**2. Materials and methods**

440 Selected Topics in DNA Repair

damage is estimated based on the ratio of DNA percentage in head and in tail of a comet. Some researchers prefer tail moment as the most reliable marker of DNA damage, because it combines measurements of tail length and percentage of DNA in tail (Ashby et al., 1995; Hellman et al., 1995; 1997; Mc Kelvey-Martin et al., 1998). Collins (2004) emphasizes the advantage of TI considering that the percentage of the tail DNA reflects the real DNA damage. Comet assay can also detect apoptotic and necrotic cells. Apoptotic cells show small comet head, and most of DNA is spread in tail in the shape of a cloud (Fairbairn et al., 1995; Olive, 1999). Comet assay is also a valuable technique to study the kinetics of primary DNA damage. It enables to estimate the DNA damage level immediately after the exposure, even when the exposure included very small dose in very short exposure period (Tice et al., 1990; Plappert et al., 1995). Fast repair can represent a problem in DNA damage evaluation in populations occupationally exposed to low doses of ionising radiation and therefore the development of sensitive methods is necessary for those experiments. Most of the primary DNA damage is repaired 30 minutes after the exposure to ionising radiation (Frankenberg-Schwager, 1989), and 2 hours after the exposure to dose of 2 Gy, most of the damaged DNA

Polymorphism by definition is expression of different phenotypes in the same species due to the change/s in genotype. They usually include loss (deletion) of small or bigger part of DNA molecule, insertion of specific number of nucleotides or repetition of di-, three-, or oligonucleotides in variant number. The number or repeating differs among individuals. Variations in human genome are usually caused by variations in DNA sequence, that is based on the change of only one nucleotide (one from the four nucleotides; A-adenine, Tthymine, C-cytosine or G-guanosine is replaced by the other) usually known as SNP polymorphism (*single nucleotide polymorphism*). Among almost 15 million of SNPs in human

The connection of the change in only one nucleotide (that happens once in every 1000nucleotides in human genome) with the complex aetiology of malignant diseases is poorly investigated (Bonassi et al., 2005). More than 7 millions of well known SNPs in human genome appear with the allelic frequency higher than 5% of the entire population (Hinds et al., 2005). More than 70% of SNPs in human population have the frequency less than 5 % and those SNPs are called rare SNPs (Shastry, 2009). The results of new experiments have shown the connection between gene polymorphisms and risk association with disease developing (Norpa, 2004), especially in polymorphisms of DNA repair genes and folic acid metabolism. Polymorphisms can lead to different gene expression (decreased or increased) and through this process can influence on cell repair mechanisms (Hung et al., 2005; Parl, 2005; Weiss et al., 2005; Kotsopoulos et al., 2007). Variations in DNA repair

Among 130 genes involved in DNA repair mechanisms, 80 of them are carriers of more than 400 SNPs (Mohrenweiser et al., 2003). DNA damage and repair correlate with the radiation sensitivity and are important in radiation protection and radiotherapy (Ross et al., 2000). Due to individual variations, some persons have higher sensitivity when compared with general population (Berwick, 2000). It has been estimated that 10 -15 percent of healthy people have phenotype that shows decreased possibility for successful DNA damage repair (Mohrenweiser & Jones, 1998; Hu et al., 2002a). Higher risk of mutations, genome instability and malignant tumours have been observed among persons

genome, 50.000 to 100. 000 of them can change the function or gene expression.

capacity have been also observed among healthy individuals (Setlow, 1983).

is totally repaired (Plappert et al., 1997).

This study included 126 subjects, 70 medical workers occupationally exposed to low doses of ionising radiation (gastroenterologists, cardiologists, anaesthesiologists, surgeons, radiologists, radiology technicians, nurses) of both gender (45 females, 25 men; mean age was 40 years, from 20-60 years old) and 56 individuals in control group who were not exposed to neither ionising radiation nor to chemical mutagens (14 women and men; mean age 40 years, from 23 to 60 years old) (Table 1).


Table 1. Characteristics of the control and exposed group considering the gender, age, years of exposure, smoking status and alcohol consumption.

