Copyright ©1999 by Jim Shull All photographs by the author. All rights reserved. Amherst Media, Inc. Buffalo, N.Y. 14226 Fax: 716-874-4508 Publisher: Craig Alesse Senior Editor/Project Manager: Richard Lynch Associate Editor: Michelle Perkins ISBN: 0-936262-70-2 Library of Congress Card Catalog Number: 98-71750 Printed in the United States of America.
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Aflatoxin genotoxicity is associated with a defective dna damage response bypassing p53 activationLiver International ISSN 1478-3223 A£atoxin genotoxicity is associated with a defective DNA damageresponse bypassing p53 activation Ozge Gursoy-Yuzugullu1,2, Haluk Yuzugullu1,2, Mustafa Yilmaz2 and Mehmet Ozturk1,2 1 Centre de Recherche INSERM, Institut Albert Bonniot, Universit ´e Joseph Fourier U823, Grenoble, France2 Department of Molecular Biology and Genetics, BilGen Genetics and Biotechnology Research Center, Bilkent University, Ankara, Turkey aflatoxin – checkpoint – DNA damage Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer response – liver cancer – p53 deaths. Aflatoxins, which may play a causative role in 5–28% of HCCsworldwide, are activated in liver cells and induce principally G ! T muta- tions, including the TP53 codon 249(G ! T) hotspot mutation. The DNA AFB1, aflatoxin B1; DMSO, dimethyl damage checkpoint response acts as an antitumour mechanism against sulphoxide; HCC, hepatocellular carcinoma.
genotoxic agents, but its role in aflatoxin-induced DNA damage is unknown.
Aim: We studied the DNA damage checkpoint response of human cells to aflatoxin B1 (AFB1). Methods and results: The treatment of HepG2 hepatoma Mehmet Ozturk, PhD, Centre de Recherche cells with mutation-inducing doses (3–5 mmol/l) of AFB1 induced DNA adducts, INSERM/UJF U823, Institut Albert Bonniot,Universit ´e Joseph Fourier U823, Grenoble, 8-hydroxyguanine lesions and DNA strand breaks that lasted several days.
Persistent phospho-H2AX and 53BP1 foci were also detected, but cell growth Tel: 133476549410 was not affected. AFB1-exposed HepG2 cells formed phospho-H2AX and 53BP1 Fax: 133476549413 foci, but failed to phosphorylate both Chk1 and Chk2. Huh7 hepatoma and HCT116 colorectal cancer cell lines also exhibited a similarly incompletecheckpoint response. p53 phosphorylation also failed, and AFB1-exposed cells Received 14 September 2010 did not show p53-dependent G1 arrest or a sustained G2/M arrest. These Accepted 10 January 2011 observations contrasted sharply with the fully functional DNA damage responseof cells to Adriamycin. Cotreatment of cells with AFB1 did not inhibit p53 and p21Cip1 accumulation induced by Adriamycin. Thus, the deficient checkpointresponse to AFB1 was not due to an inhibitory effect, but could be explained by aninefficient activation. Conclusion: Genotoxic doses of AFB1 induce an incompleteand inefficient checkpoint response in human cells. This defective response maycontribute to the mutagenic and carcinogenic potencies of aflatoxins.
More than 600 000 people die each year from hepatocel- toxins (4 20 mg/kg/day) with aflatoxicosis rarely occurs (5).
lular carcinoma (HCC), mostly (4 80%) in developing In contrast, 4 90% of people at a high risk for aflatoxin- countries (1). Dietary exposure to aflatoxins and infec- caused HCC are exposed to very low doses (0.01–0.3 mg/kg/ tion with the hepatitis B virus are the major risk factors day), but the exposition is chronic (3, 5).
for HCC, the most frequent liver cancer in these areas Aflatoxin B1 (AFB1), the major aflatoxin product, is (2). According to a recent study, about 25 200–155 000 of metabolized mainly in the liver to AFB1-8,9-exo-epoxide global HCCs may be attributable to aflatoxin exposure.
and 8,9-endo-epoxide. The exo-epoxide form of AFB1 Most cases occur in sub-Saharan Africa, Southeast Asia binds to DNA to form the predominant 8,9-dihydro- and China, where populations suffer both from a high 8-(N7-guanyl)-9-hydroxy AFB1 adduct, leading to a more hepatitis B virus prevalence and largely uncontrolled stable imidozole ring-opened AFB1–formamidopyrimidine aflatoxin exposure in food. Thus, aflatoxins may play a adduct (5). The pseudo-half-life for loss of 8,9-dihydro-8- causative role in 5–28% of all global HCC cases (3).
(N7-guanyl)-9-hydroxy AFB1 is short, but AFB1–formami- Aflatoxins are potent liver toxins, lethal when consumed dopyrimidine adducts are stable, accumulate for several in large doses. Sublethal exposures can induce chronic days and remain detectable for several weeks in rat liver (6, toxicity, and low levels of chronic exposure can result in 7). The initial 8,9-dihydro-8-(N7-guanyl)-9-hydroxy AFB1 neoplasia, primarily HCC, in many animal species (4).
adduct and AFB1–formamidopyrimidine adduct, individu- Aflatoxin exposure in humans may occur at high or low ally or collectively, represent the likely chemical precursors levels, depending on the level of dietary Aspergillus con- responsible for the genotoxic effects of AFB1 (8). In tamination. Acute exposure to high levels leads to lethal addition, common oxidative DNA damage, leading to 8- aflatoxicosis associated with liver necrosis. Chronic expo- hydroxydeoxyguanosine lesions, was observed in rat hepatic sure to low levels of aflatoxin is not lethal, but highly DNA following exposure to AFB1 (4, 9). AFB1 induces hepatocarcinogenic. Acute exposure to high levels of afla- mainly G:C to T:A transversions (4). We (10, 11) and others Liver International (2011) 2011 John Wiley & Sons A/S DNA damage response to aflatoxin B1 Gursoy-Yuzugullu et al.
(12) have identified a hotspot G ! T mutation at codon complete cell culture medium. DMSO (o 103 v/v dilu- 249 of the TP53 gene (encoding the mutant p53ser249 tion) and distilled water were used for negative control protein) in HCC tissues in patients exposed to aflatoxins.
experiments. AFB1 treatment was performed in the pre- Later studies demonstrated that this mutation was also sence of the S9-activation system for all HCT116, detectable in non-tumour liver samples (13), as well as in HCT116–p53/ and some HepG2 experiments for enzy- the plasma of 6% of healthy individuals, 15% of cirrhotic matic activation into the AFB1-8,9-exo-epoxide form. The patients and 40% of HCC patients living in aflatoxin- S9 activation system was prepared as described previously contaminated areas (14). Thus, the AFB1-specific G ! T (18, 19), with minor changes. Briefly, the S9-activation mutation of TP53 is frequently present in people exposed to mixture was prepared with 0.20 g/l S9 fraction from aflatoxins before any clinically detectable liver tumour.
