Genotoxicity and induction of DNA damage responsive genes by food-borne heterocyclic aromatic amines in human hepatoma HepG2 cells

Food and Chemical Toxicology 59 (2013) 386–394

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Genotoxicity and induction of DNA damage responsive genes by food-borne heterocyclic aromatic amines in human hepatoma HepG2 cells Marko Pezdirc, Bojana Zˇegura, Metka Filipicˇ ⇑ National Institute of Biology, Department for Genetic Toxicology and Cancer Biology, Vecˇna pot 111, 1000 Ljubljana, Slovenia

a r t i c l e

i n f o

Article history: Received 1 February 2013 Accepted 18 June 2013 Available online 28 June 2013 Keywords: Cell-cycle Gene expression Genotoxicity HepG2 cells Heterocyclic aromatic amines Metabolic transformation

a b s t r a c t Heterocyclic aromatic amines (HAAs) are potential human carcinogens formed in well-done meats and fish. The most abundant are 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-Amino-3,8dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-Amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-Amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ). HAAs exert genotoxic activity after metabolic transformation by CYP1A enzymes, that is well characterized, however the genomic and intervening responses are not well explored. We have examined cellular and genomic responses of human hepatoma HepG2 cells after 24 h exposure to HAAs. Comet assay revealed increase in formation of DNA strand breaks by PhIP, MeIQx and IQ but not 4,8-DiMeIQx, whereas increased formation of micronuclei was not observed. The four HAAs up-regulated expression of genes encoding metabolic enzymes CYP1A1, CYP1A2 and UGT1A1 and expression of TP53 and its downstream regulated genes CDKN1A, GADD45a and BAX. Consistent with the up-regulation of CDKN1A and GADD45a the cell-cycle analysis showed arrest in S-phase by PhIP and IQ, and in G1-phase by 4,8-DiMeIQx and MeIQx. The results indicate that upon exposure to HAAs the cells respond with the cell-cycle arrest, which enables cells to repair the damage or eliminate them by apoptosis. However, elevated expression of BCL2 and down-regulation of BAX may indicate that HAAs could suppress apoptosis meaning higher probability of damaged cells to survive and mutate. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Heterocyclic aromatic amines (HAAs) are dietary carcinogens formed in protein-rich food at high temperatures. In cooked meats their concentrations vary depending on temperature and duration of cooking as well as the type of meat. At temperatures commonly used for preparing meat HAAs can be found in traces or up to 500 lg/kg (Sinha et al., 1995, 1998a, 1998b). The most abundant HAA in cooked meat is 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), followed by 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-Amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8DiMeIQx) whereas 2-Amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) and 2-Amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (MeIQ) are often below the analytical quantification limit (Salmon et al., 2006; Polak et al., 2009). HAAs have been shown to be carcinogenic in rodents inducing tumors in multiple organs and tissues (Kato et al., 1988; Ohgaki et al., 1991; Turesky, 2010). Based on current evidence the International Agency for Research on Cancer (IARC) classified 2-Amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) as a ⇑ Corresponding author. Tel.: +386 5 923 28 61; fax: +386 1 257 38 47. E-mail address: metka.fi[email protected] (M. Filipicˇ). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.06.030

probable human carcinogen (class 2A) and eight other HAAs as possible human carcinogens (class 2B) (IARC, 1993). Human epidemiological studies indicate association of well-done meat intake with increased cancer risk (Zheng and Lee, 2009) however the evidence is insufficient to ascribe the increased risk specifically to HAAs. Several HAAs belong to the most potent mutagens however, in the mammalian cell based genotoxicity assays as well as in the long-term carcinogenicity assays their potency is much weaker (Turesky, 2010). They are pro-carcinogens and have to be metabolically activated to intermediates that form DNA adduct, leading to DNA strand breaks, chromosomal aberration, mutations and carcinogenicity. In humans the bioactivation pathway is initiated by oxidation of exocyclic amine group by cytochrome P450 enzymes (CYPs) to produce the genotoxic N-hydroxy-HAA metabolites (Schut and Snyderwine, 1999; Alaejos et al., 2008). The N-hydroxylation is catalyzed primarily by CYP1A2 and CYP1A1 but other CYPs are involved too (Boobis et al., 1994; Hammons et al., 1997; Crofts et al., 1998; Turesky et al., 1999; Hirata et al., 2008). The N-hydroxy-derivatives can react with DNA. However, further metabolism of N-hydroxy-derivatives by O-esterification catalyzed by phase II metabolic enzymes, mainly by N-acetyltransferases (NATs) and sulfotransferases (SULTs),

