EFFECT OF VERY LOW DOSE FAST NEUTRONS ON THE DNA OF RATS ’ PERIPHERAL BLOOD MONONUCLEAR CELLS AND LEUKOCYTES

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Paper

EFFECT OF VERY LOW DOSE FAST NEUTRONS ON THE DNA OF RATS ’ PERIPHERAL BLOOD MONONUCLEAR CELLS AND LEUKOCYTES

Sherif S. Nafee,*†Abdu Saeed,*‡Salem A. Shaheen,* Sufian M. El Assouli,§**

M-Zaki El Assouli,§ and Gehan A. Raouf††‡‡

Abstract—The effect of very low dose fast neutrons on the chromatin and DNA of rats’ peripheral blood mononuclear cells (PBMC) and leukocytes has been studied in the present work using Fourier transform infrared (FTIR) and single-cell gel electrophoresis (comet assay). Fourteen female Wistar rats were used;

seven were irradiated with neutrons of 0.9 cGy (²⁴¹Am-Be, 0.02 cGy h⁻¹), and seven others were used as control. Second derivative and curve fitting were used to analyze the FTIR spectra.

In addition, hierarchical cluster analysis (HCA) was used to clas- sify the group spectra. Meanwhile, the tail moment and percentage of DNA in the tail were used as indicators to sense the breaking and the level of damage in DNA. The analysis of FTIR spectra of the PBMC of the irradiated group revealed a marked increase in the area of phosphodiesters of nucleic acids and the area ratios of RNA/DNA and phosphodiesters/carbohydrates. A sharp significant increase and decrease in the areas of RNA and DNA ribose were recorded, respectively. In the irradiated group, leukocytes with different tail lengths were observed. The distributions of tail moments and the percentage of DNA in the tail of irradiated groups were heterogeneous. The mean value of the percentages of DNA in the tail at 0.5 h post-irradiation repre- sented low-level damage in the DNA. Therefore, one can conclude that very low dose fast neutrons might cause changes in the DNA of PBMC at the submolecular level. It could cause low-level damage, double-strand break, and chromatin fragmentation of DNA of leukocytes.

Health Phys. 110(1):50–58; 2016

Key words: DNA; dose assessment; dose, low; neutron dosimetry

INTRODUCTION

IONIZING RADIATIONinteractions with atoms and molecules within cells result in changes at the sub-molecular level.

Such changes may lead to cell injury, either directly or subsequent to attempts for the cell to repair changes. About a third of cell damage from low-linear energy transfer (LET) (x rays, gamma rays, and electrons) radiations is thought to result from direct ionization, whereas the remainder of damage is“indirect” (caused by interactions of radiation- induced species with cellular components). The vast ma- jority of the damage from high-LET (alpha particles, fast neutrons, and heavy ions) radiations appears from direct effects (Jones and Karouia 2008). The DNA molecule may suffer damage from either direct or indirect effects.

In the case of direct effects, a strand break in DNA is caused by an ionization of the molecule itself, while with an indirect effect, a strand break results when the hydroxyl (OH•) free radical from the radiolysis of the hydration shell attacks a DNA sugar at a later time (between nearly 10⁻¹²and 10⁻⁹s) (Turner 2007).

When biological cells are exposed to ionizing radiations, they may have one of four fates: full recovery to a state before irradiation; partial recovery with repair of damage but with decreased functionality; mutations caused by in- complete repair; or cell death (Jones and Karouia 2008).

Mutations may occur within the cell and be propagated dur- ing cell division. These mutations, if not repaired, may lead to initiation and promotion of tumors, either benign or ma- lignant. Damage subsequent to ionization or excitation of an atom on the DNA molecule may prevent the transmission of genetic information to the next generation (Cember and Johnson 2009).

