Republic of Iraq Ministry of Higher Education And Scientific Research University of Baghdad College of Science
Radiation Induced DNA damage
A Thesis Submitted to the Council of the College of Science University of Baghdad in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Physics By
Kharman Akram Faraj
Prof. Dr. Mazin M. Elias
…… 2008
and
Dr. Asia H. Al-Mashhadani
…….. 1429
The experiments were performed at the Cellular and Molecular Laboratory, Nuclear Research Center (SCK.CEN), MOL- Belgium, under a direct supervision of Dr. Sarah Baatout. Head of the Radiobiology Department.
Certificate We certify that the preparation of this thesis, entitled "Radiation Induced DNA damage" was made under our supervision by Kharman Akram Faraj at the College of Science University of Baghdad in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physics (Radiobiology).
Signature:
Signature:
Name: Prof. Mazin M. Elias
Name: Dr. Asia H. Al-Mashhadani
Title: Professor, University of Baghdad
Title: Assist. Professor, University of Baghdad
Date:
Date:
In view of the available recommendations, I forward this thesis for debate by the Examining Committee.
Signature: Name: Title: Head of Physics Department, College of Science Date
And say: Work; so Allah will see your work and (so will) His Messenger and the believers; and you shall be brought back to the Knower of the unseen and the seen, then He will inform you of what you did {105} And others are made to await Allah's command, whether He chastise them or whether He turn to them (mercifully), and Allah is Knowing, Wise{106}
Dedication
To:
Memory of my parents
My brother and sisters
Acknowledgements This project underlying this thesis emerged from collaboration between university of Baghdad and Belgian Nuclear research Center "SCK.CEN" in Belgium. First and foremost, I would to thank my first supervisor Prof. Dr. Mazin M. Elias for his guidance, patience, support, insightful advice and very useful thoughts throughout my Ph.D study. I learned a lot from him I appreciate his enthusiasm about science in general. I consider myself extremely fortunate to have a supervisor like him. A very special thank to my second supervisor Dr. Asia H. Al-Mashhadani for her support and encouragement. My deep and sincere gratitude goes to my supervisor Dr. Sarah Baatout in Belgium for offering me to go to there and start this work at the SCK.CEN. I would like to thank her for her help, scientific knowledge and her guidance. It is really an honor to work with a person like her. I am deeply grateful to the department of physics, college of science, university of Baghdad for their cooperation. I would like to thank all the people in the lab. especially Dr.Hanane Derradji which she provided me with technical help and support, she learned me a lot about working in the biology lab. I also appreciate all the volunteers in the center and all the staff in the medical service of the center which they took the blood from the volunteers for me. My deep gratitude goes to Ludo Melis and Bart Marlein for their technical assistance with the irradiation procedures. I also appreciate the financial support from university of Sulaimanya and SCK.CEN.
I would to acknowledge the dean of college of science university of Sulaimanya Dr.Parikhan Jaff for her support, help and her encouragement during my study. My sincere thanks to my friend Myriam Ghardi for her help, encouragement and fruitful discussions during my staying in Belgium. Finally, I sincerely appreciate my family, it was their prayers, love, support, and encouragement that carried me through these years.
Abstract The aim of this study was to investigate the effect of low-LET radiations; x rays and 60Co gamma rays on human cells. For this purpose, DNA damage (8oxoguanuine) and apoptosis were detected in normal human cells (Peripheral blood mononuclear cells PBMCs). For each detection, three different experiments were performed; for x rays the cells were irradiated with doses 0.25, 0.5, 1, 2 and 4 Gy at dose rate of 0.28 Gy/min and 15.6, 31.3, 62.5, 125 and 250 mGy at dose rate of 20 mGy/min, and for 60Co gamma rays the cells were irradiated with 0.25, 0.5, 1, 2 and 4 Gy at dose rat of 2 Gy/hr. Human blood samples each of 40 ml were taken from healthy nonsmoking donors in heparinized vacutainer tubes. Mononuclear cells were separated from the blood by centrifugation over histopaque 1077. After separation the cells washed twice with PBS. A 7 ml of culture medium (RPMI, supplemented with 20% FBS and 1% of penicillin and streptomycin) were added to pellet in the tube, then (5-10)x 106 cells/ml were prepared by counting with hemocytometer and this number of cells was transferred to each of 6 flask and then 5 ml of the culture medium were added to each of them. X-irradiation of the cells was performed with a Pantak HF420 RX machine operating to generate 208keV x-rays mean energy, while gamma irradiation of cells was performed with 60Co source, the mean energy of 1.25MeV. The irradiated cells were incubated for 24 hrs at 37oC in 5% CO2. For DNA damage (8-oxoguanine) detection, the cells were fixed with 2% paraformaldehyde and in ice-cold 70% ethanol. The day after flourescein isothiocyanate (FITC) labeled conjugate was added, then the amount of fluorescence- FITC in 8-oxoguanine was read by flow cytometry (cloture Epics XL-MCL). After calibration with sphero calibrated particles, the number of 8-oxoguanine molecules/cell was calculated. Relative granularity and size of mononuclear cells were determined.
For apoptosis detection, the cells were labeled with FITC-conjugate and the amount fluorescence FITC of activated caspase 3 was read by flow cytometry. The percentage of apoptotic and lymphocytes and monocyte cells were then calculated. Moreover, relative granularity and size of apoptotic cells were determined. To compare between the control and irradiation samples, t-test was used and a mathematical model was suggested to describe the dependence of DNA damage (8-oxoguanine molecules /cell) and apoptosis on x and gamma rays doses and dose rates. The results showed that the sensitivity of the donors varies between individuals, for x-irradiation at dose rate 0.28Gy/min with doses 0.25, 0.5, 1, 2 and 4 Gy. The number of 8-oxoguanine molecules/cell was increased significantly with dose. The percentages of increasing were 49%, 50%, 53%, 58% and 81% for doses 0.25, 0.5, 1, 2 and 4 Gy respectively. At dose rate of 20 mGy/min with doses 15.6, 31.3, 62.5, 125 and 250 mGy, the percentage of damage was 15%, 10%, 15%, 18% and 34% respectively. A significant difference at low doses 15.6, 31.3 and 62.5 mGy were observed. For
60
Co
gamma ray irradiation the percentage of the damage was 21%, 17%, 21%, 58% and 53% for doses 0.25, 0.5, 1, 2 and 4 Gy respectively. From dose response curves we found that high doses with high dose rate of x-ray (0.28 Gy/min.) have more effect on DNA damage (8-oxoguanine molecules/cell) than low doses at low x-ray dose rate (20mGy/min) and high doses at low 60Co gamma dose rates (2Gy/hr). The effect of high doses with low dose rate of
60
Co gamma ray were more effective than low doses with
low dose rate of x-ray (20 mGy/min). Changing in relative granularity and size of the cells were observed in all experiments. The results on inducing apoptosis by irradiation showed that at dose rate 0.28 Gy/min of x-ray doses 0, 0.25, 0.5, 1, 2 and 4 Gy, the amount of
fluorescence in the activated caspase 3 in apoptotic cells was increased and a significant difference was observed. The percentage number of apoptotic cells was increased significantly with dose, and lymphocyte (live) cells were decreased with dose. At dose rate 20 mGy/min of x-ray with doses 15.6, 31.3, 62.5, 125 and 250 mGy, the amount of fluorescence of caspase 3 was increased with dose but only significant at doses 62.5, 125 and 250 Gy. The percentage number of apoptotic cells was increased with dose but the increment
was
statistically
insignificant.
Moreover,
decreasing
of
lymphocytes with dose was insignificant. For 60Co gamma ray irradiation at dose rate of 2 Gy/hr with doses 025, 0.5, 1, 2 and 4 Gy, the amount of fluorescenc of caspase 3 in apoptotic cells was increased significantly with dose. The percentage number of apoptotic cells was increased with dose and a significant difference was observed at 1, 2 and 4 Gy. Number of lymphocytes cells was decreased with increasing dose but that was only significant at 4 Gy. From the dose response curves, inducing of apoptosis by high x-ray doses with dose rate 0.28 Gy/min was observed, the same as with high 60Co gamma ray doses at dose rate 2Gy/min. The effect at these two dose rates was more than the effect of low x-ray doses at dose rate of 20 mGy/min. At dose rate of 0.28Gy/min of x-rays, the relative mean of granularity of cells were increased with dose. The increasing was not linear, and the size of cells were decreased linearly with dose. At dose rate of 20 mGy/min of xrays, the granularity was increased except for 250 mGy, and the size was not decreased except at 250 mGy. For
60
Co gamma ray doses at dose rate of
2Gy/hr, the granularity was increased and the size was decreased with doses but these changes were not linear. For all experiments the changing in the granularity and size was statistically insignificant. We concluded that high dose with high dose rate of x-ray, and high dose with low dose rate of
60
Co gamma ray induce significant DNA damage and
apoptosis. Low dose with low dose rate of x-ray induce significant DNA damage but not significant apoptosis. Inducing of DNA damage depends on dose and dose rate but inducing of apoptosis depends on dose but not on dose rate. The DNA damage and apoptosis are good molecular biological markers of radiation response and changing of granularity and size of apoptotic cells is larger at high radiation doses.
List of Contents
Chapter One: Introduction and Literature Review
Page No.
1-1 General Introduction -------------------------------------------------------1 1-2 Ionizing Radiation----------------------------------------------------------2 1-3 Low-LET Radiation--------------------------------------------------------3 1-4 Structure of DNA and Oxyradicals--------------------------------------4 1-5 Apoptosis and Caspases---------------------------------------------------8 1-6 Biological Effect of Ionizing Radiation--------------------------------10 1-6-1 Introduction-----------------------------------------------------10 1-6-2 Radiation interaction with human cells-----------------------------10 1-7 Mononuclear Cells -------------------------------------------------------12 1-8 Radiation-Induced Biological Markers and Literature Review ----13 1-9 The Aim of the Present Work-------------------------------------------18 1-10 Outline of the Thesis----------------------------------------------------19 Chapter Two: Materials and Methods 2-1 Materials-------------------------------------------------------------------23 2-1-1 Chemicals-------------------------------------------------------------23 2-2-2 Kits-------------------------------------------------------------------- 24 2-2-3 Instruments------------------------------------------------------------24 2-2 Methods--------------------------------------------------------------------25 2-2-1 Procedure of mononuclear cell separation from the blood-----25 2-2-2 Preparation of mononuclear cells for irradiation----------------25 2-3 Irradiation of Blood Samples-------------------------------------------26 2-3-1 X-ray irradiation-----------------------------------------------------26 2-3-2 60Co gamma ray irradiation-----------------------------------------26
2-4 Assay of DNA Damage (8-oxoguanine)------------------------------27 2-4-1 Principle of the assay-----------------------------------------------27 2-4-2 Method---------------------------------------------------------------27 2-4-2-1 Fixation----------------------------------------------------------27 2-4-2-2 Staining with FITC---------------------------------------------28 2-5 Assay of Apoptosis------------------------------------------------------29 2-5-1 Principle of the assay-----------------------------------------29 2-5-2 Method---------------------------------------------------------------29 2-6 Flow cytometry------------------------------------------------------------29 2-7Analysis by Flow cytomtery--------------------------------------------31 2-7-1 Measuring DNA damage (8-oxoguanine) fluorescence-------32 2-7-2 Measuring fluorescence, FITC and the numbers of apoptotic cells----------------------------------------------------------------------------34 2-8 Statistical Analysis-------------------------------------------------------35 Chapter Three: X and γ rays Induced DNA Damage: Results and Discussion 3-1 Introduction ---------------------------------------------------------------37 3-2 X-ray Induced DNA Damage (8-oxoguanine)-----------------------38 3-2-1 X-ray induced DNA damage with dose rate 0.28Gy/min-----38 3-2-2 X-ray induced DNA damage (8-oxoguanine) with dose rate 20mGy/min-------------------------------------------------------------------44 3-3 γ ray Induced DNA Damage (8-oxoguanine)------------------------49 3-4 Dose Response Model----------------------------------------------------54 3-5 Measurement of Relative Granularity and Size of Mononuclear Cells------------------------------------------------------------------------------57 Chapter Four: X and γ Rays Induced Apoptosis: Results and Discussion 4-1 Introduction----------------------------------------------------------------60 4-2 X-ray Induced Apoptosis------------------------------------------------61
4-2-1 Measuring amount of fluorescence, FITC of caspase 3 for dose rate 0.28Gy/min----------------------------------------------------------------------61 4-2-2 Measurement of percentage number of lymphocytes, apoptotic and monocytes cells at dose rate of 0.28 Gy/min----------------------68 4-2-3 Measurement of Fluorescence, FITC of activated caspase 3 with dose rate 20mGy/min--------------------------------------------------------75 4-2-4 Measurement of percentage number of lymphocyte, apoptotic and monocyte cells at dose rate of 20mGy/min---------------------------80 4-3 60Co γ-ray Induced Apoptosis-------------------------------------------87 4-3-1 Measurement of amount of fluorescence, FITC of caspase 3 with dose rate of 2Gy/hr-----------------------------------------------------------87 4-3-2 Measurement of percent number of lymphocytes, apoptotic and monocytes cells at dose rate of 2Gy/hr------------------------------------93 4-4 Dose Response Model---------------------------------------------------100 4-4-1 Dose response curve for fluorescence in activated caspase 3, FITC in apoptotic cells----------------------------------------------------100 4-4-2 Dose response curves for percentage number of apoptosis----103 4-4-3 Dose response curve for the percentage number of lymphocyte--------------------------------------------------------------------------------------103 4-5 Measuring of Granularity and Size of Apoptotic Cells--------------109 Chapter Five: Conclusions and Future Work 5-1 Conclusions----------------------------------------------------------------112 5-2 Future Work---------------------------------------------------------------114
List of Tables Table No.
Title
Page No.
2.1
Presents the chemical used for blood samples preparation.
23
2.2
Presents the instruments used in the study.
24
3.1
Effect of x-ray dose on mononuclear blood cells in donor
40
1. Dose rate is 0.28 Gy/min. 3.2
Effect of x-ray dose on mononuclear blood cells in donor
40
2. Dose rate is 0.28 Gy/min. 3.3
Effect of x-ray dose on mononuclear blood cells in donor
41
3. Dose rate is0.28 Gy/min. 3.4
Effect of x-ray dose with on mononuclear blood cells in
41
donor 4. Dose rate is 0.28Gy/min 3.5
Percentage of 8-oxoguanine molecules/cell with x-ray dose
43
after normalization for all donors. Dose rate is 0.28 Gy/min. 3.6
Effect of x-ray dose on mononuclear blood cells in donor
45
1. Dose rate is 20mGy/min. 3.7
Effect of x-ray dose on mononuclear blood cells in donor
45
2. Dose rate is 20mGy/min. 3.8
Effect of x-ray dose on mononuclear blood cells in donor
46
3. Dose rate is 20 mGy/min. 3.9
Effect of x-ray dose on mononuclear blood cells in donor
46
4. Dose rate is 20mGy/min. 3.10
Percentage of 8-oxoguanine molecules/cell with x-ray dose
48
after normalization for 4 donors. Dose rate is 20 mGy/min. 3.11
Effect of 60Co γ-ray dose on mononuclear blood cells in donor 1. Dose rate is 2Gy/hr
50
3.12
Effect of 60Co γ-ray dose on mononuclear cells in donor
50
2.Dose rate 2Gy/hr. 3.13
Effect of 60Co γ-ray dose on mononuclear blood cells in
51
donor 3. Dose rate is 2Gy/hr. 3.14
Effect of 60Co γ-ray doses on mononuclear blood cells in donor
51
4. Dose rate is 2 Gy/hr.
3.15
Percentage of 8-oxoguanine molecules/cell with 60Co γ-ray
53
dose after normalization for 4 donors. Dose rate is 2Gy/hr. 3.16
The values of fitting parameters a and b in our
55
mathematical model for the three experiments. R is the correlation coefficient and c=100 which is the amount of fluorescence of 8-oxoguanine, FITC at 0 Gy after applied normalization for donors. 3.17
Mean relative granularity and size of mononuclear cells
58
with x-ray dose at dose rate of 0.28Gy/min. 3.18
Mean relative granularity and size of mononuclear cells
58
with x-ray dose at dose rate of 20mGy/min. 3.19
Mean relative granularity and size of mononuclear cells
59
with 60Co gamma ray dose at dose rate of 2Gy/hr 4.1
Amount of fluorescence of activated caspase 3, FITC with
64
x-ray dose in lymphocyte, apoptotic and monocyte cells for donor 1. Dose rate is 0.28 Gy/min. 4.2
Amount of fluorescence of activated caspase 3, FITC with
64
x-ray dose in lymphocyte, apoptotic and monocyte cells for donor 2. Dose is rate 0.28 Gy/min. 4.3
Amount of fluorescence of activated caspase 3, FITC with x-ray dose in lymphocyte, apoptotic and monocyte cells for donor 3. Dose rate is 0.28 Gy/min.
65
4.4
Amount of fluorescence of activated caspase 3, FITC with
65
x-ray dose in lymphocyte, apoptotic and monocyte cells for donor 4. Dose rate is 0.28 Gy/min. 4.5
Amount of fluorescence activated caspase 3, FITC with x-
67
ray dose in lymphocyte, apoptotic and monocyte cell after normalization for all donors. Dose rate is 0.28 Gy/min. 4.6
Percentage number of lymphocytes, apoptotic and
70
monocytes cells with dose in donor 1. Dose rate is 0.28 Gy/min. 4.7
Percentage number of lymphocytes, apoptotic and
70
monocytes cells with x-ray dose in donor 2. Dose rate is 0.28 Gy/min. 4.8
Percentage number of lymphocytes, apoptotic and
71
monocytes cells with x-ray dose in donor 3. Dose rate is 0.28 Gy/min. 4.9
Percentage number of lymphocytes, apoptotic and
71
monocytes cells with x-ray dose in donor 4. Dose rate is 0.28 Gy/min. 4.10
Mean of percentage number of apoptotic cells with x-ray
73
dose for 4 donors. Dose rate is 0.28 Gy/min. 4,11
Mean of percentage number of lymphocytes cells with x-
73
ray dose for 4 donors. Dose rate is 0.28 Gy/min. 4.12
Amount of fluorescence of activated caspase 3, FITC with
76
x-ray dose in lymphocyte, apoptotic and monocyte for donor 1. Dose rate is 20m Gy/min. 4.13
Amount of fluorescence of activated caspase 3, FITC with x-ray dose in lymphocyte, apoptotic and monocyte for donor 2. Dose rate is 20m Gy/min.
76
4.14
Variation of amount of fluorescence of activated caspase 3,
77
FITC with x-ray dose in lymphocyte, apoptotic and monocyte for donor 3. Dose rate is 20m Gy/min. 4.15
Amount of fluorescence of activated caspase 3, FITC with
77
x-ray dose in lymphocyte, apoptotic and monocyte for donor 4. Dose rate is 20 mGy/min. 4.16
Amount of fluorescence of activated caspase 3, FITC with
78
x-ray dose in lymphocyte, apoptotic and monocyte for donor 5. Dose rate is 20 mGy/min 4.17
Amount of fluorescence of activated casspase 3, FITC with
79
x-ray dose in lymphocyte, apoptotic, and monocyte after normalization for 5 donors. Dose rate is 20 mGy/min. 4.18
Percentage number of lymphocyte, apoptotic and
81
monocyte cells with x-ray dose in donor 1. Dose rate is 20 mGy/min. 4.19
Percentage number of lymphocyte, apoptotic and
81
monocyte cells with x-ray dose in donor 2. Dose rate is 20 mGy/min. 4.20
Percentage number of lymphocyte, apoptotic and
82
monocyte cells with x-ray dose in donor 3. Dose rate is 20 mGy/min. 4.21
Percentage number of lymphocyte, apoptotic and
82
monocyte cells with x-ray dose in donor 4. Dose rate is 20 mGy/min. 4.22
Percentage number of lymphocyte, apoptotic and
83
monocyte cells with x-ray dose in donor 5. Dose rate is 20 mGy/min. 4.23
Mean of percentage number of apoptotic cells with x-ray dose for 5 donors. Dose rate 20 is mGy/min
85
4.24
Mean of percentage number of lymphocytes cells with x-
85
ray dose for 5 donors. Dose rate is 20 mGy/min 4.25
Amount of fluorescence of activated caspase 3, FITC with 60
90
Coγ-ray dose in lymphocyte, apoptotic and monocyte, for
donor 1. Dose rate is 2 Gy/hr
4.26
Amount of fluorescence of activated caspase 3, FITC with 60
90
Coγ-ray dose in lymphocyte apoptotic and monocyte, for
donor 2. Dose rate is 2Gy/hr. 4.27
Amount of fluorescence of activated caspase 3, FITC with 60
91
Coγ-ray dose in lymphocyte apoptotic and monocyte, for
donor 3. Dose rate is 2 Gy/hr. 4.28
Amount of fluorescence of activated caspase 3, FITC with 60
91
Coγ-ray dose in lymphocyte, apoptotic and monocyte,
for donor 4. Dose rate is 2Gy/hr. 4.29
Amount of fluorescence of activated caspase 3, FITC with 60
92
Coγ-ray dose in lymphocyte, apoptotic and monocyte,
for 4 donors after normalization. Dose rate is 2 Gy/hr. 4.30
Percentage number of lymphocyte, apoptotic and
95
monocyte cells with 60Co gamma ray dose in donor 1. Dose rate is 2 Gy/hr. 4.31
Percentage number of lymphocyte, apoptotic and
95
monocyte cells with 60Co gamma ray dose in donor 2. Dose rate is 2 Gy/hr 4.32
Percentage number of lymphocyte, apoptotic and
96
monocyte cells with 60Co gamma ray dose in donor 3. Dose rate is 2 Gy/hr. 4.33
Percentage number of lymphocyte, apoptotic and monocyte cells with 60Co gamma ray dose in donor 4. Dose rate is 2 Gy/hr
96
4.34
Mean of percentage number apoptotic cells with 60Co
98
gamma ray dose for all donors. Dose rate is 2 Gy/hr. 4.35
Mean of percentage number lymphocyte cells with 60Co
98
gamma ray dose for all donors. Dose rate is 2 Gy/hr. 4.36
The values of fitting parameters a and b in our
101
mathematical model for the three experiments. R is the correlation coefficient and c is the amount of activated caspase 3 at 0 Gy after applied normalization for donors. 4.37
The values of parameters in our model for the percentage
104
number of apoptotic for three experiments. 4.38
The values of fitting parameters in the model for
107
percentage number of lymphocytes (survival) for the three conditions. 4.39
Mean relative granularity and size of apoptotic cells with
110
x-ray dose at dose rate of 0.28Gy/min 4.40
Mean relative granularity and size of apoptotic cells with
110
x-ray dose at dose rate of 20mGy/min. 4.41
Mean relative granularity and size of apoptotic cells with 60
Co gamma ray dose at dose rate of 2Gy/hr.
