The Role of Polymorphisms of Key Genes of DNA Base Excision Repair in Terms of Lung Cancer Predisposition in "Mayak" Workers.

An association between polymorphic variants of key genes of base excision repair (BER) and lung cancer was studied in "Mayak" workers occupationally exposed to prolonged external y-rays and internal α-radiation from incorporated (239)Pu. The study was "case-control". The group of "cases" consists of 75 "Mayak" workers with the verified diagnosis of "lung cancer". At the moment of diagnosis the mean total absorbed dose from external y-rays to whole body was 1.19 Gy; the mean total absorbed dose from internal α-radiation due to incorporated (239)Pu in lung was 0.31 Gy. The group of "controls" includes "Mayak" workers matched by sex and birth year without lung cancer and other cancers during the study period (141 individuals). Increased lung cancer risk was revealed in workers-carriers of homozygous minor genotype of genes OGG] Ser326Cys (OR - 4.67, p = 0.007), APEI Asp148Glu (OR = 1.82, p = 0.063) and XRCC1 Gln399Arg (OR = 2.86, p = 0.026). Increased lung cancer risk was revealed in carriers of different pairwise combinations of minor genotypes of the studied genes of BER or in carriers of pairwise combinations with one homozygous minor mm-genotype and the other homozygous major ww genotype. Thus, OR of lung cancer in carriers of pairwise genotypes of mm genes OGG] Ser326Cys and APE] Asp 148Glu was 12.17.

[Molecular mechanisms of lung cancer development at its different stages in nuclear industry workers].

OBJECTIVE: to assess mutational events in exons 5, 7, and 8 of the p53 gene and to reveal mutant p53 protein in verified cases of morphologically altered (proliferative and precancerous changes, lung cancer) and histologically unaltered, lung tissues in workers exposed to occupational radiation. MATERIAL AND METHODS: The investigation used formalin-fixed paraffin-embedded unaltered and altered lung tissue blocks (FFPBs) obtained from the human radiobiological tissue repository. The shelf-life of FFPBs was 5-31 years. An immunohistochemical technique using mouse antibodies against p53 protein (<>, Denmark), stained with diaminobenzidine (DAB) chromogen, was employed to determine p53 protein. DNA was isolated from lung tissue FFPBs with QIAmp DNA FFPE Tissue Kit, (<>, USA). Polymerase chain reaction (PCR) was performed to amplify the p53 gene exons 5, 7, and 8 selected for examination, by applying the sequences of genes and primers, the specificity of which was checked using the online resource (http://www.ncbi.nlm.nih.gov/blast). PCR products were detected by temporal temperature gradient gel-electrophoresis and the Sanger sequencing method. The obtained DNA fragments were analyzed on a sequencer ABI Prism 3100 Genetic Analizer (<>, USA). Computer-aided DNA analysis was made using the BLAST program. A package of applied Statistica 6.0 programs was employed for statistical data processing. Results. Immunohistochemical analysis showed that mutant p53 protein was absent in the cells of unaltered lung tissue and the number of cells with mutant p53 protein increased in all the patients with proliferative and precancerous changes and lung cancer, suggesting p53 protein dysfunction. The total number of p53 gene mutations in exons 5, 7, and 8, if there were proliferative and precancerous lung tissue changes and lung cancer, were 25, 20, and 40%, respectively. All the found mutations were transversions (the substitution of purine for pyrimidine or, conversely), indicating the action of exogenous mutagens. CONCLUSION: The results of this investigation have confirmed other investigators' data showing that p53 gene mutations in lung cancer are observed in 40-70% of cases. The differences in the number of cases of altered lung tissue with mutations in the p53 gene (not more than 40%) and in those of p53 protein expression were found in 100%, suggesting the regulation of p53 gene function in the cell at multiple levels.

[Possibility of DNA isolation from archived autopsy tissues for molecular genetic studies].