The present study aims to investigate the effect of very low dose fast neutrons on the DNA of rats (PBMC) and leukocytes using (FTIR) spectroscopy and comet assay, respectively. FTIR spectroscopy has been used in the present study because of its capability of detecting the molecular structural changes of hematological disorders (Dovbeshko et al. 2000) and to investigate DNA damage in assessing cell

*Physics Department, Faculty of Science, King Abdulaziz Uni- versity, Jeddah 20589, KSA;†Physics Department, Faculty of Science, Alexandria University, Alexandria 21121, Egypt;‡Physics Depart- ment, Thamar University, Thamar, Yemen; §King Fahd Medical Re- search Centre, King Abdulaziz University, Jeddah, KSA; **Biology Department, Faculty of Science, King Abdulaziz University, Jeddah 20589, KSA;††Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 20589, KSA;‡‡Spectroscopy Department, Physics Division, National Research Center, Cairo 12311, Egypt.

The authors declare no conflicts of interest.

For correspondence contact: Sherif S. Nafee, King Abdulaziz University Jeddah, Saudi Arabia, or email at[email protected].

(Manuscript accepted10September2015) 0017-9078/16/0

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damage after radiation (Lipiec et al. 2013), whereas the comet assay technique was used to estimate the DNA single- and double- strand breaks. This technique is simple, rapid, and sensitive to detect DNA damage at the individual cell level (Speit and Rothfuss 2012).

MATERIALS AND METHODS Experimental animals

Fourteen female Wistar rats weighing 220–270 g at 3 mo of age (Breeding Laboratory at King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia) were allowed to acclimatize for 15 d. The experimental animals adapted to an imposed 24‐h cycle (12 h day–12 h night) under standard vivarium conditions (temperature of 22 ± 1°C) with a relative humidity of 70%. Animals were maintained in box cages (three rats/

cage for the FTIR test; four rats/cage for the comet assay test) and were given food and water ad libitum. The present study was approved by the Experimental Animal Research and Ethics Committee, King Fahd Medical Research Cen- ter, King Abdulaziz University. Animals were randomly di- vided into two main groups: seven rats (three rats for the FTIR test and four for the comet assay test) were irradiated, and seven rats (three rats for the FTIR test and four for the comet assay test) were sham-irradiated as control.

Irradiation

A very low dose rate from a 185 GBq (5 Ci)²⁴¹Am-Be neutron source capsule X.14 (code AMN.24) (Amersham International PLC, Buckinghamshire, England) was applied to the posterior portions of the rats. The²⁴¹Am-Be source emits neutrons with energies ranging from 0 up to 11 MeV (Amersham 1976; National Research Council 2008; Medkour Ishak-Boushaki et al., 2012) and with an average energy of around 4.5 MeV (Amersham 1976;

El-Sersy et al. 2004; Cember and Johnson 2009). The neutron emission of the source was 1.110⁷n s⁻¹with a tolerance of around 10%. Dosimetry measurements were similarly performed (posterior portion) using a neutron mon- itor NM2 (Nuclear Enterprise, Edinburgh, UK) provided by the Nuclear Engineering Department, Faculty of Engineer- ing, King Abdulaziz University. The NM2 consists mainly of a thermal neutron detector surrounded by a polyethylene moderator with a boron trifluoride (BF₃) detector. The rats were irradiated at a dose rate of 0.2 mGy h⁻¹to a total neutron dose of 9 mGy. Gamma rays and other electromagnetic radiations that emit from the neutron source were shielded using lead bricks; lead thickness was nominally 9.6 cm.

Isolation of PBMC and FTIR measurements

For the FTIR test, each rat of the relevant groups was anaesthetized under ether. Six milliliters (6 mL) of peripheral blood samples were collected through the retro-orbital plexus

into 4‐mL anticoagulant dipotassium ethylenediaminetetraacetic acid (K₂EDTA) tubes using capillary tubes with a di- ameter of 1.5 mm. Within 1 h of collection, the samples were processed. Blood samples were applied to Histopaque 1077 gradients (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer's protocol to separate plasma, mononuclear cells, and erythrocytes. Sample gradients were centrifuged at 400g for 30 min at room temperature to remove plasma and to separate the mononuclear cells.

Mononuclear cells were collected and washed three times with sodium phosphate- buffered saline (PBS) [137 mM Sodium Chloride (NaCl), 2.7 mM potassium chloride (KCl), 10 mM disodium hydrogen phosphate (Na₂HPO₄.2H₂O), 2.0 mM monopotassium phosphate (KH₂PO₄), and pH 7.4].

