All of the authors, except Dr. M. Matilde Marques, are US government employees and performed this work as part of their official duties.
The opinions expressed in this paper do not necessarily represent those of the U.S. Food and Drug Administration.
The authors have quantified DNA adducts and the induction of micronuclei and mutations in newborn mice to elucidate the mechanisms by which the food contaminant acrylamide induces tumors. Their data indicate that a major pathway for mutation induction involves the metabolic conversion of acrylamide to the reactive electrophile glycidamide.
Acrylamide (Fig. 1), a water-soluble α,β-unsaturated amide, is a contaminant in baked and fried starchy foods, including French fries, potato chips and bread,1, 2 as a result of Maillard reactions involving asparagine and reducing sugars.3, 4 Other dietary sources of acrylamide include coffee, Postum coffee substitute, olives and certain breakfast cereals.5 The mean daily dietary intake of acrylamide in the United States is estimated to be ∼0.4 μg per kg body weight, with children between the ages of 2 and 5 consuming approximately twice this quantity.5 Similar estimates of dietary intake have been made for European countries.6 Additional sources of acrylamide exposure include cigarettes (∼3.1 μg per kg body weight per day),7 laboratory procedures involving polyacrylamide gels (∼1.4 μg per kg body weight per day)7 and various occupations (e.g, monomer production and polymerization processes).8
Acrylamide is a carcinogen in laboratory animals; for example, oral administration of acrylamide to A/J mice caused dose-related increases in lung adenomas.9 Topical, oral or intraperitoneal treatment with acrylamide followed by topical application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in skin tumors,9 and, in a similar experiment, Swiss-ICR mice treated orally with acrylamide and then topically with TPA had increased incidences of lung and skin tumors.10 The oral administration of acrylamide to F344 rats induced central nervous system tumors, thyroid gland follicular adenomas and adenocarcinomas, peritesticular mesotheliomas, and mammary gland fibroadenomas and adenocarcinomas.11, 12 Acrylamide was classified as a Group 2A carcinogen by a working group at the International Agency for Research on Cancer based upon sufficient evidence for the carcinogenicity of acrylamide in experimental animals and evidence of genotoxicity in various experimental systems.8 The same working group concluded that the evidence for carcinogenicity in humans was insufficient.8 A more recent review of epidemiological data reached a similar conclusion.13
Acrylamide is metabolized to the epoxide glycidamide (Fig. 1),8, 13–15 and although acrylamide will react very slowly with DNA,16 glycidamide reacts to a much greater extent and gives rise to a number of DNA adducts, including N7-(2-carbamoyl-2-hydroxyethyl)deoxyguanosine, 3,N4-(2-hydroxypropanoyl)deoxycytidine, and 1,N6-(2-hydroxypropanoyl)deoxyadenosine, N3-(2-carbamoyl-2-hydroxyethyl)deoxyadenosine and N1-(2-carboxy-2-hydroxyethyl)deoxyadenosine.17–19 Glycidamide also is considered to be the agent responsible for the induction of micronuclei in mice and rats administered acrylamide.20 A similar conclusion was reached concerning the induction of sister chromatid exchanges in mammalian cells by acrylamide.21
In mice, the oxidation of acrylamide to glycidamide is catalyzed by cytochrome P450 2E1 (CYP 2E1),22 and mice devoid of CYP 2E1 and administered acrylamide show much reduced levels of male germ cell mutagenicity,23 micronuclei24 and glycidamide-DNA adducts25 compared to their wild-type counterparts. In other experiments, adult Big Blue mice treated with equimolar doses of acrylamide and glycidamide produced comparable increases in mutant frequencies.26 Furthermore, the pattern of mutations observed in vivo from treatment with either acrylamide or glycidamide26 was consistent with the mutations induced by glycidamide in vitro in Big Blue mouse embryonic fibroblasts15 and with the types of DNA adducts formed from glycidamide.17–19 The totality of these data indicates that acrylamide exerts its genotoxicity and tumorigenicity through conversion to glycidamide.
