Toxicology Mechanisms and Methods

ISSN: 1537-6516 (Print) 1537-6524 (Online) Journal homepage: http://www.tandfonline.com/loi/itxm20

Protective Effect of Amifostine on Busulfan Induced DNA Damage in Human Hepatoma Cells Nasrin Ghassemi-Barghi, Mahmoud Etebari & Abbas Jafarian-Dehkordi To cite this article: Nasrin Ghassemi-Barghi, Mahmoud Etebari & Abbas Jafarian-Dehkordi (2016): Protective Effect of Amifostine on Busulfan Induced DNA Damage in Human Hepatoma Cells, Toxicology Mechanisms and Methods, DOI: 10.1080/15376516.2016.1243601 To link to this article: http://dx.doi.org/10.1080/15376516.2016.1243601

Accepted author version posted online: 24 Oct 2016.

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Date: 24 October 2016, At: 23:43

Protective Effect of Amifostine on Busulfan Induced DNA Damage in Human Hepatoma Cells Author Information Nasrin Ghassemi-Barghi1, Mahmoud Etebari1*, Abbas Jafarian-Dehkordi1 Affiliation 1: Department of Pharmacology and Toxicology, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran, Isfahan, Iran (the Islamic Republic of) *Email: [email protected]

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Abstract

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Busulfan is one of the most effective chemotherapeutic agents used for the treatment of chronic myeloid leukemia. However, as a bifunctional alkylating agent, during clinical use several side effects may occur. In addition in several in vivo and in vitro studies busulfan has shown a range of genotoxic effects including DNA strand break and inhibition of DNA synthesis. Amifostine, an organic thiophosphate compound, has been shown to exert an important cyto-protective effects in many tissues. The aim of this study was to explore whether amifostine protects against busulfan-induced genotoxicity in HepG2 cell line. Our results showed that amifostine reduced the genotoxic effects of busulfan significantly in both type of experiment conditions, as measured via comet assay. Furthermore, amifostine decreased the intracellular ROS generation induced by busulfan and also increased the intracellular GSH levels in HepG2 cells. Altogether, our results suggest a protective action of amifostine against busulfan cytotoxicity and genotoxicity via various pathways. The most protective effect was observed with amifostine when it was administrated 24 h before busulfan treatment.

1. Introduction

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Induction of senescence has been considered to be an underlying cellular mechanism of organismal aging (Bartkova et al. 2006; Meng, Wang, Van Zant, et al. 2003). Besides, it can cause normal tissue injury induced by various cancer therapies (Schmitt et al. 2002; Campisi and di Fagagna 2007). This is because senescent cells, being permanently arrested, cannot proliferate to produce progeny that are normally required for the maintenance of normal tissue homeostasis and for the repair of damaged tissues (Campisi 2005; Miller and Streit 2007). Moreover, senescent cells increased levels of proinflammatory cytokines, matrix metalloproteases and epithelial growth factors that are known to participate in the pathogenesis of pulmonary fibrosis and other normal tissue injuries after chemotherapy and/or ionizing radiation (Rodier et al. 2009; Millis et al. 1992; Meng, Wang, Van Zant, et al. 2003). Busulfan is one of the most effective chemotherapeutic agents used for the treatment of chronic myeloid leukemia (CML) and for myeloablation prior to bone marrow transplantation (de Lima et al. 2004). However, the therapeutic efficacy of busulfan is compromised by harmful effects in normal tissues, such as bone marrow suppression, chronic lung fibrosis, and hepatic veno-occlusive disease (VOD) (Lee et al. 2005; Abid, Malhotra, and Perry 2001; Meng, Wang, Brown, et al. 2003). There are recent reports documenting that busulfan induces cellular senescence in normal mouse bone marrow hematopoietic cells and human diploid fibroblasts (WI38 cells) (Meng, Wang, Van Zant, et al. 2003; Probin, Wang, and Zhou 2007). As a potent alkylating agent, busulfan can cause DNA damage by cross-linking DNAs and DNA and proteins. The cross-links can be converted into DNA strand breaks that can subsequently cause cell death (Iwamoto et al. 2004). Therefore, busulfan has shown a wide range of damages to normal tissues (Iwamoto et al. 2004; Valdez et al. 2010). Several reports have shown that busulfan forms conjugates with glutathione (GSH) leading to GSH depletion and induction of oxidative stress (Bredschneider et al. 2002; Deleve and Wang 2000; Almog et al. 2011). In addition, a recent study found that busulfan can inhibit thioredoxin reductase, which also plays an important role in the regulation of oxidative stress in cells (Witte et al. 2005). Induction of senescence may result in busulfan-induced normal tissue damage in several different ways. Several investigations confirmed busulfan induced DNA damage through chromosome aberration tests, micronucleus assays and comet assay in various in vivo and in vitro studies (Morales-Ramírez et al. 2006; Bishop and Wassom 1986). Clearly attenuating genotoxicity could increase the application and enhance the efficacy of this drug. Amifostine (AMF, WR-2721), is a cytoprotective adjuvant used in cancer chemotherapy and radiotherapy (Kanat et al. 2003). AMF is an organic thiophosphate compound which is hydrolyzed in vivo by alkaline phosphatase to the active cytoprotective thiol metabolite, WR-1065 (Stankiewicz, Skrzydlewska, and Makiela 2002; Koukourakis 2002). The selective protection of non-malignant tissues is believed to be due to higher alkaline phosphatase activity, higher pH and vascular