All components were delivered from Sigma-Aldrich. The washed cells were centrifuged at 250g for 10 min (22°C) (Zelig et al. 2011). The washed mononuclear cells were stored at−80°C (Sheng et al. 2013) before lyophilization using an ALPHA 1-2 LD_plus freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany) at−60°C under vacuum (6610⁻³mbar; Cakmak et al.

2006; Selim et al. 2011).

Lyophilized PBMC were mixed with potassium bro- mide (KBr) (BDH Chemicals Ltd., Poole, England) for preparation as KBr discs. For each disc, lyophilized PBMC were thoroughly mixed with completely dried KBr powder in the ratio 1:100 and were then ground to a fine powder with mor- tar and pestle. The mixture was then pressed in a die at 6 t force for 60 s (Andrus and Strickland 1998; Selim et al 2011) to turn out to a clear transparent disc of 13 mm diam- eter and 0.6 mm thickness. At room temperature (25 ± 1°C), FTIR spectra were recorded using a Shimadzu FTIR-8400S spectrophotometer (Shimadzu Corporation, Tokyo, Japan).

The spectrometer was continuously purged with dry nitrogen to minimize atmospheric water vapor and carbon dioxide in- terference. Typically, 20 scans were signal-averaged for a single spectrum and at a spectral resolution of 4 cm⁻¹ and analyzed with OMNIC 8.3 software (Thermo Fisher Scien- tific Inc., Waltham, MA, USA). To minimize the difficulties arising from unavoidable shifts, the entire spectrum was baseline corrected from 4,000–400 cm⁻¹. Then the entire spectrum was min-max normalized by scaling the entire spectrum to the absorbance of amide I (around 1,654 cm⁻¹).

Preparation of slides and scoring comet assay

For the comet assay, 0.1 mL from each rat's peripheral blood was collected into anticoagulant K₂EDTA tubes under the same conditions as for the FTIR test. For the irradiated group, this procedure was carried out at intervals of 0.5, 1.5, and 3 h post-irradiation. The whole blood was processed immediately upon collection, and the comet assay was performed, with some modifications, as described in published literature (Singh et al. 1988; Hartmann and Speit

51 Effect of very low dose fast neutrons on DNAc^{S. S. N}^{AFEE ET AL}^.

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1995; Olive and Banáth 2006; ElAssouli et al. 2007). One percent low melting point agarose (LMA) (VWR Interna- tional Ltd, Lutterworth, UK) was prepared by mixing pow- dered agarose with distilled water in a glass beaker, which was put in a 100°C water bath for 10 min and then heated in a microwave at low power for short intervals (to avoid boiling of the agarose) to ensure that all agarose was dis- solved. The beaker with agarose was then put into a 40°C water bath. Whole blood (1.5mL) was mixed with 100mL of the prepared 1% LMA and added to the walls of the comet assay slides. The slides (Trevigen, US) were kept at 37°C for 5 min and then on an icy-plate for another 5 min to solidify the LMA. Then the slides were immersed in a cold lysing so- lution [2.5 M NaCI, 100 mM Ethylenediaminetetraacetic acid (EDTA), 10 mM Tris, 1% N-lauroyl-sarcosinc, pH 10, 1% Triton X‐100 and 10% dimethyl sulfoxide (DMSO) from (Merck, Darmstadt, Germany) were added fresh].

The DMSO served to scavenge iron-catalyzed reactive oxygen species, which can cause strand breaks resulting from hemolysis. The slides were kept at 4°C for 18–20 h in the re- frigerator. Slides were placed on a horizontal position electrophoresis box filled with a fresh alkaline buffer (300 mM NaOH, 1 mM EDTA, and pH 13.0) to allow for DNA un- winding and expression of alkali-labile sites for 20–25 min.

An electric current of 30 V (0.9 V.cm⁻¹) and 300 mA was applied for 20 min, conducted under dim light to prevent the occurrence of additional DNA damage. The slides were removed from the electrophoresis box and rinsed with Tris buffer (0.4 M Tris, pH7.5) to neutralize the excess alkali and then allowed to dry for 5 min; this step was repeated two times. Before comet scoring, a drop of ethidium bro- mide was added to each wall of the slide to stain the slide.

