Mutagenesis, 2015, 30, 227–235 doi:10.1093/mutage/geu062 Original Article Advance Access publication 12 November 2014
Original Manuscript
Acrylamide induces specific DNA adduct formation and gene mutations in a carcinogenic target site, the mouse lung
Division of Pathology and 1Biological Safety Research Center, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan *To whom correspondence should be addressed. Tel: +81 3 3700 9819; Fax: +81 3 3700 1425; Email:
[email protected] Received July 31 2014; Revised September 12 2014; Accepted September 17 2014.
Abstract Acrylamide (AA) is a contaminant in heated foods and is carcinogenic in multiple organs of rodents. There have been many reports regarding AA-induced DNA modification and genotoxicity. However, the data are insufficient to understand fully the relationship between the two events. A recent report demonstrated carcinogenicity in the mouse lung. The lung is advantageous for investigation of AAinduced genotoxicity because DNA adduct levels are relatively high in this organ. In the present study, reporter gene mutation assays and quantitative analyses of specific DNA adducts were performed in the lungs of mature gpt delta mice treated with AA at doses of 100, 200 and 400 p.p.m. in drinking water for 4 weeks. N7-GA-Gua was detected in all AA-treated mice in a dose-dependent manner. gpt mutant frequencies (MFs) were significantly increased in the middle- and high-dose groups. In the analysis of mutation spectra, significant increases in GC-TA transversions and single base deletion mutations were observed in the high-dose group. Spi− MFs were significantly increased in the highdose group. Analysis of Spi− mutants revealed significant increases in the frequencies of single base deletion mutation in runs of G/C and A/T. Analyses of immature mice under the same experimental conditions showed that there were no differences of susceptibility to AA-induced genotoxicity in the two age classes. The overall data clearly show the causal relationship between AA-induced DNA adducts and the gene mutations at carcinogenic target sites.
Introduction Acrylamide (AA), a water-soluble α,β-unsaturated amide, has been extensively used by the chemical industry and has received considerable attention as an occupational hazard for decades (1,2). In food, AA is readily generated from food components during heat treatment at temperatures above 120°C as a result of the Maillard reaction between asparagine, a major amino acid in potatoes and cereals, and reducing sugars such as fructose and glucose (3,4). Therefore, AA is found at relatively high concentrations in baked and fried starchy foods, including French fries, potato chips and bread (5,6). The estimated average daily intake of AA in the USA and Europe through food consumption is 0.4 µg/kg body weight (7,8). In addition, age-dependent differences in susceptibility to AA-induced testicular genotoxicity in rats were recently reported (9). Considering
the types of foods containing AA, these differences should be considered when evaluating AA-induced hazards in the paediatric population (10). AA was classified as a Group 2A carcinogen by the International Agency for Research on Cancer based on sufficient evidence for the carcinogenicity of AA in experimental animals (11). In F344 rats, oral administration of AA induced tumours in the central nervous system, the thyroid glands, peritesticular mesothelia and the mammary glands (12,13). In a recent study conducted by the National Toxicology Program, lung carcinogenicity was also observed in B6C3F1 mice (14). In particular, since lung cancer is the second most common tumour and the leading cause of cancer death in many countries (15), an understanding of AA-induced lung carcinogenesis is an urgent matter for assessment of the human risk of AA exposure.
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Yuji Ishii, Kohei Matsushita, Ken Kuroda, Yuh Yokoo, Aki Kijima, Shinji Takasu, Yukio Kodama1, Akiyoshi Nishikawa1 and Takashi Umemura*
Y. Ishii et al., 2015, Vol. 30, No. 2
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Materials and methods Chemicals and reagents AA was purchased from Sigma–Aldrich Japan (St Louis, MO, USA).
Animals, diet and housing conditions The protocols for this study were approved by the Animal Care and Utilization Committee of the National Institute of Health Sciences. Male B6C3F1 gpt delta mice carrying 80 tandem copies of the transgenic lambda EG10 in a haploid genome were raised by mating C57BL/6 gpt delta and nontransgenic C3H/He mice (Japan SLC, Inc., Shizuoka, Japan). Animals were housed in polycarbonate cages (five mice per cage) with hardwood chips for bedding in a conventional animal facility maintained under conditions of controlled temperature (23 ± 2°C), humidity (55 ± 5%), air change (12 times/h) and lighting (12 h light/dark cycle). The animals were given free access to CRF-1 basal diet (Oriental Yeast, Tokyo, Japan) and tap water and were used after a 1-week acclimation period.
