**3. Results**

444 Selected Topics in DNA Repair

Table 3. Gene, forward and reverse primers, method used for determination of

polymorphism, hybridisation temperature, restriction enzymes and the DNA fragments.

DNA repair kinetics after the exposure to gamma radiation of 2 and 4 Gy was measured on a group of 126 subjects, 70 medical workers and 56 controls (Figure 1).

The groups differed in average age, gender, smoking status and alcohol consumption. The mean values for both groups did not significantly differ, although inter-individual differences were notable. In control group higher level of DNA damage compared to exposed group was observed, but without statistical difference. The repair dynamic was the same in both groups.

After genotyping, heterozygotes and polymorphic homozygotes were grouped together to evaluate polymorphic allele appearance. Number of individuals carrying particular gene is given in Table 5. Frequency of genotyping did not differ from expected Hardy-Weinberg equilibrium.

The Influence of Individual Genome Sensitivity in DNA Damage Repair

wild type, HE-heterozygote, SNP-polymorphic homozygote).

frequency, smoking and gender.

higher TM than their homozygotes.

TM than homozygotes (Table 7).

between smokers and non smokers was insignificant.

minutes after exposure to 2 Gy and after 24 hours for the dose of 4 Gy.

(WT/HE/SNP)

Gene Group

Assessment in Chronic Professional Exposure to Low Doses of Ionizing Radiation 447

XPD 23 42/58/26 15/33/8 27/25/18 XPD 10 43/63/20 17/33/6 26/30/14 XRCC1 47/67/12 22/25/9 25/42/3 PARP1 83/37/6 33/18/5 50/19/1 HOGG1 78/43/5 42/14/0 36/29/5 APE1 43/57/26 19/27/10 24/30/16 MGMT C/T 87/34/5 39/16/1 48/18/4 XRCC3 45/61/20 23/26/7 22/35/13

Table 5. Number of individuals in three genotyping groups for all genes (WT-homozygote

Table 6. Results of χ2-test in the exposed and control group compared to genotype

The differences between exposed and control group were analysed by χ2-test. Significant difference was found for hOGG1 gene and for gender (Table 6). Observed difference

Multivariate regression analysis was used to estimate the influence of smoking, gender, age, years of exposure and genotypes on comet assay parameters immediately after and 120

Immediately after irradiation with the 2 Gy dose, exposed group had significantly lower amount of DNA damage than control group. Individuals with polymorphic variants of XRCC3 gene had higher TL than their homozygotes. Polymorphic variants of hOGG1 gene had higher TI than homozygotes. Polymorphic variants of hOGG1 gene had significantly

Tail length values significantly differed between the two groups both immediately after and also 120 minutes after irradiation with 2 Gy. Polymorphic variants of APE1 and XPD10 genes had higher TI and TM compared to homozygotes 120 min after irradiation with 2 Gy, and polymorphic variants of MGMT C/T and XRCC3 genes had significantly lower TI and

Variable st χ 2 p XRCC1 1 0.1697 0.6804 **hOGG1** 1 7.3298 **0.0068**  APE2 1 0.0018 0.9665 XPD21 1 0.6372 0.4247 PARP2 1 2.1624 0.1414 MGMT C/T 1 0.0167 0.8971 XRCC3 1 1.6433 0.1999 smoking 1 1.7174 0.19 **sex** 1 13.2499 **0.0003** 

Control (WT/HE/SNP)

Exposed (WT/HE/SNP)

Fig. 1. Graphical results of DNA repair. Red colour - control group; blue - exposed group. a-TL after 2 Gy irradiation, b-TI after 2 Gy, c-TM after 2 Gy, d- TL after 4 Gy irradiation, e- TI after 4 Gy, f- TM after 4 Gy.

a b

c d

e f

Fig. 1. Graphical results of DNA repair. Red colour - control group; blue - exposed group. a-TL after 2 Gy irradiation, b-TI after 2 Gy, c-TM after 2 Gy, d- TL after 4 Gy irradiation, e- TI

after 4 Gy, f- TM after 4 Gy.


Table 5. Number of individuals in three genotyping groups for all genes (WT-homozygote wild type, HE-heterozygote, SNP-polymorphic homozygote).


Table 6. Results of χ2-test in the exposed and control group compared to genotype frequency, smoking and gender.

