Arch Toxicol (2010) 84:641–650 DOI 10.1007/s00204-010-0535-7
GENOTOXICITY AND CARCINOGENICITY
Beauvericin and ochratoxin A genotoxicity evaluated using the alkaline comet assay: single and combined genotoxic action Maja Sˇegvic´ Klaric´ • Dina Darabosˇ • Ruzˇica Rozgaj • Vilena Kasˇuba • Stjepan Pepeljnjak
Received: 23 September 2009 / Accepted: 9 March 2010 / Published online: 30 March 2010 Ó Springer-Verlag 2010
Abstract This study was aimed at investigating the genotoxic potential of single beauvericin (BEA) and ochratoxin A (OTA) as well as their interaction in porcine kidney epithelial PK15 cells and human leukocytes using the alkaline comet assay. IC50 of BEA (5.0 ± 0.6) and OTA (15.8 ± 1.5) estimated by MTT reduction assay shows that BEA is three times more toxic than OTA. BEA (0.1 and 0.5 lM) and OTA (1 and 5 lM) were applied alone or in combination of these concentrations for 1 and 24 h in PK15 cells and human leukocytes. Genotoxicity of these toxins to PK15 cells was time- and concentration dependent. After 1 h, significant increase in tail length, tail intensity, tail moment, and abnormal sized tails (AST) was noted upon exposure to 1 lM of OTA alone and BEA ? OTA combinations. Single BEA (0.5 lM) and OTA (1 and 5 lM) and their combinations evoked significant DNA damage in PK15 cells, considering all comet tail parameters measured after 24 h of treatment. Human leukocytes were slightly concentration but not time dependent. After 1 h of exposure, there were no significant changes in the tail length. Tail intensity, tail moment, and/ or incidence of AST were significantly higher in cells treated with single OTA or BEA and their combinations than in control cells. DNA damage in leukocytes was
M. Sˇ. Klaric´ (&) D. Darabosˇ S. Pepeljnjak Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Schrottova 39, 10000 Zagreb, Croatia e-mail:
[email protected] R. Rozgaj V. Kasˇuba Mutagenesis Unit, Institute of Medical Research and Occupational Health, Zagreb, Croatia
significantly higher after 24 h of exposure to single toxins and their combinations, considering all comet tail parameters, but these changes were less pronounced than in PK15 cells. Combined toxins showed additive and synergistic effects in PK15 cells, while only additive effects were observed in human leukocytes. Combined prolonged exposure to BEA and OTA in subcytotoxic concentrations through food consumption could induce DNA damage contributing to the carcinogenicity in animals and humans. Keywords Genotoxicity Comet assay Mycotoxin synergism Carcinogenicity
Introduction Beauvericin (BEA) and ochratoxin A (OTA) are mycotoxins that contaminate maize and maize-based products worldwide (Domijan et al. 2005; Jurjevic et al. 1999, 2002). They cause a great number of pathological changes in vivo and in vitro. Beauvericin (BEA) is a cyclic hexadepsipeptide, which possesses antimicrobial, insecticidal, cytotoxic, ionophoric, immunosuppressive, and apoptotic properties (Hamill et al. 1969; Grove and Pople 1980; Ojcius et al. 1991; Castelbury et al. 1999; Dombrink-Kurtzman 2003). BEA increases ion permeability in biological membranes by forming a complex with essential cations (Ca2?, Na?, K?) and/or by forming cation-selective channels in lipid membranes, which may affect ionic homeostasis (Kouri et al. 2003). There are only a few literature reports on BEA mutagenicity and genotoxicity. In the Ames assay, BEA indicated non-mutagenic activity (Fotso and Smith 2003), but in porcine kidney PK15 cells BEA showed genotoxic potential by inducing formation of micronuclei (MN), nuclear buds (NBs), and nucleoplasmic bridges (NPBs) in a
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dose-dependent manner (Sˇegvic´ Klaric´ et al. 2008a). However, the mechanism of BEA genotoxic action is still unknown. Ochratoxin A induces nephrotoxic, neurotoxic, immunosuppressive, cytotoxic, genotoxic, mutagenic, teratogenic, and carcinogenic effects (IPCS 2001). Some studies have indicated that long-term exposure to OTA in humans is associated with a high incidence of progressive nephropathy (referred to as Balkan endemic nephropathy, BEN) and urinary tract tumors (UTTs) (Plesˇtina et al. 1990; Nikolov et al. 1996; Petkova-Bocharova et al. 2003; PfohlLeszkowicz et al. 2002). Genotoxic effects of OTA have been demonstrated in different in vitro and in vivo studies, although with inconsistent results. OTA did not induce chromosome aberrations in CHO cells (Kuiper-Goodman and Scott 1989) but induced sister chromatid exchange in porcine urinary bladder epithelial cells as well as chromosome aberrations in human lymphocytes (Manolova et al. 1990; Fo¨llmann et al. 1995). Induction of micronuclei (MN) by OTA, with predominantly clastogenic effects, was reported for seminal vesicle OSV cells, Syrian hamster embryo SHE fibroblasts, HepG2 cells, as well as for porcine kidney epithelial cells PK15 (Degen et al. 1997; Dopp et al. 1999; Ehrlich et al. 2002; Robbiano et al. 2004; Sˇegvic´ Klaric´ et al. 2008a). Currently, the mode of genotoxic action by OTA is unknown. Pfohl-Leszkowicz and Manderville (2007) proposed that OTA genotoxicity may be divided into direct (covalent DNA adduction) and indirect (oxidative DNA damage) mechanisms of action. The indirect mechanism hypothesis involves oxidative stress and is supported by OTA-induced lipid peroxidation, formation of reactive oxygen species (ROS) and consequent oxidative DNA damage, and decreasing intracellular glutathione (GSH) level in vitro and in vivo. Prolonged combined exposure to OTA and BEA in subcytotoxic concentration through food can cause DNA damage, contributing to the nephrotoxicity and carcinogenicity in animals and humans. An earlier study showed that combined treatment of PK15 with BEA ? OTA induced mostly additive effect on micronuclei formation (Sˇegvic´ Klaric´ et al. 2008a). The aim of this study was to investigate the genotoxic potential of single OTA and BEA as well as their combinations in PK15 cells and human lymphocytes using the single-cell gel electrophoresis (Comet) assay.
