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Febuxostat Mitigates Genotoxicity Induced by Ionizing Radiation in Human Normal Lymphocytes.

INTRODUCTION

Ionizing radiation (IR) produces free radicals and reactive oxygen species (ROS) when passing into cells. These reactive substances destroy cellular organelles mostly DNA. Extensive DNA damages lead to cell death. However, the killing effect of IR has benefit for cancer treatment, and it limits normal organs that are surrounded by tumor tissue. IR causes side effects in patients during radiotherapy for their cancer treatment (1). It is important to find a radioprotective agent that is able to protect patients or people against side effects induced by IR. DNA is a crucial and sensitive macromolecule to IR, and its protection is important. Antioxidant and anti-inflammatory properties are mechanisms involved in radioprotective agents (1, 2).

Febuxostat (FB) is a xanthine oxidase inhibitor that has been used to treat chronic gout. It is an orally active and selective xanthine oxidase inhibitor that is used for treatment of hyperuricemia or gout. Gout is the most common cause of inflammatory arthritis (3). Anti-inflammatory effect is one of the mechanisms of this drug. FB markedly protects doxorubicin-induced cardiotoxicity (4), lipopolysaccharide (LPS)-induced lung inflammation (5), and my-ocardial ischemia/reperfusion injury (6) in animals. Since IR causes oxidative stress and inflammation in cells, we hypothesized that FB could also have beneficial effects against genotoxicity induced by IR in normal lymphocytes in addition to its anti-gout indication.

MATERIAL AND METHODS

Materials

FB was obtained from Chimidaru, Iran. Cytochalasin B was purchased from Sigma Chemicals Co., USA. Phyto-hemagglutinin (PHA), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco Co., USA. Giemsa stain, methanol, and acetic acid were obtained from Merck, Germany.

Blood Treatment

The study was approved by the research and ethical committees of Mazandaran University of Medical Sciences (ID no. 3178, 2017-7-30). Three healthy, non-smoking male volunteers aged 20-24 years old participated in the present study. Ten milliliter of whole blood was collected from participants using a heparinized syringe, transferred into tubes, and then divided into microtubes at 0.9 mL each. One hundred microliter solutions of FB at final concentrations of 1, 10, 50, or 100 [micro]M (6, 7) were added to blood samples. The plasma concentration of FB was 10-12 [micro]M after oral ingestion of FB at a dose of 80 mg in healthy humans (8), whereas it was 15-25 [micro]M at an oral dose of 120 mg (9, 10). FB as powder was dissolved in dimethyl sulfoxide (DMSO) as solvent and diluted in RPMI culture medium. Control samples received DMSO (0.1%) the same as other treated groups.

Irradiation Protocol and Micronucleus Assay

Microtubes containing blood samples were kept on a plastic box containing water as phantom, and then samples were irradiated with X-ray (6 MV) beam produced by a linear accelerator (Siemens, Primus, Germany) at a dose of 1.5 Gy. After irradiation, half milliliter of each sample was then added to 4.4 mL of RPMI 1640 culture medium, which was completed with 10% FBS, and then PHA (100 [micro]L) was added to the mixture. All samples were in duplicate. All culture samples were incubated at 37[degrees]C in a cell culture incubator. After culturing for 44 h, cytochalasin B was added in the cell culture medium. After culturing for 72 h, blood samples were centrifuged and removed from the supernatant medium. The cellular sediment was re-suspended in cold potassium chloride, and then lymphocytes were collected from blood samples. Lymphocytes were immediately fixed in methanol/acetic acid (5/1 v/v) as a fixative solution. The fixed lymphocytes were smeared onto clean microscopic slides. The cells were stained with Giemsa solution (10%). All slides were observed under a microscope with 1000x magnification to determine the frequency of micronuclei in the cytokinesis-blocked binucleated lymphocytes. Overall, 10 control and treated groups were evaluated in this experiment.

