ToxSci Advance Access originally published online on November 6, 2006
Toxicological Sciences 2007 95(2):383-390; doi:10.1093/toxsci/kfl155
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Cytogenetic Damage Induced by Acrylamide and Glycidamide in Mammalian Cells: Correlation with Specific Glycidamide-DNA Adducts
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* Department of Genetics, Faculty of Medical Sciences, New University of Lisbon, 1349-008 Lisboa, Portugal
Faculty of Pharmacy, University of Lisbon, Avenida das Forças Armadas, 1649-019 Lisboa, Portugal
Section of Molecular Carcinogenesis, The Institute of Cancer Research, Sutton, Surrey SM2 5NG, United Kingdom
Centro de Química Estrutural, Instituto Superior Técnico, 1049-001 Lisboa, Portugal
¶ Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079
2 To whom correspondence should be addressed at Department of Genetics, Faculty of Medical Sciences, New University of Lisbon, Rua da Junqueira 96, 1349-008 Lisboa, Portugal. Fax: +351 213622018. E-mail: jgaspar.gene{at}fcm.unl.pt.
Received August 11, 2006; accepted October 25, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acrylamide (AA) is a suspected human carcinogen generated in carbohydrate-rich foodstuffs upon heating. Glycidamide (GA), formed via epoxidation, presumably mediated by cytochrome P450 2E1, is thought to be the active metabolite playing a central role in AA genotoxicity. In this work we investigated DNA damage induced by AA and GA in mammalian cells, using V79 Chinese hamster cells. For this purpose, we evaluated two cytogenetic end points, chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs), as well as the levels of specific GA-DNA adducts, namely, N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade) using high-performance liquid chromatography coupled with tandem mass spectrometry. GA was more cytotoxic and clastogenic than AA. Both AA and GA induced CAs (breaks and gaps) and decreased the mitotic index. GA induced SCEs in a dose-responsive manner; with AA, SCEs were increased at only the highest dose tested (2mM). A linear dose-response relationship was observed between the GA concentration and the levels of N7-GA-Gua. This adduct was detected for concentrations as low as 1µM GA. N3-GA-Ade was also detected, but only at very high GA concentrations (
250µM). There was a very strong correlation between the levels of N7-GA-Gua in the GA- and AA-treated cells and the extent of SCE induction. Such correlation was not apparent for CAs. These data suggest that the induction of SCEs by AA is associated with the metabolism of AA to GA and subsequent formation of depurinating DNA adducts; however, other mechanisms must be involved in the induction of CAs. Key Words: acrylamide; glycidamide; DNA adducts; chromosomal aberrations; sister chromatid exchanges.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acrylamide (AA; Fig. 1) is an important industrial chemical that has been produced for about 50 years in Europe, Japan, and the United States. AA has numerous applications; it has been used as starting material for the synthesis of polyacrylamide polymers, which are employed mainly as flocculating agents in water treatment (drinking and waste waters), as flow control agents in oil well operations, in pulp and paper processing, and in mining and mineral processing. AA is also used as an ingredient in several cosmetic formulations and in molecular biology research laboratories (IARC, 1994
; Manière et al., 2005).
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AA was recently found to be generated during the heating of carbohydrate-rich foodstuffs, predominantly from the precursor asparagine (Stadler et al., 2002
). This finding has refocused the interest in this genotoxicant, especially because appreciable amounts of AA are present in Western diets. In fact, some foods (e.g., French fries, potato crisps, bread and breakfast cereals, and coffee) may contain up to 3 ppm of AA (Dybing and Sanner, 2003). The average daily intake of AA has been estimated at about 0.5–1.0 µg/kg body weight in adults and up to twofold higher in 13-year-old children consuming a normal Western diet (Dybing and Sanner, 2003). Until 2002, AA was mainly regarded as an industrial or occupational toxicant, and the foremost routes of exposure were considered to be dermal absorption and inhalation of aerosols in the workplace; the new data suggest that oral consumption of AA may be a key element for global risk assessment.
