Sulforaphane mitigates genotoxicity induced by radiation and anticancer drugs in human lymphocytes

Mutation Research 758 (2013) 29–34

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Sulforaphane mitigates genotoxicity induced by radiation and anticancer drugs in human lymphocytes Omika Katoch, Arun Kumar, Jawahar S. Adhikari, Bilikere S. Dwarakanath, Paban K. Agrawala ∗ Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Brig. SK Mazumdar Road, Timarpur, Delhi 110054, India

a r t i c l e

i n f o

Article history: Received 28 January 2013 Received in revised form 21 August 2013 Accepted 26 August 2013 Available online 1 September 2013 Keywords: Sulforaphane Radiation protection Micronucleus HDAC inhibitor

a b s t r a c t Sulforaphane, present in cruciferous vegetables such as broccoli, is a dietary anticancer agent. Sulforaphane, added 2 or 20 h following phytohemaglutinin stimulation to cultured peripheral blood lymphocytes of individuals accidentally exposed to mixed  and ␤-radiation, reduced the micronucleus frequency by up to 70%. Studies with whole blood cultures obtained from healthy volunteers confirmed the ability of sulforaphane to ameliorate -radiation-induced genotoxicity and to reduce micronucleus induction by other DNA-damaging anticancer agents, such as bleomycin and doxorubicin. This reduction in genotoxicity in lymphocytes treated at the G0 or G1 stage suggests a role for sulforaphane in modulating DNA repair. Sulforaphane also countered the radiation-induced increase in lymphocyte HDAC activity, to control levels, when cells were treated 2 h after exposure, and enhanced histone H4 acetylation status. Sulforaphane post-irradiation treatment enhanced the CD 34+ Lin− cell population in culture. Sulforaphane has therapeutic potential for management of the late effects of radiation. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Development of radioprotective agents is of interest for applications in defence, the nuclear power industry, radiation accident response, space flight, and reducing damage to normal tissues during cancer radiotherapy. Progress has been made in development of prophylactic agents that reduce biological damage when administered prior to exposure, although amifostine (an amino thiol; WR-2712, originally developed at Walter Reed Army Institute, USA) remains the only radioprotective agent approved by the US FDA [1]. Therapeutic management of radiation-exposed persons makes use of standard supportive care drugs and growth factors, generally administered following the appearance of symptoms. The development of approaches for mitigation [2] of radiation damage has received attention only recently. An ideal radiomitigating agent would provide benefit against both acute and delayed effects of ionizing radiation when administered orally, soon after exposure. Acute and late effects of ionizing radiation arise due to macromolecular damage, especially DNA double strand breaks. Cellular repair and recovery involve multiple damage response pathways regulating DNA repair, cell cycle perturbations, cell death, etc., activated soon after damage induction, and determining subsequent

∗ Corresponding author. Tel.: +91 11 23905187; fax: +91 11 23914390. E-mail addresses: [email protected], [email protected] (P.K. Agrawala). 1383-5718/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrgentox.2013.08.009

survival, transformation, and mutation. The accessibility of DNA in chromatin, an important determinant of response, is regulated by post-translational modifications (especially acetylation) of histones, among other factors. Acetylation and deacetylation of histone and non-histone proteins are tightly regulated by the opposing effects of HATs (Histone Acetyltransferase) and HDACs (Histone Deacetylase), respectively [3,4]. Modifiers of HATs and HDACs affect cellular responses to radiation [4]. HDAC inhibitors have shown radioprotective activity in animal models, especially against late effects of radiation [3,5]. Sulforaphane (SFN) is an HDAC inhibitor present in broccoli [4], SFN also affects the Nrf-2-Keap system [6,7]. Here, we report the ability of SFN to mitigate radiation-induced genotoxicity under in vitro conditions, in cultured peripheral blood samples from a cohort of accidentally radiation-exposed individuals. The blood samples used in this study were obtained from persons referred to INMAS for the assessment of radiation doses received by individuals who were exposed due to the negligent dismantling of a Co-60 gamma source in Delhi [8,9]. The gamma irradiator was imported to India in 1968, with an estimated initial strength of approximately 3600 Ci, and was disposed in March 2010. The scrap dealer tried to dismantle the instrument, leading to the exposure of workers and other persons. Some individuals spent 12–14 h per day near the source, which led to a relatively higher exposure and subsequent radiation sickness symptoms, prompting hospitalization of 5–7 persons. The highest estimated