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permeation of normal tissues (Kouvaris, Kouloulias, and Vlahos 2007). According to the several reports, inside the cell, protective effects of amifostine appear to be mediated by scavenging free radicals, hydrogen donation, the release of endogenous nonprotein sulfhydryl groups (mainly glutathione) from their bond with cell proteins, and the formation of mixed disulphides to protect normal cells (Torres and Simic 2012). The WR-1065 has shown remarkable radio- and chemoprotective effects in vitro and in vivo and it is currently approved for clinical use as a protective agent against renal toxicity induced by cisplatin in patients being treated for ovarian cancer and against xerostomia induced by ionizing radiation in patients with head and neck cancer (Arany and Safirstein 2003; Hartmann et al. 2000; Antonadou et al. 2002; Santini and Giles 1999). Preclinical studies have shown that administration of WR-2721 before irradiation can protect against radiation clastogenesis, mutagenesis and carcinogenesis (Sanderson and Morley 1986; Damron et al. 2000). Amifostine is able to inactivate electrophilic substances and scavenge free radicals (Marzatico et al. 2000). In addition several studies has been shown that amifostine protect against cardiotoxicity, nephrotoxicity and genotoxicity result from chemotherapeutic agents (Dragojevic-Simic et al. 2004; Hartmann et al. 2000; Buschini et al. 2002; Gloc et al. 2002). Single cell gel electrophoresis (comet assay) is a simple and sensitive method for measuring DNA strand breaks in eukaryotic cells (Silva et al. 2000). The concept of microgel electrophoresis was first introduced in 1984 by Ostling and Johanson, as a method to measure DNA single-strand breaks that caused relaxation of DNA supercoils (Collins 2004). A modified version was published by Singh and colleagues in 1988, which used alkaline condition (Siddique et al. 2005). Much of the interest in this method comes from its potential applications in human biomonitoring and in ecological assessment of sentinel organisms exposed to environmental contaminants (Tice et al. 2000; Collins et al. 1997). In addition, recently, the assay was applied to different strains of Drosophila melanogaster proficient and deficient in DNA repair (Azqueta et al. 2014; Mukhopadhyay et al. 2004). The aim of present study was to investigate the protective effect of amifostine against busulfan induced genotoxicity. For this purpose we measure the DNA damage level with comet assay in HepG2 cell line treated with busulfan and amifostine in different experimental conditions. We also measured intracellular ROS generation and GSH levels in cells treated with busulfan and amifostine in pre-treatment condition.

2. Materials and methods 2.1. Chemicals Busulfan was purchased from sigma-Aldrich (France). Amifostine was obtained from MedImmune Pharma BV. (Poland). EDTA, H2O2, NaCl, NaOH, Na2CO3, NaH2PO4, Tris, and Triton X-100 were acquired from Merck Co. (Germany). Low melting point agarose (LMA), Na2HPO4, KCl and ethidium bromide were from Sigma Co. (USA). Normal melting point agarose (NMA) was supplied by Cinnagen Co (Iran). The RPMI 1640 medium, fetal bovine serum (FBS) and antibiotic were purchased from biosera (France). DCFH-DA probe and mBCl were from sigma Aldrich (USA) and, HepG2 cells came from Pasture Institute (Iran). All other chemicals used were of analytical grade.

