Abstract

The recent discovery that the potent carcinogen acrylamide (AA) is present in a variety of fried and baked foods raises health concerns, particularly for children, because AA is relatively high in child-favoured foods such as potato chips and French fries. To compare the susceptibility to AA-induced genotoxicity of young versus adult animals, we treated 3- and 11-week-old male gpt delta transgenic F344 rats with 0, 20, 40 or 80 p.p.m. AA via drinking water for 4 weeks and then examined genotoxicity in the bone marrow, liver and testis. We also analysed the level of N7-(2-carbamoyl-2-hydroxyethyl)-guanine (N7-GA-Gua), the major DNA adduct induced by AA, in the liver, testis and mammary gland. At 40 and 80 p.p.m., both age groups yield similar results in the comet assay in liver; but at 80 p.p.m., the bone marrow micronucleus frequency and the gpt-mutant frequency in testis increased significantly only in the young rats, and N7-GA-Gua adducts in the testis was significantly higher in the young rats. These results imply that young rats are more susceptible than adult rats to AA-induced testicular genotoxicity.

Introduction

Acrylamide (AA) is a low molecular weight vinyl compound commonly used in industries and laboratories. Because individuals are exposed to AA in the workplace, health concerns originally centred on occupational exposure (1). A recent study, however, reported that low levels of AA are formed in many heat-processed foods, especially starchy ones such as potato chips, crackers and French fries (2,3), as a result of asparagine reacting with sugars (Maillard reaction) (4,5). This finding raises concerns that AA poses health risks for the general population (6).

Many animal studies have demonstrated that AA induces neurotoxicity, testicular toxicity and reproductive toxicity (7–9). AA also causes cancers such as mammary fibroadenomas, thyroid follicular cell adenomas and testicular mesotheliomas in rats (10–12). In mice, it induces gene mutations in liver, micronuclei in haematopoietic cells (13,14) and chromosome aberrations in spermatids and spermatocytes (15,16). Thus, AA is clearly genotoxic in vivo, although its in vitro genotoxicity remains unclear because it is not metabolically activated in standard in vitro systems (17,18). AA is metabolised to glycidamide (GA), presumably by cytochrome P450 2E1 (CYP2E1), which quickly reacts with cellular DNA and protein (6,19,20). Two major GA–DNA adducts—N7-(2-carbamoyl-2-hydroxyethyl)-guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)-adenine (N3-GA-Ade)—have been identified in mice and rats treated with AA or GA (21–23), with the level of N7-GA-Gua being 100 times as high as the level of N3-GA-Ade in the organs (22). Individual GA to AA ratios, which can be used as an indicator of the extent of AA metabolism, are highly variable, suggesting that some individuals or populations may be more susceptible than others to AA-induced genotoxicity (24). Other issues are AA intake and metabolism in children compared with adults. Children generally consume larger amounts of food relative to their body mass than adults and favour foods such as French fries and potato chips that have relatively high AA concentrations (25). These issues should be considered when evaluating the susceptibility of the paediatric population in genotoxic and carcinogenic risk assessments (26).

In the present study, to compare the susceptibility to AA-induced genotoxicity of young versus, adult age groups, we treated 3- and 11-week-old male gpt delta transgenic F344 rats with 0, 20, 40 or 80 p.p.m. of AA via drinking water for 4 weeks and examined genotoxicity in the bone marrow, liver and testis. We also analysed the level of N7-GA-Gua in the liver, testis and mammary gland.

Materials and methods

Animals, diet and housing

We purchased 20 male with 10-week-old and 15 pregnant female F344 gpt delta transgenic rats from Japan SLC (Shizuoka, Japan). The pregnant animals were time-mated at 10 weeks of age and arrived on gestational Day 12 or 13 to our facility. After delivery, we obtained >14 male pups from the pregnant rats. All animals were housed three to five rats in polycarbonate cage with sterilised wood chip bedding and maintained under specific pathogen-free standard laboratory conditions: room temperature, 24 ± 1°C; relative humidity, 55 ± 5%; 12-h light–dark cycle; basal diet (CRF-1; Oriental Yeast Company, Tokyo, Japan) and tap water ad libitum until parturition.