The differences between exposed and control group were analysed by χ2-test. Significant difference was found for hOGG1 gene and for gender (Table 6). Observed difference between smokers and non smokers was insignificant.

Multivariate regression analysis was used to estimate the influence of smoking, gender, age, years of exposure and genotypes on comet assay parameters immediately after and 120 minutes after exposure to 2 Gy and after 24 hours for the dose of 4 Gy.

Immediately after irradiation with the 2 Gy dose, exposed group had significantly lower amount of DNA damage than control group. Individuals with polymorphic variants of XRCC3 gene had higher TL than their homozygotes. Polymorphic variants of hOGG1 gene had higher TI than homozygotes. Polymorphic variants of hOGG1 gene had significantly higher TM than their homozygotes.

Tail length values significantly differed between the two groups both immediately after and also 120 minutes after irradiation with 2 Gy. Polymorphic variants of APE1 and XPD10 genes had higher TI and TM compared to homozygotes 120 min after irradiation with 2 Gy, and polymorphic variants of MGMT C/T and XRCC3 genes had significantly lower TI and TM than homozygotes (Table 7).

The Influence of Individual Genome Sensitivity in DNA Damage Repair

**TL-4 Gy-0'** 

**TI-4 Gy-0'** 

**TM-4 Gy-0'** 

**TL-4 Gy-24h** 

**TI-4 Gy-24h** 

**TM-4 Gy-24h** 

**3.1 Discussion** 

results are shown in the Table).

indirectly in the repair process.

Assessment in Chronic Professional Exposure to Low Doses of Ionizing Radiation 449

Variable PE SE Parc R2 F p

Smoking 4.10520 2.19325 3.50 0.0642 0.0267 SHMT1 -3.74806 2.11391 3.14 0.0793 0.0297

Gender 1.37561 0.94228 2.13 0.1475 0.0199 SHMT1 -1.65022 0.95455 2.99 0.0870 0.0294 PARP1 -1.63931 1.02226 2.57 0.1120 0.0257

SHMT1 -0.14041 0.07716 3.31 0.0718 0.0304 PARP1 -0.13713 0.08267 2.75 0.1003 0.0260

Gender 0.00036220 0.00017016 4.53 0.0361 0.0468 MTR 0.00047723 0.00017978 7.05 0.0094 0.0499 APE1 0.00036597 0.00018268 4.01 0.0483 0.0329 MGMT C/T -0.00034719 0.00017942 3.74 0.0562 0.0359

Gender 0.11087 0.04594 5.82 0.0180 0.0390 Age 0.00387 0.00203 3.63 0.0601 0.0376 TS -0.07933 0.05406 2.15 0.1461 0.0741 MTHFR4 0.09859 0.04499 4.80 0.0313 0.0372 PARP1 -0.10466 0.04861 4.64 0.0342 0.0267

Gender 2.03821 0.60467 11.36 0.0011 0.0869 Age 0.05384 0.02783 3.74 0.0565 0.0349 MTHFR4 1.59011 0.62442 6.48 0.0127 0.0635 MTR 0.97502 0.62696 2.42 0.1238 0.0224 PARP1 -1.21130 0.67510 3.22 0.0765 0.0313 Table 8. Results of *stepwise* procedure in multivariate regression analysis for three comet assay parameters as a depended variable in the entire population immediately after radiation with 4 Gy dose and 24 hours after irradiation (only the statistically significant

Development of new methods in genotoxicology, especially on molecular level, has greatly improved the knowledge and understanding of processes that follow after the organism was exposed to ionising radiation. In the same time, better radiation protection, that includes more sophisticated handling of radiation sources, better education of personnel who are operating on those sources, precise dosimetry and the use of protection equipment have reduced health risk in specific population occupationally exposed to ionising radiation. Recent investigations in the field of radiation protection have focused on individual differences in radiosensitivity. It has been shown that DNA repair capability is regulated with different mechanisms that include great number of genes involved directly or


Table 7. Results of *stepwise* procedure in multivariate regression analysis for three comet assay parameters as a depended variable in the entire population immediately after radiation with 2 Gy dose and 120 minutes after irradiation (only the statistically significant results are shown in the Table).

Immediately after 4 Gy irradiation, multivariate regression analysis showed influence of smoking on TL. Polymorphic variants of SHMT1 genes had lower TL than their homozygotes. Gender had significant influence on TI. Polymorphic variants of SHMT1 and PARP1 genes had lower TI and TM when compared to their homozygotes.

