Affiliation of authors: A. Besaratinia, G. P. Pfeifer, Division of Biology, Beckman Research Institute of the City of Hope National Medical Center, Duarte, CA.
Correspondence to: Ahmad Besaratinia, Ph.D., Division of Biology, Beckman Research Institute of the City of Hope National Medical Center, 1450 East Duarte Rd., Duarte, CA 91010 (e-mail: ania{at}coh.org).
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ABSTRACT |
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INTRODUCTION |
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Recently, two independent groups showed that acrylamide can be generated from food components during heating processes as a result of the Maillard reaction between amino acids and reducing sugars (6,7). The implication of this finding for human cancer epidemiology has prompted a resurgence of investigations into the genotoxicity of acrylamide and its mode of action in mammalian cells. To date, acrylamide has consistently had no effect in bacterial gene mutation assays with various strains of Salmonella, Escherichia coli (E. coli), and Klebsiella pneumoniae, in either the presence or the absence of an exogenous activation system. By contrast, glycidamide, the epoxy derivative of acrylamide, induced mutations in Salmonella strains TA100 and TA1535 (8,9). However, in in vitro mammalian gene mutation assays, acrylamide has inconsistently induced mutations in mouse lymphoma cells at the thymidine kinase and hypoxanthineguanine phosphoribosyl transferase loci (811). Likewise, in vivo in MutaMouse, acrylamide weakly induced mutations in the lacZ gene (12), whereas in the morphological specific-locus test and mouse spot test, acrylamide increased meiotic mutations in male germ cells (13) and induced spots of genetic relevance indicative of point mutations/chromosomal loss/somatic recombination in the (T x HT)F1 embryos, respectively (14). In carcinogenicity experiments, acrylamide administered via different routes increased the incidence of lung adenomas and initiated skin tumorigenesis in mice and induced scrotal mesotheliomas, thyroid adenomas, mammary gland tumors, uterine adenocarcinomas, clitoral gland adenomas, and oral papillomas in rats (8,9).
Given the non-conclusiveness of the data on acrylamide genotoxicity and the ambiguity of its mechanism(s) of action, an appealing hypothesis is that acrylamide triggers mutagenesis by damaging DNA. To test this hypothesis, we sought a methodology to determine whether acrylamideDNA adduct formation could induce mutations at the gene level. A transgenic mouse model was chosen to allow us to examine mutation induction and DNA adduct formation in a single-test system. The in vitro system consists of Big Blue mouse embryonic fibroblasts with recoverable shuttle vectors that carry the mutational target, the cII transgene (15). This system has several major advantages over similar systems, including the ability to control the experimental conditions, easy recovery of the target transgene, and the cost-effectiveness of DNA sequence analysis and DNA adduct mapping of the entire cII gene (294 base pairs [bp]) (15).
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MATERIALS AND METHODS |
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Acrylamide (acrylamide for electrophoresis, purity: 99.9%; Boehringer Mannheim, Indianapolis, IN) was dissolved in double-distilled water and sterilized by passage through a 0.2-µm filter.
Early-passage embryonic fibroblasts of Big Blue mice (prepared from 13.5-day-old embryos) were grown to monolayer confluence in Dulbeccos modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. After the cells reached confluence, the medium was removed and replaced with serum-free DMEM for several hours before the experimental treatments were begun. The cells were treated with various doses of acrylamide or control solvent (i.e., double-distilled water) in serum-free DMEM for 4 hours. For some experiments, the cells were harvested with trypsin immediately or 24 hours after the treatments to be used for the analysis of cell viability and DNA adduct formation. Cell viability was determined with the trypan blue exclusion assay. After the treatments, the medium was removed and replaced with complete growth medium and the cells were grown for an additional 8 days for use in mutation analyses. Each experimental condition was assessed in triplicate, and each experiment was done two or three times. In a separate experiment, cells were treated with increasing concentrations (0.01 µM, 0.1 µM, 1 µM) of the known carcinogen benzo[a]pyrene diol epoxide (BPDE; Midwest Research Institute, Kansas City, MO) or control solvent (dimethyl sulfoxide) for 45 minutes in the dark. The cells were subsequently handled using the same protocol as indicated above for mutation analyses.
