Genetic polymorphisms and expression of minisatellite mutations in a 3–generation population around the Semipalatinsk nuclear explosion test-site, Kazakhstan

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Genetic polymorphisms and expression of minisatellite mutations in a 3–generation population around the Semipalatinsk nuclear explosion test-site, Kazakhstan N.K. Bolegenova a, B.O. Bekmanov a, L.B. Djansugurova a, R.I. Bersimbaev a,b,, S.A. Salama c, W.W. Au c, a

Institute of General Genetics and Cytology, Almaty, Kazakhstan Eurasian National University, 010008 Astana, Munaitpasova Str. 5, Kazakhstan c The University of Texas Medical Branch, Galveston, Texas, USA b

a r t i c l e in f o

a b s t r a c t

Article history: Received 9 March 2009 Received in revised form 9 June 2009 Accepted 8 July 2009

We have reported previously that a population near the Semipalatinsk nuclear explosion test site had significantly increased minisatellite mutations (MM), suggesting increased germ-line mutation rates from the exposure in 3 generations. We hypothesize that the MM can be used as a surrogate biomarker for functional genetic alterations, e.g. gene mutations and chromosome aberrations. Therefore, we have investigated the influence of polymorphisms in genes on the expression of MM in the same two populations (247 and 172 individuals, for exposed and control, respectively, in 3 generations), and their relationships with radiation exposure. We have chosen the analyses of three polymorphic DNA - repair genes (XRCC1, XRCC1 and XRCC3) and two xenobiotic detoxification genes (GSTT1 and GSTM1). Among the exposed and in comparison with the wild-type gene, the functionally active XRCC1 Arg194Trp was significantly associated with low MM and over-represented in the exposed compared with the control populations. In a similar analysis, the functionally deficient XRCC1 Arg399Glu and XRCC3 Trp241Met were associated with increased and significantly reduced MM, respectively, but these variant genes were under-represented in the exposed population. Both GSTT1 and GSTM1 nulls were significantly associated with increased MM. The former was under-represented but the latter was significantly over-represented in the exposed compared with the control populations. In summary, the data indicate that the expected enzymatic functions of the polymorphic genes are consistent with the MM expression, except the XRCC1 Arg399Glu variant gene. In addition, the variant genes were retained in the three generations in association with their useful function, except for the GSTM1 null. However, the MM frequencies in the exposed were not consistently and significantly higher than those in the control populations, radiation exposure may therefore not have been the only cause for the high MM frequency among the exposed individuals. Since we studied three generations of citizens, the over- and under-representations of variant genes in the exposed population indicate their persistence and elimination, respectively, from the exposed individuals, suggesting their functional influence on survivability. The latter observation also indicates the complexity of gene and environmental interactions, e.g. the GSTM1 null was significantly over-represented in the exposed population. & 2009 Elsevier GmbH. All rights reserved.

Keywords: Irradiation Semipalatinsk nuclear test site DNA repair genes DNA repair gene polymorphism Detoxification genes Minisatellite mutation rates Genetic susceptibility

Introduction The former Soviet Union tested over 450 nuclear explosions at the Semipalatinsk nuclear polygon in Kazakhstan from 1949 to

 Corresponding author at: Eurasian National University, 010008 Astana, Munaitpasova Str. 5, Kazakhstan. Tel./fax: +7 717 2 35 39 01.  Correspondence to: Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, 700 Harborside Drive, Galveston, TX 77555-1110, USA. Tel.: +1 409 772 1545; fax: +1 409 772 9108. E-mail addresses: [email protected] (R.I. Bersimbaev), [email protected] (W.W. Au).