M. Pezdirc et al. / Food and Chemical Toxicology 59 (2013) 386–394

produce N-acetoxy HAAs derivatives that after heterocyclic cleavage produce DNA reactive nitrenium ion (Turesky, 2010). The N-hydroxy and N-acetoxy HAAs derivatives are also substrates of enzymes belonging to superfamilies of UDP glucuronosyltransferases (UGTs) and glutathione S-transferases (GSTs) that catalyze conjugation of reactive species subsequently enabling their elimination (Boobis et al., 1994; Hammons et al., 1997; Jägerstad and Skog, 2005). Besides conventional genotoxicity assays, alterations in gene expression have been shown to provide an early and global answer to toxic events (Brown and Botstein, 1999; Ellinger-Ziegelbauer et al., 2005). Genotoxic stress triggers a variety of cellular responses, including transcriptional activation of genes regulating DNA repair, cell-cycle arrest and apoptosis (Hollander and Fornace, 1995). The tumor-suppressor TP53 plays the central role in the cellular response to DNA damage by activating the transcription of several essential genes controlling cell-cycle arrest/DNA repair, senescence, differentiation and apoptosis (Vogelstein et al., 2000). Recent studies showed that changes in the expression of tumor suppressor TP53 and the TP53-regulated DNA damage response genes such as CDKN1A (cyclin-dependent kinase inhibitor), GADD45a (growth arrest and DNA-damage-inducible gene), and MDM2 (E3 ubiquitin ligase), can be considered as markers of genotoxic and carcinogenic stress (Ellinger-Ziegelbauer et al., 2005; Hreljac et al., 2008; Petkovic´ et al., 2010; Štraser et al., 2011; Zˇegura et al., 2008). The data on the effect of HAAs on the expression of DNA damage responsive genes is currently very limited and is available only for PhIP. Elevated expression of P53 and CDKN1A by PhIP has been observed in human mammary epithelial MCF10A cells (Creton et al., 2005) and in TK6 human lymphoblastoid cells (Gooderham et al., 2002), and recently, Wei et al. (2011) reported up-regulation of CDKN1A and to lesser extent GADD45a in rat liver following 16 week oral exposure to PhIP. The in vitro experimental model with human hepatoma HepG2 cells is frequently used in toxicology and gene expression studies. These cells have retained inducibility and activities of several phase I and phase II xenobiotic metabolising enzymes, and have been proven to be suitable for detection of different classes of indirect acting genotoxic agents (Knasmuller et al., 2004). HepG2 cells also express wild-type tumor suppressor TP53 (Bressac et al., 1990) making them an appropriate model for studying P53 regulated response to DNA damage at the level of gene transcription and translation (van Delft et al., 2004). In this study we used HepG2 cells to investigate the effects of PhIP, 4,8-DiMeIQx, MeIQx and IQ on the modulation of the expression of TP53 regulated DNA damage responsive (CDKN1A, GADD45a, MDM2) and apoptotic genes (BAX and BCL2), their genotoxic potencies and the effect on cell-cycle. To confirm inducibility of HAAs metabolizing enzymes in HepG2 cells gene expressions of CYP1A1, CYP1A2, SULT1A1, NAT2, GSTA1 and UGT1A1 were determined after the exposure to HAAs.

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2.2. Cell culture and treatment HepG2 cells were a gift from Dr. Firouz Darroudi (Leiden University Medical Centre, Department of Toxicogenetics, Leiden, Netherlands). The cells were grown in William’s medium E containing 15% of FBS, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin at 37 °C in 5% CO2 atmosphere. Prior to the treatment the cells were seeded onto 96-well plates for the MTS assay, 12-well tissue culture treated plates for the comet assay or 25 cm2 culture flask (all Corning Costar Corporation, New York, USA) for CBMN assay, QRT-PCR gene expression analysis, Western blotting and cell-cycle analysis, and incubated for 24 h to attach. The medium was then replaced with fresh complete medium containing graded doses of HAAs and incubated for 24 h. The cytotoxicity of MeIQx and IQ was determined at the concentration range from 50–1000 lM, while PhIP and 4,8-DiMeIQx were due to low solubility tested at concentrations up to 200 lM. For the Comet assay, CBMN, cell-cycle analysis and gene expression analysis the cells were exposed to non-cytotoxic concentrations of HAAs: 0, 50, 100 and 200 lM PhIP or 4,8-DiMeIQx and 0, 50, 100 and 250 lM MeIQx or IQ. The protein expression analyses were performed in cells exposed to 100 lM of HAAs. BaP, a well established pro-carcinogen that needs metabolic activation by CYP1A enzymes was used as the positive control in the genotoxicity tests and gene expression analysis. The concentration (30 lM) was chosen, on the basis of our previous research as non-cytotoxic concentration that induces expected genotoxic effects in HepG2 cells (Viegas et al., 2012). DMSO (1%) was used as the vehicle control. Final concentration of DMSO in the medium was adjusted to 1% in all experiments at all tested concentrations.

2.3. MTS viability assay Cytotoxicity of HAAs was determined with MTS assay (Cell Titer 96 AQueous NonRadioactive Cell Proliferation Assay; Promega, Madison, USA) according to the manufacturer’s instructions. Cell viability was determined by comparing the OD of the wells containing the cells treated with HAAs with those of the vehicle control. Two independent experiments were performed, each with five replicates per treatment point.

2.4. Comet assay At the end of the exposure to HAAs comet assay was performed as described by Singh et al. (1988) with minor modifications (Zˇegura and Filipicˇ, 2004). The slides were stained with ethidium bromide (5 lg/ml) and analyzed using a fluorescence microscope (Nikon, Eclipse 800) with the image analysis software (Comet IV, Perceptive Instruments). Fifty nuclei were analyzed per experimental point in each of the three independent experiments. The results from three independent experiments are expressed as % DNA in the comet tail.

2.5. Cytokinesis-block micronucleus (CBMN) cytome assay After the treatment, the cells were washed twice with 1x PBS buffer, the medium containing cytochalasin B (2 lg/ml) was added and the cells were incubated for additional 26 h at 37 °C. The medium containing cytochalasin B was then removed, the cells were washed with 1 PBS buffer, harvested and the slides were prepared as described by Štraser et al. (2011). The slides were stained with acridine orange (20 lg/ml), and examined under the fluorescence microscope (Eclipse 800, Nikon, Japan). For each experimental point micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBs) were counted in 1000 binucleated cells (BNC) per experimental point at 400 magnification according to the criteria published by (Fenech, 2000). The nuclear division index (NDI) was estimated by scoring 500 cells with one to four nuclei. The NDI was calculated using the formula [M1 + 2M2 + 3(M3 + M4)]/500, where M1, M2, M3 and M4 represent the number of cells with one to four nuclei, respectively. The experiments were repeated three times independently.

