Mutant frequencies and loss of heterozygosity induced by N-ethyl-N-nitrosourea in the thymidine kinase gene of L5178Y/TK+/--3.7.2C mouse lymphoma cells

Chen, Tao; Harrington‐Brock, Karen; Moore, Martha M.

doi:10.1093/mutage/17.2.105

Cited by 28 publications

(11 citation statements)

References 28 publications

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“…Our results extend previous studies in which mutagens such as MNU have been reported to induce LOH (39,(43)(44)(45)(46)(47)(48). However, these studies used nonmammalian systems or tumor-derived cell lines or were limited to only one or two loci.…”

Section: Loh As a Somatic Mutation: Implications For The Carcinogenicsupporting

confidence: 86%

Carcinogens induce genome-wide loss of heterozygosity in normal stem cells without persistent chromosomal instability

Donahue

Lin

Cao

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Widespread losses of heterozygosity (LOH) in human cancer have been thought to result from chromosomal instability caused by mutations affecting DNA repair͞genome maintenance. However, the origin of LOH in most tumors is unknown. The present study examined the ability of carcinogenic agents to induce LOH at 53 sites throughout the genome of normal diploid mouse ES cells. Brief exposures to nontoxic levels of methylnitrosourea, diepoxybutane, mitomycin C, hydroxyurea, doxorubicin, and UV light stimulated LOH at all loci at frequencies ranging from 1-8 ؋ 10 ؊3 per cell (10 -123 times higher than in untreated cells). This greatly exceeds the frequencies at which these agents have been reported to induce point mutations and is comparable to the rates of LOH observed in ES cells lacking the gene responsible for Bloom syndrome, an inherited DNA repair defect that results in greatly increased risk of cancer. These results suggest that LOH contributes significantly to the carcinogenicity of a variety of mutagens and raises the possibility that genome-wide LOH observed in some human cancers may reflect prior exposure to genotoxic agents rather than a state of chromosomal instability during the carcinogenic process. Finally, as a practical matter, chemically induced LOH is expected to enhance the recovery of homozygous recessive mutants from phenotype-based genetic screens in mammalian cells.cancer etiology ͉ cancer genetics ͉ carcinogenesis ͉ genome stability

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Section: Loh As a Somatic Mutation: Implications For The Carcinogenicsupporting

confidence: 86%

Carcinogens induce genome-wide loss of heterozygosity in normal stem cells without persistent chromosomal instability

Donahue

Lin

Cao

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Genomic DNA was extracted by digesting the cells in lysis buffer [10 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 1% (v/v) Triton X-100, and 1% (v/v) Tween 20] with 200 g/ml proteinase K at 60°C for 90 min, followed by inactivation of proteinase K at 95°C for 10 min. The procedure for the PCR analysis of LOH at the Tk locus was previously described (Chen et al, 2002a). For PCR analysis of LOH at other loci, the amplification reactions were carried out in a total volume of 30 l by using 2ϫ PCR master mix (Promega, Madison, WI) and a pair of primers for a specific locus obtained from The Jackson Laboratory (Bar Harbor, ME; http:// www.informatics.jax.org).…”

Section: Identification and Quantitation Of Products Of 4-hne(ac)mentioning

confidence: 99%

Mutagenic Effects of 4-Hydroxynonenal Triacetate, a Chemically Protected Form of the Lipid Peroxidation Product 4-Hydroxynonenal, as Assayed in L5178Y/Tk+/– Mouse Lymphoma Cells

