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Sudan I induces genotoxic effects and oxidative DNA damage in HepG2 cells
Mutation Research 627 (2007) 164–170
Sudan I induces genotoxic effects and oxidative DNA damage in HepG2 cells Yu An a , Liping Jiang b , Jun Cao a , Chengyan Geng b , Laifu Zhong a,∗ a
Department of Toxicology, Dalian Medical University, 465 Zhongshan Road, Dalian, 116027 Liaoning, China b China-Japanese Joint Institute for Medical and Pharmaceutical Science, Dalian Medical University, 465 Zhongshan Road, Dalian, 116027 Liaoning, China Received 15 August 2006; received in revised form 8 November 2006; accepted 13 November 2006 Available online 22 December 2006
Abstract Sudan I, a synthetic lipid soluble azo pigment, is widely used in various industrial fields. However, Sudan I has not been approved at any level of food production, since there are many inconclusive reports relating to its genotoxicity and carcinogenicity in humans. The aim of this study was to assess the genotoxic effects of Sudan I and to identifiy and clarify the reaction mechanisms by use of human hepatoma HepG2 cells. To study the genotoxic effects of Sudan I, the comet assay and micronucleus test (MNT) were used. In the comet assay and MNT, we found increase of DNA migration and of the micronuclei frequencies at all tested concentrations (25–100 M) of Sudan I in a dose-dependent manner. The data suggest that Sudan I caused DNA strand breaks and chromosome breaks. To elucidate the underlying mechanism of this difference, we monitored the level of reactive oxygen species (ROS) production with the 2,7-dichlorofluorescein diacetate assay. The level of the oxidative DNA damage and lipid peroxidation was evaluated using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid–reactive substances (TBARS). Significantly increased levels of ROS, 8-OHdG and TBARS were observed in HepG2 cells at higher concentrations, the doses being 100, 50–100 and 50–100 M, respectively. We conclude that Sudan I causes genotoxic effects, probably via ROS-induced oxidative DNA damage at the higher doses. © 2006 Elsevier B.V. All rights reserved. Keywords: Sudan I; Micronucleus test; Comet assay; 8-Hydroxydeoxyguanosine; Lipid peroxidation; HepG2 cells
1. Introduction Sudan I (1-phenylazo-2-naphthol, C16 H12 ON2 ) is a synthetic lipid soluble azo pigment [1,2], and it is extensively used in hydrocarbon solvents, oils, fats, waxes, plastics, printing inks, shoe, floor polishes, cellulose ester varnishes, styrene resins, gasoline and soap [3]. It has also been adopted for coloring various foodstuffs,
∗ Corresponding author. Tel.: +86 411 8472 0583; fax: +86 411 8472 0661. E-mail address: [email protected] (L. Zhong).
1383-5718/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2006.11.004
particularly in those containing chilli powders, because of their intense red-orange color [4]. Nevertheless, on the basis of toxicological data, Sudan I was considered to be unsafe for use in food. In 1975, the International Agency for Research on Cancer (IARC), assessed Sudan I as a Group 3 carcinogen, for which the evidence of carcinogenicity was inadequate in humans and inadequate or limited in experimental animals [5]. Moreover, with respect to Sudan I genotoxicity data, positive results were observed both in vivo and in vitro upon metabolic activation. The comet assay showed increased DNA-migration in the stomach and colon of male ddY mice after oral administration of 1000 mg/kg
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Sudan I [6], and an increased frequency of micronuclei (MN) was reported in bone marrow cells and peripheral blood reticulocytes of F344 rats exposed to 1000 mg/kg Sudan I [6,7]. In addition to the tests in vivo, positive results were also observed in Salmonella typhimurium mutagenicity tests with S9 activation [8,9]. Sudan I also increased the frequency of sister chromatid exchanges in Chinese hamster ovary cells [10]. Therefore, its use was not permitted in foodstuffs for any purpose and at any level [11]. In February 2005, Sudan I was detected in Worcester sauce produced in the United Kingdom and food products containing the sauce were recalled [12]. Following the discovery of Sudan I in food products, the evidence of genotoxicity and carcinogenicity of Sudan I were reviewed. In earlier studies of Sudan I in vitro, Zeiger et al. [8,9] added an S9 mix instead of the metabolic enzyme. This practice has many drawbacks [13]. Besides the high toxicity to mammalian cells, S9 mix is subject to variations in individual preparations [14,15]. In this study, we selected a metabolically competent human hepatoma line (HepG2), which retains many of the functions of normal liver cells [16] and expresses the activities of several phases I and II xenobiotic metabolising enzymes [17]. The liver is the