Stained slides were covered with a cover slip and kept in a humidified box. The slides were analyzed using the green light of a fluorescence microscope with an excitation filter of 546 nm and dichroic mirrors (barrier filters) of 590 nm (Olympus BH-2, Tokyo, Japan). The image of the comet was captured by a charge-coupled device (CCD) camera QICAM FAST (QImaging Corporation, Burnaby, BC,

Canada) and automatically analyzed by an LAI Automated Comet Assay (Loats Associates Inc., Westminster, MD, USA) system to evaluate the comet parameters. The tail moment is considered to be the most significant indicator for the detection of DNA breaks in comets (Testard and Sabatier 2000). According to the method of Anderson and his group, damage to cells can be graded into five cat- egories corresponding to the percentage of DNA in the tail:

<5% no damage, 5–20% low level damage, 20–40% me- dium level damage, 40–95% high level damage, and >95%

total damage (Anderson et al. 1994). This study used changes in tail moment and the percentage of DNA in the tail to estimate the DNA breakage and the damage level of the cells, respectively. Pictures of randomly selected cells (at least 100 cells, 50 cells from each of two replicate slides) were analyzed from each slide at 40X objective lens magnification.

RESULTS FTIR spectra of PBMC

Fig. 1 shows the average of PBMC spectra of control and irradiated groups in the range 1,300–900 cm⁻¹, which includes the nucleic acids bands. Table 1 gives the most ab- sorption bands of nucleic acids of the PBMC cells along with their assignments (Cakmak et al. 2006; Lu et al.

2011; Zelig et al. 2011). The spectra of the irradiated group compared to the control are nearly superimposed (apart from slight changes in certain band intensities in the range Fig. 1. Average of FTIR spectra exhibit rats’PBMC obtained from control and irradiated by fast neutron very low dose.

Table 1. Band assignments of the FTIR spectra of rat PBMC in range 1,250–900 cm⁻¹according to Cakmak et al. (2006), Lu et al. (2011), Zelig et al. (2011).^a

Wave number (cm⁻¹) Band assignments

1,236 n^as(PO2−): mostly nucleic acids and phospholipids 1,169 nas(COOC⁻): glycomaterials and proteins 1,084 ns(PO2−): mainly phosphodiester nucleic acids

974 C–N⁺–C stretch: nucleic acids

aNote.n= bond stretch; s = symmetric vibration; as = asymmetric vibration.

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of 1,070–970 cm⁻¹). Baseline-corrected curve fitting and second derivatives were carried out for FTIR spectra from 1,180.35–950.84 cm⁻¹, the region with the most information on the nuclei acids; sub-bands associated with the spe- cific components of the DNA molecule were resolved (Fig. 2). The areas of the sub-bands of the irradiated and control groups after curve fitting are listed in Table 2. The most important change was a marked increase in the area of thens(PO₂−) band that belongs to the phosphodiesters of nucleic acids. A sharp increase in the area of then(C– O) band that belongs to the RNA ribose was detected in the irradiated groups. In addition, a significant decrease in the n(C–O) of DNA deoxy ribose was recorded. Mean- while, no significant changes were recorded in other bands.

Table 3 gives the band area ratios 1,126 cm⁻¹/1,025 cm⁻¹ and 1,085 cm⁻¹/1,046 cm⁻¹as the biomarkers of RNA/DNA and phosphodiesters/carbohydrates, respectively (Andrus and Strickland 1998; Ramesh et al. 2001; Bogomolny et al.

2008). Sharp significant increases in the band area ratios of RNA/DNA and phosphodiesters/carbohydrates of the nucleic acid of the irradiated group have been detected.

Figs. 2 and 3 show the greatest variations in the band posi- tions and their intensities that were detected by curve fitting and second derivative, respectively, in the average spectra for all samples. HCA was used to derive the second derivative of FTIR spectra data from 1,200-1,000 cm⁻¹to evaluate differences between the DNA of PBMCs from control and irradiated rats (Fig. 4). This is confirming differences in the DNA structure of the PBMCs due to neutron irradiation.

As is evident in Figs. 2 and 3, the peak of then(C–O) RNA ribose band shifted to higher frequency and the phosphate stretching symmetric bandns(PO₂−) in the average spectra shifted to lower frequencies.