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absorbed dose reported for one of the victims was 3.1 Gy. One of the accidentally exposed individuals succumbed to hematopoietic syndrome [9]. Our results show that addition of SFN to blood cultures 2 or 20 h post-PHA stimulation (i.e., G0 or G1 stage of the lymphocytes [10]) significantly decreased micronucleus (MN) formation in the peripheral blood lymphocytes of these radiation-exposed subjects. These observations prompted us to investigate further the potential of SFN as an antimutagen, using blood from healthy volunteers exposed to agents such as -radiation [11], a known clastogen, bleomycin [11], a known clastogen, and the radiomimetic agent doxorubicin [12], a DNA-intercalating agent and topo-II inhibitor. 2. Material and methods

cytochalasin-B 44 h after PHA stimulation until harvesting, i.e., 72 h after PHA stimulation. Cells were washed with PBS twice and fixed with methanol: acetic acid (3:1). The fixative was removed by centrifugation (300 g; 10 min) and fresh fixative was added to the cell pellet to remove lysed red blood cells and retain only the nucleated cells. Fixed cells were kept at 4 ◦ C until slide preparation. 2.6. Slide preparation and scoring Two or three drops of cell suspension were dropped onto precleaned-chilled slides and allowed to air dry. Slides were stained with Giemsa diluted in phosphate buffer, pH 6.8 m for 10–12 min. Excess stain was washed away with the same buffer and slides were observed under 40× magnification under a light microscope. The criteria described earlier [13] were followed, for scoring micronuclei and determination of nuclear division index (NDI). A minimum of 1000 binucleated cells from three or four slides were scored for each sample and the slides were coded before scoring.

2.1. Chemicals

2.7. Calculation of nuclear division index

Sources of materials were as follows: dl-sulforaphane, doxorubicin, cytochalasin-B, RPMI-1640, IDM, penicillin-streptomycin solution, BCA kit: Sigma–Aldrich (St. Louis, MO); bleomycin: obtained locally; FBS and PHA: Life Technologies (India); HDAC assay kit: BioVision, USA (San Francisco, CA); PathScan® acetyl-histone H4 kit: Cell Signaling Technology, USA (Danvers, MA); PE-Cy-7labeled CD-34 antibody: e-Biosciences, USA (San Diego, CA); FITC-labeled lineage cocktail: BD Biosciences; 7-AAD: Molecular Probes, USA (Eugene, OR). All other chemicals were purchased locally and were of analytical grade. Sodium-heparin vacutainers were purchased from Becton Dickinson, USA (San Jose, CA).

The nuclear division index (NDI), a measure of cell division kinetics, was calculated by scoring cells for the presence of one, two, three or more nuclei. The NDI was calculated as follows: NDI = (M1 + 2 × M2 + 3 × M3 + 4 × M4)/N, where M1–M4 indicates the number of cells with one to four nuclei, and N is the total number of cells scored.

