Butyrate reduces the frequency of micronuclei in human colon carcinoma cells in vitro

Toxicology in Vitro 23 (2009) 1028–1033

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Butyrate reduces the frequency of micronuclei in human colon carcinoma cells in vitro Galina Hovhannisyan a, Rouben Aroutiounian a, Michael Glei b,* a b

Department of Genetics and Cytology, Biological Faculty, State University, Alex Manoukian Street 1, Yerevan 375025, Armenia Institute for Nutrition, Department of Nutritional Toxicology, Biological-Pharmaceutical Faculty, Friedrich Schiller University Jena, Dornburger Str. 24, 07743 Jena, Germany

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Article history: Received 21 January 2009 Accepted 15 June 2009 Available online 17 June 2009 Keywords: Butyrate Ferric nitrilotriacetate (Fe-NTA) Hydrogen peroxide (H2O2) Human colon carcinoma cells Cytokinesis-block micronucleus (CBMN) test

a b s t r a c t Butyrate, formed by bacterial fermentation of plant foods, has been shown to protect human colon cells from selected genotoxic substances. The mechanism for this effect could be the enhancement of toxicological defence leading to an increased detoxification of genotoxic risk factors and thus to a reduction of DNA and chromosome damage. Previous protective properties of butyrate against DNA damage induction in colon cells were demonstrated using the comet assay. In the present study the effect of butyrate on chromosome damage induced by ferric nitrilotriacetate (Fe-NTA) and hydrogen peroxide (H2O2) (suggested to be putative risk factors of colorectal carcinogenesis) was investigated using the cytokinesisblock micronucleus (CBMN) test. It was possible to reveal that pre-treatment of HT29 colon carcinoma cells with butyrate (2 and 4 mM) for 15 min caused a reduction of micronuclei induced with H2O2 (75 lM; p < 0.01) and Fe-NTA (500 and 1000 lM; p < 0.05). The decrease in the level of Fe-NTA- and H2O2-induced micronuclei was also confirmed in most of the corresponding variants of 24 h pre-treatment of cells with butyrate. The results obtained demonstrate for the first time protective properties of butyrate against chromosome damage induced by H2O2 and Fe-NTA in human colon carcinoma cells. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction By-products of the bacterial fermentation of dietary fibre, the short-chain fatty acids (SCFA), play an important role in colon physiology and colonocyte metabolism (Cummings et al., 1996; Velazquez et al., 1997; Mortensen and Clausen, 1996). One of the major SCFA, butyrate, has been shown to protect colon cells from genotoxic damage by genotoxic/mutagenic substances, a property associated with lowering cancer risk (Boutron et al., 1991). Mechanisms of such effects are not yet completely understood, but it has been hypothesised that particularly an up regulation of enzymes (e.g. glutathione S-transferases) which detoxify the genotoxic compounds are involved (Ebert et al., 2001, 2003). The consumption of red meat (basic source of dietary iron) is considered as one of the risk factors of developing colorectal cancer (Wiseman, 2008). The greatest risk of iron-overload to the gut comes from dietary iron that remains unabsorbed and passes into the large intestine for elimination. This large fraction of dietary

Abbreviations: CBMN test, cytokinesis-block micronucleus test; Fe-NTA, ferric nitrilotriacetate; H2O2, hydrogen peroxide; MMC, mitomycin C; MN, micronuclei; NDI, nuclear division index; ROS, reactive oxygen species. * Corresponding author. Tel.: +49 3641 949670; fax: +49 3641 949672. E-mail address: [email protected] (M. Glei). URL: http://www.uni-jena.de/biologie/ieu/et (M. Glei). 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.06.011