2. Materials and methods

2.6. QRT-PCR gene expression analysis

2.1. Chemicals

The mRNA isolation from HepG2 cells and QRT-PCR were performed as described by Štraser et al. (2011). The following Taqman Gene Expression Assays were used: CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), Hs00153120_m1; CYP1A2 (cytochrome P450, family 1, subfamily A, polypeptide 2), Hs01070374_m1; NAT2 (N-acetyltransferase 2), Hs00605099_m1; SULT1A1 (sulfotransferase family, cytosolic, 1A, phenol-preferring, member 1), Hs00419411_m1; UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), Hs02511055_s1; GSTA1 (glutathione S-transferase alpha 1), Hs00275575_m1; TP53 (tumor protein P53), Hs00153349_m1; MDM2 (Mdm2, ‘transformed 3T3 cell double minute 2’, p53 binding protein gene), Hs00234753_m1; GADD45a (‘growth arrest and DNA damage-inducible gene, alpha’), Hs00169255_m1; CDKN1A (‘cyclin-dependent kinase inhibitor 1A’), Hs00355782_m1; BAX (BCL2 associated X protein), Hs99999001_m1; BCL2 (B-cell CLL/lymphoma 2), Hs00608023_m1. Amplification of GAPDH probe (Human Endogenous Controls, Cat. No.: 4310884E, Applied Biosystems, USA) was performed as an internal control. The data obtained from Taqman

William’s medium E and trypsin were obtained from Sigma, St. Louis, USA; penicillin/streptomycin, foetal bovine serum (FBS), L-glutamine, phosphate buffered saline (PBS) from PAA, Pasching, Austria; PhIP (CAS-No. 105650-23-5, 98% purity), MeIQx (CAS-No. 77500-04-0, 98% purity), 4,8-DiMeIQx (CAS-No. 95896-78-9, 98% purity) and IQ (CAS-No. 76180-96-6, 98% purity) from Toronto Research Chemicals Inc., North York, CA. Benzo(a)pyrene (BaP, CAS-No. 50-32-8, >96% purity) was from Sigma, St. Louis, USA. PhIP, 4,8-DiMeIQx, MeIQx, IQ and BaP were dissolved in DMSO (Sigma, St. Louis, USA). Stock concentrations of PhIP and 4,8-DiMeIQx were 20 mM due to their poor solubility at higher concentrations. Stock concentrations of MeIQx and IQ were 100 mM and BaP 10 mM. High Capacity cDNA Archive Kit, and Taqman Gene Expression Assays were obtained from Applied Biosystems, Forest City, CA; TaqMan Universal PCR Master Mix from Applied Biosystems, Branchburg, USA and Human GAPDH from Applied Biosystems, Warrington, UK.

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Gene Expression Assays were analyzed using the DDCt algorithm. The expression levels of target mRNAs were normalized to the GAPDH mRNA level. Three independent experiments were performed each time in two parallels. 2.7. Western blotting Expressions of the tumor suppressor TP53 protein and its downstream regulated proteins CDKN1A, BAX and BCL2 were determined by western blotting in cells exposed to 100 lM PhIP, 4,8-DiMeIQx, MeIQx or IQ as described above (Section 2.2). 10 lg of isolated total protein were subjected to SDS/PAGE electrophoresis with 40% polyacrylamide gel. Resolved proteins were transferred to Immun-BlotÒ PVDF-Membrane (162-0218, Bio-Rad). The membrane was probed with the mouse monoclonal primary antibody against TP53 (1:500, ab1101, Abcam, UK), rabbit polyclonal primary antibody against CDKN1A (1:1000, ab102013, Abcam, UK), rabbit polyclonal primary antibody against BAX (1:1000, ab7977, Abcam, UK) and rabbit polyclonal primary antibody against BCL2 (1:500, ab59348, Abcam, UK). GAPDH rabbit polyclonal primary antibody (1:2000, ab9485, Abcam, UK) served as a control for equal loading. Then the membrane was incubated with anti-mouse (1:2500, W402, Promega, USA) or anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:2500, W401B, Promega, USA). Chemiluminescence was developed by Amersham ECL™ detection system (GE Healthcare, UK) according to the manufacturer’s instructions. 2.8. Cell-cycle analysis At the end of exposure of HepG2 cells as described in Section 2.2 floating and adherent cells were collected by trypsinization. Cells were centrifuged at 800 rpm for 5 min at 4 °C, washed twice with ice cold 1x PBS, resuspended in 0.5 ml cold 1 PBS and ethanol (1.5 ml) was added dropwise into the cell pellet, while vortexing. The cells were fixed at 4 °C overnight and stored at 20 °C till analysis. Fixed cells were centrifuged at 1200 rpm for 10 min, washed twice with ice cold 1 PBS and stained with 0.5 ml propidium iodide/RNAse staining buffer (BD Biosciences Pharmingen™, San Diego, CA, USA) for 15 min at room temperature according to the manufacturer’s recommendations. Flow cytometric analysis was carried out on a FACSCalibur flow cytometer (BD Biosciences Pharmingen™, San Diego, CA, USA). Changes in the distribution of cells through the phases of the cell-cycle were analyzed in the FL2 channel, where 104 events were recorded for each sample. The percentage of cells in G0/G1, S, and G2/M phases of the cell-cycle were determined from FL2-A histograms using Cylchred (Cardiff University, Cardiff, UK). Analysis was performed on single cells, by elimination of cell aggregates by gating FL2-W versus FL2-A using WinMDI (Scripps Institute, La Jolla, USA). 2.9. Statistics The statistical analysis was performed by the one way analysis of variance (ANOVA) with GraphPad Prism 5 software. In the case of a significant ANOVA Dunnett’s test was used for multiple comparisons versus the vehicle control; p < 0.05 was considered as statistically significant.