1438-4639/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijheh.2009.07.001

1962 (Ministry of the Russian Federation for Atomic Energy and Ministry of Defense of Russian Federation, 1996). The polygon is located about 150 km west of the Semipalatinsk city with more than 1.5 million people in the region (East Kazakhstan around Semipalatinsk, the Pavlodar regions of Kazakhstan and the Altay region of the Russian Federation). These individuals have therefore been repeatedly exposed to ionizing radiation from the radioactive cloud or environmental fallout for many years. However, the extents and dosimetry of their exposure, and health risk from the exposure are largely unknown. To gain insight into the health concerns of the exposed populations, we have initiated a series of biodosimeter studies to estimate health risk in a

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3-generation sample from the Semipalatinsk population. We have previously reported a 1.8-fold increase in the minisatelite mutation rate (MM) in the exposed P0 generation (Dubrova et al., 2002; Bersimbaev et al., 2002a, b). In addition, the germline mutation rate showed a negative correlation with the year of birth of the exposed F1 generation. These results indicated that the radiation exposure from the nuclear tests has caused the elevated minisatellite mutation rates and germ-line mutations. Based on this evidence and from our review of the literature, we believe that MM determination is a highly sensitive biomarker for the detection of radiation-induced germline effect, much more than that of traditional biomarkers such as gene mutations and chromosome aberrations. In addition, other studies indicate that transgenerational germline effects are not limited to a specific sub-set of DNA lesions, such as double strand breaks, and are probably triggered by a stress-like response to a generalized DNA damage (Barber et al., 2006; Dubrova et al., 2008). Therefore, we hypothesize that MM is a surrogate biomarker to indicate the existence of functional genetic alterations in this radiationexposed population. In this study, we have investigated the influence of polymorphisms in DNA repair and xenobiotic detoxification genes on the expression of MM in the same two populations (247 and 172 individuals, for the exposed and controls, respectively, in 3-generations: P0, F1, F2), and the relationship between radiation exposure and MM expression. We found that the expected enzymatic functions of the polymorphic genes are consistent with the MM expression, except the XRCC1 Arg399Glu variant gene. In addition, our observation suggests that radiation exposure was not the only cause for the expression of MM in the exposed population. Furthermore, significant gene-gene and gene-environmental interactions influenced the retention of certain susceptibility genes, the health risk and, possibly, survival of the exposed population.

Materials and methods Biological samples from the exposed and control populations The archived specimens from the earlier studies (Bersimbaev et al., 2002a, b; Dubrova et al., 2002; Salomaa et al., 2002; Lindholm et al., 2004) which were stored in the Laboratory of Molecular Genetics, Institute of General Genetics and Cytology, Almaty, Kazakhstan were retrieved and used for the current study. The selected samples contained 247 specimens from the exposed (from 30 three-generation families) and 172 from the matched control (from 21 three-generation families) populations. A brief summary of the three-generation of exposed individuals who were selected for our study is shown in Table 1. From the original studies, the exposed population was recruited from the Semipalatinsk region (Eastern Kazakhstan). Since the entire region has been extensively contaminated by the numerous nuclear explosion tests, everyone who lived in the region were expected to be exposed. Therefore, no specific