Treatments of animals

The protocol for this study was approved by the Animal Care and Utilisation Committee of the National Institute of Health Sciences. We randomly divided 14 and 20 of the 3- and 11-week-old rats into four groups of 3–5 animals, treated them for 4 weeks with AA (Wako Pure Chemical Co., Tokyo, Japan) at 0, 20, 40 or 80 p.p.m. in drinking water and monitored clinical signs, body weight and food and water consumption. At the end of the treatment period, we anaesthetized and killed the animals, and we excised organs for the gpt mutation assay (liver, testis), comet assay (liver), DNA adducts analysis (liver, testis, mammary gland and thyroid) and micronucleus (MN) test (bone marrow).

MN test

We removed bone marrow from the femur, mixed it with foetal calf serum, placed it on an acridine orange-coated glass slide, covered it with a coverslip and stained it supravitally (27). We analysed 2000 polychromatic erythrocytes per animal with a fluorescence microscope and recorded the number of micronucleated polychromatic erythrocytes, which fluoresced greenish yellow.

Alkaline comet assay

We performed the comet assay using the procedure recommended by the comet assay working group of the International Workshop on Genotoxicity Testing (IWGT) (28,29), except that we used a MAS-coat type slide glass (Matsunami Glass Ind. Ltd, Tokyo, Japan) instead of a conventional agarose bottom layer (30). We prepared cell suspensions from the livers, mixed them with 0.5% w/v low-melting agarose, and spotted an aliquot of the mixture onto the slide. After electrophoresis, we stained the cells with SYBR-Gold (cat. # S-11494; Molecular Probes, Invitrogen, Tokyo, Japan), and examined at least 100 cells per animal using a fluorescence microscope (BX50 and BX51; Olympus Corporation, Tokyo, Japan) connected to the comet assay scoring system (Comet IV; Perceptive Instruments Ltd, Suffolk, UK), which quantified the result as %tail intensity.

gpt mutation assay

We extracted high molecular weight genomic DNA from the liver and testis using a Recover Ease DNA Isolation Kit (Stratagene, La Jolla, CA, USA), rescued lambda EG10 phages using Transpack Packaging Extract (Stratagene) and conducted the gpt mutation assay as previously published (31). We calculated the gpt-mutant frequency (gpt-MF) by dividing the number of 6-thioguanine-resistant colonies by the number of colonies with rescued plasmids.

DNA adduct assay

As a standard for liquid chromatography tandem mass spectrometry analysis, N7-GA-Gua and [15N5]-labelled N7-GA-Gua were synthesised as described previously (18,22). We extracted DNA from the liver, testis, mammary gland and thyroid using a DNeasy 96 Blood & Tissue Kit (QIAGEN, Düsseldorf, Germany), incubated it at 37°C for 48 h for deprination. We added an aliquot of the labelled standard to each sample and filtered through an ultrafiltration membrane to remove DNA. The eluted solution was evaporated thoroughly and dissolved in water and then the solutions were subsequently quantified by a Quattro Ultima Pt triple stage quadrupole mass spectrometer (Waters-Micromass, Milford, MA, USA) equipped with a Shimadzu LC system (Shimadzu, Japan). We analysed the liver and testis for each individual rat but pooled the mammary and thyroid glands for each treatment group because the tissue yields were too small to be examined individually.

Statistical analysis

We used the Student’s t-test to determine the statistical significance of the difference in the results of the gpt mutation assay and the DNA adduct assay between the treated and negative control groups and between the young and adult groups. We examined variances in body weight and results of the MN and comet assays by one-way analysis of variance using the Dunnett’s test to compare the differences between the control and treated groups.

Results

Clinical signs, body weight and AA intake

We observed no clinical abnormality in either the young or adult rats during the 28-day treatment period. We found no significant differences in body weight or food and water consumption between the adult treatment groups, although we did observe a slight but statistically insignificant suppression of body weight in the young, 80-p.p.m. treatment group (Table I). The table shows average daily food, water and AA intake of the young and adult treatment groups and their mean body weights. The average daily intakes of AA are calculated as 3.01, 5.95 and 12.19 mg/kg body weight for 20, 40 and 80 p.p.m. group, respectively, in young rats and as 1.83, 3.54 and 7.05 mg/kg body weight for 20, 40 and 80 p.p.m. group, respectively, in adult rats.