In spite of this evidence, it has been noted that the metabolism of acrylamide to glycidamide does not account fully for the mutagenicity of acrylamide in some in vitro mammalian mutation assays.27–30 However, these experiments were conducted in cells that appear to have no CYP 2E1 activity and in some instances the levels of acrylamide necessary to produce a mutagenic response far exceeded the levels that could be obtained in vivo. It has also been noted that the tissue and organ distribution of glycidamide-DNA adducts does not always correlate with acrylamide-induced tumors in rats,31 although recent data have demonstrated the presence of glycidamide-DNA adducts in all tissues examined in rats, including the tumor target tissues.32 Nonetheless, it is unlikely that the differences in the levels of DNA adducts alone will account for the observed organ specificity for tumor induction. This has prompted suggestions of alternate mechanisms for acrylamide tumorigenicity, including alterations in signal transduction pathways and/or prolonged hormonal changes.31, 33
In a previous study, we noted marked differences in glycidamide-DNA adduct formation when comparing adult and neonatal mice.19 When adult male C3H/HeNMTV or female C57BL/6N mice were given a single intraperitoneal dose of 50 mg acrylamide or glycidamide per kg body weight, similar glycidamide-DNA adduct levels were obtained with both compounds. In contrast, when three-day-old B6C3F1 mice were administered a single dose of either 50 mg acrylamide or glycidamide per kg body weight, the DNA adduct levels from glycidamide were 5- to 7-fold greater than those observed from acrylamide. These results imply that neonatal mice are deficient in the cytochrome P-450 activity necessary to oxidize acrylamide to an electrophile, which is similar to what has been observed with other carcinogens.34 These observations, plus the fact that tumors can be induced in neonatal mice by a variety of carcinogens,34–37 suggest that by treating newborn mice with acrylamide and glycidamide, valuable information could be obtained regarding the mechanisms of acrylamide tumorigenesis. If metabolism to glycidamide and the consequent glycidamide-DNA adduct formation are necessary steps in the tumorigenicity of acrylamide, we hypothesized that glycidamide will be much more tumorigenic than acrylamide in newborn mice. As a first step in testing this hypothesis, we determined the extent of DNA adduct formation in B6C3F1/Tk mice treated neonatally with acrylamide and glycidamide, and compared these levels with the induction of 3 biomarkers for tumor induction by a genotoxic mode of action: micronuclei in peripheral red blood cells and lymphocyte Tk and Hprt mutations.
AA, acrylamide; ANOVA, analysis of variance; BrdUR, 5-bromodeoxyuridine-resistant; ENU, N-ethyl-N-nitrosourea; ES-MS/MS, electrospray tandem mass spectrometry; GA, glycidamide; HPLC, high performance liquid chromatography; Hprt, hypoxanthine-guanine phosphoribosyltransferase (gene); LOH, loss of heterozygosity; MN-NCEs, micronucleated normochromatic erythrocytes; MN-RETs, micronucleated reticulocytes; N3-GA-Ade, N3-(2-carbamoyl-2-hydroxyethyl)adenine; N7-GA-Gua, N7-(2-carbamoyl-2-hydroxyethyl)guanine; PCR, polymerase chain reaction; PND(s), postnatal day(s); RETs, reticulocytes; 6-TGR, 6-thioguanine-resistant; Tk, thymidine kinase (gene); TPA, 12-O-tetradecanoylphorbol-13-acetate.
Material and methods
Acrylamide (stated purity ≥99%, CAS 79-06-01) and N-ethyl-N-nitrosourea (ENU; CAS 759-73-9) were purchased from Sigma Chemical, St. Louis, MO. Glycidamide (stated purity >98%, CAS 5694-00-08) was obtained from Toronto Research Chemicals, North York, ON. The purity and identity of the acrylamide and glycidamide were confirmed by gas chromatography coupled with electron impact mass spectrometry, nuclear magnetic resonance spectroscopy and gas chromatography using flame ionization detection.