Materials and methods Chemicals Dulbecco’s modified Eagle’s medium, fetal bovine serum (FBS), trypsin (EDTA), and phosphate-buffered saline
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(PBS; Ca2? and Mg2? free) were obtained from the Department of Immunology (Zagreb, Croatia). Amphotericin B was from Roche Diagnostics (Mannheim, Germany); penicillin and streptomycin were from Pliva (Zagreb, Croatia). Beauvericin and ochratoxin A, MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], agarose normal melting point (NMP), agarose low melting point (LMP), Triton X-100, Tris buffer, and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich (Deisenhofen, Germany). All other chemicals including HCl, isopropanol, NaCl, Na2EDTA, and NaOH were from Kemika (Zagreb, Croatia). Cell culture Porcine kidney epithelial cells PK15 (American Type Culture Collection, Rockville, Md USA) were grown in 75-cm2 flasks in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 IU/mL; 1 U & 67.7 lg/mL), streptomycin (100 lg/mL), and amphotericin B (2.5 lg/ mL) at 37°C in a 5% CO2. Ochratoxin A (1 mg/mL) and beauvericin (1 mg/mL) stock solutions were prepared by dissolving OTA and BEA in ethanol (100%). The final concentrations of the mycotoxins were obtained by dilution with the culture medium. A sample of human blood in the volume of 10 mL was taken by venipuncture from a 50-year-old female volunteer (non-smoker), and whole blood was mixed with DMEM serum free (1:1). MTT assay The cell viability was measured by MTT reduction assay. PK15 cells were seeded at plating density 104 per well, in a 96-well flat-bottomed microplate. Afterward 36 h of incubation growth medium was replaced by the medium without FBS, and cells were incubated for the next 12 h. To determine IC50 for both toxins, PK15 cells were treated for 24 h with BEA at concentrations 0.1, 0.5, 1, 3, 6, 10, and 15 lM or OTA at concentrations 1, 2, 5, 6, 10, 18, 26, 34, and 46 lM. Cells were also treated with BEA ? OTA combinations (0.1 ? 1; 0.1 ? 5; 0.5 ? 1; 0.5 ? 5 lM). Ethanol was added to control cells in the concentration of 1.6%. At the end of the treatment, the medium was removed, and 100 lL of MTT reagent diluted in growth medium without FCS (0.5 mg/mL) was added in each well. After 3.5 h of incubation, MTT reagent was replaced with 200 lL of 0.04 M HCl diluted in isopropanol, and cells were incubated at room temperature on rotary shaker for 15 min. The absorbance was measured at wavelength of 595 nm using a microplate reader. All tests were performed on one 96-well plate in four replicates. The results were expressed as percentage of control values.