655

dosimetric measurements were conducted. The control population was recruited from non-contaminated areas near Almaty (Southern Kazakhstan) which is more than 1000 km away and upwind from the exposed region. Prospective volunteers were interviewed based on a questionnaire on age, gender, ethnicity, job exposure, smoking and alcohol consumption habits, medical history, etc. The exposed and the control individuals were frequency-matched based on age, gender, smoking and alcohol consumption habits. Individuals who were ill, had occupational exposure to hazardous agents or had cancer history were excluded. The protocol for the human study was approved by the universities in Kazakhstan. In addition, each individual provided consent to the interview and to the donation of biological samples. These are the individuals we have used for our study. DNA isolation and genotyping Genomic DNA was extracted from archived (frozen) lymphocytes from peripheral blood by a standard phenol-chloroform method. Quantitative and qualitative estimations of DNA samples were conducted by electrophoresis and spectrophotometry. The water-diluted samples were used for genotyping of DNA-repair and chemical-detoxification genes by PCR using specific primers. Restriction and/or gel analyses were used for the identification of gene polymorphisms. For determination of the polymorphism XRCC1 Arg194Trp, 100 ng genomic DNA was amplified in a total volume of 50 mL containing 0.2 mM of the following primer pairs: forward, 50 GCCCCGTCCCAGGTA-30 , reverse, 50 -AGC CCC AAG ACCC TTT-30 , 1  PCR buffer (150 mM Tris-HCl, pH 0.8, 500 mM KCl), 2.5 mM MgCl2, 0.2 mM each deoxynucleoside triphosphate (dNTP), and 1 U Taq polymerase. The PCR amplification condition consisted of the initial denaturation step at 95 1C for 2 min, followed by 40 cycles of 94 1C for 15 sec, 57 1C for 45 sec, 72 1C for 45 sec, and final extension step at 72 1C for 5 min. The PCR products (490 bp) were digested overnight with the restriction enzyme PvuII. The restricted products of XRCC1 codon 194 Arg/Arg, Arg/Trp, and Trp/Trp genotypes had band sizes of 490, 490/294/196, and 294/ 196 bp, respectively [Au et al., 2003]. For the determination of XRCC1 gene (Arg399Gln) polymorphism [Matullo et al., 2001a], 100 ng genomic DNA was amplified in a total reaction volume of 50 mL containing 0.2 mM of each of the forward primer, 50 -CAAGTACAGCCAGGTCCTAG-30 , reverse primer, 50 -CCTTCCCTCA TCTGGAGTAC-30 , 1xPCR buffer (150 mM Tris-HCl, pH 0.8, 500 mM KCl), 1.5 mM MgCl2, 0.2 mM each dNTP, and 1 U Taq polymerase. The PCR amplification condition consisted of the initial denaturation step at 95 1C for 2 min, followed by 40 cycles of 94 1C for 15 sec, 55 1C for 30 sec, 72 1C for 45 sec, and the final extension step at 72 1C for 5 min. The 248-bp PCR products were digested with NciI (Promega, Madison, WI); only the Arg allele was cut into 89 and 159 bp fragments. The XRCC3 Thr241Met polymorphism was determined using the primers (forward) 50 -GCCTGGTGGTCATCGACTC-30 and (reverse) 50 -ACAGGGCTCTGGAAGGCACTGCTCAGCTCACGCACC-30 . The PCR

Table 1 Distribution of study subjects in three generations in our study. Groups (# of families)

Exposed (30) Control (21)

Po

F1

F1

F2

Total # citizens selected

Fathers

Mathers

sons

wives

daughters

husbands

grandsons

grandaughters

30 21

30 21

56 39

17 15

38 28

11 3

32 25

33 20

247 172

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condition consisted of the initial denaturation step at 95 1C for 2 min, followed by 40 cycles of 94 1C for 15 sec, 60 1C for 30 sec, 72 1C for 45 sec, and the final extension step at 72 1C for 5 min. The 136-bp PCR product was digested with NcoI (Promega); only the Met allele was cut into 39- and 97-bp fragments. A multiplex PCR procedure was used for the simultaneous analysis of the GSTT1, GSTM1 and b-globin (as internal control) genes [Garte et al., 2001]. For the GSTT1, GSTM1 genotyping 50 ng genomic DNA was amplified in a total reaction volume of 20 mL containing 0.2 mM of each of the forward primers-GSTM1: 50 -GAA CTC CCT GAA AAG CTA AAG C-30 n 50 -GTT GGG CTC AAA TAT ACG GTG G-30 n GSTT1: 50 -CCT TAC TGG TCC TCA CAT CTC-30 ; 50 -TCA CCG GAT CAT GGC CAG CA -30 , 10  PCR buffer (100 mM Tris-HCl, pH 0.9, 500 mM KCl), 10 mM each dNTPb and 0,5 U. Taqpolymerase (Sigma, USA). The primers for the b-globin gene (268 bp) were: 50 -CAA CTT CAT CCA CGT TCACC-30 ; 50 -G AAG AGC CTAGGACAGGTAC-30 . The PCR amplification condition consisted of the initial denaturation step at 94 1C for 5 min, followed by 35 cycles of 94 1C for 2 min, 59 1C for 1 min, 72 1C for 1 min, and the final extension step at 72 1C for 10 min. The presence of PCR product 480 bp identified GSTT1 positive genotype and 215 bp identified GSTM1 positive genotype. Genotype results were verified by repeated analyses.