Sprague–Dawley rat liver (Xenotech, Lenexa, Kansas, Taken together, these observations provide strong evidence USA), 10.5 mmol/l isocitric acid (Sigma) and 1.8 mmol/l that low levels of AFB1 are highly mutagenic in people b-nicotinamide adenine dinucleotide phosphate sodium chronically exposed to this hepatocarcinogenic agent.
salt hydrate (Sigma). This mixture was filtered (0.45 mm) Eukaryotic cells have developed a powerful DNA and used at a 1:10 dilution in the cell culture medium.
damage response system to protect their genome integ-rity. DNA damage induces several cellular responses that Aflatoxin B1-DNA adduct and 8-hydroxy-deoxyguanosine enable the cell either to eliminate the damage or to immunoperoxidase assays activate senescence and apoptosis processes. DNA da-mage checkpoint proteins play a central role in co- Aflatoxin B1-DNA adducts and 8-hydroxydeoxyguanosine ordinating repair and cell cycle progression to prevent lesions were detected by immunoperoxidase assays, as mutation. Several kinases, including ATM, ATR, Chk1 described previously (20), with minor changes. Briefly, and Chk2, adaptor proteins such as 53BP1 and down- cells were treated with AFB1 or DMSO on coverslips, stream cell cycle control proteins such as p53 and Cdc25, washed with phosphate-buffered saline and then fixed in are involved in damage sensing and cell cycle control.
ice-cold methanol. AFB1-DNA adducts were detected DNA repair mechanisms include direct repair, base using a monoclonal antibody (6A10) against an imidazole excision repair, nucleotide excision repair, double-strand ring-opened persistent form of the major N7-guanine break repair and cross-link repair (15, 16).
adduct of AFB1 (21). Before the immunoperoxidase assay 8,9-Dihydro-8-(N7-guanyl)-9-hydroxy AFB1 and AFB1– of AFB1 adducts, cells were treated with a buffer containing formamidopyrimidine adducts appear to be removed pri- 15 mmol/l Na2CO3 and 30 mmol/l NaHCO3 (pH 9.6) for marily by nucleotide excision repair in mammalian cells, 2 h at room temperature. For the AFB1 adducts and but other repair systems have also been implicated in 8-hydroxydeoxyguanosine lesions, cells were treated with bacteria and yeast (8). The mechanisms of the DNA RNAse (100 mg/ml) in Tris buffer (10 mmol/l Trizma Base, damage checkpoint response to AFB1 are poorly known.
1 mmol/l EDTA and 0.4 mol/l NaCl; pH 7.5) for 1 h at Here, we explored the DNA damage checkpoint response of 37 1C. After washing with phosphate-buffered saline, pro- wild-type p53 human cells to AFB1 exposure. Our findings teinase K (10 mg/ml) treatment was carried out for 7 min at indicate that the checkpoint response to genotoxic and room temperature. After rinsing with phosphate-buffered mutagenic doses of AFB1 is incomplete. AFB1-exposed cells saline, DNA was denatured with 2N HCl for 10 min and failed to activate p53 and did not undergo cell cycle arrest cells were neutralized by soaking coverslips in 50 mmol/l or apoptosis, despite the presence of DNA adducts and the Tris base for 5 min. After blocking for 1 h, cells were accumulation of DNA strand breaks.
incubated with mouse 6A10 (Santa Cruz, Trevigen, France)or Material and methods France) monoclonal antibody overnight at 4 1C. Anti-mouse HRP-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) were used for 30 min for primary HepG2 and Huh7 cell lines were cultivated as described antibody detection. Cells were stained with diaminobenzi- previously (10). HCT116 and HCT116–p53/ cell lines dine solution (Dako, Carpinteria, CA, USA), counter- (17), gifts from B. Vogelstein, were cultivated in McCoy's stained with haematoxylin (Sigma), mounted with 80% cell growth medium (Gibco) supplemented with 10% glycerol and observed under an Olympus light microscope.
heat-inactivated fetal calf serum and 1% penicillin andstreptomycin solution (Gibco).
Post-treatment cell survival – colony-forming abilityassay Cell survival was determined by assessing cell growth in Aflatoxin B1 (Sigma, St Louis, MO, USA) was dissolved in 100 mm dishes after exposure to AFB1 or Adriamycin.
dimethyl sulphoxide (DMSO, Carlo Erba, Milano, Italy).
HepG2 cells were seeded in six-well plates and semicon- Adriamycin (Sigma) and hydroxyurea (Sigma) were dis- fluent cells were exposed to AFB1 (0–50 mmol/l) in the solved in distilled water. Aliquots were stored at 20 1C.
presence of the S9-activation system for 4 and 24 h Working dilutions were prepared fresh and added in a respectively. Control cells were exposed to Adriamycin Liver International (2011) 2011 John Wiley & Sons A/S Gursoy-Yuzugullu et al.
DNA damage response to aflatoxin B1 (0–5 mmol/l) in parallel experiments. Following expo- saponine (Sigma) and 0.3% Triton X-100 (Sigma). After sure, 104 cells were seeded into 100 mm dishes. After 10 blocking for 1 h, cells were incubated overnight at 4 1C, days of cell culture, colonies were fixed in cold methanol, with antibodies against Ser139-phosphorylated H2AX stained with Crystal Violet (Sigma) and counted in (phospho-H2AX; Millipore) or against 53BP1 (Abcam, triplicate experiments. Cell survival was calculated as the Paris, France). After incubation with Alexa 568-conjugated percent ratio of cell numbers in treated vs untreated cells.
secondary antibodies (Invitrogen), cells were counter- Survival parameters were determined by plotting survival stained with 40,6-diamidino-2-phenylindole (Roche) and data on a semi-log plot.
observed using an Apotome (Zeiss) microscope. Imageswere captured with an Axiocam HRc colour CCD camera Western immunoblotting (Zeiss) and digitally saved using AXIO VISION software (Zeiss).
These experiments were carried out as described previously(22). Proteins were subjected to electrophoresis using 10% Cell cycle analysis and bromodeoxyuridine incorporation or 4–12% Bis-Tris NuPAGE Novex Mini gel systems (Invitrogen), according to the manufacturer's instructions.