Polymorphic variants of APE1 gene showed positive correlation with TL 24 hours after radiation with 4 Gy. Polymorphic variants of MGMT C/T gene had lower TL than homozygotes.

With the increase of age, significant increase in TI was measured 24 hours after exposure to 4 Gy. Polymorphic variants of PARP1 gene had lower values of TI than homozygotes. Age has shown positive correlation with TM. Lower values for TM were observed among polymorphic variants of PARP1 gene when compared to homozygotes (Table 8).

The Influence of Individual Genome Sensitivity in DNA Damage Repair Assessment in Chronic Professional Exposure to Low Doses of Ionizing Radiation 449


Table 8. Results of *stepwise* procedure in multivariate regression analysis for three comet assay parameters as a depended variable in the entire population immediately after radiation with 4 Gy dose and 24 hours after irradiation (only the statistically significant results are shown in the Table).

#### **3.1 Discussion**

448 Selected Topics in DNA Repair

Variable PE SE Parc R2 F p

Group -0.10669 0.04189 0.0468 6.49 0.0124 XRCC3 0.09351 0.04390 0.0312 4.54 0.0357 MTR 0.07742 0.04335 0.0291 3.19 0.0772

hOGG1 0.14426 0.07300 0.0509 3.91 0.0376

TS 0.16398 0.10332 0.1157 2.52 0.0243 hOGG1 0.16083 0.09018 0.0776 3.18 0.0223

Group 0.00069854 0.00019567 0.0914 12.74 0.0006 XPD 10 0.00037667 0.00020220 0.0274 3.47 0.0655 XRCC3 0.00031641 0.00020077 0.0220 2.48 0.1183

Group 0.16440 0.04433 0.0004 13.75 0.1012 APE1 0.08068 0.04704 0.0896 2.94 0.0217 XPD10 0.09252 0.04627 0.0484 4.0 0.0239 MGMT C/T -0.07375 0.04608 0.1128 2.56 0.0217 XRCC3 -0.09915 0.04763 0.0401 4.33 0.0262

Group 0.48944 0.12713 0.0002 14.82 0.1075 APE1 0.23214 0.13490 0.0885 2.96 0.0217 XPD10 0.26994 0.13268 0.0447 4.14 0.0247 MGMT C/T -0.20599 0.13216 0.1224 2.43 0.0204 XRRC3 -0.29847 0.13659 0.0313 4.77 0.0289 Table 7. Results of *stepwise* procedure in multivariate regression analysis for three comet assay parameters as a depended variable in the entire population immediately after radiation with 2 Gy dose and 120 minutes after irradiation (only the statistically significant

Immediately after 4 Gy irradiation, multivariate regression analysis showed influence of smoking on TL. Polymorphic variants of SHMT1 genes had lower TL than their homozygotes. Gender had significant influence on TI. Polymorphic variants of SHMT1 and

Polymorphic variants of APE1 gene showed positive correlation with TL 24 hours after radiation with 4 Gy. Polymorphic variants of MGMT C/T gene had lower TL than

With the increase of age, significant increase in TI was measured 24 hours after exposure to 4 Gy. Polymorphic variants of PARP1 gene had lower values of TI than homozygotes. Age has shown positive correlation with TM. Lower values for TM were observed among

PARP1 genes had lower TI and TM when compared to their homozygotes.

polymorphic variants of PARP1 gene when compared to homozygotes (Table 8).

**Log TL-2 Gy-0'**

**Log TI-2 Gy-0'** 

**Log TM-2 Gy-0'**

**Log TL-2 Gy-120'**

**TI-2 Gy-120'** 

**TM-2 Gy-120'** 

homozygotes.

results are shown in the Table).

Development of new methods in genotoxicology, especially on molecular level, has greatly improved the knowledge and understanding of processes that follow after the organism was exposed to ionising radiation. In the same time, better radiation protection, that includes more sophisticated handling of radiation sources, better education of personnel who are operating on those sources, precise dosimetry and the use of protection equipment have reduced health risk in specific population occupationally exposed to ionising radiation. Recent investigations in the field of radiation protection have focused on individual differences in radiosensitivity. It has been shown that DNA repair capability is regulated with different mechanisms that include great number of genes involved directly or indirectly in the repair process.