2.2. Subjects and sample collection Blood from healthy volunteers (3–4 ml; n = 6, 4 male and 2 female, ages 25–38 y) and suspected radiation-exposed individuals (n = 32, 26 male and 6 female, ages 12–80) were drawn into Na-heparin-containing vacutainers by trained medical technologists of the institute. All subjects provided written informed consent. From questionnaire data, it was found that only ten male individuals (ages 28–56 y) among the 32 individuals were actually involved in work with the irradiator or spent some time in its vicinity. The data presented here are from those ten individuals only. Other donors were relatives who had never visited the site of the irradiator. The guidelines provided by the Indian Council for Medical Research (ICMR), the Government of India, and the institutional ethics committee were strictly followed during this study, which was duly approved by the Institutional Regulatory Board (IRB Approval letter INM/TS/IEC/01/1012). 2.3. Whole blood culture Whole blood for each sample was cultured using a standard procedure. Briefly, whole blood (0.5 ml) was added to medium (4.5 ml) containing 20% FBS and antibiotics, within 2 h of collection of the sample, and stimulated with 100 ␮l PHA (stock solution 1 mg/ml in PBS). Culture flasks were placed in a humidified CO2 incubator at 37 ◦ C until harvesting, with intermittent shaking of the flasks every day. Sulforaphane (400 nM) was added to each culture either when the lymphocytes were at G0 (within 2 h of PHA treatment) or G1 (20 h after PHA treatment) phase of the cell cycle [10]. 2.4. In vitro irradiation, genotoxic chemical, and sulforaphane treatment Blood samples collected from healthy volunteers were cultured as described above. Cultures were exposed to Co-60 -radiation (0.25, 0.50, 1, or 2 Gy at 1.17 Gy/min) from a Cobalt-60 teletherapy machine (Bhabhatron-II, Panacea Medical Technologies, Bengaluru, India). Standard chemical dosimetry (Fricke’s method) and ion-chamber dosimetry were followed for dose calibration. Whole blood cultures were treated with bleomycin (15 ␮g/ml) or doxorubicin (25 ␮g/ml) in incomplete medium (without FBS) for 1 h and washed twice with sterile PBS, before adding complete medium to promote growth and expression of micronuclei. The treatment conditions were designed to mimic the accidental exposure of individuals as closely as possible; i.e. test agents were added to whole blood before mitogenic stimulation. Sulforaphane 400 nM) was added 2 or 20 h after the addition of PHA, so as to ensure exposure of lymphocytes to sulforaphane at either the G0 (2 h) or G1 (20 h) stage of the cell cycle [10]. Fig. 1(a–c) depicts schematically the treatment protocols for each experiment performed. The dose of SFN was based on our preliminary observation of HUVEC cell survival assessed by the XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2Htetrazolium hydroxide} assay (unpublished data). 2.5. Cytochalasin-B-blocked micronucleus assay The MN assay using cytochalasin-B block was performed essentially according to the method described earlier [13]. Briefly, all cultures were treated with 5 ␮g/ml

2.8. Peripheral blood HSC assay Blood lymphocytes were isolated using Histopaque and cultured in IDM media containing 20% FBS and antibiotics, as described earlier [14]. Sets of lymphocyte cultures (control, 2 Gy, 400 nM SFN, and 2 Gy + 400 nM SFN) were evaluated for CD34+ Lin− cells between 4 and 48 h. 7-AAD was used to eliminate dead cells from analysis. A minimum of 50,000 cells from each sample were acquired on a LSR-II flowcytometer equipped with suitable optics. 2.9. HDAC assay Blood lymphocytes were isolated using Histopaque and cultured in RPMI media containing 20% FBS and antibiotics. Nuclear protein samples (10 ␮g) from four sets of triplicate lymphocyte cultures (control, 2 Gy, 400 nM SFN, and 2 Gy + 400 nM SFN) were evaluated for deacetylated lysine content using the HDAC colorimetric assay lit (K331-100; BioVison, San Francisco, CA, USA) at 405 nM, using a Spectra Max 2 multimodal plate reader (Molecular Devices, Eugene, OR, USA), as per the manufacturer’s instructions. The values are presented as the amount of deacetylated lysine equivalent per ␮g nuclear protein, as estimated from a standard curve prepared using the kit deacetylated lysine standard. Acetylation status of histone H4 was assayed using PathScan® acetyl histone H4 sandwich ELISA following the manufacturer’s instructions. Cell lysates containing equal amount of protein from each treatment groups were used after dilution and absorbance was recorded at 450 nm. Total protein in the nuclear extract and cell lysate were quantified using the BCA assay kit (Sigma–Aldrich), with BSA standard. 2.10. Statistical analysis Data presented are mean ± SD of all samples in the experiment. MN frequencies were compared using students’ t-test and P < 0.05 was considered significant.

