Micronucleus formation and induction of apoptosis by different isothiocyanates and a mixture of isothiocyanates in human lymphocyte cultures

Mutation Research 582 (2005) 1–10

Micronucleus formation and induction of apoptosis by different isothiocyanates and a mixture of isothiocyanates in human lymphocyte cultures Carmela Fimognaria,∗ , Fausto Bertia , Renato Iorib , Giorgio Cantelli-Fortia , Patrizia Hreliaa b

a Dipartimento di Farmacologia, Universit` a di Bologna, Via Irnerio 48, 40126 Bologna, Italy Istituto Sperimentale per le Colture Industriali, MiPAF, Via di Corticella 133, 40129 Bologna, Italy

Received 4 March 2004; received in revised form 14 October 2004; accepted 12 November 2004 Available online 18 January 2005

Abstract Isothiocyanates (ITCs) are the main sulfur-containing metabolites found in cruciferous vegetables. There is evidence that some ITCs may act as chemopreventive agents against different tumor types and induce apoptosis and modulate cell-cycle progression of highly proliferative cancer cells. However, there are also studies reporting genotoxic or co-carcinogenic effects for some ITCs, such as benzyl ITC and phenyl ITC. Since selectivity for transformed cells and absence of genotoxicity for healthy cells are important pre-requisites for new chemopreventive agents, we investigated micronucleus formation and induction of apoptosis by 4-(methylthio)butylisothiocyanate (MTBITC), sulforaphane and a mixture of ITCs in human T-lymphocyte cultures. We demonstrate that MTBITC, sulforaphane and the mixture of ITCs did not induce micronuclei. Moreover, sulforaphane induced a dose-dependent increase in the number of apoptotic cells, which was significant at the highest concentration tested (30 ␮M) (41% versus 18% in the untreated samples, P < 0.05). The mixture of ITCs presented a trend similar to that found for sulforaphane. In fact, the mixture of ITCs was able to induce a dose-dependent increase in the percentage of apoptotic cells, which reached a maximum value at the concentration of 13 ␮g/ml (46% versus 19% in control samples, P < 0.05). Induction of apoptosis was not observed in cultures treated with MTBITC. Our results suggest that different ITCs can have different effects. Moreover, although the mixture of glucosinolates (GLs) used in the present study does not reflect the exact composition of broccoli, our findings demonstrate that the quantitative effects of a single, specific ITC can be significantly different from those of an ITC mixture, where other ITCs of the mixture contribute to the outcome observed. © 2004 Elsevier B.V. All rights reserved. Keywords: Isothiocyanates; Genotoxicity; Micronucleus formation; Apoptosis; Human lymphocytes

∗

Corresponding author. Tel.: +39 051 209 56 36; fax: +39 051 209 56 24. E-mail address: [email protected] (C. Fimognari).

1383-5718/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2004.11.019

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1. Introduction Glucosinolates (GLs) are sulfur-containing glycosides found in various quantities and ratios in seeds, root, stem and leaves of cruciferous vegetables, such as watercress, Brussels sprouts, broccoli and cabbage [1]. GLs constitute a rather homogenous class of molecules with a common functional group (␤-d-thioglucoside) and a variable side-chain of aliphatic, aromatic or heteroaromatic residues. In intact cells, GLs are maintained separate from the endogenous enzyme myrosinase (Myr), a ␤-thioglucoside glucohydrolase (EC 3.2.3.1) that catalyzes their hydrolysis. Breakdown products are formed upon exposure of GLs to myrosinase during food preparation, cooking and chewing, and include isothiocyanates (ITCs), thiocyanates and nitriles depending on the substrate and the reaction conditions used [2]. Recently, most attention has been focused on the cancer preventive potential of ITCs. Several lines of evidence show that some ITCs may act as chemopreventive agents against different tumor types, such as colorectal [3] and lung cancers [4,5]; this is probably due to the inhibition of phase 1 enzymes (mainly cytochrome P-450) or induction of phase 2 enzymes [glutathione S-transferase (GST)] that cause activation and detoxification of carcinogens, respectively [6]. Moreover, it was shown that ITCs induce apoptosis and modulate cell-cycle progression of actively proliferating cancer cells [7–9]. However, there are also studies reporting genotoxic [10–14] or co-carcinogenic effects [15] for some ITCs, such as phenylethyl isothiocyanate (PEITC), benzyl isothiocyanate, allyl isothiocyanate and methyl isothiocyanate. Our previous studies already demonstrated interesting biological activities for sulforaphane and 4(methylthio)butylisothiocyanate (MTBITC), two ITCs derived from purified GLs, glucoraphanin (GRA) and glucoerucin (GER), respectively. We found that both these compounds caused apoptosis in Jurkat leukemia T-cells [8,16]. However, since selectivity for transformed cells and absence of genotoxicity for healthy cells are important pre-requisites for new chemopreventive agents, in the present study, we investigated the induction of apoptosis and the formation of micronuclei by MTBITC and sulforaphane in phytohaemoagglutinin(PHA)-stimulated human Tlymphocytes, which represent the healthy counterpart of Jurkat T-cells. Moreover, to better reproduce the cir-