target site of Sudan I toxicity [18] and CYP1A1 is assumed to play a role in the oxidative metabolism of Sudan I in this organ [19], hence HepG2 cells were considered to be more suitable for detecting the genotoxicity of Sudan I in vitro. The aim of this study was to assess the genotoxic effects of Sudan I in vitro and to elucidate the mechanism in these cells. We used the comet assay in addition to the micronucleus test (MNT) to study the genotoxic effects of Sudan I in HepG2 cells. In short-term genotoxicity assays, the comet assay and MNT are sensitive and easy to perform. The comet assay, which can successfully be used to demonstrate genotoxicity in HepG2 [20,21], is a very reliable and rapid process for the quantification of DNA lesions and is based on the measurement of DNA migration in an electric field [22]. MN reflect chromosome breakage and/or chromosome loss [23]. Since the molecular mechanism may involve the generation of various reactive oxygen species (ROS), we measured the level of intracellular ROS by use of the 2,7-dichlorofluorescein diacetate (DCFH-DA) assay. 8Hydroxydeoxyguanosine (8-OHdG) is a reliable marker for oxidative DNA damage caused by ROS [24] and in the present experiment immunoperoxidase staining for 8-OHdG was applied. In addition, lipid peroxidation as the measure of cell oxidative injury, was determined by
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measurement of thiobarbituric acid–reactive substances (TBARS). 2. Materials and methods 2.1. Chemicals, materials and mediums Sudan I (CAS No.842-07-9) was purchased from Sigma–Aldrich (Germany: purity >97%). Dimethylsulphoxide (DMSO), RNAase A, Cytochalasin B and DCFH-DA were commercially obtained from Sigma (St. Louis, MO). Monoclonal 8-OHdG antibody and Ultrasensitive Streptavidinperoxidase Kit were obtained from JaICA (Fukuroi, Japan) and Maixin-Bio (FuJian, China). Normal melting point (NMP) agarose and low melting point (LMP) agarose were bought from Gibco BRL, Life Technologies (Paisley, UK). All tissue culture reagents, i.e. minimum essential Eagle’s medium, fetal bovine serum, antibiotics (penicillin, streptomycin) and trypsin-EDTA solution were supplied by Gibco BRL–Life Technologies (Grand Island, NY). 2.2. Cell culture and treatment The human hepatoma line (HepG2) was purchased from the American Type Culture Collection (ATCC, HB-8065). The HepG2 cells were grown in 25 cm2 tissue flasks as a monolayer in minimum essential Eagle’s medium (ATCC), supplemented with 10% (v/v) fetal bovine serum and antibiotics [penicillin (100 IU/ml) and streptomycin (100 g/ml)]. The cultures were incubated at 37 ◦ C and 5% CO2 /95% air. Sudan I was prepared as 10 mM stock solution in filter-sterilized DMSO and stored at −20 ◦ C for the aqueous insolubility. The solubility limit of the highest dose is defined as the lowest concentration producing evident precipitate in the test medium [25]. Therefore, we selected the dose of no evident particle in the final medium as the highest concentration of Sudan I (100 M), viewed by naked eye. Exponentially growing cells were exposed to different concentrations of Sudan I (final concentration: 0–100 M), DMSO (for solvent control, 1%). Sudan I was dissolved in the culture medium just before use. 2.3. Comet assay The protocol comet assay described by Singh and Stephens [26] was used with slight modifications in the study. Aliquots of HepG2 samples (1 × 106 cells/ml) were treated with Sudan I (0, 25, 50, 100 M) and hydrogen peroxide (H2 O2 ) (for positive control, 20 M) for 1 h. Subsequently, the cells were washed with phosphate-buffered saline (PBS) twice to suspend in 300 l PBS. In order to determine necrosis and apoptosis in HepG2 cells, we respectively, mixed trypan blue (50 g/ml) and Hoechst 33342 (8 g/ml) in the cell suspensions (50 l). After 15-min treatment the cells were observed under a fluorescent microscope (U-MWU2 filters). Only cell suspensions with viabilities >90% and no apoptotic cell were used to determine DNA migration. Then the cell suspension in 1% LMP
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agarose dissolved in PBS was spread onto microscope slides pre-coated with 1.5% NMP agarose. The following procedures were carried out according to the guidelines for comet assay [26]. For evaluation of DNA damage, 50 randomly selected cells from each experiment were photographed at ×400 magnification under an Olympus BX-51 fluorescent microscope (excitation filter 549 nm, barrier filter 590 nm). Image analysis was performed using Comet Assay Software Project casp-1.2.2 (University of Wroclaw, Poland). The extent of DNA damage was quantified by the increase of the tail length (m), tail DNA/head DNA (%) and tail moment (TM). We performed the experiment in triplicate, and 150 randomly selected comets from the microscope slides were analyzed.