Cells were washed twice in phosphate-buffered saline and For the detection of phosphorylated proteins, cell lysates fixed in ice-cold ethanol for 10 min. After two phosphate- were prepared according to the protocol provided by the buffered saline washes, cells were incubated with 20 mg/ml supplier using the following lysis buffer: 20 mmol/l Tris of RNase A (Fermentas, Leon-Rot, Germany) at 37 1C for (pH 7.5), 150 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l 10 min and stained with propidium iodide (10 mg/ml; EGTA, 1% Triton X-100, 1 mmol/l Na3VO4, 1 mg/ml leu- Sigma). Cell cycle distribution was determined by flow peptin and 1 mmol/l phenylmethylsulphonyl fluoride. Fol- cytometry using FACSCAN and CELLQUEST software (Becton lowing electrophoresis, proteins were transferred onto Dickinson, Franklin Lakes, NJ, USA). Cell cycle analysis nitrocellulose membranes and analysed using antibodies combined with the bromodeoxyuridine incorporation against cleaved caspase-3 (Cell Signaling, Danvers, MA, assay was performed using cells first labelled with USA), total p53 (Santa Cruz), p21Cip1 (Calbiochem, Darm- 10 mmol/l bromodeoxyuridine (Sigma) for 2 h before each stadt, Germany), phospho-H2AX (Millipore, Billerica, testing time. Cells were incubated with FITC-conjugated MA, USA), phospho-Chk2, phospho-p53ser15, phospho- antibromodeoxyuridine antibody (BD Bioscience, Franklin p53ser20 (all from Cell Signaling) and Calnexin (Sigma).
Lakes, NJ, USA) at room temperature in the dark, follow-ing DNA denaturation with 4N HCl for 30 min (26).
Senescence-associated b-galactosidase assay Senescence-associated b-galactosidase activity was de- tected as described previously (22), using a senescent cell Induction of DNA adducts, 8-hydroxydeoxyguanosine staining kit (Sigma).
lesions and DNA breaks by aflatoxin B1 in HepG2 cells The human hepatoma line HepG2 has retained the activ- Single-cell gel electrophoresis (comet) assay ities of various phase I and phase II enzymes that play a Single- and double-strand DNA breaks were detected crucial role in the activation and detoxification of genotoxic using alkaline and neutral comet assays respectively (23, procarcinogens. It has been used successfully for genotoxi- 24). The alkaline comet was performed exactly as de- city assays for various classes of environmental carcinogens scribed (23). The neutral comet assay was conducted as including aflatoxins, nitrosamines, aromatic and hetero- described (24), using the lysis protocol described by cyclic amines and polycyclic aromatic hydrocarbons, as well Chandna (25). Following electrophoresis, slides were as for antimutagenicity studies (27). Furthermore, HepG2 rinsed, stained with 5 mg/ml 40,6-diamidino-2-phenylin- has retained the wild-type activity of the p53 gene, a well- dole (Roche, Mannheim, Germany) and analysed under established DNA damage response gene (28, 29). Therefore, an Apotome (Zeiss, Germany) microscope. Images were we first used the HepG2 cell line to test the genotoxic effects captured with an Axiocam HRc colour CCD camera of AFB1. Cells were treated with 3–5 mmol/l of AFB1 in the (Zeiss) and digitally saved using absence or the presence of the S9-activating system that AXIO VISION software (Zeiss). Data were analysed by CASP (Comet Assay allows the activation of AFB1 into AFB1-8,9 epoxide (18).
Software Project), which measures tail moment, using Following 24 h of exposure, cells were subjected to the DNA content in the tail and head along with the immmunoperoxidase assays to detect the imidazole distance between the means of the head and tail distribu- ring-opened persistent form of the major N7-guanine tions At least 30 nuclei were ana- adduct of AFB1 and 8-hydroxydeoxyguanosine DNA lysed for each experimental condition.
lesions. Our results verified the detection of AFB1 adductsin the nuclei of most cells with 3 or 5 mmol/l AFB1 (Fig.
S1A). Adduct detection levels were quite similar between these two doses, indicating that AFB1 was capable of Cells were fixed with 4% formaldehyde and permeabilized inducing highly abundant DNA adducts when tested at with phosphate-buffered saline supplemented with 0.5% micromolar levels. We also observed the detection of Liver International (2011) 2011 John Wiley & Sons A/S DNA damage response to aflatoxin B1 Gursoy-Yuzugullu et al.
Fig. 1. Induction of persistent single- and double-strand DNA breaks in HepG2 cells following AFB1 exposure. (A) HepG2 cells were exposedto DMSO, AFB1 (5 mmol/l) or Adriamycin (0.5–1 mmol/l) as a positive control for 24 h, followed by a culture in the absence of test chemicals forup to 72 h, and subjected to alkaline comet or neutral comet assays to detect single- and double-strand breaks respectively. (B) Quantitativeanalysis of AFB1-induced DNA breaks by automated tail moment measurement. Black, white and grey columns indicate cells exposed toDMSO, AFB1 and Adriamycin respectively. Error bars indicate SD. AFB1- and Adriamycin-treated cells displayed significantly increased tailmoments at all time-points tested (P o 0.0001). AFB1, aflatoxin B1; DMSO, dimethyl sulphoxide; SD, standard deviation.
8-hydroxydeoxyguanosine-positive nuclear foci (Fig. S1B).
increase in tail moments with both chemicals that lasted The same results were obtained in the presence or in the at least 48 h after the removal of chemicals from the cell absence of the S9-activating system (Fig. S1).
culture medium (Fig. 1B, right). Tail moments obtained The genotoxic effects of AFB1 were studied by alkaline with the neutral comet were nearly 10-fold fewer than and neutral comet assays that detect single- and double- those obtained with the alkaline comet (Fig. 1B). Thus, strand DNA breaks respectively (23, 24). Examples of AFB1 induced many more single-strand breaks than comet assay results are shown in Figure 1A. Both AFB1- and Adriamycin-exposed cells, tested by an alkalinecomet assay at 72 h post-exposure time, displayed a Lack of significant growth inhibition in response to statistically significant increase in comet tail moments aflatoxin B1 exposure (P o 0.0001), indicating the presence of abundant sin-gle-strand DNA breaks (Fig. 1B, left). A neutral comet Next, we studied the cellular response to AFB1-induced assay also detected a statistically significant (P o 0.0001) genotoxicity using cell growth, senescence and apoptosis Liver International (2011) 2011 John Wiley & Sons A/S Gursoy-Yuzugullu et al.