The Influence of Individual Genome Sensitivity in DNA Damage Repair

damage that can lead to permanent damage and genome instability.

smoking status, gender, age and years of occupational exposure.

the exposed one.

Assessment in Chronic Professional Exposure to Low Doses of Ionizing Radiation 451

exposed and control group. The results indicate the existence of small amount of DNA damage that is still present even after 24 hours from the exposure, independently of the dose level. The results could be explained by the difference in the repair of single stranded and double stranded DNA breaks. Single stranded DNA breaks are usually formed after the exposure to gamma or X-ray (90 %), while only small part of damage is created by double stranded breaks (10 %) (Cornetta et al., 2006). The repair of double stranded breaks after the exposure to ionising radiation usually lasts 12 or 16 hours (Vodicka et al., 2004), that is considerably longer than the repair of single stranded breaks. The amount of unrepaired damage in this experiment did not statistically differ from the control values before irradiation for both control and the exposed group. The difference between exposed and nonirradiated samples was higher for 2 Gy dose. The existence of small amount of unrepaired damage shows the importance of assessment of exposure to ionising radiation, especially to low doses and suggesting possible accumulation of primary, unrepaired

When the unrepaired amount of damage was compared, the results are implicating the existence of adaptive response in professionally exposed individuals to low doses of ionising radiation. Adaptive response in human lymphocytes has been described by other authors (Olivieri & Bosi, 1990; Sankaranarayanan et al., 1989; Shadley &Wolff, 1987; Wiencke et al., 1986; Wolff et al., 1990; Wolff, 1992, 1996). Aka et al. (2004) reported the existence of residual damage in tail intensity 60 and 120 minutes after the exposure to dose of 2 Gy. In their experiment, tail intensity values were also higher in the control group than

According to our results, despite the difference in the amount of damage, kinetics of repair after the exposure to both doses was similar to the one for the nonirradiated group. Unexpectedly, the difference between control and exposed group was significantly higher after 2 Gy than after 4 Gy exposure. These results were not in agreement with Wojcik et al. (1996) who claimed that kinetics of repair was faster in the exposed group. Aka et al. (2004) used comet assay for DNA repair assessment in human lymphocytes of male nuclear power plant workers and also for the control group (N=31). After the exposure, there was not significantly different repair kinetics between exposed and control group. Our results were comparable, indicating that healthy persons use the same repair mechanism, no matter if they are professionally exposed to ionising radiation or not. High standard deviation, also seen in the results of other author (DeMeo et al., 1991; Maluf et al., 2001; Maluf, 2004; Tice, 1995), implicated on the influence of interindividual differences. Since the comet assay measures primary DNA damage after the exposure, those results can be a marker of different gene activity. Besides the gene activity, the DNA repair can be influenced by the

Smoking can also cause DNA damage when heavy and non heavy smokers were compared (Maluf et al., 2001; Maluf, 2004). Marcon et al. (2003) showed that heavy smokers can repair DNA damage after the exposure to 2 Gy faster than non smokers or non- heavy smokers. Olivieri et al. (1984) and Shadley (1994) suggested the existence of adaptive response that protects lymphocytes from oxidative damage. Smokers also showed lower levels of oxidised pyrimidinic bases in lymphocytes when compared to non-smokers (Berasati et al., 2001). Touil et al. (2002) showed that chemicals from the smoke can create covalent bonds between DNA and proteins, that can result with lower migration of DNA during electrophoresis,

DNA damage kinetics of primary damage can give us first information about the level of the damage. Estimation of damage after exposure to low doses of ionising radiation is possible in a very short time period (Olive et al., 1990, 1995; Tice et al., 1990). Most of the DNA breaks can be repaired during 30 minutes from the exposure to ionising radiation (Frankenberg-Schwager, 1989), and two hours from the exposure, almost all the damage is repaired (Plappert et al., 1997). Since the repair is so quick, estimation of DNA damage represents a major problem in studies of occupationally exposed professionals and demands developing of sensitive methods for detecting the level of DNA damage after exposure to low doses of ionising radiation.