subsequently quantified using a multiparameter image analysis program, Image-Pro Plus 4.5.1. The staining data represented the average absorbance multiplied by 1000. 2.7. Determination of lipid peroxidation HepG2 cells were seeded at 1 × 105 cells into 25 cm2 tissue flasks, and treated with the indicated compounds Sudan I (0, 25, 50, 100 M) and H2 O2 (for positive control). Lipid peroxidation was monitored in the cells as previously described by Leal et al. [31]. The absorbance was measured at 535 nm with a BIO-RAD microplate reader Model 3550. 2.8. Statistical analysis
2.4. MNT The MNT was performed following the protocol of Natarajan and Darroudi [27] with some modification. Studies were initiated by seeding 5.0 × 105 cells onto 25 cm2 tissue flasks and growing for 24 h prior to any chemical treatments. Subsequently, cells were treated with Sudan I (0, 25, 50, 100 M) for approximately 24 h, cyclophosphamde (CP) (for positive control, 800 M) for 1 h. After washing twice with PBS, the cytokinesis blocking agent cytochalasin B (final concentration: 4.5 g/ml) was added in fresh medium for anther 24 h. Following exposure to cold hypotonic solution (5.6 g/l KCl) for 20 min, the cells were gently fixed in a methanol-glacial acetic acid (3:1) solution twice. After air-drying on conventional slides, the cells were stained with 2% Giemsa solution. According to the established criteria [28], MN was scored in 1000 binucleated cells (BNC) for each concentration of the experiment. Three independent experiments were performed (n = 3). 2.5. Measurement of intracellular ROS The formation of intracellular ROS was measured by use of the DCFH-DA method [29]. Briefly, after a treatment with Sudan I (0, 25, 50, 100 M) and H2 O2 (for positive control, 20 M) at 37 ◦ C for 1 h, cells were washed twice with cold PBS and then suspended in PBS at 5 × 105 cells/ml, and incubated with DCFH-DA at a final concentration of 5 M for additional 40 min at 37 ◦ C in darkness. The fluorescent intensity of the cell suspensions was then monitored with a fluorescence spectrophotometer (HITACHI 650-60, Tokyo, Japan, excitation wavelength of 485 nm, emission wavelength 530 nm). 2.6. Immunoperoxidase staining for 8-OHdG HepG2 cells were incubated with Sudan I (0, 12.5, 25, 50, 100 M) and H2 O2 (for positive control, 20 M) for 3 h on a coverslip and washed with PBS twice. Immunoperoxidase staining for 8-OHdG was performed according to the procedure described by [30]. The images were recorded by a microscope (Olympus BX-51, Omachi, Japan), and the relative intensity of the nuclear staining of 50 randomly chosen cells per group was
Results are expressed as means ± standard deviation (S.D.). The statistical significance of differences among groups was performed with one-way analysis of variance (ANOVA), followed by least significant difference (LSD) for multiple comparison, as a post hoc test. The level of significance was set at P < 0.05 and 0.01 for all statistical analysis.
3. Results 3.1. Effect of Sudan I on DNA breakage Table 1 shows the results for DNA migration in HepG2 cells exposed to different concentrations of Sudan I (0–100 M) and the positive control (H2 O2 , 20 M) for 1 h. In all groups, no apoptosis was observed, and the cell viabilities were consistently >90% (data not shown). As shown in Table 1, H2 O2 caused a significant increase of the DNA migration (P < 0.01). Sudan I increased the DNA migration in a dose-dependent manner (Table 1). All the tested concentrations, i.e. (25, 50, 100 M), caused a significant increase (P < 0.05 or 0.01) in DNA damage. 3.2. Effect of Sudan I on MN frequencies The positive control (CP, 800 M) showed a significant increase (P < 0.01) (data not shown). Twenty-four hours exposure of the cells to different concentrations of Sudan I (25–100 M) resulted in a slight but significant (P < 0.05 or 0.01), and dose-dependent increase in MN frequencies. At the highest dose (100 M), the MN frequency was approximately two-fold higher than that in the solvent control (Fig. 1). 3.3. Induction of intracellular ROS by Sudan I A statistically significant increase of DCF fluorescence intensity (25.64 ± 2.21 versus 8.56 ± 1.65,
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Table 1 Effect of Sudan I on DNA migration in HepG2 cells Sudan I (M)
Tail length (m)
0 25 50 100 H2 O2
5.83 17.71 33.64 48.00 19.34
* **
± ± ± ± ±
1.94 3.84** 6.89** 12.44** 4.01**
Tail DNA/head DNA (%) 9.88 35.14 45.26 93.85 37.56
± ± ± ± ±
2.04 13.76** 17.63** 29.88** 8.72**
Tail moment (m) 0.39 4.93 12.92 20.57 5.41
± ± ± ± ±
0.10 2.10* 1.30** 8.99** 0.45**
P < 0.05, significantly different from cells exposed to 0 M Sudan I. P < 0.01, significantly different from cells exposed to 0 M Sudan I.
P < 0.01) was observed in HepG2 cells treated with H2 O2 (20 M) as the positive control. When HepG2 cells were incubated for 1 h with Sudan I (25–100 M), the intracellular ROS level was not significantly increased at 25 or 50 M, whereas a clear increase occurred at 100 M (P < 0.01) as shown in Fig. 2. The DCF fluorescence intensity in HepG2 cells at the highest dose of Sudan I (100 M) was about two-fold higher compared to the solvent control. 3.4. Effect of Sudan I on 8-OHdG formation With H2 O2 (20 M) as the positive control for 8-OHdG, the result showed a significant difference (20.43 ± 5.25 versus 0.29 ± 0.15, P < 0.01) in the immunoassay. Fig. 3 shows the result of immunoperoxidase staining for 8-OHdG in HepG2 cells exposed to different concentrations of Sudan I (0, 25, 50, 100 M). It can be seen that the staining intensity of 8-OHdG at the two highest tested concentrations of Sudan I (50, 100 M) (P < 0.01) was different from that observed in the solvent control, whereas no effect was found with lower concentrations.