Statistical Analysis

The micronuclei frequency is presented as mean[+ or -]standard deviation. One-way analysis of variance (ANOVA), as well as post hoc Tukey's multiple comparison tests, was used for statistical analysis using Prism 7 software (2016; GraphPad Software Inc., La Jolla, CA, USA).

RESULTS

Binucleated lymphocyte with the presence of the micronucleus is shown in Figure 1. In the present study, the mean percentage of micronuclei in irradiated blood samples without any drug treatment was 7.63[+ or -]0.08, whereas it was 0.41[+ or -]0.15 in non-irradiated control samples. Exposure of blood samples to IR significantly increased the percentage of the micronucleus (18-fold increase) in irradiated lymphocytes (p<0.001) (Table 1). In FB pre-treatment and irradiated samples, the percentages of micronuclei at various concentrations of FB as 1, 10, 50, or 100 [micro]M were 6.7[+ or -]0.1%, 6.4[+ or -]0.3%, 5.4[+ or -]0.1%, and 4.4[+ or -]0.2%, respectively (Table 1). The findings demonstrated that pre-treated blood with FB at all concentrations had significantly lower percentages of micronuclei than irradiated samples without FB treatment (p<0.01). Total micronuclei frequencies in irradiated samples pre-treated with FB were lower at 11%, 16%, 30%, and 43% at concentrations of 1, 10, 50, or 100 [micro]M, respectively, than those in irradiated samples alone. The maximum protection of lymphocytes was observed at a concentration of 100 [micro]M with FB treatment. The percentages of micronuclei were significantly lower in irradiated samples treated with FB at concentrations of 50 and 100 [micro]M than in irradiated samples treated with FB at concentrations of 1 and 10 [micro]M (p<0.01). The frequency of micronuclei was insignificant between FB 1+IR and FB 10+IR groups. Non-irradiated samples with FB treatments at 1 [micro]M and 10 [micro]M have not shown any increased genotoxicity as compared with non-irradiated control group, whereas it was significant at 50 [micro]M and 100 [micro]M as compared with the control group (p<0.05)

DISCUSSION

In the present study, the radioprotective effect of FB was demonstrated against DNA damage induced by IR in normal human lymphocytes. A dose relevance between FB concentrations and its radioprotective effect was observed. The maximum radioprotection was observed at a concentration of 100 [micro]M of FB with 43% efficacy. However, FB showed a significant radioprotective effect at a concentration of 100 [micro]M, and this dose of FB exhibited higher micronucleus than control. Hence, the percentage of the micronucleus in FB treatment samples (100 [micro]M) was <1%. The micronucleus is a gene toxicity biomarker that is increased in oxidative stress condition in cells. However, IR-produced free radicals and ROS are the most toxic substances involved in cellular damage, and it is a crosstalk between oxidative stress and inflammation process in the cellular environment. IR-produced inflammation activates signaling pathways into the cell that induces DNA damage (2). Mefenamic acid exhibited a radioprotective effect through anti-inflammatory property (11). In the present study, FB exhibited radioprotective effect, but its mechanism is unclear. Several studies have been reported that FB had protective effects on normal tissues against high dose of drugs or toxic agents. FB markedly ameliorated the doxorubicin-induced cardiac toxicity in rats. It prevented the reduction of non-protein thiol levels through increased manganese superoxide dismutase (SOD) level and reduced cardiac damage markers and malondialdehyde (MDA) level as a lipid peroxidation marker. It also exhibited an anti-inflammatory effect through decreasing the expression of nuclear factor kappa and tumor necrosis factor (TNF)-[alpha]. It exerted an anti-apoptotic effect through increased Bcl-2 (a cell survival protein) expression and decreased caspase-3 and Bax expressions (4). It was able to decrease LPS-induced lung inflammation and edema. It attenuated the elevated levels of TNF-[alpha] in the lung tissue of LPS-treated rats. In addition, it exhibited an antioxidant activity through decreasing MDA levels in the lung tissue (3.4-fold) as well as enhancing SOD activity (by 34%) (5). It inhibited acute pulmonary inflammation triggered by lung injury induced by acid injection. Moreover, it inhibited inflammation in the liver in response to acetaminophen-induced hepatic injury (12). The free radical scavenging and reducing power activities of FB were evaluated by diphenyl-picrylhydrazyl radical and ferric reducing antioxidant power methods, respectively. FB did not exhibit any antioxidant activity in vitro up to 200 [micro]M, whereas a percentage of >95% antioxidant activity with ascorbic acid were observed (data not shown). Then, free radical scavenging and reducing power activities are not possibly contributed in the radioprotection of FB. Since the anti-inflammatory effects of FB have been approved in the above stated studies, then this mechanism is mostly involved in the radioprotection of FB. The maximum radiation protection of FB (100 [micro]M) was 43% efficacy in our study. In a previous study, mefenamic acid (100 [micro]M) with mainly anti-inflammatory effect was 38% (11).