Male C3H/HeNMTV mice and female C57BL/6N/Tk+/− mice were obtained from colonies maintained at the National Center for Toxicological Research. The derivation of the C57BL/6N/Tk+/− mice has been described (Von Tungeln et al.,38 and references cited therein). Female C57BL/6N/Tk+/− mice were mated with male C3H/HeNMTV mice to produce B6C3F1/Tk mice (i.e., a combination of B6C3F1/Tk+/+ and B6C3F1/Tk+/− mice). Upon delivery, pups were pooled by sex and 8 pups of the same sex were randomly assigned to a foster mother before being treated with acrylamide or glycidamide.
Two experiments were conducted. In the first, male and female B6C3F1/Tk mice were treated intraperitoneally on postnatal days (PNDs) 1, 8 and 15 with 0.0, 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. This treatment schedule corresponds to a protocol typically used in newborn mouse bioassays.39 To minimize stress upon the pups, historical body weights for mice of this age were used to determine the amount of acrylamide and glycidamide administered. The 0.70 mmol per kg body weight dose corresponds to approximately 50 mg per kg body weight, which is similar to the dose used when treating three-day-old B6C3F1 mice in our previous study.19 The 0.14 mmol per kg body weight dose allowed dose-response relationships to be investigated. The compounds were dissolved in deionized water and administered in 5, 10 and 20 μL on PNDs 1, 8 and 15, respectively. On PND 9, small samples of tail tissue were obtained to establish the genotype of the mice (i.e., to distinguish between B6C3F1/Tk+/+ and B6C3F1/Tk+/− mice).38 On PND 16, the B6C3F1/Tk+/+ mice were anesthetized with CO2, and blood was obtained by cardiac puncture to assess the induction of micronuclei. The liver, spleen and lungs were excised and frozen; bone marrow was aspirated from the femurs and frozen. These tissue samples were subsequently processed for DNA adduct analyses. Three weeks after the last treatment, the B6C3F1/Tk+/− mice were killed to determine the mutant frequency in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) and thymidine kinase (Tk) genes. This experiment was performed in 2 independent assays and the results from the 2 assays were combined. Each assay contained additional B6C3F1/Tk+/− mice that were injected on PND 15 with a single intraperitoneal dose of 40 mg ENU per kg body weight to serve as a positive control for the lymphocyte mutagenesis assays.
In the second experiment, male and female B6C3F1/Tk mice were treated intraperitoneally on PNDs 1–8 with 0.0, 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. This treatment schedule corresponds to the one we have used to assess the mutant frequency in the Hprt and Tk genes of newborn B6C3F1/Tk+/− mice administered anti-retroviral drugs.38, 40 As in the first experiment, historical body weights were used to determine the amount of acrylamide and glycidamide administered daily. Tail samples were taken on PND 8 to establish the genotype of the mice. The B6C3F1/Tk+/+ mice were killed on PND 9 to determine the induction of micronuclei in peripheral blood and DNA adduct levels in lung, liver and spleen. The B6C3F1/Tk+/− mice were killed 3 weeks after the last treatment to determine the mutant frequency in the Hprt and Tk genes. An ENU-treated positive control group was included in the experiment.
All experiments described in this manuscript were reviewed and approved by the Institutional Animal Care and Use Committee at the National Center for Toxicological Research.
DNA adduct analyses
Nuclei were prepared from liver and lungs, and DNA was isolated from liver nuclei, lung nuclei, spleen and bone marrow as outlined in Gamboa da Costa et al.41
High performance liquid chromatography (HPLC) coupled with electrospray tandem mass spectrometry (ES-MS/MS) was used to assess the levels of N7-(2-carbamoyl-2-hydroxyethyl)deoxyguanosine and N3-(2-carbamoyl-2-hydroxyethyl)deoxyadenosine after thermally hydrolyzing the DNA to effect the depurination and release of N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade) (Fig. 1). The methods for hydrolyzing the DNA, as well the HPLC-ES-MS/MS analysis of N7-GA-Gua and N3-GA-Ade, have been described in detail.32 The analyses were conducted with 10–250 μg DNA.