Fig. 1. Effect of different concentrations of Sudan I (0–100 M) on MN formation in HepG2 cells exposed to the compound for 24 h. Each bar represents the mean ± S.D. of the MN frequencies in three cultures (in each culture 1000 BNC were evaluated). Symbols (*) and (**) indicate statistical significance as compared to solvent control (DMSO), P < 0.05 and 0.01.
Fig. 2. Effect of Sudan I on ROS production in HepG2 cells monitored by using the DCFH-DA assay. Cells were exposed to increasing concentrations of Sudan I (0–100 M) for 1 h. Each bar represents mean ± S.D. of three independent experiments (n = 3). Symbol (**) indicates statistical significance as compared to solvent control (DMSO), P < 0.01.
3.5. Effect of Sudan I on lipid peroxidation As presented in Fig. 4, the result indicates the increase in TBARS formation was not significant with Sudan
Fig. 3. Effect of Sudan I on oxidative DNA damage evaluated by the determination of the staining intensity of 8-OHdG. HepG2 cells were exposed to different concentrations of the compound (0–100 M) for 3 h. The line represents the mean values ± S.D. of three independent experiments (n = 3). Symbol (**) indicates statistical significance as compared to solvent control (DMSO), P < 0.01.
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Fig. 4. Effect of Sudan I on lipid peroxidation production in HepG2 cells evaluated by measuring TBARS. Cells were treated with Sudan I (0–100 M) for 24 h. Values are means ± S.D. Symbol (**) indicates statistical significance as compared to solvent control (DMSO), P < 0.01.
I (25 M) used in the lowest concentration of this study, but a significant increase was observed at the higher concentrations (50–100 M) (P < 0.01), which led to a slight, but significant increase in comparison to the solvent control, whereas the highest concentration (100 M) caused a 3.1-fold increase. 4. Discussion In the present study, we investigated the genotoxic effects of Sudan I and estimated the ability of this compound to induce a cellular oxidative stress. In the comet assay and MNT, a dose-dependent increase of DNA migration and of the MN frequencies was found after treatment with the test compound. Sudan I is metabolized by several routes, such as oxidation by cytochrome P-450 monooxygenases [32] and peroxidase [33]. Upon redox cycling of Sudan I, ROS are produced, i.e. superoxide radical anion (O2 •− ) and hydroxyl radical (OH•− ) [2]. Overproduction of these ROS induces cell oxidative injury, such as DNA damage, oxidation of proteins and lipid peroxidation [34]. To explore the mechanism of Sudan I genotoxic activity, the level of ROS in HepG2 cells was monitored with the DCFH-DA assay. Our result clearly indicates the formation of intracellular ROS was significantly increased in Sudan I-treated cells exposed to higher concentration (100 M). Therefore, Sudan I causes genotoxicity probably by an indirect mechanism, for instance via production of ROS. Furthermore, it was observed that Sudan I at the higher concentrations (50–100 M) exposure caused increase in the levels of TBARS as an index of cellular lipid peroxidation in HepG2 cells, suggesting that the formation of ROS.
Immunocytochemistry staining for 8-OHdG is a reliable marker for oxidative DNA damage in vivo [35] and in vitro [36]. The results also indicated only Sudan I, at a higher concentration, was able to increase the levels of the oxidative product 8-OHdG in HepG2 cells. It is inferred that Sudan I at higher dose (50–100 M) caused a significant oxidative damage through 8-OHdG formation in HepG2 cells which was related to ROS formation. In the present experiment, we found Sudan I induced genotoxic effects at all tested concentrations; but no formation of ROS was found at lower dose. This indicates that various mechanisms may contribute to Sudan I-induced genotoxicity. For example, the formation of the adduct, i.e. the stable 8-(phenylazo)guanine adduct formed by the reacting benzenediazonium ion (BDI) as one of the electrophilic species derived from Sudan I with DNA in vitro may play an important role [37]. In addition, the Federal Institute for Risk Assessment presumed the effects of Sudan I, including its genotoxicity, may be attributable to the release of amines and their ensuing metabolic activation [38]. Aniline, which is the reductive product of Sudan I, is known to cause genotoxicity in vitro and in vivo while BDI which is the oxidative product, was positive in the Ames assay [37,39]. In conclusion, our investigation shows that Sudan I induced genotoxic effects in HepG2 cells. Furthermore, significantly increased levels of ROS were observed. The formation of ROS could lead to DNA strand breaks, chromosome breaks and 8-OHdG formation in Sudan Itreated cells at higher concentration. It may be suggested that the genotoxic effects of Sudan I in HepG2 cells probably depend on the ROS-induced oxidative DNA damage occurring at higher doses. Acknowledgements The authors are grateful to Shuxian Qu, Haibo Cheng in Central Laboratory of Dalian Medical University for instrumental assistance. We thank Dr. Newman L. Stephens from the Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada, for language correction of the manuscript. References [1] F. Puoci, C. Garreffa, F. Iemma, R. Muzzalupo, U.G. Spizzirri, N. Picci, Molecularly imprinted solid-phase extraction for detection of Sudan I in food matrices, Food Chem. 93 (2005) 349–353. [2] P. Møller, H. Wallin, Genotoxic hazards of azo pigments and other colorants related to 1-phenylazo-2-hydroxynaphthalene, Mutat. Res. 462 (2000) 13–30.