Comet assay of leukocytes

There are two main factors concerning the detection of the DNA strand breaks in the comet assay: the size of the DNA fragments that migrate from the nucleus to the anode and the number of broken ends that can migrate a short distance from the comet head (ElAssouli et al. 2007). Fig. 5a–d shows micrographs of the rat leukocytes of the control group (a) and the irradiated groups (b, c, and d) at time Fig. 2. Curve fitting of average FTIR spectra of rats’PBMC DNA in region (1,180–950 cm⁻¹); (a) Control group; (b) Irradiated group.

Table 2. A summary of the results of curve fitting of FTIR spectra expressed as a function of areas of nucleic acid region and their band assignments for PBMC of control and irradiated groups.

Wavenumber

(cm⁻¹) Band assignment

Area^a Control Irradiated 1,024–1,026 n(C–O) DNA deoxy ribose^b 1.12 ± 0.07 1.02 ± 0.04^c 1,045–1,046 –C–O–stretching of

carbohydrates^d

2.96 ± 1.21 2.02 ± 0.26 1,085–1,086 ns(PO2−) phosphodiesters of

nucleic acids^e

6.60 ± 1.31 9.90 ± 0.44^f 1,125–1,126 n(C–O) RNA ribose^g 0.17 ± 0.08 0.45 ± 0.05^h 1,153–1,157 n(C–O) carbohydratesⁱ 0.29 ± 0.07 0.26 ± 0.00 1,169–1,170 C–O stretching of serine,

threonine and tyrosine (protein kinase)^j

0.16 ± 0.03 0.14 ± 0.03

aValues are expressed as the mean ± SD.

b(Andrus and Strickland 1998; Ramesh et al. 2001; Bogomolny et al. 2008).

cSignificant change to the control value (p < 0.05).

d(Ramesh et al. 2001; Di Giambattista et al. 2010).

e(Ramesh et al. 2001; Gault and Lefaix 2003; Cakmak et al. 2006; Bogomolny et al. 2008; Di Giambattista et al. 2010; Lu et al. 2011; Mahmoud et al. 2011;

Zelig et al. 2011).

fSignificant change to the control value (p < 0.05).

g(Andrus and Strickland 1998; Ramesh et al. 2001; Meade et al. 2010).

hSignificant change to the control value (p less than 0.05).

i(Zelig et al. 2011).

j(Cakmak et al. 2006; Lu et al. 2011; Zelig et al. 2011; Sheng et al. 2013).

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intervals of 0.5 h, 1.5 h, and 3 h post-irradiation, respectively, after the alkaline electrophoresis. As can be seen from Fig. 5a, most cells in the control group had no detectable comet tail, whereas in the irradiated group, comets with different tail lengths were observed in the micrographs of Fig. 5b–d. A large tail far from the head was observed in the microphotograph 5b at 0.5 h post-irradiation.

Meanwhile, the micrograph in Fig. 5d at 3 h after irradiation had a shorter tail than that in microphotograph in Fig. 5c at 1.5 h post-irradiation. The histograms in Figs. 6 and 7 display the distributions of the tail moments and the percentage of DNA in the comet tail, respectively, for all samples. The results show that the longer the post- irradiation time, the less the tail moment and the percentage of DNA in the tail. Table 4 gives the mean values and the standard error of the mean (SEM) of tail moments for 400 cells (100 cells rat⁻¹) from tested groups, whereas Table 5 represents the mean values and SEM of the percentage of DNA in the tail. Results in Tables 4 and 5 show significant differences in the tail moment and the percentage of DNA in the tail that were recorded in all cases of post-irradiation compared to the control case. The effect of post-irradiation time on repair affecting the DNA damage distribution (tail moment and the percentage of DNA in the tail) was achieved by comparison between control and post-irradiation groups using the non-parametric non-paired one-way ANOVA;

P < 0.05 was considered significant. The statistical analyses

were performed using ORIGIN 8.0951 software (OriginLab Corporation, Northampton, MA, USA).