iron may increase the production of hydrogen peroxide (H2O2) and hydroxyl radicals (Babbs, 1990). A high level of oxidative stress leads to mutations of the DNA sequence and DNA strand breakage which are among the main causes of cancer initiation and progression (Ames et al., 1995). Alternatively, iron may be involved in the conversion of procarcinogens to carcinogens within the lumen of the colon (Kelly, 2002). Ferric nitrilotriacetate (Fe-NTA) is a useful model to simulate the type of iron-overload, which may occur in the gut lumen (Glei et al., 2006c) after intake of Fe-supplements or bleedings. We recently demonstrated that Fe-NTA damaged DNA of human colon cells in different stages of malignant transformation (Glei et al., 2002; Knöbel et al., 2007), inhibited cell growth and induced formation of reactive oxygen species (ROS) (Knöbel et al., 2006). H2O2 is a suited model for ROS and widely used to study consequences of oxidative damage in colon cells. Measurements of H2O2-induced DNA damage in different colon cell lines (human and animal) demonstrated that the potential target cells of colon cancer induction are indeed susceptible to the oxidative activity of H2O2 (Pool-Zobel and Leucht, 1997; Liegibel et al., 2000; Schaeferhenrich et al., 2003; Oberreuther-Moschner et al., 2005; Glei et al., 2006a). In previous investigations the protective activity of butyrate against DNA damage in colon cells was demonstrated applying the comet assay. Using this technique it was found that pretreatment of freshly isolated rat colon epithelial cells with butyrate

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resulted in significant protection against oxidative DNA damage induced by H2O2 (Abrahamse et al., 1999). Protective properties of butyrate against DNA damage induced by H2O2 and bile acids in human normal colonocytes and colon tumour cells were also demonstrated (Rosignoli et al., 2001, 2008). Furthermore it was shown that dietary red meat increased in rats (in vivo) the level of DNA strand breaks in colonocytes and dietary fibre protects against this damage, probably through increased butyrate production (Toden et al., 2007). However, only a small amount of induced DNA damage detected by the comet assay eventually leads to fixed mutations. These chromosome and/or genome mutations can be quantified by determining micronuclei (MN) formation in interphase cells. MN may originate from acentric fragments or chromosomes that are unable to migrate following the mitotic spindle. They appear after division of the cell as small membrane-bound DNA fragments. The current methodology is based on the cytokinesis-block micronucleus (CBMN) test in which the divided cells are recognized by their binucleate appearance after cytokinesis-block with cytochalasin B (Fenech, 2000). Comparison of the comet assay and MN results allows estimating the amount of DNA breakage translated into chromosome and/or genome mutations. Since the CBMN method detects chromosomal aberrations resulting from non-repaired lesions and the comet assay detects DNA damage, which include repairable DNA lesions (Vrzoc and Petras, 1997), a combination of the comet assay and CBMN test was recommended for optimal genotoxicity testing (He et al., 2000). From the results of our previous experiments using the comet assay, it is evident that butyrate is capable of reducing the level of DNA damage in colon cells. These experiments also permitted to determine the range of genotoxic concentrations of Fe-NTA (Glei et al., 2002, 2006c; Knöbel et al., 2007) and H2O2 (Pool-Zobel and Leucht, 1997; Schaeferhenrich et al., 2003; Oberreuther-Moschner et al., 2005) and conditions in which butyrate manifests protective properties in colon cells of different origin (Abrahamse et al., 1999; Knoll et al., 2005). In the present work for the first time the protective activity of butyrate on H2O2- and Fe-NTA-induced chromosome damage in human colon tumour cells HT29 with the CBMN test was evaluated. Normal colonic cells are not suitable for MN test owing to the lack of convenient culture methods. Because of this, the HT29 colon carcinoma cell line was used as a colon tissue model with regular and well established cell cycle, since the MN can only be analyzed in cells which completed nuclear division in vitro. Results were discussed in comparison to previously obtained corresponding data on DNA damage measured with the comet assay.