Analysis of micronucleated reticulocytes and normochromatic erythrocytes
The frequency of reticulocytes (RETs), micronucleated reticulocytes (MN-RETs) and micronucleated normochromatic erythrocytes (MN-NCEs) was determined by flow cytometry as described in Von Tungeln et al.42
Hprt and Tk lymphocyte mutagenesis assays in B6C3F1/Tk+/− mice
The frequency of Hprt mutants was determined by measuring the ability of splenic T-lymphocytes to grow in the presence of 6-thioguanine [i.e., 6-thioguanine-resistant (6-TGR) T-lymphocytes], as described by Meng et al.43 The frequency of Tk mutants was assessed by determining the ability of splenic T-lymphocytes to grow in the presence of 5-bromodeoxyuridine [i.e., 5-bromodeoxyuridine-resistant (BrdUR) T-lymphocytes], as described by Dobrovolsky et al.,44 with the modifications indicated in Von Tungeln et al.40
Loss of heterozygosity (LOH) in BrdUR T-lymphocyte clones was measured by allele-specific polymerase chain reaction (PCR),44 as outlined in Von Tungeln et al.40
Statistical analyses of the micronucleus data, mutant frequencies and mutant fractions were conducted by analysis of variance (ANOVA), with pairwise comparisons performed by the Student–Newman–Keuls test. When necessary, the data were log-transformed before the analyses to maintain a homogeneous variance, normal distribution or both. When the log-transformed data did not have a homogeneous variance or normal distribution, the data were analyzed by a Kruskal–Wallis ANOVA, with pairwise comparisons conducted by Dunn's test.
DNA adduct data were analyzed by repeated-measures ANOVA, with pairwise comparisons conducted by the Student–Newman–Keuls test.
Statistical analyses of LOH and intragenic mutant frequencies were conducted by the hypergeometric test of Adams and Skopek,45 using a program written by Cariello et al.46 Bonferroni-type adjustments were applied to correct for multiple comparisons.47
Data are reported as the mean ± SEM. The number of samples for each analysis is presented in the tables or figure legends. p-Values of <0.05 were considered significant.
DNA adduct levels in neonatal B6C3F1/Tk+/+ mice treated with acrylamide or glycidamide
In the first experiment, B6C3F1/Tk mice were injected intraperitoneally on PNDs 1, 8 and 15 with 0.0, 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. The treatments were well tolerated, with >90% of the mice surviving the dosing period.
The levels of N7-GA-Gua and N3-GA-Ade were assessed by HPLC-ES-MS/MS in DNA obtained on PND 16 from lung, liver, spleen and bone marrow of the B6C3F1/Tk+/+ mice. Both adducts were readily detected in mice treated with acrylamide or glycidamide. Representative chromatograms are shown in Figure 2.
The levels of N7-GA-Gua did not differ significantly between the male and female mice for any of the tissues (Fig. 3). The highest levels of N7-GA-Gua were found in lung DNA, followed by the liver, spleen and bone marrow DNA; each of these tissues differed significantly from one another. Treatment with 0.70 mmol glycidamide per kg body weight gave significantly higher levels of N7-GA-Gua in all tissues examined than did injection with 0.70 mmol acrylamide per kg body weight. Likewise, treatment with 0.70 mmol acrylamide per kg body weight gave significantly higher levels of N7-GA-Gua than injection with 0.14 mmol acrylamide per kg body weight or 0.14 mmol glycidamide per kg body weight, which did not differ from one another.
The levels of N3-GA-Ade did not differ between the sexes and were approximately 100-fold lower than the levels of N7-GA-Gua (Fig. 3). The relative ranking of N3-GA-Ade levels within tissues was similar to that observed with N7-GA-Gua, with the highest levels being in the lung, followed by the liver, spleen and bone marrow. Likewise, treatment with 0.70 mmol glycidamide per kg body weight gave the highest levels of N3-GA-Ade. This was followed by dosing with 0.70 mmol acrylamide per kg body weight and then 0.14 mmol glycidamide per kg body weight and 0.14 mmol acrylamide per kg body weight, which did not differ from one another. N3-GA-Ade was not detected in bone marrow DNA from mice administered 0.70 mmol acrylamide per kg body weight or 0.14 mmol glycidamide or acrylamide per kg body weight.
Neither N7-GA-Gua (<1.0 adducts/108 nucleotides) nor N3-GA-Ade (<1.5 adducts/108 nucleotides) was detected in DNA from mice treated with the solvent.