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2.2. Cell culture

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Human hepatoma (HepG2) cells were grown as monolayer culture in RPMI 1640 medium supplemented with 10% FBS, 1% of mixture of penicillin (100 IU/ml) and streptomycin (100 µg/ml) incubated at 37 ◦C in an atmosphere of 5% CO2–95% air mixture. Busulfan was dissolved in pure dimethylsulfoxide (DMSO). The studied concentrations selected after range findings, minimum effective and safe concentrations and then were prepared in DMSO in final volumes that represent under 1% of the total medium volume. Amifostine was dissolved in the cell culture medium. We have chosen untreated cells as control. Cells were seeded on 24-well culture plates at 25×10 4 cells/well, after overnight growth, cells treated with studied concentrations of amifostine (1, 5 and 10 mg/ml) 24 h prior to busulfan treatment (24.63 µg/ml or 100 µM) for 1 h at 37 ◦C (Etebari, Ghannadi, et al. 2012; Etebari, Zolfaghari, et al. 2012).

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2.3. Single-cell gel electrophoresis (SCGE, the comet assay)

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The comet assay procedure has been described in previous studies (Etebari, Ghannadi, et al. 2012; Ghassemi-Barghi et al. 2016). Briefly, incubated cell suspensions (1 × 106 cells/ml) were mixed with 1% LMP agarose at 37 ºC, were placed on the precoated slides (1% NMP agarose), and covered by cover glasses for 5 min at 2-8 °C. The slides were incubated with lysis solution (pH=10.0) for 40 min and rinsed with distilled water to remove excess lysis solution. In the next step, slides were incubated with electrophoresis buffer (pH> 13.0) for 40 min. Electrophoresis was conducted for 40 min at 25 V with an electricity current adjusted to 300 mA. After this stage, the slides were rinsed with distilled water to remove excess alkaline buffer and were placed in neutralization solution (pH=7.5) for 10 min. The slides were covered by sufficient dye solution (20 μg/ml ethidium bromide) for 5 min and washed with distillated water. Finally comets were visualized under × 400 magnification using fluorescence microscope with an excitation filter of 510‐560 nm and barrier filter of 590 nm. For each condition 100 randomly selected comets for each concentration (from the 3 replicate experiments) can be scored,

and % DNA in tail, tail length, and tail moment were determined by Comet score software (TriTek Corporation, USA, Version 1.5). Usually, % DNA in tail is preferred for assessing the DNA damage. All stages of comet assay were performed in dark conditions and all solutions were prepared freshly and used cool (Etebari, Ghannadi, et al. 2012). 2.4. Measurement of Oxidative Stress

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Approximately 4 × 104 cells per well were cultured for 24 h in 96-wellplates (blackwall/clear-bottom). Thereafter, the medium was aspirated, and the cells were washed twice with HBSS. The cells were then treated with studied concentrations of amifostine (1, 5 and 10 mg/ml) 24 h prior busulfan treatment (24.63 µg/ml) for 1 h at 37 ◦C. After the treatment, cells were washed twice with HBSS and incubated in 2 ml of fresh culture medium without FBS. 2, 7- Dichlorodihydrofluorescein diacetate was added at a final concentration of 10 µM and incubated for 20 min. The cells were then washed twice with PBS and maintained in 1 ml of culture medium. ROS were assessed by immediately analyzing cells by fluorescence plate reader using the 488 nm for excitation and detected at 535 nm. We have chosen untreated cells as a negative control and cells treated with 0.1 mM H2O2 as a positive control (Wang et al. 2004) 2.5. Measurement of intracellular GSH levels

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HepG2 cells were plated in a 96-well plate at 50,000 cells/well. After overnight growth, they were treated with amifostine for studied concentration (1 ,5 and 10 mg/ml) and busulfan (24.63 µg/ml) in pretreatment condition, then incubated with monochlorobimane (mBCI, 40 μM) in a staining solution (5 mM glucose, 1 mM CaCl2, 0.5 mM MgSO4, 5 mg/ml BSA) for 30 min at 37°C in the dark. Although mBCI is a nonfluorescent probe, it forms a stable fluorescent adduct with GSH in a reaction catalyzed by the GSH S-transferases. The mean fluorescent intensity of the fluorescent GSH-bimane adduct was measured using a Spectra fluorescent plate reader at λex=380 nm and λem=460 nm to detect GSH (Hedley and Chow 1994) . 2.6. Statistical analysis Each experiment was carried out at least three times separately. Data are expressed as means ± SEM. Statistical comparison between different groups were done using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc, to detect the difference between various groups. Differences for which the p value was