Genomic DNA Isolation
Genomic DNA was isolated using a standard phenol and chloroform extraction and ethanol precipitation protocol (16). The DNA was dissolved in TE buffer (10 mM TrisHCl, 1 mM EDTA, pH 7.5) and kept at -80 °C until further analysis.
Terminal Transferase-Dependent Polymerase Chain Reaction for Mapping of DNA Adducts
The entire length of the cII gene was subjected to terminal transferase-dependent polymerase chain reaction (TD-PCR) as described (17), with some modifications. Briefly, genomic DNA (1 µg) was used as a template, and single-stranded products were made by repeated primer extensions. The extension protocol consisted of primer U1: 5'-AATCGAGAGTGCGTTGCTT-3' (Tm = 49.9 °C) in a mixture of Vent (exo) DNA polymerase (New England Biolabs, Beverly, MA); and a thermocycler setting of 2 minutes at 95 °C, 2 minutes at 53 °C, 3 minutes at 72 °C, nine cycles (in which one cycle consisted of 45 seconds at 95 °C, 2 minutes at 53 °C, and 3 minutes at 72 °C), 45 seconds at 95 °C, 2 minutes at 53 °C, and 10 minutes at 72 °C. The resulting product was precipitated with ethanol and a salt solution containing 10 M ammonium acetate, 0.5 M EDTA (pH 8.0), and glycogen at 20 mg/mL, and then subjected to the homopolymeric ribotailing and adapter ligation. The ligated fragments were PCR-amplified using primer U2: 5'-GCGTTGCTTAA CAAAATCGCAATGCT-3' (Tm = 63.1 °C). The thermocycler was set for 2 minutes at 95 °C, 2 minutes at 62 °C, 3 minutes at 72 °C, 18 cycles (in which one cycle consisted of 45 seconds at 95 °C, 2 minutes at 62 °C, and 3 minutes at 72 °C), 45 seconds at 95 °C, 2 minutes at 62 °C, and 10 minutes at 72 °C. Final primer extension of the PCR products was done using a fluorescence infrared dye-labeled primer (IRD-700; LI-COR, Lincoln, NE) U3: 5'-GCAATGCTTGGAACTGAGAAGACAGC-3' (Tm = 61.4 °C). The thermocycler setting was 2 minutes at 95 °C, 2 minutes at 65 °C, 3 minutes at 72 °C, six cycles (in which one cycle consisted of 45 seconds at 95 °C, 2 minutes at 65 °C, and 3 minutes at 72 °C), 45 seconds at 95 °C, 2 minutes at 65 °C, and 10 minutes at 72 °C. The labeled reaction products were subjected to gel electrophoresis (5% acrylamide/urea) in an IR2 Long Ranger 4200 system with simultaneous detection (LI-COR). The sites of DNA adduct formation were identified as the locations in which the presence of the lesions stopped the DNA polymerase from progressing, resulting in an intense dark band (dependent on the lesion frequency) in the sequencing gel.
cII Mutation Frequency Analysis
The cII mutation frequency was quantified by using the Select-cII Mutation Detection System for Big Blue Rodents (Stratagene, La Jolla, CA). The assay system is based on the ability of the
phage to multiply either lytically or lysogenically in E. coli host cells (15). The commitment of the
phage to lysis or lysogeny upon infection of the host is regulated by a chain of events, of which cII transcription is a determiner (18). The cII protein activates the transcription of cI repressor and
integrase, both of which obligate the phage to undergo lysogenization (18). Only
LIZ shuttle vectors carrying mutant cII can enter the lytic pathway and, as a result, form visible plaques on an E. coli lawn (15). The
LIZ vector, however, harbors a cI857 temperature sensitive (ts) mutation that makes the cI(ts) protein labile at temperatures exceeding 32 °C (19). Hence, under nonselective conditions, e.g., incubation at 37 °C, all
LIZ phages, regardless of their cII mutant/nonmutant status, multiply lytically in the host E. coli (15).