In the second experiment, B6C3F1/Tk mice were injected intraperitoneally on PNDs 1–8 with 0.0, 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. This treatment schedule had a marked effect upon survival. In the control group, 79% of the mice survived the dosing period. This survival contrasted with 66 and 43% in the 0.14 and 0.70 mmol acrylamide per kg body weight groups and 57 and 14% in the 0.14 and 0.70 mmol glycidamide per kg body weight groups. Because of the poor survival in the group treated with 0.70 mmol glycidamide per kg body weight, these mice were not used in subsequent analyses, with the exception of a single mouse that was used for DNA adduct analyses.
The adduct levels in the second experiment (Fig. 4) showed more variation than those in the first experiment (Fig. 3); nonetheless, the values were qualitatively similar to the first experiment, with N7-GA-Gua being formed at approximately 100 times the level of N3-GA-Ade. Furthermore, the levels of N7-GA-Gua were highest in the lung, followed by the liver, and then the spleen. Because of the small size of the mice, bone marrow was not collected in the second experiment.
Frequency of MN-RETs and MN-NCEs in neonatal B6C3F1/Tk+/+ mice treated with acrylamide or glycidamide
In the first experiment, in which mice were treated on PNDs 1, 8 and 15, the administration of 0.70 mmol glycidamide per kg body weight caused slight, but statistically significant, increases in MN-RETs and MN-NCEs in peripheral blood at PND 16 (Table I). In the second experiment, in which mice were dosed on PNDs 1–8, treatment with 0.14 mmol glycidamide per kg body weight caused a statistically significant increase in MN-NCEs in peripheral blood at PND 9 (Table I). None of the other treatments affected the percentage of MN-RETs or MN-NCEs.
Table I. Frequency of RETs, MN-RETs and MN-NCEs in Peripheral Blood of Male and Female B6C3F1/Tk+/+ Mice after Treatment on PNDs 1, 8 and 15 or PNDs 1–8 with Acrylamide or Glycidamide1
Dose (mmol per kg)
Male and female B6C3F1/Tk+/+ mice were injected intraperitoneally on PNDs 1, 8 and 15 or PNDs 1–8 with 0.0, 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. Measurements of RETs, MN-RETs and MN-NCEs in peripheral blood were made on samples obtained on PND 16 from mice treated on PNDs 1, 8 and 15, and on samples obtained on PND 9 from mice treated on PNDs 1–8. The data are expressed as the mean ± SEM for the number of mice (n) indicated.
Significantly different (p < 0.05) from the control.
Only one B6C3F1/Tk+/+ mouse survived PNDs 1–8 treatment with 0.70 mmol glycidamide per kg body weight. This animal was not included in the analyses.
Tk and Hprt mutant frequencies in B6C3F1/Tk+/− mice
In both experiments, splenic T-lymphocytes were isolated from the B6C3F1/Tk+/− mice three weeks after the last injection for determination of the mutant frequencies in the Tk and Hprt genes (Table II). In the first experiment (PNDs 1, 8 and 15 dosing), none of the treatments affected the cloning efficiencies or the Tk mutant frequencies of the T-lymphocytes. Pairwise comparisons to the control group indicated that 0.70 mmol glycidamide per kg body weight caused a significant increase in the Hprt mutant frequency. Increases in the Tk and Hprt mutant frequencies were also observed with the positive control ENU. The ENU-induced mutant frequencies were not analyzed statistically due to the small number of mice treated.
Table II. Cloning Efficiency, Tk Mutant Frequency, and Hprt Mutant Frequency in Splenic T-Lymphocytes of Male and Female B6C3F1/Tk+/− Mice after Treatment on PNDs 1, 8 and 15 or PNDs 1–8 with Acrylamide or Glycidamide1
Dose (mmol per kg)
Cloning efficiency (%)
Tk mutant frequency (BrdUR lymphocytes/106 cells)
Hprt mutant frequency (6-TGR lymphocytes/106 cells)
Male and female B6C3F1/Tk+/− mice were injected intraperitoneally on PNDs 1, 8 and 15 or PNDs 1–8 with 0.0, 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. The frequency of Tk and Hprt mutants was determined in splenic T-lymphocytes three weeks after the last treatment of each group. The data are expressed as the mean ± SEM for the number of mice (n) indicated.