DNA damage response to aflatoxin B1 Detectable effects of AFB1 were observed only when cellswere exposed for 24 h at doses reaching 50 mmol/l (Fig. 2,bottom, inset).
We noticed that both AFB1 and Adriamycin displayed genotoxic effects that caused DNA breaks at comparableintensities (Fig. 1), but their effects on cell survival werehighly different (Fig. 2). DNA damage usually triggers astrong cytotoxic response as observed here with Adria-mycin (Fig. 2, open circles). This was not the case forAFB1-induced DNA damage that resulted in only a weakcolony-inhibitory effect (Fig. 2, closed circles). In con-firmation of these observations, 3 days of exposure toAFB1 did not induce a senescence response as tested by asenescence-associated b-galactosidase assay (Fig. S2A)nor apoptosis as tested by an activated caspase-3 assay(Fig. S2B). These findings prompted us to further explorethe DNA damage response of HepG2 cells to AFB1.
DNA damage checkpoint foci induction by aflatoxin B1 To test the checkpoint response, we first used 53BP1 andphospho-H2AX foci assays (15) by immunofluorescence.
Both AFB1 and Adriamycin induced 53BP1 and phos-pho-H2AX foci that were detectable after 3 days ofculture, but AFB1-induced foci formation appeared tobe less strong (Fig. 3). These findings provided evidencefor a double-strand DNA break response (15, 16) toboth agents, although the response appeared to beslightly different. We tested the statistical significance of Fig. 2. The effects of AFB1 (closed circles) or Adriamycin (open circles) AFB1-induced foci formation by counting cells with treatment of HepG2 cells for 4 h (top) or 24 h (bottom) on cell survivalcolony-forming ability. Cell survival was calculated as the per cent ratio 53BP1-positive foci (4 5 foci/cell). Cells exposed to of cell numbers in treated vs untreated cells (n = 3). Survival parameters AFB1 between 1 and 72 h showed a progressive and were determined by plotting survival data on a semi-log plot. Insets: statistically significant (P o 0.0001) accumulation of cell survival at higher AFB1 (up to 50 mmol/l) and Adriamycin (up to 53BP1 foci (Fig. S3). To test the duration of 53BP1 foci 5 mmol/l) doses. Error bars: SD. cell survival was determined by following a fixed time of exposure to AFB1, cells were assessing cell growth in 100 mm dishes after exposure to AFB1 or first treated with AFB1 for 24 h and then cultivated in the Adriamycin. HepG2 cells were seeded in six-well plates and absence of chemical treatment for up to 120 h. Cells with semiconfluent cells were exposed to AFB1 (0, 1, 3, 5, 10 and 50 mmol/l) positive 53BP1 foci were detected by an indirect immu- or Adriamycin (0, 0.1, 0.3, 0.5, 1 and 5 mmol/l) for 4 or 24 h. Following nofluorescence assay (Fig. S4A) and then counted. As exposure, 104 cells were seeded into 100-mm dishes and colonies shown in Fig. S4B, the accumulation of 53BP1 foci were counted 10 days later. AFB1, aflatoxin B1; SD, standard deviation.
peaked at 48 h of post-treatment, with 40% positive cells.
A residual foci activity with 15–20% positive cells was assays. Cell survival was determined by assessing colony detectable for at least 120 h in cells no longer exposed to growth in 100 mm dishes after exposure to AFB1 or AFB1. In contrast, cells exposed to DMSO only displayed Adriamycin. HepG2 cells were seeded in six-well plates low foci activity (o 5%) throughout the experiment, indicating that increased foci formation was because of (0–50 mmol/l) in the presence of the S9-activation sys- AFB1 exposure. Western blot analysis of the total 53BP1 tem. Control cells were treated with Adriamycin protein demonstrated its higher expression in cells ex- (0–5 mmol/l). Following 4 and 24 h of exposure, 104 cells posed to AFB1 for at least 72 h (Fig. S4C). Taken were seeded into 100-mm dishes and colonies were together, our findings indicated that following exposure counted 10 days later. Cell survival was calculated as the to AFB1, HepG2 cells develop persistent 53BP1 foci that percent ratio of cell numbers in treated vs untreated cells.
are compatible with a double-strand DNA break re- Survival parameters were determined by plotting survival sponse lasting for several days.
data on a semi-log plot. AFB1 did not affect colonysurvival after 4 or 24 h of treatment at doses 5 mmol/l Effects of aflatoxin B1 on HepG2 cell cycle progression (Fig. 2, closed circles). In contrast, Adriamycin displayeda strong inhibition of colony survival, even with 50 times Based on observations indicating a defective growth less concentrated molar doses (Fig. 2, open circles).
response to AFB1 (Fig. 2), despite the formation of Liver International (2011) 2011 John Wiley & Sons A/S DNA damage response to aflatoxin B1 Gursoy-Yuzugullu et al.
Fig. 3. Induction of 53BP1 and phospho-H2AX foci following AFB1 exposure in HepG2. Cells were treated with AFB1 (3 mmol/l) for 3 days andthen subjected to 53BP1 and phoshpo-H2AX foci detection by indirect immunofluorescence. Control cells were exposed to DMSO only.
Adriamycin (0.1 mmol/l) was used as a positive control. Scale bar = 20 mm. ADR, Adriamycin; AFB1, aflatoxin B1; DMSO, dimethyl sulphoxide.
persistent AFB1-DNA adducts (Fig. S1), DNA strand Incomplete DNA damage checkpoint response to breaks (Fig. 1) and DNA damage foci (Figs. 3, S3 and aflatoxin B1 in different cell types S4), we performed time-course studies on cell cycleprogression of HepG2 cells following AFB1 exposure. As p53-dependent response to DNA damage is a strong shown in Figure 4, AFB1 exposure resulted in a transient mechanism protecting cells against the accumulation of accumulation of cells at the S phase (up to 26% from deleterious mutations (15, 17, 30). Based on the current 13%, one-fold increase) at 24 h, followed by a return to model for p53 activation upon DNA damage (15), we control levels at 48 and 72 h. These changes were accom- tested the status of critical components of DNA damage panied by a slight increase (40%) in G2/M-phase cells at signalling after AFB1 exposure. Adriamycin and hydro- 48 and 72 h, together with a slight decrease (18–26%) in xyurea were used for control experiments. As shown in G1-phase cells. These observations provided evidence for Figure 5, Adriamycin treatment induced a typical dou- a transient and weak growth inhibition in HepG2 cells ble-strand break response in HepG2 cells by induced following AFB1 exposure (Fig. 4). The lack of a total cell phosphorylations of H2AX, Chk2 and p53ser15, together cycle block under AFB1 exposure was compatible with a with a weak induction of p53ser20 phosphorylation.
nearly complete colony survival after 5 mmol/l AFB1 Hydroxyurea treatment resulted in a weak phosphoryla- exposure (Fig. 2). Of particular interest, AFB1-exposed tion of Chk1. As expected, we noted time-dependent HepG2 cells did not undergo G1 arrest despite the differences in these responses. The response to AFB1 was expression of wild-type p53, strongly suggesting that globally weak or even absent. The only detectable AFB1-induced DNA damage did not trigger a p53- response was observed with H2AX phosphorylation that dependent DNA damage response in these cells.
was detectable after 24 h of AFB1 exposure, as well as Liver International (2011) 2011 John Wiley & Sons A/S Gursoy-Yuzugullu et al.