111
List of figures
Figure No.
Title
Page No.
1.1
Excitation and ionization processes by photon.
3
1.2
Structure of pyrimidines.
5
1.3
Structure of purines.
5
1.4
Polynucleotide structure.
6
1.5
Double helix of DNA.
6
1.6
8-oxoguanine molecule.
7
1.7
Apoptosis (programmed cell death).
8
1.8
Direct and indirect interaction of photon with DNA.
13
2.1
60
27
2.2
Flow cytometry; schematic of a typical flow cytometer setup. Fluorescence intensity (FITC) in arbitrary units was
31
2.3
Co gamma ray source used in the present work.
33
plotted in histograms by flow cytometry 2.4
Calibration of MEFL with fluorescence intensity.
34
2.5
Variation of amount 8-oxoguanine, FITC with known
34
MEFL. Errors are within the points size. 2.6
Fluorescence intensity of caspase 3, (FITC) measured
36
directly by flow cytometry and show by histogram. 3.1
Variation of 8-oxoguanine molecules/cell with x-ray dose,
42
Dose rate is 0.28Gy/min. 3.2
Variation of percentage of 8-oxoguanine molecules/cell with x-ray dose, after normalization for 4 donors. Dose rate is 0.28 Gy/min.
43
Figure No. 3.3
Title
Page No.
Variation of 8-oxoguanine molecules/cell with x-ray dose, 47 Dose rate is 20 mGy/min.
3.4
Variation of percentage of 8-oxoguanine molecules/cell 48 with x-ray dose after normalization for 4 donors. Dose rate is 20 mGy/min.
3.5
Variation of 8-oxoguanine molecules/cell with x-ray 52 doses. Dose rate is 0.28 Gy/min.
3.6
Variation of percentage of 8-oxoguanine molecules/cell 53 with γ-ray doses after normalization for 4 donors. Dose rate is 2Gy/hr.
3.7
Dose response curve for high doses of x-ray at dose rate 56 of 0.28Gy/min.
3.8
Dose response curve for low doses of x-ray at dose rate of 56 20 mGy/min.
3.9
Dose response curve for low doses of 60Co gamma ray at
56
dose rate of 0.033Gy/min. 4.1
Histogram of fluorescence intensity of activated caspase
62
3, FITC for dose 0, 0.25, 0.5, 1, 2, 4 Gy. 4.2
Variation of amount of fluorescence of activated caspase
66
3, FITC with x-ray dose in apoptotic cells after normalization for 4 donors. Dose rate is 0.28 Gy/min. 4.3
Variation of mean of percentage number of apoptotic cells
72
with x-ray dose. Dose rate of 0.28 Gy/min. 4.4
Variation of mean of percentage number of lymphocytes cells with x-ray dose. Dose rate of 0.28 Gy/min.
72
Figure No.
Title
Page No.
4.5
Variation of amount of fluorescence of activated caspase
77
3, FITC with x-ray dose in apoptotic cells after normalization for all donors. Dose rate of 20 mGy/min. 4.6
Variation of mean of percentage number of apoptotic cells
83
with x-ray dose for all donors. Dose rate is 20mGy/min 4.7
Variation of mean of percentage number of lymphocytes
83
cells with x-ray dose for all donors. Dose rate is 20 mGy/min. 4.8
Histogram of fluorescence of activated caspase 3, FITC
85
for 60Co gamma ray dose 0, 0.25, 0.5, 1, 2, 4 Gy. 4.9
Variation of
60
Co gamma ray dose with amount of
88
fluorescence activated caspase 3, FITC in apoptotic cells after normalization for all donors. Dose rate is 2 Gy/hr. 4.10
Variation of mean of percentage number apoptotic cells
93
with 60Co gamma ray dose. Dose rate is 2 Gy/hr. 4.11
Variation of mean of percentage number lymphocyte cells
93
with 60Co gamma ray dose. Dose rate is 2 Gy/hr. 4.12
Dose response curve for high doses of x-ray with dose
96
rate of 0.28 Gy/min. 4.13
Dose response curve for low doses of x-ray with dose rate
96
of 20 mGy/min. 4.14
60
Co gamma ray
96
Dose response curve for high doses of x-ray with dose
99
Dose response curve for low doses of with dose rate of 0.033 mGy/hr.
4.15
rate of 0.28 Gy/min with apoptotic cells.
Figure No.
Title
Page No.
4.16
Dose response curve for low doses of x-ray with dose rate
99
of 20 mGy/min with percentage number of apoptotic cells. 4.17
Dose response curve for low doses of
60
Co gamma ray
99
with dose rate of 0.033 Gy/min with percentage number of apoptosis 4.18
Dose response curve of high dose of x-ray with
102
percentage number of lymphocytes cells. Dose rate is 0.28 Gy/min. 4.19
Dose response curve of low dose of x-ray with percentage
102
number of lymphocyte cells. Dose rate is 20 mGy/min. 4.20
Dose response curve of low dose of 60Co gamma ray with percentage number of lymphocytes cells. Dose rate is 0.033 Gy/min.
102
Abbreviations A
: Adenine
PCR: Polymerase chain reaction
BER : Base excision repair C
RNA: Ribonucleic acid
: Cytosine
ROS: Reactive oxygen specie
cGy : Centigray
SLDR: Sub-low dose rate
CHO-K1: Chines hamster cells
SSBs: Single strand breaks
DNA: Deoxyribonucleic acid
SSC: Side scatter light
DSBs : Double strand breaks
T: Thymine
FACS : Fluorescence activate cell sorting FBS : Fetal bovine serum
TM: Tail moment
FITC : Fluorescence isothiocyanate
U: Uracil
FSC : Forward scatter light G
: Guanine
Gy : Gray (SI unit of absorbed dose =1J/kg) H⋅
: Hydrogen free radical
H2O2: Hydrogen peroxide HDR: High dose rate Hela-Hep : Human adenocarcinoma cells HO2⋅ : Hydroperoxyl radical LDR: Low dose rate LET : Linear energy transfer MEFL : Molecular of equivalent fluorescent MOLT-4: Human leukemia cells NER: Nucleotide excision repair OH- : Negative water molecule "heavy water" OH⋅
:
TL: Tail length
Hydroxyl free radical
PBMCs : Peripheral blood mononuclear cells PBS : Phosphate buffer saline
XX
Chapter One
Introduction and Literature Reviews
1-1 General Introduction During their lifetime, humans are exposed to physical, chemical, and biological agents. Among the physical agents, ionizing radiations can produce damage to molecular systems [1]. Ionizing radiations have been shown to induce a broad spectrum of genetic effects, including gene, minisatellite mutations, micronucleus formation, chromosome aberrations, ploidy changes DNA (deoxyribonucleic acid) strand breaks and chromosome instability [2]. It has long been known to be deleterious after high dose exposure (>100mSv), predominantly inducing cancer although very high dose exposures yield to tissue damage and ultimately death [1]. Furthermore, ionizing radiation has been called the "universal carcinogen" in that it might induce cancer in most tissues of most species at all ages, including the fetus [3]. The hazards of exposure of ionizing radiation were recognized shortly after Roentgen's discovery of the x-ray in 1895. Acute skin reactions were observed in many individuals working with early x-ray generators, and by 1902 the first radiation-induced cancer was reported arising in an ulcerated area of the skin. Then, a few years later, a large number of cancers were
observed, and the first report of leukemia in five radiation workers appeared in 1911. Indeed, Marie Curie and her daughter Irene are both thought to have died of radiation-induced leukemia. Since that time, so many experimental and epidemiological studies have confirmed the oncogenic effects of radiation in many tissues of many species [3]. Radiation is perceived by the public as a major carcinogen, despite convincing evidence that it contributes only for a small amount to the overall cancer burden since the vast majority of people are only exposed to very low doses. This perception likely comes from images of wartime uses of nuclear weapons and, more recently, reactor accidents such as Chernobyl [4]. Although radiation is a universal carcinogen, it is a weak one, in part because it is an especially good killer of cells. In general, we live in a sea of low-level natural radiation from terrestrial and cosmic sources, and our bodies have developed repair mechanisms to correct damage following such exposures [4]. 1-2 Ionizing Radiation The absorption of energy from radiation in biologic material can lead to excitation or to ionization. The raising of an electron in an atom or molecule to a higher energy level without actual ejection of electron is called excitation. If the radiation has sufficient energy to eject one or more orbital electrons from the atom or molecule, the process is called ionization and radiation is named ionizing radiation. Fig. (1.1) shows excitation and ionization processes.
Excitation
Ionization
Fig (1.1): Excitation and ionization processes by photon [5].
The important characteristic of ionizing radiation is localized release of large amounts of energy. The energy dissipated per ionizing event is about 33 eV, which is more than enough to break a chemical bond (for example, the energy associated with a C=C bond is 4.9 eV) [5]. Ionizing radiations are conveniently categorized into four general types: 1-Fast electrons: include beta particles (positive or negative) emitted in nuclear decay, as well as energetic electrons produced by any other process. 2-Heavy charged particles denote a category that encompasses all energetic ions with mass of one atomic mass unit or greater, such as alpha particles, protons, fission products, or the product of many nuclear reactions. 3-The electromagnetic radiation includes x-rays emitted in the rearrangements of electrons shells of atoms, when fast electrons hit materials and gamma (γ) rays originate from transitions within the nucleus itself. 4-Neutrons generated in various nuclear processes, often divided into slow and fast neutrons [6]. 1-3 Low –LET Radiation Low-linear energy transfer (LET) radiation include electrons and electromagnetic waves such as x- and γ rays. Emission of γ ray is a result of radioactive decay and may follow other modes of disintegration such as β or α emission in order to allow an unstable nucleus to recover its ground state
level. The energy of γ ray is variable and usually ranges from 10keV to 3MeV. X-ray can be produced in an accelerator, by electronic impact, or in atoms in which vacancies in the K and L shells have been produced after electron capture, internal conversion or photoelectric absorption. The interaction of low-LET with matter depends on their nature (particles or photons) and their energy. Photons mainly interact with their biological target via the Compton process if their energy between 20keV and several MeV. For the lowest energetic photons ranging from 0.2 to 5 keV, photoelectric effect absorption is the preponderant process involved. Both modes of interaction result in excited and ionized matter. The trajectory of incident photons is deflected after each interaction, and dislodged electrons are emitted with a defined scattering angle and energy. The interaction of photons with biological material sets other electrons in motion, which in turn interact with matter, until their energy falls below 10eV. As a consequence of their mode of interaction, the ionizations and excitation produced by photons and electrons are sparsely produced a large targeted volume and over a wide range. Thus, the LET of such radiation is low, with a range values from <0.5keV/µm for 60Co γ-rays to a few keV/ µm for x-rays [7]. 1-4 Structure of DNA and Oxyradicals There are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the genetic material that organisms inherit from their parents. A DNA molecule is very long and usually consists of hundreds or thousand of genes. When a cell reproduces itself by dividing, its DNA is copied and passed along from one generation of cells to the next. Nucleic acids are polymers of monomers called nucleotides. Each nucleotide is composed of three parts: an organic part called nitrogenous base, a pentose (five-carbon sugar) and a phosphate group. There are two families of nitrogenous bases: pyrimidines and purines. A pyrimidines has a six-
member ring of carbon and nitrogen atom. The members of the pyrimidine family are cytosine (C), thymine (T) and uracil (U), as shown in Fig. (1.2). Purines are larger, with the six member ring fused to a five-member ring. The purines are adenine (A) and guanine (G), as shown in Fig. (1.3).
Fig. (1.2): Structure of pyrimidines [8]
Fig. (1.3): Structure of purines [8]
In polynucleotides, nucleotides are joined by covalent bonds called phosphodiester linkages between the phosphate of one nucleotide and the sugar of the next. This bonding results in a backbone with a repeating pattern of sugar-phosphate-sugar-phosphate, as shown in Fig. (1.4).
Fig. (1.4): Polynucleotide structure [5]
The DNA molecules of cells actually consist of two polynucleotides that spiral around an imaginary axis to form a double helix. The two strands are held together by hydrogen bonds between the paired bases. Only certain bases in the double helix are compatible with others, A always pairs with T, and G always pairs with C [9] (Fig. 1.5).
Fig. (1.5): Double helix of DNA [8]
Oxidative DNA damage plays a critical role in several biological processes such as mutagenesis, aging and a variety of diseases. DNA is continuously exposed to a number of environmental agents including reactive oxygen species (ROS) such as superoxide, hydrogen peroxide and hydroxyl radical [10]. ROS is generated by the action of ionizing radiation, chemical mutagens and carcinogens, also normal cellular metabolism is well established as the source of endogenous ROS, and it is these ( normally non-pathogenic) cellular processes that account for the background levels of oxidative DNA damage detected in normal tissue [11,12]. Recent investigation have established that guanine is the main target for ROS in DNA, with 8-oxoguanine being the most frequent lesion. Therefore, formation of 8-oxoguanine is an important biomarker of oxidative damage to DNA [7, 11]. The molecule of 8-
oxoguanine formed as a 7, 8-dihydro-8-oxoguanine by addition of oxygen atom on the C8 atom of guanine. ( the addition of oxygen O8 at the C8, hybridization of the N7 and transforming the double bond C8-N7 into single one) as shown in Fig. [1.6].
Fig. (1.6): 8-oxoguanine molecule [13]
1-5 Apoptosis and Caspases Apoptosis is a naturally occurring form of programmed cell death. It is essential in many physiological processes, including the embryonic development and the maturation of the immune system. Apoptosis is morphologically characterized by increased cytoplasmic granularity, cell shrinkage, chromatin condensation, membrane blebbing, and the formation of distinctive nuclear bodies as shown in Fig (1.7). Although apoptosis occurs spontaneously, it can also be induced by various physiological conditions and external stimuli such as ionizing radiation. It has been shown that tumour cells are susceptible to death by apoptosis in response to drugs and /or radiation treatment.
Fig. (1.7): Apoptosis (programmed cell death [14].
The interest for using apoptosis as a possible measure of radiosensitivity has increased substantially both with regard to the possibilities of using the extent of apoptosis as a biological dosimeter as well as for estimating the radiosensitivity of cancer cells before radiotherapy. The status and level of expression of proteins that regulate apoptosis have even been proposed to serve as radiation exposure indicators or sensors [15-17]. Two distinct but interconnected apoptotic pathways have been characterized, the cell surface death recepter pathway (extrinsic) and the mitochondria initated (intrinsic) pathway. Both pathway terminate in activation of effector caspases, which mediate the proteolytic events characterizing apoptosis [18-20]. Caspases (cysteine-proteases) are synthesized as a single chain polypeptides called zymogens. Caspase types which have essential role in apoptosis are: initiators in apoptosis ( caspase-2, 8, 9 and 10) and excutioners in apoptosis (caspase-3, 6 and 7). All initiator and excutioner caspases have either a direct or indirect role in the processing, propagation and amplification of apoptotic signales that results in the destruction of cellular structures [20].
In mammals, caspases (principally caspase 3) appear to be activated in a protease cascade that leads inappropriate activation or rapid disablement of key structural protiens and important signaling, homeostatic and repair enzymes. Caspase 3 is a frequently activated death protease. It has been revealed that caspase 3 is important for cell death in a remarkable tissue, cell type or death stimulus-specific manner, and is essential for some of the charcteristic changes in cell morphology and certain biochemical events associated with the execution and completion of apoptosis [21]. Several sensitive methods have been developed for the rapid detection of apoptosis in human lymphocytes, due to its speed, flow cytometry has become a popular method for detecting apoptosis [15].
1-6 Biological Effects of Ionizing Radiation 1-6-1 Introduction Any cells that are immature, undifferentiated and actively dividing (i.e., stomach mucosa, basal layer of skin, stem cells) are usually radiosensitive. They respond by exhibiting some effect from radiation exposure that causes cell injury or cell death. Cells that are mature, differentiated and not actively dividing (i.e., neurons) are more radioresistant. The interaction of radiation with cells is a probability function. Because cell repair usually takes place, permanent damage will not necessarily result from an interaction of ionizing radiation with living tissue. Energy deposition to a cell occurs quickly, in some 10-18 s, with the energy being deposited in the cell in a random fashion. All interactions happen on a cellular level, which in turn may affect the organ and the entire system. In addition, there is no unique cellular damage associated with radiation. Any damage to a cell due to radiation exposure may also happen due to chemical, heat, or physical damage. After radiation exposure to a cell, there is a latent period before any observable response. The latent period could be decades for low radiation doses, but only minutes or
hours for high radiation exposure [22]. The resulting biological effect on mammalian cells is influenced by the dose, dose rate and quality of the radiation. The reason is that the biological effectiveness depends on the spatial distribution of the energy imparted and the density of ionization per unit path length of ionization particles [23]. 1-6-2-Radiation interaction with human cells Ionizing radiation interacts with a cell in 2 different ways: direct or indirect interaction. 1-Direct interaction In the direct interaction, a cell's macromolecules (proteins or DNA) are hit by the ionizing radiation, which affects the cell as a whole, either killing the cell or mutating the DNA. Target and cell survival studies show that it is harder to permanently destroy or break double-stranded DNA than singlestranded DNA. Although humans have 23 pairs of double-strand chromosomes, some cells react as if they contain single-stranded, non-paired chromosomes and therefore more radiosensitive. Many different types of direct hits can occur, and the type of damage that occurs determines whether the cell can or can not repair itself. Generally, if a direct hit which causes a complete break in DNA or some other permanent damage, the cell dies immediately or dies eventually. Fig. (1.8) shows direct and indirect interaction between ionizing radiation and DNA [22]. 2-Indirect interaction Indirect interaction occurs when radiation energy is deposited in the cell and that the radiation interacts with cellular water rather than with macromolecules within the cell as shown in Fig. (1.8). Most of primary
interactions of radiation in tissue results in the ionization of simpler molecules and the creation of chemically active free radicals.
Fig. (1.8): Direct and indirect interaction of photon with DNA [24].
Ionization of a water molecule produces a free electron and a positively charged molecule: H2O+ +e-
H2O + radiation
The released electron is most likely to be captured by another water molecule converting it into a negative ion: e- + H2O
H2O-
Both these ions are unstable and dissociate as follows: H2O+
H+ + OH·
H2O-
H· + OH-
The free radical H· tend to combine with oxygen, creating hydroperoxyle radicals. The hydroperoxyle radicals may cause biologic damage directly or break down to form hydrogen peroxide and oxygen: H· + O2 HO2·
HO2· H2O2 +O2
Two hydroxyle radicals can combine to form hydrogen peroxide: OH· + OH·
H2O2
About two thirds of all biologic damage is caused by the hydrogen peroxide from these processes. By abstracting hydrogen from organic molecules, which can represent as RH, these free radicals can generate organic free radicals (R·): RH + OH· RH + H·
R· + H2O R· + H2
The organic free radicals may react with and disrupt other molecules that may be part of a biologically more complex system, such as a chromosome, possibly disabling it and leading to the death of a cell. Alternatively, they may modify the genetic information that is passed on to future generation (genetic mutation) [22, 25, 26]. 1-7 Mononuclear Cells Mononuclear cells consist of lymphocytes and monocytes with relatively clear cytoplasm. There are several kinds of lymphocytes with different function to perform. The most common types are: • B-lymphocytes (B-cells). These are responsible for making antibodies. • T-lymphocytes (T-cells). There are several subsets of these: -inflammatory T-cells that recruit macrophages and neutrophils to the site of infection or other tissue damage. -cytotoxic T-lymphocytes that kill virus-infected and, perhaps, tumour cells. -helper T-cells that enhance the production of antibodies by B- cells. Monocytes leave the blood and become macrophages and dendritic cells. Macrophages are large, phagocytic cells that engulf dead and dying cells of the body or any foreign material (antigens) that enter the body [27, 28].
1-8 Radiation-Induced Biological Markers and Literature Review Molecular biological markers of radiation response could potentially be of use for monitoring the progress of radiation therapy, even for predicting outcome early in a treatment regimen. Biomarkers of radiation exposure could also be an important tool for potentially exposed populations after a radiological accident or a "dirty bomb" incident [29]. Although still in its infancy as a scientific discipline, the study of radiation biomarkers could include DNA mutation, chromosome aberration, apoptosis and gene expression array technologies [30]. Radiation-induced DNA damage was explained by some scientists. Significant clustering of DNA damage occurs when ionizing radiation traverse the 30 nm chromatin fibres with generation of heavily damaged DNA regions with an average size of about 2 kilo base pair (kbp) [31]. Ogawa et al.[32] examined the radiation and the timing of oxidative DNAdamage induced in human peripheral T-lymphocytes following x-ray irradiation, they demonstrated oxidative DNA damage (8-oxoguanine) occur in dose dependent manner after irradiation the cells with (0, 2, 5, 20) Gy at dose rate 3.5 Gy/min. In another study they found that ROS was occured immediately after 5 Gy of x-irradiation, continued for several hours, and resulted in oxidative DNA damage [33]. Balkely et al.[34] showed that the analysis of multitarget nucleic acid biomarkers, using the multiplex fluorogenic 5'-nuclease polymerase chain reaction (PCR) assay, has beneficial applications in radiation epidemiology, radiation therapy and biodosimertry . Niedzwiedz and Cebulska [35] observed an increase in DNA damage in linear and non-linear quadratic manner, with increasing doses of x-ray (from 0 to 1.4 Gy) and 252Cf neutrons (from 0 to 0.8 Gy) respectively. Singh et al.[36] showed a significant increase in the length of DNA migration to x-ray dose (0.25-2) Gy, linear increase in the length of DNA
migration was observed for doses between 0.25 and 1 Gy, by 2Gy the length of migration appeared to plateau. Mozdarani et al.[37] obtained that an almost linear increasing the amount of radiation induced DNA damage in mouse blood leukocytes with increasing γ radiation dose (1 to 4Gy). Chaubey et al.[38] reported their observation on the low dose ( 1.25, 2.5, 5, 10) cGy effects of
60
Co gamma rays on DNA damage in human peripheral
blood leukocytes, they observed dose dependent increase in tail length (TL) with increasing doses of gamma rays and the lowest dose of 1.25 cGy showed significant increase in DNA damage. In another work [39] they irradiated human and mouse whole blood with different doses of γ-rays (2, 4 and 8Gy) at a dose rate of 0.668 Gy/min b. Analysis of their data revealed heterogeneity in the response of leucocytes to γ-ray induced DNA damage both in human as well as in mouse and a dose-dependent increase in tail moment (TM) and TL at all the irradiation doses (2-8Gy) both in human and mouse leukocytes. However, there was a difference in the nature of dose response curves for human and mouse leucocytes. In human leucocytes, a linear increase in TM and TL was observed up to the highest radiation dose of 8 Gy. However in the case of mouse leucocytes, a sharp increase in TM and TL was observed only up to 4 Gy, and there after saturation ensued. Vijayalaxmi et al.[40] irradiated lymphocytes, granulocytes and unfractionated leukocytes with low doses of gamma rays (0.05-0.5) Gy from 137
Cs source, they observed a linear and dose dependent increase in DNA
damage in all 3 cell populations, with a significant increase being detected at 0.05 Gy. The dose dependent increase for DNA migration was not significantly different between separated lymphocytes and granulocytes, but their response were significantly elevated over that obtained for leukocytes irradiated in whole blood .