cumstances of dietary contact with ITCs following consumption of brassicas, we decided to study the effects of the mixture of GER or GRA and Myr to produce MTBITC and sulforaphane, respectively. However, dietary exposure to GLs actually occurs by eating vegetables containing a mixture of GLs. It is unknown whether the effects of a GL within such a mixture are similar to or different from those exerted when the same GL is administered as a single agent. We, therefore, analyzed also a mixture of ITCs obtained by Myr-mediated hydrolysis of a mixture of GLs isolated from ripe seeds of broccoli. We tested genotoxicity, in terms of micronucleus (MN) formation, induced by MTBITC, sulforaphane and the mixture of ITCs on human lymphocytes. DNA or chromosome damage is only one of several critical events that happen following exposure to xenobiotic agents [17]. The probability of a cell surviving exposure to a DNA-damaging agent is dependent on the propensity of that cell to undergo apoptosis. In order to obtain an accurate description of the mechanism of action and measurement of cellular sensitivity to the ITCs, we also evaluated the induction of apoptosis on the same preparations used for the MN analysis.

2. Materials and methods 2.1. Chemicals GER (Fig. 1) was isolated to a high degree of purity from the ripe seeds of rocket (Eruca sativa Miller) [18] according to a well-defined protocol [19]. The purity of GER was assessed by HPLC analysis of the desulfo-derivative according to the ISO 9167-1 method [20], and the compound was fully characterized by 1 H and 13 C NMR and mass spectrometry. GRA (Fig. 1) was obtained by a recently developed procedure [18], whereby a quantitative yield is obtained starting from GER. The transformation of GER into GRA is based on the oxidation reaction of sulfides into their corresponding sulfoxides. The semi-synthetic GRA was purified according to the method reported by Visentin et al. [19]. Purity was assessed by HPLC analysis of the desulfo-derivative according to the ISO 9167-1 method [20]. The mixture used in the present study was isolated from ripe seeds of broccoli supplied by SUBA and UNICO (Longiano, Italy), according to the method proposed by Thies [21] with some important modifi-

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Fig. 1. Hydrolysis of glucoerucin (GER) and glucoraphanin (GRA) by myrosinase (Myr).

cations reported by Barillari et al. [22]. HPLC analyses of desulfo derivatives according to the ISO 9167-1 method [20] were performed to measure the GL content of the mixture. Myr (E.C.3.2.3.1) used in the present study was isolated from ripe seeds of white mustards (Sinapis alba L.), as reported by Pessina et al. [23]. The Myr stock solution had a specific activity of ca. 60 units/mg of soluble protein. One Myr unit is defined as the amount of enzyme able to hydrolyze 1 ␮mol of sinigrin/min at pH 6.5 and temperature 37 ◦ C. The Myr solution was stored at 4 ◦ C in sterile distilled water until use. MTBITC (Fig. 1), sulforaphane (Fig. 1) and the mixture of ITCs were generated in situ by Myrcatalyzed hydrolysis of GER, GRA and a mixture of GLs, respectively. In our experimental conditions of cell culture (pH 7.4 at 37 ◦ C), ITCs are the only enzymatic breakdown products. The formation of ITCs was, thus, quantitative, as confirmed by GC–MS techniques [24]. 2.2. Isolation and cultivation of human lymphocytes Six healthy, non-smoking males were recruited from AVIS (Italian Association of Voluntary Blood Donors); donors provided written, informed consent at the time