Significantly different (p < 0.05) from the control.
Male and female B6C3F1/Tk+/− mice were given a single intraperitoneal injection of 40 mg ENU per kg body weight on PND 8 or 15 to serve as a positive control.
Only one B6C3F1/Tk+/− mouse treated with 0.70 mmol glycidamide per kg body weight survived until weaning at PND 22. This animal was not included in the analyses.
In the second experiment (PNDs 1–8 dosing), there were no treatment-related differences in the cloning efficiencies (Table II). The administration of 0.14 or 0.70 mmol acrylamide per kg body weight or 0.14 mmol glycidamide per kg body weight caused a significant increase in the Tk mutant frequency, whereas dosing with 0.14 or 0.70 mmol acrylamide per kg body weight resulted in a significant increase in the Hprt mutant frequency. Increases in the Tk and Hprt mutant frequencies were also observed with the ENU control.
LOH analysis of BrdUR lymphocyte clones
The Tk gene of BrdUR clones was analyzed for LOH using allele-specific PCR. The data are summarized in Table III. Clones were classified as having LOH when the intensity of the PCR product amplified from the wild-type Tk+ allele was greatly diminished compared to the PCR product amplified from the disrupted Tk− allele or as having intragenic mutation (e.g., a point mutation or frameshift) when the PCR products from both alleles had a similar intensity. Only a single clone could not be classified.
Table III. Loss of Heterozygosity (LOH) Analysis of BrdUR Clones from Splenic T-Lymphocytes of Male and Female B6C3F1/Tk+/− Mice after Treatment on PNDs 1, 8 and 15 OR PNDs 1–8 with Acrylamide or Glycidamide
The Tk gene of BrdUR clones was analyzed for LOH or intragenic mutation using allele-specific PCR as described in Von Tungeln et al.40 The data are presented as the number of clones displaying LOH and intragenic mutation, with the percentage being given in parentheses.
The LOH mutant frequency and intragenic mutant frequency are the products of mutant frequency (Table II) and the relative contribution of LOH and intragenic mutation, respectively, to the overall mutant frequency. The data (BrdUR lymphocytes/106 cells) are expressed as the mean ± SEM.
The proportion of LOH and intragenic mutation differed significantly from the proportion observed in the respective control.
Significantly different from the respective control.
Analysis of BrdUR clones from B6C3F1/Tk+/− mice that had been treated on PNDs 1, 8 and 15 indicated that 0.70 mmol glycidamide per kg body weight increased the mutant frequency resulting from LOH (Table III). The other treatments did not alter the mutant frequency resulting from LOH and none of the treatments affected the mutant frequency resulting from intragenic mutation. When B6C3F1/Tk+/− mice were treated with acrylamide or glycidamide on PNDs 1–8, each of the treatments caused a significant increase in the mutant frequency resulting from LOH (Table III), whereas the intragenic mutant frequency was not affected.
A number of possible mechanisms have been proposed for the tumorigenicity of acrylamide, including genotoxicity through conversion to glycidamide, alterations in signal transduction pathways and prolonged hormonal dysregulation.13–15, 31, 33 Because neonatal mice are particularly sensitive to tumor induction by genotoxic carcinogens,34–37 we employed this cancer model to elucidate the mechanisms of acrylamide carcinogenesis. As a first step, we compared the DNA adduct levels and the micronuclei and mutant frequencies induced in neonatal mice exposed to acrylamide and glycidamide.
In a previous study, we found that neonatal (PND 3) mice were relatively insensitive to glycidamide-DNA adduct formation (i.e., N7-GA-Gua or N3-GA-Ade) by acrylamide as compared to glycidamide, a finding that we attributed to a reduced level of CYP 2E1 activity in neonatal mice.19 In our current study, we noted a similar reduced sensitivity to DNA adduct formation with acrylamide, compared to glycidamide, after intraperitoneal injections on PNDs 1, 8 and 15. At a dose of 0.70 mmol per kg body weight, which was similar to the 50 mg per kg body weight dose used in our previous study,19 approximately 2- to 3-fold higher DNA adduct levels were formed by treatment with glycidamide than with acrylamide.