Rossler et al.[41] exposed human whole blood to x and γ rays (0.5-3.0Gy), they evaluated three DNA damage parameters for x and γ (137Cs and
60
Co):
%DNA in the tail, TM and the TL, they found no difference in the induced initial radiation damage for the parameters, all radiation qualities showed a linear dose-effect relationship with the same slope when best–fit analyses performed with the data. They observed that x-rays induced a significantly different dose response than the γ-rays, at 1.0-1.5Gy the x-ray dose response showed saturation. Chen et al.[42] irradiated MOLT-4 human leukaemia cells and CHO-K1 to 0, 0.2, 0.4, 0.6 and 1.0 Gy of γ-rays. They detected the amounts of 8oxoguanine and total DNA in the cell nucleus by fluorescein-isothiocyanate (FITC), labeled avidin, which binds specifically and directly to 8-oxoguanine, and propidium iodide, respectively. They found an apparent dose-dependent increase in the amount of 8-oxoguanine accumulated in the cells of both lines. Courtemanche et al.[43] showed that DNA damage and apoptosis induced in T lymphocytes after irradiation the cells with different doses of gamma ray. Sutherland et al.[44] showed that irradiation of human monocytes with xray doses (0-1) Gy at rate 0.7 Gy/min.induce pyrimidine cluster in DNA of the cells. Some studies confirmed that apoptosis is induced by ionizing radiations. Boreham et al.[45] have tested the possibility of using apoptosis in human peripheral blood lymphocytes as a short-term biological dosimeter. They irradiated human peripheral blood lymphocytes to 0, 0.2, 0.4, 0.6, 0.8, 1 Gy of x and gamma rays with different dose rate, they observed x and gamma rays induced similar levels of apoptosis at similar doses and induction of apoptosis was dose dependent. Ogawa et al.[46] found in their study after 5Gy irradiation with 10 MeV x-ray from a linear accelerator that the percentages of apoptotic T cells
estimated are approximately 5, 10, 20, 35 and 70% at 0, 3, 6, 10 and 20 h after irradiation. Payne et al.[47] showed that irradiation of normal human lymphocytes in vitro with x-ray doses (0, 2.5, 5, 7.5, 10, 15, 20) Gy induce apoptosis and significant increase at low dose was observed. Wilkins et al.[48] examined the in vitro effect of x-ray doses ( 0, 0.02, 0.05, 0.1, 0.3, 0.5, 1, 1.5) Gy on the apoptotic response in whole blood, granulocytes, mononuclear cells, and mononuclear cell subpopulations ( B, NK, and CD4+ and CD8+ T-cells), data are presented on the variability of the apoptotic response, in both irradiated and unirradiated white blood cell subpopulations, between and within blood donors. In all cases, the dose response curve between 0 and 0.5 Gy demonstrated a high degree of linearity as demonstrated by high R2-values. Induced apoptosis in mononuclear cells and increase with 37Cs gamma ray dose (0.1, 0.2, 0.5, 1, 1.5, 2) Gy with dose rate 0.95 Gy/min and time after exposure also studied by Wilkins, et al [15]. Holl, et al.[49] showed irradiation of mice to x-ray doses (0.2-4) Gy occurring apoptosis in spleen lymphocytes cells was dose dependent and reaches a plateau at 3Gy. The generation of apoptotic signalling by interaction of ionizing radiation with cellular membrane was studied by Haimovitz-Friedman et al.[50]. The induction of apoptosis in a mouse T-cell hybridoma following x-irradiation was studied by Warters [51]. Ozsahin et al.[52] obtained, in their study on CD4 and CD8 T – lymphocyte, that apoptosis is induced in these cells after irradiation by 8 Gy x-rays. The increase of apoptotic index (percentage of apoptotic cells) with increasing radiation dose and time after irradiation by x-rays and fast neutron were observed by Wang et al.[53].
Vral et al.[54] investigated the effectiveness of low LET
60
Co γ-rays to
induce apoptosis in lymphocytes. They showed that for the dose-response analyses doses ranging from 0.05 to 5Gy at 1.5Gy/min, the dose-response curve characterized by an initial fast increase in the number of apoptotic cells below 1Gy, with flattening of the curves at higher doses towards 5Gy. Vávrová et al.[55] compared the effect of γ-radiation with sub-low doserate (SLDR) 1.8 mGy/min, low dose-rate (LDR) 3.9 mGy/min and high doserate (HDR) 0.6 Gy/min on human leukemic cell lines. They conclude that the effect of irradiation of human T-lymphocytes leukemia cells MOLT-4 is not significantly affected by dose rate, the dose rate has significant effect on HL60 cells. Belyaev et al.[56] detected that irradiation of human peripheral lymphocytes in Go phase with (1-5) Gy of 137Cs gamma ray at dose rate 10.6 Gy/min. produced apoptosis and saturation between (2-3) Gy with relatively weak increase at higher doses. Kim et al.[57] suggested that ionizing radiation induced apoptosis by activation of various signalling pathways including caspase family in a time and dose dependent manner. Peña et al.[58] demonstrated from their study that ionizing radiation induces early endothelial cell apoptosis through the central nervous system. Ghosh et al.[59] irradiated Chines hamster V79 to different gamma ray doses, their study indicated that the radiation can induce apoptosis in Chinese hamster V79. They also showed that cells irradiated to 0.58Gy, 10% of the cells have apoptotic morphology. This number increased to 29% at 3.5Gy. Irradiation of Hela Hep2 cells to 60C0 gamma ray dose (0.5, 1, 2, 5, 10 and 15) Gy at dose rate 0.80 Gy/min were done by Homa et al.[60], they observed radiation doses below 2 Gy did not cause any significant apoptosis, but between 5 and 15 Gy significant apoptosis was observed, with peak value at 5 Gy. They irradiated also the cells to the radiation dose 2, 5 and 10 Gy at
dose rate 0.072 Gy/min. The cells that achieved a dose below 2 Gy did not present increased apoptosis. At above 2 Gy, however the cells again demonstrated significant apoptosis. Inducing of apoptotic cells in T-lymphocytes leukemic cells, MOLT-4 was done by Aleš et al.[61], they showed after 24hr, 30.7, 76.9, and 94.4% of cells were apoptotic when irradiated by 1.5, 3.0, and 7.5 Gy of gamma radiation respectively. Collins et al.[62] observed increasing of DNA damage ( % DNA in tail and TL) after irradiation of human lymphocytes with x-ray doses (0-10) Gy. Liegler et al.[63] studied effect of
137
Cs gamma radiation doses (0-45)Gy
on the human lymphocytes, with increasing exposure to irradiation dose and length of postirradiation incubation, a decrease in the number of peripheral lymphocytes is observed. Loss of cell viability is both dose and time dependent. Correspondingly the number of apoptotic cells increase with radiation dose. 1-9 The Aim of the Present Work Living organisms are constantly exposed to a shower of ionizing radiation from the natural sources such as cosmic rays, radionuclides present in the earth's crust, artificial man-made medical and industrial radiation sources, nuclear exposures and industrial accidents etc… . Ionizing radiation is thus an integral part of our life [64]. Ionizing radiation produce through direct and indirect effects a variety of DNA lesions, such SSBs, DSBs, a variety of base modification, sugar modification, and DNA-DNA and DNA-protein cross links. DSB is generally thought to be the main lesion involved in cell killing and formation of chromosomal aberrations [65, 66].
The aim of this study is to investigate the low-LET radiations (x and γ) rays that can induce damage in human cells. For this purpose the following processes are designed: 1- Detection of DNA damage (8-oxoguanine) in normal human cells (Peripheral blood mononuclear cells PBMCs). 2- Detection of apoptosis in human blood lymphocytes. 3-The granularity and size of mononuclear and apoptotic cells were measured. 4-Different doses and dose rates are going to be considered. 1-10 Outline of the Thesis This thesis contains five chapters. The first, deals with introduction and literature review. Chapter Two deals with materials and methods. Chapter Three and Four deal with the result and discussion of DNA damage (8oxoguanine) and apoptosis respectively which induced by low-LET radiations (x and
60
Co gamma) rays. Finally, conclusions extracted from the present
results are presented in chapter Five in addition to some future works.
Chapter Two
Materials and Methods
2- Materials and Methods All the experiments were performed at Belgian Nuclear Center (SCK.CEN) in Mol, Belgium. Human blood samples each of 40 ml were taken from healthy non- smoking donors in heparinized vacutainer tubes from medical service in SCK.CEN. The donors were of age between (25-50) years. 2-1 Materials The chemicals, kits and instruments used in the present work are as follows: 2-1-1 Chemicals Table (2.1) shows the chemicals which are used in our study. Table (2.1): Presents the chemical used for blood samples preparation. Chemicals
Source
Histopaque 1077
Sigma, Germany
Phosphate buffer Saline (PBS)
Gibco, UK
RPMI (1640+Guloamax)
Gibco, UK
Fetal bovin serum (FBS)
Gibco, UK
L-glutamine
Gibco, Belgium
penicilline-streptomycine
Gibco, Belgium
Paraformaldehyde
Gibco, Belgium
Ethanol
Merck, Belgium
Fluorescence activated cell sorting (FACS) fluid
Beckman coulter, France
Sphero calibrate particles (bead)
Spherotech Inc., USA
2-2-2 Kits 1- OxyDNA kit fluorometric (calbiochem, U.S.A). The kit contained the following materials: a- Concentrated wash solution, diluted by 1:25 with distill water. b- Concentrated block solution, diluted by 1:10 with wash solution. c- Concentrated flourescein isothiocyanate (FITC) - conjugate, diluted by 1:10 with wash solution. 2- CaspGLOWTM fluorescein active caspase-3 staining kit (MBL, U.S.A): The kit contained: a- Wash buffer. b- FITC. 2-2-3 Instruments The instruments which are used in this study present in the following table. Table (2.2): Presents the instruments used in the study. Instruments
Source
Heparinzed vacutainer tubes
Escapo, Belgium
Conical centrifuge tubes
Greiner-bio-one, Belgium
Culture flask (volume 25cm3)
Greiner-bio-one, Belgium
Hemocytometer
Paul Marienfeld, Germany
Centrifuge.
Eppendorf, Germany
Flowcytometry tubes.
BD Falcon, Belgium
Incubator
Binder GmbH, Germany
Flow cytometry (cloture Epics XL-MCL).
Beckman coulter, U.S.A
Different type and size of pipettes.
Greiner-bio-one, Belgium
2-2 Methods Methods employed for separation and preparation of mononuclear cells are described as follows: 2-2-1 Procedure of mononuclear cell separation from the blood The procedure for separation of mononuclear cells from human blood can be summarized by the following steps [63, 67]. 1- A 10 ml of the histopaque was added to 25 ml conical centrifuge tubes. 2-The whole blood 10 ml was layered carefully onto the histopaque into each tube, and centrifuged at 1800g for 30 min. 3-After centrifugation, the opaque interfaces containing the mononuclear cells from all the tubes were transferred carefully by Pasteur pipette to a clean conical centrifuge tube. 4-To this tube, 10 ml of the PBS solution was added and mixed by gentle aspiration, and centrifuged at 800g for 10min. 5-The supernatant was then discarded 6-The cells pellet was resuspended with 10 ml of PBS solution, mixed by gentle aspiration, and centrifuged at 800g for 10 min. 7-The previous steps were done for each tube of the donor. 2-2-2 Preparation of mononuclear cells for irradiation 1- After mononuclear cells separation and two washings with PBS, 7 ml of culture medium (RPMI, supplemented with 20% FBS and 1% of penicillin and streptomycin) were added to the pellet inside the tube. 2- 5-10x 106 cells/ml were prepared by counting with hemocytometer. 3- From the tube which contains cell culture, 5-10x106cells/ml were transferred to each 6 flask and then 5 ml of the culture medium were added to each of them.
2-3 Irradiation of Blood Samples 2-3-1 X-ray irradiation X-irradiation of the cells was performed with a Pantak HF420 RX machine operating at 250 kV, 1mm Cu filtering, 1mA with dose rate of 20 mGy/min, or 15mA with dose rate of 0.28 Gy/min, the mean energy was 208keV. Dosimetry was performed on a regular basis with a 0.6 cm3 ionizing chamber (NE 2571), which was connected to a dosimeter (Farmer dosimeter 2570). The champer was placed in parallel to the irradiated cell flasks. Dose homogenecity was evaluated as being<1.5%. Control cultures were not irradiated but otherwise treated like irradiated cultures. The cells from each donor were irradiated with 0.25, 0.5, 1, 2, and 4 Gy with dose rate 0.28 Gy/min., and with 15.6, 31.3, 62.5, 125 and 250 mGy with dose rate of 20 mGy/min. and incubated for 24 hrs at 37oC in 5% CO2. 2-3-2 60Co gamma ray irradiation Gamma irradiation of the cells was performed with
60
Co of 1.25 MeV
mean energy with dose rate 2Gy/hr for doses (0.25, 0.5, 1, 2, 4) Gy. Fig. (2.1) shows the γ source used in the present work. Its active diameter is d=15mm, outer diameter is D=18.2 mm and height is I=36.8 mm.
Fig. (2.1):
60
Co gamma ray source used in the present work.
2-4 Assay of DNA Damage (8-oxoguanine) 2-4-1 Principle of the assay The OxyDNA assay is based upon the direct binding of a fluorescent probe to 8-oxoguanine moieties in the DNA of fixed cells. The amount of fluorescence in 8-oxoguanine (FITC) was measured by flow cytometry and 8oxoguanine molecules/cells was calculated using the calibration curve.
2-4-2 Method The procedure for fixation and staining with FITC can be summarized by following steps [32, 33]. 2-4-2-1 Fixation 1-After 24hr. of incubation, the cells (3-4x106) were harvested and transferred to conical centrifuge tubes (15ml) then centrifuged at 230g for 5min. The supernatant was discarded. 2-The cells were resuspended in 2ml PBS contain (0.9%NaCl, pH 7.4), and centrifuged at 230g for 5min after which the supernatant was discarded. 3- A 500 µl of PBS was added to the cells and mixed gently. Then 500µl of 2% paraformaldehyde was added, and the samples were mixed gently again. The tubes were incubated on ice for 15min. 4-The cells were washed twice with 2ml of the PBS and centrifuged at 230g for 5min then the supernatant was discarded. 6-The cells were resuspended in 1ml ice-cold 70% ethanol, and mixed gently. The cell suspension was stored at -20C° for 24 hrs. 2-4-2-2 Staining with FITC 1- The day after, fixed ethanol-treated cells were centrifuged at 230g for 5min. and the supernatant was discarded. 2-The cells were washed with 2ml of the PBS, and resuspended in 3ml of wash solution, then centrifuged at 230g for 5min.to remove the supernatant.
3- A 50µl freshly prepared Blocking solution was added, and incubate 1hr at 37C°. 4-The cells were washed twice with 3ml of wash solution, and centrifuged at 230g for 5min. The cells then were incubated with 100µl of FITC-conjugate for 1hr in the dark room. 5-The cells were washed twice with 3ml of wash solution, and then the cells washed with 2ml of the PBS. 6-The cells were resuspended in FACS fluid, then transferred to the flowcytometry tubes and kept in the dark on ice, then read by the flow cytomtery. 2-5 Assay of Apoptosis 2-5-1 Principle of the assay Caspases are crucial mediators of programmed cell death (apoptosis). Among them, caspase 3 is frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins. Pathway to caspase 3 activation has been identified that are either dependent on or independent of mitochondrial cytochrome c release and caspase 9 functions [21]. The kit provides a convenient and sensitive means for detecting activated caspase-3 in situ in living cells. The FITC label allows for direct detection of activated caspases in apoptotic cells by flow cytometry [68].
2-5-2 Method 1-From 106 cell/ml cell suspension, 300µl from the irradiation and control cultures were aliquoted to flow cytometry tubes, 1µl of FITC were added into each tube and incubated for 1hr at 37°C with 5% CO2. 2-After incubation the cells centrifuged at 1800g for 5min., and then the supernatant were removed.
3-The cells were resuspended twice in 0.5ml of wash buffer, centrifuged at 1800g for 5min. then the supernatant removed. 4-The cells were resuspended in 300µl of wash buffer and kept on ice, then analyzed by flow cytometry.
2-6 Flow Cytometry Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. The properties measured include a particle’s size, granularity or internal complexity, and fluorescence intensity. These characteristics are determined using an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence. A flow cytometer is made up of three main systems: fluidics, optics, and electronics. • The fluidics system transports particles in a stream to the laser beam for interrogation. • The optics system consists of lasers to illuminate the particles in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors. • The electronics system converts the detected light signals into electronic signals that can be processed by the computer. In the flow cytometer, particles are carried to the laser intercept in a fluid stream. Any suspended particle or cell from 0.2–150 micrometers in size is suitable for analysis. Cells from solid tissue must be disaggregated before analysis. The portion of the fluid stream where particles are located is called the sample core. When particles pass through the laser intercept, they scatter laser light. The scattered and fluorescent light is collected by appropriately positioned lenses. Forward-scattered light (FSC) is proportional to cellsurface area or size and side-scattered light (SSC) is proportional to cell
granularity or internal complexity. A combination of beam splitters and filters steers the scattered and fluorescent light to the appropriate detectors. The detectors produce electronic signals proportional to the optical signals striking them. List mode data are collected on each particle or event. The characteristics or parameters of each event are based on its light scattering and fluorescent properties. The data are collected and stored in the computer [69]. This data can be analyzed to provide information about subpopulations within the sample fig. (2.2).
Fig. (2.2): Flow cytometry; schematic of a typical flow cytometer setup [70].
2-7Analysis by Flow cytomtery Flow cytometry allows detection of altered light scattering and fluorescent behavior of nucleoids after cellular irradiation. These may be related to structural change within the nucleus induced by the radiation. The use of flow
cytometry compared to other method allows a rapid analysis of nuclear damage within individual cells [71].
2-7-1 Measuring DNA damage (8-oxoguanine) fluorescence OxyDNA assay is specific for 8-oxoguanine which is formed during free radical damage to DNA and is a sensitive and specific indicator of oxidative DNA damage. After the cells were fixed and FITC labeled protein was added. Binding to the 8-oxoguanine moiety present in the 8-oxoguanosine of oxidized DNA ensued. The presence of oxidized DNA was indicated by green fluorescence that read directly by flowcytomtery. Fluorescence intensity (FITC) in arbitrary units was plotted in histograms as shown in fig. (2.3), and the mean fluorescence intensity was calculated using software system II version 3.0. To evaluate the molecular of equivalent fluorescent (MEFL) for DNA damages sphero calibration particles (RCP-30-5) were used.
Fig.(2.3): Fluorescence intensity (FITC) in arbitrary units as plotted in histograms by flow cytometry.
We used sphero calibration particles (beads) with known MEFL values which permitted the calibration and the conversion the channel to MEFL as a unit of fluorescence intensity as shown in Fig.(2.4). We used triplicate and took the mean of 8-oxoguanine fluorescence, FITC, then the value of MEFL was drawn versus FITC as shown in Fig.(2.5)
Fig.(2.4): Calibration of MEFL with fluorescence intensity.
350000 y = 4608.6x - 3047.8
300000 MEFL
250000 200000 150000 100000 50000 0 0
20
40
60
80
8-oxoguanine fluore se cnce ,FITC
Fig.(2.5): Variation of amount 8-oxoguanine, FITC with known MEFL. Errors are within the points size.
We calculated MEFL (8-oxoguanine molecules) for each dose using the curve fitting equation: Y=4608.6X-3047.8 where : Y= MEFL X=8-oxoguanine fluorescence, FITC for each dose. The number of 8-oxoguanine molecules/cell was obtained after dividing MEFL by 4 because each 8-oxoguanin molecules bind with 4 FITC.
2-7-2 Measuring fluorescence, FITC and the numbers of apoptotic cells Flow cytometry is a much faster method for detecting apoptosis, allowing thousands of cells to be analyzed, which increases the sensitivity of the assay [15]. After the cell were labeled with FITC analyzed by flow cytometry. Fluorescence intensity (FITC) in arbitrary units was plotted in histograms as shown in fig. (2.6), and the mean fluorescence intensity was calculated
using software system II version 3.0. The number of apoptotic, lymphocytes, and monocytes were recorded, and then their percentage was calculated.
Fig.(2.6): Fluorescence intensity of caspase 3, FITC as measured directly by flow cytometry..
2-8 Statistical Analysis Results were always represented as mean± standard error of the mean (SEM) of four or five independent experiments with triplicate measurements. The t-test was performed to compare between the control and irradiation samples and values of p<0.05 were considered significant, and those of p<0.001 reflect high significance.
