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Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin
Journal of Radiation Research and Applied Sciences xxx (2017) 1e7
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Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin Fatmah M. Alshammari a, Sonia M. Reda a, b, *, Magdy M. Ghannam c a
Department of Physics, Faculty of Science, Hail University, KSA Department of Physics, Faculty of Science, Zagazig University, Egypt c Department of Biophysics, Faculty of Science, Cairo University, Egypt b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 November 2016 Received in revised form 27 January 2017 Accepted 28 January 2017 Available online xxx
Cancer therapy has progressed dramatically in recent years. In order to decrease the dose and side effects of the anticancer drug, the therapeutic options for patients with cancer include increasingly complex combinations of chemotherapy and radiotherapy. This combination may cause overlapping interaction between the two types of treatment and affect the stability of the anticancer drug. In this study, the effect of gamma irradiation on the stability and therapeutic activity of one anticancer drug (Doxorubicin) was studied. For this purpose, doxorubicin drug characterized by two methods, at first, in-vitro study, before and after drug irradiation with different doses of gamma rays (2, 5, 20, 100 GY) which achieved through measuring the dielectric relaxation and absorption spectrum of drug solution. Secondly, in-vivo studies, where the unirradiated and the drug, which later exposed to gamma rays, were injected respectively into 6 groups of mice (3 mice in each group). The dielectric relaxation and absorption spectrum were studded for hemoglobin of the injected mice. The results for the in-vitro study indicate that the values of dielectric parameters show unnoticeable change for drug molecules before and after irradiation as compared with the control. The results for in-vivo study indicated an increase in the values of relaxation time and ColeCole parameter, that may as a result of changes in the conformational structure in hemoglobin molecules which may affect their properties and hence RBC's physiological functions. The absorption spectra of hemoglobin molecules show an increase in the amplitude of the characteristic bands with irradiation dose indicate an increase of the oxygen binding capacity with hemoglobin. It was concluded that combination between the drugs and gamma irradiation can be used as a powerful conjunction that may give us the benefit chemo and radiotherapy treatment. © 2017 The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Keywords: Radiotherapy Chemotherapy Gamma radiation Doxorubicin Overlapping Stability of anticancer drug
1. Introduction Cancer therapy has progressed dramatically in recent years, and tremendous progress has been made in reducing the morbidity and mortality from many forms of cancer. The therapeutic options for patients with cancer include increasingly complex combinations of chemotherapy, radiotherapy and surgical intervention. (Chantal, Waston, Drake, Fenton, & Mcloughlin, 2008; Edward et al., 2004; Schultz, Beck, Stava, et al., 2003).
* Corresponding author. Department of Physics, Faculty of Science, Zagazig University, Egypt E-mail address: [email protected] (S.M. Reda). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications.
Radiotherapy is a crucial component of anticancer treatment; up to 50% of cancer patients receive radiotherapy. Apart from surgery, radiotherapy is the major method applied with curative intent for cancer patients. Radiotherapy plays a major part in the palliation of symptoms. (Stephen, 2009). On the other hand, cancer chemotherapy refers to the administration of cytotoxic chemicals, with the aim to, eradicate the tumour or reduce the tumour-related symptoms. Most chemotherapeutic drugs cause damage to DNA, which leads to programmed cell death (apoptosis) (Peter, 2001; Jaishree and Geoff, 2009). Doxorubicin is an anti-cancer drugs widely used in the treatment of various types of cancers. It belongs to antitumor antibiotics. The cytotoxicity of the drug is due to its ability to interact with DNA and plasma membrane and to participate in various oxidation reduction reactions. (Varshney & Dodke, 2004). It is intrinsically fluorescent which makes it convenient for
http://dx.doi.org/10.1016/j.jrras.2017.01.001 1687-8507/© 2017 The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Alshammari, F. M., et al., Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin, Journal of Radiation Research and Applied Sciences (2017), http://dx.doi.org/10.1016/j.jrras.2017.01.001
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F.M. Alshammari et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e7
probing and visualization with various microscopic imaging technologies (Dai et al., 2008). Combined modality treatment with radiotherapy and chemotherapy is used increasingly for the primary management of a variety of human tumors, with the aim of improving both local and distant control, decreasing distant metastasis, and improving survival without excessively increasing normal tissue injury. (Franco, Cortes-Funes, & Wasserman, 1978; Phillips & Karen, 1978; Ian, 1989; Karen, 1985; Maurice, Arriagada, & Cosset, 1985). In this combined treatment, radiation interacts with drugs and may effect on their quality. It was concluded that combination between the drugs and gamma irradiation can be used as a powerful conjunction that may give us the benefit chemo and radiotherapy treatment. This highly versatile system can be used in the testing of drug e organ disease interaction and so will aid in improving the clinical treatment protocol and fights against serious diseases. This work is aimed to evaluate the effects of gamma irradiation on the stability of anticancer drugs. Moreover, study the overlapping interaction between radiotherapy and chemotherapy during cancer fitting is the main target of the presented work. For this purpose, doxorubicin, one of the most widely used anticancer drugs was chosen. The stability of the drug upon application of gamma irradiation will be achieved through measurements of electrical properties. Dielectric measurements can provide valuable significant information about the molecular arrangement of the drugs as well as the electrical conduction mechanism (Grant, Sheppard, & South, 1978). Furthermore, using UVeVisible spectroscopy can derive useful information on the biological molecules. This information can lead to valuable structural proposals (Pavia, Lampman, Kriz, & Vyvyan, 2009). The overlapping interaction between radiotherapy and the used drug will be achieved by studying the effect of irradiation on the therapeutic activity of the used chemotherapeutic drug.