DISCUSSION DNA of PBMC

Changes in the FTIR spectra that were recorded in the irradiated group in the range of 1,200–950 cm⁻¹are attributed to the nucleic acid structures, in which the decrease in Table 3. Area ratios characteristics of the biomarkers derived from

curve fitting of FTIR spectra from rat PBMC nucleic acid of control and irradiated groups.

Biomarkers Band

Ratio^a Control Irradiated

RNA/DNA 1,126/1,025 0.15 ± 0.08 0.44 ± 0.05^b

Phosphodiesters/carbohydrates 1,085/1,046 2.23 ± 0.51 4.91 ± 0.68^b

aValues are expressed as the mean ± SD.

bSignificant change to the control value (p less than 0.05).

Fig. 3. Second derivative of average FTIR spectra of rats’PBMC DNA in region 1,200–950 cm⁻¹obtained from control and irradiated groups.

Fig. 4. HCA of second derivative spectra in the 1,200 to 1,000 cm⁻¹ region of PBMCs from three control rats and three irradiated rats.

HCA was according to Wards' method, Euclidean distance linkage.

C and I indicate control and irradiated, respectively.

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the detectable area ofn(C–O) DNA ribose is consistent with results reported by Gault and Lefaix (2003), who suggested conformational changes and/or rearrangement of nucleic acid structures. The decrease in the absorbance intensity of the DNA vibrational mode at 1,025 cm⁻¹was detected after irradiation, probably because the DNA passed through various structural modifications, such as conformational changes and inactivation of different genes involved in DNA repair (Bogomolny et al. 2008). The symmetric stretching of the phosphate band of the phosphodiesters in the nucleic acid backbone reflects the degree of intermolecular interactions in the nucleic acids (Fung et al. 1996; Gault and Lefaix 2003). When this band shifted toward lower

wave numbers, the intensity of intermolecular nucleic acid interactions increased, suggesting that the intermolecular packing of the nucleic acids had tightened (Fung et al.

1996; Gault and Lefaix 2003). This band centered at 1,086.716 cm⁻¹for the control group and then shifted to 1,085.878 cm⁻¹for the irradiated group, suggesting a decrease in the intensity of intermolecular interactions in the nucleic acids and that the intermolecular packing of the nucleic acids had slightly loosened. This agrees with some discussions that considered the shifting of the phosphate symmetric stretching mode bands to lower frequencies as evidence of a double strand break (Dovbeshko et al. 2000;

Meade et al. 2010). These results agree with Lipiec et al.

Fig. 5. Micrographs of rat leukocytes from control and irradiated by fast neutron very low dose subjected to comet assay. The microphotograph in (a) was from control. The micrographs in (b), (c), and (d) were from rats irradiated at 0.5, 1.5, and 3 h post-irradiation, respectively.

Fig. 6. Distributions of comet tail moments of rats’leukocytes from control and irradiated by fast neutron, very low dose, at time interval post- irradiation 0.5, 1.5, and 3 h.

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(2013), who attributed this shift to the increase of molecular degrees of freedom after chromatin fragmentation due to apoptosis. The significant increase in the RNA/DNA ratio evident in the irradiated group compared to the control may suggest an increase in the activity of nucleic acid that took place after the initial DNA damage by neutron irradiation (Bogomolny et al. 2008). The band that centered at 1,125 cm⁻¹was attributed to the C–O stretching vibration RNA (Andrus and Strickland 1998; Ramesh et al. 2001;

Meade et al. 2010). The shifting in this band could be discussed by Lipiec et al. (2013), who suggested that the changes in the RNA molecule structure induced by irradiation could lead to the formation of modified or inactive proteins as a result of incorrect translation processes (Lipiec et al. 2013). The significant increase in the phosphodiesters/carbohydrates ratio of the irradiated group

compared to the control may occur when the irradiation by neutron causes disordering in the synthesis of nucleic acids.

DNA of leukocytes

Micrographs of rat leukocytes in Fig. 4a–d revealed that rats irradiated with very low dose fast neutrons might exhibit DNA fragmentation. The results in Tables 4 and 5 showed clearly that the greater the time post-irradiation, the more similar the distributions of the irradiated group to that of the control group. At the end of 3 h post- irradiation, the distributions of tail moments and the percentages of DNA in the tail of the irradiated group approach those of the control group, suggesting that although DNA repair begins directly after irradiation, more than 3 h post- irradiation are needed for maximum repair. Moreover, the results in Tables 4 and 5 revealed the heterogeneity of the Fig. 7. Distributions of percentage of DNA in the tail of rats’leukocytes from control and irradiated by fast neutron, very low dose, at time interval post-irradiation 0.5, 1.5, and 3 h.