In this study the influence of gene polymorphisms on DNA repair after exposure to 2 and 4 Gy of gamma radiation was investigated. The analysis of comet assay parameters did not give consistent results. Immediately after the irradiation with the dose of 2 Gy, values for tail intensity, which is recently considered the most reliable parameter for DNA damage estimation (Collins, 2004), were higher than the values of other authors for the same dose (Cornetta et al., 2006). The exposed and control group also had significantly higher TL and TM for all observed time intervals when compared to the control values before irradiation in both groups. On the other hand, TI significantly decreased after 30 minutes from the exposure. After 60 minutes from the exposure, the values did not significantly differ from the control values before irradiation in both control and exposed group. Those results were different that the ones found by Cornetta et al. (2006) during DNA repair assessment after the exposure to the dose of 2 Gy. They have shown that even after 60 minutes from the exposure, TI were still significantly higher than before radiation. The differences in significance in TL and TI in our research showed different sensitivity of those two parameters. Tail length values implicate the existence of small DNA fragments that have created comet tail shape during electrophoresis and showed the length of travelling of small fragments from the nucleus. On the other hand, TI show the amount of damaged DNA in comet tail. Damaged DNA can be seen as small DNA fragments or relaxed DNA loops from the comet head created during electrophoresis. Tail moment shows the ratio of DNA in comet tail. Our results have shown that most of the DNA damage has been repaired during 30 minutes from the exposure to 2 Gy. The amount of the unrepaired damage did not significantly differ from the amount of the damage in samples that were not irradiated, but were also investigated in DNA repair process. The results showed that TI was better marker of DNA damage than TL. Those findings are in agreement with results of Kumaravel & Jha (2006), who have also estimated the reliability of comet assay parameter after the exposure of peripheral blood samples to gamma irradiation with the doses of 0, 1, 2, 4 and 8 Gy. Besides the fact that TI and TM showed greater reliability for DNA damage estimation, they have also showed strong correlation with the received dose of irradiation.

After 4 Gy irradiation, the values for all three parameters in both control and exposed groups were higher than after irradiation with 2 Gy. Tail length values in irradiated samples were significantly higher than non-irradiated samples for the time periods of 0, 15, 30, 60 and 120 minutes, but not after 24 hours for both exposed and control group.

Values for TI and TM in irradiated samples were significantly different from the values of non-irradiated samples for time period of 0, 15, 30 and 60 minutes, suggesting that most of DNA damage has been repaired during that period, although the values measured 120 minutes and 24 hours after the exposure did not reach the values before the exposure in

DNA damage kinetics of primary damage can give us first information about the level of the damage. Estimation of damage after exposure to low doses of ionising radiation is possible in a very short time period (Olive et al., 1990, 1995; Tice et al., 1990). Most of the DNA breaks can be repaired during 30 minutes from the exposure to ionising radiation (Frankenberg-Schwager, 1989), and two hours from the exposure, almost all the damage is repaired (Plappert et al., 1997). Since the repair is so quick, estimation of DNA damage represents a major problem in studies of occupationally exposed professionals and demands developing of sensitive methods for detecting the level of DNA damage after exposure to low doses of

In this study the influence of gene polymorphisms on DNA repair after exposure to 2 and 4 Gy of gamma radiation was investigated. The analysis of comet assay parameters did not give consistent results. Immediately after the irradiation with the dose of 2 Gy, values for tail intensity, which is recently considered the most reliable parameter for DNA damage estimation (Collins, 2004), were higher than the values of other authors for the same dose (Cornetta et al., 2006). The exposed and control group also had significantly higher TL and TM for all observed time intervals when compared to the control values before irradiation in both groups. On the other hand, TI significantly decreased after 30 minutes from the exposure. After 60 minutes from the exposure, the values did not significantly differ from the control values before irradiation in both control and exposed group. Those results were different that the ones found by Cornetta et al. (2006) during DNA repair assessment after the exposure to the dose of 2 Gy. They have shown that even after 60 minutes from the exposure, TI were still significantly higher than before radiation. The differences in significance in TL and TI in our research showed different sensitivity of those two parameters. Tail length values implicate the existence of small DNA fragments that have created comet tail shape during electrophoresis and showed the length of travelling of small fragments from the nucleus. On the other hand, TI show the amount of damaged DNA in comet tail. Damaged DNA can be seen as small DNA fragments or relaxed DNA loops from the comet head created during electrophoresis. Tail moment shows the ratio of DNA in comet tail. Our results have shown that most of the DNA damage has been repaired during 30 minutes from the exposure to 2 Gy. The amount of the unrepaired damage did not significantly differ from the amount of the damage in samples that were not irradiated, but were also investigated in DNA repair process. The results showed that TI was better marker of DNA damage than TL. Those findings are in agreement with results of Kumaravel & Jha (2006), who have also estimated the reliability of comet assay parameter after the exposure of peripheral blood samples to gamma irradiation with the doses of 0, 1, 2, 4 and 8 Gy. Besides the fact that TI and TM showed greater reliability for DNA damage estimation, they

have also showed strong correlation with the received dose of irradiation.

and 120 minutes, but not after 24 hours for both exposed and control group.