Table I

Body weight, food and water consumption and AA intake of young and adult rats

Group AA dose (p.p.m.) No. of animals Initial body weight (g) mean ± SD Final body weight (g) mean ± SD Food consumption (mg/rat/day) Water consumption (ml/rat/day) Intake of AA (mg/kg/day) 
Young 40.5 ± 2.7 168.9 ± 14.3 11.2 17.9 
20 37.1 ± 3.5 164.7 ± 17.8 11.3 16.9 3.01 
40 38.2 ± 2.1 165.1 ± 3.6 11.1 16.7 5.95 
80 40.6 ± 2.5 157.4 ± 8.2 11.0 16.9 12.19 
Adult 249.8 ± 10.0 301.3 ± 11.5 16.4 25.8 
20 249.8 ± 8.2 299.9 ± 7.3 16.1 25.4 1.83 
40 250.5 ± 8.7 302.4 ± 12.2 16.2 24.8 3.54 
80 249.1 ± 7.7 306.6 ± 5.4 16.8 24.6 7.05 
Group AA dose (p.p.m.) No. of animals Initial body weight (g) mean ± SD Final body weight (g) mean ± SD Food consumption (mg/rat/day) Water consumption (ml/rat/day) Intake of AA (mg/kg/day) 
Young 40.5 ± 2.7 168.9 ± 14.3 11.2 17.9 
20 37.1 ± 3.5 164.7 ± 17.8 11.3 16.9 3.01 
40 38.2 ± 2.1 165.1 ± 3.6 11.1 16.7 5.95 
80 40.6 ± 2.5 157.4 ± 8.2 11.0 16.9 12.19 
Adult 249.8 ± 10.0 301.3 ± 11.5 16.4 25.8 
20 249.8 ± 8.2 299.9 ± 7.3 16.1 25.4 1.83 
40 250.5 ± 8.7 302.4 ± 12.2 16.2 24.8 3.54 
80 249.1 ± 7.7 306.6 ± 5.4 16.8 24.6 7.05 

MN test

While no AA dose induced MN in adult rat bone marrow, the highest dose (80 p.p.m.) significantly increased the MN frequency in young rat bone marrow (Figure 1a). Because of the large standard deviation, however, the difference between young and adult rats was not significant (Figure 1a).

Fig. 1

(a) MN frequency in bone marrow of AA-treated young (open bars) and adult (closed bars) gpt delta rats. (b) Tail intensity (%) in the comet assay in liver of AA-treated young (open bars) and adult (closed bars) gpt delta rats. The values represent the mean of experiments ± standard deviations. *is statistically significant experiment compared with the untreated control (P < 0.05)discussion.

Fig. 1

(a) MN frequency in bone marrow of AA-treated young (open bars) and adult (closed bars) gpt delta rats. (b) Tail intensity (%) in the comet assay in liver of AA-treated young (open bars) and adult (closed bars) gpt delta rats. The values represent the mean of experiments ± standard deviations. *is statistically significant experiment compared with the untreated control (P < 0.05)discussion.

Alkaline comet assay

DNA damage induced by AA in liver was evaluated by the comet assay under alkaline conditions (Figure 1b). The comet tail intensities increased in a dose-dependent manner in both young and adult rats with no statistically significant differences between the two groups. AA significantly induced DNA damage at 40 and 80 p.p.m. in the adult rat liver and at 80 p.p.m. in the young rat liver.

gpt mutation assay

Figure 2 shows the gpt mutation assay results. The gpt-MF of control (0 p.p.m.) young and adult rat livers was 1.57 ± 0.72 (×10−6) and 3.66 ± 2.14 (×10−6), respectively. The control gpt-MF of the young rat liver was lower than that of the adult rat liver, but not significantly. AA did not increase the gpt-MF in the liver of either age group at any dose; but at 80 p.p.m., it approximately doubled the gpt-MF in the testis of both young and adult rats, but the increase in adult rats was not statistically significant.

Fig. 2

gpt Mutation frequency in liver (a) and testis (b) of AA administered young (open bars) and adult (closed bars) gpt delta rats. The values represent the mean of experiments ± standard deviations. *is statistically significant experiment compared with the untreated control (P < 0.05).

Fig. 2

gpt Mutation frequency in liver (a) and testis (b) of AA administered young (open bars) and adult (closed bars) gpt delta rats. The values represent the mean of experiments ± standard deviations. *is statistically significant experiment compared with the untreated control (P < 0.05).