of Saudi Arabia with activity of 0.44 Gy/sec using gamma cell 220 manufactured by NORDION (Canada). Physical methods were taken to characterize the drugs include dielectric relaxation and absorption spectrum of samples. 2.1.2. Dielectric relaxation Electric measurements were investigated in the frequency range from 20 Hz up to 3 MHz (a & b dispersions of biological materials) using a WAYNE KERR precision component analyzer model 6440B (UK). The sample cell has two squared platinum black electrodes with cell constant k ¼ 1 cm1. The measurements were performed at room temperature. For a dielectric material placed between two parallel plates capacitor, the measured values of the capacitance, C and resistance R were used to calculate the conductivity s, as well as the real, ε0 , and imaginary, ε00 , parts of the complex permittivity and the relaxation t time through the following equations: C 13 C i) ε0 ¼ CCo ¼ εCo k ¼ εCo Ad ¼ 8:8510 12 100 ¼ 1:13 10 εo is the permittivity of free space (Foster & Schwan, 1996). 00
ii) Loss tangent factor D ¼ εε0 ¼ 2p1RCf 1 1 iii) The conductivity s ¼ R1A ¼ Rk ¼ 100 R ðU m Þ ð dÞ 00 iv) The dielectric loss ε ¼ Dε0 v) The dielectric increment Dε ¼ ε1 εo vi) Relaxation time t ¼ 2p1f , fc is the critical frequency correc sponding to the midpoint of the dispersion curve. vii) The plot ε0 Vs ε00 (Cole-Cole plot) will produced a semi-circle. It was shown by Cole and Cole that the angle traced between the circle radius and ε0 axis is q ¼ ap/2. This in turn enable to estimate the Cole-Cole parameter a, experimentally, so a ¼ 2q/p (Koji Asami, 2002).
2. Materials and methods In this work, the study of the interaction between gamma radiations with anti-cancer drugs was divided into two main parts: 2.1. In-Vitro studies 2.1.1. Drug Doxorubicin is an anthraxcycline antibiotic with antineoplastic activity. The molecular stucture of the drug is shown in Fig. 1. Doxorubicin, isolated from the bacterium Streptomyces peucetius var. caesius, is the hydroxylated congener of daunorubicin. Doxorubicin intercalates between base pairs in the DNA helix, thereby preventing DNA replication and ultimately inhibiting protein synthesis. Additionally, doxorubicin inhibits topoisomerase II which results in an increased and stabilized cleavable enzyme-DNA linked complex during DNA replication and subsequently prevents the ligation of the nucleotide strand after double-strand breakage. Doxorubicin also forms oxygen free radicals resulting in cytotoxicity secondary to lipid peroxidation of cell membrane lipids; the formation of oxygen free radicals also contributes to the toxicity of the anthracycline antibiotics, namely the cardiac and cutaneous vascular effects. Doxorubicin hydrochloride drug (Adriblastina), was purchased from Pharmacia Italia S.P.A. (Italy). The drug concentration (2 mg/ ml) was diluted to 0.1 mg/ml, and divided into five test tubes, the first is used as control and the others is exposed to 2Gy, 5Gy, 20Gy, and 100Gy respectively. The samples were diluted to a five different concentrations. The studied samples were irradiated by 60Co gamma rays presented at King Saud University, College of Science, Riyadh, Kingdom
2.1.3. The absorption spectrum The absorption spectrum of the samples were measured using UV/Visible double beam spectrophotometer type 1650 PC manufactured by Shimatzu (Japan), in the wavelength range from 200 to 800 nm. 2.2. In-Vivo studies 2.2.1. Experimental design Normal SWR/J male mouse, 8e10 weeks old and weighing 28±3g were used throughout the study. Animals were maintained under standard laboratory at a temperature of 24 ± 1 C, a relative humidity of 45 ± 5% and photoperiod cycle of 12/12 h. Mice food (commercially available in Saudi Arabia) and water were offered ad libitum. Male mice were grouped into six groups each group
Fig. 1. Structure of doxorubicin.
Please cite this article in press as: Alshammari, F. M., et al., Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin, Journal of Radiation Research and Applied Sciences (2017), http://dx.doi.org/10.1016/j.jrras.2017.01.001
F.M. Alshammari et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e7
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Fig. 4(aec) shows the Cole- Cole plot (ε00 Vs. ε0 ) for the studied drug samples before and after exposure to 5, and100Gy of gamma irradiation. The dielectric increment Dε, the relaxation frequency fc, the relaxation time t, the Cole- Cole parameter a, and the deviation angle q were calculated for all studied drug samples and summarized in Table 1. It is clear from the figures and table that, the electrical conductivity s still constant for all the samples, the dielectric increment does not changes for the irradiated samples. The critical frequency and relaxation time remain constant for all the samples before and after irradiation. 3.2. In-Vivo studies
Fig. 2. The variation of electrical conductivity s (U. m)1 as a function of frequency f (Hz) for all drug samples.
containing three mice: the first group were used as control (without drug or radiation), second group were injected with 1 ml of unirradiated doxorubicin drug. Groups from third to sixth were injected with 1 ml of doxorubicin and exposed to different gamma doses of 2, 5, 20, and 100Gy respectively after one hour of injection. 2.2.2. Blood preparation After one hour of the injection, blood samples were directly collected from all the groups in EDTA tubes and prepared for analysis as following: 1 Erythrocytes (RBCs) were isolated from the whole blood by centrifugation at 2500 r.p.m for 10 min at room temperature. Three layers are obtained: red blood cells (RBCs), buffy coat (BC) layer (the second layer) and platelet-poor plasma (PPP). 2 The plasma was removed and RBCs stored for further investigation. 3 The buffy coat (BC) layer was pulled and stored in another tube with acetone, then re-centrifuged to extract the chromophore, to be used in the fluorimetric measurements. 4 The packed red blood cells were washed with 5 vol of normal saline solution and gently agitated for 2 min, then recentrifuged to separate the washed RBCs. This step was repeated three times. 5 The washed RBCs were lysed with two volumes of distilled water to extract HB, and kept two hours to insure lysis of cells. 6 The mixture was then centrifuged at 2500 r.p.m for 10 min at room temperature. The supernatant (HB-solution) were used for the dielectric and spectrophotometer measurements. 7 Before the measurements the (HB-solution) was diluted to a desired concentration by using the relation C1 V1 ¼ C2 V2 , where C1, V1, and C2, V2 are concentrations and volumes before and after dilution respectively.