Chapter Three
X and γ rays Induced DNA Damage: Results and Discussion
3-1 Introduction Radiation can penetrate into living cells and results in the transfer of radiation energy to the biological material. The absorbed energy can increase the reactive oxygen species and break chemical bonds and cause ionization of different biologically essential macromolecules, such as DNA, membrane lipids and proteins. The DNA damage induced by radiation such as base alteration, cross-linking, strands break or chromosomal aberration which may in turn lead to mutation [72]. Oxidation of DNA can result in damage to all four bases and the deoxyribose. Among oxidative base damages in DNA, a C8-oxidation form of guanine, 7, 8-dihydro-8-oxoguanine (8-oxoG) is one of the major base lesions and is often used as a key indicator of cellular oxidative stress. Oxidative damage to DNA induced exogenously by radiation or chemical agents or endogenously by free radicals released during respiration [10, 73]. In this chapter we present our experimental results on 8-oxoguanine fluorescence, and calculated 8-oxoguanine molecules/cell after calibration using sphero calibrate particles (beads), also relative granularity and size of mononuclear cells were determined, which they are induced by:
1- X- ray doses 0.25, 0.5, 1, 2, 4 Gy at dose rate 0.28Gy/min. 2- X –ray doses 15.63, 31.3, 62.5, 125, 250 Gy at dose rate 20mGy/min. 3- 60Co gamma ray doses 0.25, 0.5, 1, 2, 4 Gy at dose rate 2Gy/hr. Based on our results, a dose response model is formulated and discussed. 3-2 X-ray Induced DNA Damage (8-oxoguanine) 3-2-1 X-ray induced DNA damage with dose rate 0.28Gy/min The results with dose rate 0.28 Gy/min for doses 0, 0.25, 0.5, 1, 2 and 4 Gy for four donors are summarized in Tables (3.1-3.4). Significant difference of 8-oxoguanine/cell were observed between control and irradiated samples for all donors. Variation of the number of 8-oxoguanin molecules /cell with doses was observed inside each donor. This variation is due to the differential radiosensitivity of mononuclear populations [74- 80] and due to their repair capacity. Healthy individuals also differ in intrinsic repair capacity [81], which also Singh et al.[36] explained that the bulk of the repair occurred within 15min after irradiation of human lymphocytes with 200 rad (2Gy) of xray. Because mononuclear cells are a heterogeneous mixture of cells, as regarding their life-span and sensitivity, some difference may be due to different cell populations [82]. Oxidized DNA base lesion are removed by essentially two types of activity; base excision repair (BER), involving removal of single lesion by glycosylase action; and a more complex process involving the removal of a lesion-containing olignucleotid, nucleotide excision repair (NER) [12]. Niedźwiedź and Cebulska, observed from their study the efficiency of the resealing of the DNA damage might suggest an existence of two phases in the kinetics of the DNA damage repair. The first phase might represent the restoration of the DNA damage that could be rejoined by the simple action of ligases or BER (base excision repair) mechanism which is very fast. On the second phase which is much slower, the NER mechanism might take place and much more complex DNA damage
might repaired. Thus the nucleotide excision repair mechanism could be responsible for the slight increase of DNA damage [35]. Fig. (3.1) shows variation x- ray doses 0, 025, 0.5, 1, 2, 4 Gy with the number of 8-oxoguanine molecules/cell, with dose rate of 0.28 Gy/min. for four donors. After normalization for the donors the number of 8-oxoguanine molecules /cell increase with increasing doses and significant for all doses except for 4 Gy as shown in Table (3.5), the percentages of increasing were 49%, 50%, 53%, 58%, 81% for doses 0.25, 0.5, 1, 2, 4 Gy respectively. Our results of the normalization shown in Fig.(3.2) agree with the result of Singh et al.[36] which show a linear increase in the length of DNA migration (DNA damage) in human lymphocytes for doses between 25 and 100 rad (0.25-1) Gy, and by 200 rad (2) Gy the length of migration appeared to plateau and with Rössler et al.[41] which they showed that irradiation of human whole blood to x- ray and
60
Co gamma ray with doses( 0.5-3) Gy the DNA damage parameter
increases with doses and at 1-1.5Gy the x-ray dose response showed a saturation behavior. Also Collins et al.[62] showed that increasing of DNA breakage after irradiation of human lymphocytes with x-ray doses (0-10) Gy at dose rate 1.38Gy/min.
Table (3.1): Effect of x-ray dose on mononuclear blood cells in donor 1. Dose rate is 0.28 Gy/min. Mean of 8oxoguanine fluorescence
SEM
8-oxoguanine molecules/cell
SEM
p -value
0 (Control)
1.20
0.04
621
42
-
0.25
1.59
0.01
1074
10
0.0004**
0.5
1.74
<0.01
1239
4
0.0042*
1
1.68
0.05
1174
58
0.0014*
2
1.71
0.03
1212
33
0.0003**
4
2.15
0.26
1715
303
0.0231*
X-ray dose (Gy)
*Significant difference in comparison with control p<0.05. ** High significant difference in comparison with control p<0.001.
Table (3.2): Effect of x-ray dose on mononuclear blood cells in donor 2. Dose rate is 0.28 Gy/min. Mean of 8oxoguanine fluorescence
SEM
8-oxoguanine molecules/cell
SEM
p -value
0 (Control)
2.07
0.04
1619
43
-
0.25
2.72
0.02
2368
23
1x10-04**
0.5
2.61
0.01
2249
8
1.3x10-04**
1
3.02
0.02
2714
21
2.24x10-05**
2
3.71
0.01
2894
8
8.4x10-06**
4
2.35
0.01
1949
10
1.7x10-03*
X-ray doses (Gy)
*Significant difference in comparison with control p<0.05. ** High significant difference in comparison with control p<0.001.
Table (3.3): Effect of x-ray dose on mononuclear blood cells in donor 3. Dose rate is0.28 Gy/min. X-ray doses (Gy)
Mean of 8oxoguanine fluorescence
SEM
8-oxoguanine molecules/cell
SEM
p- value
0 (Control)
1.68
<0.01
1170
4
-
0.25
1.86
0.01
1377
10
4.43x10-05**
0.5
1.85
0.01
1373
10
4.78x10-05**
1
1.95
0.02
1485
18
6.29x10-05**
2
1.83
0.01
1343
8
3.6x10-05**
4
1.99
0<0.01
1527
4
3.2x10-07**
** High significant difference in comparison with control p<0.001.
Table (3.4.): Effect of x-ray dose with on mononuclear blood cells in donor 4. Dose rate is 0.28Gy/min. X-ray doses (Gy)
Mean of 8oxoguanine fluorescence
SEM
8-oxoguanine molecules/cell
SEM
p -value
0 (Control)
1.77
0.01
1274
10
-
0.25
2.37
0.01
1965
14
2.27x10-06**
0.5
2.19
0.01
1761
7
2.29x10-06**
1
2.04
0.01
1585
8
1.64E-05**
2
2.19
0.01
1765
10
4.35x10-06**
4
2.76
0.01
2422
27
2.34x10-06**
** High significant difference in comparison with control p<0.001.
3500
8-oxoguanine /cell
3000 2500
Donor 1
2000
Donor 2
1500
Donor 3 Donor 4
1000 500 0 0
1
2
3
4
5
X-ray dose (Gy)
Fig. (3.1): Variation of 8-oxoguanine molecules/cell with x-ray dose (dose rate is 0.28Gy/min). The results represent mean±SEM for each donor.
Table (3.5): Percentage of 8-oxoguanine molecules/cell with x-ray dose after normalization for all donors. Dose rate is 0.28 Gy/min. Doses (Gy)
8oxoguanine molecules/cell in donor 1 100
8-oxoguanine molecules/cell in donor 2 100
8-oxoguanine molecules/cell in donor 3 100
8-oxoguanine molecules/cell in donor 4 100
8-oxoguanine molecules/cell mean 100
SEM
p-value
0
-
173
146
118
159
149
12
0.01*
0.5
200
139
117
142
150
18
0.03*
1
189
168
127
128
153
15
0.01*
2
195
179
115
143
158
18
0.02*
4
276
120
131
196
181
36
0.07
0 (Control) 0.25
*Significant difference in comparison with control p<0.05.
Oxoguanine molecules/cell after normalization
250
200 150
100 50
0 0
1
2
3
4
5
X-ray dose (Gy)
Fig. (3.2): Variation of percentage of 8-oxoguanine molecules/cell with x-ray dose, after normalization for 4 donors. Dose rate is 0.28 Gy/min.
3-2-2 X-ray induced DNA damage (8-oxoguanine) with dose rate 20mGy/min The results with dose rate 20 mGy/min. for doses 0, 15.625, 31.25, 62.5, 125, 250 mGy for five donors are summarized in Tables (3.6- 3.9). Increasing of DNA damage (8-oxoguanine/cell) was observed with increasing dose. The damage was significant only in donors 1 for all doses, but in donors 2, 3 and 4 the damage was not significant for all doses which depends on the sensitivity of individuals and repair capacity inside each donor. After normalization increasing in DNA damage observed with dose and significant at lowest doses 15.6, 31.3, 60.5 mGy. As comparison, Chaubey et al. observed a significant increase in DNA damage in human leukocytes even at lowest dose 1.25cGy than (2.5, 5, 10) cGy [38]. Figure (3.3) shows the effect of x-ray dose 0, 15.6, 31.3, 62.5, 125, 250 mGy on the number of 8-oxoguanine/cell at dose rate 20 mGy/min for five donors. Table (3.10) shows the percentage of 8-oxoguanine molecules/cell x-ray dose at dose rate of 20mGy/min after normalization for all donors. The results are shown in fig.(3.4 ) as the percentage of DNA damage were 15%, 10%, 15%, 18%, 34% for doses 15.6, 31.3, 62.5, 125, 250 mGy respectively.
Table (3.6): Effect of x-ray dose on mononuclear blood cells in donor 1. Dose rate is 20mGy/min. X-ray dose (mGy)
Mean of 8oxoguanine fluorescence
SEM
8-oxoguanine molecules/cell
0 (Control)
3.71
0.01
3509
17
-
15.6
4.07
0.02
3931
19
7.75x10-05**
31.3
4.13
0.01
3996
7
1.11x10-05**
62.5
4.31
0.03
4208
33
4.84x10-05**
125
5.33
0.02
5375
20
2.39x10-07**
250
6.32
0.16
6520
186
8.7x10-05**
SEM
p value
** High significant difference in comparison with control p<0.001.
Table (3.7): Effect of x-ray dose on mononuclear blood cells in donor 2. Dose rate is 20mGy/min. X-ray dose (Gy)
Mean of 8oxoguanine fluorescence
SEM
8-oxoguanine molecules/cell
SEM
p -value
0 (Control) 15.6
2.92
0.04
2598
47
-
3.28
0.01
3013
10
<0.001**
31.25
3.20
0.01
2925
7
0.02*
62.5
3.16
0.003
2883
4
0.03*
125
2.99
0.01
2679
8
0.2
250
3.62
0.003
3405
4
0.003*
*Significant difference in comparison with control p<0.05. ** High significant difference in comparison with control p<0.001.
Table (3.8): Effect of x-ray dose on mononuclear blood cells in donor 3. Dose rate is 20 mGy/min. X-ray dose (mGy)
Mean of 8oxoguanine fluorescence
SEM
8-oxoguanine molecules/cell
SEM
p- value
0 (Control)
2.10
0.02
1661
20
-
15.63
2.36
0.03
1953
37
0.002*
31.3
2.16
0.01
1723
14
0.07
2.26
0.02
1842
20
0.003*
125
2.21
0.01
1780
17
0.01*
250
2.27
0.01
1850
8
<0.001**
62.5
*Significant difference in comparison with control p<0.05. ** High significant difference in comparison with control p<0.001.
Table (3.9): Effect of x-ray dose on mononuclear blood cells in donor 4. Dose rate is 20mGy/min. X-ray dose (mGy)
Mean of 8oxoguanine fluorescence
SEM
8-oxoguanine
0 (Control)
2.88
15.625
p- value
molecules/cell
SEM
0.06
2556
70
-
3.16
0.02
2883
21
0.01*
31.25
3.07
0.01
2779
8
0.08
62.5
3.31
0.01
3048
10
0.02*
125
3.03
0.01
2729
12
0.07
250
3.03
0.01
2733
14
0.07
*Significant difference in comparison with control p<0.05.
8-oxoguanine molecules/cell
7000 6000 5000 Donor 1 4000
Donor 2
3000
Donor 3 Donor 4
2000 1000 0 0
50
100
150
200
250
300
X-ray dose (m Gy)
Fig. (3.3): Variation of 8-oxoguanine molecules/cell with x-ray dose, (Dose rate is 20 mGy/min.). The results represent mean±SEM for each donor.
Table (3.10): Percentage of 8-oxoguanine molecules/cell with x-ray dose after normalization for 4 donors. Dose rate is 20 mGy/min. X-ray dose (mGy) 0 (Control)
8oxoguanine molecules/ cell in donor 1
8oxoguanine molecules/ cell in donor 2
8oxoguanine molecules/ cell in donor 3
8oxoguanine molecules/ cell in donor 4
8oxoguanine molecules/ cell (Mean)
SEM
p-value
100
100
100
100
100
0
112
116
118
113
115
1
3.12x1005 **
114
113
104
109
110
2
0.005*
120
111
111
119
115
3
<0.001*
153
103
107
107
118
12
0.19
107
134
18
0.112
15.63 31.3 62.5 125 250
186 131 111 *Significant difference in comparison with control p<0.05.
** High significant difference in comparison with control p<0.001.
Oxoguanine molecules/cell after normalization
160 140 120 100 80 60 40 20 0 0
50
100
150
200
250
300
X-ray dose (m Gy)
Fig. (3.4): Variation of percentage of 8-oxoguanine molecules/cell with x-ray dose after normalization for 4 donors. Dose rate is 20 mGy/min.
3-3 60Co γ ray Induced DNA Damage (8-oxoguanine) In γ-ray experiments the dose rate taken was 2 Gy/hr (0.033 Gy/min) and the results for doses 0, 0.25, 0.5, 1, 2, 4 Gy for 4 donors are summarized in Tables (3.11-3.14). Significant difference was observed between control and irradiated samples for all donors. Also variation
of
8-oxoguanine
was
observed
due
to
different
radiosensitivity between individuals [83, 84]. This variation in radiosenstivity between individual may be a consequence of differences in DNA repair capacity due to specific mutation or polymorphisms in DNA repair genes or alternatively, may be linked to cell cycle arrest [74]. The heterogenecity in response to gamma ray is a function of cell type and the repair capacity of the cell [38]. Figure (3.5) shows the effect of these γ- ray doses on the number of 8oxoguanine molecules/cell for the four donors. Table (3.15) shows variation of 8-oxoguanine molecules/cell with γ ray dose, dose rate 2Gy/hr after normalization for the donors, and a significant difference was observed for all doses with variation in 8-oxoguanine/cell due to heterogenecity between individuals and Fig. (3.6) shows the effect of γ- ray dose on the number of 8-oxoguanine molecules/cell after normalization
which
the
percentage
of
DNA
damage
(8-
oxoguanine/cell) were 21%, 17%, 21%, 58%, 53% for doses 0.25, 0.5, 1, 2, 4 Gy respectively. Increasing of DNA damage was with dose also observed by Mozdarani et al.[37] which they found that radiation significantly increase DNA damage in leukocytes of mouse in a dose dependent manner.
Table (3.11): Effect of 60Co γ-ray dose on mononuclear blood cells in donor 1. Dose rate is 2 Gy/hr γ-ray doses (Gy)
Mean of 8-oxoguanine fluorescence
SEM
8-oxoguanine molecules/cell
SEM
p- value
0 (Control)
5.24
0.06
5279
68
-
0.25
5.52
0.07
5594
80
3.9x10-02*
0.5
5.53
0.03
5606
33
1.2x10-02*
1
6.60
0.07
6842
75
1x10-04**
2
8.11
0.02
8586
27
1.42x10-06**
4
7.65
0.05
8048
63
7.4x10-06**
*Significant difference in comparison with control p<0.05. ** High significant difference in comparison with control p<0.001.
Table (3.12): Effect of 60Co γ-ray dose on mononuclear blood cells in donor 2. Dose rate is 2 Gy/hr.
SEM
Mean of 8-oxoguanine molecules/cell
SEM
p-value
1.78
0.01
1293
8
-
2.13
0.01
1688
10
6.41x10-06**
2.10
0.01
1661
10
8.48x10-06**
1.96
0.01
1496
13
1.9x10-04**
2.72
0.02
2372
24
1.77x10-06**
2.33 0.01 1926 ** High significant difference in comparison with control p<0.001.
10
9.77x10-07**
γ-ray doses(Gy) 0 (Control) 0.25
Mean of 8-oxoguanine fluorescence
0.5 1 2 4
Table (3.13): Effect of 60Co γ-ray dose on mononuclear blood cells in donor 3. Dose rate is 2Gy/hr. γ-ray doses
Mean of 8-oxoguanine
(Gy)
fluorescence
SEM
molecules/cell
SEM
p- value
0 (Control)
2.38
0.02
1976
28
-
0.25
2.47
0.02
2084
24
4.3x10-03**
0.5
2.92
0.01
2602
12
3.1x10-05**
1
2.88
0.02
2556
18
6.02x10-05**
2
3.63
<0.01
3424
4
2x10-04**
4
3.13
0.01
2844
7
6.91x10-06**
8-oxoguanine
** High significant difference in comparison with control p<0.001.
Table (3.14): Effect of 60Co γ-ray doses on mononuclear blood cells in donor 4. Dose rate is 2 Gy/hr. γ-ray doses (Gy)
Mean of 8-oxoguanine fluorescence
SEM
8-oxoguanine molecules/cell
SEM
p -value
0 (Control)
3.73
0.03
3539
30
-
0.25
5.04
0.01
5049
17
1.6x10-06**
0.5
3.85
0.01
3674
13
1.4x10-02*
1
3.96
0.02
3804
28
2x10-03**
2
4.14
0.01
4012
10
1x10-04**
4
5.78
0.04
5894
43
1.49x10-06**
*Significant difference in comparison with control p<0.05. ** High significant difference in comparison with control p<0.001.
8 -o x o g u a n in e m o le c u le s /c e ll
10000 9000 8000 7000
Donor !
6000
Donor 2
5000
Donor 3
4000
Donor 4
3000 2000 1000 0 0
1
2
3
4
5
Co60 gamma ray dose (Gy)
Fig.(3.5): Variation of 8-oxoguanine molecules/cell with x-ray doses. (Dose rate is 0.28 Gy/min.) The results represent mean±SEM for each donor.
Table (3.15): Percentage of 8-oxoguanine molecules/cell with after normalization for 4 donors. Dose rate is 2 Gy/hr.
Doses(Gy) 0 (Control) 0.25
60
Co γ-ray dose
8-oxoguanine molecules/cell in donor 1
8-oxoguanine molecules/cell in donor 2
8-oxoguanine molecules/cell in donor 3
8-oxoguanine molecules/cell in donor 4
8-oxoguanine molecules/cell mean
SEM
p-value
100
100
100
100
100
0
106
131
105
143
121
11
106
129
132
103
117
9
6.2x1002 * 5.9x1002 *
130
116
129
107
121
6
163
183
173
113
158
18
167
153
6
0.5 1 2 4 152 149 144 *Significant difference in comparison with control p<0.05.
** High significant difference in comparison with control p<0.001.
Oxoguanine molecules/cell after normalization
200 180 160 140 120 100 80 60 40 20 0 0
1
2
3
4
5
Co60 gam m a ray (Gy)
Fig. (3.6): Variation of percentage of 8-oxoguanine molecules/cell with γ-ray doses after normalization for 4 donors. Dose rate is 2 Gy/hr.
9x10-03* 9.5x1003 * 3.46x1005 **
3-4 Dose Response Model To describe the dependence of DNA damage (number of 8-oxogunanie molecules /cell) on the dose and dose rates, we suggested a mathematical model of the form: Y= a Db +c … where
(3.1)
Y= number of 8-oxoguanine molecules/cell a=The amplitude parameter. D=The radiation dose. b= A shape parameter. c= A constant, which represents background of the variable Y.
The values of the parameters are given in Table (3.16). We observed from the value of parameter (a) that high doses with high dose rate of x- ray (0.28 Gy/min.)
have
more
effect
on
the
DNA
damage
(8-oxoguanine
molecules/cell) than low doses at low x-ray dose rate (20mGy/min) and high doses at low
60
Co gamma dose rates (2Gy/min). That can be related to the
effectiveness of low-LET radiation which can be reduced by a factor 2-4 fold at low dose rates [85]. A number of studies have demonstrated that, in general, the biological response to radiation exposure decreases at low dose rate compared with the response at high dose rate. The effects of dose rate seem to differ according to the quality of the radiation, the range of doses and dose rate, the biological response being measured. These many observations suggest that biological response to ionizing radiation are highly complex processes that depend on both dose and dose rate [86]. The effect of high doses with low dose rate of 60Co gamma ray (2Gy/hr) more than low doses with low dose rate of x-ray (20mGy/min), because the majority of DNA lesion by low doses of ionizing radiation are repaired
rapidly and efficiently [38]. From our results we observed that DNA damage depends on dose and dose rate in agreement with Mozdarani et al.[37], Chaubey et.al [38, 39], Vijayalaxma et al. [40] and Rossler et al. [41]. From the value of b one can find the damage slowly increases with the dose in high doses with high dose rate of x-ray than low doses with low dose rate of x-ray and low doses with low dose rate of 60Co gamma ray. This might be because the response of the cells at low dose rate is more than high dose rate this mean that at low dose rate the damage was fast at the same time the rate of repairing was also fast. Figs. (3.7, 3.8, 3.9) show the fitting curves for the three experiments. The value of c=100 which represents the number of 8oxoguanine/cell at 0 Gy after appling normalization for donors. Table (3.16): Fitting parameters a and b in our mathematical model for the three
experiments. R is the correlation coefficient and c=100 which is the amount of fluorescence of 8-oxoguanine, FITC at 0 Gy after normalization.
Type of irradiation
Parameter a ±Std error
Parameter b ±Std error
R2
High x-ray dose with dose rate 0.28Gy/min.
57.09±2.72
0.19±0.05
0.961
Low dose of x-ray with dose rate 20mGy/min.
2.47±1.55
0.46±0.13
0.878
High dose Co60 gamma ray with dose rate 0.033Gy/min.
30.82±5.31
0.46±0.16
0.834
8-oxoguanine molecules/cell
200 150 100 50 0 0
100
200
300
x-ray dose (mGy)
8-oxoguanine molecules/cell
Fig.(3.7): Dose response curve for high doses of x-ray at dose rate of 0.28 Gy/min.