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1 h) and in Dulbecco’s Modified Eagle Medium (DMEM, Gibco BRL, Eggenstein, Germany) for long treatment (24 h) of cells. 2.2. Cell line and culture conditions HT29 cells were isolated from a colon adenocarcinoma of a female Caucasian (Fogh and Trempe, 1975) and originated from an adenoma colon tissue. They were purchased from the American Tissue Culture Collection (Rockville, MD). The cells were grown in tissue culture flasks with DMEM supplemented with 10% foetal calf serum (FCS) and 1% penicillin/streptomycin at 37 °C in a (95%) humidified incubator (5% CO2). Under these conditions, doubling time for HT29 cells was about 24 h. For this study we used passages 30–50. A mycoplasma test (VenorÒGeM, Minerva Biolabs, Berlin, Germany) was performed at regular interval which showed the absence of mycoplasma contamination. 2.3. Cell treatment with butyrate, H2O2 and Fe-NTA Cells were treated with H2O2 and Fe-NTA at concentrations that induced a significant increase of DNA damage without affecting the viability and growth rates of the cells (Glei et al., 2002, 2006b; Knöbel et al., 2006). Prior to treatment with the genotoxic agents cells were pre-treated with medium or with butyrate at concentrations based on the proliferation inhibitory concentration ranges EC50 and EC25 for cell survival (Ebert et al., 2001), that in previous studies had been shown to effectively decrease the level of induced DNA damage in the comet assay (Abrahamse et al., 1999; Knoll et al., 2005). HT29 cells were grown in 6-well tissue culture plates (Corning, New York, USA) at a start concentration of 0.75  106 cells/ml (3.0 ml/well). After 24 h of incubation, the medium was removed from each well, and replenished with 3 ml of butyrate at concentrations of 2, 4, 10, 15 mM for 15 min or 24 h. After the end of the treatment, butyrate was removed, cells were washed with PBS and incubated with potentially physiologically relevant concentrations of H2O2 (37, 75 and 150 lM for incubation periods of 30 min and 1 h) or Fe-NTA (500 or 1000 lM for 15 min), followed by the measurement of cell viability and the evaluation of MN according to the protocol described below. Positive (MMC, 3 lg/ml) and negative (DMEM) controls were included in each experiment. Solvent control (NTA) at 500 and 1000 lM were included in experiments with Fe-NTA. The concentration of MMC was determined on the base of a positive control dose range-finding study (0.1–6 lg/ml, data not shown). The dose-dependent increase of level of MN was recorded and finally the dose, which induced a moderate level of MN in HT29 cells (about 20–30 micronuclei in 1000 binucleated cells) was selected.

2. Materials and methods 2.4. Analysis of cell viability 2.1. Chemicals H2O2. was obtained as aqueous solution from Merck, Darmstadt, Germany. Fe-NTA was prepared from ferric nitrate (40 mg) [Fe(NO3)3] (Sigma–Aldrich, Deisenhofen, Germany) dissolved in 10 ml double distilled water and the chelating agent disodium nitrilotriacetate (23.5 mg) (NTA) (Sigma–Aldrich, Deisenhofen, Germany) was added. The pH of the solution was adjusted to 7.4 by sodium hydrogen carbonate (NaHCO3). The final iron concentration of the stock solution was 10 mM. A sodium salt of butyrate was available from Boehringer Ingelheim Bioproducts (Heidelberg, Germany). Mitomycin C (MMC) was obtained from Sigma–Aldrich (Deisenhofen, Germany). All compounds were dissolved till final concentrations in phosphate-buffered saline solution (PBS) without Ca2+ and Mg2+ obtained from Life Technologies (Eggenstein/ Leopoldshafen, Germany) for short treatment (15 min, 0.5 and

The trypan blue exclusion test was used to determine membrane integrity as a reflection of cell viability. 2.5. Cytokinesis-block micronucleus test The CBMN test was done following the recommendations for performing the in vitro micronucleus assay with cell lines (Kirsch-Volders et al., 2003; Fenech, 2000) with some modifications for adaptation of the method to HT29 cells. Immediately after the treatment with butyrate and H2O2 or Fe-NTA, the medium was removed from each well and cytochalasin B (3 lg/ml) were added to the cells for 36 h incubation. Cells were then washed and detached by a trypsin-EDTA solution (Gibco BRL, Paisley, UK). After that, cells were collected with PBS and fixed twice with ethanol/ acetic acid (3:1) for 20 and 10 min, respectively. Cells were

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dropped on glass slides (two slides per concentration), air-dried and stained with Giemsa (Sigma–Aldrich) (4%) for 15 min.

37, 75 and 150 lM for 0.5 h. Noteworthy a 1 h treatment with H2O2 had no stronger effect. The frequency of MN increased in a dose-dependent mode.