Briefly, the LIZ shuttle vectors were recovered from Big Blue mouse embryonic fibroblast genomic DNA (
5 µg) and packaged into viable phage particles by Transpack packaging extract, according to the manufacturers instructions (Stratagene). The phages were then pre-adsorbed to G1250 E. coli, and the bacteria were plated on TB1 agar plates. The plates were incubated for 48 hours at 24 °C or overnight at 37 °C (regarded as selective and nonselective conditions, respectively). The cII mutation frequency was expressed as the ratio of the number of plaques formed on the selective plates to the number formed on the nonselective plates. As recommended by the manufacturer (Stratagene), a minimum of 3 x 105 rescued phages was screened for each experimental condition. For quality assurance, control phage solutions containing a mixture of
cII+ and
cII- with known mutant frequencies (Stratagene) were assayed in all runs.
cII Mutational Spectrum Analysis
The cII plaques containing putative mutations were all verified after being replated under the selective conditions on a second TB1 agar plate. The verified plaques were subsequently amplified in a PCR by using a Select-cII sequencing primer (Stratagene), according to the manufacturers recommended protocol. The PCR products were purified with a QIAquick PCR Purification Kit (Qiagen GmbH, Hilden, Germany) and sequenced by a Big Dye terminator cycle sequencing kit on an ABI Prism 377 DNA Sequencer (Applied BioSystems, Foster City, CA).
Statistical Analysis
Results are expressed as means and 95% confidence intervals (CIs). Mutation frequency data were analyzed by one-way analyses of variance (ANOVA). Mutational spectra were analyzed with the hypergeometric test of Adams and Skopek (20) and the chi-square test, where appropriate. All statistical tests were two-sided. Values of P.05 were considered statistically significant. The statistical software packages included Statview SM+ Graphics (Abacus Concepts, Amsterdam, The Netherlands) and DNA Mutation Analysis Application (21) available at: ftp://anonymous@sunsite.unc.edu/pub/academic/biology/.
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RESULTS |
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We first determined whether acrylamide was cytotoxic to Big Blue mouse embryonic fibroblasts. The cells were exposed to increasing concentrations of acrylamide (32 nM, 320 nM, 3.2 µM, 32 µM, 320 µM, 3.2 mM, 16 mM, 160 mM, and 320 mM) for 4 hours, and cell viability was determined by trypan blue dye exclusion over a period of 24 hours (Fig. 1). The cytotoxic effects appeared only when the cells were treated with concentrations of acrylamide in the millimolar range and were time- and dose-dependent. The cytotoxicity and reduction in the number of viable cells at doses of 3.2 mM or greater were accompanied by almost negligible cell division in the surviving cells (data not shown).
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To determine whether acrylamide induced the formation of DNA adducts, cells were exposed to 3.2 mM and 16 mM acrylamide or control solvent (double-distilled water) for 4 hours. Genomic DNA was subsequently isolated and subjected to TD-PCR, a method that allows the mapping of polymerase-blocking lesions at nucleotide resolution (16). Our preliminary work showed that the TD-PCR results were more quantitative for samples treated with millimolar rather than micromolar doses of acrylamide (data not shown). Preferential adduct formation was observed at specific nucleotide positions along the cII gene in DNA isolated from acrylamide-treated cells when compared with DNA isolated from the control-treated cells (Fig. 2). The formation of DNA adducts at most nucleotide positions was dose-dependent.
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We next determined whether acrylamide induced specific mutations in the cII gene. The cells were treated with incremental concentrations of acrylamide (32 nM, 320 nM, 3.2 µM, 32 µM, 320 µM, 3.2 mM, and 16 mM) for 4 hours and then allowed to grow for an additional 8 days. This time frame was chosen to permit multiple population doublings and fix any potential mutations into the genome. After 8 days, the cells were analyzed to determine the mutation frequency and the mutational spectrum of the cII gene.