200 150 100 50 0 0
1 60
2
3
4
5
Co gamma ray dose (Gy)
8-oxoguanine molecules/cell
Fig.(3.8): Dose response curve for low doses of x-ray at dose rate of 20 mGy/min. 250 200 150 100 50 0 0
1
2
3
4
5
X-ray dose (Gy)
Fig.(3.9): Dose response curve for low doses of Gy/min.
60
Co gamma ray at dose rate of 0.033
3-5 Measurement of Relative Granularity and Size of Mononuclear Cells Granularity and size of the mononuclear cells were measured using flow cytometry, since FSC is proportional to size and SSC is proportional to cell granularity. Values relative to control were determined. The effect of x-ray doses 0, 0.25, 0.5, 1, 2, 4 Gy at dose rate 0.28Gy/min on the granularity and size of mononuclear cells is presented in Table (3.17). The relative size and granularity of the cells were larger than control for all doses. However, the increasing was not linear, might be because mononuclear subpopulation cells have different size and properties. Table (3.18) shows the effect of x-ray doses 15.6, 31.3, 62.5, 125, 250 mGy on the relative of granularity and size of the cell when dose rate was 20 mGy/min.. At all doses the relative values were increased for both granularity and size except at 15.63 Gy dose The results for
60
Co gamma ray are presented in Table (3.19). For all doses the
relative granularity was more than the control and also the relative size was also larger than control except at 0.5 Gy dose. In all the three experiments from p values (p>0.05) we observed changing in the relative of granularity and size of mononuclear cells were insignificant.
Table (3.17): Mean relative granularity and size of mononuclear cells with x-ray dose at dose rate of 0.28Gy/min.
Mean relative granularity of mononuclear cells±SEM
p-value
Mean relative size of mononuclear cells±SEM
0
-
0
0.25
6.35±0.05
0.77
7.29±0.04
0.65
0.5
1.98±0.05
0.92
7.32±0.04
0.64
1
1.19±0.07
0.95
6.06±0.05
0.68
2
4.17±0.05
0.85
4.53±0.05
0.78
4
6.94±0.04
0.76
7.54±0.03
0.65
X-ray dose (Gy) 0 (Control)
p-value
Table (3.18): Mean relative granularity and size of mononuclear cells with x-ray dose at dose rate of 20mGy/min.
p-value
Relative size of mononuclear cells±SEM
p-value
-
-
0
-
15.6
-1.51±0.06
0.94
-0.80±0.07
0.97
31.3
6.53±0.01
0.78
6.34±0.05
0.79
62.5
13.9±0.06
0.63
11.00±0.005
0.7
125
9.38±0.04
0.72
5.82±0.02
0.82
250
6.53±0.01
0.78
4.00±0.04
0.87
X-ray dose (mGy) 0 Control
Relative Granularity of mononuclear cells±SEM
Table (3.19): Mean relative granularity and size of mononuclear cells with
60
Co gamma
ray dose at dose rate of 2Gy/hr. Relative granularity of mononuclear cells±SEM
p-value
Relative size of mononuclear cells±SEM
p-value
0
-
0
-
0.25
5.28±0.01
0.66
2.66±0.01
0.86
0.5
6.24±0.03
0.57
-2.11±0.02
0.88
1
7.19±0.02
0.53
0.22±0.01
0.99
2
16.07±0.04
0.14
1.58±0.05
0.9
4
26.14±0.03
0.05
1.85±0.03
0.89
60
Co gamma ray dose(Gy) 0 Control
Chapter Four
X and γ rays induced apoptosis: Results and Discussion
4-1 Introduction When cells are exposed to ionizing radiation, they respond to a variety of ways that differ quantitatively and qualitatively according to the absorbed dose and the cell type, and that generally reflect damage caused to welldefined cellular components and molecular structures. Apoptosis represents an extreme and highly complex response to ionizing radiation since it requires an active participation of the cell in its own death [49]. Apoptotic cell death is regarded as one of the major cell death forms after the cells are exposed to ionizing radiation [87]. Majority of hematopoietic cells die by apoptosis after irradiation with ionizing radiation [88]. A number of caspases are known to play a pivotal role in the execution of apoptosis. Especially caspase 3 is generally accepted to be a key protease that is activated during the apoptotic process [89]. We detected apoptosis cells by activation of caspase 3 using CaspGLOWTM fluorescein active caspase-3 staining kit. In this chapter we present our experimental results on the amount of fluorescence intensity of activated caspase 3 and determined percentage number of apoptosis cells (cell death), percentage number of lymphocytes
(living cells) and relative granularity and size of apoptotic cells which they are induced by: 1- X- ray dose 0.25, 0.5, 1, 2, 4 Gy with dose rate 0.28Gy/min. 2- X –ray dose 15.63, 31.3, 62.5, 125, 250 Gy with dose rate 20mGy/min. 3- 60Co gamma ray dose 0.25, 0.5, 1, 2, 4 Gy with dose rate 2Gy/hr. Based on our results, a dose response model is formulated and discussed.
4-2 X-ray Induced Apoptosis 4-2-1 Measuring amount of fluorescence of caspase 3 for dose rate 0.28Gy/min. Effect of x-ray doses 0, 0.25, 0.5, 1, 2, 4 Gy with amount of fluorescence of activate caspase 3 are summarized in the Tables (4.1- 4.4) for donors 1, 2, 3 and 4 respectively. Fluorescence intensity is proportional to the activated of caspsae 3, this proportionality also was recorded by Maher et al. [90] as the intensity of the fluorescence signal is proportional to the amount of antibody bound per cell. The tables show the amount of fluorescence of activated caspase 3 in lymphocytes, apoptotic and monocyts cells. Figure (4.1) shows a histogram of fluorescence of caspase 3 for all doses which were measured by flow cytometry.
Fig.(4.1): A flow cytometry histogram of fluorescence intensity of activated caspase 3, FITC for dose 0, 0.25, 0.5, 1, 2, 4 Gy (A, B, C, D), the peak in the left is due to the lymphocyte( living) cells which decreases with the dose while the peak in the right is due to the apoptotic cells which increases with the dose.
The fluorescence intensity of activated caspase 3 in apoptotic cells was increased with dose and a significant difference was observed between control and irradiated samples for all donors which reflects the mount of apoptosis. Table (4.5) shows data of normalization of lymphocytes, apoptotic cells, and monocytes cells for 4 donors. Increasing of the fluorescence in apoptotic cells was significant for all doses and dose dependent which indicated increasing of apoptotic cells with the radiation doses. This activation of caspase 3 in apoptotic cells agrees with that obtained by Kim et al. [57] who suggested that ionizing radiation induced apoptosis is mediated by the activation of various signaling pathways including caspase family in a time and dose depend manner. Figure (4.2) shows the results for the normalization for apoptotic cells.
Table (4.1): Amount of fluorescence of activated caspase 3 with x-ray dose in lymphocyte, apoptotic and monocyte cells for donor 1. Dose rate is 0.28 Gy/min. Dose (Gy) 0 (Control) 0.25
Fluorescence of caspas3 in lymphocyte
p-value
Fluorescence of caspas3 in apoptotic
p-value
Fluorescence of caspas3 in monocyte
SEM
SEM
SEM
p-value
0.91
0.01
-
3.15
0.14
-
9.68
0.28
-
1.22
0.15
0.11
4.93
0.18
0.001*
11.17
0.98
0.22
1.18
0.12
0.09
5.12
0.29
0.003*
10.90
0.70
0.18
1.56
0.08
0.001**
5.73
0.34
0.002*
12.90
0.21
0.0007**
1.40
0.11
0.01*
6.04
0.24
0.0004**
12.23
0.29
0.003*
1.51
0.06
0.0007**
6.40
0.13
0.00007**
12.57
0.38
0.003*
0.5 1 2 4 *Significant difference in comparison with control p<0.05 ** High significant difference in comparison with control p<0.001.
Table (4.2): Amount of fluorescence of activated caspase 3 with x-ray dose in lymphocyte, apoptotic and monocyte cells for donor 2. Dose is rate 0.28 Gy/min.
Dose (Gy) 0 (Control) 0.25
Fluorescence of caspas3 in lymphocyte
p-value
Fluorescence of caspas3 in apoptotic
SEM
0.86
Fluorescence of caspase3 in monocyte
SEM
SEM
p-value
0.05
-
2.44
0.14
-
9.31
0.43
-
0.61
0.02
0.01*
3.15
0.04
0.008*
9.20
0.25
0.83
0.76
0.02
0.13
3.34
0.09
0.006*
9.42
0.19
0.83
0.90
0.02
0.46
4.62
0.17
0.0005**
11.47
0.23
0.01*
0.98
0.05
0.17
5.21
0.20
0.0003**
11.30
0.26
0.02*
0.11
0.00004**
12.63
0.48
0.01*
p-value
0.5 1 2 4 1.24 0.05 0.005* 6.00 *Significant difference in comparison with control p<0.05.
** High significant difference in comparison with control p<0.001.
Table (4.3): Amount of fluorescence of activated caspase 3 with x-ray dose in lymphocyte, apoptotic and monocyte cells for donor 3. Dose rate is 0.28 Gy/min. Fluorescence of Caspase3 in lymphocyte
SEM
0.70
0.25
p-value
Fluorescence of caspase3 in apoptotic
SEM
0.04
-
0.72
0.64
0.03
0.23
0.5
0.85
0.09
1
0.89
2
0.93
Dose (Gy) 0 (Control)
p-value
Fluorescence of caspase3 in monocyte
SEM
p-value
0.02
-
8.29
0.27
-
0.84
0.02
0.02*
7.98
0.26
0.46
0.18
1.04
0.06
0.008*
9.57
0.53
0.10
0.06
0.06*
1.46
0.01
0.000008**
9.51
0.25
0.03*
0.03
0.01*
2.04
0.20
0.003*
10.70
0.23
0.003*
0.10
0.00003**
10.77
0.12
<0.001*
4 0.97 0.04 0.006* 2.89 *Significant difference in comparison with control p<0.05.
** High significant difference in comparison with control p<0.001.
Table (4.4): Amount of fluorescence of activated caspase 3 with x-ray dose in lymphocyte, apoptotic and monocyte cells for donor 4. Dose rate is 0.28 Gy/min. fluorescence of Caspase3 in lymphocyte
SEM
0.83
p-value
fluorescence of caspase3 in monocyte
SEM
p-value
0.04
-
10.27
0.12
-
1.51
0.07
0.02*
10.38
0.43
0.81
0.10
1.66
0.08
0.01*
10.18
0.26
0.78
0.02
0.35
1.80
0.10
0.01*
10.60
0.15
0.16
0.01
0.0004**
3.73
0.18
0.0001**
15.07
0.43
0.0004**
1.13 0.02 0.0043* 4.06 *Significant different in comparison with control p<0.05
0.16
0.00007**
12.60
0.31
0.0020*
Dose (Gy) 0 (Control) 0.25
p-value
fluorescence of caspase3 in apoptotic
SEM
0.05
-
1.18
0.96
0.11
0.36
0.94
0.01
0.89 1.37
0.5 1 2 4
** High significant difference in comparison with control p<0.001.
Table (4.5): Amount of fluorescence activated caspase 3 with x-ray dose in lymphocyte, apoptotic and monocyte cell after normalization for all donors. Dose rate is 0.28 Gy/min.
Dose (Gy) 0 (Control) 0.25
Amount of fluorescence of caspase 3 in lymphocyte
SEM
100.00
p-value
Amount of fluorescence of caspase 3 in apoptotic
p-value
Amount of fluorescence of caspase 3 in monocyte
SEM
SEM
p-value
0
-
100.00
0
-
100.00
0
-
103.02
13.81
0.83
132.56
8.46
0.008*
102.89
4.28
0.53
113.17
8.93
0.19
146.14
5.68
0.0001**
107.09
4.07
0.13
127.61
15.45
0.12
181.64
10.62
0.0002**
118.60
6.38
0.03*
141.43
11.33
0.0106*
251.18
29.15
0.002*
130.88
5.52
0.001*
43.39
0.004*
129.53
2.66
0.00003**
0.5 1 2 4 146.21 6.79 0.0004** 296.20 *Significant difference in comparison with control p<0.05.
Amount of fluorescence of activated caspase 3 in apoptotic cells after normalization
** High significant difference in comparison with control p<0.001.
400 350 300 250 200 150 100 50 0 0
1
2
3
4
5
X-ray dose (Gy)
Fig.(4.2): Variation of amount of fluorescence of activated caspase 3 with x-ray dose in apoptotic cells after normalization for all donors. Dose rate is 0.28 Gy/min.
4-2-2 Measurement of percentage number of lymphocytes, apoptotic and monocytes cells at dose rate of 0.28 Gy/min Effect of X-ray doses 0.25, 0.5, 1, 2, 4 Gy on the percentage number of apoptotic, lymphocyte and monocyte cells are summarized in Tables (4.64.9) for donors 1, 2, 3 and 4 respectively. The percentage number of apoptotic cells increases significantly with the dose for all donors while lymphocyte decreases with dose. However, this decreasing is not significant at 0.25Gy for all donors and at 0.5Gy only for donor 2. Tables (4.10) and (4.11) show the mean of percentage number apoptotic and percentage number lymphocytes respectively for the donors. The percentage number of apoptotic cells were 5.57%, 6.14%, 7.60%, 8.91%, 11.72% and 16.51% for 0, 0.25, 0.5, 1, 2 and 4 Gy respectively. We observed increasing of mean of percentage number of apoptotic cells were dose dependent and significant at 1, 2, 4 Gy. The present results agree well with others, like inducing of apoptotic cells by irradiation investigated by Payne et al.[47] which observed that irradiation of human lymphocytes with x-ray doses (0-20) Gy induced apoptosis cells and the significant difference was observed at low dose irradiation (2.5-5) Gy. However irradiation of CD4+ and CD8+ T-lymphocytes with x-ray doses (0-1.5) Gy was done by Wilkins et al. [48] as the dose response curves were linear up to 0.5Gy and thereafter the response became saturated. Also Holl et al.[95] showed irradiated of mice to x-ray doses (0.2-4) Gy apoptosis occurring in spleen cells was dependent on dose, and a dose dependent induction of apoptosis that was significant observed by Boreham et al.[45] in the case of irradiation of human lymphocytes to x-ray doses (0, 0.2, 0.4, 0.6, 0.8, 1) Gy . Our results also showed decreasing of percentage number of lymphocytes with dose but they are only significant at 4Gy. The results agree with that Nomura et al. [91] despite they were working on mice, they showed that irradiation of different types of mice strains to x-ray doses (0-2.5) Gy
Survival rate of white blood cells and splenic lymphocytes decreased with increasing dose. The mean of percentage number of apoptotic and lymphocytes cells with dose is sketched in Figs. (4.3) and (4.4) respectively.
Table (4.6): Percentage number of lymphocytes, apoptotic and monocytes cells with dose in donor 1. Dose rate is 0.28 Gy/min. Dose (Gy) 0 (Control) 0.25
% lymphocytes
SEM
p-value
% apoptotic
SEM
p-value
% monocytes
SEM
pvalue
75.30
0.50
-
4.15
0.20
-
20.53
0.32
-
74.44
0.12
0.17
5.22
0.32
0.05*
18.66
1.98
0.40
72.80
0.55
0.03*
6.60
0.12
0.0004**
20.58
0.59
0.94
71.75
0.07
0.02*
8.56
0.19
0.00009**
20.53
0.66
1
69.94
0.27
0.0007**
11.05
0.36
0.00007**
18.99
0.46
0.05
60.89 0.37 0.00002** 16.61 0.70 *Significant difference in comparison with control p<0.05. ** High significant difference in comparison with control p<0.001.
0.00007**
22.49
0.63
0.05
0.5 1 2 4
Table (4.7): Percentage number of lymphocytes, apoptotic and monocytes cells with xray dose in donor 2. Dose rate is 0.28 Gy/min. Dose (Gy) 0 (Control)
% lymphocytes
SEM
p-value
% apoptotic
SEM
p-value
% monocytes
SEM
p-value
79.11
0.65
-
4.84
0.52
-
16.02
0.15
-
0.25
78.56
0.35
0.50
5.37
0.16
0.39
16.05
0.47
0.96
0.5
77.22
0.52
0.09
6.25
0.20
0.06
16.50
0.47
0.38
1
75.11
0.42
0.007*
7.63
0.17
0.006*
17.23
0.37
0.039*
2
70.55
0.50
0.0004**
12.06
0.30
0.0002**
17.38
0.48
<0.05
4 61.19 0.52 0.00003** 19.26 0.45 *Significant difference in comparison with control p<0.05. ** High significant difference in comparison with control p<0.001.
0.00003**
19.53
0.48
0.002*
Table (4.8): Percentage number of lymphocytes, apoptotic and monocytes cells with xray dose in donor 3. Dose rate is 0.28 Gy/min. Dose (Gy) 0 (Control)
% lymphocytes
p-value
% apoptotic
86.47
0.25
p-value
% monocytes
0.35
-
5.89
0.30
-
7.59
0.12
-
85.41
0.44
0.13
6.68
0.06
0.06
7.90
0.43
0.53
0.5
84.95
0.24
0.02*
7.24
0.34
0.04*
7.78
0.14
0.37
1
83.35
0.45
0.005
8.89
0.26
0.001*
7.75
0.30
0.65
2
80.50
0.27
0.0001**
11.22
0.22
0.0001**
8.80
0.05
0.0008**
0.19
0.00002**
9.21
0.38
0.02*
SEM
SEM
4 77.17 0.50 0.0001** 14.10 *Significant difference in comparison with control p<0.05.
SEM
p-value
** High significant difference in comparison with control p<0.001.
Table (4.9): Percentage number of lymphocytes, apoptotic and monocytes cells with xray dose in donor 4. Dose rate is 0.28 Gy/min.
Dose (Gy)
% lymphocytes
SEM
p-value
% apoptotic
SEM
p-value
% monocytes
SEM
p-value
0 (Control)
76.88
0.06
-
7.38
0.61
-
14.71
0.51
-
0.25
76.34
0.39
0.25
8.28
0.32
0.26
15.35
0.52
0.43
0.5
72.95
0.20
0.00004**
10.31
0.10
0.009*
16.72
0.10
0.02*
1
73.09
0.15
0.00002**
10.55
0.38
0.01*
16.35
0.23
0.04*
2
70.63
0.13
0.00001**
12.55
0.23
0.001*
16.80
0.12
0.02*
4 65.75 0.50 0.00002** 16.06 *Significant difference in comparison with control p<0.05.
0.34
0.0002**
17.86
0.18
0.004*
** High significant difference in comparison with control p<0.001.
Table (4.10): Mean of percentage number of apoptotic cells with x-ray dose for all donors. Dose rate is 0.28 Gy/min. % apoptotic cells in donor 1
% apoptotic cells in donor 2
% apoptotic cells in donor 3
% apoptotic cells in donor 4
% apoptotic cells (mean)
SEM
p-value
4.15
4.84
5.89
7.38
5.57
0.70
-
0.25
5.22
5.37
6.68
8.28
6.14
0.86
0.44
0.5
6.6
6.25
7.24
10.31
7.60
0.93
0.13
1
8.56
7.63
8.89
10.55
8.91
0.61
0.011*
2
11.05
12.06
11.22
12.55
11.72
0.35
0.0002**
16.06
16.51
1.06
0.0001**
Dose (Gy) 0 (Control)
4 16.61 19.26 14.1 *Significant difference in comparison with control p<0.05.
** High significant difference in comparison with control p<0.001.
Table (4.11): Mean of percentage number of lymphocytes cells with x-ray dose for all donors. Dose rate is 0.28 Gy/min. %lymphocytes cells in donor 1
%lymphocytes cells in donor 2
%lymphocytes cells in donor 3
%lymphocytes cells in donor 4
%lymphocytes cells (mean)
SEM
75.3
79.11
86.47
76.88
79.44
2.47
74.44
78.56
85.41
76.34
78.69
2.39
0.83
72.8
77.22
84.95
72.95
76.98
2.85
0.54
71.75
75.11
83.35
73.09
75.81
2.61
0.50
69.94
70.55
80.5
70.63
72.91
2.54
0.11
60.89 61.19 77.17 *Significant difference in comparison with control p<0.05.
65.75
66.25
3.81
0.03*
Dose (Gy) 0 (Control) 0.25
p-value
0.5 1 2 4
Mean of percentage number of apoptotic cells
20 18 16 14 12 10 8 6 4 2 0 0
1
2
3
4
5
X-ray dose (Gy)
Fig.(4.3): Variation of mean of percentage number of apoptotic cells with x-ray dose. The results represent mean±SEM for four independent experiments. Dose rate of 0.28 Gy/min.
Mean of percetage number of lymphocytes
85 80 75 70 65 60 55 50 0
1
2
3
4
5
X-ray dose (Gy)
Fig.(4.4): Variation of mean of percentage number of lymphocytes cells with x-ray dose The result represent mean±SEM for four independent experiments. Dose rate of 0.28 Gy/min.
4-2-3 Measurement of Fluorescence of activated caspase 3 with dose rate 20mGy/min Effect of x-ray doses 0, 15.6, 31.3, 62.5, 125, 250 mGy on amount of fluorescence of activated caspase 3 are summarized in Tables (4.12- 4.16) for donors 1, 2, 3, 4 and 5 respectively. The tables show amount of fluorescence of activated caspase 3 in lymphocytes, apoptotic and monocytes cells. The fluorescence in apoptotic cells is increased with doses but not significant for all doses and donors, because at low doses the effect is less and varies between individuals. Table (4.17) shows data of normalization of amount of fluorescence of activated caspase 3 in lymphocytes, apoptotic cells, and monocytes cells for 5 donors. The amount of fluorescence in apoptotic cells increase linearly with the dose and significant different was observed at 62.3, 125 and 250 mGy only. Fig.(4.5) shows this variation of fluorescence in apoptotic cells with x-ray dose after normalization for the donors.