3. Results 3.1. In vitro CBMN assay A dose response curve for the induction of micronuclei was generated following in vitro irradiation of human lymphocytes, using the CBMN assay, in the absorbed dose range 0–2 Gy (Fig. 2). This curve was used to extrapolate the possible dose received by the individuals. These individuals did not have a history of smoking, chronic alcohol consumption, or recent fever. The estimated doses in nearly ten individuals were in the range 0.25–1.7 Gy, based on the in vitro dose-response plot (Fig. 2). Addition of SFN (400 nM) either 2 or 20 h after irradiation resulted in a significant decrease in MN frequency, which was not strictly dependent on the time of addition of SFN (Fig. 3). Under the present experimental conditions, SFN reduced bleomycin- and doxorubicin-induced micronuclei by 30–44% in the whole blood cultures obtained from healthy volunteers (Fig. 4). Bleomycin is a radiomimetic agent that can induce

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Protein Isolation for HDAC assay and H4 Acetylation status

Fig. 1. A schematic representation of different experimental protocols showing the timings of treatments like (a) whole blood irradiation with respect to PHA stimulation, SFN treatment and harvesting for fixation, (b) carcinogen (doxorubicin or bleomycin) treatment of whole blood with respect to PHA stimulation, SFN treatment and harvesting for fixation and (c) isolated lymphocyte irradiation with respect to PHA stimulation, SFN treatment and harvesting for HDAC activity assay. The schematic representation is not to scale and details are given in the text.

clustered DNA damage; doxorubicin is a topo-II poison capable that can cause DNA strand breaks. Treatment with 400 nM SFN, either 2 or 20 h after PHA stimulation, significantly (P < 0.05) reduced the radiation-induced MN frequency in the lymphocytes of the ten subjects who worked in the contaminated area (Table 1). Similar results were obtained with in vitro irradiation of the blood from normal volunteers (Fig. 3). The concentration of SFN (400 nM) used here did not compromise the nuclear division index (NDI), either in case of the in vitro irradiation of whole blood from healthy volunteers or the blood cultures from suspected exposed individuals (Table 1).

3.2. CD 34+ Lin− cell population in peripheral blood A significant decrease in the population of CD 34+ Lin− cells was observed in the group receiving 2 Gy irradiation, at all time points studied, as compared to the control group (Fig. 5). The SFN and irradiation plus SFN groups were comparable to the control group at corresponding time points. 3.3. HDAC activity assay Irradiation (2 Gy -radiation) of the lymphocytes significantly (P < 0.05) increased the amount of deacetylated lysine in the nuclear

250 2

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Radiation Dose (Gy) Fig. 2. Heparinized blood from healthy six volunteers was cultured and exposed to 0–2 Gy Co-60 gamma rays before PHA stimulation. Slides were prepared for MN observation as described in the text. MN frequencies in all treatment groups were plotted as a function of radiation dose. The curve obtained without SFN treatment was used as the standard curve for absorbed dose estimation in the accidentally exposed individuals, the dotted lines shows upper and lower confidence limits (95% confidence interval).

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Fig. 3. Heparinized blood from healthy volunteers was cultured and exposed to 0–2 Gy Co-60 gamma rays before PHA stimulation. Whole blood cultures exposed to identical radiation dose were treated with 400 nM SFN either 2 or 20 h post-PHA stimulation. 72 h after culture, slides were prepared for MN observation as described and MN frequencies in all treatment groups were plotted as a function of radiation dose. The curve obtained without SFN treatment was used for absorbed dose estimation in the accidentally exposed individuals. Values presented are mean ± SD of blood from six healthy volunteers. Table 1 Effect of SFN treatment of radiation induced MN frequency and NDI. MN frequency (Percent)

Fig. 4. Whole blood samples from healthy volunteers were treated with chemical mutagens like bleomycin or doxorubicin for 1 h, washed with sterile PBS and treated with PHA. SFN was administered at a concentration of 400 nM at either 2 or 20 h post PHA stimulation and MN frequency was estimated as described in the text. Values presented are mean ± SD of blood from six healthy volunteers.