Statistical analysis All statistical tests were performed with the Software GraphPad Instat tm Copyrigh (V. 2.04. Ralf Stahlman, Purdue University). For the assessment of distribution differences between cases and controls we used the Chi-square (w2) and the Hardy-Weinberg tests. The tests were performed for each generation separately. Because of the family cluster effect, it is not appropriate to stack the entire population (P0+F1+F2) for the analysis. Power analysis was performed to evaluate the sufficiency of the current sample size. The analysis was based on the expected prevalence of the polymorphic genes in the control population and the expected frequencies in the cases. To determine the association between each genotype and MM rates, we used traditional method of variation statistics – comparison between mean frequencies of MM in different genotypes cohorts (Rokitski, 1973, 1978). The odds ratios (Ors) and 95% confidence intervals (CIs) were calculated according to Jedrychowski and Maugeri (2000). To determine the association between each genotype and minisatellite mutation rates we used traditional method of variation statistics – comparison between mean frequencies of minisatellite mutations in different genotypes cohorts [Jedrychowski and Maugeri, 2000].

Results Distributions of the polymorphic genes The genotypes of all individuals from the exposed and control populations were categorized as follows: XRCC1(194) (Arg/Arg, Arg/Trp; Trp/Trp); XRCC1(399)( Arg/Arg, Arg/Gln; Gln/Gln); XRCC3(241) (Trp/Trp, Trp/Met; Met/Met); GSTM1 (+/+; +/; /) and GSTT1 (+/+; +/; /). The distributions did not differ significantly (po0.05) from those expected by Hardy-Weinberg equilibrium for polymorphisms XRCC1 (194) and GSTM1 gene. The distributions for other polymorphisms are not consistent with Hardy-Weinberg equilibrium for our Kazakh population. In order to conduct meaningful statistical evaluation of the genotype distributions in the two populations, the heterozygous and homozygous variants for each gene were combined into one group for the analysis. This group will be known as the variant gene group. The genotype data are summarized in Table 2. As shown in the Table, the variant versions of XRCC1 Arg194Trp was significantly overrepresented in the exposed compared to the control populations (OR ¼ 1.69; CI ¼ 1.03 – 2.76; Po0.03). There were no significant differences in the distributions of the remaining two polymorphic DNA repair genes (XRCC1 Arg399Gln and XRCC3 Trp241Met) in the two populations. The GSTT1 null genotype was distributed evenly among the two populations. However, the GSTM1 null genotype was preferentially over-represented in the exposed population (OR ¼ 11.14; CI ¼ 6.09 – 20.37; po0.001). Association between minisatellite mutation frequencies and variant genes The association was performed for each generation and the summary of the evaluation is shown in Table 3. As shown in the table, the ORs for XRCC1 Arg194Trp and GSTM1 null are consistent across all three generations. The consistencies are less for other variant genes. By combining the generations into a summary group, the associations became clarified (Table 4). For the functional XRCC1 Arg194Trp gene, the MM frequencies among the variant gene group were significantly lower than those of the wild-type (9.3171.85 vs. 29.9572.91; po0.001). The significantly different relationship was consistent for the exposed and the control populations. In addition, this MM frequency for the variant gene group in the exposed population (9.3171.85) is significantly higher than that in the matched control population (1.1670.82) (tSt ¼ 4.03, Po0.003). For the functionally deficient XRCC1 Arg399Gln gene, the MM frequency was not significantly higher than that of the wild-type in the exposed population (20.2472.55 vs. 16.6072.38; p40.05). However, the frequency was significantly higher among the controls (16.2872.81 vs. 6.3971.86; po0.02). In addition, this MM frequency in the

Table 2 Variant genotype distributions and ORs between the exposed and controls. Genes

Genotype

The control n (%)

Exposed n (%)

XRCC1 (194)

GSTT1

Arg/Arg Arg/Trp; Trp/Trp Arg/Arg Arg/Gln; Gln/Gln Trp/Trp Trp/Met; Met/Met +/+

143 29 62 110 97 75 56 116

184 63 103 144 155 92 65 182

GSTM1

+/+

XRCC1 (399) XRCC3 (241)

7; / 7; /

(83) (17) (36) (64) (56) (44) (33) (67)

72 (42) 100 (58)

OR

CI (95%)

P

(74) (26) (42) (58) (63) (37) (26) (74)

1.69

1.033-2.76

0.03

0.79

0.53-1.18

0.24

0.77

0.52-1.14

0.19

1.35

0.88-2.07

0.16

15 (6) 232 (94)