The dielectric measurements are made for hemoglobin (Hbsolution) at suitable concentration which includes the Hb before and after the injection with drug and before and after exposed to different gamma doses. The variation of the electrical conductivity s in (U. m)1 as a function of frequency f in Hz for the Hb sample before injection (control) and after injection (exposed to gamma irradiation dose of 0, 2, 5, 20, and 100Gy respectively) are shown in Fig. 5. The variation of relative permittivity ε0 as a function of frequency f in Hz for the Hb sample before injection, and the Hb samples after injection (exposed to gamma irradiation dose of 0, 2, 5, 20, and 100Gy respectively) is shown in Fig. 6. Fig. 7(aed) show the Cole- Cole plot (ε00 Vs. ε0 ) for Hb sample before injection, and the Hb samples after injection exposed to gamma irradiation dose of 0, 5, and 100Gy respectively. The dielectric parameters for all studied blood samples are summarized in Table 2. It is clear from the figures and table that, the dielectric increment, and critical frequency are decreased after injection of drug and increase at 2 Gy irradiation dose and decreased as the irradiation dose increases. The Cole-Cole parameter is increased with radiation dose. Ultravioletevisible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in the ultravioletevisible spectral region. In this part of the work, the spectroscopic measurements were recorded in the wavelength range from 200 up to 800 nm. The absorption spectra of the drug samples at desired concentrations for exposed samples to gamma doses of 0, 2, 5, 20, and 100Gy is shown in Fig. 8. Then, the variation of the absorbance at
3. Results 3.1. In-Vitro studies The variation of the electrical conductivity sin (U. m)1 as a function of frequency f (Hz), for all the drug samples is shown in Fig. 2. The variation of relative permittivity ε0 as a function of frequency f(Hz) for all the drug samples is shown in Fig. 3. From Fig. 3, the relaxation frequency fc was measured and then the relaxation time t was calculated. Furthermore, the dielectric increment Dε was calculated.
Fig. 3. The variation of relative permittivity ε0 as a function of frequency f (Hz) for all drug samples.
Please cite this article in press as: Alshammari, F. M., et al., Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin, Journal of Radiation Research and Applied Sciences (2017), http://dx.doi.org/10.1016/j.jrras.2017.01.001
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F.M. Alshammari et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e7 Table 1 The dielectric increment Dε, the relaxation frequency fc, the relaxation time t, the angle q and Cole- Cole parameter a, for all the studied drug samples. Exposure dose (Gy)
Dε 108
fc (Hz)
t (msec)
q
a ¼ 2pq
Unexposed (Control) 2 5 20 100
4.46 4.51 4.46 4.47 4.47
510 510 510 510 510
0.312 0.312 0.312 0.312 0.312
23 25 23 22 22
0.256 0.278 0.256 0.244 0.244
Fig. 5. The variation of electrical conductivity s (U. m)1 as a function of frequency f (Hz) for all Hb samples.
The characteristic absorbance bands of HB are shown in Fig. 10. The absorbance value of the characteristic bands and the different absorbance ratios for all samples of hemoglobin are shown in Table 4. The results indicate that, the absorbance at characteristic bands is increased, and the absorbance ratio is decreased as the irradiation dose increases. 4. Discussion The effects of Gamma irradiation on anticancer and other drugs are remarkably increased in recent years. The purposes of exposure
Fig. 4. The variation of ε00 Vs. ε0 (Cole - Cole plot) for a-unexposed drug sample, b-exposed to 5Gy, c-exposed to 100 Gy of gamma dose.
specific wavelength (l ¼ 480 nm) as a function of concentration is shown in Fig. 9. The molar absorption coefficients (ε) for the studied samples are calculated from these figures by using Beer-Lambert law, these coefficients are summarized Table 3. It is clear from the results that the values of the molar absorption coefficient are remained constant for all studied samples. These measurements were also applied with HB samples at desired concentration. The absorption spectrum of the HB have a five characteristic absorbance bands: Globin band at 273 nm, Globin band at 346 nm (Globin-heme interaction), Soret band at 416 nm, Hemo band at 542 nm, and Hemo band at 578 nm (Hemeheme interaction).
Fig. 6. The variation of electrical relative permittivity ε0 as a function of frequency f (Hz) for the all Hb sample.
Please cite this article in press as: Alshammari, F. M., et al., Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin, Journal of Radiation Research and Applied Sciences (2017), http://dx.doi.org/10.1016/j.jrras.2017.01.001
F.M. Alshammari et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e7
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Fig. 7. The variation of ε00 Vs. ε0 (Cole - Cole plot) for the Hb samples: a- Before injection, b- Exposed to 0Gy, c- Exposed to 5Gy, d- Exposed to100 Gy of gamma dose respectively.
Table 2 The dielectric parameters for all the studied Hb samples. Exposure dose (Gy)
Dε 104
fc (Hz)
t (msec)
q
a ¼ 2pq
Pure blood Unexposed (Control) 2 5 20 100
8.26 3.76 9.26 3.57 3.97 4.56
120 80 100 80 50 80
1.32 1.99 1.59 1.99 3.18 1.99
24 21 28 23 29 27
0.267 0.233 0.311 0.256 0.322 0.3
Fig. 8. The absorption spectrum of the drug samples at desired concentration.
of these drugs to radiation were to enhance their functional activity on tumour cells (Smeltzer et al., 2015; Sung et al., 2013), for sterilization purposes (Maksimenko et al., 2008; Varshney & Dodke, 2004) or to reduce their immunological toxicity (Kim et al., 2009). The objective of this study was to evaluate the effect of gamma irradiation on the structural stability of one of the most famous anticancer drugs, doxorubicin, (DOXO) (Varshney & Dodke, 2004). DOXO is an anticancer drug used in the treatment of various types of cancers. The cytotoxicity of the drug is due to its ability to interact with DNA and plasma membrane and to participate in
Fig. 9. The variation of the absorbance as a function of concentration.