of donation for the use of their blood sample in this study. In each experiment, only one compound was tested. We performed two independent experiments (using two different donors, respectively) for each compound. For each subject, treatments were done in duplicate and separate cultures were set up for each treatment. Lymphocytes were separated by density gradient (Histopaque, Sigma, St. Louis, MO, USA), and after two washes in phosphate-buffered saline (PBS), cultured at a concentration of 2 × 106 /5 ml in Roswell Park Memorial Institute (RPMI) 1640 medium containing 15% fetal calf serum (FCS, Sigma), 1% PHA (Sigma) and 1% penicillin–streptomycin solution (Sigma), and incubated at 37 ◦ C in a wet atmosphere for 72 h. 2.3. Cell treatment GER, GRA and the mixture of GLs were dissolved in 0.9% NaCl. Myr (0.23 units) was directly added to 5 ml of the complete cell culture medium containing increasing concentrations of GLs. In particular, the following ranges of concentration were used: 0.1–10.0 ␮M for GER, 0.3–30.0 ␮M for GRA and 0.4–39.0 ␮g/ml for the mixture of GLs. The concentrations of the mixture were selected to provide levels of GRA comparable with those used for GRA in these

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experiments and to better compare the effects of the mixture with those of single GLs. 2.4. MN assay The MN test performed in this study included the use of cytochalasin-B (Sigma), which was added after 44 h at a final concentration of 6 ␮g/ml [25]. At the end of the 72-h incubation period, the lymphocytes were fixed with ice-cold methanol/acetic acid (1:1). The fixed cells were put directly on slides using a cytospin centrifuge and were stained with May Gr¨unwald-Giemsa. All slides were coded prior to scoring. The criteria for scoring MN were as described by Fenech [26]. At least 2000 binucleated lymphocytes were examined per concentration (two cultures per concentration) for the presence of one, two or more MN. As described by Kirsch-Volders et al. [27], compounds used as positive controls included a clastogen, ethylmethanesulfonate (EMS, Sigma), at a final concentration of 120 ␮g/ml, and an aneugen, colchicine (COL, Sigma) at a final concentration of 0.02 ␮g/ml.

boundaries as well as cells exhibiting nuclear fragmentation into smaller bodies within an intact cytoplasm/cytoplasmic membrane were classified as apoptotic. Apoptotic cells were easily distinguished from necrotic cells, which exhibit a pale cytoplasm or a loss of cytoplasm, numerous vacuoles, and a damaged/irregular nuclear membrane with a partially intact nuclear structure [29]. Five hundred cells were counted for each sample. 2.7. Statistical analysis All results are expressed as the mean ± S.D. Data were statistically analyzed with the F-test for analysis of variance (ANOVA) and the significance of differences between the negative control and a series of treated groups was determined with Dunnett’s t-test. Statistical analysis was performed using GraphPad InStat Version 3.00 for Windows 95, GraphPad Software, San Diego, CA, USA. P < 0.05 was considered significant.

2.5. Replicative Index

3. Results

Replicative Index (RI), a measure of cell division kinetics, was calculated by scoring at least 500 cells per sample for the presence of one, two, three or more nuclei [28]. Nuclear division was not affected by the addition of cytochalasin B, but cell division was arrested. Cells that underwent one division have two nuclei and cells that underwent two divisions have three or four nuclei. Cells that responded to PHA stimulation but did not complete one division had only one nucleus. Cells that did not undergo mitogen stimulation, as judged by their size and the density of DNA-positive material, were not included in the count. The RI was calculated as follows: RI = (M1 + 2M2 + 3M3 + 4M4 )/N, where M1 –M4 indicate the number of cells with one to four nuclei, and N indicates the total number of cells scored (viable and non-viable).