3. Results 3.1. Genotoxicity of PhIP, 4,8-DiMeIQx, MeIQx and IQ in HepG2 cells The effect of each HAA on cell viability was evaluated after 24 h exposure to concentrations ranging from 50 lM to maximal soluble

concentration with PhIP and 4,8-DiMeIQx and from 50–1000 lM with MeIQx and IQ. PhIP and 4,8-DiMeIQx at concentrations from 50 to 200 lM and MeIQx and IQ at concentrations from 50 to 250 lM did not decrease the cell viability by more than 20% (Fig. 1). MeIQx and IQ at higher concentrations (500–1000 lM) caused > 30% dose dependent decrease in cell viability (Fig. 1B). At the highest tested concentration the viability of cells exposed to MeIQx or IQ was reduced by 57% and 65%, respectively. Based on these results comet and CBMN assays, cell-cycle analysis and the gene expression analysis were performed at concentration range from 50 to 200 with PhIP and 4,8-DiMeIQx, and 50–250 lM with MeIQx and IQ. Using comet assay an increase of DNA damage was found after 24 h exposure of HepG2 cells to PhIP, MeIQx or IQ, but not 4,8DiMeIQx (Fig. 2). PhIP and MeIQx induced dose dependent increase in % DNA in the comet tail. IQ induced significant increase in % DNA in the comet tail at all tested concentrations; however the response was not dose dependent. The results of the CBMN cytome assay showed that exposure of HepG2 to PhIP, 4,8-DiMeIQx, MeIQx or IQ for 24 h did not result in any increase in the frequency of MNi, NPBs or NBs compared to non-treated control (Table 1). The positive control, BaP (30 lM) is an indirect acting genotoxic compound that similarly as HAAs has to be metabolically activated (by CYP1A enzymes) to induce DNA-adducts and formation of MNi. BaP induced significant, more than 2-fold increase in MNi formation compared to vehicle treated control cells, confirming metabolic activity of HepG2 cells. 3.2. Effect of PhIP, 4,8-DiMeIQx, MeIQx and IQ on the expression of genes encoding HAAs metabolizing enzymes After 24 h exposure all four tested HAAs induced significant upregulation of mRNA expression of CYP1A1 and CYP1A2 at all tested concentrations (Table 2). The levels of mRNA of these two enzymes in cells exposed to MeIQx, 4,8-DiMeIQx and IQ were much higher than in cells exposed to PhIP. The changes in the mRNA expression of phase II metabolic enzymes were less pronounced than changes in the expression of phase I metabolic enzymes. The expression of NAT2 was significantly down-regulated by IQ at all tested concentrations, while the other three HAAs did not induce significant changes in the expression of this gene. The expression of GSTA1 was strongly down-regulated by 4,8-DiMeIQx and MeIQx at all concentrations, while IQ down-regulated the expression of this gene only at the highest tested concentration. The mRNA expression of UGT1A1 was up-regulated by all four HAAs at all concentrations, 4,8-DiMeIQx being the strongest inducer and PhIP the weakest. The up-regulation of UGT1A1 correlates with the up-regulation of

Fig. 1. The effect of PhIP and 4,8-DiMeIQx (A) and MeIQx and IQ (B) on the viability of HepG2 cells. Viability was determined with the MTS assay after the exposure to different concentrations of HAAs for 24 h. The asterisk () denotes a significant difference between vehicle control and treated cells (One way ANOVA; Dunnett’s test, p < 0.01). The viability of cells in the vehicle control cells did not differ significantly from that of non-treated control cells (data not shown).

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Fig. 2. PhIP, 4,8-DiMeIQx, MeIQx and IQ induced DNA strand breaks in HepG2 cells. The cells were exposed to different concentrations of PhIP, 4,8-DiMeIQx, MeIQx or IQ for 24 h. BaP (30 lM) was used as a positive control. DNA damage was assessed with the comet assay and is expressed as % DNA in the comet tail. Fifty cells were analyzed per experimental point in each of the three independent experiments. The asterisk () denotes a significant difference between HAAs-treated cells and the vehicle control (One way ANOVA; Dunnett’s test, p < 0.01). The % tail DNA of the vehicle control cells did not differ significantly from that of non-treated control cells (data not shown).

CYP1A1 (Pearson r = 0.795; p < 0.0002) and to lesser extent also with CYP1A2 (Pearson r = 0.630; p = 0.009). The mRNA expression of SULT1A1 was detected neither in control nor in HAA-exposed cells (data not shown). 3.3. Effect of PhIP, 4,8-DiMeIQx, MeIQx and IQ on the expression of DNA damage responsive genes The mRNA expression of TP53 was by IQ at 100 lM up-regulated, by 4,8-DiMeIQx it was down-regulated at all concentrations, while it was not affected by PhIP and MeIQx. All four HAAs induced

elevated mRNA expression of CDKN1A (Table 3). The strongest inducer was 4,8-DiMeIQx followed by MeIQx, PhIP and IQ. Up-regulation of CDKN1A by different HAAs correlates with corresponding up-regulation of CYP1A1 (Pearson r = 0.741, p < 0.001). PhIP, MeIQx and 4,8-DiMeIQx also up-regulated the expression of GADD45a with MeIQx being the strongest inducer, whereas IQ did not significantly change the expression of this gene (Table 2). The expression of MDM2 was by IQ down-regulated and by 4,8-DiMeIQx up-regulated, while PhIP and MeIQx did not induce significant changes in the expression of this gene (Table 2). Its expression negatively correlated with the expression of p53 (Pearson r = 0.753, p < 0.01).

Table 1 Micronuclei (MN), nucleoplasmic bridges (NPB), nuclear buds (NB) and nuclear division index (NDI) in HepG2 cells exposed to PhIP, 4,8-DiMeIQx, MeIQx and IQ for 24 h.