2.6. Counting of micronuclei and statistical analysis 3.4. MN in HT29 cells treated with butyrate and H2O2 For MN identification, the criteria of Fenech (2000) were followed which are essentially based on the diameter of the MN and their staining properties. MN were scored in 1000 binucleated cells with two nuclei of approximately equal size per culture (2000 cells per test compound). The nuclear division index (NDI) was calculated by screening 500 cells per culture for the frequency of cells with one or more nuclei using the formula: NDI = [M1 + 2(M2) + 3(M3) + 4(M4)]/N, where M1–M4 is the number of cells with 1–4 nuclei and N is the total number of cells scored (Fenech, 2000). The average frequency of MN per 1000 binucleated (BN) cells and standard deviations (SD) from two independent experiments were evaluated. This accepted, but limited set of measurements can be the reason for relatively high SD, which was also found by e.g. Gebel et al. (1997). Differences between mean values were tested for significance using the Student’s t-test. A statistically significant difference was set at p < 0.05. 3. Results

Treatment with 75 lM H2O2 for 0.5 h (with moderate outcome of MN formation) was selected to estimate protective activity of butyrate. The damage level (18 MN/1000 BN cells) induced with H2O2 was statistically significant and decreased after 15 min pretreatment of the cells with butyrate. Almost all concentrations (2, 4, and 10 mM) used were effective (Table 2). The pre-treatment of HT29 cells with butyrate (4 mM) for 24 h also decreased the H2O2-induced MN from 17.5 ± 2.12 to 8.50 ± 3.54 per 1000 BN cells (p < 0.05) (data not shown). 3.5. MN in HT29 cells treated with Fe-NTA Treatment of HT29 cells with Fe-NTA alone at 500 and 1000 lM resulted in an increase of the MN level (Table 3). The effects were statistically significant for both concentrations compared with negative (DMEM) and solvent (NTA) controls. Also it was shown that NTA used as solvent control, at 1000 lM slightly but significantly increased the level of MN, too.

3.1. Viability of the cells 3.6. MN in HT 29 cells treated with butyrate and Fe-NTA The test substances used in this study had no influence on the viability of the HT29 cells (Tables 1–3). The trypan blue exclusion test showed that at most only 17% of the cells were damaged. Viability of HT29 cells was higher than 83% in samples treated with H2O2 and butyrate and higher than 86% in samples treated with Fe-NTA and butyrate. The cell viability in the negative, solvent and in positive controls was higher than 94%, 88% and 87%, respectively. In general, this means that the reported mutagenic activity of the test substances was found in the absence of cytotoxical effects.

Pre-treatment of cells with butyrate at 2 and 4 mM (15 min) resulted in a significant decrease of Fe-NTA-induced MN (Table 3). Furthermore, pre-treatment with butyrate (4 mM) for 24 h resulted in a decrease of the level of MN, induced by Fe-NTA (500 lM) from 23.0 ± 5.7 to 5.0 ± 1.4 per 1000 BN cells, (p < 0.05). Twenty-four hours pre-treatment with butyrate at 2 and 4 mM also significantly reduced the level of MN induced with 1000 lM Fe-NTA from 19.0 ± 1.4 to 3.5 ± 2.1 (p < 0.01) and 11.0 ± 1.4 per 1000 BN cells (p < 0.05), respectively (data not shown).

3.2. MN in HT29 cells treated with butyrate

3.7. Nuclear division index

The MN frequency of cells treated with butyrate alone in all experiments did not differ from negative controls (p > 0.05) (Tables 2 and 3), that means up to 15 mM butyrate were not genotoxic (clastogenic or aneugenic).

The cytotoxic effect of all substances investigated was determined also by calculating the NDI (Tables 1–3). The NDI values of all treatments were not significantly different from controls. This data is in accordance with the results of viability evaluation using the Trypan blue exclusion test presented above.