Table (4.12): Amount of fluorescence of activated caspase 3 with x-ray dose in lymphocyte, apoptotic and monocyte for donor 1. Dose rate is 20 mGy/min. fluorescence of caspase 3 in lymphocyte
SEM
1.08
SEM
pvalue
fluorescence of caspase 3 in monocyte
SEM
pvalue
2.49
0.22
-
12.03
0.12
-
0.01*
2.56
0.35
0.87
12.00
0.06
0.81
<0.01
0.001*
2.55
0.22
0.86
11.17
0.19
0.02*
1.20
0.02
0.007*
2.54
0.05
0.85
11.20
0.10
0.006*
1.50
0.02
0.00008**
3.11
0.15
0.08
13.17
0.18
0.006*
1.44 0.08 0.01* 4.72 *Significant difference in comparison with control p<0.05
0.23
0.002*
12.87
0.41
0.12
Dose (mGy) 0 (Control) 15.6
p-value
fluorescence of caspase 3 in apoptotic
0.01
-
1.22
0.03
1.18
31.3 62.5 125 250
** High significant difference in comparison with control p<0.001.
Table (4.13): Amount of fluorescence of activated caspase 3 with x-ray dose in lymphocyte, apoptotic and monocyte for donor 2. Dose rate is 20 mGy/min.
Dose (mGy) 0 (Control)
fluorescence of caspase 3 in lymphocyte
SEM
p-value
fluorescence of caspase 3 in apoptotic
SEM
p-value
fluorescence of caspase 3 in monocyte
SEM
p-value
0.78
0.02
-
1.04
0.04
-
11.97
0.47
-
15.6
0.74
0.02
0.36
1.07
0.12
0.80
11.60
0.61
0.66
31.3
0.70
0.03
0.08
1.06
0.14
0.89
11.37
0.29
0.34
62.5
0.94
0.01
0.003*
1.32
0.02
0.002*
12.87
0.72
0.36
125
0.95
0.06
0.05*
1.58
0.20
0.06
13.20
1.05
0.34
250 1.19 0.05 0.002* 2.02 *Significant difference in comparison with control p<0.05.
0.09
0.0006**
16.97
0.03
0.01**
** High significant difference in comparison with control p<0.001.
Table (4.14): Variation of amount of fluorescence of activated caspase 3 with x-ray dose in lymphocyte, apoptotic and monocyte for donor 3. Dose rate is 20 mGy/min. fluorescence of caspase 3 in lymphocyte
SEM
1.08
15.63
p-value
fluorescence of caspase 3 in apoptotic
p-value
fluorescence of caspase 3 in monocyte
SEM
SEM
p-value
0.08
-
4.24
0.36
-
13.13
0.15
-
1.27
0.08
0.17
5.34
0.30
0.08
13.70
0.06
0.02*
31.3
1.66
0.11
0.013*
5.95
0.17
0.01
15.33
0.27
0.002*
62.5
1.33
0.09
0.10
5.15
0.48
0.20
12.70
0.31
0.27
125
1.06
0.06
0.80
4.55
0.10
0.45
12.73
0.29
0.29
250 0.93 0.05 0.18 4.62 *Significant difference in comparison with control p<0.05.
0.16
0.39
11.47
0.12
0.0009**
Dose (mGy) 0 (Control)
** High significant difference in comparison with control p<0.001.
Table (4.15): Amount of fluorescence of activated caspase 3 with x-ray dose in lymphocyte, apoptotic and monocyte for donor 4. Dose rate is 20 mGy/min. fluorescence of caspase 3 in lymphocyte
SEM
1.26
15.63
p-value
fluorescence of caspase 3 in apoptotic
SEM
0.06
-
2.89
1.60
0.10
0.038*
31.3
1.49
0.08
62.5
2.23
125
1.70
Dose (mGy) 0 (Control)
p-value
fluorescence of caspase 3 in monocyte
SEM
p-value
0.19
-
17.47
0.50
-
3.29
0.29
0.31
19.37
0.74
0.10
0.09
3.39
0.15
0.12
19.13
0.48
0.08
0.11
0.002*
4.34
0.19
0.006*
22.53
0.88
0.008*
0.12
0.03*
4.18
0.22
0.01*
20.63
0.75
0.02*
0.26
0.002*
20.73
0.46
0.009*
250 1.86 0.10 0.007* 5.23 *Significant difference in comparison with control p<0.05.
Table (4.16): Amount of fluorescence of activated caspase 3 with x-ray dose in lymphocyte, apoptotic and monocyte for donor 5. Dose rate is 20 mGy/min.
Dose (mGy) 0 (Control)
fluorescence of caspase 3 in lymphocyte
SEM
pvalue
fluorescence of caspase 3 in apoptotic
p-value
fluorescence of caspase 3 in monocyte
SEM
SEM
pvalue
1.71
0.18
-
4.21
0.06
-
22.47
1.16
-
1.42
0.12
0.26
4.18
0.01
0.64
20.47
1.47
0.35
1.69
0.14
0.93
4.56
0.04
0.007*
20.10
0.25
0.12
1.37
0.09
0.17
4.64
0.09
0.02*
18.87
1.07
0.08
1.85
0.23
0.64
5.44
0.22
0.006*
22.83
0.41
0.78
1.88
0.06
0.43
5.57
0.05
0.00006**
21.67
0.64
0.58
15.6 31.3 62.5 125 250
*Significant difference in comparison with control p<0.05.
** High significant difference in comparison with control p<0.001.
Table (4.17): Amount of fluorescence of activated casspase 3 with x-ray dose in lymphocyte, apoptotic, and monocyte after normalization for all donors. Dose rate is 20 mGy/min.
Dose (mGy)
amount of fluorescence of caspase 3 in lymphocyte
SEM
100.0
p-value
amount of fluorescence of caspase 3 in apoptotic
P-value
amount of fluorescence of caspase 3 in monocyte
SEM
SEM
P-value
0
-
100.0
0
-
100.0
0
-
107.1
8.0
0.40
109.0
4.9
0.11
100.6
3.3
0.86
114.0
11.0
0.24
114.1
7.1
0.08
100.7
5.3
0.89
122.4
15.7
0.19
122.2
8.2
0.03*
102.1
7.7
0.80
120.4
7.7
0.03*
131.6
7.8
0.003*
107.3
3.7
0.08
125.9
12.4
0.07
160.5
17.6
0.01*
110.2
9.5
0.310
0 15.63
31.3 62.5 125 250
*Significant difference in comparison with control p<0.05.
Amount of fluorscence ofcaspase 3 in apoptotic cells after normalization
200 180 160 140 120 100 80 60 40 20 0 0
50
100
150
200
250
300
X-ray dose (m Gy)
Fig. (4.5): Variation of amount of fluorescence of activated caspase 3 with x-ray dose in apoptotic cells after normalization for donors. (Dose rate of 20 mGy/min.)
4-2-4 Measurement of percentage number of lymphocyte, apoptotic and monocyte cells at dose rate of 20mGy/min Effect of x-ray doses 0, 15.6, 31.25, 62.5, 125 and 250 mGy on the percentage number of the cells are summarized in Tables (4.18-4.22) for donors 1, 2, 3, 4, and 5. The percentage number of apoptotic cells was increased but not significantly with all the doses and changing of percentage number of lymphocytes was not always regularly and significant. Tables (4.23) and (4.24) show the means of percentage number of apoptotic and lymphocyte cells for the donors respectively. The mean percentage number of the apoptotic cells was 4.50%, 4.56%, 5.06%, 5.21%, 5.28% and 5.50% for the dose 0, 15.6, 31.3, 62.5, 125 and 250 mGy respectively. The percentage number of apoptotic cells was increased linearly but it is not significant, because most of the damage repaired at low doses and radiosensitivity varies between subpopulation of lymphocytes. However, the effect of low dose rate on apoptosis was less clear in agreement with results of ref. [92]. Our results agree with that Zheng et al. [94] which they showed that after irradiation of whole body of mice with x-ray doses (0.025-4) Gy, a dose dependent increases in apoptosis in thymocytes with a dose above 0.5Gy. The percentage number of lymphocytes was decreased but decreasing also not significant. Fig. (4.6) and (4.7) show these results for apoptotic and lymphocytes cells respectively.
Table (4.18): Percentage number of lymphocyte, apoptotic and monocyte cells with xray dose in donor 1. Dose rate is 20 mGy/min. %lymphocyte
SEM
p-value
% apoptotic
SEM
p-value
%monocyte
SEM
p-value
74.88
0.42
-
2.46
0.15
-
23.54
0.37
-
73.85
0.44
0.17
2.33
0.07
0.48
23.77
0.48
0.72
75.36
0.21
0.37
2.88
0.19
0.16
21.75
0.35
0.024*
75.24
0.32
0.54
2.78
0.19
0.27
21.97
0.49
0.06
75.04
0.27
0.77
3.19
0.21
0.05
21.75
0.07
0.009*
76.46 0.57 0.09 3.17 *Significant difference in comparison with control p<0.05.
0.26
0.08
20.36
0.30
0.003*
Dose (mGy) 0 (Control) 15.6 31.3 62.5 125 250
Table (4.19): Percentage number of lymphocyte, apoptotic and monocyte cells with xray dose in donor 2. Dose rate is 20 mGy/min. Dose (mGy) 0 (Control) 15.6
%lymphocyte
SEM
p-value
%apoptotic
SEM
p-value
%monocyte
SEM
p-value
83.53
0.38
-
7.37
0.22
-
9.10
0.17
-
82.70
0.63
0.32
7.32
0.44
0.92
10.20
0.39
0.06
82.71
0.19
0.12
7.35
0.22
0.96
9.93
0.36
0.11
82.64
0.31
0.14
7.09
0.09
0.30
10.26
0.33
0.04*
83.44
0.22
0.84
7.49
0.14
0.66
9.06
0.11
0.83
0.22
0.83
10.04
0.15
0.01*
31.3 62.5 125 250 82.50 0.31 0.10 7.44 *Significant difference in comparison with control p<0.05.
Table (4.20): Percentage number of lymphocyte, apoptotic and monocyte cells with x-ray dose in donor 3. Dose rate is 20 mGy/min. Dose (mGy) 0 (Control 15.6
%Lymphocyte
SEM
p-value
%apoptotic
SEM
p-value
%monocyte
SEM
p-value
81.89
0.65
-
3.76
0.07
-
14.46
0.54
-
82.99
0.34
0.21
3.83
0.25
0.79
13.17
0.09
0.08
82.16
0.72
0.80
4.50
0.04
0.0009**
13.33
0.75
0.29
83.24
0.53
0.18
4.89
0.28
0.018*
11.85
0.36
0.02*
82.46
0.65
0.57
4.87
0.31
0.027*
12.65
0.34
<0.05*
0.0002**
14.15
0.61
0.72
31.3 62.5 125 250 80.06 0.64 0.12 5.77 0.15 *Significant difference in comparison with control p<0.05. ** High significant difference in comparison with control p<0.001.
Table (4.21): Percentage number of lymphocyte, apoptotic and monocyte cells with x-ray dose in donor 4. (Dose rate is 20 mGy/min). Dose (mGy) %lymphocyte
SEM
p-value
%apoptotic
SEM
p-value
%monocyte
SEM
p-value
85.58
0.22
-
4.83
0.23
-
9.57
0.06
-
85.96
0.46
0.50
4.49
0.22
0.30
9.54
0.34
0.93
82.87
0.37
0.006*
5.81
0.12
0.02*
11.30
0.46
0.02*
84.14
0.62
0.09
6.20
0.27
0.01*
9.68
0.32
0.76
83.75
0.48
0.025*
5.75
0.26
0.06
10.15
0.49
0.31
86.67 0.11 0.011* 5.19 *Significant difference in comparison with control p<0.05.
0.15
0.26
8.12
0.16
0.001*
0 (Control) 15.6 31.3 62.5 125 250
Table (4.22): Percentage number of lymphocyte, apoptotic and monocyte cells with x-ray dose in donor 5. Dose rate is 20 mGy/min. %lymphocyte
SEM
p-value
%apoptotic
SEM
p-value
%monocyte
SEM
pvalue
88.94
0.34
-
4.10
0.30
-
6.95
0.05
-
87.55
0.42
0.06
4.81
0.27
0.15
7.62
0.15
0.01*
87.92
0.30
0.09
4.78
0.24
0.15
7.26
0.32
0.38
87.00
0.38
0.02*
5.09
0.06
0.03*
7.91
0.31
0.04*
87.82
0.21
0.05*
5.11
0.01
0.08
7.06
0.21
0.63
86.41 0.40 0.009* 5.94 *Significant difference in comparison with control p<0.05.
0.51
0.04*
7.62
0.38
0.16
Dose (mGy) 0 (Control) 15.6 31.3 62.5 125 250
Table (4.23): Mean of percentage number of apoptotic cells with x-ray dose for all donors. Dose rate 20 is mGy/min.
Dose (mGy) 0 (control) 15.6
%number apoptotic cells in donor 1
%number apoptotic cells in donor 2
%number apoptotic cells in donor 3
%number apoptotic cells in donor 4
%number apoptotic cells in donor 5
Mean% number apoptotic cells
SEM
p-value
2.46
7.37
3.76
4.83
4.10
4.50
0.81
-
2.33
7.32
3.83
4.49
4.81
4.56
0.81
0.97
2.88
7.35
4.50
5.81
4.78
5.06
0.74
0.62
2.78
7.09
4.89
6.20
5.09
5.21
0.73
0.54
3.19
7.49
4.87
5.75
5.11
5.28
0.70
0.48
3.17
7.44
5.77
5.19
5.94
5.50
0.69
0.38
31.3 62.5 125 250
Table (4.24): Mean of percentage number of lymphocytes cells with x-ray dose for all donors. Dose rate is 20 mGy/min.
Dose (mGy) 0 (control) 15.6
%number lymphocytes cells in donor1
%number lymphocytes cells in donor2
%number lymphocytes cells in donor3
%number lymphocytes cells in donor4
%number lymphocytes cells in donor5
Mean %number lymphocytes cells
SEM
p-value
74.88
83.53
81.89
85.58
88.94
82.96
2.34
-
73.85
82.70
82.99
85.96
87.55
82.61
2.37
0.91
75.36
82.71
82.16
82.87
87.92
82.20
2.00
0.81
75.24
82.64
83.24
84.14
87
82.45
1.95
0.87
75.04
83.44
82.46
83.75
87.82
82.50
2.08
0.88
76.46
82.5
80.06
86.67
86.41
82.42
1.94
0.86
31.3 62.5 125 250
Mean of percentage number of apoptoic cells
7 6 5 4 3 2 1 0 0
50
100
150
200
250
300
X-ray dose (m Gy)
Fig. (4.6): Variation of mean of percentage number of apoptotic cells with x-ray dose for all donors. Dose rate is 20mGy/min
Mean of percentage number of lymphoctes
90
80
70
60
50 0
50
100
150
200
250
300
X-ray dose (m Gy)
Fig. (4.7): Variation of mean of percentage number of lymphocytes cells with x-ray dose for all donors. Dose rate is 20m Gy/min.
4-3 60Co γ-Ray Induced Apoptosis 4-3-1 Measurement of amount of fluorescence of caspase 3 with dose rate of 2Gy/hr Effect of γ-ray doses 0, 0.25, 0.5, 1, 2, 4 Gy on amount of fluorescence of activated caspase 3 in apoptotic, lymphocytes and monocytes are summarized in Tables (4.25-4.28) for donors 1, 2, 3 and 4 respectively. The tables show amount of fluorescence of activated caspase 3 in lymphocytes, apoptotic and monocytes. Fig. (4.8) shows the histogram of fluorescence of activated caspase 3, FITC for all doses. The results showed that fluorescence of activated caspase 3 in apoptotic cells increases with dose and a significantly difference was observed between control and irradiated samples except for donors 1 and 2 at dose 0.25 Gy due to the radiosensitivity dependence between individuals. Table 4.29 presents data for normalization fluorescence of activated caspase 3 in lymphocytes, apoptotic cells and monocytes cells for 4 donors. The amount of fluorescence of activated caspase 3 in apoptotic cells was dose dependent. Significant difference was observed between control and all doses. The results agree with Ismail et al. [94] which they showed increasing of relative signal with gamma radiation dose. Fig. (4.9) shows the normalization of apoptotic cells.
A
B
C
E
D
F
Fig.(4.8): Histogram of fluorescence of activated caspase 3 for 60Co gamma ray dose 0, 0.25, 0.5, 1, 2, 4 Gy , the peak in the left is due to the lymphocytes( living) cells which decrease with the dose while the peak in the right is due to the apoptosis which increase with the dose.
Table (4.25): Amount of fluorescence of activated caspase 3 with
60
Coγ-ray dose in
lymphocyte, apoptotic and monocyte, for donor 1. Dose rate is 2 Gy/hr.
Dose (Gy) 0 (Control) 0.25
fluorescence of caspase 3 in lymphocyte
SEM
1.11
p-value
fluorescence of caspase 3 in apoptotic
p-value
fluorescence of caspase 3 in monocyte
SEM
SEM
p-value
0.02
-
2.06
0.09
-
10.33
0.15
-
0.92
0.05
0.02*
2.16
0.09
0.47
8.99
0.16
0.004*
1.02
0.01
0.007*
2.88
0.27
0.05*
9.72
0.29
0.13
1.43
0.02
0.0003**
3.90
0.38
0.009*
11.87
0.28
0.009*
1.13
0.01
0.40
4.25
0.10
0.00008**
10.67
0.23
0.29
0.13
0.00009**
10.70
0.15
0.16
0.5 1 2 4 1.22 0.07 0.20 4.60 *Significant difference in comparison with control p<0.05.
** High significant difference in comparison with control p<0.001.
Table (4.26): Amount of fluorescence of activated caspase 3 with 60Coγ-ray dose in lymphocyte apoptotic and monocyte, for donor 2. Dose rate is 2Gy/hr.
Dose (Gy) 0 (Control) 0.25
fluorescence of caspase 3 in lymphocyte
SEM
1.57
p-value
fluorescence of caspase 3 in apoptotic
p-value
fluorescence of caspase 3 in monocyte
SEM
SEM
pvalue
0.05
-
2.67
0.10
-
15.40
0.51
-
1.56
0.09
0.90
3.45
0.33
0.08
14.60
0.78
0.44
1.33
0.06
0.03*
4.10
0.50
0.05*
13.93
0.34
0.08
1.53
0.08
0.69
5.32
0.38
0.002*
14.77
0.32
0.35
1.37
0.06
0.06
5.55
0.43
0.003*
14.63
0.41
0.31
0.33
0.0005**
14.00
0.32
0.08
0.5 1 2 4 1.54 0.07 0.71 6.11 *Significant difference comparison with control p<0.05.
** High Significant difference in comparison with control p<0.001.
Table (4.27): Amount of fluorescence of activated caspase 3 with 60Coγ-ray dose in lymphocyte apoptotic and monocyte, for donor 3. Dose rate is 2 Gy/hr.
Dose (Gy) 0 (Control) 0.25
fluorescence of caspase 3 in lymphocyte
SEM
1.04
0.03
1.43
0.02
1.48
fluorescence of caspase 3 in apoptotic
SEM
1.79
0.10
0.0004**
2.57
0.19
0.03
0.0004**
3.13
1.84
0.05
0.0001**
1.57
0.02
0.0001**
p-value
fluorescence of caspase 3 in monocyte
SEM
11.73
0.34
0.03*
14.47
0.13
0.002*
0.25
0.008*
15.23
0.49
0.004*
3.73
0.07
0.0001**
17.77
0.79
0.002*
3.70
0.09
0.0001**
16.20
0.49
0.002*
0.30
<0.001*
18.00
1.10
0.005*
p-value
pvalue
0.5 1 2 4 1.86 0.10 0.0011* 4.48 *Significant difference in comparison with control p<0.05
** High significant difference in comparison with control p<0.001.
Table (4.28): Amount of fluorescence of activated caspase 3 with 60Coγ-ray dose in lymphocyte, apoptotic and monocyte, for donor 4. Dose rate is 2Gy/hr.
Dose (Gy) 0 (Control) 0.25
fluorescence of caspase 3 in lymphocyte
SEM
1.26
0.06
1.85
0.11
1.74
fluorescence of caspase 3 in apoptotic
fluorescence of caspase 3 in monocyte
SEM
SEM
4.08
0.21
14.73
0.38
0.009*
6.11
0.39
0.01*
19.23
0.66
0.004**
0.13
0.026*
6.24
0.10
0.0007**
18.97
0.72
0.006*
1.57
0.10
0.06
6.27
0.17
0.001*
18.57
0.62
0.006*
1.47
0.02
0.03*
6.97
0.16
0.0004**
17.97
0.42
0.005*
1.57
0.08
0.03*
7.09
0.15
0.0003**
17.60
0.42
0.007*
pvalue
p-value
p-value
0.5 1 2 4
*Significant difference in comparison with control p ** High significant difference in comparison with control p<0.001.
Table (4.29): Amount of fluorescence of activated caspase 3 with 60Coγ-ray dose in lymphocyte, apoptotic and monocyte, for all donors after normalization. Dose rate is 2 Gy/hr. Dose (Gy) 0 (Control) 0.25
Amount of fluorescence of caspase 3 in lymphocyte
Amount of fluorescence of caspase 3 in apoptotic
SEM
SEM
100.0
0
100.0
0
116.6
15.2
0.32
131.9
10.0
114.3
15.1
0.38
155.3
132
16.5
0.10
114.2
13.7
0.34
pvalue
Amount of fluorescence of caspase 3 in monocyte
SEM
100.0
0
0.02*
108.9
10.6
0.43
7.3
0.0002**
110.8
10.7
0.35
187.7
12.0
0.0003**
122.1
11.6
0.11
197.9
9.0
0.0004**
114.6
9.7
0.18
16.2
0.0003**
116.9
13.5
0.26
p-value
p-value
0.5 1 2 4 127.9 17.8 0.17 219.1 *Significant difference in comparison with control p<0.05.
** High significant difference in comparison with control p<0.001.
Fluorscnce of activated casase 3 in apoptotic after normalization
250
200 150
100 50
0 0
1
2
3
4
5
Co60 gam m a ray dose (Gy)
Fig. (4.9): Variation of
60
Co gamma ray dose with amount of fluorescence activated
caspase 3 in apoptotic cells after normalization for all donors. Dose rate is 2 Gy/hr.