In addition to its well-known neurotoxicity (LoPachin, 2004), the toxicological hazards associated with AA exposure include germ cell mutagenicity (Dearfield et al., 1995) and cancer (Rice, 2005) in rodents. AA has been classified as a probable human carcinogen by IARC (group 2A) (IARC, 1994). This classification is based on experimental rodent models that have shown AA to be carcinogenic, causing tumors at multiple organ sites in both male and female mice and rats, including follicular thyroid tumors, adrenal pheochromocytomas, scrotal mesotheliomas, mammary gland tumors, lung adenomas and carcinomas, glial brain tumors, oral cavity papillomas, and uterine adenocarcinomas (reviewed in IARC, 1994; Rice, 2005). Evidence for the induction of malignant neoplasia by AA in humans is inadequate, mainly because it is quite difficult to associate dietary consumption of AA with a specific cancer outcome. Moreover, occupational studies have failed to show that AA is carcinogenic to industrial workers. It is therefore extremely important to obtain data on the mechanisms of action of AA, in order to understand how this genotoxicant may affect the human genome.
AA is metabolized to glycidamide (GA; Fig. 1) by an epoxidation reaction, presumably mediated by cytochrome P450 (CYP) 2E1 (Ghanayem et al., 2005a,b; Glatt et al., 2005). This metabolic conversion appears to be critical for the genotoxicity of AA because when the mutagenicity of AA and that of GA have been compared, GA has typically been more potent (Baum et al., 2005; Besaratinia and Pfeifer, 2004; Koyama et al., 2006; Manjanatha et al., 2006). Recently, a number of DNA adducts have been characterized from the interaction of GA with DNA. These adducts include N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua, Fig. 1), N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade; Fig. 1), and N1-(2-carboxy-2-hydroxyethyl)-2'-deoxyadenosine (N1-GA-dA) (Gamboa da Costa et al., 2003; Segerbäck et al., 1995). In this work we have compared the extent of GA-DNA adduct formation induced by AA and GA with the genotoxicity of AA and GA using two different mechanistically based cytogenetic assays: the induction of chromosomal aberrations (CA) assay and the sister chromatid exchange (SCE) assay. These assays were performed in V79 Chinese hamster cells, a widely used nontransformed mammalian cell line devoid of CYP activity (Doehmer et al., 1988; Glatt et al., 2005).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
5-Bromo-2'-deoxyuridine (BrdU), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), trypsin, Ham's F-10 medium, Hoechst 33258, 30% hydrogen peroxide (w/w), newborn calf serum, mitomycin C, phosphate-buffered saline, pH 7.4 (PBS), ribonuclease A, and penicillin-streptomycin solution were purchased from Sigma-Aldrich (St Louis, MO). Dimethylsulfoxide (DMSO), methanol, acetic acid, potassium chloride, sodium chloride, and Giemsa dye were obtained from Merck (Darmstadt, Germany). Colchicine and AA (CAS Registry Number 79-06-1,
99.5% pure) were purchased from Fluka (Buchs, Switzerland). GA (CAS Registry Number 5694-00-8, > 98.5% pure, containing 
1% AA) was obtained from Toronto Research Chemicals (North York, Ontario, Canada).
MTT cytotoxicity assay.
Approximately 5 x 103 V79 Chinese hamster cells (MZ) (kindly provided by Prof. H. R. Glatt, German Institute of Human Nutrition, Nuthetal, Germany) were cultured in 200 µl of culture medium per well (Ham's F-10 medium, supplemented with 10% newborn calf serum and 1% antibiotic solution [penicillin-streptomycin]) in 96-well plates and incubated at 37°C under a 5% CO2 atmosphere. The cells were grown for 16 h and then exposed to different concentrations of AA and GA (dissolved in PBS, pH 7.4), ranging from 100 to 10,000µM, for a 24-h period. Hydrogen peroxide (250µM) was used as a positive control. The cells were washed with culture medium, incubated with MTT (500 µg/ml) for a further period of 4 h, and then carefully washed with PBS. At the end of the incubation period, the medium was discarded and DMSO (200 µl) was added to each well. Absorbance was read at 595 nm in a Zenyth 3100 microplate reader. Four independent experiments were performed and eight individual cultures were used for each GA or AA concentration in each independent experiment.
CA assay.
Twenty-four–hour cultures (approximately 5 x 105 cells), growing in 25-cm2 culture flasks, were exposed to different concentrations of AA and GA, ranging from 1 to 2000µM, for a period of 16 h. Mitomycin C (750nM) was used as the positive control. The cells were subsequently washed with fresh culture medium, and colchicine was added at a final concentration of 600 ng/ml; the cells were incubated for a further period of 2.5 h and then harvested by trypsinization, submitted to a hypotonic treatment, and fixed, and the slides were stained according to Oliveira et al. (2005) and scored (Dean and Danford, 1984; Swierenga et al., 1991).
For the quantification of the DNA damage induced by both AA and GA, the index percentage of aberrant cells excluding gaps (%ACEG) was used. This index represents the frequency of metaphases containing CAs excluding gaps and is the standard indicator for the CA assay. The types of aberrations considered for this index were breaks (chromatid and chromosome), dicentric chromosomes and rings, chromatid-type rearrangements (triradial and quadriradial), other complex rearrangements, and multiaberrant cells (MA) (cells with more than 10 aberrations, including heavily damaged pulverized cells). The presence of chromatid and chromosome gaps in AA- and GA-exposed cultures was also evaluated. The index percentage of aberrant cells including gaps (%ACIG) was calculated as mentioned for the %ACEG, including, however, the metaphases containing gaps.
The evaluation of cell proliferation was carried out using the mitotic index (MI). For this index, 1000 V79 cells were scored for each independent experiment and the number of metaphases recorded.
SCE assay.
Twenty-four–hour cultures (approximately 5 x 105 cells), growing in 25-cm2 culture flasks, were exposed to different concentrations of AA and GA, ranging from 1 to 2000µM. BrdU was also added at a final concentration of 6µM. Mitomycin C (750nM) was used as a positive control. After a period of 27 h, the cells were washed with fresh culture medium and colchicine (600 ng/ml) was added. The cells were then incubated for a further 2.5-h period and then harvested by trypsinization, as described before.
Differential staining of BrdU-substituted sister chromatids was performed according to the fluorescence-plus-Giemsa method (Perry and Wolff, 1974). Briefly, the slides were stained for 12 min with the fluorescent dye Hoechst 33258 (10 µg/ml) in 2% KCl (w/v), exposed to UV (254 nm) for 9 min, and then stained with 4% Giemsa ([v/v] in 10mM phosphate buffer, pH 6.8) for 10 min.
SCEs per cell were scored in 30-s metaphases for each dose level in each independent experiment. At least two independent experiments were performed. The evaluation of cell proliferation was carried out using the MI, as described above. At least 100 metaphases per culture for each dose level, in each independent experiment, were scored for the replication index, calculated according to Krishna and Theiss (1995).
DNA adducts—chemical exposure and DNA extraction.
Twenty-four–hour cultures (approximately 8 x 106 cells), growing in 75-cm2 culture flasks, were exposed to different concentrations of AA (0–2000µM) and GA (0–2000µM) during two different time periods, 18 h (corresponding to parallel cultures of the CAs assay) and 29 h (corresponding to parallel cultures of the SCE assay). The cells were then harvested by trypsinization as described above and washed with PBS, and the cell suspensions were immediately stored at – 20°C. DNA was extracted from the cell suspensions using the QIAamp DNA mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions, with minor modifications done in order to prevent depurination of the DNA adducts (Doerge et al., 2005a). Cell suspensions (200–250 µl) were lysed with 20 µl of proteinase K, provided by Qiagen, and ribonuclease A (200 µg) for 1 h at 37°C, and at the end of the chromatographic process, DNA samples were eluted in water (200 µl) and stored at – 20°C for subsequent DNA quantification and determination of DNA adducts.
DNA quantification.
Quantification of DNA was carried out using a PicoGreen dsDNA quantitation kit (Molecular Probes, Eugene, OR). 
Phage DNA (100 µg/ml) was used as the standard. The DNA concentration in the standard curve ranged from 0 to 300 ng/ml. Briefly, 10 µl of final DNA eluate was mixed with 190 µl of Tris–ethylenediaminetetraacetic acid (EDTA) (10mM Tris-HCl, 1mM EDTA, pH 7.5) diluted with PicoGreen reagent. Fluorescence intensity was measured in a Zenyth 3100 microplate reader at excitation and emission wavelengths of 485 and 535 nm, respectively. The yield of DNA extracted from each cell suspension was in the range of 20–40 µg, in accordance with the manufacturer's standard yields of DNA for cultured cells.
Quantification of DNA adducts.
GA-DNA adducts, specifically N7-GA-Gua and N3-GA-Ade (Fig. 1), were released from the DNA by neutral thermal hydrolysis and quantified by high-performance liquid chromatography coupled with tandem mass spectrometry, essentially as described in Gamboa da Costa et al. (2003). Briefly, aliquots of DNA solutions (5 µg), containing the 15N-labeled adducts as internal standards, were heated at 100°C for 15 min and then filtered through a prewashed 3-kDa molecular weight cutoff spin filter. The adducts were separated on a 2 x 150-mm C18 analytical column (Luna C18(2), Phenomenex, Torrance, CA) with 2% acetonitrile in water and quantified by tandem mass spectrometry in the multiple reaction–monitoring mode, using a Quattro Ultima triple quadrupole mass spectrometer (Waters, Milford, MA) equipped with an electrospray source.
Statistical analyses.
Dose-related effects were assessed using linear regression analysis. One-way ANOVA, followed by Dunnett's test, was used to compare specific treatment levels with the control group. Two-way ANOVA, followed by Dunnett's test, was used to compare AA and GA treatments. Pearson's product moment test was used to assess correlations between variables.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A wide range of AA and GA concentrations were tested in a 24-h incubation MTT cytotoxicity assay protocol. The average survival values obtained from four independent experiments with V79 cells treated with AA and GA are depicted in Figure 2. Previous experiments revealed no changes in the survival frequency of V79 cells using a 3-h incubation period with both compounds (data not shown). It is clear from Figure 2 that both AA and GA induced dose-dependent cell death, as measured by the MTT assay. Moreover, GA was clearly more cytotoxic than AA, causing lower survival rates at all the equimolar concentrations studied. Very low (< 5%) survival values occurred at very high concentrations of GA (
4mM) and AA (10mM).
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Both AA and GA induced CAs (Table 1) and decreased the MI, evaluated as a measure of cell proliferation associated with this cytogenetic end point. This antiproliferative effect was more pronounced in GA exposure, with the MI being zero at 2000µM, which prevented the cytogenetic evaluation at this dose level (Table 1).
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AA and its metabolite GA increased the %ACEG, especially for the higher concentrations evaluated (1000 and 2000µM, for GA and AA, respectively). As observed in the survival assays, GA had, as expected, a more pronounced effect than AA at equimolar concentrations (Table 1).
For both AA- and GA-exposed cultures, the CA pattern consisted mainly of chromatid breaks, also a few chromatid-type rearrangements (e.g., triradial/quadriradial) were also found for GA. Dicentric and ring chromosomes and MA were nearly absent.
Gaps are generally considered to be a minor class of aberrations, and their real biological significance has been a matter of discussion. These events are usually recorded separately from the other aberrations (Dean and Danford, 1984; Swierenga et al., 1991); however, it is clear that both AA and GA are very efficient inducers of chromatid gaps, leading to a consistent dose-response effect (p < 0.001) (Table 1). In view of this, we also calculated the %ACIG and the number of gaps per cell (Table 1). The highest values of %ACIG were about 30% for both the AA-exposed (2000µM) and GA-exposed (1000µM) cultures. Moreover, the gaps/cell index revealed a maximum value of 0.2 for AA (2000µM) and 0.3 for GA (1000µM) treatment (Table 1), showing the importance of these aberrations.
Table 2 presents data on the two cytogenetic indices associated with the SCE assay (SCE/cell and SCE/chromosome), as well as the proliferation indices (mitotic and replicative) associated with exposure to AA and GA. A wide range of GA (1–1000µM) and AA (250–2000µM) concentrations were included in this study. The results clearly show that GA consistently induces SCEs for concentrations 10µM (see Table 2), increasing the background level of SCEs by about 10-fold, to levels of 60 SCE/cell at the highest concentration tested (1000µM). For AA-exposed cultures, a significant increase in SCE/cell was only observed for the highest dose tested (Table 2), and this effect can be considered as mild since it represents only an 1.6-fold increase over background.
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The levels of N7-GA-Gua and N3-GA-Ade in V79 cell cultures exposed to AA and GA for 18 and 29 h (corresponding to parallel cultures of the CA and SCE assays, respectively) are presented in Table 3. These data show that GA is a potent inducer of N7-GA-Gua, with a linear dose-response dependence (p < 0.001). For both periods of exposure, the detection of N7-GA-Gua was observed for doses as low as 1µM GA. In addition, the levels of N7-GA-Gua did not show any significant differences between the two exposure periods tested (Table 3). In fact, these levels were in the same range for all the concentrations tested, except for the highest concentration of GA (2000µM), which showed an approximately twofold increase in the 29-h exposure period when compared with the 18-h period; however, this difference did not reach statistical significance.
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N3-GA-Ade was only detected for GA concentrations higher than 250µM, with a dose-response effect. In addition, the levels of this adduct were in all circumstances two orders of magnitude lower than those of N7-GA-Gua, which is fully consistent with previous data from DNA modifications in vitro and from AA and GA administration to mice (Gamboa da Costa et al., 2003
). As observed for N7-GA-Gua, the levels of N3-GA-Ade were independent of the exposure time period, although a twofold to threefold decrease was apparent at 29 h for the highest GA doses tested (1000 and 2000µM), compared with the 18-h incubation period (Table 3). AA exposure led to very low levels of N7-GA-Gua, which were only observed for concentrations higher than 1000µM. The adduct levels detected at 2000µM AA were comparable to those observed for 1µM GA. N3-GA-Ade was not detected in AA-exposed cultures at any dose level. Negative controls, corresponding to cells not exposed to either AA or GA, did not present any detectable levels of GA-DNA adducts.
The levels of N7-GA-Gua were compared with the levels of cytogenetic damage in AA- and GA-exposed cultures, at the periods of time corresponding to parallel cultures for the SCE and CA assays. A very strong correlation was observed between the levels of N7-GA-Gua and SCE/cell (r = 0.987; p = 1.25 x 10–12).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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AA is metabolized to GA in mice, rats, and humans (Boettcher and Angerer, 2005
; Doerge et al., 2005b,c). The conversion of AA to GA is apparently saturable in rodents (Bergmark et al., 1991), and both compounds are detoxified by conjugation with glutathione; in addition, GA can also be detoxified by epoxide hydrolase (IARC, 1994).
The cytotoxic potential of both AA and GA was investigated in this work in order to select the range of concentrations to be tested in the subsequent studies using different cytogenetic end points (CAs and SCEs). The results show that, with our experimental conditions, GA is clearly more cytotoxic than AA for all the concentrations evaluated. Additionally, at concentrations up to 4mM for AA and up to 1mM for GA, the cell survival was clearly above 50%, indicating that the cytotoxicity of both compounds would not hinder the cytogenetic studies (Fig. 2). These results are in agreement with data recently reported by other groups, using different cell survival end points (Baum et al., 2005; Koyama et al., 2006).
The genotoxicity of AA has been evaluated in several systems (reviewed in Besaratinia and Pfeifer, 2004). Positive results for the induction of CAs and SCEs in Chinese hamster V79H3 cells at concentrations in the millimolar range were reported by Tsuda et al. (1993). These results are in general agreement with the results reported in the present work. Our data showed that AA induced CAs in a dose-response manner, with chromatid gaps and breaks being the typical features observed. However, if we exclude the gap-type aberrations, the genotoxicity observed at the highest AA concentration tested (2mM) can be considered moderate. Since CYP2E1 activity is not detectable in V79 cells (Doehmer et al., 1988; Glatt et al., 2005), the clastogenicity observed, about 10% ACEG, must be related to mechanisms other than metabolic conversion to GA.
AA may undergo Michael-type additions, in particular with thiols, thus potentially depleting the levels of glutathione, a molecule protecting the cell against endogenous oxidants and electrophiles (Glatt et al., 2005). Michael-type addition reactions, which proceed at very low rates, have also been reported between AA and DNA to yield a series of depurinating and nondepurinating adducts (Solomon et al., 1985). Additionally, there is some evidence of the involvement of free radicals in AA genotoxicity, leading to oxidative modification of pyrimidines (Blasiak et al., 2004). These mechanisms might, to some extent, explain the clastogenic activity of AA observed in this study. SCEs were induced at only the highest AA dose evaluated (2mM), which was similar to what was observed for the induction of CAs and also consistent with previously reported data (Glatt et al., 2005; Tsuda et al., 1993). The same aforementioned reasons for the results of the CA assay may explain the slight increase in the frequency of SCEs observed in 2mM AA–treated cultures.
Our results concerning the induction of CAs by GA showed that this compound is approximately twofold more clastogenic than AA and, as observed for AA, chromatid gaps and breaks were the most common features observed (Table 1). In addition, the induction of chromatid gaps was observed to be dose dependent. Considering that gaps might be a consequence of DNA breaks (Savage, 2004), these data are in agreement with the results obtained by other authors using the comet assay, where GA induced alkali-labile sites (Koyama et al., 2006; Johansson et al., 2005; Puppel et al., 2005). The comparison of the genotoxicity of AA and GA in human lymphoblastoid TK6 cells in three different end points (comet assay, micronucleus test, and thymidine kinase assay) also suggested that GA is more genotoxic than AA (Koyama et al., 2006), which is in agreement with the data obtained in our cytogenetic end points.
There are only a few studies comparing, in the same experimental conditions, the genotoxic activity of AA with that of its reactive metabolite, GA. In addition, only limited information is available concerning cytogenetic end points for both compounds. To our knowledge, this is the first study reporting data from SCEs and CAs for both compounds in the same experimental conditions. Moreover, it should be noted that the serum levels of AA and GA observed in animals exposed to AA are in the same range of concentrations used in this study. In fact, a single oral dose of 50 mg/kg AA in mice produced peak serum concentrations of AA and GA of 450 and 200µM, respectively (Twaddle et al., 2004), and a repeat dosing through drinking water of 1 mg/kg/day produced steady-state serum concentrations of 500nM in rats for both AA and GA (Doerge et al., 2005a).
The data concerning the levels of N7-GA-Gua after exposure to AA showed that this adduct was only detected for doses higher than 1mM, but at very low levels (Table 3). Since the V79 cells used in our study are essentially devoid of CYP2E1 activity, the low levels of N7-GA-Gua stemming from AA exposure might be related to either residual metabolism of AA in V79 cells or to a small extent of spontaneous nonenzymatic oxidation to GA, under the aerobic conditions used for the incubations (Besaratinia and Pfeifer, 2004).
In cells treated with GA, the measurement of the N7-GA-Gua levels was much more dose sensitive than the determination of the cytogenetic end points evaluated in this study. In fact, N7-GA-Gua was detected for concentrations as low as 1µM GA at two different exposure periods. However, the detection of N3-GA-Ade was only possible for exposure to doses higher than 250µM, which is consistent with the data previously reported by Gamboa da Costa et al. (2003). In that study, the levels of N7-GA-Gua in mice treated with AA were found to be considerably higher than those of N3-GA-Ade, and a similar result was obtained from in vitro incubations of GA with DNA. The levels of N7-GA-Gua in V79 cultures, corresponding to parallel cultures of the CA and SCE assays (18 and 29 h, respectively), were within the same range. Since the half-life of N7-GA-Gua in DNA was determined to be 42 h at 37°C (Gamboa da Costa et al., 2003), a depurination-related decrease in adduct levels between the 18- and 29-h incubation periods would not be expected to exceed 16%, which is consistent with our observations. Moreover, the absence of a net increase in the levels of N7-GA-Gua at 29 h further suggests that the GA concentrations in the incubation media might be essentially depleted at 18 h, presumably through hydrolysis. Likewise, considering that the half-life of N3-GA-Ade in DNA was estimated to be 14 h at 37°C (Gamboa da Costa et al., 2003) and assuming no mechanisms are involved other than spontaneous depurination, a decrease of 42% in the N3-GA-Ade levels would be expected in the 11-h period separating the 18- and 29-h incubations. This is compatible with the apparent decrease in adduct levels observed for N3-GA-Ade at 29 h.
This study shows that GA induced SCEs, in a linear dose-response manner, with a 10-fold increase being observed at 1mM GA (Table 2). Moreover, GA was approximately two orders of magnitude more potent than AA, for which SCEs were only induced at the highest dose tested (2mM). The DNA adducts investigated in the present work are depurinating lesions, known to be formed upon direct reaction of GA with DNA, even at low GA concentrations (Gamboa da Costa et al., 2003). The fact that there is an excellent correlation (r = 0.987; p = 1.25 x 10–12) between the levels of N7-GA-Gua in the GA- and AA-treated cells and the extent of SCE induction strongly suggests that the metabolism of AA to GA and the ensuing formation of depurinating DNA lesions (Ghanayem et al., 2005a,b) are responsible for the SCE induction.
The repair of the lesions induced by GA was recently associated with the small patch of base excision repair pathway (Johansson et al., 2005). In addition, the authors also noted that GA is a strong inducer of single-strand breaks (SSB). It is well known that base excision repair can lead to the formation of DNA breaks (Friedberg et al., 1995). Likewise, depurination produces abasic sites that can initiate DNA breaks (Friedberg et al., 1995). Therefore, DNA breakage may to some extent explain the higher clastogenic effect of GA, compared with AA.
DNA breaks can also be repaired by homologous recombination. This type of repair is important for the double-strand break repair in late S and in G2 phases of the cell cycle (Jackson, 2002). However, in the case of an unrepaired SSB, conversion into a double-strand break can occur. This event may take place during replication, collapsing the replication fork and leaving one free DNA end that is a substrate for homologous recombination (Helleday, 2003). This newly created double-strand break may initiate an SCE by homologous recombination after two subsequent mitotic steps. There is growing evidence that SCEs are formed from persisting SSB; for example, cells deficient in SSB repair have increased levels of SCEs (Helleday, 2003).
It should be stressed that while only a moderate increase in clastogenicity was observed with GA, compared with AA, there were substantial differences (two to three orders of magnitude) in the levels of N7-GA-Gua between cells treated with equimolar doses of AA and GA. Thus, while there appears to be a causal relationship between depurinating adduct levels and SCEs/cell, other mechanisms must be involved in the induction of the other cytogenetic end points measured in this study. For example, while GA should be intrinsically less reactive than AA with free radicals, due to the absence of the olefinic double bond, it is still a potent electrophile that may contribute to glutathione depletion, thus increasing vulnerability of the cells to oxidative damage, as suggested for AA (Blasiak et al., 2004). Additionally, nondepurinating GA adducts (e.g., N1-GA-dA) and depurinating and nondepurinating adducts from direct reaction of AA with DNA (Solomon et al., 1985) might play a role in the cytogenetic responses.
In summary, these results are consistent with the conclusion that the induction of SCEs by AA is associated with the metabolism of AA to GA and subsequent formation of depurinating DNA adducts. Other mechanisms, however, must be involved in the formation of CAs. The elucidation of these mechanisms warrants further investigation.
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NOTES |
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1 Both authors contributed equally to this work.
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ACKNOWLEDGMENTS |
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This research was supported by Fundação para a Ciência e a Tecnologia (FCT) and Grant 69405 from Fundação Calouste Gulbenkian. A Ph.D. grant from FCT to M.P. (SFRH/BD/22612/2005) is also acknowledged. The opinions expressed in this paper do not necessarily represent those of the U.S. Food and Drug Administration.
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