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Comet assay Cells were seeded in 6-well plates (105–106 cells/mL) and treated with single OTA (1 and 5 lM) and BEA (0.1 and 0.5 lM), as well as with their combinations (0.1 ? 1; 0.1 ? 5; 0.5 ? 1; 0.5 ? 5 lM, respectively) for 1- and 24 h. Prior to mycotoxin addition, cells were incubated in serum-free media for 12 h. Whole blood samples were exposed to the same concentrations of mycotoxins also for 1- and 24 h. Ethanol was added to control cells in the concentration of 1.6%. The comet assay was carried out according to Singh et al. (1988). After exposure to mycotoxins, cells were detached using trypsin (EDTA) and resuspended in 5 mL PBS. A single-cell suspension was centrifuged at 600 g for 8 min, at 4°C. The supernatant was removed, and pellet was resuspended in 5 mL PBS and centrifuged again. The supernatant was removed, and cells were resuspended in 100 lL of PBS. Aliquots of 20 lL of this suspension were mixed with 100 lL 0.5% LMP, and 100 lL of agarose-cell suspension was spread onto a fully frosted slide (Surgipath, Richmond, Il, USA) pre-coated with 1.5% NMP (in Ca- and Mg-free PBS buffer). The slides were allowed to solidify on ice for 10 min. After overnight lysis at 4°C in a mixture of 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris (pH 10) supplemented with 1% Triton-X, the slides were placed in denaturation and electrophoresis buffer (10 mM NaOH, 200 mM Na2EDTA, pH 13), incubated for 20 min, and electrophoresed for 20 min at 25 V and 300 mA. DNA was neutralized with a neutralization solution (0.4 M Tris/ HCl, pH 7.5) three times 5 min each. The slides were kept in a humid atmosphere in a dark box at 4°C until further analysis. For image analysis, DNA was stained with 100– 250 lL ethidium bromide solution (20 lg/mL) per slide for 10 min. Whole human blood in the culture media was prepared in the same manner with minor modifications. Aliquots of 8 lL cell suspension were mixed with 100 lL of 0.5% LMP, dropped on slides pre-coated with a 300-lL layer of 0.6% NMP agarose, and laid on ice for 10 min. After removing the coverslips, 100 lL 0.5% LMP was added on each slide and left to solidify. Further procedure was carried out as described previously. Slides were scored using an image analysis system (Comet assay II, Perceptive instruments Ltd., UK) connected to a fluorescence microscope (Zeiss, Germany). All experiments were performed in duplicate, and in each experiment images of 200 randomly selected cells (100 cells from each of the two replicate slides) were measured. Only comets with a defined head were scored. Comet parameters considered in this study were the tail length, the proportion of DNA in the comet tail (tail DNA
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or tail intensity), and tail moment, which was calculated as the product of the fraction of DNA in the comet tail and the tail length. Statistical analysis The results of cytotoxicity test are represented as mean ± SD (standard deviation) and were statistically analyzed by one-way analysis of variance (ANOVA) followed by a multiple comparison procedure (Tukey test). The level of P \ 0.05 was considered statistically significant. The results of tail length, tail intensity, and tail moment are represented as mean ± SE (standard error of mean), minimum and maximum. The Kolmogorov–Smirnov test was used to verify whether the results of comet parameters were normally distributed. Data were logarithmically transformed to normalize distribution. ANOVA followed by Tukey’s multiple comparison test was used for statistical analysis of logarithmically transformed values of tail length, tail intensity, and tail moment. Comets were also classified as damaged or undamaged regarding to the 95th percentile of comet parameters (abnormal size tails, AST) in control (Gabianelli et al. 2003). AST values were statistically analyzed using Pearson’s v2 test. The level of P \ 0.05 was considered statistically significant for all statistical calculations. To compare the expected values of MTT test as well as expected values of tail moment (as the best indicator of DNA damage) after combined toxin treatment with measured values, the expected values were calculated by adding the mean obtained after exposure to BEA alone to the mean obtained after exposure to OTA (Weber et al. 2005). For example: mean ðexpected for OTA + BEAÞ ¼ mean ðOTAÞ þ mean ðBEAÞ mean ðcontrolÞ Calculation of expected standard error of means (SEM): SEM ðexpected for OTA + BEAÞ h i1=2 ¼ ðSEM for OTAÞ2 þðSEM for BEAÞ2 The significance of difference between the expected and measured values was calculated using an unpaired t-test. P \ 0.05 was considered statistically significant. The results were interpreted as follows: (i) an additive effect was recorded if the measured values were not significantly above or below the expected values; (ii) a synergistic effect was recorded if the measured values were significantly above the expected values; and (iii) an antagonistic effect was recorded if the measured values were significantly below the expected values.