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Sudan I induces genotoxic effects and oxidative DNA damage in HepG2 cells Yu An a , Liping Jiang b , Jun Cao a , Chengyan Geng b , Laifu Zhong a,∗ a
Department of Toxicology, Dalian Medical University, 465 Zhongshan Road, Dalian, 116027 Liaoning, China b China-Japanese Joint Institute for Medical and Pharmaceutical Science, Dalian Medical University, 465 Zhongshan Road, Dalian, 116027 Liaoning, China Received 15 August 2006; received in revised form 8 November 2006; accepted 13 November 2006 Available online 22 December 2006
Abstract Sudan I, a synthetic lipid soluble azo pigment, is widely used in various industrial fields. However, Sudan I has not been approved at any level of food production, since there are many inconclusive reports relating to its genotoxicity and carcinogenicity in humans. The aim of this study was to assess the genotoxic effects of Sudan I and to identifiy and clarify the reaction mechanisms by use of human hepatoma HepG2 cells. To study the genotoxic effects of Sudan I, the comet assay and micronucleus test (MNT) were used. In the comet assay and MNT, we found increase of DNA migration and of the micronuclei frequencies at all tested concentrations (25–100 M) of Sudan I in a dose-dependent manner. The data suggest that Sudan I caused DNA strand breaks and chromosome breaks. To elucidate the underlying mechanism of this difference, we monitored the level of reactive oxygen species (ROS) production with the 2,7-dichlorofluorescein diacetate assay. The level of the oxidative DNA damage and lipid peroxidation was evaluated using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid–reactive substances (TBARS). Significantly increased levels of ROS, 8-OHdG and TBARS were observed in HepG2 cells at higher concentrations, the doses being 100, 50–100 and 50–100 M, respectively. We conclude that Sudan I causes genotoxic effects, probably via ROS-induced oxidative DNA damage at the higher doses. © 2006 Elsevier B.V. All rights reserved. Keywords: Sudan I; Micronucleus test; Comet assay; 8-Hydroxydeoxyguanosine; Lipid peroxidation; HepG2 cells
1. Introduction Sudan I (1-phenylazo-2-naphthol, C16 H12 ON2 ) is a synthetic lipid soluble azo pigment [1,2], and it is extensively used in hydrocarbon solvents, oils, fats, waxes, plastics, printing inks, shoe, floor polishes, cellulose ester varnishes, styrene resins, gasoline and soap [3]. It has also been adopted for coloring various foodstuffs,
∗ Corresponding author. Tel.: +86 411 8472 0583; fax: +86 411 8472 0661. E-mail address: [email protected] (L. Zhong).
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particularly in those containing chilli powders, because of their intense red-orange color [4]. Nevertheless, on the basis of toxicological data, Sudan I was considered to be unsafe for use in food. In 1975, the International Agency for Research on Cancer (IARC), assessed Sudan I as a Group 3 carcinogen, for which the evidence of carcinogenicity was inadequate in humans and inadequate or limited in experimental animals [5]. Moreover, with respect to Sudan I genotoxicity data, positive results were observed both in vivo and in vitro upon metabolic activation. The comet assay showed increased DNA-migration in the stomach and colon of male ddY mice after oral administration of 1000 mg/kg
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Sudan I [6], and an increased frequency of micronuclei (MN) was reported in bone marrow cells and peripheral blood reticulocytes of F344 rats exposed to 1000 mg/kg Sudan I [6,7]. In addition to the tests in vivo, positive results were also observed in Salmonella typhimurium mutagenicity tests with S9 activation [8,9]. Sudan I also increased the frequency of sister chromatid exchanges in Chinese hamster ovary cells [10]. Therefore, its use was not permitted in foodstuffs for any purpose and at any level [11]. In February 2005, Sudan I was detected in Worcester sauce produced in the United Kingdom and food products containing the sauce were recalled [12]. Following the discovery of Sudan I in food products, the evidence of genotoxicity and carcinogenicity of Sudan I were reviewed. In earlier studies of Sudan I in vitro, Zeiger et al. [8,9] added an S9 mix instead of the metabolic enzyme. This practice has many drawbacks [13]. Besides the high toxicity to mammalian cells, S9 mix is subject to variations in individual preparations [14,15]. In this study, we selected a metabolically competent human hepatoma line (HepG2), which retains many of the functions of normal liver cells [16] and expresses the activities of several phases I and II xenobiotic metabolising enzymes [17]. The liver is the target site of Sudan I toxicity [18] and CYP1A1 is assumed to play a