3.3. MN in HT29 cells treated with H2O2 4. Discussion Table 1 summarizes the genotoxic effects of different concentrations of H2O2 in HT29 cells. The average frequency of MN significantly increased after treatment with H2O2 at concentrations of

Finding an optimal balance/proportion between potential risk and protective dietary factors is essential for the gut health. There-

Table 1 Frequencies of micronuclei in HT29 cells treated with hydrogen peroxide (H2O2). Results are expressed as mean ± SD of the mean values from 2000 cells of two separate experiments. The significance was calculated by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001 significantly different from negative control). MNCB, the average frequency of micronuclei (MN) per 1000 binucleated cells; NDI, nuclear division index; MMC, mitomycin C. Treatment

MNCB (mean ± SD)

NDI (mean ± SD)

Viability (%) (mean ± SD)

H2O2 (lM), 0.5 h exposure 37 75 150

11.0 ± 2.83* 22.5 ± 0.71*** 25.5 ± 3.54*

1.53 ± 0.12 1.50 ± 0.11 1.47 ± 0.04

93.19 ± 0.28 93.35 ± 0.77 85.04 ± 0.21

H2O2 (lM), 1 h exposure 37 75 150

11.5 ± 3.55* 19.5 ± 0.71*** 22.5 ± 2.12**

1.52 ± 0.11 1.51 ± 0.06 1.43 ± 0.11

89.36 ± 0.15 84.19 ± 2.35 83.63 ± 2.15

Positive control (MMC, 3 lg/ml) Negative control (DMEM)

35.5 ± 3.54** 3.5 ± 0.71

1.34 ± 0.21 1.50 ± 0.11

87.32 ± 3.05 94.61 ± 0.98

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Table 2 Effect of pre-treatment with butyrate on micronuclei induced by hydrogen peroxide (H2O2) in HT29 cells. Results are expressed as mean ± SD of the mean values from 2000 cells of two separate experiments. The significance was calculated by Student’s t-test (**p < 0.01 significantly different from negative control, *p < 0.01 significantly different from samples treated with H2O2). MNCB, the average frequency of micronuclei (MN) per 1000 binucleated cells; NDI, nuclear division index; MMC, mitomycin C. Treatment

MNCB (mean ± SD)

NDI (mean ± SD)

Viability (%) (mean ± SD)

Butyrate (mM), 15 min treatment 2 4 10 15 H2O2 (75 lM), 0.5 h exposure

3.5 ± 0.71 4.5 ± 0.71 5.5 ± 2.12 4.5 ± 3.54 18 ± 0.00**

1.51 ± 0.03 1.41 ± 0.01 1.49 ± 0.10 1.50 ± 0.02 1.49 ± 0.13

89.81 ± 0.51 90.29 ± 1.28 90.57 ± 1.73 90.03 ± 0.88 93.28 ± 4.72

Butyrate (mM) + H2O2 (lM) 2 + 75 4 + 75 10 + 75 15 + 75 Positive control (MMC, 3 lg/ml) Negative control (DMEM)

5.5 ± 0.71* 9.0 ± 1.41* 9.5 ± 0.71* 10.5 ± 3.54 21.5 ± 2.12** 2.5 ± 0.71

1.50 ± 0.08 1.53 ± 0.07 1.52 ± 0.01 1.46 ± 0.21 1.39 ± 0.01 1.59 ± 0.03

88.42 ± 1.05 86.51 ± 6.07 89.10 ± 0.92 89.29 ± 4.89 88.80 ± 3.54 94.65 ± 0.75

Table 3 Effect of pre-treatment with butyrate on micronuclei induced by ferric nitrilotriacetate (Fe-NTA) in HT29 cells. Results are expressed as mean ± SD of the mean values from 2000 cells of two separate experiments. The significance was calculated by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001 significantly different from negative control,  p < 0.01 significantly different from solvent control,   p < 0.05 significantly different from samples treated with Fe-NTA). MNCB, the average frequency of micronuclei (MN) per 1000 binucleated cells; NDI, nuclear division index; MMC, mitomycin C. Treatment

MNCB (mean ± SD)

NDI (mean ± SD)

Viability (%) (mean ± SD)

