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Molecular Analysis of Hprt Gene Mutations in Skin Fibroblasts of Rats Exposed in Vivo to N-Methyl-N-Nitrosourea Or N-Ethyl-N-Nitrosourea'

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Molecular Analysis of Hprt Gene Mutations in Skin Fibroblasts of Rats Exposed in Vivo to N-Methyl-N-Nitrosourea Or N-Ethyl-N-Nitrosourea' ICANCER RESEARCH54, 2478-2485, May 1, 19941 Molecular Analysis of hprt Gene Mutations in Skin Fibroblasts of Rats Exposed in Vivo to N-Methyl-N-nitrosourea or N-Ethyl-N-nitrosourea' Jacob G. Jansen,2 George it Mohn, Harry Vrieling, Come M. M. van Teijlingen, Paul H. M. Lohman, and Albert A. van Zeeland MGC-Department ofRadiation Genetics and Chemical Mutagenesis, State University ofLeiden, Wassenaarseweg 72, 2333 AL Leiden (J. G. J., H. V., C. M. M. v. T., P. H. M. L, A. A. v. Z.J; Laboratory of Carcinogenesis and Mutagenesis, National Institute of Public Health and Environmental Protection, Bilthoven [C. R. M.]; and J. A. Cohen Institute, Interuniversity Research Institute for Radiopathology and Radiation Protection, Leiden (H. V., A. A. v. Z.J, the Netherlands ABSTRACT apart from 06-guanine alkylation, MNU methylates preferentially the N-7 position of guanine and at a lower frequency the N-3 The granuloma pouch assay in the rat is a model system in which position of adenine, while ENU alkylates mostly phosphodiesters; relative frequencies of genetic and (pro-) neoplastic changes induced in vivo by carcinogenic agents can be determined within the same target and at lower frequencies the @2position of thymine and the N-7 tissue. The target is granuloma pouch tissue and consists of a population position of guanine. Ethylation by ENU at the N-3 position of of (transient) proliferating fibroblasts which can be cultured in vitro.hprt adenine, the 0― position of thymine, and the @2position of gene mutations were studied in granuloma pouch tissue of rats treated cytosine occurs at much lower frequencies. It is believed that with single doses of direct acting alkylating agents N-methyl-N-nitro alkylation at oxygen residues of DNA bases results in mutagenic mum (MNU) or N-ethyl-N-nitrosourea (ENU). Both agents showed an events while N-alkylation leads to cytotoxic lesions (6). exposure-dependent increase in the hprt mutant frequency. Thirty-seven Most of the mutational spectra generated by MNU or ENU in MNU (60 mgfkg)- and 43 ENU (100 mgfkgJ-induced hprt mutant cell clones were analyzed at the molecular level. Twenty-two MNU-induced Escherichia coli, in yeast, and in vitro in eukaryotic cells show that and 36 ENU-induced mutants carried a single base pair change in exon the predominant base pair alterations induced by these mutagens are sequences ofthe hprt gene. The predominant base pair alterations induced GC to AT transitions (7—12).In carcinogenicity studies, MNU was by MNU were GC to AT transitions (18 of22), which are probably caused shown to induce various types of tumors in rats and mice, which by O'-methylguanine lesions. For most of the GC to AT transitions (16 of contained activated ras oncogenes with mutations predominantly at 18), the G was located in the nontranscribed strand, suggestinga strand codons 12 and 13 (GC to AT transitions) and to a minor extent at bias in the repair of O'-methylguanine lesions. ENU-induced mutations occurred predominantly at AT base pairs (28 of 36), beIng mostly AT to codon 61 (AT to TA transversions) (3, 13—15).These types of muta TA and AT to CG transversions, and are probably caused by O@-ethyI tions in tumors were similar to those observed after MNU exposure in thymidine. Also here, DNA repair processes seem to act with different in vitro mutation assays. However, ENU showed a different picture. rates/efficiencies on DNA adducts in the 2 strands of the hprt gene, since Rats exposed transplacentally to ENU developed neuroblastomas all the 24 transversions observed at AT base pairs had the thymidine which contained the neu oncogene activated by mutations predomi residue in the nontranscribed strand. GC to AT transitions were only nantly at AT base pairs (16). Furthermore, it was shown that the present at a low frequency among ENU-induced mutations, suggesting development of lung tumors in mice upon exposure to ENU was that O@-ethylguaninelesions were repaired efficiently before mutations were fixed during replication. The mutational spectra of MNU- and accompanied by the activation of ras protooncogenes as a result of ENU-induced hprt mutant clones were different from spontaneously oc mutations mostly