Animal treatment Forty male gpt delta mice (3 and 11 weeks old) were randomly divided into four groups, and groups of 10 mice were given an AA solution at concentrations of 0, 100, 200 and 400 p.p.m. in the drinking water, 7 days a week for up to 4 weeks. Considering the neurotoxicity of AA, the dose of 400 p.p.m. was selected based on a mutagenic dose reported in an in vivo mutation assay in the livers of Big Blue mice (20). AA is stable in water for at least 1 week and the dosing solutions were prepared and changed weekly (21). General signs were observed daily, and body weight and food consumption per cage were measured once a week. Six immature mice
in the group of 400 p.p.m. AA treatment died after 2 weeks of exposure because of the neurotoxicity of AA. At the end of the period, all mice were killed under isoflurane anaesthesia and the lungs were removed immediately. Lungs and livers from the first five animals in each group were fixed in 10% buffered formalin for haematoxylin and eosin staining. Lungs and livers from the remaining five animals in each group were stored at −80°C for DNA adduct analysis and in vivo mutation assays. In addition, to examine the mutation spectra of Spi− mutants observed in the lungs of mice treated with 400 p.p.m. AA, additional animal experiments using mature mice were performed. Ten of the 11-week-old male gpt delta mice were randomly divided into two groups, and groups of five mice were given AA solution at concentrations of 0 and 400 p.p.m. in the drinking water, 7 days a week for up to 4 weeks. At the end of the period, all mice were killed under isoflurane anaesthesia and the lungs were removed immediately. Lungs were stored at −80°C for Spi− assay.
Measurement of GA-specific DNA adducts As a standard for liquid chromatography with tandem mass spectrometry analysis, N7-GA-Gua and 15N5-N7-GA-Gua were synthesised as described previously (19). Sample preparation was performed according to the methods of Koyama et al. (9). Briefly, DNA was extracted from the lung with a DNeasy 96 Blood & Tissue Kit (QIAGEN, Dusseldorf, Germany), followed by incubation at 37°C for 48 h for depurination. An aliquot of the 15N-labeled N7-GA-Gua was added to each sample that was then passed through an ultrafiltration membrane to remove DNA. The eluted solution was evaporated thoroughly and dissolved in water. The solutions were subsequently quantified with a Quattro Ultima Pt triple stage quadrupole mass spectrometer (Waters Micromass, Milford, MA, USA) equipped with an Agilent 1100 LC system (Agilent technologies, Palo Alto, CA, USA).
In vivo mutation assays 6-TG and Spi− selections were performed as previously described (22). Briefly, genomic DNA was extracted from lungs, and lambda EG10 DNA (48 kb) was rescued as the lambda phage by in vitro packaging. For 6-TG selection, the packaged phage was incubated with Escherichia coli YG6020 that expressed Cre recombinase and converted to a plasmid carrying gpt and chloramphenicol acetyltransferase. Infected cells were mixed with molten soft agar and poured onto agar plates containing chloramphenicol and 6-TG. In order to determine the total number of rescued plasmids, 3000-fold diluted phages were used to infect YG6020, and the suspension was poured on plates containing chloramphenicol without 6-TG. The plates were then incubated at 37°C for selection of 6-TG-resistant colonies. Positively selected colonies were counted on Day 3 and collected the following day. The mutant frequency (MF) was calculated by dividing the number of gpt mutants by the number of rescued phages. For Spi− selection, the packaged phages were incubated with E.coli XL-1 Blue MRA for survival titration and E.coli XL-1 Blue MRA P2 for mutant selection. Infected cells were mixed with molten lambda-trypticase soft agar and poured onto lambda-trypticase agar plates. The next day, plaques (Spi− candidates) were punched out with sterile glass pipettes and the agar plugs were suspended in SM buffer. In order to confirm the Spi− phenotype of candidates, the suspensions were spotted on three types of plates on which XL-1 Blue MRA, XL-1 Blue MRA P2 or WL95 P2 strains were spread with soft agar. Real Spi− mutants, which made clear plaques on every plate, were counted.
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AA is capable of forming specific DNA adducts due to the electrophilic form of the epoxide derivative, glycidamide (GA), which is formed via the action of CYP2E1 (16). GA forms several specific DNA adducts due to reactions with the N7-positions of guanine or the N3- and N1-position of adenine (17–19). Especially, N7-GAGua is found in several organs, such as the liver, lung, kidney, testis, mammary gland and thyroid gland of mice or rats treated with AA (9,19). GA is also considered to be responsible for most of the genotoxicity of AA, which might explain the inconsistent results in in vitro genotoxicity studies of AA. In this respect, in vivo mutation assays using reporter gene transgenic rodents capable of considering pharmacokinetics of the test compound are likely effective tools to investigate AA-induced genotoxicity. In addition, this assay has the advantage of measuring the amount of DNA adducts and mutation frequency in the reporter genes of the identical organs. A recent study using gpt delta rats demonstrated the levels of GA adducts in the liver, testis, mammary gland and thyroid glands as well as gpt mutation frequencies in the liver and testis (9). However, there have been no reports of the relationship between the two events at the carcinogenic target tissue of the same animal. It is likely that analysis of the lung, which harbours relatively high levels of DNA adducts (19), might provide detailed information about AA-induced gene mutations. To clarify the causal relationship between AA-specific DNA adduct formation and gene mutation in the lung, this study undertook quantitative analysis of the DNA adduct N7-GA-Gua and reporter gene mutation assays in the lungs of B6C3F1 gpt delta mice. In addition, in vivo mutagenicity and DNA adduct formation in the lungs of immature gpt delta mice were also examined to compare the susceptibility to AA-induced genotoxicity in the two age groups.