Butyrate (mM), 15 min treatment 2 4

4.5 ± 0.71 2.5 ± 2.12

1.63 ± 0.07 1.92 ± 0.06

94.90 ± 0.24 87.80 ± 0.46

Fe-NTA (lM), 15 min exposure 500 1000

16.0 ± 1.41**,  21.5 ± 0.71***, 

1.67 ± 0.01 1.63 ± 0.07

88.29 ± 2.39 89.51 ± 2.31

Butyrate (mM) + Fe-NTA (lM) 2 + 500 2 + 1000 4 + 500 4 + 1000

8.0 ± 1.41   4.5 ± 2.12   12.5 ± 0.71   11.0 ± 4.24  

1.61 ± 0.03 1.61 ± 0.01 1.69 ± 0.01 1.64 ± 0.00

89.02 ± 1.41 88.12 ± 0.18 86.18 ± 1.47 88.09 ± 2.47

Solvent control (NTA) (lM) 500 1000 Positive control (MMC, 3 lg/ml) Negative control (DMEM)

6.0 ± 1.41 7.0 ± 1.41* 21.5 ± 0.71*** 3.5 ± 0.71

1.57 ± 0.07 1.65 ± 0.01 1.33 ± 0.14 1.71 ± 0.23

90.49 ± 0.87 88.69 ± 0.68 87.85 ±0.49 94.25 ± 2.64

fore it is necessary to reveal relevant associations. The main aim of this study was to test the hypothesis that butyrate, a well known fermentation product in the gut, can protect human colon cells from Fe-NTA- and H2O2-induced chromosome damage in vitro. Butyrate has been suggested to enhance toxicological defence in colon cells particularly by reducing the level of DNA damage measured with the comet assay. Comparing the comet assay results with other parameters of DNA damage (e.g., MN, mutations, chromosome aberrations or cell killing) is necessary to interpret their biological relevance (Olive and Banáth, 2006). MN test is widely used for evaluation of genotoxicity of various substances including food ingredients in vitro and in vivo (Loprieno et al., 1991; Tucker et al., 1993; Fenech and Rinaldi, 1995; Fenech, 2002, 1998). Our experiments were designed to evaluate if physiologically relevant concentrations of butyrate (2–15 mM) have the potential to preclude or diminish Fe-NTA- and H2O2-induced chromosome damage, considering former results for the same substances using the comet assay. Butyrate can be found in the human gut after ingestion of dietary fibre at concentrations of about 10–20 mM (Schröder and Stein, 1997; Alles et al., 1999; Jenkins et al., 1999; Topping and Clifton, 2001). Comparable, but also lower concentrations inhibit cell proliferation and induce apoptosis and differentiation in transformed cell lines (Barnard and Warwick, 1993; Scheppach, 1994; Hague and Paraskeva, 1995) as well as induce glutathione S-transferases in human colon cells (Ebert et al., 2001).

We found that butyrate alone has no genotoxic potential in the CBMN test at concentrations of up to 15 mM. We also confirmed the DNA damaging effects of H2O2 and Fe-NTA in HT29 cells. Furthermore, our results demonstrate clearly the protective effects of butyrate against the genotoxicity of H2O2 and Fe-NTA in HT29 cells in CBMN test. In comparison to the negative control (DMEM), the solvent control NTA (1000 lM) also induced little but significantly higher formation of MN. Earlier it was shown that the chelating agent NTA is able to increase the level of ROS formation (Knöbel et al., 2006). The effect observed here might be explained by the damaging action of ROS on chromosomes. Butyrate alone was not genotoxic towards HT29 cells according to the data obtained both by CBMN test in the present study and the comet assay (Knoll et al., 2005). Comparison of the data on H2O2- and Fe-NTA-induced DNA damage in the comet assay (unpublished data, Glei, M., 2008) and chromosomal damage in the CBMN test in HT29 cells revealed agreement between results obtained for equal concentrations and time of treatment of both substances investigated. Our results obtained by CBMN method on protective activity of butyrate are in accordance with data of former comet assay investigations (Rosignoli et al., 2001), when 15 min pre-treatment with butyrate also significantly reduced H2O2-induced DNA damage in HT29 cells. The protective action of butyrate against H2O2 in HT29 cells in case of 24 h pre-treatment was shown by using CBMN test and was not revealed in

