Use of shuttle vectors to study the molecular processing of defined carcinogen-induced DNA damage: mutagenicity of single O<sup>4</sup>-ethylthymine adducts in HeLa cells

Klein, Johanna; BIeeker, M.J.; Lutgerink, Jan T.; Dijk, Willem J. van; Brugghe, Humphrey F.; Elst, H.den van; Marel, G.A.der van; Boom, J. H. van; Westra, J.G.; Bems, A.J.M.; Kriek, E.

doi:10.1093/nar/18.14.4131

Cited by 69 publications

(48 citation statements)

References 36 publications

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“…Correction for the slightly lower proportion of A-T base pairs in the lacZ gene results in a revised estimate of 73% mutations in A-T base pairs, which would not affect these comparisons. The most prominent classes of mutations induced by ENU in this study (Table 3) are (i) AT -* TA transversions, presunmably induced by 02-ethylthymine (32), (ii) G-C --AT transitions in non-5'-CpG sites, thought to be caused by 06-ethylguanine adducts (27), and (iii) A-T --G-C transitions, reported to be caused by 04-ethylthymine adducts (33). Mutant frequency is a function of a number of interrelated variables including the level of adduct deposition, DNA repair, and DNA replication (13).…”

Section: Discussionmentioning

confidence: 69%

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

Douglas

Jiao

Gingerich

et al. 1995

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

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The lacZ transgenic mouse (Muta mouse) model was used to examine the timing of ethylnitrosourea (ENU)-induced mutations in germ cells. The spectrum of mutations was also determined. Animals received five daily treatments with ENU at 50 mg/kg and were sampled at times up to 55 days after treatment. In mixed germ-cell populations isolated from seminiferous tubules, there was little increase in the mutant frequency 5 days after treatment; subsequently, there was a continuous increase until the maximum (17.5-fold above background) was reached by -35 days. In the spermatozoa, an increase in mutant frequency was not seen until 20 days after treatment, with the maximum (4.3-fold above background) being achieved no sooner than -35 days. Based on the timing of sampling, these data demonstrate the detection of both spermatogonial and postspermatogonial mutations. The most prominent feature of the ENU-induced basepair mutations in testicular germ cells sampled 55 days after treatment is that 70%o are induced in AT base pairs, compared to only 16% in spontaneous mutations. These findings are consistent with comparable data from ENU studies using assays for inherited germ-cell mutations in mice. This study has demonstrated the utility and potential of the transgenic mouse lacZ model (Muta mouse) for the detection and study of germ-cell mutations and provides guidance in the selection of simplified treatment and sampling protocols.in bacteria, A-T base pairs are the predominate source of mutations in mammalian species in vivo (13). This change is related to the reduced capacity of DNA alkyltransferase to remove ethyl adducts other than 06-ethylguanine from mammalian cells (13). ENU induces both spermatogonial stem cell and postspermatogonial mutations in mice (14). The former are characterized by base-substitution mutations in A-T base pairs (15-19); the latter have been shown to result in relatively more multilocus lesions (20) and, if adducts are induced late in development, mosaic mutations in the progeny result (21).In this study we have used a lacZ transgenic mouse (Muta mouse) model to determine the timing of ENU-induced mutation induction in a mixed population of germ cells isolated from seminiferous tubules and in spermatozoa isolated from the vas deferens. The mutation spectrum for mutations from mixed germ cells was also determined. It is anticipated that such information will be influential in determining the acceptability of transgenic mouse models for the study of germ-cell mutagenicity, in suggesting appropriate experimental protocols for future studies, and in alleviating the need to conduct costly experiments on the offspring of exposed animals when it is not necessary. It is envisaged that such a transgenic mouse germ-cell assay, once validated, may become a companion to the dominant lethal test in terms of its role in hazard identification.The establishment of transgenic mouse models for the quantitative detection of gene mutations (1, 2) has provided unprecedented access to the study of mutagenesis ...

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Section: Discussionmentioning

confidence: 69%

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

Douglas

Jiao

Gingerich

et al. 1995

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

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“…ENU reacts with oxygen in DNA and results in modification of the O 6 position of guanine and the O 4 and O 2 positions of thymine. O 6 -Ethylguanine induces G:C 3 A:T transitions [Ellison et al, 1989], O 4 -ethylthymine induces A:T 3 G:C transitions [Klein et al, 1990], and O 2 -ethylthymine induces A:T 3 T:A transversions .…”

Section: Discussionmentioning

confidence: 99%

Mutant frequency and mutational spectra in the Tk and Hprt genes of N‐ethyl‐N‐nitrosourea‐treated mouse lymphoma cells†