at the AT base pair in codon 61 (17). These results curling hprt mutant clones. These results indicate that MNU and ENU indicate that ENU induces a quite different pattern of gene mutations induce different mutational spectra in vivo and that DNA repair systems in vivo than in vitro. remove O'-methylguanlne, (Y, and/or O@-ethylthymldinemuch faster Several studies show that mutational spectra generated by in vitro from the transcribed strand than the nontranscribed strand of the hprt assays may not always be representative for the in vivo situation. gene in these rat fibroblasts. Recently, spectra of mutations induced by MNU or ENU in Drosoph ila melanogaster, rodents, and monkeys have been reported and clear INTRODUCTION differences were seen when these spectra were compared with in vitro The monofunctional alkylating agent MNU3 and its ethyl ana results (18—21).These studies indicated that less frequent lesions such logue ENU have been studied extensively with regard to their as O@-and 04-ethylthymine are important mutagenic lesions induced DNA-alkylating properties. Both compounds react with DNA in by ENU in vivo, since mutations at AT base pair were found at high vitro and in vivo at more than 12 different nucleophilic sites (1). frequencies. Among other adducts, MNU and ENU alkylate the @6position of An assay allowing the direct comparison of short term effects (e.g., guanine, which is an important premutagenic and precarcinogenic gene mutations) and long term effects (fibrosarcomas) within the event (2, 3) giving rise to GC to AT transitions, with relative high same target tissue is the granuloma pouch assay (22). The target tissue efficiencies (2, 4, 5). However, the proportions of the various DNA consists of skin fibroblasts which proliferation is induced by injecting adducts induced by these compounds show marked differences: sterile air s.c. into the backs of young adult male rats. Shortly after mutagen exposure of the rats, skin fibroblasts can be isolated from the Received 12/13/93; accepted 3/3/94. pouch and subcultured in vitro for the analysis of different genetic end Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage points, including gene mutations at the hprt locus (23). Mutations in charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the coding sequence of the rat hprt gene can be analyzed at the 1 Parts of these studies were supported by grants from the Commission of the European molecular level, since the hprt cDNA from the rat has been cloned and Communities Environmental Programme (Contracts EV4V-0047-NL and EV5V-CT'91- its nucleotide sequence determined (24—26).In this paper, the induc 0012) and from the Dutch Cancer Society (Project IKW-89.14). 2 To whom requests for reprints should be addressed. tion of mutations in skin fibroblasts of rats following exposure in vivo 3 The abbreviations used are: MNU, N-methyl-N-nitrosourea; ENU, N-ethyl-N-nitro to MNU or ENU is presented. In addition to comparative hprt mu sourea; hprt, hypoxanthine-guanine phosphoribosyltransferase; eDNA, complementary DNA; PCR, polymerasechain reaction;AGT, alkylguanine-DNAalkyltransferase; tagenesis studies, this study reports the molecular analysis of 37 MNNG, N-methyl-N'-nitro-N-nitrosoguanidine. MNU- and 43 ENU-induced hprt mutant cell clones. 2478 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1994 American Association for Cancer Research. AT BASE PAIRS ARE TARGEF FOR ENU-INDUCEDMUTAGENESISIN VIVO vivoCompoundTreatment Table 1 hprt mutant frequencies induced byMNU or ENU in rat skin fibroblasts in of of cells no. of clone-forming of animals isolated cells tested for 6@TGr frequency 10_6)ENU0 mg/kg mmol/kgModeadministrationaNo. testedNo. (X 10@)CEb (%)Total mutants X106ç@C (%)Mutant (x 10_6)Range(X ±4.0― ±15 ±6.7 30 0.256 4 7.5 7.2 ±4.6 2.3 29 ±3 49 ±17 27—69 60 0.512 i.p. 6 12.1 7.6 ±5.3 3.2 24 ±10 103 ±51 48—167 80 0.680 i.p. 3 3.9 7.6 ±2.4 2.9 39 ±5 109 ±70 68—190 100 0.854 i.p. 4 9.9 6.9 ±4.1 1.6 24 ±4 135 ±71 64—232 160 1.366 i.p. 6 7.2 8.9 ±4.6 2.9 23 ±8 206 ±61 152—319 153—447MNU6.52500 2.135i.p. i.p.15 629.0 9.311.7 7.4 ±4.511.1 2.930 24 ±107.1 284 ±1171.5—25 ±5.1 ±10 ±2 15 0.146 i.p. 2 3.6 7.6 1.2 31 113 109—116 30 0.291 i.p. 9 15.4 12.3 ±4.9 7.3 35 ±9 126 ±53 45—223 45 0.438 i.p. 2 4.1 7.8 1.6 40 194 177—210 51—430MNU6.5600.063 0.582i.p. i.p.3 86.0 13.216.2 9.3 ±4.42.6 6.532 36 ±148 262 ±1316.1—10 ±3.0 ±7 ±37 13 0.126 p.o. 4 6.8 7.2 ±3.1 3.3 31 ±14 68 ±61 3—146 30 0.291 p.o. 4 8.8 6.5 ±3.4 2.3 22 ±16 102 ±99 27—244 600.063 0.582p.o.
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