Optimal conditions were defined for DNA isolation from formalin-fixed and paraffin-embedded (FFPE) archived autopsy lung tissue slices. The quality of DNA preparations isolated from the FFPE archived slices was assessed and their suitability for further molecular genetic analysis estimated. DNA isolated from the FFPE slices stored less and more than 10 years was suitable for molecular genetic studies in 100 and 66.7% of cases, respectively.

[Analysis of genome instability in offspring of "Mayak" workers families: minisatellite CEB1].

Genome instability transmission in offspring was analyzed in order to evaluate the risk of delayed genetic effects of exposure in 95 family triplets in which only fathers experienced prolonged occupational radiation exposure. The mean total preconceptive absorbed dose (TPAD) of external gamma radiation in the paternal gonads was 1.65 ± 0.08 Gy (dose range of 0.57-5.70 Gy), and the mean TPAD of internal alpha radiation from incorporated plutonium-239 in.the gonads was 0.0015 ± 0.0003 Gy (dose range 0.000-0.015 Gy). The control group consisted of 50 family triplets in which parents were not occupationally exposed. The mutation process was studied using PCR based on hypervariable minisatellite marker CEB 1 (chromosome 2, 2q37.3). The paternal type of inheritance of mutations for minisatellite CEB 1 was found in 80% of cases. The analysis revealed a statistically significant increase in minisatellite CEB1 mutations in the common group of families in which fathers experienced prolonged occupational radiation exposure and in the group of families in which fathers were exposed to radiation in a dosage range of 0.5-1.0 Gy as compared to the control, reaching a significance level of p = 0.109 and p = 0.058, respectively. The dose threshold of mutation detection in the off-spring of Mayak PA workers was estimated.

[Role of gene polymorphisms of phase II of xenobiotic biotransformation from glutathione-S-transferase and N-acetyltransferase families in susceptibility to lung cancer among Mayak workers].

An association between polymorphous (allelic) gene variants of phase II of enzymatic xenobiotic biotransformation (EXB) of multigene families of glutathione-S-transferase (GSTs) GSTM1*0, GSTT1*0, GSTP1*B Ile105Val, and N-acetyltransferase (NAT) NAT2*6 590G>A, NAT2*5 481C>T, as well as lung cancer in Mayak workers exposed occupationally to prolonged external γ-rays and internal α-radiation from incorporated 239Pu was studied. Analysis of the population frequency of genotypes and alleles of the studied genes in the cohort of Mayak workers revealed their compliance with the Hardy-Weinberg principle and with the corresponding frequency in the European population. The study was based on the case-control method. A case-group consisted of 49 Mayal workers with a verified diagnosis of lung cancer. The mean total absorbed dose from external γ-rays at the moment of diagnostics was 1.03 Gy; the mean total absorbed dose from internal α-radiation from incorporated 239Pu to lung was 0.35 Gy. Control consisted of 172 Mayak workers matched by the year of birth, gender, and age at the moment of employment at one of the main facilities with no lung cancer registered within the study period. No increase in the relative risk of lung cancer (odds ratio, OR) was revealed among the individuals with deletion variants of genes GSTM1*0 and GSTT1*0 (pp genotype, complete absence of gene products) as compared to the individuals with ww or wp genotype, which was determined in total for these genes (normal or partly decreased gene activity). An increase in OR of lung cancer in 1.849 times (p = 0.239) and in 2.439 times (p = 0.075) was found in the carriers with a complete absence of the product of genes GSTP1*B and NAT2*6 590G>A, correspondingly (pp genotype). A statistically significant decrease in OR of lung cancer was found in the wp genotype carriers of gene GSTP1*B (OR = 0.50, p = 0.041). Three variants of paired combinations of gene alleles were established in the carriers with a statistically significant increase in OR of lung cancer (ww GSTP1*B + pp GSTM1*0; ww GSTP1*B + pp NAT2*6 590G>A; pp GSTP1*B + pp NAT2*5 481C>T), and one combination in the carriers with a statistically significant decrease in OR of lung cancer (wp GSTP1*B and ww +wp GSTT1*0).