*

Compound

MNed cells/103 BNC

MN/103 BNC

NPB/103 BNC

BUDS/103 BNC

NDI

PhIP (lM) 0 50 100 200

16.3 ± 8.7 22.0 ± 5.0 18.3 ± 5.5 19.0 ± 6.6

16.7 ± 9.3 23.0 ± 6.0 19.7 ± 5.8 20.0 ± 7.2

2.7 ± 0.6 1.0 ± 1.0 1.0 ± 0.0 1.7 ± 1.5

43.3 ± 6.7 44.3 ± 28.0 64.0 ± 19.3 56.0 ± 20.9

1.8 ± 0.1 1.7 ± 0.1 1.8 ± 0.1 1.7 ± 0.1

4,8-DiMeIQx (lM) 0 50 100 200

11.3 ± 5.1 9.7 ± 6.4 7.7 ± 0.6 9.0 ± 4.4

11.7 ± 5.7 10.0 ± 7.0 8.0 ± 0.0 9.0 ± 4.4

1.0 ± 1.0 0 0.3 ± 0.6 0.7 ± 1.2

37.7 ± 12.7 35.0 ± 5.6 40.7 ± 19.9 32.0 ± 18.3

1.9 ± 0.1 1.9 ± 0.03 1.7 ± 0.1* 1.6 ± 0.1*

MeIQx (lM) 0 50 100 250

14.0 ± 4.0 19.3 ± 4.9 18.0 ± 1.0 17.3 ± 3.2

15.0 ± 4.6 21.7 ± 4.6 20.0 ± 3.0 17.7 ± 3.8

3.3 ± 4.0 3.3 ± 1.5 2.7 ± 2.1 2.7 ± 3.1

36.0 ± 21.3 46.3 ± 14.0 28.3 ± 19.9 31.7 ± 13.4

1.8 ± 0.1 1.8 ± 0.1 1.8 ± 0.1 1.6 ± 0.1*

IQ (lM) 0 50 100 250

14.0 ± 7.5 19.3 ± 4.9 18.0 ± 1.0 17.3 ± 3.2

18.0 ± 9.0 20.3 ± 6.0 21.7 ± 4.6 20.0 ± 3.0

3.3 ± 4.0 3.3 ± 1.5 2.7 ± 2.1 2.7 ± 3.1

36.0 ± 21.3 46.3 ± 14.0 28.3 ± 19.9 31.7 ± 13.4

1.8 ± 0.1 1.8 ± 0.1 1.8 ± 0.1 1.7 ± 0.1

BaP (lM) 30

37.7 ± 12.5*

38.9 ± 14.3*

1.7 ± 2.6

46.3 ± 21.4

1.6 ± 0.1*

Significant difference from the vehicle control – One way ANOVA with Dunnett’s post test; p < 0.05.

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Table 2 Modulation of mRNA expression of selected genes involved in HAA metabolism in HepG2 cells after 24 h exposure to PhIP, 4,8-DiMeIQx, MeIQx, or IQ. HAA (lM)

Phase I

Phase II

CYP1A1

CYP1A2

NAT2

GSTA1

UGT1A1

PhIP 50 100 200

4.0 ± 2.3 6.3 ± 3.1⁄ 13.3 ± 1.9⁄

3.7 ± 1.1⁄ 7.9 ± 1.1⁄ 15.3 ± 3.0⁄

1.0 ± 0.2 1.0 ± 0.1 2.0 ± 0.4

0.8 ± 0.1 1.1 ± 0.04 0.8 ± 0.04

1.5 ± 0.1⁄ 1.7 ± 0.02⁄ 2.9 ± 0.2⁄

4,8-DiMeIQx 50 100 200

302.0 ± 68.0⁄ 330.7 ± 61.2⁄ 436.7 ± 34.2⁄

59.8 ± 5.3⁄ 65.3 ± 5.6⁄ 52.6 ± 8.9⁄

1.0 ± 0.03 1.1 ± 0.3 1.2 ± 0.1

0.2 ± 0.1⁄ 0.1 ± 0.04⁄ 0.1 ± 0.01⁄

3.1 ± 0.6⁄ 4.4 ± 0.3⁄ 4.9 ± 1.0⁄

MeIQx 50 100 250

45.6 ± 6.8⁄ 80.0 ± 45.9⁄ 163.3 ± 37.0⁄

28.1 ± 3.8⁄ 56.2 ± 1.9⁄ 114.3 ± 6.3⁄

1.1 ± 0.02 1.3 ± 0.1 0.9 ± 0.01

0.3 ± 0.03⁄ 0.2 ± 0.07⁄ 0.1 ± 0.01⁄

2.3 ± 0.4⁄ 2.9 ± 0.3⁄ 3.1 ± 1.1⁄

IQ 50 100 250

47.9 ± 4.7⁄ 42.7 ± 3.4⁄ 68.7 ± 5.2⁄

75.3 ± 15.1⁄ 74.7 ± 7.6⁄ 88.4 ± 10.4⁄

0.4 ± 0.7⁄ 0.6 ± 0.2⁄ 0.4 ± 0.2⁄

0.8 ± 0.3 0.7 ± 0.4 0.5 ± 0.2⁄

2.9 ± 0.9⁄ 2.8 ± 0.8⁄ 3.9 ± 0.6⁄

BaP 30

314.3 ± 141.5⁄

14.7 ± 7.8⁄

1.1 ± 0.7

0.2 ± 0.04⁄

1.9 ± 0.7⁄

The results are expressed as relative mRNA expression normalized to the vehicle control. Data are means ± SD of three independent experiments. * Significant difference between HAAs-treated groups and the vehicle control (One way ANOVA with Dunnett’s post test; p < 0.05). Bold values indicate the up-regulation of genes.

Table 3 Modulation of mRNA expression of selected DNA damage responsive and apoptotic genes in HepG2 cells after 24 h exposure to PhIP, 4,8-DiMeIQx, MeIQx or IQ. HAA (lM)