After 4 Gy irradiation, the values for all three parameters in both control and exposed groups were higher than after irradiation with 2 Gy. Tail length values in irradiated samples were significantly higher than non-irradiated samples for the time periods of 0, 15, 30, 60

Values for TI and TM in irradiated samples were significantly different from the values of non-irradiated samples for time period of 0, 15, 30 and 60 minutes, suggesting that most of DNA damage has been repaired during that period, although the values measured 120 minutes and 24 hours after the exposure did not reach the values before the exposure in

ionising radiation.

exposed and control group. The results indicate the existence of small amount of DNA damage that is still present even after 24 hours from the exposure, independently of the dose level. The results could be explained by the difference in the repair of single stranded and double stranded DNA breaks. Single stranded DNA breaks are usually formed after the exposure to gamma or X-ray (90 %), while only small part of damage is created by double stranded breaks (10 %) (Cornetta et al., 2006). The repair of double stranded breaks after the exposure to ionising radiation usually lasts 12 or 16 hours (Vodicka et al., 2004), that is considerably longer than the repair of single stranded breaks. The amount of unrepaired damage in this experiment did not statistically differ from the control values before irradiation for both control and the exposed group. The difference between exposed and nonirradiated samples was higher for 2 Gy dose. The existence of small amount of unrepaired damage shows the importance of assessment of exposure to ionising radiation, especially to low doses and suggesting possible accumulation of primary, unrepaired damage that can lead to permanent damage and genome instability.

When the unrepaired amount of damage was compared, the results are implicating the existence of adaptive response in professionally exposed individuals to low doses of ionising radiation. Adaptive response in human lymphocytes has been described by other authors (Olivieri & Bosi, 1990; Sankaranarayanan et al., 1989; Shadley &Wolff, 1987; Wiencke et al., 1986; Wolff et al., 1990; Wolff, 1992, 1996). Aka et al. (2004) reported the existence of residual damage in tail intensity 60 and 120 minutes after the exposure to dose of 2 Gy. In their experiment, tail intensity values were also higher in the control group than the exposed one.

According to our results, despite the difference in the amount of damage, kinetics of repair after the exposure to both doses was similar to the one for the nonirradiated group. Unexpectedly, the difference between control and exposed group was significantly higher after 2 Gy than after 4 Gy exposure. These results were not in agreement with Wojcik et al. (1996) who claimed that kinetics of repair was faster in the exposed group. Aka et al. (2004) used comet assay for DNA repair assessment in human lymphocytes of male nuclear power plant workers and also for the control group (N=31). After the exposure, there was not significantly different repair kinetics between exposed and control group. Our results were comparable, indicating that healthy persons use the same repair mechanism, no matter if they are professionally exposed to ionising radiation or not. High standard deviation, also seen in the results of other author (DeMeo et al., 1991; Maluf et al., 2001; Maluf, 2004; Tice, 1995), implicated on the influence of interindividual differences. Since the comet assay measures primary DNA damage after the exposure, those results can be a marker of different gene activity. Besides the gene activity, the DNA repair can be influenced by the smoking status, gender, age and years of occupational exposure.

Smoking can also cause DNA damage when heavy and non heavy smokers were compared (Maluf et al., 2001; Maluf, 2004). Marcon et al. (2003) showed that heavy smokers can repair DNA damage after the exposure to 2 Gy faster than non smokers or non- heavy smokers. Olivieri et al. (1984) and Shadley (1994) suggested the existence of adaptive response that protects lymphocytes from oxidative damage. Smokers also showed lower levels of oxidised pyrimidinic bases in lymphocytes when compared to non-smokers (Berasati et al., 2001). Touil et al. (2002) showed that chemicals from the smoke can create covalent bonds between DNA and proteins, that can result with lower migration of DNA during electrophoresis,

The Influence of Individual Genome Sensitivity in DNA Damage Repair

cutting of 8-oxodeoxiguanosine when compared to heterozygote.

homozygotes and polymorphic variants showed significant differences.

comet assay parameters 120 min after exposure to 2 Gy of gamma radiation.

hours after the exposure to 4 Gy.

after the exposure to 2 Gy.