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Results Cytotoxicity assay Figures 1 and 2 show the results of MTT reduction assay. BEA and OTA reduced viability of PK15 cells in concentration-dependant manner. Compared to control, metabolic activity significantly dropped after exposure to 1 lM of BEA (82%) and 2 lM of OTA (68%), respectively (Fig. 1a, b). IC50 for BEA and OTA obtained by MTT assay was 5.0 ± 0.6 and 15.8 ± 1.5 lM, respectively. To estimate cytotoxic and genotoxic interactions of these toxins, two concentrations of BEA (0.1 and 0.5 lM) and OTA (1 and 5 lM) below IC50 were used. After 24 h of combined treatment, cell viability dropped to 50 and 40% of control. An additive effect was observed for BEA ? OTA (0.1 ? 5 and 0.5 ? 5 lM), while synergism was noted for combinations BEA ? OTA 0.1 ? 1 and 0.5 ? 1 lM (Fig. 2). Comet assay Tables 1 and 2 show the results of the alkaline comet assay on PK15 cells and human lymphocytes following exposure
Cell viability (% of control + SD)
(a)
100 90 80 70 60 50 40 30 20 10 0
*
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
BEA concentrations ( µM)
Cell viability (% of control + SD)
(b)
100 90 80 70 60 50 40 30 20 10 0
*
0
5
10
15
20
25
30
35
40
45
50
OTA concentrations (µM) Fig. 1 Cytotoxicity effects of a BEA (0.1, 0.5, 1, 3, 6, 10, and 15 lM) and b OTA (1, 2, 5, 6, 10, 18, 26, 34, and 46 lM) on PK15 cells after 24 h of exposure. Data are expressed as mean ± SD % of control cells of independent experiments (n = 4). Control cells were exposed to vehicle only, and value was taken as 100%. *(P \ 0.05), when compared to the control value
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Cell viability (% of control + SE)
100 80 60
* *
40 20
0 OTA µM BEA µM
1 0.1
5 0.1
1 0.5
5 0.5
Fig. 2 Cell viability of PK15 cells after 24 h of combined treatment with BEA and OTA. White bars represent the expected values and gray bars the measured values. *Represents significant synergistic effect (P \ 0.05)
to single BEA and OTA as well as their combinations. The incidence of comets with abnormal size tails (AST) and cutoff values (95th percentile) for the comet tail parameters are shown in Tables 3 and 4. Genotoxic activity of toxins alone and their combinations on PK15 cells was concentration- and time dependent. After 1 h of treatment, significant increase in tail length, tail intensity, and tail moment was noted upon exposure to 1 lM of OTA alone and BEA ? OTA combinations when compared to control cells (P \ 0.05). The combination BEA ? OTA 0.5 ? 5 lM evoked significant increase in these comet parameters comparing to BEA and OTA alone (P \ 0.05). Four percent of control cells had abnormal size tail with respect to all three comet parameters while AST values varied between 6 and 13% in cells exposed to single toxins and between 12 and 24% in cells treated with toxin combinations. The incidence of AST was significantly higher in cells treated with toxin combinations than in control (P \ 0.05). BEA alone at 0.5 lM evoked a significantly lower DNA damage than BEA and OTA applied at 0.5 ? 5 lM (P \ 0.05). The combination of BEA ? OTA 0.5 ? 5 lM produced synergism, while other three toxin combinations showed additive effects as assessed by statistical calculation of tail moment (Fig. 3). After 24 h of exposure to BEA and OTA, the values of all four comet parameters (tail length, tail intensity, tail moment, and AST) were significantly higher in all experimental groups of PK15 cells than in control cells (P \ 0.05), except in cells treated with the lowest concentration of BEA. Comet parameters were also significantly higher in almost all PK15 cells exposed to toxin combinations (P \ 0.05). The combination BEA ? OTA (0.1 ? 1 lM) did not evoke significant DNA damage compared to 1 lM of OTA alone. The combinations of BEA ? OTA in the concentrations of 0.1 ? 1 lM and 0.1 ? 5 lM showed additive effects, while higher concentrations of 0.5 ? 1 lM and 0.5 ? 5 lM produced synergism (Fig. 4).