Singh¹,

Chen²,

Chen³

et al. 2005

The Journal of Pharmacology and Experimental Therapeutics

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The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) is cytotoxic and genotoxic at superphysiological concentrations. To characterize the mechanism of action of 4-HNE, we assessed genotoxic damage by 4-HNE and by 4-HNE triacetate [4-HNE(Ac) 3 ] using the mouse lymphoma assay that measures the mutant frequency in the Tk gene. As a strong electrophile, 4-HNE reacts readily with nucleophilic centers on cellular components. When added extracellularly, it may react preferentially with proteins in culture medium or on the cell surface and not reach deeper cellular targets such as nuclear DNA. Therefore, 4-HNE(Ac) 3 , a protected form of 4-HNE that is metabolically converted to 4-HNE in cells (Neely MD, Amarnath V, Weitlauf C, and Montine TJ, Chem Res Toxicol 15: 40 -47, 2002), was assayed in addition to 4-HNE. When added in serum-containing medium, 4-HNE was not mutagenic in the mouse lymphoma assay up to 38 M (cytotoxicity ϭ 13%). In contrast, exposure to 4-HNE(Ac) 3 , which mimics intracellular formation of 4-HNE, resulted in dose-dependent induction of mutations. At 17 M 4-HNE(Ac) 3 (cytotoxicity ϭ 33%), the mutant frequency was 719 ϫ 10 Ϫ6 (Ͼ7-fold higher than the spontaneous mutant frequency). Loss of heterozygosity analysis in the Tk mutants revealed that the majority of mutations induced by 4-HNE(Ac) 3 resulted from clastogenic events affecting a large segment of the chromosome. The results indicate that, in the presence of serum that approximates physiological conditions, 4-HNE generated intracellularly but not extracellularly is a strong mutagen via a clastogenic action at concentrations that may occur during oxidative stress.The production of partially reduced forms of dioxygen such as superoxide, hydrogen peroxide, or the hydroxyl radical (reactive oxygen species or ROS) is an inevitable consequence of aerobic life. Because of their high reactivity, ROS and/or their downstream products such as peroxynitrite (Crow, 2000) can attack all cellular constituents. It is often tacitly assumed that functionally relevant effects of ROS are due to damage to proteins and nucleic acids because such damage results in immediate functional consequences or mutations. However, ROS also target lipids. The hydroxyl radical can initiate a chain reaction that produces multiple lipid hydroperoxide molecules from a single initial event (Gutteridge and Halliwell, 1990). Since lipid hydroperoxides are oxidants capable of damaging cellular constituents, the chain reaction is in effect amplifying the initial oxidative insult. Peroxida-

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“…The MLA, using the thymidine kinase (Tk) gene as the selection marker, was chosen as the preferred mutation assay instead of the various assays using the hypoxanthine guanine phosphoribosyltransferase (Hprt) gene because of the MLA ability to detect point mutations and a wide variety of chromosomal mutations [Applegate et al, 1990;Dearfield et al, 1991;Chen et al, 2002]. Because of the autosomal location of the Tk gene, it detects chromosomal mutations that cannot be detected using the X-linked Hprt gene [Moore et al, 1989].…”

Section: In Vitro Mammalian Assaysmentioning

confidence: 99%

Use of genetic toxicology information for risk assessment

Dearfield

Moore

2005

Environ. Mol. Mutagen.

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Genetic toxicology data are used worldwide in regulatory decision-making. On the 25th anniversary of Environmental and Molecular Mutagenesis, we think it is important to provide a brief overview of the currently available genetic toxicity tests and to outline a framework for conducting weight-of-the-evidence (WOE) evaluations that optimize the utility of genetic toxicology information for risk assessment. There are two major types of regulatory decisions made by agencies such as the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA): (1) the approval and registration of pesticides, pharmaceuticals, medical devices, and medical-use products, and (2) the setting of standards for acceptable exposure levels in air, water, and food. Genetic toxicology data are utilized for both of these regulatory decisions. The current default assumption for regulatory decisions is that chemicals that are shown to be genotoxic in standard tests are, in fact, capable of causing mutations in humans (in somatic and/or germ cells) and that they contribute to adverse health outcomes via a "genotoxic/mutagenic" mode of action (MOA). The new EPA Guidelines for Carcinogen Risk Assessment [Guidelines for Carcinogen Risk Assessment, USEPA, 2005, EPA Publication No. EPA/630/P-03/001F] emphasize the use of MOA information in risk assessment and provide a framework to help identify a possible mutagenic and/or nonmutagenic MOA for potential adverse effects. An analysis of the available genetic toxicity data is now, more than ever, a key component to consider in the derivation of an MOA for characterizing observed adverse health outcomes such as cancer. We provide our perspective and a two-step strategy for evaluating genotoxicity data for optimal use in regulatory decision-making. The strategy includes integration of all available information and provides, first, for a WOE analysis as to whether a chemical is a mutagen, and second, whether an adverse health outcome is mediated via a mutagenic MOA.

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Mutant frequencies and loss of heterozygosity induced by N-ethyl-N-nitrosourea in the thymidine kinase gene of L5178Y/TK+/--3.7.2C mouse lymphoma cells

Cited by 28 publications

References 28 publications

Carcinogens induce genome-wide loss of heterozygosity in normal stem cells without persistent chromosomal instability

Carcinogens induce genome-wide loss of heterozygosity in normal stem cells without persistent chromosomal instability

Mutagenic Effects of 4-Hydroxynonenal Triacetate, a Chemically Protected Form of the Lipid Peroxidation Product 4-Hydroxynonenal, as Assayed in L5178Y/Tk+/– Mouse Lymphoma Cells

Use of genetic toxicology information for risk assessment

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