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the comet assay (unpublished data, Glei, M., 2008). The mentioned example of discrepancy between results of the two approaches can be explained by having different mechanisms of formation. Here it is reasonable to note that the protective effect of butyrate after 24 h pre-treatment was shown in CBMN test also against Fe-NTA treatment. Butyrate prevented the genotoxic effects of H2O2 also in rat colon cells in vitro (Abrahamse et al., 1999) and in vivo (Toden et al., 2007) and in human colonocytes (Rosignoli et al., 2001, 2008) according to data of the comet assay. An increase of resistance of HT29 cells pre-treated with butyrate against genotoxicity of 4-hydroxy-2-nonenal, a product of endogenous lipid peroxidation, and a genotoxic risk factor for carcinogenesis, was also described (Knoll et al., 2005). To our knowledge so far nothing is known about the effect of butyrate on Fe-NTA induced DNA damage. Data on agreement and discrepancy of comet and MN assay results in case of their concurrent application are presented in the literature both for substances with genotoxic (Andrighetti-Fröhner et al., 2006; Kalantzi et al., 2004; Maffei et al., 2005; Tafazoli and Kirsch-Volders, 1996) and antigenotoxic (Devipriya et al., 2008; Pasquini et al., 2002) activity. One example of discrepancy between results of the comet and CBMN assays is similar to our results on protective activity of butyrate in case of 24 h pre-treatment against H2O2. Antigenotoxic effect of some of aqueous extracts of Agaricus blazei Murill against mutagenicity induced by methyl methanesulfonate in V79 cells was shown using the MN test and not revealed in the comet assay (Martins de Oliveira et al., 2002; Guterrez et al., 2004). Whilst our results on the genotoxicity of H2O2 and Fe-NTA obtained by application of comet and MN tests are positively correlated, the data on antigenotoxic activity not always coincided, because of the complexity of mutagenic and antimutagenic mechanisms on DNA and chromosome level. Further investigations are needed to elucidate the effects observed. It seems likely that butyrate may act by many different mechanisms. Butyrate is known as a compound able to induce structural modifications in the chromatin and arrest of cell proliferation through hyperacetylation of nucleosomal core histones (Kruh, 1982). The mechanism of butyrate activity was investigated particularly in colon cells. Butyrate has been shown to increase glutathione S-transferases (GSTs) in primary, premalignant LT97 and tumour HT29 colon cells, which protect from products of oxidative stress and may contribute to the detoxification of dietary carcinogens (Ebert et al., 2003; Pool-Zobel et al., 2005a,b). Comparison of butyrate-induced inhibition of growth of cells in different stages of malignant transformation demonstrated its higher activity in LT97 than in HT29 cells. These findings indicate that butyrate protects by reducing survival of colon cells in an early transformation stage (Kautenburger et al., 2005). However, butyrate may not only suppress proliferation in transformed cells, but also directly enhance cell proliferation in normal cells (Lupton and Kurtz, 1993). SCFA can modulate the level of apoptosis in the gastrointestinal tract in mouse (Augenlicht et al., 1999). It was also shown that apoptosis may be increased in transformed cells, but can be inhibited in normal cells when butyrate is present (Hague et al., 1995). Theoretically each of the mentioned mechanisms of butyrate could be involved in formation of effects observed in the present study. We did not find the delay of cell proliferation in cells treated with butyrate on the base of NDI estimation. We can propose that the conformational changes in the chromatin could increase the access for repair enzymes. However, a few examples of enhancement of the mutagenic response to daunorubicin and X-rays in cells pre-treated with butyrate (Pani et al., 1984; Sankaranarayanan et al., 1985, 1990) allow assuming that structural modifications in the chromatin also could be responsible for an increased accessibility of DNA to genotoxic agents. The mechanism related