4-3-2 Measurement of percentage number of lymphocytes, apoptotic and monocytes cells at dose rate of 2 Gy/hr Effect of γ-ray doses 0.25, 0.5, 1, 2, 4 Gy on the percentage number of the cells is summarized in the Tables (4.30-4.33) for donors 1, 2, 3 and 4 respectively. The percentage number of apoptotic cells was increased significantly with dose except for donor 2 at doses 0.25 and 0.5 Gy and for donor 3 at dose 0.25 Gy. That is because radiation induced apoptosis of lymphocytes subpopulations differs among individuals, which was also explained by Schmitz et al. [95]. Tables (4.34) and (4.35) present the mean of percentage number of apoptotic and lymphocytes respectively. The percentage mean number ofapoptotic cells were 4.97%, 5.74%, 6.72%, 8.15%, 10.89% and 14.16% for 0, 0.25, 0.5, 1, 2 and 4 Gy respectively. Increasing of apoptotic cells was dose dependent and significant for all doses except at 0.25Gy. This property of dose dependent was observed by Vral et al.[54], Wilkins et al [15], Belyaev et al.[56] and Liegler et al. [63] Decreasing of lymphocytes was also observed in our results and it was dose dependent. It was significant at 2 and 4 Gy, which agrees well with Liegler et al.[63]. They found that; with increasing exposure to irradiation dose and the length of postirradiation incubation, the number of peripheral lymphocytes is decreased. The variation of the dose with the mean of percent number of apoptotic and lymphocyte is shown in Figs.(4.10) and (4.11) respectively. In all experiments we observed that the radiosensitivity of cell killing varies among individuals which agree with Mei et al.[96].
Table (4.30): Percentage number of lymphocyte, apoptotic and monocyte cells with 60 Co gamma ray dose in donor 1. Dose rate is 2 Gy/hr. Dose (Gy)
%lymphocyte
SEM
p-value
% apoptotic
SEM
p-value
% monocyte
SEM
p-value
0 (Control)
73.29
0.47
-
6.02
0.10
-
20.68
0.44
-
0.25
71.68
0.02
0.07
6.54
0.14
0.04*
21.76
0.12
0.08
0.5
70.88
0.67
0.04*
7.75
0.26
0.004*
21.35
0.45
0.34
1
70.47
1.01
0.06
8.33
0.13
0.0001**
21.19
0.93
0.64
2
70.90
0.99
0.09
9.10
0.49
0.003*
19.98
0.57
0.39
0.21
0.00001**
20.93
0.75
0.79
4 64.31 2.29 0.02* 12.08 *Significant difference in comparison with control p<0.05.
** High significant difference in comparison with control p<0.001.
Table (4.31): Percentage number of lymphocyte, apoptotic and monocyte cells with 60 Co gamma ray dose in donor 2. Dose rate is 2 Gy/hr. Dose (Gy)
%lymphocyte
SEM
p-value
%apoptotic
SEM
p-value
% monocyte
SEM
p-value
0 (Control)
79.21
0.40
-
5.29
0.43
-
15.49
0.18
-
0.25
77.11
0.51
0.03*
6.20
0.61
0.29
16.67
0.53
0.14
0.5
76.79
0.52
0.02*
6.84
0.46
0.07
16.35
0.21
0.03*
1
74.45
0.39
0.001*
8.72
0.29
0.003*
16.80
0.15
0.005**
2
69.46
1.07
0.001*
12.91
0.57
0.0004**
17.40
0.74
0.07
4 62.71 0.04 0.0005** 18.84 *Significant difference in comparison with control p<0.05.
0.54
0.00004**
18.43
0.50
0.005*
** High significant difference in comparison with control p<0.001
Table (4.32): Percentage number of lymphocyte, apoptotic and monocyte cells with 60Co gamma ray dose in donor 3. Dose rate is 2 Gy/hr. Dose (Gy)
%lymphocyte
SEM
p-value
%apoptotic
SEM
p-value
%monocyte
SEM
pvalue
0 (Control)
81.53
0.75
-
4.72
0.36
-
13.73
0.41
-
0.25
80.31
0.89
0.36
5.37
0.58
0.40
14.28
0.48
0.43
0.5
78.81
1.38
0.16
6.48
0.27
0.02*
13.70
0.60
0.97
1
78.12
0.65
0.03*
7.65
0.38
0.005**
14.22
0.31
0.40
2
73.99
0.36
0.0008**
11.25
0.28
0.0001**
14.66
0.28
0.13
4 71.29 0.16 0.0002** 12.55 *Significant difference in comparison with control p<0.05.
0.17
0.00004**
16.14
0.04
0.03*
** High significant difference in comparison with control p<0.0
Table (4.33): Percentage number of lymphocyte, apoptotic and monocyte cells with 60
Co gamma ray dose in donor 4. Dose rate is 2 Gy/hr.
Dose (Gy)
%lymphocyte
SEM
p-value
%apoptotic
SEM
p-value
%monocyte
SEM
pvalue
0 (Control)
83.54
0.35
-
3.85
0.09
-
12.60
0.27
-
0.25
82.88
0.19
0.17
4.86
0.10
0.002*
12.28
0.17
0.38
0.5
82.59
0.51
0.20
5.79
0.27
0.002*
11.61
0.34
0.09
1
78.73
0.33
0.0005**
7.89
0.08
0.000005**
13.36
0.32
0.14
2
76.38
0.45
0.0002**
10.28
0.29
0.00003**
13.34
0.67
0.36
4 74.38 0.37 0.00005** 13.15 *Significant difference in comparison with control p<0.05.
0.31
0.000009**
12.45
0.09
0.63
** High significant difference in comparison with control p<0.001.
Table (4.34): Mean of percentage number apoptotic cells with 60Co gamma ray dose for all donors. Dose rate is 2 Gy/hr. Dose (mGy) 0 (Control)
%apoptotic in donor 1
%apoptotic in donor 2
%apoptotic in donor 3
%apoptotic in donor 4
Mean of % apoptotic
SEM
p-value
6.02
5.29
4.72
3.85
4.97
0.46
-
0.25
6.54
6.20
5.37
4.86
5.74
0.38
0.24
0.5
7.75
6.84
6.48
5.79
6.72
0.41
0.03*
1
8.33
8.72
7.65
7.89
8.15
0.24
0.0008**
2
9.10
12.91
11.25
10.28
10.89
0.81
0.0006**
4 12.08 18.84 12.55 13.15 *Significant difference in comparison with control p<0.05.
14.16
1.58
0.001*
** High significant difference in comparison with control p<0.001.
Table (4.35): Mean of percentage number lymphocyte cells with 60Co gamma ray dose for all donors. Dose rate is 2 Gy/hr.
Dose (Gy) 0 (Control) 0.25
%lymphocyte in donor 1
%lymphocyte in donor 2
%lymphocyte in donor 3
%lymphocyte in donor 4
73.29
79.21
81.53
83.54
71.68
77.11
80.31
70.88
76.79
70.47 70.90
Mean of %lymphocyte SEM
p-value
79.39
2.22
-
82.88
78.00
2.41
0.69
78.81
82.59
77.27
2.45
0.54
74.45
78.12
78.73
75.44
1.91
0.23
69.46
73.99
76.38
72.68
1.55
0.05*
74.38
68.17
2.75
0.02*
0.5 1 2 4 64.31 62.71 71.29 *Significant difference in comparison with control p<0.05.
M ean o f p ercen tag e n u m b er o f ap o p to tic cells
18 16 14 12 10 8 6 4 2 0 0
1
2
3
4
5
Co60 gamma ray dose (Gy)
Fig.(4.10): Variation of mean of percentage number apoptotic cells with 60Co gamma ray dose. Dose rate is 2 Gy/hr.
Mean of percentage number of lymphocytes
85 80 75 70 65 60 55 50 0
1
2
3
4
5
Co60 gam m a ray dose (Gy)
Fig.(4.11): Variation of mean of percentage number lymphocyte cells with 60Co gamma ray dose. Dose rate is 2 Gy/hr.
4-4 Dose Response Model To describe the dependence of amount of fluorescence of activated caspase 3, percentage number of apoptosis (death cell) and percentage number of lymphocyte (living cells) on the dose, we suggested a mathematical model of the form: Y= a Db +c …
(4.1)
where Y= The variable a= The amplitude parameter, D= The radiation dose b= A shape parameter. c= A constant represents the background of the variable Y.
4-4-1 Dose response curve for fluorescence in activated caspase 3 in apoptotic cells From the dose response curves we observed the amount of caspase 3 activated in apoptotic cells increase with doses for the three different experiments as shown in Figs. (4.12-4.14). The value of fitting parameters a and b are given in Table 4.36. From the value of (a) we observed that the amount of caspase 3 activated in apoptotic cells was more for high doses of x-ray than low doses of x-ray and high doses of 60
Co gamma ray and the amount was more in high doses of 60Co gamma
ray than low doses of x-ray. The value of b is higher for low doses and low radiation energy. The value of c=100 which is the amount of activated caspase 3 for controls after applied normalization for donors.
From the present results we observed that at low dose of x-ray the activation of caspase 3 is very low in comparison with other two experiments. This confirms that at low doses inducing of apoptosis is insignificant.
Table (4.36): The values of fitting parameters a and b in our mathematical model for the three experiments. R is the correlation coefficient and c is the amount of activated caspase 3 at 0 Gy after applied normalization for donors.
Type of irradiation
Parameter a ±Std error
Parameter b ±Std error
R2
High x-ray dose with dose rate 0.28Gy/min. Low dose of x-ray with dose rate 20mGy/min. High dose gamma ray with dose rate 0.033 mGy/hr.
85.27±6.44
0.63±0.07
0.981
0.995±0.27
0.74±0.053
0.991
73.63±4.8
0.38±0.06
0.963
Amount of fluorescence of activated caspase 3 in apoptotic cells
400 300 200 100 0 0
1
2
3
4
5
X-ray dose (Gy)
Amount of fluorescence of activated caspase 3 in apoptotic cells
Fig. (4.12): Dose response curve for high doses of x-ray with dose rate of 0.28 Gy/min.
200 180 160 140 120 100 80 0
100
200
300
X-ray dose (mGy)
Amount of fluorescence of activated caspase 3 in apoptotic cells
Fig. (4.13): Dose response curve for low doses of x-ray with dose rate of 20 mGy/min. 250 200 150 100 50 0 0
1 60
2
3
4
5
Cogamma ray dose (Gy)
Fig. (4.14): Dose response curve for low doses of 0.033 mGy/hr.
60
Co gamma ray with dose rate of
4-4-2 Dose response curves for percentage number of apoptosis The fitting parameters a, b and c of our model equation for percentage number of apoptotic cells are given in Table 4.37. From the value of (a) the percentage number of apoptotic cells induced by high dose with high dose rate of x-ray ( 0.28 Gy/min.) was almost the same for high doses with dose rate ( 0.033 Gy/min.) of 60Co gamma ray. We observed from the percentage number of apoptotic cells from each experiment which were (5.57%, 6.14%, 7.60%, 8.91%, 11.72% and 16.51%) for x-ray and (4.97%, 5.74%, 6.72%, 8.15%, 10.89% and 14.16) for
60
Co gamma ray. our result agree with Boreham et al.[45]
which they showed that x-ray and gamma ray exposures induced similar levels of apoptosis at similar doses At low doses and low dose rate (20 mGy/min.) of x-ray the percentage number of apoptosis was very few in comparable with the other two experiments which percentage number of apoptosis was (4.50%, 4.56%, 5.06%, 5.21%, 5.28% and 5.50%). This might be at this range of low doses the irradiation induce another types of lesion other than apoptosis like DNA damage which we observed in the first part of our study, because apoptosis occur as a result of DSBs and usually DSBs are not repair easily and soon. From the value of parameter (b) we noticed that for both high doses of x-ray with high dose rate and high doses of 60Co gamma ray with low dose rate the increasing of percentage number of apoptosis with doses more rapid than low doses with low dose rate of x-ray. From these observation we noticed that percentage number of apoptosis induced by x-ray with high dose rate (0.28 Gy/min.) and 60Co gamma rays with low dose rate (0.033 Gy/min.) is independent on dose
rate which agree with Vral et al.[62] which they conclude that the ability of lymphocytes to undergo apoptosis independent on dose rate. Also our results agree with that of Fujikawa et al.[97], which they obtained that radiation–induced apoptosis in lymphocytes is a dose-rate independent event, and with Pecaut et al.[98], which observed that the irradiation of whole body of mice C57BL/6 to gamma radiation (0, 0.5, 1.5 and 3) Gy with dose rate 1cGy/min. and 80 cGy/min changed in the number of leukocytes and lymphocytes numbers on lymphoid and organs were highly dependence on the dose, but not dose rate . A simple hypothesis concerning dose-rate independent of apoptosis after irradiation is as follows; in lymphocytes, apoptosis occurs so rapidly after the production of DSBs by radiation that most cells with DSBs undergo apoptosis before the DSBs are repaired. The recombinational repair of DSBs in mammalian cells occurs only after cells with DSBs enter the S phase, because the Rad51 protein, indispensable for this repair, is synthesized in the S-G2 phases and degraded in the M phases [97]. The dose response curves shown in Figs (4.15- 4.17).
Table (4.37): The values of parameters in our model for the percentage number of apoptotic for three experiments.
Type of irradiation
High x-ray dose with dose rate 0.28Gy/min. Low dose of x-ray with dose rate 20mGy/min. High dose gamma ray with dose rate 0.033 mGy/hr.
Parameter b ±Std error
Parameter c ±Std error
R2
3.48±0.31
0.84±0.06
5.43±0.24
0.997
0.11±0.12
0.41±0.18
4.46±0.18
0.875
3.41±0.38
0.74±0.07
4.78±0.29
0.995
Parameter a ±Std error
Percentage of apoptotic cells
20 15 10 5 0 0
1
2
3
4
5
X-ray dose (Gy)
Percentage of apoptotic cells
Fig. (4.15): Dose response curve for high doses of x-ray with dose rate of 0.28 Gy/min with apoptotic cells.
8 6 4 2 0 0
100
200
300
X-ray dose (mGy)
Percentage of apoptotic cells
Fig. (4.16): Dose response curve for low doses of x-ray with dose rate of 20 mGy/min with percentage number of apoptotic cells.
20 15 10 5 0 0
1 60
2
3
4
5
Co gamma ray dose (Gy)
Fig. (4.17): Dose response curve for low doses of 60Co gamma ray with dose rate of 0.033 Gy/min with percentage number of apoptosis.
4-4-3 Dose response curve for the percentage number of lymphocyte The parameters of our mathematical model that describe the dose response for the percentage number of lymphocytes cells are given in Table (4.38). The minus sign of the parameter (a) indicates that the number of lymphocyte cells is decreased. From this value decreasing of percentage number of lymphocytes for high doses of x-ray with high dose rate (0.28 Gy/min.) was much more than low doses of x-ray with low dose rate (20 mGy/min) and almost the same with high doses with low dose rate (0.033 Gy/min.) of
60
Co gamma ray, however, the
decreasing for high doses of x-ray with high dose rate shows high linearity than that for 60Co gamma rays as shown in the Fig.(4.18- 4.20). Decreasing of number of cells was observed by Pecaut et al [98] which found decreasing in leukocytes and lymphocytes numbers occurred with increasing dose in blood and spleen. This was also observed by Gridely et al.[ 99] where significant dose inverse dependenancy was seen in erythrocyte and blood leukocytes counts. We noticed that percentage number of lymphocytes is not significantly affected by low doses of x-ray, which means tiny increasing of percentage number of apoptotic. That is because the primary cause of radiation-induced apoptosis is DSBs in DNA and DSBs are more difficult to repair compared to base damage and SSBs which was also explained by Fujikawa et al.[98] and Ismail et al. [94].
Table (4.38): The values of fitting parameters in the model for percentage number of lymphocytes (survival) for the three experimental conditions.
Type of irradiation
Parameter a ±Std error
Parameter b ±Std error
Parameter c ±Std error
R2
High x-ray dose with dose rate 0.28Gy/min. Low dose of xray with dose rate 20mGy/min. High dose gamma ray with dose rate 0.033 mGy/hr.
-3.52±0.46
0.94±0.08
79.36±0.35
0.996
-0.48±0.36
0.02±0.15
82.96±0.17
0.718
-3.93±0.14
0.76±0.02
79.42±0.11
0.999
Percentage of lymphocyte cells
90 80 70 60 50 0
1
2
3
4
5
X-ray dose (Gy)
Fig. (4.18): Dose response curve of high dose of x-ray with percentage number of
Percentage of lymphocyte cells
lymphocytes cells. Dose rate is 0.28 Gy/min. 90 80 70 60 50 0
100
200
300
X-ray dose (mGy)
Percentage of lymphocyte cells
Fig. (4.19): Dose response curve of low dose of x-ray with percentage number of lymphocyte cells. Dose rate is 20 mGy/min. 90 80 70 60 50
0
1 60
2
3
4
5
Co gamma ray dose (Gy)
Fig. (4.20): Dose response curve of low dose of 60Cogamma ray with percentage number of lymphocytes cells. Dose rate is 0.033 Gy/min.
4-5 Measuring of Granularity and Size of Apoptotic Cells Apoptosis has been characterized morphologically by increased cytoplasmic granularity, a reduction in cell size, cell shrinkage and nuclear and chromosomal condensation, membrane blebbing and the formation of distitinctive nuclear bodies [15, 19, 48, 51, 100]. We determined the relative granularity and size of the apoptotic cells for the three experiments. For high x-ray dose with dose rate of 0.28Gy/min, the mean relative of granularity of the cells was increased with dose. However, the increasing was not linear, while the size of the cell decreased linearly with dose. For both granularity and size the effect was not significant. The results are presented in Table (4.39). The effect of low dose of x-ray with dose rate of 20mGy/min is shown in the Table (4.40) as the granularity and size increased except for 250 mGy dose. The effect of
60
Co gamma ray dose present is given in
Table (4.41) as granularity was increased and the size was decreased with dose. The changes were not linearly and statistically insignificant.
Table (4.39): Mean relative granularity and size of apoptotic cells with x-ray dose at dose rate of 0.28Gy/min.
X-ray dose (Gy)
Mean relative granularity of apoptotic cells±SEM p-value
0 (control) 0.25
Mean relative size of apoptotic cells±SEM p-value
0
-
0
-
2.180±0.006
0.81
-0.732±0.003
0.97
1.910±0.009
0.83
-2.489±0.015
0.89
1.634±0.008
0.85
-3.484±0.006
0.84
2.997±0.003
0.76
-4.597±0.015
0.81
3.542±0.010
0.7
-5.534±0.012
0.77
0.5 1 2 4
Table (4.40): Mean relative granularity and size of apoptotic cells with x-ray dose at dose rate of 20mGy/min.
X-ray dose (mGy) 0 (control) 15.6
Mean relative granularity of apoptotic cells±SEM
Mean relative size of apoptotic cells±SEM p-value
p-value
0
-
0
-
0±0.006
1
0.15±0.0001
0.99
0.670±0.006
0.97
0.479±0.002
0.97
1.786±0.003
0.93
0.209±0.0003
0.99
1.786±0.004
0.93
0.03±0.006
0.99
-10.045±0.045
0.56
-9.485±0.046
0.43
31.3 62.5 125 250
Table (4.41): Mean relative granularity and size of apoptotic cells with 60Co gamma ray dose at dose rate of 2Gy/hr.
60
Co gamma ray doses (Gy) 0 (control) 0.25
Mean relative granularity of apoptotic cells±SEM
p-value
Mean relative size of apoptotic cells±SEM
p-value
0
-
0
-
1.739±0.007
0.3
-0.913±0.01
0.70
3.188±0.001
0.16
-1.995±0.007
0.38
4.928±0.003
0.06
-1.725±0.009
0.45
4.348±0.02
0.2
-3.652±0.009
0.13
6.377±0.007
0.04
0.5 1 2 4 -4.024±0.008
0.10
Chapter Five
Conclusions and Future Work
5-1 Conclusions Radiobiological studies have shown for some time that the effects of ionizing radiation on cells are mainly explained by modification of the DNA. Numerous studies over past 50 years have accumulated clear evidence of the cause-effect relationship between damage to DNA and the cytotoxic and mutagenic effects of ionizing radiation. DNA is an important target for the damaging effects of ionizing radiation and endogenously induced reactive oxygen species. Ionizing radiation produces through direct and indirect a variety of DNA lesion, such as SSBs, DSBs, a variety of base modification, sugar modification, and DNA-DNA cross-link and DNA-protein cross-link. Mononuclear cells are sensitive to ionizing radiation. They are a good tool to assessment the risk of ionizing radiation, its subpopulations (monocytes and lymphocytes) have different susceptibility to DNA damage and cell killing. Also subpopulations of lymphocytes are different from their response to ionizing radiation. Different studies
showed that the sensitivity to ionizing radiation is also different between individuals. Several biological indicators are available for the assessment of radiation risk. In biological dosimetry DNA damage, chromosome aberrations and apoptosis are biological endpoints that have been used to asses radiation damage to a cell. However, additional studies are needed to validate candidate biomarkers for applied biological dosimetry applications and that could provide early and rapid information after exposure to radiation. In this respect, flow cytometry assay technology is a promising tool. Through our study we used two molecular biological markers DNA damage and apoptotic in human cells to show the effect of high and low dose and dose rate of LET. Depending on our results we can conclude that: 1-High doses with high dose rate of x-ray induce significant DNA damage and apoptosis. 2- Low doses with low dose rate of x-ray induce significant DNA damage. Their effect is insignificant for apoptosis. 3-High doses with low dose rate of 60Co gamma rays induce significant DNA damage and apoptosis. 4-Inducing of DNA damage by x rays and 60Co gamma rays depends on dose and dose rate. 5- Inducing of apoptosis by x rays and
60
Co gamma rays depends on
dose but not on dose rate. 6- DNA and apoptosis are good molecular biological markers of radiation response. 7- Changing of granularity and size of apoptotic cells are more at high doses than low doses.
8- The granularity and size of mononuclear cells change by irradiation the cells with x rays and 60Co gamma rays. 5-2 Future Work 1- Investigation of chromosome aberrations (tricentric dicentric, ring and fragments), we have already started with the effect of LET on unstable chromosomal aberrations. 2- Study on repair of DNA damage after irradiation using different time of incubation or different agent like phytohemagglutinin (PHA). 3- Study on DNA damage and apoptosis in human cells using other types of ionizing radiation like alpha particles, neutrons and beta particles. 4- Study on inducing apoptosis by ionizing radiation using another techniques like Annexin V and Propidium Iodide. 5- Detection of DNA damage by ionizing radiation by measuring the amount of the DNA in G1 and G2 of cell cycle. 6- Comparsion between the ability of low and high LET radiation on damage of human cells.