Please cite this article in press as: Alshammari, F. M., et al., Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin, Journal of Radiation Research and Applied Sciences (2017), http://dx.doi.org/10.1016/j.jrras.2017.01.001
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F.M. Alshammari et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e7 Table 3 The molar absorption coefficients (ε) for all the studied drug samples. Dose (Gy)
ε (L.mg1.cm1)
0 2 5 20 100
0.023 0.024 0.023 0.023 0.023
Fig. 10. The hemoglobin absorption spectrum at desired concentration for different samples.
various oxidationereduction reactions. The obtained result will be of great value to assess the overlapping interaction between radiotherapy and chemotherapy during cancer treatment. The molecular structure of doxorubicin was studied through measurements of dielectric relaxation of the drug molecules in the frequency range 20 Hz e 3 MHz which in a and b range in addition to measurements of absorption spectrum. It is clear from the data that the permittivity (ε0 ) passed through a dielectric dispersion in the frequency range demonstrated corresponding to alpha relaxation region and the decrease in the values of (ε0 ) was accompanied by an increase in the values of conductivity (s). The low frequency a dispersion is associated with ionic diffusion processes at the site of the cellular membrane (Foster & Schwan, 1996). The values of dielectric parameters of dielectric increment (Dε),
conductivity (s) and relaxation time t, show unnoticeable change for drug molecules before and after irradiation with different gamma dose as compared with control indicating a stable behavior of the drug under different irradiation dose. In this context, Varshney and Dodke (2004) studied the effect of gamma radiation on doxorubicin for sterilization purpose. They have shown that the concentration of radiation degradation product of the drug is estimated to be less than 0.01% at 30 kGy. Moreover, no significant changes were observed in the colour of the irradiated DOXO with respect to its original orange-red colour. It is well known that the dielectric dispersion in the frequency range used for biological cells is mainly due to counter-ion polarization of the studied molecules (Pethig, 1979). Therefore, the increased values of permittivity (ε0 ) and conductivity (s) of hemoglobin indicate an increase of the counter ion molecules intensities for hemoglobin rate exposed to gamma irradiation along with anticancer drug treatment. Hence an increase in the whole surface charge and charge distribution are expected and may a result of changes in the metabolic functions. Moreover, the results indicate an increase in the values of relaxation time (t) and Cole- Cole parameter a as compared with control, this changes indicate to the changes in the conformational structure in hemoglobin molecules which may affect their properties and hence RBC's physiological functions (Ali et al., 2003). The absorption spectra of hemoglobin molecules show an increase in the amplitude of the characteristic bands with irradiation dose in addition to decrease in the ratio of (A578/A542) indicate an increase of the oxygen binding capacity with hemoglobin. It is clear from the results that the molar absorption coefficient is still constant for all the drug samples. Moreover, the increase of the Hemo band (578 nm) and Globin band (273 nm) confirm the alteration of stabilization and conformational of the hemoglobin molecules. Based on the above results, our finding indicates that gamma irradiation clearly decreases the toxicity of doxorubicin on nontarget cells. These results are in agreement with findings of Sung et al. (2013) and Kim et al., (2009), who concluded that gamma irradiation could be regarded as a potential method for reducing the immunological toxicity of anti-cancer drugs including doxorubicin. Therefore, the gamma irradiation could be considered as a useful technology for the reduction of the toxicity of the anticancer drug lectin without compromising its bioactivity.
5. Conclusion The dielectric data of the studied drug indicates that they are characterized by a dielectric a dispersion. After drug injection
Table 4 The absorbance values of the characteristic bands and the different absorbance ratios for all studied HB samples. Dose (Gy)
Globin band 273 nm
Globin band 346 nm
Soret band 416 nm
Soret band width (nm) FWAHM
Hemo band 542 nm
Hemo band at 578 nm
Ratio A578/ A542
395e430 Dl ¼ 35 nm 393e430 Dl ¼ 37 nm 395e430 Dl ¼ 35 nm 395e430 Dl ¼ 35 nm 397e427 Dl ¼ 30 nm 395e430 Dl ¼ 35 nm
0.065 (541)
0.069 (576.5)
1.0713
0.099 (541.5)
0.104 (576.5)
1.0453
0.117 (541)
0.123 (576.5)
1.0531
0.119 (541)
0.124 (576.5)
1.0498
0.123 (541)
0.129 (576.5)
1.0464
0.173 (541)
0.179 (576.5)
1.0353
Pure blood (without 0.163 (274.5) drug) Unexposed (with drug) 0.262 (274.5)
0.126 (345)
0.604 (414)
0.200 (345)
0.886 (414)
2Gy
0.307 (274.5)
0.232 (345.5)
1.044 (414)
5Gy
0.308 (274.5)
0.235 (345)
1.035 (414)
20Gy
0.334 (274)
0.249 (345.5)
1.106 (414)
100Gy
0.474 (274)
0.349 (345.5)
1.439 (414)
Please cite this article in press as: Alshammari, F. M., et al., Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin, Journal of Radiation Research and Applied Sciences (2017), http://dx.doi.org/10.1016/j.jrras.2017.01.001
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there is a change in the measured parameters, so the drug affects the blood and consequently affects the organ metabolism. The combination between the probable drug and gamma irradiation can be used as a powerful conjunction that may give us the benefit of chemo and radiotherapy treatment. This highly versatile system will aid in improving the clinical treatment against serious diseases. References Ali, Fadel M., Mohamed, Wael S., & Mohamed, Mostafa R. (2003). Effect of 50 Hz, 0.2 mT magnetic fields on RBC properties and heart function of albino rats. Bioelectromagnetic, 24, 535e545. Chantal, Simon, Waston, Max, Drake, Anne, Fenton, Audrey, & Mcloughlin, Caroline (2008). Principles of cancer treatment. Oxford Journals, 1(2), 107e118. Dai, Xiaowen, Yue, Zhilian, Eccleston, Mark E., Swartling, Johannes, Slater Nigel, K. H., & Kaminski Clemens, F. (2008). Fluorescence intensity and lifetime imaging of free and micellar encapsulated doxorubicin in living cells. Nanomedicine: Nanotechnology, Biology, and Medicine, 4, 49e56. Edward, T. H. Yeh, Tong, Ann T., Lenihan, Daniel J., Wamique Yusuf, S., Swafford, Joseph, Champion, Christopher, et al. (2004). Cardiovascular complications of cancer therapy (diagnosis, pathogenesis, and management). Circulation, 109, 3122e3131. Foster, K. R., & Schwan, H. P. (1996). Dielectric properties of tissues. In C. Polk, & E. Postow (Eds.), A review in Handbook of biological effect of electromagnetic field (2nd ed., pp. 25e102). pages: New York CRC Press. Franco, M. Muggia, Cortes-Funes, Hernan, & Wasserman, Todd H. (1978). Radiotherapy and chemotherapy in combined clinical trials: Problems and promise. International Journal Radiation Oncology Biology Physics, 4, 161e171. Grant, E. H., Sheppard, R. J., & South, G. P. (1978). Dielectric behaviour of biological molecules in solution (Oxford). Ian, F. Tannock (1989). Combined modality treatment with radiotherapy and chemotherapy. Radiotherapy and Oncology, 16(2), 83e101. Jaishree, Bhosle, & Geoff, Hall (2009). Principles of cancer treatment by