11.14

6.09-20.37

6,43e-19

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Table 3 Association between variant genes and minisatellite mutations in three-generations. Genes Po (1 generation) XRCC1 -194 XRCC1-399 XRCC3-241 F1 (2 generation) XRCC1 -194 XRCC1-399 XRCC3-241 F2 (3 generation) XRCC1 -194 XRCC1-399 XRCC3-241 Total(Po+F1+F2) XRCC1 -194 XRCC1-399 XRCC3-241 Po (1 generation) GSTT1 GSTM1 F1 (2 generation) GSTT1 GSTM1 F2 (3 generation) GSTT1 GSTM1 Total(Po+F1+F2) GSTT1 GSTM1

Genotypes

Control, n (%)

Exposed, n (%)

Arg/Arg Arg/Trp+Trp/Trp Arg/Arg Arg/Gln+Gln/Gln Trp/Trp Trp/Met+Met/Met Number of people Arg/Arg Atr/Trp+Trp/Trp Arg/Arg Arg/Gln+Gln/Gln Trp/Trp Trp/Met+Met/Met Number of people Arg/Arg Arg/Trp+Trp/Trp Arg/Arg Arg/Gln+Gln/Gln Trp/Trp Trp/Met+Met/Met Number of people Arg/Arg Atr/Trp+Trp/Trp Arg/Arg Arg/Gln+Gln/Gln Trp/Trp Trp/Met+Met/Met Number of people +/+ (7)+(/) +/+ (7)+(/) Number of people +/+ (7)+(/) +/+ (7)+(/) Number of people +/+ (7)+(/) +/+ (7)+(/) Number of people +/+ (7)+(/) +/+ (7)+(/)

34(81) 8(19) 15(36) 27(64) 26( 62) 16(38) 85 70(82 ) 15(18 ) 38(45) 47(55 ) 50(59 ) 35(41) 45 39(87) 6(13) 9(20) 36(80) 21(47) 24(53) 172 143(83) 29(17) 62(36) 110(64) 97(56) 75(44) 42 16 (38) 26 (62) 20 (48) 22 (52) 85 34 (40) 51(60) 34 (40) 51(60) 45 6 (13) 39 (87) 18 (40) 27 (60) 172 56 (33) 116 (67) 72 (42) 100 (58)

46(77) 14(23) 25(42) 35(58 ) 35(58 ) 25(42) 122 93(76) 29(24) 51(42) 71(58) 78(64) 44(36) 65 45(69) 20(31) 27 (42) 38(58) 42(65) 23(35) 247 184(74) 63(26) 103(42) 144(58) 155(63) 92(37) 60 15 (25) 45 (75) 3 ( 5) 57(95) 122 32 (26) 90 (74) 6 (5) 116 ( 95) 65 18 (28) 47 (42) 6 (9) 59 (91) 247 65 (26) 182 (74) 15 (6) 232 (94)

exposed population (20.2472.55) is not significantly different from that of the control population (16.2872.81) (tSt ¼ 1,04, P40.05). For the functionally deficient XRCC3 Trp241Met gene, the MM frequency was significantly lower compared to the wild-type gene in the expose (14.1771.27 vs. 25.1072.76; Po0.001), but not significant among the control populaitons. The difference between the exposed and control populations is not significant (P40.05). From the analysis of xenobiotics detoxification genes (GSTT1 and GSTM1), the MM frequencies for those with each of the mutant genotypes (+/, /) were significantly higher than in those with wild-type genotypes (po0.001) for both the exposed and control populations, except for GSTM1 (+/, /) among the controls.

Discussion We have conducted an investigation on 3 generations of radiation-exposed individuals in Kazakhstan to elucidate the relationship between genetic susceptibility and expression of MM (as a surrogate biomarker of functional genetic alterations), and health risk from the exposure. For XRCC1 Arg194Trp, several

OR

CI (95%)