References 1. K. M. Prise, New advances in radiation biology, Occupational Medicine 56, 156-161 (2006). 2. K. Bishay, K. Ory, M.F. Olivier, J. Lebeau, C. Levalois, S. Cherillaed, DNA damage-related RNA expression to asses individual sensitivity to ionizing radiation, Carcinogenesis, 22, 1179-1183 (2001). 3. D. Kufe, R. Pollock, R. Weichselbaum, R. Bast Jr, T. Ganster, J. Holland, "Cancer Medcine", BC Decker Inc. Hamilton, London (2003). 4. D. B John., D. I. Jr. Peter., "Leukemia", W.B.Saunders (1996), chapter 11, 195-209. 5. E. J. Hall, A. J. Giaccia. "Radiobiology for the radiologist", Lippincott Williams and Wilikins, New York (2006). 6. G. F. Knoll, "Radiation detection and measurement", John Wiley and Sons, New York (2000). 7. J.-P. Pouget, S. J. Mather, General aspect of the cellular response to low-and high-LET radiation, European Journal of Nuclear Medicine 28 (2001). 8. H. Lodish, A.Berk, S. L. Zipursky, P. Matsudaira, D. Battimore, J. Darneh, "Molecular cell biology", W.H. freeman and company, New York (2003). 9. N. A. Campbell, J. Reece, L. G. Mitchell, "Biology" Addison Wesley Longman Inc., New York (1999). 10. H. Ishida, Molecular dynamics simulation of 7, 8-dihydro-8oxoguanine DNA, Biomolecular Structure and Dynamics 19, 839-851 (2002). 11.V. I. Bruskov, L. V. Malakhova, Z. K. Masalimov, A. V. Chernkov, Heat-induced formation of reactive oxygen species and 8-oxoguanine, a
biomaker of damage to DNA, Nucleic Acid Research 30, 1354-1363 (2002). 12. M. S. Cooke, M. D. Evans, M. Dizaroglu, J. Lunec. Oxidative DNA damage: mechanisms, mutation, and disease, The FASEB Journal 17, 1195-1214 (2003). 13. L. J. Marnett, Oxyradical and DNA damage, Carcinogenesis 21, 361370 (2000). 14.V.S. Cruchten, V. D. W. Broeck, Morphological and biochemical aspects of apoptosis, oncosis and necrosis, Anat Histol Embryol 31,214223 (2002). 15. R. C.Wilkins, B. C. Kutzner, M.Truong, J.Sanchez-Dardon, J.R.N McLean, Analysis of radiation-induced apoptosis in human lymphocyes: flow cytometry using annexin V and propidium iodide versus the nuteral comet assay, Cytometry 48, 14-19 (2002). 16. S. Baatout, H. Derradji, P. Jacquet, D. Ooms, A. Michaux, M. Mergeay, Enhanced radiation-induced apoptosis of cancer cell lines after treatment with resveratrol, International Journal of Molecular Medicine 13, 895-902 (2004). 17. A. G. Renehan, G. Booth, C. S. Potten, What is apoptosis, and why is it important?, BMJ 322, 1536-1538 (2001). 18. C. J. Zeiss, Review article the apoptosis-nicrosis continuum: insights from genetically altered mice, Vet Pathol, 40, 481-495 (2003). 19. S. Elmore, Apoptosis: a review of programmed cell death, Toxologic Pathology 35, 495-516 (2007). 20. A. Zhang, Y. Wu, Helen W. L. Lai, David T. Yew, Apoptosis- a brief review, Neuroembryology 5, 47-59 (2004). 21. A. G., Porter, R. U. Jänicke, Emerging roles of caspase-3 in apoptosis, Cell Death and Differentiation 6, 99-104 (1999)
22. N. E. Bolus, Basic review of radiation biology and terminology, J Nucl. Med. Technol. 29, 67-73 (2001). 23. E. Schmid, T. Schrader, Different biological effectiveness of ionizing and non-ionizing radiations in mammalian cells, Adv. Radio Sci. 5, 1-4 (2007). 24. G. G. Steel, "Basic Clinical Radiobiology", Arnold, London (2002). 25. J. S. Lilley, "Nuclear physics principles and applications", John Willy and sons, New York (2006). 26. S. B. Dowed, E. R. Tilson, "Practical Radiation Protection and Applied Radiobiology", Saunders, USA (1999). 27. A. Davies, A. G. H. Blakeley, C. Kidd, "Human physiology", Harcourt , London (2001). 28. K. S. Saladin, "Anatomy and Physiology", McGraw-Hill, New York (2001). 29. S.A. Amundson, M.B. Grace, C.B. Mcleland, M.W. Epperly, A. Yeager, Q. Zhan, J.S. Greenberger, A.J. Fornace,Jr, Human In vivo radiation-induced biomarkers: Gene expression changes in radiotherapy patients, Cancer Research 64, 6368-6371 (2004). 30. P.G. S Prasanna, J.M Muderhwa, A.C Miller, M.B Grace, C.A Salter, and W.F Blakely, Diagnostic biodosmetry response for radiation disasters: current research and service activities at AFRRI, papers presented at the RTO HFM symposium on "NATO medical purveilance and response, research and technology opportunities and option", held in Budapest, Hungary,??? 31. B. Rydberg, Radiation-induced DNA damage and chromatin structure, Acta Oncologica 40, 682-685 (2001). 32.Y. Ogawa, T. Kobayashi, A. Nishika, S. Kariya, S. Hamasato, H. Seguchi, S. Yoshida, Radiation-induced oxidative DNA damage, 8-
oxoguanine, in human peripheral T cells, International Journal of Molecular Medicine 11, 27-32 (2003). 33. Y. Ogawa, T. Kobayashi, A. Nishika, S. Kariya, S. Hamasato, H. Seguchi, S. Yoshida, Radiation-induced reactive oxygen species formation prior to oxidative DNA damage in human peripheral T cells, International Journal of Molecular Medicine 11, 149-152 (2003). 34.W. F.Balkely, A. C. Miller, L. Luo, J. Lukas, Z. D. Hornby, C. J.C. Hamel, M. J. T. Nelson, N. E. Escalada, P. G.S. Prasanna, Nuleic acid molecular biomarkers for diagnostic biodosmetery applications: use of the fluorogenic 5'-nuclease polymerase chain reaction assay, Military Medicine 167, 16-19 (2002). 35.W.Niedzwieedz, A.Cebulska-Wasilewska, DNA damage induction and repair evaluated in human lymphocytes irradiated with X-rays and and neutrons, Report No. 1866/B (2000) , www.ifj.edu.pl/reports/2000.html. 36. N. P. Singh, M. T. McCoy, R. R.Tice, E. L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Expermiental Cell Research 175, 184-191 (1988). 37. H. Mozdarani, B. Nasirian, S. A. Haeri, In vivo γ-rays induced initial DNA damage and the effect of famotidine in mouse leukocytes as assayed by alkaline comet assay, J. Radiat. Res. 48, 129-134 (2007). 38. R.C. Chaubey, H.N. Bhilwade, R. Rajagopalan, Effect of low doses and low dose-rates of gamma radiation on DNA damage in human peripheral blood leukocytes using comet assay. Int. J. Low Radiation 2, 71-83 (2003). 39. R.C.Chaubey, H.N. Bhilwade, R.Rajagopalan, S.V. Bannur, Gamma ray induced DNA damage in human and mouse leucocytes measured by SCGE-Pro: a software developed for automated image analysis and data
processing for comet assay, Mutation Research/Genetic Toxology and Enviromental Mutagenesis 490, 187-197 (2001). (Abstract). 40.Vijayalaxim, Strauss GH, Tice RR, An analysis of gamma-rayinduced DNA damage in human blood leukocytes, lymphocytes and granulocytes, Mutat Res. 292, 123-128 (1993). (Abstract) 41. U. Rossler, S. Homrnhardt, C. Seidl, E. Muller-Laue, L. Walsh, W. Panzer, E. Schmid, R. Senekowitsch-Schmidtke, M. Gomolka, The sensitivity of alkaline comet assay in detecting DNA lesions induced by x rays, gamma rays and alpha particles, Radiation Protection Dosimetry 122, 154-159 (2006). 42. S.K. Chen, M. H. Tsai, C.H. lin, J. J. Hwang, W. P. Chan, Determention of 8-Oxoguanine in individual cell nucleus of gammairradiated mammalian cells, Radiation Research 155, 832-836 (2001). 43. C. Courtemanche, A. C. Huang, I. Elson-Schwab, N. Kerry, Bernice Y. NG, B. N. Ames, Folate deficiency and ionizing radiation cause DNA breaks in primary human lymphocytes: comparison, Faseb 18, 209-211 (2004). 44. B. M. Sutherland, P. V. Bennett, N. Cintron-Torres, M. Hada, J. Trunk, D. Mouteleone, J. C. Sutherland, J. Lavel, M. Stanislaus, A. Gewirtz, Clustemred DNA damages induced in human hematopoietic cells by low doses of ionizing radiation, J Radiant. Res. 43, 149-152 (2002). 45. D. R. Boreham, K.L. Gale , S.R. Maves, J.A. Walker, D.P. Morrison, Radiation –induced apoptosis in human lymphocytes:potential a biological dosimeter, Health Phys. 71, 685-691 (1996). 46. Y. Ogawa, T. Kobayashi, A. Nishika, S. Kariya, S. Hamasato, H. Seguchi, S. Yoshida. Mitocjondrial cytochrome c release in radiation-
induced apoptosis of human peripheral T cell, International Journal of Molecular Medicine 10, 263-268 (2002). 47. C. M. Payne, C. G. Bjore, Jr., D. A. Schultz, Change in the frequency of apoptosis after low-and high-dose X-irradiation of human lymphocytes, J. of Leukocyte Biology 52,433-440 (1992). 48. R.C.Wilkins, D.Wilkinson, H. P. Maharaj, P. V. Bellier, M. B. Cybulski, J. R. N. McLean, Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells, Mutation Research 513, 27-36 (2002). 49. V. Holl, D. Coelho, D. Weltin, J. M. Denis, P. Dufour, J. Gueulette, P. Bischoff, Ex vivo determination of the effect of whole-body exposure to fast neutrons on murine spleen cell viability and apoptosis, Radiation Research, 154, 301-306 (2000). 50. A. Haimovitz-Friedman, C.-C. Kan, D. Ehleiter, R. S. Persaud, M. McLoughiln, Z. Fuks, R. N. Kolesnick. Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis, J. Exp. Med. 180, 525-535 (1994). 51. R. L.Warters, Radiation-induced apoptosis in a murine T-cell hybridoma, Cancer Research 52, 883-890 (1992). 52. M. Ozsahin, N. E.A. Crompton, S. Gourgou, A. Kramar, L. Li, Y. Shi, W. J. Sozzi, A. Zouhair, R. O. Mirimanoff, D. Azria, CD4 and CD8 T-lymphocyte apoptosis predict radiation-induced late toxicity: a prospective study in 399 patients, Clin Cancer Res 11, 7426-7433 (2005). 53. L. P. Wang, K. Liang, Y. Shen, W. B. Yin, G. Hans, Y. J. Zeng, Neutron-induced apoptosis of HR8348 cells in vitro,World J. Gastroentero 7, 435-439 (2001).
54. A. Vral, M. Cornelissen, H. Thierens, H. Louagie, J. Philippe, K. Strijckmans, L. De Ridder, Apoptosis induced by fast neutrons versus 60
Co gamma-rays in human peripheral blood lymphocytes, International
J. of Radiation Biology 73, 289-295 (1998). (Abstract). 55. J. Vávrová, M. Řezáčová, D. Vokurková, J. Psutka, Cell cycle alteration, apoptosis and response of leukemic cell lines to gamma radiation with high and low dose rate, Physiol. Res 33, 335-342 (2004). 56. I. Y. Belyaev, S. Czene, M. Harms-Ringdahl, Changes in chromatin conformation during radiation-induced apoptosis in human lymphcytes, Radiation Research 156, 355-364 (2001).(Abstract). 57. H.J. Kim, S.K. Moon, J.H. Lee, S.R.Moon, A study on apoptotic signaling pathway in HL-60 cells induced by radiation, J. Korean Soc. Ther Radiol Oncol, 19, 153-162 (2001). 58. L. A. Peña, Z. Fuks, R. N. Kolesnick, Radiation-induced apoptosis of endothelial cells in murinecentral nervous system:protection by fibroblast growth factor and sphingomyelinase deficiency, Cancer Reaserch 60,321-327 (2000). 59. R. Ghosh, S. Sengupta, N.P. Bhattacharyya, Induction of apoptosis by ionizing radiation in Chinese hamster V79 cells and a radioresistant cell strain derived from V79, Indian J. of Experimental Biology 34, 863867 (1996). 60. M. J. Homa., E. Davied, S Ali., J. Amanda, L. Per-Olov, J. Lennart, S. Torgny, Apoptosis induced by low-dose and low-dose rate radiation, Cancer 94, 1210-1214 (2002). 61. T.Aleš , Z. Darina, Ř.Martina, V. Jiřina, V.Doris, P. Jaroslav, V. Zdena, C. Jaroslav, Ö. Jan, Gamma radiation induced ATM dependent signaling in human T-lymphocyte leukemic cells, MOLT-4, Acta Biochimica Polonica 54, 282-287 (2007).
62. A. R. Collins, M. Dušinská, C. M. Gedik, R. Štětina, Oxidative damage to DNA: Do we have a reliable biomarker?, Environmental Health Perspectives 104, 465-469 (1996). 63. T. J. Liegler, W. Hyun, T. S. Benedict Yen, D. P. Sttes, Detection and quantification of live, apoptotic, and necrotic human peripheral lymphocytes by single-laser flow cytometry, Clinical and Diagnostic Laboratory Immunology 2, 369-376 ( 1995). 64. C. M. Pathak, P. K. Avti, S. Kumar, K. L. Khanduja, S. C. Sharma, Whole body exposure to low-dose gamma radiation promotes kidney antioxidant status in balb/c mice, J. Radiat. Res. 48, 113-120 (2007). 65. D. P. Hayes, Non-problemyic risks from low dose radiation induced clustered, Dose Response 6, 30-52 (2008). 66. M. Gulston, C. de Lara, T. Jenner, E. Davis, P. O′Neill, Processing of clustered DNA damage generates additional double-strand breaks in mammalian cells post-irradiation, Nucleic Acids Research 32 (2004). 67. J. Zempleni and D. M. Mock, Uptake and metabolism of biotin by human peripheral blood mononuclear cells, Am. J Physiol Cell Physiol, 275,383-388 (1998). 68. Medical and biological labortories co.,LTD, MBL, CasGLOWTM fluorescein active caspase-3 staining kit catalogue (2008). 69. "Introduction to flow cytometry a learning guide". Becton, Dickinson and Company, USA (2002). 70. M. Rahman, "Introduction to Flow Cytometry", Serotec Ltd, UK, (2006). 71. A. E. Milner, A. T. M. Vaughan, I. P. Clark, Measurement of DNA damage in mammalian cells Using flow cytometry, Radiation Research, 110, 108-117 (1987).
72. R. G. Ahmed, Damage pattern as function of various types of radiations, Medical J. of Islamic World Academy of Science 15, 135147 (2005). 73. A.E. Aust, J. F. Eveleigh, Mechanisms of DNA oxidation, Society for Experimental Biology and Medicine 222, 246-252 (1999). 74. R. E. Anderson, W. L. Williams, Radiosensivity of T and B lymphocytes, Am. J. Pathol 89,367-378 (1977). 75. G. B. Olson, R. E. Anderson, P. H. Bartels, Computer analysis of defined populations of lymphocytes irradiated in vitro, Human Pathology 10:179-190 (1979). 76. J. Wasserman, B.Petrini, H. Blomgrn,
Radiosensitivity of T-
lymphocytes subpopulations, J. Clin. Lab. Immunol 7,139-140 (1982). 77. D. K. Kwan, A. Norman, Radiosensitivity of larg human monocytes, Radiation Research 75, 556-562 (1976). 78. A. Facchini , N. M. Maraldi, S. Bartoli , A. Farulla , F. A. Manzoli, Changes in membrane receptors of B and T human lymphocytes exposed to 60Co gamma rays, Radiation Research 68, 330-348 (1976). 79. M. Mori, M. A.Benotmane, I. Tirone , E. L. Hooghe-Peters, C. Desaintes, Transcriptional response to ionizing radiation in lymphocyte subsets, Life Sci. 62, 1489-1501 (2005). 80. D.Vokurková, J.Šinkora, J.Vávrová, M.Řezáčvá, J.Knížek , J. Östereicher , CD8+ natural killer cells have a potential of a sensitive and reliable biodosmetric m in vitro, Physiol. Res. 55, 689-698 (2006). 81. M. Francesce, A. Cristina ,R.Sabrina , V.Alessandra , G. Rossella , C. Riccardo, Assessment of individual sensitivity of ionizing radiation and DNA repair efficiency in a healthy population, Mutation Research 541,1-8 (2003).
82. N. Kopjar, D. Zelijezic, V. Garaj-Vrhovac, Evaluation of DNA damage in white blood cells of healthy human volunteers using the alkaline comet assay and the chromosome aberration test, Acta Biochimica Polonica 53, 321-336 (2006). 83. M. Di Giorgio, M. Sardi , E. Busto , M.B.Vallerga, M.R.Taja, L. Mairal, Assessment of individual radiosensitivity in human lymphocytes using micronucleus and microgel electroresis " comet" assays. 11th International Congress on the International Radiation Protection Association. Madrid, Espana, 23-28 May (2004). 84. V. Smart , G. B. Curwen, C. A. Whitehouse, A. Edwards , E. J. Tawn, Chromosomal radiosensitivity : a study of chromosomal G2 assay in human blood lymphocytes indicating significant inter-individual variability, Mutation Research 528,105-110 (2003). 85. J. Tacker, Radiation-induced mutation in mammalian cells at low doses and low dose rates, Adv. Radiant. Biol., 16,77-124 (1992). 86. H. Ogata, C. Furukawa, Y. Kawakami, J. Magae, A quantitative model for the evaluation of dose rates effects following exposure to lowdose gamma radiation, Radioprotection 40, 191-202 (2005). 87. N. Shinomiya, New concept in radiation-induced apoptosis: 'premitotic apoptosis' and 'postmitotic apoptosis', J. Cell. Mol. Med. 5, 240-253 (2001). 88. M. Marekova, J. Vavrova, D. Vokurkova, Monitoring of premitotic and postmitotic apoptosis in gamma –irradiated HL-60 cells by the mitochondrial membrane protein-specific monoclonal antibody AP02.7, Gen. Physiol. Biophys 22, 191-200 (2003). 89. M. Cornelissen, A. Vral, H. Therenns, L. de. Ridder, The effect of caspase-inhibitors on radiation induced apoptosis in human peripheral
blood lymphocytes: an electron microscopic approach, Apoptosis 4, 449-454 (1999). 90. K. J. Maher, M. A. Fletcher, Quantitative flow cytometry in the clinical laboratory, Clinical and Applied Immunology Review 5,353372 (2005). 91. T. Nomura, M. Kinuta, T. Hangyo, H. Nakajima, T. Hatanmaka, Programmed cell death in whole body and systems by low dose radiation, J. of Radiation Research 33, 109-123 (1992). 92. A. E. Meijer , A. B. B. Saeidi , A. Zelenskaya, S.Czene, F.Granath, M. Harms-Ringhahl, Influence of dose rate, post irradiation incubation time and growth factors on interphase cell death by apoptosis and clonogenic survival of human peripheral lymphocytes, Int J Radiat Biol 75:1265-1273 (1999).(Abstract). 93. L. S.Zheng, Z. Y. Chun, M. Ying, S. Xu, L. J.Xiang, Thymocyte apoptosis in response to low-dose radiation, Mutat. Res., 358, 185-191 (1996). 94. I. H. Ismail, T. I. Wadhra, O. Hammarsten, An optimized method for detecting
gamma-H2AX
in
blood
cells
reveals
a
significant
interindividual variation in the gamma-H2AX response among humans, Nucleic Acid Research 35, 1-10 (2007). 95. A. Schmitz, J. Bayer, N. Dechamps, G. Thomas, Intrinsic susceptibility to radiation-induced apoptosis of human lymphocyte subpopulations, Int. J. Radiation Oncology Boil. Phys. 57, 769-778 (2003). 96. N. Mei, H. Imada, S. Nomoto, N. Kunugita, T. Norimura, Individual variation and age dependency in the radiosensitivity of peripheral blood T-lymphocytes from normal donors, J. Radiat. Res. 37, 235-245 (1996).
97. K. Fujikawa, Y. Hasegawa, S.Matsuzawa, A. Fukunaga, T. Itoh , S. Kondo, Dose and dose-rate effects of X-rays and fission neutrons on lymphocyte apoptosis in p53(+/+) and p53(-/-) mice, J. Radiat. Res. 41, 113-127 (2000). 98. M.J Peacut , G.A.Nelson , D.S.Gridely, Dose and dose rate effects of whole-body gamma-irradiation:I. Lymphocytes and lymphoid organs, In Vivo, 15,195-208 (2001).(Abstract). 99. D.S. Gridley , M.J.Pecaut , G.M.Miller , M.F.Moyers , G.A. Nelson, Dose
and
dose
rate
effects
of
whole-body
gamma-
irradiation:II,Hematological variables and cytokines, In Vivo 15,209216 (2001).(abstract). 100. J. C. Reed, Mechanisms of apoptosis, Am. J. of Pathology, 157,1415-1430 (2000).
×a‹ÉÜa@óîŠíé»@ ðáÝÉÜa@szjÜaì@ðÜbÉÜa@âïÝÉnÜa@òŠaŒì@ †a‡Íi@óÉàbu@@@@@@@@@@@@@@@@@@ ãíÝÉÜa@óïÝØ@@@@@@@@@@@@@@@@@@@ @ @ @
Ë@bÉ’ýbi@sna DNA@ñìíåÜa@àb¨a@À@ÒÝnÜa@@ @ @ @
óà‡Õà@óyì‹ c@ †a‡Íi@óÉàbu@@@O@ãíÝÉÜa@óïÝØ@¶a@ óÑÝÐ@òaŠín؆@óuŠ†@Þïä@pbjÝnà@æà@öu@ðèì@ öbîïÑÜa@À@ @ ÞjÔ@æà@ @
x‹Ð@ã‹Øc@çbàŠ@ê‚@
@ Óa‹’di@ @ ðäa‡é“¾a@‡¼@bïeN†NãNc@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@‘bïÜc@Þï÷íäbà@çŒbàN†Nc@ @ 2008M@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@1430@@@M@@@@@@@ @