3.1. HPLC analysis of the GL mixture

2.6. Evaluation of apoptosis The analysis of apoptosis was performed on the same slides prepared for the analysis of MN, as suggested by Fenech et al. [29]. Cells with chromatin condensation and intact cytoplasmic and nuclear

HPLC analyses (Fig. 2) showed that GRA was the predominant GL in the mixture, representing about 55% of the total GLs. The percentages of glucoiberin and GER were 35 and 7%, respectively. The total amount of the other four minor GLs was only 3%. 3.2. Effect of ITCs and GLs on lymphocyte RI Isolated lymphocytes were treated with different concentrations of MTBITC, sulforaphane or a mixture of ITCs in presence and absence of Myr. The RI of MTBITC-treated human lymphocyte cultures showed a gradual dose-dependent decrease (Fig. 3A). In fact, no decrease in the RI was observed at the concentrations 0.1 and 1.0 ␮M of MTBITC. At 3 and 10 ␮M, MTBITC caused a decrease in the RI by about 32 and 62%, respectively. Since the concentration of 10 ␮M exhibited more than 60% cytotoxicity, the analysis of MN formation induced by MTBITC was conducted at concentrations of up to 3 ␮M [27].

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Fig. 2. Glucosinolates present in the mixture as revealed by HPLC analysis.

Fig. 3. Replicative Index (RI) of human lymphocytes after treatment with 4-(methylthio)butylisothiocyanate (MTBITC) (A), sulforaphane (B) or a mixture of ITCs (C), generated in situ by myrosinase-catalyzed hydrolysis of glucoerucin (GER), glucoraphanin (GRA) and a mixture of glucosinolates (GLs), respectively. Each data point represents the means ± S.D. (deviation bars, except when smaller than the symbol size) of two independent experiments.

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The RI of sulforaphane-treated human lymphocyte cultures showed a similar profile (Fig. 3B): a gradual dose-dependent decrease, with the highest effect recorded at 30 ␮M, where sulforaphane reduced the RI by more than 60%. The analysis of MN formation upon exposure to sulforaphane was, therefore, conducted at concentrations of up to 10 ␮M. Treatment of human lymphocyte cultures with the mixture of ITCs caused a decrease in the RI (Fig. 3C), which was particularly marked at the concentration of 39 ␮g/ml (reduction by 70%). We, therefore, analyzed the MN induction by the mixture of ITCs in the concentration range 0.4–13 ␮g/ml. Treatment of lymphocyte cultures with GER or GRA or the mixture of GLs without Myr did not affect the RI (data not shown). Similar results were obtained in cultures treated with Myr alone (data not shown). 3.3. Effect of ITCs and GLs on MN frequency Fig. 4 presents data concerning the induction of MN in cytokinesis-blocked human lymphocytes by MTBITC, sulforaphane and the mixture of ITCs, respectively. Fig. 4A shows MN frequency in MTBITC-treated lymphocytes. No increase was observed up to the highest concentration tested. No MN induction was found in lymphocytes treated with 3 ␮M GER in absence of Myr nor with Myr alone, where the frequency of MN was comparable to that in the control. Treatment with EMS and COL significantly (P < 0.01) increased the MN frequency compared with the control. In Fig. 4B, data from sulforaphane-treated cultures are shown. Sulforaphane did not induce MN under the conditions used in our experiments, where only a slight, not significant, increase in MN frequency compared with the control was found at 1.0 ␮M sulforaphane. Similarly, no effects on MN frequency were recorded in cultures treated with 10 ␮M GRA without Myr. Treatment of lymphocyte cultures with the mixture of ITCs in the presence or absence of Myr did not induce any significant increase in the MN frequency in the range of concentrations tested (0.4–13.0 ␮g/ml) (Fig. 4C). It is important to note that, although the MN frequencies in the control lymphocyte cultures were lower than the range of the MN frequencies for healthy adults