NDI

No SFN

SFN

Fold change

No SFN

SFN

Fold change

1.30 1.69 1.40 1.20 1.40 0.90 5.15 4.45 4.10 4.65 24.90 17.30 8.70 5.50 2.90 4.50

1.20 1.20 1.15 1.11 1.25 0.90 2.80 3.57 3.14 3.60 7.20 10.00 7.20 3.10 2.20 3.25

0.92 0.71 0.82 0.92 0.89 1.00 0.54 0.80 0.77 0.77 0.29 0.57 0.29 0.56 0.75 0.72

1.56 1.55 1.56 1.45 1.48 1.56 1.58 1.60 1.55 1.56 1.46 1.44 1.46 1.55 1.48 1.51

1.50 1.56 1.56 1.50 1.51 1.52 1.44 1.67 1.49 1.48 1.53 1.45 1.53 1.52 1.49 1.50

1.04 0.99 1.00 1.03 1.02 0.97 1.09 0.96 1.04 1.05 0.95 1.006 0.95 0.98 1.006 1.006

Whole blood cultures from accidentally radiation exposed individuals were treated with 400 nM SFN 2 h after PHA stimulation. First six samples are from healthy volunteers and remaining are from the cohort of accidentally exposed individuals. Micronuclei frequency in binucleated lymphocytes and nuclear division index were estimated as described in Section 2. Effect of SFN was designated in terms of fold change (ratio of MN frequency or NDI with out SFN and with SFN treatment groups).

extract as compared to the untreated samples (controls), at 2 h post-SFN treatment (Fig. 6a). SFN alone did not alter the total deacetylated lysine level while treatment with 400 nM SFN 2 h after 2 Gy irradiation restored the deacetylated lysine level comparable to the untreated controls (Fig. 6a). Fig. 6b depicts the changes in acetylation status of histone H4 as a function of time. SFN treatment 2 h after 2 Gy irradiation was observed to enhance H4 histone acetylation status until 24 h, as compared to the control and radiation-alone groups. 4. Discussion Fig. 5. The percentage of live circulating hematopoietic stem cells were studied using antibodies for CD 34 and Lin markers and 7-AAD as described in Section 2. Values presented are mean ± SD of blood from six healthy volunteers.

The mutagenic effects of physical and/or chemical agents can be ameliorated by multiple mechanisms, broadly classified as modification (prevention) of the induction of genomic damage and enhancement of DNA repair capability. Several compounds can

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Treatments Fig. 6. (a): HDAC activity (␮M deacetylated lysine generated per ␮g nuclear protein) in the lymphocyte nuclear protein obtained from various treatment groups as depicted in the figure was assayed as described in Section 2. Values presented are mean ± SD of blood from six healthy volunteers. (b) The acetylation status of histone H4 was studied using PathScan acetyl H4 sandwich ELISA as described by the manufacturer. Values presented are mean ± SD of blood from six healthy volunteers.

antagonize the effects of mutagenic agents, and these are grouped based on their mechanism and site of action. For example, inhibitors of tumor initiation are called blocking agents of mutagenesis, while suppressing agents are inhibitors of cancer promotion and progression [15]. On the other hand, desmutagens inactivate mutagens before they can attack DNA and antimutagens interfere with the fixation of DNA damage [16]. The ICPEMC Expert Group on Antimutagens and Desmutagens made a distinction between stage1 inhibitors, acting extracellularly, and stage-2 inhibitors, acting intracellularly [17]. Antimutagens act at the cellular level as modulators of DNA repair and replication [18]. The protective effects of antimutagens can be mediated by increases in the fidelity of DNA replication, by stimulating error-free repair of DNA damage, or by inhibiting error-prone repair systems. As was evident from the in vitro irradiation studies, SFN ameliorated radiation-induced MN formation under various treatment regimens, implying its potential application as an antimutagen. MN formation is a result of DNA strand breaks caused by radiation and other clastogenic agents. Amelioration of MN formation by SFN administered 2 or 20 h following PHA stimulation (Figs. 3 and 4) suggests that its anti-mutagenic effect could arise either due to enhanced repair or reduced manifestation of the damage. Reduction in the micronuclei by SFN was not due to reduced proliferation (an important pre-requisite for MN expression), as revealed by the