DNA damage response to aflatoxin B1 Fig. 4. The effects of AFB1 on HepG2 cell cycle distribution. Cells were treated with either 5 mmol/l AFB1 or DMSO up to 72 h, and cell cycledistribution was analysed by flow cytometry at 24, 48 and 72 h. AFB1, aflatoxin B1; DMSO, dimethyl sulphoxide.
after 24 h post-exposure. We performed additional stu- obtained with HepG2 cells. The great majority of cells dies with Huh7, an HCC cell line with retained activities stained positive for 8-hydroxydeoxyguanosine lesions for AFB1 activation (31), but displaying a homozygous following drug exposure (Fig. S6A) and displayed DNA p53 mutation (28). As shown in Fig. S5, Huh7 cells double-strand breaks as tested by a neutral comet assay responded to Adriamycin by upregulation of phospho- (Fig. S6B). Furthermore, HCT116 cells exposed to H2AX levels only, and there was no phospho-Chk1 5 mmol/l AFB1 for 24 h displayed a statistically significant phosphorylation in response to hydroxyurea treatment.
(P o 0.0001) increase in both 53BP1 and phospho- AFB1 did no affect phosphorylations of Chk1, chk2 or H2AX-positive foci that lasted at least 48 h post-expo- p53ser15, and the effect on H2AX phosphorylation was sure, independent of TP53 status (Fig. S7). Western blot weakly detectable at 24 h of exposure. Taken together, analysis of critical components of DNA damage check- these studies indicated that, apart from H2AX phosphor- point response also provided results quite similar to that ylation, critical components of DNA damage checkpoint of HepG2. As shown in Fig. S8 and in comparison with proteins were not affected in hepatoma cells. In addition, Adriamycin and hydroxyurea, AFB1 treatment induced the induction of p53 phosphorylation in response to only a weak upregulation of phospho-H2AX levels at 24 h DNA damage by Adriamycin appeared to be dependent of exposure, with a more pronounced increase at 24 h of on the wild type of the mutant status of p53 gene (see Fig. 5 in comparison with Fig. S5).
Taken together, our observations with three different In order to further investigate the role of AFB1 in DNA cell lines indicated that AFB1 induced a weak and delayed damage response induction, we decided to explore wild- accumulation of phospho-H2AX. The phosphorylation type p53-expressing HCT116 colorectal cancer cells and of H2AX strongly suggested that AFB1-induced DNA their p53 knockout HCT116–p53/ derivatives (17). We damage triggered ATM activation by double-strand DNA performed all AFB1 experiments in these cell lines in the breaks (15, 33). However, this ATM response was not presence of the S9-activating system to allow the trans- accompanied by phosphorylations of Chk1, Chk2 or p53, formation of AFB1 into epoxy-AFB1 (32). First, we three key proteins involved in the DNA damage check- assessed the formation of DNA lesions following expo- point response. The lack of Chk1 phosphorylation after sure to AFB1 (5 mmol/l) and Adriamycin (1 mmol/l). The AFB1 exposure also suggested that the ATR/Chk1 path- response of HCT116 cells to both AFB1 and Adriamycin way response was also inactive against AFB1-induced treatment was not different from the observations Liver International (2011) 2011 John Wiley & Sons A/S DNA damage response to aflatoxin B1 Gursoy-Yuzugullu et al.
Fig. 5. Incomplete DNA damage checkpoint response of HepG2cells to aflatoxin B1 (AFB1). HepG2 cells were treated with dimethylsulphoxide (DMSO) or AFB1 (5 mmol/l) for 4 and 24 h, and testedimmediately (410 h and 2410 h) or after 24 h of incubation withouttreatment (24124 h). HepG2 cells treated with 0.5 mmol/l Fig. 6. Comparative analysis of the wild-type p53 response of Adriamycin (ADR) or 5 mmol/l hydroxyurea (HU) were used as HCT116 cells to AFB1 and Adriamycin treatment indicates that AFB1 positive controls for experiments shown in (A) and (B) respectively.
cannot induce effective p53 activation. (A) Wild-type 53 HCT116 and Total cell lysates were subjected to western blot analysis. Calnexin p53-deficient HCT116-p53/ cells were treated with AFB1 (3 mmol/l) was used as a loading control. p-H2AX, phospho-H2AX; p- or DMSO (in the presence of the S9-activating system) or Adriamycin p53ser15, phospho-p53ser15; p-p53ser20, phospho-p53ser20; (ADR; 1 mmol/l) for 24 h, followed by an additional cell culture in the p-Chk2, phospho-Chk2; p-Chk1, phospho-Chk1.
absence of this chemical for another 24 h. (B and C) HCT116 (B) andHCT116-p53/ (C) cells were cotreated with Adriamycin (ADR, The mechanism of the inefficient DNA damage response increasing doses: 0, 0.1, 0.5 and 1 mmol/l respectively) in the absence (DMSO) or in the presence of 3 mmol/l AFB1, as described in (A) for24 h (24 h pre-exposure to AFB1, followed by 24 h of co-exposure).
As we observed similar responses of HCT116 and hepatoma Total cell lysates were used for western blot using anti-p53 and anti- cells to both AFB1 and Adriamycin treatment, we decided p21Cip1 antibodies. Calnexin was used as a loading control. AFB1, to further explore AFB1 effects using the isogenic HCT116 aflatoxin B1; DMSO, dimethyl sulphoxide.
model, allowing us to better define its potential implicationsin p53-mediated DNA damage response. Before testing ofAFB1 effects, we first examined the cell cycle responses of Our cell cycle studies with AFB1 treatment in the same HCT116 and HCT116–p53/ cells to Adriamycin. HCT116 cell lines are shown in Fig. S10. Unlike HepG2 cells, the cells displayed G1 and G2/M arrests in response to Adria- HCT116 cell lines did not display a significant increase mycin, associated with low levels of apoptosis (subG1 peak) in S-phase cells. However, they displayed a weak decrease and polyploidy formation at higher doses (Fig. S9A). There in the G1 phase, in parallel to a weak increase in G2/M was also a depletion of S-phase cells as an indication of cells, as observed with HepG2 cells. The response of DNA synthesis inhibition that lasted at least 48 h following HCT116–p53/ cells to AFB1 exposure was not remark- the removal of Adriamycin from the cell culture medium.
able either, except for a slight increase in G2/M cells.
