The metabolically competent human lymphoblastoid cell line MCL-5 was treated with a panel of mutagens to assess the induction of DNA damage. Treatment effects were observed by monitoring cell proliferation and by single-cell gel electrophoresis (SCGE). The direct-acting mutagens benzo[a]pyrene-7,8-diol 9, 10-epoxide (BPDE) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), as well as pro-mutagens requiring metabolic activation, i.e. benzo[a]pyrene (BaP), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyri-dine (PhIP), 4-N-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and cigarette-smoke condensate (CSC), were assayed by SCGE. Assay schemes were adapted for the MCL-5 cell line and for low levels of strand break induction, by inclusion of the DNA synthesis inhibitors cytosine arabinoside and hydroxyurea, and by extending the electrophoresis time. For all mutagens tested, dose-dependent increases of median and average tail moment values among 50 nucleoids per slide were observed. The determining factors for selecting the treatment doses for mutation-induction experiments were the solubility of BaP and PhIP in the exposure medium, and the cytotoxicity exhibited by BPDE, MNNG and CSC. Induction of DNA strand breaks was obtained at mutagen concentrations permitting sufficient cell proliferation, except in the case of MNNG.
AmesB.N., DurstonW.E., YamasakiE., & LeeF.D. (1973). Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proceedings of the National Academy of Sciences, USA70, 2281–2285.
2.
HaneltS., HelbigR., HartmannA., LangM., SeidelA., & SpeitG. (1997). A comparative investigation of DNA adducts, DNA strand breaks and gene mutations induced by benzo[a]pyrene-7,8-diol 9,10-oxide in cultured human cells. Mutation Research390, 179–188.
3.
FairbairnD.W., OliveP.L., & O'NeillK.L. (1995). The comet assay: a comprehensive review. Mutation Research339, 37–59.
4.
SinghN.P. (2000). Microgels for estimation of DNA strand breaks, DNA protein crosslinks and apoptosis. Mutation Research455, 111–127.
5.
KumaravelB., & KumaravelT.S. (1999). Comet Assay Interest Group Website. Web site: www.cometassay.com (Accessed 01/07/01).
6.
CrespiC.L., GonzalezF.J., SteimelD.T., TurnerT.R., GelboinH.V., PenmanB.W., & LangenbachR. (1991). A metabolically competent human cell line expressing five cDNAs encoding procarcinogen-acti-vating enzymes: application to mutagenicity testing. Chemical Research in Toxicology4, 566–572.
7.
KrauseG., GargantaF., VrielingH., & SchererG. (1999). Spontaneous and chemically induced point mutations in HPRT cDNA of the metabolically competent human lymphoblastoid cell line, MCL-5. Mutation Research431, 417–428.
8.
WolzL., KrauseG., & SchererG. (2001). Oncogenic mutations in a human lymphoblastoid cell line after treatment with environmental mutagens. Naunyn Schmiedeberg's Archives of Pharmacology363, R155.
9.
MartinF.L., ColeK.J., OrmeM.H., GroverP.L., PhillipsD.H., & VenittS. (1999). The DNA repair inibitors hydroxyurea and cytosine arabinoside enhance the sensitivity of the alkaline single-cell gel electrophoresis (“comet”) assay in metabolically-competent MCL-5 cells. Mutation Research445, 21–43.
10.
KrauseG., WolzL., & SchererG. (2001). Performing the “comet assay” for genetic toxicology applications. Life Science News7, 7–8.
11.
TiceR.R., AgurellE., AndersonD., BurlinsonB., HartmannA., KobayashiH., MiyamaeY., RojasE., RyuJ-C., & SasakiY.F. (2000). Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environmental and Molecular Mutagenesis35, 20–221.
12.
OliveP.L., BanathJ.P., & DurandR.E. (1990). Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the “comet” assay. Radiation Research122, 86–94.
13.
AndersonD., YuT-W., PhillipsB.J., & SchmezerP. (1994). The effect of various antioxidants and other modifying agents on oxygen-radical-generated DNA damage in human lymphocytes in the COMET assay. Mutation Research307, 261–271.
14.
