Assessment of DNA Damage in Lymphocytes of RadiationWorkers at Al-Tuwaitha Site using Nuclear Division Index and Hypoxanthine guanine phosphoribosyl transferase (HPRT)

 

Zainab A. Ouda¹, Abdlamer N. Kaloub¹, Abdulsahib K. Ali²

¹Al- Mustansiriyah University, Science College, Biology Dep.

²Ministry of Science and Technology, Central Laboratories Directorate

 

ABSTRACT:

Radiation of its different types is one of the major well known mutagens and carcinogens. Ionizingradiation (IR) imposes risks to human health and the environment. Man is exposed to different types of radiation of  which those used in diagnosis of diseases,  treatment of cancer and occupationally exposed to ionizing radiation . Radiation exposure induces damage on normal cells.IR at low dosesand low dose rates has the potency to initiate carcinogenesis. Genotoxic environmental agents such as IR trigger a cascade of signal transduction pathways for cellular protection. Iraqi radiation workers in radioactive waste treatment and management at Al-Tuwaithasite.This study included 30 Iraqi radiation workers in radioactive waste treatment and management at Al-Tuwaitha site , 20 were non-smokers and 10 smokers, aged (25 - 55 year), as well as 20 male blood samples, aged (26 - 54 year)  which are 13 were non-smokers and 7 smokers as control.  Three cytogenetic parameters were studied to determine genotoxic effects of occupational exposure of low ionizing radiation doses in peripheral blood lymphocytes of from radiation workers at radioactive waste treatment and management at Al-Tuwaitha site in Baghdad .Investigations were carried out by using  Hypoxanthine guanine phosphorribosyl transferase (HPRT) mutation assay  were  performed on peripheral blood  lymphocytes for workers and control groups.

 

 

 

1. INTRODUCTION:

In physics, radiation is defined as electromagnetic waves or as moving subatomic particles, especially high energy particles which cause ionization. Radiation is energy in the form of waves of particles. Radiation whether natural orusedfor treatmentisharmless, the damage starts at the cellular level when radiation is absorbed in a cell so that it targets cell components the most important of which is the DNA causing cell death, mutation, and carcinogenesis⁽¹⁾.

 

Radiation may impact the DNA directly, causing its ionization⁽²⁾, or cause an indirect actionin this case, the radiation interacts with non-critical target atoms or molecules, usually water which results in the production of free radicals.

 

These free radicals can then attack critical targets such as the DNA⁽³⁾. Radiationis considered carcinogenic. Exposure to ionizing radiation (IR) produces several forms of cellular DNA damage, including single-strand breaks (SSBs), alkali-labile sites, double-strand breaks (DSBs), DNA–DNA and DNA–protein cross-links and damageto purine and pyrimidine bases⁽⁴⁾.

 

Several studies of HPRT (hypoxanthine--guanine phosphor ribosyl transferase) gene mutations in human cultured cells and lymphocytes in vivo have provided evidence for age, exposure and genetics to influence mutation frequency. An increased mutation frequency with increasing age in normal healthy people is generally observed⁽⁵⁾. The approach for somatic mutation analysis in human has been widely used to determine in vivo background as well as acquired somatic cell mutation frequencies in pediatric and adult populations exposed to know and unknown environmental mutagens⁽⁶⁾. Moreover, certain occupational exposures and life style factors such as smoking have been associated with an increased mutation frequency, while the intake of specific dietary items seems to have a protective effect against mutations and cancer⁽⁷⁾ .

 

A gene can have more than one promoter, resulting in RNAs that differ in how far they extend in the 5'end⁽⁸⁾. The process of producing a biologically functional molecule of either RNA or protein is called gene expression⁽⁹⁾, several steps in the gene expression process may be modulated, including the transcription step and translation step and the post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis, the versatility and adaptability of any organism⁽¹⁰⁾ .

 

2. METHODS:

Population Studies:

The present study included collected 20 male blood samples, (aged 32 - 59 year and duration of employment more than 5 years) from radiation workers in radioactive waste treatment and management at Al-Tuwaitha site. Three genetic parameter were studied by using themicronucleus assay, nuclear division index, and mitotic index , as well as 20 male blood samples as control group which including ( aged 29 -55 year).They were asked to fill in extensive questionnaire which included the following criteria (age, sex, smoking drink alcohol, X-ray, abortion, duration of exposure in these area, kind of their work).

 

Blood Sampling:

Five ml of human peripheral blood from all select subjects were collected and placed into sterile plain tube that contained lithium heparin.For hematological analysis, blood samples were collected into EDTA bulbs. Automatic electronic blood cells counter (Erma, Japan) model PCE 170 was employed to analyses the sample. However while computing the table, only four major parameters (Total WBC, Total RBC, Total platelet counts and hemoglobin percentage) has been considered.