Table 4. Effects of fast neutron very low dose radiation on the DNA in rat leukocytes using the alkaline comet assay. Mean ± SEM of tail moment of control and irradiated groups at various time postirradiation.

Control (tail moment^a) 400 cells (100 cells/rat)

Irradiated (tail moment^a) Time postirradiation 30 min 400 cells

(100 cells/rat)

1.5 h 400 cells (100 cells/rat)

3 h 400 cells (100 cells/rat) 0.17 ± 0.11 5.63 ± 1.02^b 4.65 ± 0.85^b 2.92 ± 0.35^b

aValues are expressed as the mean ± SEM.

Table 5. Effects of fast neutron very low dose radiation on the DNA in rat leukocytes using the alkaline comet assay. Mean ± SEM of percentage of DNA in the tail of control and irradiated groups at various time postirradiation.

Control (percentage of DNA in the tail^a) 400 cells (100 cells/rat)

Irradiated (percentage of DNA in the tail^a) Time postirradiation (hour) 30 min 400 cells

(100 cells/rat)

1.5 h 400 cells (100 cells/rat)

3 h 400 cells (100 cells/rat) 1.30 ± 0.53 14.9 ± 1.56^b 14.44 ± 1.59^b 10.80 ± 0.95^b

aValues are expressed as the mean ± SEM.

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level of DNA breakage. This heterogeneity is attributable to the DNA damage induced at the level of a single rat’s leukocytes by neutron irradiation. Because of the very low dose, the heterogeneity of the distribution with the variation of DNA damage can be explained by suggesting that some nuclei might have been hit by different neutrons and then re- ceived different doses, whereas some nuclei did not interact with any neutron. This has been observed in several studies carried out using different irradiation particles at low doses (Pöller et al. 1996; Testard and Sabatier 2000). The maximum mean of DNA percentages in the tail was 14.897 at 30 min post-irradiation, which gives an idea about the im- pact distribution in a population if the values were in the range 0 to 85. This value also plays an important role in the damage assessment, which represents a low-level damage according to the method of Anderson et al. (1994).

CONCLUSION

Based on results that were described previously, this study concluded that the fast neutron at very low doses might cause changes in the DNA of PBMC at a submolecular level. It could cause a single, double strand break and chromatin fragmentation of DNA of PBMC. FTIR results revealed that the most changes induced by very low dose in the nucleic acids were in the C–O stretching vibration at 1,125 cm⁻¹of RNA. Comet assay results revealed low-level damage in the DNA of leukocytes induced by fast neutrons at very low doses. Thus, this study deduced that a dose 0.09 Gy of fast neutrons could damage and break the DNA molecule. In addition, comet assay results showed that the DNA repair in leukocytes occurs in the first 3 h, confirming that repairs in the chromatin of DNA take place after irradiation.

Acknowledgments—This work was financially supported by King Abdulaziz City for Science and Technology (KACST) under grant number A-S‐12‐ 1011. The authors would like to express their deepest appreciations to the head of the nuclear engineering division at King Abdulaziz University for providing the neutron monitoring throughout this work. In addition, great thanks are extended for the Radiation Safety Committee for carrying out safety proce- dures. Moreover, the authors are grateful for Lisa Karam, Chief of the Radiation Measurements Division, National Institute of Standards and Technology (NIST), Gaithersburg, MD, for reviewing the language through the manuscript and for suggestions.

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EFFECT OF VERY LOW DOSE FAST NEUTRONS ON THE DNA OF RATS ’ PERIPHERAL BLOOD MONONUCLEAR CELLS AND LEUKOCYTES

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EFFECT OF VERY LOW DOSE FAST NEUTRONS ON THE DNA OF RATS ’ PERIPHERAL BLOOD MONONUCLEAR CELLS AND LEUKOCYTES

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