The tissues from mice treated on PNDs 1, 8 and 15 that were examined for adduct formation included the liver and lungs, presumptive target tissues for tumorigenicity in newborn mouse bioassays,34–37 and the spleen and bone marrow, tissues that are sources of the T-lymphocytes used in the mutagenesis assays and the RETs and NCEs monitored in the micronucleus assays.48, 49 N7-GA-Gua was detected in all tissues with each of the treatments. N3-GA-Ade also was detected in the same tissues; however, in each instance, the levels of N3-GA-Ade were approximately 100-fold lower than those of N7-GA-Gua, which is similar to what we have observed previously in vitro and in vivo.19, 21, 25, 32 The low levels of N3-GA-Ade, coupled with the fact that the amount of DNA was at times quite limited, precluded the detection of this adduct with certain samples, in particular bone marrow DNA. Although 0.70 mmol glycidamide per kg body weight produced significantly more DNA binding than an equimolar dose of acrylamide, both agents produced similar levels of DNA binding at 0.14 mmol per kg body weight. We speculate that the metabolism of acrylamide at this lower concentration was not limited by the amount of CYP 2E1 activity in the neonatal mice.
In mice treated on PNDs 1, 8 and 15, only treatment with 0.70 mmol glycidamide per kg body weight, the treatment that produced the greatest levels of DNA adducts, resulted in increased genotoxicity, specifically, significant increases in Hprt mutant frequency and in MN-RET and MN-NCE frequency. The 0.70 mmol glycidamide per kg body weight dose also produced an increase in Tk mutant frequency, although the increase was not significant, and a significant increase in the frequency of Tk mutants produced by LOH. The micronucleus frequencies and the Tk and Hprt lymphocyte mutant frequencies in mice treated with 0.14 mmol glycidamide per kg body weight and with both doses of acrylamide were very similar to those of the negative control animals. These observations suggest that micronucleus and lymphocyte mutation induction by acrylamide in neonatal mice are limited by the low extent of acrylamide conversion to glycidamide and subsequent DNA adduct formation in this model. In addition, the relatively low levels of DNA adducts in bone marrow in comparison with lung and liver suggest that the genotoxicity responses measured in our assays may be an underestimation of the genotoxic potential of acrylamide and glycidamide in the target tissues of this carcinogenesis model.
The results from the experiment conducted with PND 1–8 dosing were not as straightforward to interpret. The PND 1–8 dosing schedule was considerably more toxic than the PND 1, 8 and 15 regimen, suggesting that it was more effective at producing DNA damage in the mice. Although the PND 1, 8 and 15 treatment had little effect on animal survival (all treatments resulted in survivals of >90%), dose-related decreases in survival were readily apparent in animals treated on PNDs 1–8. Furthermore, equimolar doses of glycidamide were more toxic than acrylamide, to the extent that only 14% of the mice injected with 0.70 mmol glycidamide per kg body weight survived. This observation suggests that at this dose, similarly to what was observed with the PND 1, 8 and 15 schedule, glycidamide produced more DNA damage than did acrylamide; this appeared to be confirmed in the single animal that was available for DNA adduct analyses.
In mice dosed on PNDs 1–8, a significant increase in MN-NCEs was detected only with glycidamide, as was the case with the PND 1, 8 and 15 treatment. However, with the PND 1–8 dosing, measurements could only be made with 0.14 mmol glycidamide per kg body weight, a dose that was negative with the PND 1, 8 and 15 schedule. The positive response produced at this dose after the PND 1–8 regimen is consistent with the fact that this treatment caused levels of DNA damage that were up to 2- to 3-fold greater than those obtained with the PND 1, 8 and 15 dosing. Unlike the results produced in the micronucleus assays, the Hprt and Tk mutant frequencies were similar in animals treated with acrylamide or glycidamide on PNDs 1–8. These results suggest that DNA adducts (or cellular damage) other than those produced by glycidamide may be responsible, at least partially, for Tk and Hprt mutant induction after the PNDs 1–8 treatment.