**5. Acknowledgment**

**4. Conclusion** 

**6. References** 

Assessment in Chronic Professional Exposure to Low Doses of Ionizing Radiation 453

repair pathway, while hOGG1 cuts oxidised base, XRCC1 together with ligase closes the break. Aka et al. (2004) and Matullo (2003) have described their connection. On the other hand, Cornetta et al. (2006), did not show significant difference between XRCC1, hOGG1 and XPC polymorphisms and DNA damage level measured 30 minutes from the exposure to 2 Gy dose. Yamane et al. (2004) showed that homozygotes differ for 2 fold factor in

XRCC1 polymorphism did not show the influence on DNA damage and repair, but polymorphic variants had lower levels of DNA damage for all three parameters measured. The results are not in agreement to those of Cornetta et al. (2006). Tail intensity values after the exposure to 2 Gy dose, immediatelly after, 30 minutes and 60 minutes after,

The two most common SNP polymorphisms in XPD gene are in exons 10 and 23. The results of studies of those polymorphisms are inconsistent. Naccarati et al. (2006) observed fewer number of single strand breaks in polymorphic homozygotes compared to heterozygotes and wild type of homozygotes. Similar results state Vodicka et al. (2004), analyzing chromosome aberrations frequency. Polymorphic XPD23 gene variants did not influence primary DNA damage, while polymorphic XPD10 gene variants showed positive correlation in all three

Polymorphic variants of XRCC3 in the exposed group had highest values for DNA damage. There was also positive correlation of polymorphic variants with TL immediately after the exposure to 2 Gy and negative correlation with TI and TL 120 minutes from the exposure to 2 Gy dose. Aka et al. (2004) did not find the correlation with primary damage, probably because this gene is involved in double strand breaks repair. PARP1 is important for integrity of chromosome ends and is involved in maintaining the integrity of BER and NHEJ repair. Our results showed negative correlation between polymorphic variants and TI values measured immediately after the exposure to 4 Gy and with TM values measured 24

MGMT C/T variants have higher risk of glioma (Liu et al., 2009), but there are no investigations that are connecting it with ionising radiation. In our study polymorphic variants have shown negative correlation with DNA damage level measured 120 minutes

The results indicate that in addition to individual gene polymorphisms, the influence of

This study was partly supported by the Ministry of Science, Education and Sports of the

Aka, P.; Mateuca, R.; Buchet, J.P.; Thierens, H. & Kirsch-Volders, M. (2004) Are genetic

polymorphisms in OGG1, XRCC1 and XRCC3 genes predictive for the DNA strand

combinations of polymorphic genes to DNA damage and repair should be tested.

Republic of Croatia (grant number 0022-0222148-2137).

showing false results, that is lower damage. Cebulska et al. (2007) showed negative influence of smoking on the efficiency of DNA repair in lymphocytes. On the other hand, Rzeszowska-Wolny et al. (2005) did not find significant correlation between smoking and DNA damage and DNA repair after the irradiation of human lymphocytes with the dose of 2 Gy. Our results also did not show the influence of smoking on DNA repair of primary damage, except for the tail length after the exposure to 4 Gy dose. The results are similar to Aka et al. (2004) on nuclear power plant workers.

It is still not clear whether the basal damage differ due to gender. Dusinska et al. (2004a) did not find the difference in creation of strand breaks, nor in the amount of DNA damage sensitive to action of specific enzymes such as endonuclease III, or FPG enzyme or alkali labile sites, although male had lower DNA damage after the treatment with hydrogen peroxide (Dusinska et al., 2004a, b). Bajpayee et al. (2002) showed that healthy men have more basal damage in lymphocytes than women. Our results showed that difference was only seen after the 4 Gy dose, where women had higher values for DNA damage in all three parameters observed in comet assay immediately after and 24 hours after the irradiation. There was not similar number of male and female, and that could also influence the results. Trzeciak et al. (2008) showed that there are differences in the amount of damage and the repair rate after the exposure of whole blood sample to 6.3 Gy dose. But most of the experiments did not show the gender influence on DNA repair capacity (Muller et al., 2001, 2002; Rajaee-Behbahani et al., 2001; Marcon et al., 2003).