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Table 1 Evaluation of primary DNA damage measured in PK15 cells following 1- and 24-h exposure to single OTA and BEA and their combinations Cell treatment
Mean ± SE Tail length (lm) 1h
Tail intensity (%DNA)
Tail moment
24 h
1h
1h
24 h 0.34 ± 0.04
24 h
Control (1.6% EtOH)
19.28 ± 0.13
20.24 ± 0.39
1.23 ± 0.13
1.90 ± 0.24
0.22 ± 0.02
BEA (0.1 lM)
19.41 ± 0.13
20.89 ± 0.56a
1.25 ± 0.20
3.29 ± 0.40a
0.27 ± 0.02
BEA (0.5 lM)
19.97 ± 0.17
b
26.75 ± 0.69*
,c
2.72 ± 0.17*
6.36 ± 0.42*
0.35 ± 0.02*
1.30 ± 0.12*,c
2.49 ± 0.15d
10.07 ± 0.82*,d
0.33 ± 0.02d
2.35 ± 0.20*,d
2.85 ± 0.23*
,a
0.35 ± 0.03*
1.52 ± 0.16*,a
,a
0.36 ± 0.03*
2.29 ± 0.20*,a
,b,c
0.35 ± 0.03*
OTA (1 lM)
20.44 ± 0.16*
26.90 ± 0.60*
OTA (5 lM)
20.07 ± 0.17d
32.23 ± 0.92*,d
BEA ? OTA (0.1 ? 1 lM) 20.77 ± 0.16*
28.87 ± 1,10*
,a
32.39 ± 1.01*
,a
41.44 ± 1.45*
,b,c
51.38 ± 1.79*
,b,d
BEA ? OTA (0.1 ? 5 lM) 20.17 ± 0.18 * BEA ? OTA (0.5 ? 1 lM) 20.22 ± 0.14* ,b,d
BEA ? OTA (0.5 ? 5 lM) 21.67 ± 0.26*
b
0.57 ± 0.08a
,b
1.34 ± 0.15
,b
0.29 ± 0.02
b
1.32 ± 0.09*,b
,c
6.41 ± 0.54*
7.12 ± 0.67*
2.83 ± 0.22*
10.31 ± 0.85*
2.70 ± 0.20*
11.84 ± 0.76*
,b,d
4.47 ± 0.49*
,b,d
15.14 ± 0.79*
0.70 ± 0.06*
3.25 ± 0.24*,b,c ,b,d
4.54 ± 0.29*,b,d
* Compared to control (P \ 0.05) a BEA 0.1 lM versus BEA ? OTA (0.1 ? 1 lM; 0.1 ? 5 lM) (P \ 0.05) b
BEA 0.5 lM versus BEA ? OTA (0.5 ? 1 lM; 0.5 ? 5 lM) (P \ 0.05)
c
OTA 1 lM when compared to BEA ? OTA (0.1 ? 1 lM; 0.5 ? 1 lM) (P \ 0.05)
d
OTA 5 lM when compared to BEA ? OTA (0.1 ? 5 lM; 0.5 ? 5 lM) (P \ 0.05)
Table 2 Evaluation of primary DNA damage measured in human leukocytes following 1- and 24-h exposure to single OTA and BEA and their combinations Cell treatment
Mean ± SE Tail length (lm) 1h
Tail intensity (%DNA)
Tail moment
24 h
1h
24 h
1h
24 h
Control (1.6% EtOH)
14.55 ± 0.12
14.49 ± 0.22
0.86 ± 0.10
0.85 ± 0.08
0.11 ± 0.01
0.11 ± 0.01
BEA (0.1 lM)
14.60 ± 0.11
14.78 ± 0.22a
1.29 ± 0.21a
0.93 ± 0.09a
0.17 ± 0.03
0.12 ± 0.01a
BEA (0.5 lM)
14.65 ± 0.12
b
15.10 ± 0.18
b
0.22 ± 0.03
0.19 ± 0.02*,b
OTA (1 lM)
14.31 ± 0.11c
15.31 ± 0.26c
1.56 ± 0.15*
1.58 ± 0.12*
0.20 ± 0.02
0.23 ± 0.02*
OTA (5 lM)
14.68 ± 0.17
17.31 ± 0.34*
1.87 ± 0.23*
1.65 ± 0.18*
0.25 ± 0.03*
0.24 ± 0.03*
BEA ? OTA (0.1 ? 1 lM)
14.99 ± 0.16
16.71 ± 0.28*,a,c
1.78 ± 0.21*
1.62 ± 0.16*,a
0.24 ± 0.03*
0.23 ± 0.02*,a
,a
BEA ? OTA (0.1 ? 5 lM)
15.03 ± 0.18 c
1.75 ± 0.22
1.44 ± 0.18
,a
1.95 ± 0.23*
1.68 ± 0.16*
0.26 ± 0.03*
0.24 ± 0.02*,a
,b,c
17.08 ± 0.33*
,a
BEA ? OTA (0.5 ? 1 lM)
15.25 ± 0.19
17.45 ± 0.36*
2.07 ± 0.23*
2.20 ± 0.27*
0.29 ± 0.03*
0.35 ± 0.04*,b
BEA ? OTA (0.5 ? 5 lM)
14.83 ± 0.14
17.82 ± 0.46*,b
2.24 ± 0.22*
2.37 ± 0.24*,b
0.28 ± 0.03*
0.38 ± 0.08*,b
* Compared to control (P \ 0.05) a BEA 0.1 lM versus BEA ? OTA (0.1 ? 1 lM; 0.1 ? 5 lM) (P \ 0.05) b
BEA 0.5 lM versus BEA ? OTA (0.5 ? 1 lM; 0.5 ? 5 lM) (P \ 0.05)
c
OTA 1 lM versus BEA ? OTA (0.1 ? 1 lM; 0.5 ? 1 lM) (P \ 0.05)
After 1 h of leukocyte exposure to toxins alone and their combinations, tail length did not significantly change, but the incidence of comets with abnormal sized tails was significantly higher in cells treated with 0.5 ? 1 lM BEA ? OTA (11%) than in control cells (3%). However, comet tail intensity and/or tail moment was significantly higher in leukocytes treated with OTA alone and BEA ? OTA combinations than in control (P \ 0.05). Four percent of control cells had abnormalsized tails with respect to both comet parameters, while
in cells treated with toxins alone and their combinations, AST varied between 4 and 15% and between 12 and 19%, respectively. Significantly higher AST values were also seen in leukocytes exposed to 0.1 ? 1 lM and 0.1 ? 5 lM BEA ? OTA than in cells treated with 0.1 lM BEA alone (P \ 0.05). At the same time, only the concentration of 0.1 ? 5 lM BEA ? OTA led to a significantly higher tail intensity but not significantly higher tail moment than in cells treated with a 0.1 lM BEA alone (P \ 0.05).
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Table 3 Incidence of comets with abnormal size tails (AST) and comet parameters values corresponding to the 95th percentile of tail length, tail intensity, and tail moment evaluated in PK15 cells 95th percentile
AST (%) Tail length (lm) 1h 23.31
Tail intensity (%DNA)
Tail moment
24 h 31.44
1h 4.56
24 h 8.49
1h 0.65
24 h 1.49
Control (1.6% EtOH)
4
5
4
5
4
4
BEA (0.1 lM)
6
8a
7
8a
6
8a
BEA (0.5 lM)
8b
19*,b
9b
25*,b
10b
28*,b
12
,c
11
34*,c
,d
12
45*,d
,a
14*
33*,a
,a
42*,a
OTA (1 lM) OTA (5 lM) BEA ? OTA (0.1 ? 1 lM)
,c
13*
20*
,d
12
40*
12
,a
13*
27*
20*
42*
12
,a
29*
BEA ? OTA (0.1 ? 5 lM)
15*
39*
13*
37*
14*
BEA ? OTA (0.5 ? 1 lM)
15*
58*,b,c
13*
53*,b,c
14*
BEA ? OTA (0.5 ? 5 lM)
,b
23*
,b,d
,b
70*
22*
,b,d
65*
24*
58*,b,c ,b
71*,b,d
* Compared to control (P \ 0.05) a
BEA 0.1 lM versus BEA ? OTA (0.1 ? 1 lM; 0.1 ? 5 lM) (P \ 0.05)
b
BEA 0.5 lM versus BEA ? OTA (0.5 ? 1 lM; 0.5 ? 5 lM) (P \ 0.05)
c
OTA 1 lM when compared to BEA ? OTA (0.1 ? 1 lM; 0.5 ? 1 lM) (P \ 0.05)
d
OTA 5 lM when compared to BEA ? OTA (0.1 ? 5 lM; 0.5 ? 5 lM) (P \ 0.05)
Table 4 Incidence of comets with abnormal size tails (AST) and comet parameters values corresponding to the 95th percentile of tail length, tail intensity, and tail moment evaluated in human leukocytes 95th percentile
AST (%) Tail length (lm)
Control (1.6% EtOH) BEA (0.1 lM) BEA (0.5 lM)
Tail intensity (%DNA)
Tail moment
1h 17.98
24 h 17.94
1h 4.25
24 h 3.3
1h 0.54
24 h 0.42
3
3
4
4
4
5
3 4 c
5
a
8
b
4
a
5
12* ,c
a
5
11*
13*,b
, c
11*
16*,c
15*
19*
12*,a
13*
14*
3
12*
12*
13*
OTA (5 lM)
6
26*
14*
19*
BEA ? OTA (0.1 ? 1 lM)
7
19*,a
13*,a
15*,
8
,a
,a
BEA ? OTA (0.5 ? 1 lM) BEA ? OTA (0.5 ? 5 lM)
21* ,c
11* 6
15*
,b,c
26*
16*
,b
35*
17*
8a
,b
OTA (1 lM)
BEA ? OTA (0.1 ? 5 lM)
a
a
,a
15*
19*,a
,b,c
19*
27*,b,c
,b
17*
25*,b
19*
26* 25*
,a
* Compared to control (P \ 0.05) a
BEA 0.1 lM versus to BEA ? OTA (0.1 ? 1 lM; 0.1 ? 5 lM) (P \ 0.05)
b
BEA 0.5 lM versus BEA ? OTA (0.5 ? 1 lM; 0.5 ? 5 lM) (P \ 0.05)
c
OTA 1 lM versus to BEA ? OTA (0.1 ? 1 lM; 0.5 ? 1 lM) (P \ 0.05)
After 24 h tail length and incidence of comets with abnormal tail length increased when compared to shorter exposure, while tail intensity and tail moment values remain similar. Tail length was significantly greater in leukocytes exposed to 5 lM OTA and all toxin combinations than in control lymphocytes (P \ 0.05). Tail length was also significantly greater in cells treated with toxin combinations than in cells exposed to 1 lM OTA alone or
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0.1 and 0.5 lM BEA alone (P \ 0.05). A significantly higher incidence of AST was observed in cells treated with OTA alone at the concentrations of 1 lM (12%) and 5 lM (26%), as well as in cells exposed to BEA and OTA combinations (19–35%), when compared to control (3%) (P \ 0.05). OTA at the concentration of 1 and 5 lM and BEA ? OTA combinations significantly influenced comet tail intensity compared to control (P \ 0.05). Also,
Tail moment (mean + SE)
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0.7
*
0.6 0.5 0.4 0.3 0.2 0.1
0.0 OTA µM BEA µM
1 0.1
5 0.1
1 0.5
5 0.5
Fig. 3 DNA damage represented as tail moment in PK15 cells after 1 h of combined treatment with BEA and OTA. White bars represent the expected values and gray bars the measured values. *Represents significant synergistic effect (P \ 0.05). Dashed line represents tail moment of control PK15 cells
Fig. 4 DNA damage represented as tail moment in PK15 cells after 24 h of combined treatment with BEA and OTA. White bars represent the expected values and gray bars the measured values. *Represents significant synergistic effect (P \ 0.05). Dashed line represents tail moment of control PK15 cells
0.1 ? 1 lM, 0.1 ? 5 lM, and 0.5 ? 5 lM BEA ? OTA induced higher DNA damage than 0.1 and 0.5 lM BEA alone (P \ 0.05). Tail moment varied even more than tail intensity and tail length. BEA (0.5 lM) and OTA (1 and 5 lM) as well as all toxin combinations produced a significant increase in tail moment compared to control cells (P \ 0.05). BEA alone at either concentration had a significantly lower DNA damage than BEA and OTA combinations (P \ 0.05). The incidence of AST was similar for both tail intensity and tail moment, varying from 5 to 19% in cells exposed to single toxins and from 13 to 27% in leukocytes treated with toxin combinations. BEA ? OTA combinations showed additive effects on tail moment after 1- and after 24 h of treatment (Figs. 4, 5).
Discussion Since BEA and OTA are potentially genotoxic food contaminants, the aim of this study was to see whether they would produce DNA damage and how much damage would
Fig. 5 DNA damage represented as tail moment in human leukocytes cells after 1 h of combined treatment with BEA and OTA. White bars represent the expected values and gray bars the measured values. Dashed line represents tail moment of control leukocytes
they produce alone and in combination in vitro. To do that, we used the alkaline comet assay. Prior to these experiments, we assessed IC50 using MTT reduction assay (Fig. 1a, b). The results showed that OTA (IC50 = 15.8 ± 1.5 lM) was three times less toxic to PK15 cells than BEA (IC50 = 5.0 ± 0.6 lM). Toxin combinations produced additive or synergistic effects. Our earlier cytotoxicity study performed using Trypan blue exclusion assay and lactate dehydrogenase (LDH) activity demonstrated that a significant decrease in cell viability (BEA at 6 lM and OTA at 12 lM, 24 h) preceded an increase in LDH activity (after 48 h) and suggested that apoptosis is the prevalent type of cell death in PK15 cells. Also, combinations of BEA and OTA additively affected cell membrane integrity (Sˇegvic´ Klaric´ et al. 2007, 2008b). Previously, we also demonstrated that in subcytotoxic concentrations, both mycotoxins induced formation of micronuclei (MN), nuclear buds (NBs), and nucleoplasmic bridges (NPBs) in porcine kidney PK15 cells (Sˇegvic´ Klaric´ et al. 2008a). The micronucleus assay is a non-specific genotoxicity test, which detects fixed mutations and is a good biomarker of exposure to genotoxins. On the other hand, in vitro alkaline comet assay is a sensitive method for detecting reparable DNA damage such as single- and double-strand breaks or alkaline labile sites (Kassie et al. 2000). Taking into account these facts, the results of both tests will provide more accurate data on genotoxic action of these two mycotoxins. BEA (0.1 and 0.5 lM) and OTA (1 and 5 lM) alone and their combinations caused a significant DNA damage in PK15 cells, which was dose- and time dependent. Longer exposure produced more severe DNA damage considering all comet tail parameters measured. These results correspond to our previous findings of significant MN induction in PK15 cells after treatment with similar micromolar concentrations of BEA and OTA (Sˇegvic´ Klaric´ et al. 2008a). However, our previous study on PK15 cells showed that OTA increased caspase-3 activity after 24 h of treatment with 1.2 lM, while BEA applied at 0.6 lM significantly affected this
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enzyme after 48 h of exposure. Significant morphological apoptotic changes in hematoxylin and eosin-stained cells were observed after 48 h of treatment with these toxins (Sˇegvic´ Klaric´ et al. 2008b). Therefore, despite the fact that only comets with a defined head were scored the observed increase in DNA damage could be influenced by apoptotic changes in PK15 cells because the onset of apoptotic fragmentation might appear as comets (Roser et al. 2001). Apoptotic fragmentation could take a place particularly after combined toxin treatment, which is supported by earlier findings of synergistic effects on caspase-3 activity after simultaneous treatment with BEA and OTA (Sˇegvic´ Klaric´ et al. 2008b). Human leukocytes were less sensitive to genotoxic action of BEA and OTA than PK15 cells. Comparing 1- and 24-h exposure, comet parameters were similar or slightly increased. After short time exposure, there were no significant changes in tail length compared to control cells. However, tail intensity, tail moment, and/or incidence of AST were significantly higher in cells treated with OTA or BEA alone and with their combinations than in control cells. These differences could be explained by the theory of comet tail formation. Tail length is the length of relaxed DNA loops, which migrated from the core during electrophoresis, while tail intensity is the number of DNA breaks in the loop. As the level of damage increases, the intensity of DNA staining in the tail increases rather than tail length (Collins et al. 2008). After 24 h of exposure, tail length and incidence of AST increased, while tail intensity and tail moment remain similar to 1 h treatment. The differences in sensitivity of used in vitro systems might be explained by genome instability of PK15 cell line as well as apoptotic fragmentation of DNA. Opposite to cultured cell line, leukocytes are primary cells and therefore have much stable genome (Collins et al. 2008). Also, PK15 cells are kidney cells and might be more sensitive to nephrotoxic action of OTA. Since the alkaline comet assay can detect DNA strand breaks and alkali-labile sites (Singh 2000; Miyamae et al. 1998), BEA and OTA could induce DNA damage either directly or indirectly. To our knowledge, this is the first report on BEA genotoxicity determined by the alkaline comet assay. Direct DNA damage may be associated with the hexadepsipeptide structure of BEA. We can speculate that if groups of fenilalanine in the BEA structure were positively charged, they could interfere with negatively charged phosphate groups in the DNA strand, influencing DNA migration. On the other hand, it is known that BEA increases intracellular calcium levels, which might influence endonuclease activity leading to DNA strand breaks and also to apoptosis. The third possible scenario might also involve indirect DNA damage through oxidative stress. Previously, we demonstrated that exposure of PK15
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Fig. 6 DNA damage represented as tail moment in human leukocytes after 24 h of combined treatment with BEA and OTA. White bars represent the expected values and gray bars the measured values. Dashed line represents tail moment of control leukocytes
cells to BEA resulted in decreased glutathione levels and increased lipid peroxidation (Sˇegvic´ Klaric´ et al. 2007). Pro-oxidative action of BEA is also supported by findings of time- and concentration-dependent increase in ROS production and lipid peroxidation in Chinese hamster ovary-K1 cells (Ferrer et al. 2009). Opposite to that, Dornetshuber et al. (2009) showed that BEA did not evoked any detectable increase in ROS or changes in tail intensity in human epidermal carcinoma KB-3-1 and human promyelocytic leukemia HL-60 cell lines upon exposure to apoptotic concentrations of BEA for 1 h. Such discrepancy is probably influenced by differences between used in vitro systems. The mechanism of OTA genotoxic action is still under debate. Based on current literature, OTA genotoxicity may have a direct (DNA adduct formation) and indirect (oxidative DNA damage) mechanisms of action (PfohlLeszkowicz and Manderville 2007). However, some studies showed that OTA-mediated DNA adducts (analyzed by 32Ppostlabelling method) were controlled by biotransformation enzymes such as CYP450 1B1, 2C9, cyclooxygenase (COX), and lipooxygenase (LOX) (Petkova-Bocharova et al. 2003; Pfohl-Leszkowicz et al. 2002). Comet assay performed on mouse fibroblast cell line NIH/3T3 expressing CYP2C9 and CYP3A4 also showed that biotransformation of OTA increased its genotoxicity (Doorten et al. 2006). Results of our study support the findings of OTA genotoxic properties in rat kidneys (Zˇeljezˇic´ et al. 2006). An Fpg-modified comet assay showed a significantly higher DNA damage in rat kidney than did the standard alkaline comet test (Domijan et al. 2006). The authors concluded that oxidative stress is responsible for OTA genotoxic action but some other mechanisms such as production of OTA metabolites could also be involved in OTA genotoxicity. As tail moment is tail length 9 % of DNA in the tail, or simply the low DNA damage (Faust et al. 2004), we used it to compare the expected (calculated) DNA damage with tail
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moment values measured after exposure of PK15 cells and human leukocytes to BEA and OTA combinations (Figs. 3, 4, 5, 6). Toxin combinations showed additive and synergistic effects on PK15 cells and predominantly additive effects on human leukocytes. Additive effects of BEA and OTA were also seen earlier for MN formation in PK15 cells. In conclusion, this study brings additional evidence of BEA and OTA genotoxicity. Taking into account their additive and/or synergistic action, prolonged exposure to these toxins might induce DNA damage at relatively low doses and might lead to carcinogenesis. Acknowledgments We wish to thank Professor Josip Madic´, PhD and Snjezana Kovacˇ, MSc (Veterinary Faculty, University of Zagreb, Croatia) for providing the PK15 cells. Our thanks are also due to Dado Cˇakalo for language advice. This work was financially supported by the Ministry of Science, Education, and Sports of the Republic of Croatia (Grants No. 022-0222148-2137, 006-0061117-1242).
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