DNA damage responsive genes

Apoptotic genes

P53

CDKN1A

GADD45a

MDM2

BAX

BCL-2

BAX/BCL-2

PhIP 50 100 200

1.0 ± 0.1 1.0 ± 0.1 1.0 ± 0.1

1.1 ± 0.2 1.4 ± 0.2* 1.7 ± 0.1*

1.4 ± 0.2* 1.2 ± 0.3 2.0 ± 0.2*

0.9 ± 0.1 0.8 ± 0.2 0.8 ± 0.1

1.1 ± 0.1 1.0 ± 0.2 1.0 ± 0.2

1.4 ± 0.4 1.5 ± 0.7 2.0 ± 0.5*

0.75 0.64 0.53

4,8-DiMeIQx 50 100 200

0.7 ± 0.1* 0.8 ± 0.2 0.6 ± 0.2*

1.6 ± 0.2* 3.1 ± 1.2* 6.6 ± 1.0*

1.1 ± 0.5 1.7 ± 0.5 2.0 ± 0.4*

1.2 ± 0.1 1.4 ± 0.01* 1.2 ± 0.1

0.8 ± 0.2 0.8 ± 0.2 0.6 ± 0.1*

1.4 ± 0.3 1.5 ± 0.2* 0.7 ± 0.2*

0.55 0.57 0.92

MeIQx 50 100 250

1.1 ± 0.1 1.0 ± 0.2 1.0 ± 0.1

2.0 ± 0.4* 2.8 ± 0.2* 3.1 ± 0.5*

1.3 ± 0.02* 2.1 ± 0.2* 1.2 ± 0.1

0.8 ± 0.1 1.2 ± 0.1 1.1 ± 0.1

0.9 ± 0.1 1.2 ± 0.01* 0.6 ± 0.1*

0.9 ± 0.01 1.7 ± 0.2* 1.4 ± 0.02*

0.83 0.71 0.37

IQ 50 100 250

1.0 ± 0.1 1.7 ± 0.3* 1.2 ± 0.2

1.3 ± 0.03* 1.5 ± 0.1* 0.9 ± 0.05

1.2 ± 0.03 1.2 ± 0.7 0.6 ± 0.4

0.7 ± 0.1* 0.6 ± 0.1* 0.4 ± 0.1*

1.0 ± 0.1 1.2 ± 0.2 0.5 ± 0.1*

1.2 ± 0.04* 2.3 ± 0.3* 0.8 ± 0.2

0.81 0.53 0.66

BaP 30

1.5 ± 0.5

17.0 ± 5.8*

7.1 ± 2.9*

0.6 ± 0.2*

/

/

/

The results are expressed as relative mRNA expression normalized to the vehicle control. Data are means ± SD of three independent experiments. Significant difference between HAAs-treated groups and the vehicle control (One way ANOVA with Dunnett’s post test; p < 0.05). Bold values indicate up-regulation of genes.

*

The expression of the pro-apoptotic gene BAX was significantly down-regulated by 4,8-DiMeIQx, MeIQx and IQ at the highest tested concentration, while PhIP did not affect its expression. The expression of the anti-apoptotic gene BCL2 was significantly upregulated in cells exposed to PhIP (200 lM), MeIQx (100 and 250 lM), 4,8-DiMeIQx and IQ (100 lM). It has been proposed that BAX/BCL2 ratio determines the progress of cells to apoptosis (Danial and Korsmeyer, 2004). The results showed that the BAX/BCL2 ratio tended to be < 1 after exposure to all four tested HAAs (Table 2). No difference in mRNA expression of the selected genes was observed between vehicle (1% DMSO) and media control (data not shown). The changes in the expression of TP53, CDKN1A and BAX and BCL2 at the protein level were confirmed with the Western blot after exposure to 100 lM of each of the four HAAs (Fig. 3). All four

tested HAAs increased protein levels of TP53, CDKN1A and BCL2, whereas no difference in protein level was observed for BAX.

3.4. Effect of PhIP, 4,8-DiMeIQx, MeIQx and IQ on cell-cycle The cell-cycle analysis was performed after 24 h exposure of HepG2 cells to HAAs. PhIP caused significant decrease in the number of cells in G0/G1-phase only at the highest tested concentration (200 lM), while IQ caused significant decrease in the number of cells in G0/G1 and accumulation of cells in S-phase at 50, 100 and 250 lM (Fig. 4). This indicates that PhIP and IQ induced cellcycle delay in S-phase. At the highest tested concentration 4,8DiMeIQx (at 200 lM) and MeIQx (at 250 lM) induced significant increase in the number of cells in G0/G1, reduction of number of

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Fig. 3. Western blot of TP53, CDKNA1, BAX and BCL2 in HepG2 cells after exposure to 100 lM PhIP, 4,8-DiMeIQx, MeIQx or IQ for 24 h. BaP (30 lM) was used as a positive control. The asterisk () denotes possible post-translation modification of protein TP53.

cells in S-phase, and MeIQx also in G2/M phase, indicating cell cycle arrest in G1-phase (Fig. 4). 4. Discussion In spite of extensive genotoxicity studies of HAAs there is limited information on their effect on the expression of DNA damage responsive genes and early cellular responses, which could contribute to a better understanding of the mechanisms of their potential carcinogenicity. In our study with the HepG2 cells that express several enzymes involved in activation and detoxification of HAAs we showed that

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PhIP, MeIQx, IQ but not 4,8-DiMeIQx induced DNA strand breaks, while none of them induced MNi formation. Induction of DNA strand breaks but not formation of MNi has been observed also in recently developed hepatic cell line HepaRG exposed to PhIP (Le Hegarat et al., 2010) and in HepG2 cells exposed to PhIP and MeIQx (Viegas et al., 2012). Cellular response to the genotoxicity of HAA in a great deal depends on the balance between the metabolic activation and detoxification pathways and also cell defence by DNA repair system. The analysis of the expression of genes involved in HAAs metabolism showed that expressions of CYP1A1 and CYP1A2 were in HepG2 cells up-regulated by all four HAAs in a dose dependent manner. This induction most probably occurred via aryl hydrocarbon receptor as has been recently confirmed by Dumont et al. (2010a). The strongest inducers of CYP1A1 and CYP1A2 were imidazo-quinoxalines (4,8-DiMeIQx and MeIQx), followed by imidazo-quinoline (IQ), whereas the imidazopyridine (PhIP) was the weakest. These differences may be explained by their differences in the affinity to AhR receptor. It has been shown that imidazo-quinoxalines have higher affinity to AhR receptor than imidazo-quinoline and imidazopyridine (Kleman et al., 1992). However, the extent of DNA damage induced by individual HAAs did not correlate with the gene expression of CYP1A1 and CYP1A2. The lack of correlation could be due to differences in the concurrent activation of detoxification pathways by PhIP, 4,8-DiMeIQx, MeIQx and IQ, respectively. The expression of NAT2 was not affected by the tested HAAs, and SULT1A1 was not expressed under the used experimental conditions. These data suggest that the DNA reactive nitrenium ion was probably not produced and DNA strand breaks detected by the comet assay were probably induced predominantly by

Fig. 4. Cell-cycle distribution of HepG2 after 24 h exposure to PhIP, 4,8-DiMeIQx, MeIQx or IQ. Cells were stained with propidium iodide and the DNA content analyzed with the flow cytometry as described in the materials and methods. The asterisk () denotes a significant difference between vehicle control and treated cells (One way ANOVA; Dunnett’s test, p < 0.01).