with detoxification of mutagens as a result of an increased glutathione S-transferase activity (Pool-Zobel et al., 2005a,b) can also contribute to the effect observed. The results obtained with CBMN test confirm our previous data about ability of butyrate to protect cells particularly against oxidative damage and this finding is in agreement with the assumption of Rosignoli et al. (2001) that butyrate could modify DNA repair and levels of antioxidant systems. In summary the present work has demonstrated for the first time results of CBMN test in HT29 cells. The results have clearly shown that butyrate can exert in vitro protective effects against chromosomal damage induced by Fe-NTA and H2O2. These findings confirm the beneficial role of butyrate as a protective agent for colon cells against genotoxicity of nutritional compounds. Overall, these results demonstrate the suitability of HT29 cells for detecting genotoxic effects of food related compounds in the in vitro MN test. Conflict of interest statement There is no conflict of interest involved in this study. Acknowledgements This research was carried out with the financial support by BMBF-0313829A. Galina Hovhannisyan was supported by a visiting grant of DAAD. References Abrahamse, S.L., Pool-Zobel, B.L., Rechkemmer, G., 1999. Potential of short chain fatty acids to modulate the induction of DNA damage and changes in the intracellular calcium concentration by oxidative stress in isolated rat distal colon cells. Carcinogenesis 20, 629–634. Alles, M.S., Hartemink, R., Meyboom, S., Harryvan, J.L., Van Laere, K.M.J., Nagengast, F.M., Haytvast, J.G.A.J., 1999. Effect of transgalactooligosaccharides on the composition of the human intestinal microflora and putative risk markers for colon cancer. American Journal of Clinical Nutrition 69, 980–991. Ames, B.X., Gold, X.S., Willett, W.C., 1995. The causes and prevention of cancer. Proceedings of the National Academy of Sciences of the United States of America 92, 5258–5265. Andrighetti-Fröhner, C.R., Kratz, J.M., Antonio, R.V., Creczynski-Pasa, T.B., Barardi, C.R.M., Simões, C.M.O., 2006. In vitro testing for genotoxicity of violacein assessed by comet and micronucleus assays. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 603, 97–103. Augenlicht, L.H., Anthony, G.M., Church, T.L., Edelmann, W., Kucherlapati, R., Yang, K., Lipkin, M., Heerdt, B.G., 1999. Short-chain fatty acid metabolism, apoptosis, and Apc-initiated tumorigenesis in the mouse gastrointestinal mucosa. Cancer Research 59, 6005–6009. Babbs, C., 1990. Free radicals and the etiology of colon cancer. Free Radical Biology and Medicine 8, 191–200. Barnard, J.I., Warwick, G., 1993. Butyrate rapidly induces growth inhibition and differentiation in HT-29 cell. Cell Growth Differ 4, 495–501. Boutron, M.C., Wilpart, M., Faivre, J., 1991. Diet and colorectal cancer. European Journal of Cancer Prevention: The Official Journal of the European Cancer Prevention Organisation (ECP) 1, 13–20. Cummings, J.H., Beatty, E.R., Kingman, S.M., Bingham, S.A., Englyst, H.N., 1996. Digestion and physiological properties of resistant starch in the human large bowel. British Journal of Nutrition 75, 733–747. Devipriya, N., Sudheer, A.R., Menon, V.P., 2008. Caffeic acid protects human peripheral blood lymphocytes against gamma radiation-induced cellular damage. Journal of Biochemical and Molecular Toxicology 22, 175–186. Ebert, M.N., Beyer-Sehlmeyer, G., Liegibel, U.M., Kautenburger, T., Becker, T.W., Pool-Zobel, B.L., 2001. Butyrate induces glutathione S-transferase in human colon cells and protects from genetic damage by 4-hydroxy-2-nonenal. Nutrition and Cancer 41, 156–164. Ebert, M.N., Klinder, A., Peters, W.H., Schäferhenrich, A., Sendt, W., Scheele, J., PoolZobel, B.L., 2003. Expression of glutathione S-transferases (GSTs) in human colon cells and inducibility of GSTM2 by butyrate. Carcinogenesis 24, 1637– 1644. Fenech, M., 1998. Chromosomal damage rate, aging, and diet. Annals of the New York Academy of Sciences 854, 23–36. Fenech, M., 2000. The in vitro micronucleus technique. Mutation Research 455, 81– 95. Fenech, M., 2002. Micronutrients and genomic stability: a new paradigm for recommended dietary allowances (RDAs). Food and Chemical Toxicology 40, 1113–1117.

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