[Role of ancestral genotypes of the genes of xenobiotics biotransformation in susceptibility to lung cancer among Mayak workers].

An association between polymorphous (allelic) variants of genes of the 1st Phase of enzymatic xenobiotic biotransformation (EXB) in the superfamily of cytochromes P450 gene CYP1A1 * 2A (ml polymorphism), CYP1A1 * 2C (m2 polymorphism), gene CYP2E1 * 6 (C polymorphism), and between polymorphous vari- ants of gene of the 2nd EXB Phase of mircosomal epoxide hydrolase mEPOX Tyr113His (EH3 polymor- phism), and mEPOXHis139Arg (EH4 polymorphism) and lung cancer in Mayak workers with occupational prolonged exposure was studied. Analysis of population genotype frequency and alleles of genes CYP1A1 * 2A, CYP1A1 * 2C, CYP2E1 * 6, mEPOX Tyr113His, and mEPOXHis139Arg was conducted in the group of Mayak workers (289 individuals). The study has shown their compliance with the Hardy-Weinberg equilibrium, and correspondence of the genotype frequency to their frequency in European population. The study was per- formed using case-control method. Case-group consisted of 49 Mayak workers with verified diagnosis of lung cancer. Control-group consisted of 172 Mayak workers without lung cancer registered at the moment of the research. There was an increasing trend in odds ratio (OR) of lung cancer among homozygous carriers of the mutation in pp gene CYP1A1 * 2A, heterozygous carriers of the mutation in wp gene mEPOX Tyr113His and homozygous carriers ofww gene CYP2E1 * 6; also, there was a decreasing trend in OR among heterozy- gous carriers of wp gene CYP2E1 * 6. Two variants of pair allelic combinations of genes (genotypes ww CYP1A1 * 2A/genotype wp mEPOX Tyr113His, and genotype ww CYP2E1 * 6/genotype wp mEPOX Tyr113His) were revealed, which carriers had a statistically significant increase in the odds ratio of lung can- cer. With regard to these variants of genotype combinations, OR of lung cancer was 2.01 (CI 95%: 1.04-3.91) and 2.09 (CI 95%: 1.08-4.03), correspondingly.

[Professional chronic radiation exposure: prospects for studying mutagenic effects in generations using the Southern Ural DNA bank of irradiated people and their offspring]. / Professional'noe khronicheskoe radiatsionnoe vozdeistvie: perspektivy izucheniia mutationnykh éffektov v pokoleniiakh na osnove sozdannogo na iuzhnom Urale banka DNK obluchennykh liudei i ikh potomkov.

Data on the established DNA Bank were summarized. The DNA Bank included workers of the Mayak nuclear facility in the Southern Ural, who were exposed to chronic radiation predominantly from external gamma-rays in different doses, and their families for the future study on radiation mutations in somatic cells of parents and possible transmission of genome instability through the germline. At present the DNA Bank contains genetic material from 1500 individuals, among whom there are 223 families.

[A DNA bank for people subjected to occupational irradiation. Goals and prospects]. / Bank DNK liudei podvergshikhsia professional'nomu oblucheniiu. Tseli i perspektivy.

A DNA bank was created to preserve the genetic material of staff of the first Russian atomic plant, Production Association (PO) Mayak, who were exposed to chronic external gamma-irradiation at various doses in the early years of plant operation. Some of the workers experienced combined irradiation from external and internal (incorporated plutonium-239) sources. The unique genetic material of the subjects and their children can be used to study the remote consequences of irradiation by means of modern molecular-genetic techniques. At present, the bank contains the genetic material of 500 people. A database was constructed to include medical, demographic, professional, and dose characteristics of each individual and quantitative and qualitative parameters of each DNA preparation. The bank is continuously augmented with new DNA samples of the irradiated people and members of their families.