Age influence was not seen in this experiment. That is in agreement with other authors (Betti et al., 1994; Frenzili et al., 1997). Rzeszowska-Wolny et al. (2005) did not show the influence of sex on DNA damage level and DNA repair by the comet assay after the exposure to 2 Gy dose. Maluf et al. (2001) showed positive correlation of age and DNA repair in the exposed group. Singh et al. (1988) described positive correlation between the age and DNA damage in non-smokers.

Although former results showed the existence of radio adaptive response, our results did not confirm the relationship between primary DNA damage and adaptive response. Few authors have also shown lower DNA damage levels after the repeated exposure to ionising radiation (Sankaranarayanan et al., 1989; Wang et al., 1991; Ikushima, 1992; Domingues et al., 1993).

APE1 gene is involved in BER repair. Polymorphism in exon 5 is connected with hypersensitivity during the exposure to ionising radiation (Au et al., 2006). Results are in agreement with Au et al. (2006). There was positive correlation of polymorphic variants and DNA damage level 120 minutes after the exposure to 2 Gy.

Polymorphic variants of hOGG1 gene showed positive correlation with TI and TM measured immediately after the exposure to the dose of 2 Gy. The results are in agreement with Aka et al. (2004) who showed the connection between polymorphic variants of hOGG1 Ser326Cys with lower capability of repair of oxidative DNA damage in the exposed, but not in the control group measured 60 and 120 minutes after the exposure to 2 Gy. After 120 minutes from the exposure, 71 % of individuals with high level of DNA damage had polymorphic variant of hOGG1 gene. Cornetta et al. (2006) did not show the influence of hOGG1 polymorphism on DNA damage.

Goode et al. (2002)) showed the connection between polymorphic variants of hOGG1 with higher risk of lung, head and neck cancer. hOGG1 and XRCC1 are involved in the same repair pathway, while hOGG1 cuts oxidised base, XRCC1 together with ligase closes the break. Aka et al. (2004) and Matullo (2003) have described their connection. On the other hand, Cornetta et al. (2006), did not show significant difference between XRCC1, hOGG1 and XPC polymorphisms and DNA damage level measured 30 minutes from the exposure to 2 Gy dose. Yamane et al. (2004) showed that homozygotes differ for 2 fold factor in cutting of 8-oxodeoxiguanosine when compared to heterozygote.

XRCC1 polymorphism did not show the influence on DNA damage and repair, but polymorphic variants had lower levels of DNA damage for all three parameters measured. The results are not in agreement to those of Cornetta et al. (2006). Tail intensity values after the exposure to 2 Gy dose, immediatelly after, 30 minutes and 60 minutes after, homozygotes and polymorphic variants showed significant differences.

The two most common SNP polymorphisms in XPD gene are in exons 10 and 23. The results of studies of those polymorphisms are inconsistent. Naccarati et al. (2006) observed fewer number of single strand breaks in polymorphic homozygotes compared to heterozygotes and wild type of homozygotes. Similar results state Vodicka et al. (2004), analyzing chromosome aberrations frequency. Polymorphic XPD23 gene variants did not influence primary DNA damage, while polymorphic XPD10 gene variants showed positive correlation in all three comet assay parameters 120 min after exposure to 2 Gy of gamma radiation.

Polymorphic variants of XRCC3 in the exposed group had highest values for DNA damage. There was also positive correlation of polymorphic variants with TL immediately after the exposure to 2 Gy and negative correlation with TI and TL 120 minutes from the exposure to 2 Gy dose. Aka et al. (2004) did not find the correlation with primary damage, probably because this gene is involved in double strand breaks repair. PARP1 is important for integrity of chromosome ends and is involved in maintaining the integrity of BER and NHEJ repair. Our results showed negative correlation between polymorphic variants and TI values measured immediately after the exposure to 4 Gy and with TM values measured 24 hours after the exposure to 4 Gy.

MGMT C/T variants have higher risk of glioma (Liu et al., 2009), but there are no investigations that are connecting it with ionising radiation. In our study polymorphic variants have shown negative correlation with DNA damage level measured 120 minutes after the exposure to 2 Gy.