DNA adduct formation

Figure 3 shows N7-GA-Gua DNA adduct levels in the liver, testis and mammary glands and thyroid of the young and adult rats. The adduct level increased in a dose-dependent manner in all the tissues. In the mammary glands and thyroid, adduct levels did not differ significantly between young and adult rats. In the liver and testis, on the other hand, the level was higher in the young rats than in the adult rats. In the testis, the DNA adduct level of young rats was approximately six times that of adult rats at all treatment doses.

Fig. 3

Levels of N7-GA-Gua in the liver (a) testis (b) mammary gland (c) thyroid and (d) administered AA young (open bars) and adult (closed bars) gpt delta rat. The mammary gland and thyroid were pooled and analysed in the treatment group. Data are expressed as the number of adducts in 106 nucleotides. * and **are statistically significant experiment compared with the untreated control (*P < 0.05, **P < 0.05). # and ##are statistically significant experiment compared between young and adult gpt delta rat (#P < 0.05, ##P < 0.05).

Fig. 3

Levels of N7-GA-Gua in the liver (a) testis (b) mammary gland (c) thyroid and (d) administered AA young (open bars) and adult (closed bars) gpt delta rat. The mammary gland and thyroid were pooled and analysed in the treatment group. Data are expressed as the number of adducts in 106 nucleotides. * and **are statistically significant experiment compared with the untreated control (*P < 0.05, **P < 0.05). # and ##are statistically significant experiment compared between young and adult gpt delta rat (#P < 0.05, ##P < 0.05).

Discussion

The in vivo genotoxicity of AA has been clearly demonstrated by various rodent genotoxicity tests including MN tests in peripheral blood (13,14,32) and gene mutation and comet assays in various organs (14,33,34). However, there has been no report for the comparison of genotoxicity between young and adult animals. In this study of the genotoxicity of AA in various organs of young (3-week-old) and adult (11-week-old) male rats, we showed that the testis were more vulnerable to AA genotoxicity in the young rat than in the adult rat. Especially, N7-GA-Gua DNA adduct was much higher accumulated in the testis of young rats than of adult rats (Figure 3). The daily intake of AA per weight in young rats was ∼1.5-fold of the adult rats because the younger animals drank more water. It can explain the higher accumulation of adduct in the young rat liver, but the level in testis was approximately six times high in the young rats than in the adult rats, suggesting that AA metabolism in testis is different depending on animal age. Testis is one of the target organs of AA-induced genotoxicity (15,16,35–39). We believe that this is the first report of an age difference in the effect.

AA is primarily metabolised in animals via two competing pathways: oxidation by CYP2E1 to form GA (activation) and conjugation by glutathione S-transferase (GST) with reduced glutathione (detoxification) (19,40,41). GA may subsequently undergo conjugation or hydrolysis catalysed by epoxide hydrolase. The balance between activation and detoxification probably determines AA genotoxicity in vivo. Rat testis shows CYP2E1 activity (42). Wang et al. (43) reported that the treatment of 1.4 and 7.0 mM of AA or GA via drinking water for 4 weeks induced the testicular cII mutation in Big Blue mice. The cII mutation spectra significantly differed between testis and liver, suggesting that testis may have different pathway to metabolise AA and GA. However, the developmental changes were not studied. Recently, Takahashi et al.. (44) showed that GST activity in the testis was significantly lower in young rats than in adult rats and that could explain the different age-related N7-GA-Gua adduct levels and gpt-MFs in the present study. The greater mutagenicity of aflatoxin B1 in liver of neonatal mice than of adult mice corresponds to liver GST levels (45). The GST level in the organs could be responsible for the expression of genotoxicity of AA and aflatoxin B1.

While the N7-GA-Gua adduct level in liver and testis clearly increased in a dose-dependent manner and significantly differed between young and adult rats, the gpt mutation results were not clear. We treated the rats with doses that were lower than those used in other studies (14,34,43), and these doses may have been insufficient to induce gene mutations in our study. Indication of the DNA adduct must be good biomarker to demonstrate genotoxic insult under low-dose exposure condition.

In conclusion, this finding that young rats were more susceptible than adult rats to AA-induced genotoxicity, especially in the testis, suggests that we should be concerned about the risk to children exposed to AA via ordinary foods.

Funding

Health, Labour and Wealth Science Research Grant in Japan (H21-food-general-012), Human Science Foundation in Japan (KHB1006).

The authors are grateful to Dr Miriam Bloom (SciWrite Biomedical Writing & Editing Services) for providing professional editing.

Conflict of interest statement: None declared.

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