[Genome of somatic cells of animals during prolonged radiation exposure]. / Genom somaticheskikh kletok zhivotnykh v usloviiakh dlitel'nogo radiatsionnogo vozdeistviia.

The genome of the thymus and bone marrow of the Wistar rat brain was investigated during daily repeated irradiation at a dose of 0.5 Gy in total doses of 0.5-20 Gy. The genome was assessed by a number of tests characterizing the high and low levels of DNA structural organization. Analysis was performed one day after the last fraction of irradiation. Stable changes in the primary DNA structure of thymocytes were shown by a decrease in the content of double-strand DNA using spectrofluorimetry. Changes in the primary DNA structure of the bone marrow were undetectable. Prolonged irradiation led to the formation of conformation changes in the nucleoid of cells of both tissues detectable by viscosimetric and spectrofluorimetric methods.

[A study of DNA repair in the thymocytes of irradiated rats using a spectrofluorometric method]. / Issledovanie reparatsii DNK timotsitov obluchennykh krys spektrofliuorimetricheskim metodom.

In studying DNA repair in thymocytes of irradiated rats it was shown that the increase in radiation dose from 2 to 20 Gy made DNA damages increase in number and caused changes in their spectrum and growth of irreparable damages. The one-hour study of DNA repair process exhibited its fast, median and slow phases.

[Damage to the structure of DNA and to the metabolism of nucleic acids in the thymus in relation to the dose of tritium oxide]. / Povrezhdenie struktury DNK i metabolizma nukleinovykh kislot timusa v zavisimosti ot dozy okisi tritiia.

Single administration of tritium oxide (1.1-44 MBq per 1 kg of body mass) causes a dose-dependent decrease in the level of rat thymus nucleic acids within the range of concentrations of injected tritium 5.5-44 MBq/g. Biosynthesis of nucleic acids turned out to be more sensitive to tritium oxide action. The rate of biosynthesis changed on the 1st day at the lowest dose of 1.1 MBq/g, the absorbed dose on the 1st day was 7.5 cGy. Dose-dependent disorders of DNA structure in the content of 2-strand DNA after the administration of tritium oxide (9-44 MBq/g) were established.

[Comparison of the biological effects of tritium oxide and gamma radiation based on changes in the mass of the rat thymus gland]. / Sravnenie biologicheskogo deistviia okisi tritiia i gamma-oblucheniia po izmeneniiu massy vilochkovoi zhelezy krys.

RBE of tritium oxide (cumulative doses from 0.33 to 14.7 Gy), in comparison with gamma-radiation, amounted to 2-3 as estimated by the thymus mass. As determined by the rate of injury and repair, the RBE values decreased from 4 to 1.4 with dose increasing, and from 6.5 to 1.3, by the periods of half-decrease and half-recovery of the organ mass. The 2-3-fold variations in the RBE values for various parameters of the organ mass changes were registered at low doses, whereas within the range of median and high doses under study, the differences were insignificant.

[Evaluation of the expression of damage of the DNA-structural complex of rat thymocytes based on kinetics of alkaline lysis following irradiation]. / Otsenka vyrazhennosti narushenii DNK-strukturnogo kompleksa timotsitov krys po kinetike shchelochnogo lizisa posle radiatsionnogo vozdeistviia.

Rotary viscosimeters were used to study the postirradiation destruction of the DNA-structural complex (DSC) of rat thymocyte nuclei exhibited by a change in alkaline denaturation of DSC upon lysis. The S area, limited by the characteristic viscosity values obtained during alkaline lysis of thymocyte nuclei, was used as a characteristic of DSC. Immediately after irradiation the S area changed up to 81-84 per cent at 0.5-1.5 Gy and up to 56-44 per cent at 2-10 Gy. 6 to 24 h following irradiation a change in the profile of alkaline denaturation of DSC was a function of dose and dropped from 100 down to 11 per cent at doses of 0 to 10 Gy. After 2-3 days, the changes in S were also observed but they were not a strict function of dose and were the same with the values obtained immediately after irradiation.