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Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin Fatmah M. Alshammari a, Sonia M. Reda a, b, *, Magdy M. Ghannam c a
Department of Physics, Faculty of Science, Hail University, KSA Department of Physics, Faculty of Science, Zagazig University, Egypt c Department of Biophysics, Faculty of Science, Cairo University, Egypt b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 November 2016 Received in revised form 27 January 2017 Accepted 28 January 2017 Available online xxx
Cancer therapy has progressed dramatically in recent years. In order to decrease the dose and side effects of the anticancer drug, the therapeutic options for patients with cancer include increasingly complex combinations of chemotherapy and radiotherapy. This combination may cause overlapping interaction between the two types of treatment and affect the stability of the anticancer drug. In this study, the effect of gamma irradiation on the stability and therapeutic activity of one anticancer drug (Doxorubicin) was studied. For this purpose, doxorubicin drug characterized by two methods, at first, in-vitro study, before and after drug irradiation with different doses of gamma rays (2, 5, 20, 100 GY) which achieved through measuring the dielectric relaxation and absorption spectrum of drug solution. Secondly, in-vivo studies, where the unirradiated and the drug, which later exposed to gamma rays, were injected respectively into 6 groups of mice (3 mice in each group). The dielectric relaxation and absorption spectrum were studded for hemoglobin of the injected mice. The results for the in-vitro study indicate that the values of dielectric parameters show unnoticeable change for drug molecules before and after irradiation as compared with the control. The results for in-vivo study indicated an increase in the values of relaxation time and ColeCole parameter, that may as a result of changes in the conformational structure in hemoglobin molecules which may affect their properties and hence RBC's physiological functions. The absorption spectra of hemoglobin molecules show an increase in the amplitude of the characteristic bands with irradiation dose indicate an increase of the oxygen binding capacity with hemoglobin. It was concluded that combination between the drugs and gamma irradiation can be used as a powerful conjunction that may give us the benefit chemo and radiotherapy treatment. © 2017 The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Keywords: Radiotherapy Chemotherapy Gamma radiation Doxorubicin Overlapping Stability of anticancer drug
1. Introduction Cancer therapy has progressed dramatically in recent years, and tremendous progress has been made in reducing the morbidity and mortality from many forms of cancer. The therapeutic options for patients with cancer include increasingly complex combinations of chemotherapy, radiotherapy and surgical intervention. (Chantal, Waston, Drake, Fenton, & Mcloughlin, 2008; Edward et al., 2004; Schultz, Beck, Stava, et al., 2003).
* Corresponding author. Department of Physics, Faculty of Science, Zagazig University, Egypt E-mail address: [email protected] (S.M. Reda). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications.
Radiotherapy is a crucial component of anticancer treatment; up to 50% of cancer patients receive radiotherapy. Apart from surgery, radiotherapy is the major method applied with curative intent for cancer patients. Radiotherapy plays a major part in the palliation of symptoms. (Stephen, 2009). On the other hand, cancer chemotherapy refers to the administration of cytotoxic chemicals, with the aim to, eradicate the tumour or reduce the tumour-related symptoms. Most chemotherapeutic drugs cause damage to DNA, which leads to programmed cell death (apoptosis) (Peter, 2001; Jaishree and Geoff, 2009). Doxorubicin is an anti-cancer drugs widely used in the treatment of various types of cancers. It belongs to antitumor antibiotics. The cytotoxicity of the drug is due to its ability to interact with DNA and plasma membrane and to participate in various oxidation reduction reactions. (Varshney & Dodke, 2004). It is intrinsically fluorescent which makes it convenient for
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Please cite this article in press as: Alshammari, F. M., et al., Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin, Journal of Radiation Research and Applied Sciences (2017), http://dx.doi.org/10.1016/j.jrras.2017.01.001
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F.M. Alshammari et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e7
probing and visualization with various microscopic imaging technologies (Dai et al., 2008). Combined modality treatment with radiotherapy and chemotherapy is used increasingly for the primary management of a variety of human tumors, with the aim of improving both local and distant control, decreasing distant metastasis, and improving survival without excessively increasing normal tissue injury. (Franco, Cortes-Funes, & Wasserman, 1978; Phillips & Karen, 1978; Ian, 1989; Karen, 1985; Maurice, Arriagada, & Cosset, 1985). In this combined treatment, radiation interacts with drugs and may effect on their quality. It was concluded that combination between the drugs and gamma irradiation can be used as a powerful conjunction that may give us the benefit chemo and radiotherapy treatment. This highly versatile system can be used in the testing of drug e organ disease interaction and so will aid in improving the clinical treatment protocol and fights against serious diseases. This work is aimed to evaluate the effects of gamma irradiation on the stability of anticancer drugs. Moreover, study the overlapping interaction between radiotherapy and chemotherapy during cancer fitting is the main target of the presented work. For this purpose, doxorubicin, one of the most widely used anticancer drugs was chosen. The stability of the drug upon application of gamma irradiation will be achieved through measurements of electrical properties. Dielectric measurements can provide valuable significant information about the molecular arrangement of the drugs as well as the electrical conduction mechanism (Grant, Sheppard, & South, 1978). Furthermore, using UVeVisible spectroscopy can derive useful information on the biological molecules. This information can lead to valuable structural proposals (Pavia, Lampman, Kriz, & Vyvyan, 2009). The overlapping interaction between radiotherapy and the used drug will be achieved by studying the effect of irradiation on the therapeutic activity of the used chemotherapeutic drug.