P

1.29

0.49-3.43

0.60

0.78

0.35-1.75

0.54

1.16

0.52-2.60

0.71

1.46

0.73-2.92

0.28

1.13

0.64-1.97

0.67

0.81

0.46-1.43

0.45

2.89

1.05-7.92

0.03

0.35

0.69-3.86

0.01

0.48

0.22-1.04

0.06

1.69

1.03-2.76

0.03

0.79

0.59-1.18

0.24

0.77

0.52-1.14

0.19

1.85

0.79-4.34

0.16

4.67-63.9

0.001

1.88

1.04-3.39

0.03

12.89

5.09-32.61

0.001

0.40

0.15-1.11

0.07

6.56

2.34-18.36

0,0001

1.35

0.88-2.07

0.16

6.09-20.37

0.001

17.3

11.13

investigators have reported that the variant genotype has no reduction in the repair of radiation-induced DNA damage (Au et al., 2003; Vodicka et al., 2007) and inheritance of this variant gene has not been associated with the development of breast, esophagus and lung cancers (Duell et al., 2001; Butkiewicz et al., 2001; Improta et al., 2008). In our study, inheritance of this variant gene was significantly associated with reduced MM frequencies compared to the wild-type gene in both the radiation-exposed and matched control populations. In addition, exposed individuals with the variant gene had significantly more MM than those among the controls, indicating functional and exposure effects. More intriguing to us is that the variant gene was significantly over-represented in the exposed compared to the control populations. This suggests that not only does the variant gene have good repair activities towards radiation-induced DNA damage but may also have beneficial effects towards survival among the three generations of the exposed population. For XRCC1 Arg399Gln, the variant genotype have been reported to show radiosensitivity (Au et al., 2003; Vodicka et al., 2007) and inheritance of this gene has been associated with the development of breast cancer (Hu et al., 2002; Smith et al., 2003) but not

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Table 4 Association between average frequencies of minisatellite mutations and genotype groups. Polymorphism

The exposed group

Control group

XRCC1 Arg194Trp Frequency of MM tSt, P XRCC1 Arg399Gln Frequency of MM tSt, P XRCC3 Trp241Met Frequency of MM tSt, P GSTT 1

Arg/Arg 29.9572.91 5.91; 0.001 Arg/Arg 16.6072.38 1.04; not significant Trp/Trp 25.1072.76 3.59; 0.001 +/+

Arg/Trp; Trp/Trp 9.3171.85

Frequency of MM tSt, P GSTM 1

13.7672.19 3.33; 0.001 +/+

25.5172.77

Frequency of MM tSt, P

0.8170.57 11.95; 0.001

38.4673.10

Arg/Gln; Gln/Gln 20.2472.55 Trp/Met; Met/Met 14.1771.27 7;/

7;/

Arg/Arg 21.5173.13 6.30; 0.001 Arg/Arg 6.3971.86 2.93; 0.02 Trp/Trp 10.4672.33 0.51; not significant +/+

Arg/Trp; Trp/Trp 1.1670.82

5.2371.69 3.64; 0.001 +/+

17.4472.89

9.8872.27 0.85; not significant

12.7972.55

Arg/Gln; Gln/Gln 16.2872.81 Trp/Met; Met/Met 12.2172.49 7;/

7;/

 MM – minisatellite mutations

with gastric or lung cancer (Shen et al., 2000; Improta et al., 2008). In this study, the variant gene was associated with nonsignificantly higher MM frequencies among the exposed but significantly higher MM frequencies among the control populations in comparison to the wild-type gene. Furthermore, the distribution of the variant gene was similar in both the exposed and the control populations, suggesting that inheritance of this defective gene may not have detrimental effects towards survival of the exposed population. For XRCC3 Trp241Met, the variant gene has been shown to have reduced repair activities towards radiation-induced DNA damage (Au et al., 2003) and reduced DNA adducts removal (Matullo et al., 2001b), and inheritance of this variant gene is associated with lung and colorectal cancer (Improta et al., 2008) but not with lung cancer or melanoma (Beabes et al., 2001; Duan et al., 2003). In our study, the variant gene was associated with the significant reduction of MM frequencies compared to the wild-type gene in the exposed population but with higher MM frequencies in the control population. Furthermore, the distribution of the variant gene was similar in the exposed compared to the control populations, suggesting no detrimental effects on survival. We have also conducted association studies with the polymorphic xenobiotic metabolizing genes, GSTT1 and GSTM1. Both null variant genes were associated with significant increase of MM frequencies compared with the wild-type genes in both the exposed and control populations, except for the GSTM1 null among the controls. An intriguing observation was that the GSTM1 null but not the GSTT1 null genotype was significantly overrepresented in the exposed population, suggesting its beneficial effects on survival of the exposed population. The observations from our investigation can be concisely summarized into Table 5. Construction of the table was based on the following criteria. The function of the variant genes was based on reports in the literature, with A being active and D being deficient in function. The associations between the variant genes and increased or decreased MM frequencies in comparison to the wild-type genes in the exposed population were based on our current observations. The interpretation of radiation effect was based on having at least 3-fold higher MM frequencies for any genes in the exposed compared to the control populations. According to this criterion, only the exposed individuals with the variant XRCC1 Arg194Trp or the GSTM1 null genes had more than a 3X increase of MM frequencies compared with the matched controls. Gene persistence was based on having significant over-representation of the variant genotypes in the exposed compared to the control populations over three generations. As shown in the