 

Culture technique:

The MN was performed according to the description by ⁽¹¹⁾. In short, whole blood cultures were prepared by adding 0.5 ml blood to 4.5 ml culture RPMI-1640 culture medium (sigma) supplemented with 20% fetal bovine serum (sigma), 100 UI/ml penicillin (Sigma-Aldrich) and 0.1 mg/ml streptomycin (Sigma-Aldrich). Phytohemagglutinin (PHA) at a concentration of10 μl / ml was used to stimulate lymphocyte proliferation. Blood cultures were incubated at 37°C for 72 hours. Cytochalasin B (Sigma) was added 44 h after PHA stimulation at a concentration of 4.5 µg / ml to block cells at cytokinesis. The cultures were harvested after incubation for 72 h.

Treatment with hypotonic solution and fixation:

After a 72 h incubation period, cells were collected by centrifugation (1000 rpm, for 5 min), hypotonically treaded with cold 0.01M KCl for 3 min to lyse red blood cells. After removal of the supernatant, the pellet was fixed with a fixative solution containing methanol:acetic acid (3:1). The cells were washed with four further exchanges of fixative solution, After fixation, the cells were gently resuspended, dropped onto wet clean glass slides and allowed to dry. Slides were stained in 4% Giemsa solution for 8 min After fixation, the cells were gently resuspended, dropped onto wet clean glass slides and allowed to dry. Slides were stained in 4% Giemsa solution for 20 min.

 

Microscopic examination:

Stained samples were evaluated using microscope; a total of 500 binucleated cells was evaluated for the frequency of MN using 400 x magnification for surveying the slides while 1000 × magnification was used to confirm the presence or absence of MN in the cells. A total of 1000 living interphasic cells were used for assessment of mono-, bi-, and poly-nucleated cells and calculation of NDI by using the formula ⁽¹²⁾:

 

NDI = (M1 + 2 x M2 + 3 x M3 + 4 x M4) /N

Where M1 to M4 represent the number of cells with one to four nuclei and N is the total number of viable cells scored.

 

Scoring of Mf-HPRT:

The binucleated and multinucleated cells per 1000 lymphocytes in two sets of cultures were scored under light microscopy (magnification 1000X).Mutant frequency of HPRT gene (Mf-HPRT) was calculated with the following formula ( Caoet al. 2002):

 

                  Binucleated and multinucleated cells in culture

with  6per1000

Mf-HPRT =-----------------------------------------------------------×1000‰.

(‰)                      Binucleated and multinucleated cells in culture

without 6per1000

 

Data Analysis and Statistics:

The data of this study were compiled into the computerized data file and frequency, distribution and statistical description (Mean, SE) were divided using SPSS statistical software. We used statistical analysis of variance (ANOVA) test and least significantly difference (LDS) test by probability of less than 0.05 (p < 0.05) according to ⁽¹⁴⁾.

 

4. RESULTS AND DISCUSSION:

Evaluation of nuclear division index (NDI) and Mf-HPRT gene mutation:

The frequencies of NDI and Mf-HPRT gene mutationassay were performed on peripheral blood lymphocytes which were obtained from 30 male blood samples, aged ( 26 – 55) year from radiation workers in radioactive waste treatment and management at Al-Tuwaitha site, as well as 20 male blood samples as control group which age ranged (25 -55 year).The NDIassay was performed according to the description by ⁽¹⁵⁾ , the NDI was calculated binucleated, trinucleated and quadrinucleated lymphocyte cell per 1000 lymphocytes . (Fig.1 ).

 

A-                                                        -B-                                 -C-

Figure (1): Cytokinesis blocked human lymphocyte cell ,(A): Binucleated lymphocyte cell, (B): Trinucleated lymphocyte cell and (C): Qudrinucleated lymphocyte cell (1000X).

 

 

 

The average of NDI ( Mean ± SE ) for exposed groups were 1.244 ± 0.00901 when compared with the control 1.408 ±0.0196 . A significant decrease (P > 0.01) in NDI was observed in the peripheral lymphocytes among radiation workers inradioactive waste treatment and management at Al-Tuwaitha site. The ranges of NDI for radiation workers and controls were 1.135–1.327 and 1.265 –1.557 ‰, respectively . The nuclear division index as biomarker of cell proliferation in cultures which is considered a measure of general cytotoxicity and the relative frequencies of the cells may be used to define cell cycles progression of the lymphocyte after mitogenic stimulation and how this has been affected by the exposure ⁽¹⁶⁾. Therefore, this type of study may become an indicator for the need for greater control and protection against the harmful effects of radiation over occupationally exposed professionals ⁽¹⁷⁾ . The results of the present study revealed that in this group of professionals, whose work environments were not regularly inspected.