role in the oxidative metabolism of Sudan I in this organ [19], hence HepG2 cells were considered to be more suitable for detecting the genotoxicity of Sudan I in vitro. The aim of this study was to assess the genotoxic effects of Sudan I in vitro and to elucidate the mechanism in these cells. We used the comet assay in addition to the micronucleus test (MNT) to study the genotoxic effects of Sudan I in HepG2 cells. In short-term genotoxicity assays, the comet assay and MNT are sensitive and easy to perform. The comet assay, which can successfully be used to demonstrate genotoxicity in HepG2 [20,21], is a very reliable and rapid process for the quantification of DNA lesions and is based on the measurement of DNA migration in an electric field [22]. MN reflect chromosome breakage and/or chromosome loss [23]. Since the molecular mechanism may involve the generation of various reactive oxygen species (ROS), we measured the level of intracellular ROS by use of the 2,7-dichlorofluorescein diacetate (DCFH-DA) assay. 8Hydroxydeoxyguanosine (8-OHdG) is a reliable marker for oxidative DNA damage caused by ROS [24] and in the present experiment immunoperoxidase staining for 8-OHdG was applied. In addition, lipid peroxidation as the measure of cell oxidative injury, was determined by
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measurement of thiobarbituric acid–reactive substances (TBARS). 2. Materials and methods 2.1. Chemicals, materials and mediums Sudan I (CAS No.842-07-9) was purchased from Sigma–Aldrich (Germany: purity >97%). Dimethylsulphoxide (DMSO), RNAase A, Cytochalasin B and DCFH-DA were commercially obtained from Sigma (St. Louis, MO). Monoclonal 8-OHdG antibody and Ultrasensitive Streptavidinperoxidase Kit were obtained from JaICA (Fukuroi, Japan) and Maixin-Bio (FuJian, China). Normal melting point (NMP) agarose and low melting point (LMP) agarose were bought from Gibco BRL, Life Technologies (Paisley, UK). All tissue culture reagents, i.e. minimum essential Eagle’s medium, fetal bovine serum, antibiotics (penicillin, streptomycin) and trypsin-EDTA solution were supplied by Gibco BRL–Life Technologies (Grand Island, NY). 2.2. Cell culture and treatment The human hepatoma line (HepG2) was purchased from the American Type Culture Collection (ATCC, HB-8065). The HepG2 cells were grown in 25 cm2 tissue flasks as a monolayer in minimum essential Eagle’s medium (ATCC), supplemented with 10% (v/v) fetal bovine serum and antibiotics [penicillin (100 IU/ml) and streptomycin (100 g/ml)]. The cultures were incubated at 37 ◦ C and 5% CO2 /95% air. Sudan I was prepared as 10 mM stock solution in filter-sterilized DMSO and stored at −20 ◦ C for the aqueous insolubility. The solubility limit of the highest dose is defined as the lowest concentration producing evident precipitate in the test medium [25]. Therefore, we selected the dose of no evident particle in the final medium as the highest concentration of Sudan I (100 M), viewed by naked eye. Exponentially growing cells were exposed to different concentrations of Sudan I (final concentration: 0–100 M), DMSO (for solvent control, 1%). Sudan I was dissolved in the culture medium just before use. 2.3. Comet assay The protocol comet assay described by Singh and Stephens [26] was used with slight modifications in the study. Aliquots of HepG2 samples (1 × 106 cells/ml) were treated with Sudan I (0, 25, 50, 100 M) and hydrogen peroxide (H2 O2 ) (for positive control, 20 M) for 1 h. Subsequently, the cells were washed with phosphate-buffered saline (PBS) twice to suspend in 300 l PBS. In order to determine necrosis and apoptosis in HepG2 cells, we respectively, mixed trypan blue (50 g/ml) and Hoechst 33342 (8 g/ml) in the cell suspensions (50 l). After 15-min treatment the cells were observed under a fluorescent microscope (U-MWU2 filters). Only cell suspensions with viabilities >90% and no apoptotic cell were used to determine DNA migration. Then the cell suspension in 1% LMP
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agarose dissolved in PBS was spread onto microscope slides pre-coated with 1.5% NMP agarose. The following procedures were carried out according to the guidelines for comet assay [26]. For evaluation of DNA damage, 50 randomly selected cells from each experiment were photographed at ×400 magnification under an Olympus BX-51 fluorescent microscope (excitation filter 549 nm, barrier filter 590 nm). Image analysis was performed using Comet Assay Software Project casp-1.2.2 (University of Wroclaw, Poland). The extent of DNA damage was quantified by the increase of the tail length (m), tail DNA/head DNA (%) and tail moment (TM). We performed the experiment in triplicate, and 150 randomly selected comets from the microscope slides were analyzed.