The response of HCT116–p53/ cells to Adriamycin was Taken together, these observations strongly suggested essentially similar, with the noticeable absence of a G1 peak that human cells exposed to AFB1 could not develop a (Fig. S9A). Based on Fig. S9B, which compares Adriamycin- growth control response. The most likely reason for this induced cell cycle changes in wild-type and p53 knockout was a delayed and deficient checkpoint response, includ- HCT116 cells, we concluded that DNA damage induced by ing a lack of efficient phosphorylation of p53 protein.
Adriamycin is associated with a p53-dependent G1 arrest Therefore, we also compared the effects of AFB1 and and a p53-independent G2/M arrest.
Adriamycin on p53 and p21Cip1. As shown in Figure 6A, Liver International (2011) 2011 John Wiley & Sons A/S Gursoy-Yuzugullu et al.
DNA damage response to aflatoxin B1 both p53 and p21Cip1 responded to Adriamycin treatment response studies over a period of several days so that we with a dose-dependent increase in HCT116 cells. The could determine both immediate and delayed effects.
increase in p21Cip1 levels was p53-dependent, because we Our findings demonstrate that AFB1, when tested under did not observe p21Cip1 response in HCT116–p53/ cells.
conditions comparable with mutagenic and carcinogenic In contrast, AFB1 treatment did not produce any detect- exposure levels, creates DNA adducts, 8-hydroxy-deoxy- able change in p53 levels in HCT116 cells. As a result, there guanosine lesions and persistent strand breaks, but it does was no detectable increase in p21Cip1 in both HCT116 and not lead to a sustained cell cycle arrest and/or an apoptosis HCT116–p53/ cells. These findings suggested that either response. AFB1 adducts are repaired by nucleotide excision the AFB1 was actively involved in the inhibition of an repair (8); however, their removal is slow (6) and they effective DNA damage checkpoint response or the damage remain at maximum levels for several days and are inflicted by AFB1 did not reach a threshold that is detectable over several weeks in rat liver cells (6, 7). The necessary for checkpoint activation, similar to previous unusual stability of AFB1 adducts together with a slow observations with low-dose ionizing radiation (16). To test repair process could account for their strong genotoxic whether AFB1 inhibits the checkpoint response, we co- effects. The expansion of cells with unrepaired DNA lesions treated HCT116 cells with increasing doses of Adriamycin could cause mutations in their genomes. Therefore, such (0, 0.1, 0.5 and 1 mmol/l respectively) in the absence or cells are under the strict control of DNA damage check- presence of 3 mmol/l AFB1. As shown in Figure 6B and C, point proteins that block cell cycle and/or induce apoptosis the accumulation of p53 and p21Cip1 after Adriamycin (15, 16, 30). Our in vitro findings and previously reported exposure was not inhibited by AFB1 in HCT116 cells.
in vivo studies strongly suggest that cells exposed to Indeed, there was a slight increase in the accumulation mutagenic doses of AFB1 cannot develop a strong cell cycle p21Cip1 after 0.1 mmol/l Adriamycin treatment in the pre- arrest and/or apoptosis response. Our detailed analysis of sence of AFB1. The p21Cip1 response of HCT116–p53/ DNA damage checkpoint proteins provides a plausible cells to Adriamycin was not affected by AFB1, except for a explanation for the uncoupling between DNA damage weak accumulation that was observed when cells were and growth control following AFB1 exposure. AFB1- cotreated with 1 mmol/l Adriamycin and 3 mmol/l AFB1.
exposed cells displayed DNA damage foci formation with These findings showed that AFB1 did not inhibit DNA both 53BP1 and phospho-H2AX marker proteins. These damage checkpoint response under the conditions tested.
findings suggest that AFB1-induced DNA damage might Instead, AFB1 slightly stimulated the checkpoint response trigger a checkpoint response compatible with a double- to Adriamycin.
strand break-type response involving ATM. However, thisresponse was weak and delayed, as indicated by phospho-H2AX levels tested by western blot analysis. Our western blot studies for phospho-ATM levels after AFB1 exposure Hepatocellular cancer risk from aflatoxins, as well as provided inconsistent results with or without an increase aflatoxins' hepatocellular biochemistry, DNA interacting (data not shown), further indicating that ATM is not forms, the types of DNA damage and their repair by activated consistently following AFB1-induced DNA da- nucleotide excision, and their in vitro and in vivo mage. In confirmation of this hypothesis, AFB1-induced mutagenic specificity for G ! T transversions are DNA damage failed to activate Chk2 and p53ser15 phos- well-established facts (34). Here, we addressed a less phorylations. The alternative DNA damage checkpoint well-understood, but critical component of aflatoxin response mediated by ATR and Chk1 was also ineffective, genotoxicity, namely the DNA damage checkpoint re- as tested by Chk1 and p53ser20 phosphorylation. The most sponse. The in vitro experimental model system used important outcome of a deficient response to AFB1 was a here was designed after carefully considering previously lack of cell growth control. Apart from a slight and described features associated with aflatoxin-related carci- transient increase in the G2/M phase, cells did not undergo nogenicity. Human cells with a wild-type p53 expression stable cell cycle arrest, senescence and/or apoptosis. Con- were preferred because of the fact that a specific hotspot sequently, the overall cell survival was not affected even mutation of this gene was observed only in human HCC, after exposure to 5 mmol/l AFB1. It was necessary to expose not in other aflatoxin-induced mammalian tumours cells to 50 mmol/l AFB1 for at least 24 h in order to observe (35). We considered estimated chronic aflatoxin expo- a cytotoxic effect. Such a high dose represents a more than sure levels in humans (0.01–0.3 mg/kg/day) (3) and 150-fold higher value when compared with effective doses hepatocarcinogenic doses (0.015–1 ppm) in rats (36).
of Adriamycin in the same type of cells.