of Saudi Arabia with activity of 0.44 Gy/sec using gamma cell 220 manufactured by NORDION (Canada). Physical methods were taken to characterize the drugs include dielectric relaxation and absorption spectrum of samples. 2.1.2. Dielectric relaxation Electric measurements were investigated in the frequency range from 20 Hz up to 3 MHz (a & b dispersions of biological materials) using a WAYNE KERR precision component analyzer model 6440B (UK). The sample cell has two squared platinum black electrodes with cell constant k ¼ 1 cm1. The measurements were performed at room temperature. For a dielectric material placed between two parallel plates capacitor, the measured values of the capacitance, C and resistance R were used to calculate the conductivity s, as well as the real, ε0 , and imaginary, ε00 , parts of the complex permittivity and the relaxation t time through the following equations: C 13 C i) ε0 ¼ CCo ¼ εCo k ¼ εCo Ad ¼ 8:8510 12 100 ¼ 1:13 10 εo is the permittivity of free space (Foster & Schwan, 1996). 00
ii) Loss tangent factor D ¼ εε0 ¼ 2p1RCf 1 1 iii) The conductivity s ¼ R1A ¼ Rk ¼ 100 R ðU m Þ ð dÞ 00 iv) The dielectric loss ε ¼ Dε0 v) The dielectric increment Dε ¼ ε1 εo vi) Relaxation time t ¼ 2p1f , fc is the critical frequency correc sponding to the midpoint of the dispersion curve. vii) The plot ε0 Vs ε00 (Cole-Cole plot) will produced a semi-circle. It was shown by Cole and Cole that the angle traced between the circle radius and ε0 axis is q ¼ ap/2. This in turn enable to estimate the Cole-Cole parameter a, experimentally, so a ¼ 2q/p (Koji Asami, 2002).
2. Materials and methods In this work, the study of the interaction between gamma radiations with anti-cancer drugs was divided into two main parts: 2.1. In-Vitro studies 2.1.1. Drug Doxorubicin is an anthraxcycline antibiotic with antineoplastic activity. The molecular stucture of the drug is shown in Fig. 1. Doxorubicin, isolated from the bacterium Streptomyces peucetius var. caesius, is the hydroxylated congener of daunorubicin. Doxorubicin intercalates between base pairs in the DNA helix, thereby preventing DNA replication and ultimately inhibiting protein synthesis. Additionally, doxorubicin inhibits topoisomerase II which results in an increased and stabilized cleavable enzyme-DNA linked complex during DNA replication and subsequently prevents the ligation of the nucleotide strand after double-strand breakage. Doxorubicin also forms oxygen free radicals resulting in cytotoxicity secondary to lipid peroxidation of cell membrane lipids; the formation of oxygen free radicals also contributes to the toxicity of the anthracycline antibiotics, namely the cardiac and cutaneous vascular effects. Doxorubicin hydrochloride drug (Adriblastina), was purchased from Pharmacia Italia S.P.A. (Italy). The drug concentration (2 mg/ ml) was diluted to 0.1 mg/ml, and divided into five test tubes, the first is used as control and the others is exposed to 2Gy, 5Gy, 20Gy, and 100Gy respectively. The samples were diluted to a five different concentrations. The studied samples were irradiated by 60Co gamma rays presented at King Saud University, College of Science, Riyadh, Kingdom
2.1.3. The absorption spectrum The absorption spectrum of the samples were measured using UV/Visible double beam spectrophotometer type 1650 PC manufactured by Shimatzu (Japan), in the wavelength range from 200 to 800 nm. 2.2. In-Vivo studies 2.2.1. Experimental design Normal SWR/J male mouse, 8e10 weeks old and weighing 28±3g were used throughout the study. Animals were maintained under standard laboratory at a temperature of 24 ± 1 C, a relative humidity of 45 ± 5% and photoperiod cycle of 12/12 h. Mice food (commercially available in Saudi Arabia) and water were offered ad libitum. Male mice were grouped into six groups each group
Fig. 1. Structure of doxorubicin.
Please cite this article in press as: Alshammari, F. M., et al., Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin, Journal of Radiation Research and Applied Sciences (2017), http://dx.doi.org/10.1016/j.jrras.2017.01.001
F.M. Alshammari et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e7
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Fig. 4(aec) shows the Cole- Cole plot (ε00 Vs. ε0 ) for the studied drug samples before and after exposure to 5, and100Gy of gamma irradiation. The dielectric increment Dε, the relaxation frequency fc, the relaxation time t, the Cole- Cole parameter a, and the deviation angle q were calculated for all studied drug samples and summarized in Table 1. It is clear from the figures and table that, the electrical conductivity s still constant for all the samples, the dielectric increment does not changes for the irradiated samples. The critical frequency and relaxation time remain constant for all the samples before and after irradiation. 3.2. In-Vivo studies
Fig. 2. The variation of electrical conductivity s (U. m)1 as a function of frequency f (Hz) for all drug samples.
containing three mice: the first group were used as control (without drug or radiation), second group were injected with 1 ml of unirradiated doxorubicin drug. Groups from third to sixth were injected with 1 ml of doxorubicin and exposed to different gamma doses of 2, 5, 20, and 100Gy respectively after one hour of injection. 2.2.2. Blood preparation After one hour of the injection, blood samples were directly collected from all the groups in EDTA tubes and prepared for analysis as following: 1 Erythrocytes (RBCs) were isolated from the whole blood by centrifugation at 2500 r.p.m for 10 min at room temperature. Three layers are obtained: red blood cells (RBCs), buffy coat (BC) layer (the second layer) and platelet-poor plasma (PPP). 2 The plasma was removed and RBCs stored for further investigation. 3 The buffy coat (BC) layer was pulled and stored in another tube with acetone, then re-centrifuged to extract the chromophore, to be used in the fluorimetric measurements. 4 The packed red blood cells were washed with 5 vol of normal saline solution and gently agitated for 2 min, then recentrifuged to separate the washed RBCs. This step was repeated three times. 5 The washed RBCs were lysed with two volumes of distilled water to extract HB, and kept two hours to insure lysis of cells. 6 The mixture was then centrifuged at 2500 r.p.m for 10 min at room temperature. The supernatant (HB-solution) were used for the dielectric and spectrophotometer measurements. 7 Before the measurements the (HB-solution) was diluted to a desired concentration by using the relation C1 V1 ¼ C2 V2 , where C1, V1, and C2, V2 are concentrations and volumes before and after dilution respectively.