The examinees were informed of the study scope and experimental details, have filled a standardised questionnaire designed to obtain relevant information on the current health status, medical history, and lifestyle, and gave their written consent, submitted and approved by the local Ethics Committee. The questionnaire included data on the exposure to possible confounding factors: smoking, alcohol consumption, use of medicines, contraceptives, severe infections, or viral diseases over the past six months, vitamin intake, recent vaccinations, presence of known inherited genetic disorders and chronic diseases, family history of cancer, exposure to diagnostic X-rays. Subjects with history of previous radio- or chemotherapy were not included. Exposed group was under regular film dosimetry and the dose received did not exceed 20mSv/year (data not shown).

The Influence of Individual Genome Sensitivity in DNA Damage Repair

cut with restriction enzymes.

**2.3 Polymerase chain reaction-RFLP** 

(5 min) and (VI) incubation at 10 °C.

DNA polymerase (Platinum

and 1 μl of DNA sample).

Compounds for the reaction mixture are given in Table 2.

Assessment in Chronic Professional Exposure to Low Doses of Ionizing Radiation 443

diluted in 100 μL of TE buffer (10mM Tris–HCl, pH 7.4; 1 mM EDTA, pH 8.0). Purity and concentration of DNA was specified by spectrophotometric method (NanoDrop ND- 1000 spectrophotometer, NanoDrop Technologies, Thermo Scientific, Wilmington, USA). Samples were diluted till concentration of 10 ng μL-1 and kept at -20 °C till amplification. Specific polymorphisms were determined: in BER- (base excision repair) APE1- (apurinic/apirimidinic endonuclease, Asp148Glu), hOGG1 (human 8-oxoguanine DNA glycosylase, Ser326Cys), XRCC1 (X-ray repair cross-complementing protein-group 1, Arg194Trp); in NER- (nucleotide excision repair) XPD (Xeroderma pigmentosum-group D, Lys751Gln; DSBR- (double-strand-break repair) XRCC3 (X-ray repair cross-complementing protein-group 3, Thr241Met), PARP1 (poly (ADP-ribose) polymerase 1, Val762Ala); in DRR- (direct reversal repair) MGMT (O6-methylguanine-DNA methyltransferase, Leu84Phe) Genotyping was performed by either Real Time PCR (polymerase chain reaction) with Taqman assay, or after electrophoresis and fluorescence visualisation, DNA samples were

In a total volume of 10 ml, 10 ng of genomic DNA was amplified for each sample.

PCR reaction was completed in six steps: (I) incubation at 94 °C (2 min) for Taq DNA polymerase activation; (II) incubation at 94 °C (30 s) for denaturation of double stranded DNA; (III) incubation at specific temperature that depended on the specific gene (30 s), for hybridisation of primers (Table 3); (IV) incubation at 72 °C (30 s) for DNA synthesis. Steps No.2 to No. 4 were repeated for 34 times. After that there were steps: (V) incubation at 72 °C

Reaction mixture Stock solution Final concentration for PCR reaction

reH2O 1x 0.64x (for XRCC3-0.59x)

(DMSO) (1x) (for XRCC3-0.05x)

10X BUFFER 10x 1.00 x

Taq, Invitrogen) 5 U μl-1 0,03 U μl-1

Table 2. Compounds for PCR-RFLP reaction (10 μl of reaction mixture (9 μl of Master Mix

MgCl2 50 mM 2.00 mM dNTP 1.25 mM 0.11 mM Primer F 20 μM 0.30 μM Primer R 20 μM 0.30 μM

Two millilitres of venous blood was stored in heparinised vacutainers for comet assay and assessment of DNA repair kinetics and stored at +4°C before further procedure. Detailed protocol is described before (Milić et al., 2010). Five millilitres of venous blood was stored in vacutainers with EDTA (ethylenediaminetetraacetic acid) at -20°C until further DNA isolation (Milić et al., 2010). Blood samples were irradiated with 60Co (Alcyon, CGR-MeV, France). The doses used were 2 and 4 Gy, with the same distance from the source (80 cm). Irradiation field was 15 x 15 cm2. After irradiation, samples were kept at + 4°C to prevent the repair of the damage. Details are also described before (Milić et al., 2010).