Table (1): Frequency nuclear division index (NDI) and Hprt gene mutation in peripheral lymphocytes among radiation workers in radioactive waste treatment and management at Al-Tuwaitha site and control group.

Study groups	No. of sam-ples	Ages (Mean ± SE)	NDI					Mf-hprt (‰) (Mean ± SE)
			M1 (Mean ± SE)	M2 (Mean ± SE)	M3 (Mean ± SE)	M4 (Mean ± SE)	NDI (Mean ± SE)
Radiation Workers	30	43.27 ± 1.493	812.23  ± 8.583	157.23 ± 8.57	19.37 ± 1.463	10.67 ± 0.570	1.224a ± 0.00901	0.978 a±0.0337
Control	20	39.7 ±1.74	653.10 ± 13.38	300.75 ± 14.06	30.4 ± 2.484	15.75 ± 1.15	1.408 b ± 0.0196	0.909b±0.0085
LSD Value							0.061	0.0351
P Value							0.01	0.01

·        Least Significant Difference_{(NDi 0.05, 0.01) = 0.0455, 0.0610}

·        Least Significant Difference_{(Hprt 0.05, 0.01) = 0.0263, 0.0351}

·        Similar latter in a column mean there is no significant difference (p < 0.01), according to Duncan test.

 

Similarly dentists exposed to several years of ionizing radiation presented NDI levels similar to exposed and control individuals, which indicates the adoption of safety measures during occupational exposure^(15)(18). The results of HPRT gene mutation assay was performed on peripheral blood lymphocytes obtained in 30 radiation workers in radioactive waste treatment and management at Al-Tuwaitha site and 20 controls. The HPRT gene mutation assay was performed according to the description by⁽¹³⁾ . Mutant frequency of HPRT gene was calculated binucleated, trinucleated and quadrinucleated CB lymphocyte cell per 1000 lymphocytes in tissue culture with and without 6-thioguanine were identified byGiemsa staining (Fig.1). The result of Mfs-hprt gene mutation for workers and controls are shown in Table 4-6, the ranges of Mf-hprt for workers and controls were 0.89–1.08 and 0.85–0.98 %, respectively. the average Mf-HPRT (Mean±SE) for workers in workers in radioactive waste treatment and management at Al-Tuwaitha site were 0.978±0.0337 %, when compared with the control 0.909±0.0085%. The difference of Mfs-hprt between workers and controls was significant (P < 0.01). So the frequency of hprt assay have been used to detect the genetic hazard of workers occupationally exposed IR in the present investigation. The results of hprt assay assay in our experiment indicated the significant difference between the exposed group and control group. In the present study, radiation workers chronically exposed to ionizing radiation were studied cytogenetically to evaluate the frequencies of HPRT gene mutation, in comparison with control individuals. It is well known that the exposure of mammalian cells to ionizing radiation produces a variety of DNA lesions, including base alterations, DNA protein cross links, and single and double strand breaks⁽¹⁹⁾ . However, it has been reported that human populations exposed to ionizing radiation also present increased frequencies of HPRT mutant frequencies of lymphocytes^(20)(21).

 

Occupation contributed to the levels of primary DNA damage recorded by means of the NDI, hprt gene mutation and MN assay, although observed differences were not statistically significant (P < 0.01) between physicians , engineers ,Technicians groups. While the results of NDI , hprt gene mutation and MN assay showed statistically significant differences (p < 0.05) between radiation worker (Physician, Technicians and Engineers) and control groups (Table1).

Table ( 2): Mean values of NDI , Mf-hprt and MN, in peripheral lymphocytes for radiation worker employed at in radioactive waste treatment and management regarding to their occupation

♦Similar latter in a column mean there is no significant difference (p < 0.01), according to Duncan test.

 

Among radiation worker employed in radioactive waste treatment and management, the highest group mean value of MN, NDI and hprt gene mutation were recorded in peripheral blood leukocytes of physicians, following by technicians, and engineers. The lowest group mean value of NDI, hprt gene mutation and MN assay were recorded in peripheral blood leukocytes of engineers employed in radiation worker employed at in radioactive waste treatment and management (Table 1).

 

After having been validated as an in vivo biomonitor in several subject studies, the NDI, HPRT gene and CBMN, assay have been applied for large scale biomonitoring of occupationally exposed radiation workers, e.g. nuclear power plant and hospital staff, radioactive waste treatment^(13)(17). These biomonitoring studies showed the dependence of MN on the accumulated dose received over the years preceding the venipuncture. Large scale biomonitoring studies show that the micronucleus assay is able to demonstrate genetic damage at the population level for accumulated doses received occupationally exceeding 50 mGy^(18)(22) .

 

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Accepted on 17.12.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(7): 2855-2858.