Induction of mutations in the cII gene by acrylamide was initiated at a concentration of 3.2 µM (11.2 x 10-5, 95% CI = 6.2 to 16.2 x 10-5) and increased dose dependently thereafter. The induced cII mutant frequency was maximal at an acrylamide dose of 320 µM, showing a twofold increase in the number of mutations relative to control (13.8 x 10-5, 95% CI = 12.3 to 15.3 x 10-5 versus 6.9 x 10-5, 95% CI = 6.5 to 7.3 x 10-5; df = 2, 21; P<.001, ANOVA). In cells treated with millimolar doses of acrylamide, the cII mutation frequency was lower, i.e., similar to that of untreated cells (i.e., baseline) (Fig. 3).
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To analyze the mutational spectra (i.e., the kinds of mutational events), we sequenced the DNA isolated from the verified mutant cII plaques induced by acrylamide at 320 µM (number of sequenced plaques = 232) and derived spontaneously in the control-treated group (number of sequenced plaques = 173) for the entire length of the cII gene. Overall, eight (4.6%) samples in the control group and six (2.6%) samples in the acrylamide-treated group did not show any mutation throughout the cII gene. Four samples from each group harbored multiple mutations, all of these double- or triple-base substitutions or deletions. The total numbers of mutations in the control and acrylamide-treated groups were 170 and 231, respectively.
We next characterized the spontaneous and the acrylamide-induced cII mutational spectra. In both spectra, we observed three "jackpot" mutations at nucleotide positions 179184 [G insertion/deletion], 211 [G C transversion], and 221 [T
G transversion]. These jackpot mutations, already found in the cII gene in previous studies by us and by others (17,2325), are assumed to occur in the early development of the transgenic rodent and to undergo clonal expansion such that many cells from a single tissue harbor the same type of mutation (26). Methodologically, therefore, it is appropriate to exclude these jackpot mutations from the comparative spectral analysis. The jackpot mutations accounted for 21% of the spontaneous and 17% of the acrylamide-induced cII mutations (Fig. 4
, A and B). The remaining nonjackpot mutations were predominately single-base substitutions, which constituted 73% and 81% of the spontaneous and acrylamide-induced cII mutations, respectively.
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DISCUSSION |
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We found that, compared with the potent mutagen and known carcinogen BPDE (22), acrylamide was indeed a weak yet distinguishable mutagen in this test system. Our results showed that acrylamide could statistically significantly induce cII mutations only at relatively high doses (in the micromolar range) and that it produced a different spectrum of mutations from that derived spontaneously. At millimolar doses of acrylamide, however, the cII mutation frequency was at the baseline level (Fig. 3). The decrease in cII mutation frequency in cells treated with millimolar doses of acrylamide may be due to the cytotoxic effects on cell division observed in the same dose range. Alternatively, it may reflect the mechanism of acrylamide mutagenicity. Glycidamide, which is thought to be responsible for at least some of the genotoxicity of acrylamide (9), is formed by a saturable epoxidation process that follows MichaelisMenten kinetics (31). Rats that received intraperitoneal injections of low doses of acrylamide had higher ratios of glycidamideto acrylamidehemoglobin adducts than rats that received high doses (31). This result suggests that conversion of the parent acrylamide to its epoxide form may be more efficient at lower acrylamide concentrations than at higher concentrations. It is conceivable that the ratio of glycidamide to acrylamide will decline as the concentration of acrylamide exceeds the Km for its metabolizing enzyme(s). Furthermore, in mice pretreated with 1-aminobenzotriazole, an inhibitor of CYP2E1 (the isozyme involved in the epoxidation of acrylamide), the formation of dominant lethal mutations in spermatids associated with exposure to acrylamide was diminished or substantially reduced (32). Taken together, these data raise the possibility that, in the present study, micromolar doses of acrylamide might have yielded higher concentrations of glycidamide than millimolar doses, thereby more frequently inducing cII mutations. We should, however, acknowledge that this possible scenario deserves further investigation because the exact metabolic machinery of the embryonic mouse fibroblasts may differ from that of an organ (i.e., the liver) in the fully grown animal.