Studies conducted on the mutagenicity of acrylamide and glycidamide using in vitro mammalian cell models may help explain our in vivo mutagenicity responses. Both acrylamide and glycidamide are mutagenic in the cII transgene of Big Blue mouse embryonic fibroblasts and in the TK genes of mouse lymphoma Tk+/− cells and human lymphoblastoid TK6 cells, with glycidamide producing a greater response than acrylamide.27–30 Although these results appear to be consistent with the mutagenicity of acrylamide being dependent upon its conversion to glycidamide, this may not be strictly the case. With regard to the response in Big Blue mouse cells, the spectra of mutations produced by acrylamide and glycidamide are different,28 suggesting that different types of DNA damage may be responsible for the mutagenicity of the 2 compounds. Moreover, mouse lymphoma cells have no CYP 2E1 activity, and treatment of mouse lymphoma cells with acrylamide produced no glycidamide-DNA adducts.30 In addition, it should be noted that the positive mutagenicity responses produced by acrylamide in the mouse lymphoma30 and TK6 cell29 assays were obtained at acrylamide concentrations that exceeded both the maximum concentration of 10 mM established for using in vitro mammalian cell mutation assays for regulatory purposes50 and the plasma levels of acrylamide that can be achieved in vivo.51, 52
These in vitro mutagenicity responses are in sharp contrast to the mutagenic responses produced by acrylamide and glycidamide in adult Big Blue mice.26 When administered orally, both compounds increased the liver cII and splenic lymphocyte Hprt mutant frequencies in a quantitatively similar, dose-dependent manner, which probably is a reflection of the efficient metabolism of acrylamide to glycidamide in adult mice.32, 51 Importantly, the spectra of cII mutations produced by the 2 compounds differed from the control but were very similar to each other, suggesting that similar types of DNA damage accounted for the response produced by each compound. In addition, the predominant mutation detected in the cII transgene of Big Blue mice administered acrylamide or glycidamide was a G:C → T:A transversion, a mutation that could result from the formation of glycidamide-DNA adducts, such as N7-GA-Gua. A similar cII mutation spectrum has been observed in Big Blue mouse fibroblasts treated with glycidamide.15
Our results reinforce the utility of the neonatal mouse model for elucidating the mechanisms of acrylamide carcinogenesis. As concluded in other studies,20, 24 we found evidence that micronucleus induction by acrylamide was dependent upon its conversion to glycidamide. This observation is important because lymphocyte micronuclei, along with chromosome aberrations, are validated biomarkers of cancer risk in humans.53 Consistent with the observations of Manjanatha et al.26 in adult Big Blue mice, the lack of significant acrylamide mutagenicity in neonatal mice treated on PNDs 1, 8 and 15, as opposed to the response obtained with glycidamide, suggests that acrylamide mutagenicity is dependent upon its conversion to glycidamide. In fact, under the conditions of our assay, it is plausible that insufficient acrylamide was converted to glycidamide to produce a significant mutagenic response.
Acrylamide was considerably more toxic and genotoxic when administered to neonatal mice on PNDs 1–8. However, under these conditions, metabolism to glycidamide may not be responsible for the Hprt and Tk lymphocyte mutations induced by acrylamide. We conclude that in situations of limiting CYP2E1 activity (as occurs in neonatal mice, mouse lymphoma cells and possibly TK6 cells and Big Blue mouse embryonic fibroblasts), acrylamide can induce gene mutations by a pathway not involving glycidamide. This may involve the generation of reactive oxygen species and oxidative DNA damage.54, 55 However, because this alternate pathway appears to take place only with very high toxic doses of acrylamide (as occurred in the PND 1–8 treatment regimen), it may not be significant for any toxicity associated with human exposure to this agent.
The authors thank Ms. Michelle Vanlandingham, Ms. Carey Nobles, Ms. Sherry Smith and Ms. Tina Glover for helping in the treatment and care of the mice. This paper is dedicated to Cynthia A. Hartwick, a very dear friend and colleague, who died at far too young an age, from the disease we are trying to prevent.