We also considered that 30 min of exposure to 1.6 mmol/ The mechanisms of the failing checkpoint response to l AFB1 was sufficient to induce p53-249 G ! T muta- AFB1 are currently unknown. We speculate that AFB1 is tions in HepG2 cells (37) and 0.2–5 mmol/l doses induced able to induce DNA damage, without triggering an effective reporter gene mutations in mouse fibroblasts (32). Thus, damage response signal at doses 5 mmol/l. The delayed the AFB1 doses that we used here (3–5 mmol/l) were at and defective DNA damage response to AFB1 could be the upper limits of in vitro mutagenic activity in mam- related to the type of DNA and protein adducts that it malian cells and were estimably superior to carcinogenic forms in exposed cells (5, 8). AFB1 DNA adducts that are doses in humans and rats. We performed our cell known to be repaired primarily by nucleotide excision Liver International (2011) 2011 John Wiley & Sons A/S DNA damage response to aflatoxin B1 Gursoy-Yuzugullu et al.
repair (8) may not be sufficient to trigger directly a strong and State Planning Office of Turkey (DPT). Additional DNA damage response, which usually requires single- and support was provided by the Turkish Academy of double-strand DNA breaks (15, 16). Instead, the DNA Sciences. The funders had no role in the study design, breaks could occur during the repair process causing a data collection and analysis, decision to publish or delayed response, as suggested by a weak and delayed preparation of the manuscript.
occurrence of phospho-H2AX accumulation observed here.
Conflict of interest statement: none declared.
Alternatively, or in addition, adducts of AFB1 formed withcritical cellular proteins may hamper an effective damageresponse. This alternative is highly unlikely, as suggested bythe inability of AFB1 cotreatment to inhibit Adriamycin- induced accumulation of p53 and p21Cip1 as an end-pointreporter for checkpoint response. Thus, our findings favour 1. Parkin DM. The global health burden of infection-associated the hypothesis that AFB1-induced DNA damage, tested cancers in the year 2002. Int J Cancer 2006; 118: 3030–44.
here at doses 5 mmol/l, did not reach the threshold for 2. Wild CP, Montesano R. A model of interaction: aflatoxins an efficient induction of checkpoint response. At higher and hepatitis viruses in liver cancer aetiology and preven- doses, AFB1 is probably effective to trigger a DNA damage tion. Cancer Lett 2009; 286: 22–8.
response. Indeed, it has been reported previously that 3. Liu Y, Wu F. Global burden of aflatoxin-induced hepatocel- HepG2 cells exposed to 10 mmol/l AFB1 can elicit a cell lular carcinoma: a risk assessment. Environ Health Perspect cycle arrest response (38). When tested with 5 mg/kg dose, 2010; 118: 818–24.
AFB1 exposure could induce p21Cip1 upregulation in rat 4. Wang JS, Groopman JD. DNA damage by mycotoxins.
liver (39). However, as stressed earlier, cancer-causing diet- Mutat Res 1999; 424: 167–81.
ary exposure to AFB1 occurs at low levels, a condition that 5. Wild CP, Gong YY. Mycotoxins and human disease: a largely is similar to our in vitro conditions that provided evidence ignored global health issue. Carcinogenesis 2010; 31: 71–82.
for a defective checkpoint response.
6. Croy RG, Wogan GN. Temporal patterns of covalent DNA A defective or a negligent G2/M checkpoint response adducts in rat liver after single and multiple doses of to low ionizing radiation exposure has been postulated aflatoxin B1. Cancer Res 1981; 41: 197–203.
by L¨obrich and Jeggo (16) as a potential cause of genomic 7. Smela ME, Hamm ML, Henderson PT, et al. The aflatoxin instability and cancer risk. The authors also proposed B(1) formamidopyrimidine adduct plays a major role in that a master p53-dependent G1 checkpoint might causing the types of mutations observed in human hepatocel- remain effective during a negligent G2/M checkpoint for lular carcinoma. Proc Natl Acad Sci USA 2002; 99: 6655–60.
later elimination of escaping cells. Our findings strongly 8. Bedard LL, Massey TE. Aflatoxin B1-induced DNA damage suggest that the DNA damage checkpoint in response to and its repair. Cancer Lett 2006; 241: 174–83.
low doses of AFB1 is defective, negligent or delayed. In 9. Shen HM, Ong CN, Lee BL, Shi CY. Aflatoxin B1-induced addition, a p53-dependent salvage pathway is apparently 8-hydroxydeoxyguanosine formation in rat hepatic DNA.
ineffective against AFB1-induced DNA damage. The lack Carcinogenesis 1995; 16: 419–22.
of an efficient response to AFB1-induced DNA damage 10. Bressac B, Kew M, Wands J, Ozturk M. Selective G to T may be because of the type of lesion(s) induced at the mutations of p53 gene in hepatocellular carcinoma from DNA and/or protein levels by activated AFB1 in exposed southern Africa. Nature 1991; 350: 429–31.
cells. It will be interesting to further investigate these 11. Ozturk M. P53 mutation in hepatocellular carcinoma after issues in future studies.
aflatoxin exposure. Lancet 1991; 338: 1356–9.
In conclusion, our findings provide in vitro evidence 12. Hsu IC, Metcalf RA, Sun T, et al. Mutational hotspot in the for a negligent G1 and G2/M checkpoint response to p53 gene in human hepatocellular carcinomas. Nature AFB1-induced DNA damage. This defective response 1991; 350: 427–8.
may contribute to the mutagenic and carcinogenic 13. Aguilar F, Harris CC, Sun T, Hollstein M, Cerutti P.
potencies of aflatoxins.
Geographic variation of p53 mutational profile in nonma-lignant human liver. Science 1994; 264: 1317–9.
14. Kirk GD, Lesi OA, Mendy M, et al. 249(ser) TP53 mutation in plasma DNA, hepatitis B viral infection, and risk of O. G. Y. and H. Y. were supported by short-term hepatocellular carcinoma. Oncogene 2005; 24: 5858–67.
European Molecular Biology Organization (EMBO), 15. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. Mole- and O. G. Y., H. Y. and M. Y. by long-term The Scientific cular mechanisms of mammalian DNA repair and the DNA and Technological Research Council of Turkey (TUBI- damage checkpoints. Annu Rev Biochem 2004; 73: 39–85.
TAK) PhD fellowships respectively. We would like to 16. L¨obrich M, Jeggo PA. The impact of a negligent G2/M thank Stefan Dimitrov for his critical reading of the checkpoint on genomic instability and cancer induction.
manuscript and helpful suggestions.
Nat Rev Cancer 2007; 7: 861–9.
Funding: this work was supported by grants from the 17. Bunz F, Dutriaux A, Lengauer C, et al. Requirement for p53 Institut National de Cancer (France), The Scientific and and p21 to sustain G2 arrest after DNA damage. Science Technological Research Council of Turkey (TUBITAK) 1998; 282: 1497–501.