To elucidate the importance of acrylamide-induced DNA adducts in the mutagenicity of acrylamide, we sought an association between the preferential adduct formation sites and the mutational hotspots produced by this compound in the cII gene. The profile of acrylamide-induced DNA adducts did not perfectly match that of acrylamide-induced mutations. Although some of the frequently mutated sites in the cII gene co-localized with the sites of preferential DNA adduction, there was no direct relationship. This may imply that not all DNA adducts formed by acrylamide are necessarily promutagenic. The imperfect correspondence between preferential DNA adduct sites and mutational hotspots might to some extent be methodologic, because the TD-PCR is a polymerase arrest-based assay, which indiscriminately quantifies all lesions regardless of their mutagenicity (16). It needs to be taken into account that the relatively low sensitivity of the TD-PCR for quantification of DNA adducts formed at the micromolar doses of acrylamide did not permit parallel analyses of the adduct mapping and the mutational spectrum produced at the same dose of acrylamide. Therefore, we could only establish an association between DNA adduct formation data obtained at the cytotoxic (millimolar) dose of acrylamide with the mutational data found at the non-cytotoxic (micromolar) dose of this compound.
Whether the mutagenicity of acrylamide is associated with acrylamide-induced DNA adducts is unclear. Acrylamide interacts directly with DNA via a Michael-type reaction, forming various adducts, including 1-carboxyethyl adenine, 3-carboxyethyl cytosine, 7-formamidoethyl guanine, 1-carboxyethyl guanine, and N6-carboxyethyl adenine (33). Moreover, glycidamide can alkylate DNA primarily at the N7 position of guanine, thereby giving rise to N7-2-carbamoyl-2-hydroxyethylguanine (9,34). Although the mutagenicities of the different DNA adducts are yet to be determined, the glycidamide adduct may well be promutagenic, especially in light of the positive results obtained with glycidamide in some mutagenesis assays (8,9). In our study, the non-dose dependency of DNA adduct formation at specific nucleotide positions, e.g., 161, 167, and 176187, may indicate that at least some of the formed adducts are glycidamide-derived. However, the contribution of such presumed glycidamideDNA adducts to the observed mutagenicity of acrylamide is unclear. It remains to be seen if glycidamideDNA adducts indeed gave rise to the herein induced cII mutations because the more specific mutational events ascribed to acrylamide (T C + A
G transitions and G
C + C
G transversions) were not detected at nucleotide positions 161, 167, and 176187 in the treated samples. To explore the involvement of glycidamide in acrylamide mutagenicity, we are currently investigating the mutagenic effects of glycidamide on the Big Blue mouse embryonic fibroblasts. It is also worth mentioning that several of the above-identified acrylamide DNA adducts are labile DNA bases, which can be converted to abasic sites. This fact, together with the diverse pattern of mutations induced by acrylamide, supports the idea that more than one type of DNA adduct might be involved in acrylamide mutagenicity.
In summary, our data show a distinct pattern of mutagenicity for acrylamide in mammalian cells, which may potentially be ascribed to its DNA adduct-inducing property. Given a limitation of our studythat is, it was conducted at a transgene rather than a native gene levelwe must be cautious in interpreting our results, particularly in generalizing their biologic significance to humans. To this end, our findings warrant further investigations into the carcinogenicity of acrylamide in humans. Such investigations will require careful study design because acrylamide is relatively ubiquitous in the diet, is found in high concentration in tobacco smoke, and is formed endogenously. All of these properties may easily act as confounding factors and complicate the interpretation of the data (35). The interfering role played by such confounders may be best reflected in the conventional epidemiologic studies in which occupational exposure to acrylamide could not be linked to mortality from any types of cancer (36,37).
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NOTES |
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We thank Dr. Hsiu-Hua Chen for expertise in TD-PCR analysis, Steven Bates for assistance in cell culturing, and Paul Frankel for help with statistical analysis.