Table 5 Summary of the observations1. Genotypes

Function

XRCC1 Arg194Trp XRCC1 Arg399Gln XRCC3 Trp241Met GSTT1 null GSTM1 null

A D D D D

MM frequencies k m k m m

Radiation effect Y N N N Y

Gene persistence Y N N N Y

1 Information on Function is based on literature reports, A ¼ active and D ¼ deficient; MM frequencies are based on comparison with those having the wild-type genes in the exposed population.  Significant differences; Radiation effect is based on having 3 fold increase in MM frequencies in the exposed compared with those in the control populations, Y ¼ yes and N ¼ no; Survival effect is based on having significant overrepresentation in the exposed compared with the control populations.

table, the function of the variant genes is correlated with the expression of MM, except the XRCC3 Trp241Met in the exposed population (columns 2 and 3). Based on our determination, only two variant genes are correlated with radiation effect (column 2 and 4). In addition, the radiation effect is not correlated with MM frequencies. Furthermore, the MM frequencies for the exposed are not consistently and/or significantly higher than those of the controls under the different gene categories. Therefore, our observations suggest that exposure to ionizing radiation may not have been the only cause for the increase of MM frequencies in the exposed population. Indeed, other studies in this program indicate that the exposed population did not have significant increase in chromosome translocations or glycophorin A somatic cell mutations (Salomaa et al., 2002; Lindholm et al., 2004). However, these two published studies involved much smaller sample sizes than ours. As indicated by the authors, the small sample sizes reduced their ability to detect exposure to low doses of ionizing radiations. The last column in Table 3 indicates that persistence of the variant genes in three generations of the exposed population was, in general, associated with the functional usefulness of the genes, except the GSTM1 null. Their persistence in the exposed population may therefore contribute to the survival of the exposed population. The observation of preferential retention of GSTM1 null may have apparent controversy but the observed effects may be due to the effect of gene to gene and gene-environment interactions. For example, a report indicates that GSTM genotypes may influence other metabolic pathways (Vineis et al., 2004) and DNA repair activities (Slykova et al., 2007). In addition, the role of GSTM1 null on survival of the exposed population may gain more support

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Family # 25

+/+;+/-

+/+;-/-

+/+;+/-

+/+;-/-

+/+;-/-

+/+;+/-

+/+;-/-

+/+;-/-

+/+;-/-

+/+;-/-

+/+;+/-

+/+;-/-

+/+;-/-

+/+;-/-

Fig. 1. Transmission of GSTT1 and GSTM1 variants through 3 generations of a family.

from our raw data. The data showed their preferential transmission throughout the three generations, as illustrated in one family (Fig. 1). Currently, we have no further explanation for the preferential retention. However, we know that the genotype analysis has been validated by repeated evaluations. In summary, our investigation showed the role of susceptibility genes on the expression of MM in three generations of a radiationexposed population. Since these genes are involved in the formation of other genetic effects, it is possible that other functional genetic alterations exist in the exposed population. In addition, radiation did not seem to be the only source of hazardous substances of exposure for the population. The data also illustrate the complexity of gene-gene and gene-environmental interactions on health risk assessment, e.g. the GSTM1 null appears to be preferentially retained and transmitted through the three exposed generations. Therefore, further studies need to be conducted to clarify biological mechanisms towards improving the risk assessment process.

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