Mutagenicity of 11 cigarette smoke condensates in two versions of the mouse lymphoma assay

Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA.
Mutagenesis (Impact Factor: 2.79). 10/2010; 26(2):273-81. DOI: 10.1093/mutage/geq083
Source: PubMed


Cigarette smoke condensate (CSC) is genotoxic in nearly all assays in which it has been tested. In this study, we investigated the mutagenicity of 11 CSCs using the microwell and soft-agar versions of the mouse lymphoma assay (MLA). These CSCs were prepared from commercial or experimental cigarettes, 10 of them were produced using International Organisation for Standardisation (ISO) conditions and one CSC was generated using intense Massachusetts Department of Public Health (MDPH) conditions. In the presence of rat liver S9, the L5178Y/Tk(+/-) mouse lymphoma cells were treated with 11 CSCs at different concentrations (25-200 μg/ml) for 4 h. All CSCs resulted in dose-dependent increases of both cytotoxicity and mutagenicity in both versions of the MLA. The mutagenic potencies of the CSCs were calculated as mutant frequency per microgram CSC from the slope of the linear regression of the dose-response curves and showed no correlations with the tar yield of the cigarette or nicotine concentrations of the CSCs. Comparing two CSCs produced from the same commercial cigarettes using two different smoking conditions, the one generated under ISO conditions was more mutagenic than the other generated under intense conditions on a per microgram CSC basis. We also examined the loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11 for the mutants induced by 11 CSCs. The most common type of mutation observed was LOH with chromosome damage spanning less than ∼34 Mbp. These results indicate that the MLA identifies different genotoxic potencies among a variety of CSCs and that the results from both versions of the assay are comparable.

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    • "The cell pellets were quickly frozen and stored at À20 °C. The procedures for genomic DNA extraction, PCR, and agarose gel electroph oresis have been described previousl y (Guo et al., 2011 ). One band was scored as LOH and retention of two bands was scored as non-LOH at the given microsatelit e locus. "
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    ABSTRACT: 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) is a low molecular weight nitroxide and stable free radical. In this study, we investigated the cytotoxicity and genotoxicity of TEMPO in mammalian cells using the mouse lymphoma assay (MLA) and in vitro micronucleus assay. In the absence of metabolic activation (S9), 3 mM TEMPO produced significant cytotoxicity and marginal mutagenicity in the MLA; in the presence of S9, treatment of mouse lymphoma cells with 1-2 mM TEMPO resulted in dose-dependent decreases of the relative total growth and increases in mutant frequency. Treatment of TK6 human lymphoblastoid cells with 0.9-2.3 mM TEMPO increased the frequency of both micronuclei (a marker for clastogenicity) and hypodiploid nuclei (a marker of aneugenicity) in a dose-dependent manner; greater responses were produced in the presence of S9. Within the dose range tested, TEMPO induced reactive oxygen species and decreased glutathione levels in mouse lymphoma cells. In addition, the majority of TEMPO-induced mutants had loss of heterozygosity at the Tk locus, with allele loss of ⩽34 Mbp. These results indicate that TEMPO is mutagenic in the MLA and induces micronuclei and hypodiploid nuclei in TK6 cells. Oxidative stress may account for part of the genotoxicity induced by TEMPO in both cell lines.
    Toxicology in Vitro 03/2013; 27(5). DOI:10.1016/j.tiv.2013.02.019 · 2.90 Impact Factor
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    • "The Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) has recommended descriptive statistics, to characterise PM dose responses in the Ames test and IVMNT (CORESTA, 2004, 2010). The in vitro methods have also been able to quantitatively differentiate PMs from a variety of cigarettes (DeMarini et al., 2008; Guo et al., 2011; Roemer et al., 1998). Novel tobacco materials can reduce PM genotoxicity (Combes et al., 2012; McAdam et al., 2011). "
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    ABSTRACT: In vitro genotoxicity assays are often used to compare tobacco smoke particulate matter (PM) from different cigarettes. The quantitative aspect of the comparisons requires appropriate statistical methods and replication levels, to support the interpretation in terms of power and significance. This paper recommends a uniform statistical analysis for the Ames test, mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT); involving a hierarchical decision process with respect to slope, fixed effect and single dose comparisons. With these methods, replication levels of 5 (Ames test TA98), 4 (Ames test TA100), 10 (Ames test TA1537), 6 (MLA) and 4 (IVMNT) resolved a 30% difference in PM genotoxicity.
    Toxicology in Vitro 03/2013; 27(4). DOI:10.1016/j.tiv.2013.02.015 · 2.90 Impact Factor
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    • "lymphoma cell line was utilized for the mutation assay. Cells were grown according to previously published method [Guo et al., 2011]. Briefly, the basic medium was Fischer's medium for leukemic cells of mice with L-glutamine supplemented with pluronic F68 (0.1%), sodium pyruvate (1 mM), penicillin (100 U/mL), and streptomycin (100 lg/mL). "
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    ABSTRACT: Silver nanoparticles (Ag-NPs) have increasingly been used for coatings on various textiles and certain implants, for the treatment of wounds and burns, as a water disinfectant, and in air-freshener sprays. The wide use of Ag-NPs may have potential human health impacts. In this study, the mutagenicity of 5-nm Ag-NPs was evaluated in the mouse lymphoma assay system, and modes of action were assessed using standard alkaline and enzyme-modified Comet assays and gene expression analysis. Treatments of L5178Y/Tk(+/-) mouse lymphoma cells with 5-nm uncoated Ag-NPs resulted in a significant yield of mutants at doses between 3 and 6 μg/mL; the upper range was limited by toxicity. Loss of heterozygosity analysis of the Tk mutants revealed that treatments with uncoated Ag-NPs induced mainly chromosomal alterations spanning less than 34 megabase pairs on chromosome 11. Although no significant induction of DNA damage in Ag-NP-treated mouse lymphoma cells was observed in the standard Comet assay, the Ag-NP treatments induced a dose-responsive increase in oxidative DNA damage in the enzyme-modified Comet assay in which oxidative lesion-specific endonucleases were added. Gene expression analysis using an oxidative stress and antioxidant defense polymerase chain reaction (PCR) array showed that the expressions of 17 of the 59 genes on the arrays were altered in the cells treated with Ag-NPs. These genes are involved in production of reactive oxygen species, oxidative stress response, antioxidants, oxygen transporters, and DNA repair. These results suggest that 5 nm Ag-NPs are mutagenic in mouse lymphoma cells due to induction of oxidative stress by the Ag-NPs.
    Environmental and Molecular Mutagenesis 07/2012; 53(6):409-19. DOI:10.1002/em.21698 · 2.63 Impact Factor
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