The dielectric measurements are made for hemoglobin (Hbsolution) at suitable concentration which includes the Hb before and after the injection with drug and before and after exposed to different gamma doses. The variation of the electrical conductivity s in (U. m)1 as a function of frequency f in Hz for the Hb sample before injection (control) and after injection (exposed to gamma irradiation dose of 0, 2, 5, 20, and 100Gy respectively) are shown in Fig. 5. The variation of relative permittivity ε0 as a function of frequency f in Hz for the Hb sample before injection, and the Hb samples after injection (exposed to gamma irradiation dose of 0, 2, 5, 20, and 100Gy respectively) is shown in Fig. 6. Fig. 7(aed) show the Cole- Cole plot (ε00 Vs. ε0 ) for Hb sample before injection, and the Hb samples after injection exposed to gamma irradiation dose of 0, 5, and 100Gy respectively. The dielectric parameters for all studied blood samples are summarized in Table 2. It is clear from the figures and table that, the dielectric increment, and critical frequency are decreased after injection of drug and increase at 2 Gy irradiation dose and decreased as the irradiation dose increases. The Cole-Cole parameter is increased with radiation dose. Ultravioletevisible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in the ultravioletevisible spectral region. In this part of the work, the spectroscopic measurements were recorded in the wavelength range from 200 up to 800 nm. The absorption spectra of the drug samples at desired concentrations for exposed samples to gamma doses of 0, 2, 5, 20, and 100Gy is shown in Fig. 8. Then, the variation of the absorbance at
3. Results 3.1. In-Vitro studies The variation of the electrical conductivity sin (U. m)1 as a function of frequency f (Hz), for all the drug samples is shown in Fig. 2. The variation of relative permittivity ε0 as a function of frequency f(Hz) for all the drug samples is shown in Fig. 3. From Fig. 3, the relaxation frequency fc was measured and then the relaxation time t was calculated. Furthermore, the dielectric increment Dε was calculated.
Fig. 3. The variation of relative permittivity ε0 as a function of frequency f (Hz) for all drug samples.
Please cite this article in press as: Alshammari, F. M., et al., Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin, Journal of Radiation Research and Applied Sciences (2017), http://dx.doi.org/10.1016/j.jrras.2017.01.001
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F.M. Alshammari et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e7 Table 1 The dielectric increment Dε, the relaxation frequency fc, the relaxation time t, the angle q and Cole- Cole parameter a, for all the studied drug samples. Exposure dose (Gy)
Dε 108
fc (Hz)
t (msec)
q
a ¼ 2pq
Unexposed (Control) 2 5 20 100
4.46 4.51 4.46 4.47 4.47
510 510 510 510 510
0.312 0.312 0.312 0.312 0.312
23 25 23 22 22
0.256 0.278 0.256 0.244 0.244
Fig. 5. The variation of electrical conductivity s (U. m)1 as a function of frequency f (Hz) for all Hb samples.
The characteristic absorbance bands of HB are shown in Fig. 10. The absorbance value of the characteristic bands and the different absorbance ratios for all samples of hemoglobin are shown in Table 4. The results indicate that, the absorbance at characteristic bands is increased, and the absorbance ratio is decreased as the irradiation dose increases. 4. Discussion The effects of Gamma irradiation on anticancer and other drugs are remarkably increased in recent years. The purposes of exposure
Fig. 4. The variation of ε00 Vs. ε0 (Cole - Cole plot) for a-unexposed drug sample, b-exposed to 5Gy, c-exposed to 100 Gy of gamma dose.
specific wavelength (l ¼ 480 nm) as a function of concentration is shown in Fig. 9. The molar absorption coefficients (ε) for the studied samples are calculated from these figures by using Beer-Lambert law, these coefficients are summarized Table 3. It is clear from the results that the values of the molar absorption coefficient are remained constant for all studied samples. These measurements were also applied with HB samples at desired concentration. The absorption spectrum of the HB have a five characteristic absorbance bands: Globin band at 273 nm, Globin band at 346 nm (Globin-heme interaction), Soret band at 416 nm, Hemo band at 542 nm, and Hemo band at 578 nm (Hemeheme interaction).
Fig. 6. The variation of electrical relative permittivity ε0 as a function of frequency f (Hz) for the all Hb sample.
Please cite this article in press as: Alshammari, F. M., et al., Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin, Journal of Radiation Research and Applied Sciences (2017), http://dx.doi.org/10.1016/j.jrras.2017.01.001
F.M. Alshammari et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e7
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Fig. 7. The variation of ε00 Vs. ε0 (Cole - Cole plot) for the Hb samples: a- Before injection, b- Exposed to 0Gy, c- Exposed to 5Gy, d- Exposed to100 Gy of gamma dose respectively.