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N-hydroxy-HAA metabolites. This can also be the explanation for the relatively low genotoxicity of the four HAAs in this study. This assumption is supported by a number of previous studies with genetically modified mammalian cells expressing CYP1A enzymes together with NATs or SULTs, which showed that DNA adduct formation as well as mutagenicity of HAAs was higher in strains expressing NATs or SULTs, (Thompson et al., 1995; Wu et al., 1997, 2000; Metry et al., 2007; Bendaly et al., 2009). It is considered that the major HAAs detoxification pathway is UGT-mediated glucuronidation of N-hydroxy-HAAs (Turesky and Le Marchand, 2011; Yueh et al., 2001; Turesky et al., 2002; Langouët et al., 2002). In our experiments all four HAAs induced significant dose dependent increase in the expression of UGT1A1 gene indicating activation of detoxification pathway with 4,8DiMeIQx being the strongest and PhIP the weakest inducer of the expression of this enzyme. Induction of the expression of UGT1A1 may to certain extent explain relatively weak response of HepG2 to the genotoxicity of HAAs in our study. This assumption is supported by the study with genetically modified excision repair deficient CHO cells co-expressing CYP1A2 and human UGT1A1 which were highly resistant toward cytotoxicity and mutagenicity of PhIP (Malfatti et al., 2005). In addition to the detoxification by glucuronidation, other detoxification pathways of HAAs could be the reason for the lack of the correlation between HAAs induced DNA damage and expression of CYP1A enzymes. For instance, the studies of the metabolism of MeIQx in isolated human hepatocytes showed that human CYP1A2 catalyzes the C8-oxidation of MeIQx to form the carboxylic acid derivate 2-Amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH), which is considered as the major detoxification pathway of this pro-carcinogen in humans (Langouët et al., 2001). Due to chemical structural similarities between MeIQx and 4,8-DiMeIQx it is possible that also 4,8-DiMeIQx could be the substrate for the CYP1A2 mediated C8-oxidation. Another HAAs detoxification pathway is conjugation of reactive species with glutathione either spontaneously or by catalysis with GSTs (Coles et al., 1990). The gene expression of GSTA1 in HepG2 cells was by 4,8-DiMeIQx, MeIQx and IQ strongly down-regulated, while PhIP did not modulate its expression. This can be explained by the findings that HAAs are not substrates for the GSTs except the activated PhIP derivative N-acetoxy-PhIP (Coles et al., 2001). Very similar modulation of gene expression pattern of genes involved in metabolic transformation of HAAs as observed in our study has been observed also in HepaRG cells exposed to PhIP and MeIQx (Dumont et al., 2010b). As already mentioned cellular response to the exposure to genotoxic agents depends on the cellular defence, in particular DNA damage repair mechanisms, which so far, for HAAs have not been extensively studied. Thus relatively weak induction of DNA strand breaks and the lack of the induction of MNi in HepG2 cells after exposure to the tested HAAs could also be a consequence of efficient DNA damage repair. The HAA-induced DNA adducts are the substrate for nucleotide excision repair (NER) and the breaks revealed by the comet assay represent predominantly intermediates formed during the DNA repair. It has been shown that in MCL-5 cells in the absence of DNA repair inhibitors HAAs induced much lower levels of DNA strand breaks (Pfau et al., 1999). In the literature there is very little data on the kinetics of the repair of DNA adducts induced by different types of HAAs in different types of cells and tissues. Efficient repair of HAAs DNA adducts has been shown in the mammary epithelial cell culture (Fan et al., 1995). 60–80% of IQ- and PhIP-DNA adducts were removed within 24 h. The lack of the appearance of DNA strand breaks by 4,8-DiMeIQx after 24 h exposure could be either due rapid repair or the lack of excision of DNA adducts. In both cases no DNA strand breaks would be revealed by the comet assay.