Liver International (2011) 2011 John Wiley & Sons A/S Gursoy-Yuzugullu et al.
DNA damage response to aflatoxin B1 18. Dreiem A, Fonnum F. Thiophene is toxic to cerebellar developing countries. Annu Rev Public Health 2008; 29: granule cells in culture after bioactivation by rat liver enzymes. Neurotoxicology 2004; 25: 959–66.
35. Gouas D, Shi H, Hainaut P. The aflatoxin-induced TP53 19. Erexson GL, Periago MV, Spicer CS. Differential sensitivity mutation at codon 249 (R249S): biomarker of exposure, of Chinese hamster V79 and Chinese hamster ovary (CHO) early detection and target for therapy. Cancer Lett 2009; cells in the in vitro micronucleus screening assay. Mutat Res 286: 29–37.
2001; 495: 75–80.
36. Newberne PM, Wogan GN. Sequential morphologic 20. Yarborough A, Zhang YJ, Hsu TM, Santella RM. Immuno- changes in aflatoxin B carcinogenesis in the rat. Cancer Res peroxidase detection of 8-hydroxydeoxyguanosine in afla- 1968; 28: 770–81.
toxin B1-treated rat liver and human oral mucosal cells.
37. Aguilar F, Hussain SP, Cerutti P. Aflatoxin B1 induces the Cancer Res 1996; 56: 683–8.
transversion of G ! T in codon 249 of the p53 tumor 21. Hsieh LL, Hsu SW, Chen DS, Santella RM. Immunological suppressor gene in human hepatocytes. Proc Natl Acad Sci detection of aflatoxin B1-DNA adducts formed in vivo.
USA 1993; 90: 8586–90.
Cancer Res 1988; 48: 6328–31.
38. Ricordy R, Gensabella G, Cacci E, Augusti-Tocco G. Im- 22. Ozturk N, Erdal E, Mumcuoglu M, et al. Reprogramming pairment of cell cycle progression by aflatoxin B1 in human of replicative senescence in hepatocellular carcinoma- cell lines. Mutagenesis 2002; 17: 241–9.
derived cells. Proc Natl Acad Sci USA 2006; 103: 2178–83.
39. Ellinger-Ziegelbauer H, Stuart B, Wahle B, Bomann W, Ahr 23. Olive PL, Durand RE, Le Riche J, Olivotto IA, Jackson SM.
HJ. Characteristic expression profiles induced by genotoxic Gel electrophoresis of individual cells to quantify hypoxic carcinogens in rat liver. Toxicol Sci 2004; 77: 19–34.
fraction in human breast cancers. Cancer Res 1993; 53: 733–6.
24. Olive PL, Banath JP. Detection of DNA double-strand breaks Supporting information through the cell cycle after exposure to X-rays, bleomycin,etoposide and 125IdUrd. Int J Radiat Biol 1993; 64: 349–58.
Additional supporting information may be found in the 25. Chandna S. Single-cell gel electrophoresis assay monitors online version of this article: precise kinetics of DNA fragmentation induced duringprogrammed cell death. Cytometry A 2004; 61: 127–33.
Figure S1. Induction of DNA adducts and 8-hydroxy- 26. Senturk S, Mumcuoglu M, Gursoy-Yuzugullu O, et al.
deoxyguanosine lesions following AFB1 exposure in Transforming growth factor-beta induces senescence in hepatocellular carcinoma cells and inhibits tumor growth.
Figure S2. Induction of senescence arrest and apoptosis Hepatology 2010; 52: 966–74.
in HepG2 cells by Adriamycin, but not AFB1 in HepG2.
27. Knasmuller S, Parzefall W, Sanyal R, et al. Use of metaboli- Figure S3. Time-dependent increase in 53BP1 foci- cally competent human hepatoma cells for the detection positive HepG2 cells under AFB1 exposure.
of mutagens and antimutagens. Mutat Res 1998; 402: Figure S4. The duration of 53BP1 foci after 24 h of exposure to AFB1 in HepG2.
28. Puisieux A, Ji J, Guillot C, et al. P53-mediated cellular Figure S5. Incomplete DNA damage checkpoint response to DNA damage in cells with replicative hepatitis response of Huh7 hepatoma cells to AFB1.
B virus. Proc Natl Acad Sci USA 1995; 92: 1342–6.
Figure S6. Induction of 8-hydroxy-deoxyguanosine 29. Puisieux A, Galvin K, Troalen F, et al. Retinoblastoma and lesions and double-strand breaks in HCT116 isogenic p53 tumor suppressor genes in human hepatoma cell lines.
clones following AFB1 exposure.
FASEB J 1993; 7: 1407–13.
Figure S7. Increased DNA damage-induced foci detec- 30. Meek D W. Tumour suppression by p53: a role for the DNA tion after exposure of HCT116 isogenic clones to AFB1.
damage response? Nat Rev Cancer 2009; 9: 714–23.
Figure S8. Incomplete DNA damage checkpoint 31. Sivertsson L, Ek M, Darnell M, et al. CYP3A4 catalytic response of wild-type p53 HCT116 cells to AFB1.
activity is induced in confluent Huh7 hepatoma cells. Drug Figure S9. p53-dependent and p53-independent cell Metab Dispos 2010; 38: 995–1002.
cycle arrest in HCT116 isogenic clones after Adriamycin 32. Besaratinia A, Kim SI, Hainaut P, Pfeifer GP. In vitro recapitulating of TP53 mutagenesis in hepatocellular carci- Figure S10. Lack of cell cycle arrest of HCT116 isogenic noma associated with dietary aflatoxin B1 exposure. Gas- clones in response to AFB1-induced DNA damage.
troenterology 2009; 137: 1127–37, 37 e1–5.
33. Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ. ATM Please note: Wiley-Blackwell is not responsible for the phosphorylates histone H2AX in response to DNA double- content or functionality of any supporting materials strand breaks. J Biol Chem 2001; 276: 42462–7.
supplied by the authors. Any queries (other than missing 34. Groopman JD, Kensler TW, Wild CP. Protective inter- material) should be directed to the corresponding author ventions to prevent aflatoxin-induced carcinogenesis in for the article.
Liver International (2011) 2011 John Wiley & Sons A/S
Drosophila and Antioxidant Therapy F. Missirlis1, J.P. Phillips2, H. Jäckle3 and T.A. Rouault1 1Cell biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland, U.S.A. 2Molecular Biology and Genetics, University of Guelph, Ontario, Canada. 3Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany