone-1-oxide; D009153:Mutagens; D009602:Nitrosamines; D015363:Quinolones; D001564:Benzo(a)pyrene; D015112:4-Nitroquinoline-1-oxide; D015073:2-Acetylaminofluorene; D008741:Methyl Methanesulfonate; D004128:Dimethylnitrosamine