Chen

Harrington‐Brock

Moore

2002

Environ and Mol Mutagen

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The mouse lymphoma assay (MLA) utilizing the Tk gene is widely used to identify chemical mutagens. The autosomal location of the Tk gene allows for the detection of a wide range of mutational events, from point mutations to chromosome alterations. However, chemically induced point mutation spectra in the Tk gene of mouse lymphoma cells have not been characterized. In this study, we determined and compared the mutagenicity and mutational spectra of N-ethyl-N-nitrosourea (ENU) in the Tk and Hprt genes of mouse lymphoma cells. Treatment of L5178Y mouse lymphoma cells with 100 microg/ml ENU induced a Tk mutant frequency of 756 x 10(-6) and an Hprt mutant frequency of 311 x 10(-6). Sequence analysis of Tk and Hprt mutant cDNAs showed a similar overall mutation pattern in the two genes with base-pair substitutions accounting for 83% of non-loss of heterozygosity mutations in the Tk gene and 75% of all mutations in the Hprt gene. The most common point mutation induced by ENU was G:C --> A:T transition (36 and 28% of independent mutations detected in the Tk and Hprt genes, respectively). The mutation spectra induced by ENU in both the Tk and Hprt genes were different from the respective patterns produced in mutants from untreated cells. About 9% of Tk and 7% of Hprt mutations from control cells were in-frame deletions, whereas no such mutations were found among the ENU-induced Tk and Hprt mutations. Our results indicate that ENU produces a chemical-specific point mutational profile in the Tk gene of mouse lymphoma cells that is remarkably similar to that found in the X-linked Hprt gene. This study provides evidence that the MLA can be used not only to detect point mutagens but also for analysis of mutational spectra.

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“…These four DNA oxidation products are all promutagenic. 8-OxodG is a well-known DNA oxidation product, and has been shown to cause G → T transversions in both prokaryotic and eukaryotic cells (20)(21)(22). The mutagenic potential of oxazolone following DNA synthesis in vitro suggests that the formation of oxazolone in DNA mainly leads to G → T and to some extent G → C transversions (23).…”

Section: Introductionmentioning

confidence: 99%

Quantitation of Four Guanine Oxidation Products from Reaction of DNA with Varying Doses of Peroxynitrite

Venkatarangan

Wishnok

et al. 2005

Chem. Res. Toxicol.

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The oxidation products obtained from the reaction of peroxynitrite (ONOO − ) with dG includeamong others -8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG)In the present work, the formation of these products from the treatment of calf thymus DNA with varying amounts of ONOO − was studied quantitatively in vitro. 13 C-, 15 N-labeled standards were synthesized for the nucleosides of interest, and calf thymus DNA was reacted with ONOO − and digested enzymatically down to the nucleoside level. Specific modifications in the DNA were measured by HPLC separation followed by electrospray ionization tandem mass spectrometric analysis in the selected reactionmonitoring mode. Artifacts of the above four oxidation products, arising from oxidation of dG and/ or 8-oxodG during DNA digestion and subsequent work-up, were evaluated with 7-15 N-dG and/or stable-isotope labeled 8-oxodG as internal standards. Levels of artifactual 8-oxodG were about 5/10 6 nucleosides. The artifacts of spiroiminodihydantoin and guanidinohydantoin, arising from 8-oxodG, were 3.7% and 0.6% of the measured 8-oxodG values, respectively. No artifacts of oxazolone were detected. 8-OxodG and oxazolone were formed dose-dependently in DNA treated with ONOO − , while the levels of spiroiminodihydantoin and guanidinohydantoin increased significantly at low ONOO − doses, and then dropped off at higher ONOO − doses. The complexity of these doseresponse relationships is likely due to the dual role of peroxynitrite as both an oxidant and a nucleophile in competition with water.

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Use of shuttle vectors to study the molecular processing of defined carcinogen-induced DNA damage: mutagenicity of single O⁴-ethylthymine adducts in HeLa cells

Cited by 69 publications

References 36 publications

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

Mutant frequency and mutational spectra in the Tk and Hprt genes of N‐ethyl‐N‐nitrosourea‐treated mouse lymphoma cells†

Quantitation of Four Guanine Oxidation Products from Reaction of DNA with Varying Doses of Peroxynitrite

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Use of shuttle vectors to study the molecular processing of defined carcinogen-induced DNA damage: mutagenicity of single O4-ethylthymine adducts in HeLa cells

Cited by 69 publications

References 36 publications

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

Mutant frequency and mutational spectra in the Tk and Hprt genes of N‐ethyl‐N‐nitrosourea‐treated mouse lymphoma cells†

Quantitation of Four Guanine Oxidation Products from Reaction of DNA with Varying Doses of Peroxynitrite

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Use of shuttle vectors to study the molecular processing of defined carcinogen-induced DNA damage: mutagenicity of single O⁴-ethylthymine adducts in HeLa cells