DiemE., & RüdigerH.W. (1999). Mikrokerntest und Comet Assay: Ein Ergebnisvergleich bei Normalprobanden. Arbeitsmedizin Sozialmedizin Umweltmedizin34, 437–441.
15.
SomorovskaM., SzabovaE., VodickaP., TulinskaJ., BarancokovaM., FabryR., LiskovaA., RiegerovaZ., PetrovskaH., KubovaJ., RausovaK., DusinskaM., & CollinsA. (1999). Biomonitoring of genotoxic risk in workers in a rubber factory: comparison of the comet assay with cytogenetic methods and immunology. Mutation Research445, 181–192.
16.
CollinsA.R., DobsonV.L., DusinskáM., KennedyG., & StetinaR. (1997). The comet assay: what can it really tell us?Mutation Research375, 183–193.
17.
OliveP.L. (1999). DNA damage and repair in individual cells: applications of the comet assay in radio-biology. International Journal of Radiation Biology75, 395–405.
18.
RoserS., Pool-ZobelB-L., & RechkemmerG. (2001). Contribution of apoptosis to responses in the comet assay. Mutation Research497, 169–175.
19.
KleinsasserN.H., WallnerB.C., KastenbauerE.R., MuenzenriederR.K., & HarréusU.A. (2000). Comparing the genotoxic sensitivities of human peripheral blood lymphocytes and mucosa cells of the upper aerodigestive tract using the Comet assay. Mutation Research467, 21–30.
20.
BignamiM., O'DriscollM., AquilinaG., & KarranP. (2000). Unmasking a killer: DNA O6-methylgua-nine and the cytotoxicity of methylating agents. Mutation Research462, 71–82.
21.
UhlM., HelmaC., & KnasmüllerS. (1999). Single-cell gel electrophoresis assays with human-derived hepatoma (Hep G2) cells. Mutation Research441, 215–224.
22.
UhlM., HelmaC., & KnasmüllerS. (2000). Evaluation of the single cell gel electrophoresis assay with human hepatoma (Hep G2) cells. Mutation Research468, 213–225.
23.
AnnasA., BritteboE., & HellmanB. (2000). Evaluation of benzo[a]pyrene-induced DNA damage in human endothelial cells using alkaline single cell gel electrophoresis. Mutation Research471, 145–155.
24.
MorgenthalerP-M.L., & HolzhäuserD. (1995). Analysis of mutations induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human lymphoblastoid cells. Carcinogenesis16, 713–718.
MøllerP., KnudsenL.E., LoftS., & WallinH. (2000). The comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors. Cancer Epidemiology Biomarkers and Prevention9, 1005–1015.
27.
GlückU., GebbersJ-O., SutherlandB.M., & WoodheadA.D. (2000). The comet assay of nasal epithelia: measurement of DNA damage for the assessment of genotoxic air pollution. Laryngoscope110, 123–125.
28.
YangQ., HergenhahnM., WeningerA., & BartschH. (1999). Cigarette smoke induces direct DNA damage in the human B-lymphoid cell line Raji. Carcinogenesis20, 1769–1775.
29.
PryorW.A., StoneK., ZangL-Y., & BermúdezE. (1998). Fractionation of aqueous cigarette tar extracts: fractions that contain the tar radical cause DNA damage. Chemical Research in Toxicology11, 441–448.
30.
LeeJ-G., MaddenM.C., ReedW., AdlerK., & DevlinR. (1996). The use of the single cell gel electrophoresis assay in detecting DNA single strand breaks in lung cells in vitro.Toxicology and Applied Pharmacology141, 195–204.
31.
LeonardS.S., WangS., ShiX., JordanB.S., CastranovaV., & DubickM.A. (2000). Wood smoke particles generate free radicals and cause lipid peroxidation, DNA damage, NFkB activation and TNF-alpha release in macrophages. Toxicology150, 147–157.
32.
WolzL., KrauseG., SchererG., AufderheideM., & MohrU. (2002). In vitro genotoxicity assay of sidestream smoke using a human bronchial epithelial cell line. Food and Chemical Toxicology, 40, 845–850.