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REFERENCES |
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1 International Agency for Research on Cancer (IARC). IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans: some industrial chemicals. Lyon (France): IARC 1994;60:389.
2 Rosen J, Hellenas KE. Analysis of acrylamide in cooked foods by liquid chromatography tandem mass spectrometry. Analyst 2002;127:8802.[CrossRef][ISI][Medline]
3 Tareke E, Rydberg P, Karlsson P, Eriksson S, Törnqvist M. Analysis of acrylamide, a carcinogen formed in heated foodstuffs. J Agric Food Chem 2002;50:49985006.[CrossRef][ISI][Medline]
4 Weiss G. Cancer risks. Acrylamide in food: uncharted territory. Science 2002;297:27.
5 World Health Organization. FAO/WHO consultation on the health implications of acrylamide in foodsummary report; 2002 June 2527; Geneva, Switzerland. Geneva (Switzerland): WHO; 2002. p. 112.
6 Mottram DS, Wedzicha BL, Dodson AT. Acrylamide is formed in the Maillard reaction. Nature 2002;419:4489.[CrossRef][ISI][Medline]
7 Stadler RH, Blank I, Varga N, Robert F, Hau J, Guy PA, et al. Acrylamide from Maillard reaction products. Nature 2002;419:44950.[CrossRef][ISI][Medline]
8 Dearfield KL, Abernathy CO, Ottley MS, Brantner JH, Hayes PF. Acrylamide: its metabolism, developmental and reproductive effects, genotoxicity, and carcinogenicity. Mutat Res 1988;195:4577.[ISI][Medline]
9 Dearfield KL, Douglas GR, Ehling UH, Moore MM, Sega GA, Brusick DJ. Acrylamide: a review of its genotoxicity and an assessment of heritable genetic risk. Mutat Res 1995;330:7199.[ISI][Medline]
10 Knaap AG, Kramers PG, Voogd CE, Bergkamp WG, Groot MG, Langebroek PG, et al. Mutagenic activity of acrylamide in eukaryotic systems but not in bacteria. Mutagenesis 1988;3:2638.[Abstract]
11 Tsuda H, Shimizu CS, Taketomi MK, Hasegawa MM, Hamada A, Kawata KM, et al. Acrylamide; induction of DNA damage, chromosomal aberrations and cell transformation without gene mutations. Mutagenesis 1993;8:239.[Abstract]
12 Hoorn AJ, Custer LL, Myhr BC, Brusick D, Gossen J, Vijg J. Detection of chemical mutagens using Muta Mouse: a transgenic mouse model. Mutagenesis 1993;8:710.[Abstract]
13 Russell LB, Hunsicker PR, Cacheiro NL, Generoso WM. Induction of specific-locus mutations in male germ cells of the mouse by acrylamide monomer. Mutat Res 1991;262:1017.[CrossRef][ISI][Medline]
14 Neuhauser-Klaus A, Schmahl W. Mutagenic and teratogenic effects of acrylamide in the mammalian spot test. Mutat Res 1989:226:15762.[CrossRef][ISI][Medline]
15 Jakubczak JL, Merlino G, French JE, Muller WJ, Paul B, Adhya S, et al. Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target. Proc Natl Acad Sci U S A 1996;93:90738.
16 Pfeifer GP, Chen HH, Komura J, Riggs AD. Chromatin structure analysis by ligation-mediated and terminal transferase-mediated polymerase chain reaction. Methods Enzymol 1999;304:54871.[ISI][Medline]
17 Besaratinia A, Bates SE, Pfeifer GP. Mutational signature of the proximate bladder carcinogen N-hydroxy-4-acetylaminobiphenyl: inconsistency with the p53 mutational spectrum in bladder cancer. Cancer Res 2002;62:43318.
18 Herskowitz I, Hagen D. The lysis-lysogeny decision of phage lambda: explicit programming and responsiveness. Annu Rev Genet 1980;14:399445.[CrossRef][ISI][Medline]
19 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1989.