subsequently quantified using a multiparameter image analysis program, Image-Pro Plus 4.5.1. The staining data represented the average absorbance multiplied by 1000. 2.7. Determination of lipid peroxidation HepG2 cells were seeded at 1 × 105 cells into 25 cm2 tissue flasks, and treated with the indicated compounds Sudan I (0, 25, 50, 100 M) and H2 O2 (for positive control). Lipid peroxidation was monitored in the cells as previously described by Leal et al. [31]. The absorbance was measured at 535 nm with a BIO-RAD microplate reader Model 3550. 2.8. Statistical analysis
2.4. MNT The MNT was performed following the protocol of Natarajan and Darroudi [27] with some modification. Studies were initiated by seeding 5.0 × 105 cells onto 25 cm2 tissue flasks and growing for 24 h prior to any chemical treatments. Subsequently, cells were treated with Sudan I (0, 25, 50, 100 M) for approximately 24 h, cyclophosphamde (CP) (for positive control, 800 M) for 1 h. After washing twice with PBS, the cytokinesis blocking agent cytochalasin B (final concentration: 4.5 g/ml) was added in fresh medium for anther 24 h. Following exposure to cold hypotonic solution (5.6 g/l KCl) for 20 min, the cells were gently fixed in a methanol-glacial acetic acid (3:1) solution twice. After air-drying on conventional slides, the cells were stained with 2% Giemsa solution. According to the established criteria [28], MN was scored in 1000 binucleated cells (BNC) for each concentration of the experiment. Three independent experiments were performed (n = 3). 2.5. Measurement of intracellular ROS The formation of intracellular ROS was measured by use of the DCFH-DA method [29]. Briefly, after a treatment with Sudan I (0, 25, 50, 100 M) and H2 O2 (for positive control, 20 M) at 37 ◦ C for 1 h, cells were washed twice with cold PBS and then suspended in PBS at 5 × 105 cells/ml, and incubated with DCFH-DA at a final concentration of 5 M for additional 40 min at 37 ◦ C in darkness. The fluorescent intensity of the cell suspensions was then monitored with a fluorescence spectrophotometer (HITACHI 650-60, Tokyo, Japan, excitation wavelength of 485 nm, emission wavelength 530 nm). 2.6. Immunoperoxidase staining for 8-OHdG HepG2 cells were incubated with Sudan I (0, 12.5, 25, 50, 100 M) and H2 O2 (for positive control, 20 M) for 3 h on a coverslip and washed with PBS twice. Immunoperoxidase staining for 8-OHdG was performed according to the procedure described by [30]. The images were recorded by a microscope (Olympus BX-51, Omachi, Japan), and the relative intensity of the nuclear staining of 50 randomly chosen cells per group was
Results are expressed as means ± standard deviation (S.D.). The statistical significance of differences among groups was performed with one-way analysis of variance (ANOVA), followed by least significant difference (LSD) for multiple comparison, as a post hoc test. The level of significance was set at P < 0.05 and 0.01 for all statistical analysis.
3. Results 3.1. Effect of Sudan I on DNA breakage Table 1 shows the results for DNA migration in HepG2 cells exposed to different concentrations of Sudan I (0–100 M) and the positive control (H2 O2 , 20 M) for 1 h. In all groups, no apoptosis was observed, and the cell viabilities were consistently >90% (data not shown). As shown in Table 1, H2 O2 caused a significant increase of the DNA migration (P < 0.01). Sudan I increased the DNA migration in a dose-dependent manner (Table 1). All the tested concentrations, i.e. (25, 50, 100 M), caused a significant increase (P < 0.05 or 0.01) in DNA damage. 3.2. Effect of Sudan I on MN frequencies The positive control (CP, 800 M) showed a significant increase (P < 0.01) (data not shown). Twenty-four hours exposure of the cells to different concentrations of Sudan I (25–100 M) resulted in a slight but significant (P < 0.05 or 0.01), and dose-dependent increase in MN frequencies. At the highest dose (100 M), the MN frequency was approximately two-fold higher than that in the solvent control (Fig. 1). 3.3. Induction of intracellular ROS by Sudan I A statistically significant increase of DCF fluorescence intensity (25.64 ± 2.21 versus 8.56 ± 1.65,
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Table 1 Effect of Sudan I on DNA migration in HepG2 cells Sudan I (M)
Tail length (m)
0 25 50 100 H2 O2
5.83 17.71 33.64 48.00 19.34
* **
± ± ± ± ±
1.94 3.84** 6.89** 12.44** 4.01**
Tail DNA/head DNA (%) 9.88 35.14 45.26 93.85 37.56
± ± ± ± ±
2.04 13.76** 17.63** 29.88** 8.72**
Tail moment (m) 0.39 4.93 12.92 20.57 5.41
± ± ± ± ±
0.10 2.10* 1.30** 8.99** 0.45**
P < 0.05, significantly different from cells exposed to 0 M Sudan I. P < 0.01, significantly different from cells exposed to 0 M Sudan I.
P < 0.01) was observed in HepG2 cells treated with H2 O2 (20 M) as the positive control. When HepG2 cells were incubated for 1 h with Sudan I (25–100 M), the intracellular ROS level was not significantly increased at 25 or 50 M, whereas a clear increase occurred at 100 M (P < 0.01) as shown in Fig. 2. The DCF fluorescence intensity in HepG2 cells at the highest dose of Sudan I (100 M) was about two-fold higher compared to the solvent control. 3.4. Effect of Sudan I on 8-OHdG formation With H2 O2 (20 M) as the positive control for 8-OHdG, the result showed a significant difference (20.43 ± 5.25 versus 0.29 ± 0.15, P < 0.01) in the immunoassay. Fig. 3 shows the result of immunoperoxidase staining for 8-OHdG in HepG2 cells exposed to different concentrations of Sudan I (0, 25, 50, 100 M). It can be seen that the staining intensity of 8-OHdG at the two highest tested concentrations of Sudan I (50, 100 M) (P < 0.01) was different from that observed in the solvent control, whereas no effect was found with lower concentrations.