The results of the effects of HAAs exposure on the mRNA expression of DNA damage responsive genes and induction of cell-cycle arrest indicate the activation of cell defence against DNA damage in HepG2 cells. All four HAAs modulated expression of TP53 and its downstream regulated DNA damage response genes CDKN1A, GADD45a and MDM2. At the transcriptional level slight up-regulation of TP53 gene was detected only in cells exposed to IQ, whereas in cells exposed to 4,8-DiMeIQx it was even down-regulated. It is not unusual that P53 is not up-regulated at the transcription level as it is known that DNA damage activates the P53 protein, predominantly through its phosphorylation by DNA damage-responsive kinases and to a lesser extent through up-regulation of gene expression (Zhou and Elledge, 2000). This was confirmed by the Western blot analysis, which showed that all four HAAs elevated the expression of TP53 protein and its post-translation forms. The changes in the mRNA expression of MDM2 negatively correlated with the changes in the mRNA expression of TP53, which is in line with its function as a negative regulator of the expression of TP53 (Michael and Oren, 2003; Vogelstein et al., 2000). Elevated expression of CDKN1A gene and its protein was detected after exposure to all four HAAs. The most potent inducer of CDKN1A expression was 4,8-DiMeIQx, followed by MeIQx, while IQ and PhIP showed much lower potency. The expression of GADD45a was up-regulated by PhIP, MeIQx and 4,8-DiMeIQx. Previous studies showed elevated protein expression of P53 and CDKN1A by PhIP in the experimental model with human mammary epithelial MCF10A cells, co-cultured with a transfected human lymphoblastoid cell line MCL-5 expressing CYP enzymes (Creton et al., 2005) and in TK6 human lymphoblastoid cells co-cultured with irradiated metabolically competent cells (Gooderham et al., 2002; Duc and Leong-Morgenthaler, 2004). According to our best knowledge no data on the effect of 4,8-DiMeIQx, MeIQx and IQ on the expression of DNA damage responsive genes are available. CDKN1A and GADD45a are both associated with the cell-cycle arrest upon DNA damage in G1 and G2-M phase of the cell-cycle, respectively (Waldman et al., 1995; Zhan, 2005). In addition recent studies provided several lines of evidence indicating that CDKN1A also is directly involved in DNA repair, including NER (Cazzalini et al., 2010). Our study showed that all four HAAs modulated cell-cycle progression. After 24 h exposure PhIP and IQ caused cell-cycle delay in S-phase, while 4,8-DiMeIQx and MeIQx caused cell-cycle delay in G1-phase. Similar observation to our was reported by Zhu et al. (2000) in human lymphoblastoid TK6 cells exposed to metabolically activated PhIP. They proposed two mechanisms that depend on the duration of the exposure, to be involved: transient S-phase delay after shorter exposure (20 h) and prolonged cell-cycle arrest accompanied by increased apoptosis and gene mutations after longer exposure (40 h). In another study with P53 proficient and deficient TK6 cells PhIP induced cell-cycle arrest was observed in G2-M phase and was not dependent on the functionality of P53 (Duc and Leong-Morgenthaler, 2004). It is difficult to explain why PhIP and IQ caused cell-cycle arrest in S-phase, and 4,8-DiMeIQx and MeIQx in G1-phase. Possible explanation might be that the type of DNA damage induced by the two imidazo-quinoxalines (4,8-DiMeIQx and MeIQx) that were also significantly more potent inducers of CDKN1A than IQ and PhIP, was different from that induced by IQ and PhIP. It has been considered that G1-phase arrest is triggered by the presence of single strand breaks formed during NER, whereas the trigger of S-phase arrest is associated with double strand breaks (Sancar et al., 2004). P53 regulates apoptosis by regulating the transcription of antiand pro-apoptotic genes of BCL2 family (Adams and Cory, 2007; Perfettini et al., 2004; Hanahan and Weinberg, 2011). The antiapoptotic BCL2 family members such as BCL2 and BCL-XL maintain cell survival by inhibiting pro-apoptotic BCL2 family members BAX

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and BAK (Youle and Strasser, 2008). In HepG2 cells HAAs induced an increase in the mRNA expression of the anti-apoptotic gene BCL2 and its protein. The expression of pro-apoptotic BAX was by 4,8-DiMeIQx, MeIQx and IQ down-regulated while no changes were observed at the protein level. These results suggest that HAAs may suppress apoptosis. This assumption is corroborated by the results of Hayashi et al. (1996), who showed that the development of IQ-induced colorectal tumors in rats has been associated with increased expression of BCL2, and decreased expression of BAX proteins that was accompanied by inhibition of programmed cell death. Potential anti-apoptotic effects of PhIP were reported by Venugopal et al. (1999) who found significant inhibition of cell death in human mammary epithelial MCF-10A cells exposed to 1 and 5 lM N-hydroxy-PhIP or 100 lM PhIP that was accompanied by increased expression of anti-apoptotic BCL2 and BCL-XL proteins. 5. Conclusions Our study showed that PhIP, 4,8-DiMeIQx, MeIQx, and IQ did not induce MNi formation and relatively weakly induced DNA strand breaks in HepG2 cells. This can be partly explained by the lack of the induction of NAT2 and SULT1A1 gene expression that code for enzymes responsible for the formation of highly DNAreactive nitrenium ion, and by the consistent up-regulation of the expression of UGT1A1 by all four HAAs that indicates activation of detoxification via UGT-mediated glucuronidation. Despite rather weak genotoxic effects under the same exposure conditions as used in the genotoxicity assays all four HAAs induced elevated expression of P53 regulated DNA damage responsive genes CDKN1A and GADD45a, which was associated with the cell-cycle arrest. This confirms that all four HAAs induced damage to DNA to which the cells responded by activation of cellular defence systems. All four HAAs up-regulated the expression of anti-apoptotic BCL2, which together with some previous observations by other authors, may suggest that HAAs could suppress apoptosis. Cellcycle arrest activates DNA repair mechanisms and provides the cells with damaged DNA time to repair the damage or in the case of unrepaired DNA damage allows for the activation of apoptosis. If apoptosis is suppressed, as seems likely to be by HAAs, the cells will proceed to replication despite carrying excessive levels of DNA damage, which would result in an increased probability of mutation. Conflict of interest The authors declared no conflict of interest. Acknowledgments This study was supported by Slovenian Research Agency: Program P1-0245, Project J1-2054 and the young researcher grant to MP. References Adams, J.M., Cory, S., 2007. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 26, 1324–1337. Alaejos, M.S., Pino, V., Afonso, A.M., 2008. Metabolism and toxicology of heterocyclic aromatic amines when consumed in diet: influence of the genetic susceptibility to develop human cancer. A review. Food Res. Int. 41, 327–340. Bendaly, J., Metry, K.J., Doll, M.A., Jiang, G., States, J.C., Smith, N.B., Neale, J.R., Holloman, J.L., Pierce, W.M., Hein, D.W., 2009. Role of human CYP1A1 and NAT2 in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mutagenicity and DNA adducts. Xenobiotica 39, 399–406. Boobis, A.R., Lynch, A.M., Murray, S., Delatorre, R., Solans, A., Farre, M., Segura, J., Gooderham, N.J., Davies, D.S., 1994. CYP1A2-catalyzed conversion of dietary

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