Fig. 4. Effects of 4-(methylthio)butylisothiocyanate (MTBITC) (A), sulforaphane (B) and the mixture of ITCs (C) (generated in situ by myrosinase (Myr)-catalyzed hydrolysis of glucoerucin (GER), glucoraphanin (GRA) and the mixture of glucosinolates (GLs), respectively) on MN frequency. Results for MN frequency are reported as MN/1000 binucleated cells. The data presented are averaged from four measures with error bars denoting S.D. of the mean.

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identified by the HUMN Interlaboratory Comparison [30], similar MN frequencies were previously reported [28,31,32]. The inter-experimental variation was not significant. 3.4. Effect of ITCs and GLs on apoptosis In Fig. 5A, induction of apoptosis by MTBITC on healthy, PHA-stimulated human lymphocytes was presented. No significant increase in the percentage of apoptotic cells was recorded in the range of concentration tested (1–10 ␮M). Interestingly, a significant induction of apoptosis was observed in lymphocyte cultures treated with sulforaphane (Fig. 5B). Sulforaphane was able to induce a dose-dependent increase in the number of apoptotic cells, which was significant at the highest concentration tested (30 ␮M) (41% versus 18% in the untreated samples, P < 0.05). The mixture of ITCs presented a similar trend to that found for sulforaphane. In fact, the mixture was able to induce a dose-dependent increase in the percentage of apoptotic cells, which reached a maximum value at the concentration of 13 ␮g/ml (46% versus 19% in control samples, P < 0.05) (Fig. 5C). No apoptosis induction was observed for GRA or GER or the mixture of GLs without Myr, and for Myr alone (data not shown).

4. Discussion

Fig. 5. Effects of 4-(methylthio)butylisothiocyanate (MTBITC) (A), sulforaphane (B) and the mixture of ITCs (C) (generated in situ by myrosinase-catalyzed hydrolysis of glucoerucin, glucoraphanin and the mixture of glucosinolates, respectively) on apoptosis induction in human lymphocyte cultures. Results are expressed as percentage of total cell counts. The data presented are averaged from four measures with error bars denoting S.D. of the mean.

Numerous reports indicate that cruciferous vegetables and their constituents, in particular ITCs, protect experimental animals against chemically induced cancer, and induce apoptosis and modulate cell-cycle progression of actively proliferating cancer cells [4–9]. On the other hand, some authors reported that juices of cruciferous vegetables contain compounds that are direct-acting genotoxins in bacteria and mammalian cells [33]. It is likely that these effects are due to the presence of ITCs. Musk and Johnson [34] and Musk et al. [35] have repeatedly reported that ITCs, such as benzyl ITC, PEITC and phenyl ITC, induce genotoxic effects in rodent cell lines. Due to the causal relationship between genetic damage in somatic cells and the initiation of cancer, these findings suggest that some ITCs might be carcinogenic by themselves. Indeed, it