Fig. 1. Effect of different concentrations of Sudan I (0–100 M) on MN formation in HepG2 cells exposed to the compound for 24 h. Each bar represents the mean ± S.D. of the MN frequencies in three cultures (in each culture 1000 BNC were evaluated). Symbols (*) and (**) indicate statistical significance as compared to solvent control (DMSO), P < 0.05 and 0.01.
Fig. 2. Effect of Sudan I on ROS production in HepG2 cells monitored by using the DCFH-DA assay. Cells were exposed to increasing concentrations of Sudan I (0–100 M) for 1 h. Each bar represents mean ± S.D. of three independent experiments (n = 3). Symbol (**) indicates statistical significance as compared to solvent control (DMSO), P < 0.01.
3.5. Effect of Sudan I on lipid peroxidation As presented in Fig. 4, the result indicates the increase in TBARS formation was not significant with Sudan
Fig. 3. Effect of Sudan I on oxidative DNA damage evaluated by the determination of the staining intensity of 8-OHdG. HepG2 cells were exposed to different concentrations of the compound (0–100 M) for 3 h. The line represents the mean values ± S.D. of three independent experiments (n = 3). Symbol (**) indicates statistical significance as compared to solvent control (DMSO), P < 0.01.
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Fig. 4. Effect of Sudan I on lipid peroxidation production in HepG2 cells evaluated by measuring TBARS. Cells were treated with Sudan I (0–100 M) for 24 h. Values are means ± S.D. Symbol (**) indicates statistical significance as compared to solvent control (DMSO), P < 0.01.
I (25 M) used in the lowest concentration of this study, but a significant increase was observed at the higher concentrations (50–100 M) (P < 0.01), which led to a slight, but significant increase in comparison to the solvent control, whereas the highest concentration (100 M) caused a 3.1-fold increase. 4. Discussion In the present study, we investigated the genotoxic effects of Sudan I and estimated the ability of this compound to induce a cellular oxidative stress. In the comet assay and MNT, a dose-dependent increase of DNA migration and of the MN frequencies was found after treatment with the test compound. Sudan I is metabolized by several routes, such as oxidation by cytochrome P-450 monooxygenases [32] and peroxidase [33]. Upon redox cycling of Sudan I, ROS are produced, i.e. superoxide radical anion (O2 •− ) and hydroxyl radical (OH•− ) [2]. Overproduction of these ROS induces cell oxidative injury, such as DNA damage, oxidation of proteins and lipid peroxidation [34]. To explore the mechanism of Sudan I genotoxic activity, the level of ROS in HepG2 cells was monitored with the DCFH-DA assay. Our result clearly indicates the formation of intracellular ROS was significantly increased in Sudan I-treated cells exposed to higher concentration (100 M). Therefore, Sudan I causes genotoxicity probably by an indirect mechanism, for instance via production of ROS. Furthermore, it was observed that Sudan I at the higher concentrations (50–100 M) exposure caused increase in the levels of TBARS as an index of cellular lipid peroxidation in HepG2 cells, suggesting that the formation of ROS.
Immunocytochemistry staining for 8-OHdG is a reliable marker for oxidative DNA damage in vivo [35] and in vitro [36]. The results also indicated only Sudan I, at a higher concentration, was able to increase the levels of the oxidative product 8-OHdG in HepG2 cells. It is inferred that Sudan I at higher dose (50–100 M) caused a significant oxidative damage through 8-OHdG formation in HepG2 cells which was related to ROS formation. In the present experiment, we found Sudan I induced genotoxic effects at all tested concentrations; but no formation of ROS was found at lower dose. This indicates that various mechanisms may contribute to Sudan I-induced genotoxicity. For example, the formation of the adduct, i.e. the stable 8-(phenylazo)guanine adduct formed by the reacting benzenediazonium ion (BDI) as one of the electrophilic species derived from Sudan I with DNA in vitro may play an important role [37]. In addition, the Federal Institute for Risk Assessment presumed the effects of Sudan I, including its genotoxicity, may be attributable to the release of amines and their ensuing metabolic activation [38]. Aniline, which is the reductive product of Sudan I, is known to cause genotoxicity in vitro and in vivo while BDI which is the oxidative product, was positive in the Ames assay [37,39]. In conclusion, our investigation shows that Sudan I induced genotoxic effects in HepG2 cells. Furthermore, significantly increased levels of ROS were observed. The formation of ROS could lead to DNA strand breaks, chromosome breaks and 8-OHdG formation in Sudan Itreated cells at higher concentration. It may be suggested that the genotoxic effects of Sudan I in HepG2 cells probably depend on the ROS-induced oxidative DNA damage occurring at higher doses. Acknowledgements The authors are grateful to Shuxian Qu, Haibo Cheng in Central Laboratory of Dalian Medical University for instrumental assistance. We thank Dr. Newman L. Stephens from the Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada, for language correction of the manuscript. References [1] F. Puoci, C. Garreffa, F. Iemma, R. Muzzalupo, U.G. Spizzirri, N. Picci, Molecularly imprinted solid-phase extraction for detection of Sudan I in food matrices, Food Chem. 93 (2005) 349–353. [2] P. Møller, H. Wallin, Genotoxic hazards of azo pigments and other colorants related to 1-phenylazo-2-hydroxynaphthalene, Mutat. Res. 462 (2000) 13–30.
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