CONCLUSION

In the present study, FB reduced genotoxicity induced by IR in human normal lymphocytes, with regard to drug approval; it will help defense normal tissue against toxicity induced by IR in humans.

Ethics Committee Approval: This study was approved by the ethics committee of Mazandaran University of Medical Sciences, Sari, Iran (ID no. 3178, 2017-7-30).

Informed Consent: N/A.

Peer-review: Externally peer-reviewed.

Author Contributions: Conceived and designed the experiments or case: SJH. Performed the experiments or case: SA, AG. Analyzed the data: SA, SJH. Wrote the paper: SJH. All authors have read and approved the final manuscript.

Conflict of Interest: The authors declare that they have no conflict of interest.

Financial Disclosure: This research was supported by Mazandaran University of Medical Sciences, Sari, Iran (grant no. 3178).

REFERENCES

(1.) Hosseinimehr SJ. Trends in the development of radioprotective agents. Drug Discov Today 2007; 12(19-20): 794-805. [CrossRef]

(2.) Salehifar E, Hosseinimehr SJ. The use of cyclooxygenase-2 inhibitors for improvement of efficacy of radiotherapy in cancers. Drug Discov Today 2016; 21(4): 654-62. [CrossRef]

(3.) Waller A, Jordan KM. Use of febuxostat in the management of gout in the United Kingdom. Ther Adv Musculoskelet Dis 2017; 9(2): 55-64.

(4.) Krishnamurthy B, Rani N, Bharti S, Golechha M, Bhatia J, Nag TC, et al. Febuxostat ameliorates doxorubicin-induced cardiotoxicity in rats. Chem Biol Interact 2015; 237: 96-103. [CrossRef]

(5.) Fahmi AN, Shehatou GS, Shebl AM, Salem HA. Febuxostat protects rats against lipopolysaccharide-induced lung inflammation in a dose-dependent manner. Naunyn Schmiedebergs Arch Pharmacol 2016; 389(3): 269-78. [CrossRef]

(6.) Wang S, Li Y, Song X, Wang X, Zhao C, Chen A, et al. Febuxostat pretreatment attenuates myocardial ischemia/reperfusion injury via mitochondrial apoptosis. J Transl Med 2015; 13: 209. [CrossRef]

(7.) Nomura J, Busso N, Ives A, Tsujimoto S, Tamura M, So A, et al. Febuxostat, an inhibitor of xanthine oxidase, suppresses lipopolysaccharide-induced MCP-1 production via MAPK phosphatase-1-mediated inactivation of JNK. PLoS One 2013; 8(9): e75527. [CrossRef]

(8.) Chandu BR, Kanala K, Hwisa NT, Katakam P, Khagga M. Bioequivalance and pharmacokinetic study of febuxostat in human plasma by using LC-MS/MS with liquid liquid extraction method. Springerplus 2013; 2(1): 194. [CrossRef]

(9.) Zhang M, Di X, Xu L, Xu J, Yang Y, Jiang N, et al. Pharmacokinetics and pharmacodynamics of febuxostat under fasting conditions in healthy individuals. Exp Ther Med 2014; 7(2): 393-6. [CrossRef]

(10.) Zhou H, Zheng Y, Wu G, Hu X, Zhai Y, Iv D, et al. Pharmacokinetics and tolerability of febuxostat after oral administration in healthy Chinese volunteers: a randomized, open-label, singleand multiple-dose three-way crossover study. Int J Clin Pharmacol Ther 2016; 54(2): 115-24.