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has been found that allyl ITC causes bladder tumors in rats [36]. To our knowledge, no data are available for one of the most promising chemopreventive ITCs, sulforaphane, and for the more recently studied MTBITC. Moreover, no studies have been performed on ITC mixtures. It is unknown whether the effects of an ITC within a mixture is similar to or different from those observed when the same ITC is administered as a single agent. We, therefore, decided to investigate the formation of MN by MTBITC, sulforaphane and a mixture of ITCs in human lymphocytes. We demonstrated that MTBITC, sulforaphane and the mixture of ITCs do not induce MN up to the highest concentrations tested. However, the probability of a cell surviving after exposure to a DNA-damaging agent is dependent on the propensity of that cell to undergo apoptosis. We, therefore, investigated the ability of MTBITC, sulforaphane and the mixture of ITCs to induce apoptosis. In this case, we recorded a different behavior. In fact, MTBITC was not able to induce apoptosis, whereas a significant increase in the apoptotic cell fraction was recorded for both sulforaphane and the mixture of ITCs. Although the effects of the mixture of ITCs were qualitatively similar to those exerted by sulforaphane, quantitative differences were observed. In particular, it is important to underline that the mixture reached the highest activity at a concentration of 13 ␮g/ml, corresponding to 15 ␮M of sulforaphane (by far the most prominent component in the mixture, accounting for about 55% of its total ITC content). The fraction of apoptotic cells in cultures treated with 15 ␮M sulforaphane (calculated by interpolation from doseresponse curves) was significantly smaller (P < 0.05, Fisher’s exact test) than that recorded for the ITC mixture (i.e. 13 ␮g/ml) containing the same amount of sulforaphane. Although the mixture of GLs used in the present study does not reflect the exact composition of broccoli, our findings demonstrate that the quantitative effects of a single, specific ITC can be significantly different from those of an ITC mixture at realistic doses, where other ITCs in the mixture contribute to the effects observed. Apoptosis is activated by many oxidative agents [37], and Payen et al. [38] indicated a sulforaphanemediated induction of reactive oxygen species. Otherwise, Bonnesen et al. [39] demonstrated that sulforaphane is not associated with DNA damage in colon cell lines, as assessed by the comet assay. We cannot

exclude that sulforaphane induces the formation of micronucleated cells that could be eliminated by a fully efficient apoptosis effector pathway. The reasons for the different behaviors of MTBITC compared with those recorded for sulforaphane remain to be elucidated. The chemical structures of MTBITC and sulforaphane (Fig. 1) differ only in the oxidation status of the sulfur atom. Glutathione (GSH) is the principal driving force for accumulation of ITCs, while cellular GST further enhances such accumulation [40]. Many ITCs can elevate GSH levels and induce GST. Exposure of cells to ITCs results in their rapid uptake and accumulation through the GSH conjugation reaction catalyzed by GST; such accumulation then leads to an elevation of cellular GSH and GST, which in turn causes more rapid and higher accumulation of ITCs in cells [40]. However, the degree of this type of synergism may depend on specific ITCs. For example, it may be significant for sulforaphane because increases of cellular GSH level and GST activity resulted in increases of both initial uptake and long-term accumulation levels of sulforaphane [40]. Moreover, sulforaphane was previously found to be the most potent inducer of GST among several dozen of ITCs tested, and the presence of oxygen next to the sulfur atom enhances this potency [41]. In contrast, such synergism may be of limited importance for other ITCs, such as allyl isothiocyanate, benzyl isothiocyanate and PEITC [40]. No information is currently available on MTBITC in this context. It is, therefore, possible that the different abilities of MTBITC and sulforaphane to induce apoptosis are mediated by GSH and GST. It is interesting to note that sulforaphane and MTBITC have similar effects on Jurkat leukemia T-cells, because both compounds are able to induce apoptosis and block cell-cycle progression of T-lymphoblastoid cells [8,16]. Taking into account the selectivity toward leukemia cells, MTBITC appears as a conceptually promising agent in cancer therapy. Although in vitro studies do not necessarily predict in vivo outcomes, our findings also indicate that sulforaphane lacks selectivity and is active not only in transformed lymphocytes, but also in their normal counterparts. With regard to the concentrations used, quantities of ITCs up to 100 mg daily and even higher quantities of their GL precursors are widely consumed by humans [42]; 100 mg of ITCs may contain 77.2% sulforaphane [43], corresponding to 435 ␮mol. After an oral dose of

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50 ␮mol of sulforaphane, the peak plasma concentration in vivo was 20 ␮M [44], which lends clear relevance to our in vitro cell culture study, where between 0.3 and 30 ␮M GRA was used. In conclusion, our findings underline the need of more in depth studies of the toxicity profiles of different ITCs. In fact, any chemopreventive use of ITCs would have to be very carefully examined, as dietary supplementation with single, putatively anti-carcinogenic compounds is not warranted without extensive investigation of their possible harmful effects.

Acknowledgements This work was jointly supported by MIUR ex 60% and FIRB grants.

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