20 Adams WT, Skopek TR. Statistical test for the comparison of samples from mutational spectra. J Mol Biol 1987;194:3916.[ISI][Medline]
21 Cariello NF, Douglas GR, Soussi T. Databases and software for the analysis of mutations in the human p53 gene, the human hprt gene and the lacZ gene in transgenic rodents. Nucleic Acids Res 1996;24:11920.
22 International Agency for Research on Cancer (IARC). Monographs on the evaluation of the carcinogenic risk of chemicals to humans. Overall evaluations of carcinogenicity: an updating of IARC monographs volumes 160. Lyon (France): IARC; 1994.
23 Harbach PR, Zimmer DM, Filipunas AL, Mattes WB, Aaron CS. Spontaneous mutation spectrum at the lambda cII locus in liver, lung, and spleen tissue of Big Blue transgenic mice. Environ Mol Mutagen 1999;33:13243.[CrossRef][ISI][Medline]
24 Davies R, Gant TW, Smith LL, Styles JA. Tamoxifen induces G:C T:A mutations in the cII gene in the liver of lambda/lacI transgenic rats but not at 5'-CpG-3' dinucleotide sequences as found in the lacI transgene. Carcinogenesis 1999;20:13516.
25 Davies R, Gant TW, Smith LL, Styles JA, Glickman BW, Cunningham ML. Mutant frequencies and mutation spectra of dimethylnitrosamine (DMN) at the lacI and cII loci in the livers of Big Blue transgenic mice. Mutat Res 2000;452:197210.[ISI][Medline]
26 de Boer JG, Erfle H, Walsh D, Holcroft J, Provost JS, Rogers B, et al. Spectrum of spontaneous mutations in liver tissue of lacI transgenic mice. Environ Mol Mutagen 1997;30:27386.[CrossRef][ISI][Medline]
27 Swiger RR, Cosentino L, Shima N, Bielas JH, Cruz-Munoz W, Heddle JA. The cII locus in the MutaMouse system. Environ Mol Mutagen 1999;34:2017.[CrossRef][ISI][Medline]
28 Mellon I, Spivak G, Hanawalt PC. Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell 1987;51:2419.[ISI][Medline]
29 Hanawalt PC. Transcription-coupled repair and human disease. Science 1994;266:19578.[ISI][Medline]
30 Denissenko MF, Pao A, Pfeifer GP, Tang M. Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers. Oncogene 1998;16:12417.[CrossRef][ISI][Medline]
31 Bergmark E, Calleman CJ, Costa LG. Formation of hemoglobin adducts of acrylamide and its epoxide metabolite glycidamide in the rat. Toxicol Appl Pharmacol 1991;111:35263.[ISI][Medline]
32 Adler ID, Baumgartner A, Gonda H, Friedman MA, Skerhut M. 1-Aminobenzotriazole inhibits acrylamide-induced dominant lethal effects in spermatids of male mice. Mutagenesis 2000;15:1336.
33 Solomon JJ, Fedyk J, Mukai F, Segal A. Direct alkylation of 2'-deoxynucleosides and DNA following in vitro reaction with acrylamide. Cancer Res 1985;45:346570.[Abstract]
34 Segerbäck D, Calleman CJ, Schroeder JL, Costa LG, Faustman EM. Formation of N-7-(2-carbamoyl-2-hydroxyethyl)guanine in DNA of the mouse and the rat following intraperitoneal administration of [14C]acrylamide. Carcinogenesis 1995;16:11615.[Abstract]
35 Reynolds T. Acrylamide and cancer: tunnel leak in Sweden prompted studies. J Natl Cancer Inst 2002;94:8768.
36 Marsh GM, Lucas LJ, Youk AO, Schall LC. Mortality patterns among workers exposed to acrylamide: 1994 follow up. Occup Environ Med 1999;56:18190.[Abstract]
37 Granath F, Ehrenberg L, Paulsson B, Törnqvist M. Cancer risk from exposure to occupational acrylamide. Occup Environ Med 2001;58:6089.
Manuscript received December 2, 2002; revised April 1, 2003; accepted April 15, 2003.
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