[Relative biological effectiveness of tritium oxide based on DNA destruction and thymus depletion in rats]. / Otnositel'naia biologicheskaia éffektivnost' okisi tritiia po kriteriam destruktsii DNK i opustosheniia vilochkovoi zhelezy krys.

As estimated by DNA destruction and thymus depletion for 21 days of observation, relative biological effectiveness (RBE) of tritium oxide (RBE HTO), as compared to gamma-quanta (137Cs), was 3.1 +/- 0.2 and 3.1 +/- 0.3, respectively. On days 1, 3, 7, 10, 14, and 21 RBE HTO, for the same indices at minimum radiation doses, was about 6-2. As determined by T1/2 period of double-stranded DNA diminution and thymus cell depletion RBE HTO was 5.5 +/- 0.4.

[Changes in the structure and metabolism of rat thymus nucleic acids following cessation of long-term administration of different doses of tritium oxide]. / Izmeneniia struktury i metabolizma nukleinovykh kislot vilochkovoi zhelezy krys posle prekrashcheniia dlitel'nogo vvedeniia okisi tritiia v raznykh dozakh.

A study was made of the processes of restoration of metabolism and nucleic acid indices in rat thymus after the cessation of the long-term administration of various doses of tritium oxide. The restoration of the indices under study was only observed after tritium dose of 0.37 MBq(g.day)-1. At higher doses, 0.925 and 1.85 MBq(g.day)-1 the recovery was transient and the indices changed again.

[Structure of DNA from the rat thymus during prolonged exposure to tritium oxide and external gamma-irradiation]. / Struktura DNK vilochkovoi zhelezy krys pri dlitel'nom vozdeistvii okisi tritiia i vneshnego gamma-oblucheniia.

Thymus depopulation, DNA destruction as estimated according to the level of single-stranded breaks (SSB) and DNA structural derangement revealed by the viscosimetric assay of alkaline lysed thymocytes were observed in the course of a prolonged (1 mos) radiation exposure to tritium oxide (the 1st group) and equidimensional external gamma-irradiation (the 2nd group) in a summary dose of 10 Gy in rats. Depletion and destruction were 1.5-2 times more pronounced in case of HTO exposure. A study of the repair has revealed an increase in the number of hardly repairable or irrepairable breaks by the end of the study: by the 25th day the number of SSB repaired after additional test irradiation was O and 30% in the rats of the 1st and 2nd groups respectively.

[Viscosimetric analysis of the dynamics of alkaline lysis of mammalian cell nuclei as a method of studying radiation damage to DNA structures]. / Viskozimetricheskii analiz dinamiki shchelochnogo lizirovaniia iader kletok mlekopitaiushchikh kak metod izucheniia radiatsionnogo porazheniia struktury DNK.

The method is proposed for the analysis of DNA viscosity during alkaline lysis of rat thymocyte nuclei which permits to reveal early changes in the DNA structure at doses of 0.5 to 1 Gy. Doses from 0.5 to 30 Gy were chosen for the experiment. The changes in the lysis dynamics, the decrease in the intrinsic viscosity maximum [eta], and the increase in the alkaline lysis rate were shown to be a function of radiation dose.

[Nucleic acid metabolism in rat hematopoietic tissues during and after prolonged administration of different doses of tritium oxide]. / Metabolizm nukleinovykh kislot v krovetvornykh tkaniakh krys v protsesse i posle okonchaniia dlitel'nogo vvedeniia okisi tritiia v raznykh dozakh.

During the long-term administration to rats of tritium oxide in doses of 0.37, 0.925 and 1.85 MBq/g body mass the content of karyocytes and nucleic acids in the bone marrow and spleen was decreased, the rate of their biosynthesis changed, the DNA structure impaired, the content of salt-soluble polydeoxynucleotides increased, and DNAases activated. The observed changes were function of dose. After the end of the administration of the isotope the animals which had received a lesser tritium dose exhibited a more rapid and complete recovery.