(11.) Hosseinimehr SJ, Nobakht R, Ghasemi A, Pourfallah TA. Radiopro-tective effect of mefenamic acid against radiation-induced genotoxicity in human lymphocytes. Radiat Oncol J 2015; 33(3): 256-60.

(12.) Kataoka H, Yang K, Rock KL. The xanthine oxidase inhibitor Febuxostat reduces tissue uric acid content and inhibits injury-induced inflammation in the liver and lung. Eur J Pharmacol 2015; 746: 174-9.

Shiva Asemi (1,2) [iD], Arash Ghasem (3) [iD], Seyed Jalal Hosseinimehr (1) [iD]

Cite this article as: Asemi S, Ghasem A, Hosseinimehr SJ. Febuxostat Mitigates Genotoxicity Induced by Ionizing Radiation in Human Normal Lymphocytes. Erciyes Med J 2019; 41(1): 99-101.

(1) Department of Radiopharmacy, Faculty of Pharmacy Mazandaran University of Medical Sciences, Sari, Iran (2) Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran (3) Department of Radiology and Radiation Oncology, Faculty of Medicine Mazandaran University of Medical Sciences, Sari, Iran

Submitted 02.11.2018

Accepted 28.01.2019

Available Online Date 01.02.2019

Correspondence Seyed Jalal Hosseinimehr, Department of Radiopharmacy, Faculty of Pharmacy Mazandaran University of Medical Sciences, Sari, Iran Phone: +98-11-33543691 e.mail: sjhosseinim@yahoo.com, sjhosseinim@mazums.ac.ir

DOI: 10.14744/etd.2019.35762
Table 1. The frequency of micronuclei induced in vitro by 1.50 Gy X-ray
radiation in cultured blood lymphocytes from human volunteers examined
treated at different doses of febuxostat

Group       % micronuclei in binucleated
            human lymphocyte
Sample      I (#)   II     III    Mean[+ or -]SD

Control     0.45    0.25   0.54   0.41[+ or -]0.15
IR          7.67    7.54   7.68   7.63[+ or -]0.08 (a)
FB 1+IR     6.73    6.86   6.69   6.76[+ or -]0.09 (b)
FB 10+IR    6.50    6.68   5.99   6.39[+ or -]0.36 (b)
FB 50+IR    5.38    5.47   5.27   5.37[+ or -]0.09 (b,c)
FB 100+IR   4.57    4.26   4.35   4.39[+ or -]0.16 (b,c)
FB 1        0.49    0.59   0.58   0.55[+ or -]0.05
FB 10       0.74    0.87   0.76   0.79[+ or -]0.06
FB 50       0.89    0.85   0.93   0.89[+ or -]0.04 (d)
FB 100      0.94    1.03   1.02   0.99[+ or -]0.05 (d)

(#) I, II, and III are the number of volunteers who participated in the
study, and whole blood was obtained here. 1000 BN cells were examined
in each sample. (a) p<0.001 compared with control; (b) p<0.01 compared
with IR; (c) p<0.01 FB 1+IR with FB 50+IR and FB 100+IR; (d) p<0.05
compared with control (ANOVA analysis with Tukey's multiple comparison
test was applied for comparison of data). SD: Standard deviation; IR:
Ionizing radiation; FB: Febuxostat
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Title Annotation:BRIEF REPORT
Author:Asemi, Shiva; Ghasem, Arash; Hosseinimehr, Seyed Jalal
Publication:Erciyes Medical Journal
Article Type:Report
Date:Mar 1, 2019
Words:2346
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