Table 2 The dielectric parameters for all the studied Hb samples. Exposure dose (Gy)
Dε 104
fc (Hz)
t (msec)
q
a ¼ 2pq
Pure blood Unexposed (Control) 2 5 20 100
8.26 3.76 9.26 3.57 3.97 4.56
120 80 100 80 50 80
1.32 1.99 1.59 1.99 3.18 1.99
24 21 28 23 29 27
0.267 0.233 0.311 0.256 0.322 0.3
Fig. 8. The absorption spectrum of the drug samples at desired concentration.
of these drugs to radiation were to enhance their functional activity on tumour cells (Smeltzer et al., 2015; Sung et al., 2013), for sterilization purposes (Maksimenko et al., 2008; Varshney & Dodke, 2004) or to reduce their immunological toxicity (Kim et al., 2009). The objective of this study was to evaluate the effect of gamma irradiation on the structural stability of one of the most famous anticancer drugs, doxorubicin, (DOXO) (Varshney & Dodke, 2004). DOXO is an anticancer drug used in the treatment of various types of cancers. The cytotoxicity of the drug is due to its ability to interact with DNA and plasma membrane and to participate in
Fig. 9. The variation of the absorbance as a function of concentration.
Please cite this article in press as: Alshammari, F. M., et al., Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin, Journal of Radiation Research and Applied Sciences (2017), http://dx.doi.org/10.1016/j.jrras.2017.01.001
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F.M. Alshammari et al. / Journal of Radiation Research and Applied Sciences xxx (2017) 1e7 Table 3 The molar absorption coefficients (ε) for all the studied drug samples. Dose (Gy)
ε (L.mg1.cm1)
0 2 5 20 100
0.023 0.024 0.023 0.023 0.023
Fig. 10. The hemoglobin absorption spectrum at desired concentration for different samples.
various oxidationereduction reactions. The obtained result will be of great value to assess the overlapping interaction between radiotherapy and chemotherapy during cancer treatment. The molecular structure of doxorubicin was studied through measurements of dielectric relaxation of the drug molecules in the frequency range 20 Hz e 3 MHz which in a and b range in addition to measurements of absorption spectrum. It is clear from the data that the permittivity (ε0 ) passed through a dielectric dispersion in the frequency range demonstrated corresponding to alpha relaxation region and the decrease in the values of (ε0 ) was accompanied by an increase in the values of conductivity (s). The low frequency a dispersion is associated with ionic diffusion processes at the site of the cellular membrane (Foster & Schwan, 1996). The values of dielectric parameters of dielectric increment (Dε),
conductivity (s) and relaxation time t, show unnoticeable change for drug molecules before and after irradiation with different gamma dose as compared with control indicating a stable behavior of the drug under different irradiation dose. In this context, Varshney and Dodke (2004) studied the effect of gamma radiation on doxorubicin for sterilization purpose. They have shown that the concentration of radiation degradation product of the drug is estimated to be less than 0.01% at 30 kGy. Moreover, no significant changes were observed in the colour of the irradiated DOXO with respect to its original orange-red colour. It is well known that the dielectric dispersion in the frequency range used for biological cells is mainly due to counter-ion polarization of the studied molecules (Pethig, 1979). Therefore, the increased values of permittivity (ε0 ) and conductivity (s) of hemoglobin indicate an increase of the counter ion molecules intensities for hemoglobin rate exposed to gamma irradiation along with anticancer drug treatment. Hence an increase in the whole surface charge and charge distribution are expected and may a result of changes in the metabolic functions. Moreover, the results indicate an increase in the values of relaxation time (t) and Cole- Cole parameter a as compared with control, this changes indicate to the changes in the conformational structure in hemoglobin molecules which may affect their properties and hence RBC's physiological functions (Ali et al., 2003). The absorption spectra of hemoglobin molecules show an increase in the amplitude of the characteristic bands with irradiation dose in addition to decrease in the ratio of (A578/A542) indicate an increase of the oxygen binding capacity with hemoglobin. It is clear from the results that the molar absorption coefficient is still constant for all the drug samples. Moreover, the increase of the Hemo band (578 nm) and Globin band (273 nm) confirm the alteration of stabilization and conformational of the hemoglobin molecules. Based on the above results, our finding indicates that gamma irradiation clearly decreases the toxicity of doxorubicin on nontarget cells. These results are in agreement with findings of Sung et al. (2013) and Kim et al., (2009), who concluded that gamma irradiation could be regarded as a potential method for reducing the immunological toxicity of anti-cancer drugs including doxorubicin. Therefore, the gamma irradiation could be considered as a useful technology for the reduction of the toxicity of the anticancer drug lectin without compromising its bioactivity.
5. Conclusion The dielectric data of the studied drug indicates that they are characterized by a dielectric a dispersion. After drug injection
Table 4 The absorbance values of the characteristic bands and the different absorbance ratios for all studied HB samples. Dose (Gy)
Globin band 273 nm
Globin band 346 nm
Soret band 416 nm
Soret band width (nm) FWAHM
Hemo band 542 nm
Hemo band at 578 nm
Ratio A578/ A542
395e430 Dl ¼ 35 nm 393e430 Dl ¼ 37 nm 395e430 Dl ¼ 35 nm 395e430 Dl ¼ 35 nm 397e427 Dl ¼ 30 nm 395e430 Dl ¼ 35 nm
0.065 (541)
0.069 (576.5)
1.0713
0.099 (541.5)
0.104 (576.5)
1.0453
0.117 (541)
0.123 (576.5)
1.0531
0.119 (541)
0.124 (576.5)
1.0498
0.123 (541)
0.129 (576.5)
1.0464
0.173 (541)
0.179 (576.5)
1.0353
Pure blood (without 0.163 (274.5) drug) Unexposed (with drug) 0.262 (274.5)
0.126 (345)
0.604 (414)
0.200 (345)
0.886 (414)
2Gy
0.307 (274.5)
0.232 (345.5)
1.044 (414)
5Gy
0.308 (274.5)
0.235 (345)
1.035 (414)
20Gy
0.334 (274)
0.249 (345.5)
1.106 (414)
100Gy
0.474 (274)
0.349 (345.5)
1.439 (414)
Please cite this article in press as: Alshammari, F. M., et al., Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin, Journal of Radiation Research and Applied Sciences (2017), http://dx.doi.org/10.1016/j.jrras.2017.01.001
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Please cite this article in press as: Alshammari, F. M., et al., Potential effects of gamma irradiation on the stability and therapeutic activity of anticancer drug, doxorubicin, Journal of Radiation Research and Applied Sciences (2017), http://dx.doi.org/10.1016/j.jrras.2017.01.001