ND index values (Table 1). Another study [19] also demonstrated the antimutagenic potential of SFN against four known mutagens, without any adverse affects of SFN on cell division or cell death at a concentration of >10 ␮M. In our study also, SFN did not induce significant apoptotic cell death (data not shown), which otherwise could contribute to the reduction in micronuclei frequency. These observations also suggest that the SFN concentration required for mitigation (as used here) is neither toxic to cells nor has any cytostatic effect. Ex vivo SFN treatment many days to several weeks after accidental exposure to radiation was effective in mitigating radiation-induced micronuclei in the blood cells of human subjects (Table 1). It is important to note here that the individuals were exposed to low doses of gamma and beta (since the pencils were dismantled) radiation, over a period of several days intermittently before sample collection took place. The total absorbed dose and dose rate were dependent on the time spent and proximity of the individuals to the source, based on the nature of their work duties. This indicates that SFN can ameliorate micronuclei arising not only due to exposure to acute gamma radiation but also due to mixed radiation exposure over a prolonged period. If SFN merely suppressed the expression of micronuclei and not reduce the manifestation of damage, then the survival of cells is expected to be compromised under these conditions, which should affect

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mortality/morbidity of the exposed individual. Taken together, these observations suggest that SFN indeed mitigates radiationinduced damage by modifying genomic damage in cells. SFN has been reported to act via the Nfr-2 keap1 pathway to impart its anticancer properties [20]. More recently, SFN has been observed to act as a HDAC inhibitor [21], which can alter the acetylation status of histones and non-histone proteins, imparting significant effects not only on chromatin structure but also on signal transduction pathways. The current study has shown the ability of SFN to restore normal levels of deacetylated lysine in the nuclear proteins (HDAC activity), which was increased due to radiation exposure (Fig. 6a). This in turn suggests a reduced amount of acetylated histones in the irradiated samples and an increased amount of acetylated histones in the irradiated-plus-SFN-treated group. Increased acetylation in the later may facilitate the downstream events like transcription and repair of the more open DNA [22], which ultimately may result in lesser cytogenetic damage. Reduction in the MN frequency observed in the peripheral blood lymphocytes of individuals who earlier (several weeks before actual blood sample collection) were exposed to radiation accidentally suggests that the residual genomic damage is susceptible to alterations or manipulation by HDAC linked changes in chromatin status. Our further observation on the prolonged hyperacetylation status of histone H4 (Fig. 6b) also indicates an increased repair activity following SFN treatment. H4 acetylation status facilitates DNA double-strand break repair [23,24]. Whether the anticancer properties of SFN are linked to its HDAC inhibitory effects needs further investigation. Irrespective of the mechanisms underlying the reduction in cytogenetic damage, our observations suggest that SFN can reduce both acute as well as late effects related to radiation-induced genomic damage. 5. Funding The work was supported by the Defence Research and Development Organization, Government of India. Conflicts of interest No conflict of interest exists. Acknowledgments We thank V.R. Senthil and Namita Kalra for carrying out the dose calibration of the irradiator and for helping with flow cytometry. References [1] D.M. Brizel, T.H. Wasserman, M. Henke, V. Strnad, V. Rudat, A. Monnier, F. Eschwege, J. Zhang, L. Russell, W. Oster, R. Sauer, Phase III randomized trial of amifostine as a radioprotector in head and neck cancer, J. Clin. Oncol. 18 (2000) 3339–3345.

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