AA-induced DNA adduct and gene mutation, 2015, Vol. 30, No. 2
Sequence analysis of gpt mutants
follows: primer 001: 5′-CTCTCCTTTGATGCGAATGCCAGC-3′; primer 002: 5′-GGAGTAATTATGCGGAACAGAATCATGC-3′; primer 005: 5′-CGTGGTCTGAGTGTGTTACAGAGG-3′; primer 006: 5′-GTTATGCGTTGTTCCATACAACCTCC-3′; primer 012: 5′-CGGTCGAGGGACCTAATAACTTCG-3′.
Statistical evaluation Variances in the data for body and lung weights, gpt and Spi− assays and mutation spectrum analyses of gpt and Spi− mutants were evaluated for homogeneity using Bartlett’s tests. When the data were homogenous, one-way analysis of variance was applied. In heterogeneous cases, the Kruskal–Wallis test was applied. When statistically significant differences were indicated, Dunnett’s multiple tests were employed for comparisons between the control and treatment groups.
Results
Sequence analysis of Spi mutants
Body and tissue weights
Mutation spectrum analysis of Spi− mutants was performed as described in Masumura et al. (23). Briefly, the lysates of Spi− mutants were obtained by infection of E.coli LE392 with the recovered Spi− mutants, and the lambda DNA was extracted from the lysate with the Gentra Puregene DNA kit (QIAGEN K. K., Tokyo, Japan). The extracted DNA was used as a template for PCR analysis to determine the deleted regions. DNA fragments containing the deletions were amplified by PCR using primers 001-002 (5 kb in length), 005-012 (14 kb in length) or 005-006 (21 kb length), followed by sequencing analysis of the PCR products. Sequence changes within and outside the gam/redBA genes were identified by DNA sequencing analysis at Takara Bio Inc. (Shiga, Japan). For the deletion size analysis, we categorised the deletion sizes into single base pair (1 bp), 2 bp to 1 kbp and >1 kbp. Single base pair deletions were subdivided into ‘simple’ and ‘in run’; the former occurred at nonrepetitive sequences, and the latter occurred at repetitive sequences (24). The PCR primers used in this study were as
In the 400 p.p.m. group, hind-leg paralysis and sluggish movement associated with the neurotoxicity of AA were observed in immature and mature gpt delta mice 1 and 3 weeks after the beginning of treatment, respectively. Since six immature mice died between 2 and 4 weeks, immature mice treated with 400 p.p.m. AA were excluded from all examinations. Data for final body and organ weights and intake of AA are shown in Table 1. In 400 p.p.m. AA-treated mature mice, the final body and lung weights were significantly decreased, and lung/body weight ratios were significantly increased. In immature mice, the final body weights were significantly decreased in all AA-treated mice. Absolute lung weights were significantly decreased in the 200 p.p.m. group. Daily water consumption was decreased in all AA-treated animals compared to the control group value (Table 1). The average daily intakes of AA were calculated to be 22.5, 38.6 and 59.2 mg/kg body weight for 100, 200 and 400 p.p.m. groups in mature mice and 21.8 and 41.2 mg/kg body weight for 100 and 200 p.p.m. groups in immature mice, respectively.
−
Table 1. Final body, absolute and relative lung weights, water consumption and intake of AA of mature and immature gpt delta mice treated with AA for 4 weeks Age
Mature
Immature
Parameter
No. of animals Body weight (g) Lung weight Absolute (g) Relative (g%) Water consumption (ml/mouse/day) AA intake (mg/kg/day) No. of animals Body weight (g) Lung weight Absolute (g) Relative (g%) Water consumption (ml/mouse/day) AA intake (mg/kg/day)
Control
100
200
400
10 30.6 ± 1.4a
10 28.6 ± 1.7
10 28.4 ± 1.8
10 19.7 ± 2.2##
0.16 ± 0.02 0.52 ± 0.04 8.2 – 10 23.2 ± 0.8
0.17 ± 0.01 0.58 ± 0.05 6.4 22.5 10 21.5 ± 1.1*
0.16 ± 0.01 0.55 ± 0.04 5.4 38.6 10 18.1 ± 1.6**
0.13 ± 0.01## 0.65 ± 0.05## 3.8 59.2 – –
0.13 ± 0.01 0.58 ± 0.04 4.9 –
0.13 ± 0.01 0.60 ± 0.06 4.2 21.8
0.11 ± 0.02** 0.63 ± 0.08 3.0 41.2
– – – –
Data represent means ± SD (n = 10). *, **Significantly different from control group (immature) at P
