Chen C.C. (ed.) Selected Topics in DNA Repair

Подождите немного. Документ загружается.

DNA Radiosensitization: The Search for Repair Refractive

Lesions Including Double Strand Breaks and Interstrand Crosslinks

431

difficult to repair without a loss of DNA information, and/or would require major chromatin

remodeling. Indeed, Faruqi et al. (1998) designed PNAs that bind to the supFG1 mutation

reporter gene and found that 8- or 10- b.p. PNA bound to this site induces mutations at

frequencies in the range of 0.1%, well above the in vivo background. Later, the same group

(Kim et al., 2006) demonstrated that a psoralen-bis-PNA conjugate directs the formation of a

photoadduct at the 5’-TpA step of the PNA binding site to the same supFG1 gene. In

mammalian cells, the UV-generated PNA-targeted psoralen phtoadducts induced mutations

as high as 0.46%, i.e. 6.5-fold above the background.

6. Conclusion

In conclusion, there is a need to better understand the parameters which control the

formation and repair of complex DNA lesions, such as interstrand crosslinks. Such complex,

repair refractive lesions may offer a means to selectively kill tumor cells by taking advantage

of either enhanced formation or reduced repair within the tumor environment.

7. References

Aflatooni K., Gallup G. A., Burrow P.D. (1998) Electron Attachment Energies of the DNA

Bases. Journal of Physical Chemistry A, 102, 6205–6207.

Cadet J., Douki T., Ravanat J.-L. (2010) Oxidatively generated base damage to cellular DNA.

Free Radical Biology & Medicine 49, 9–21.

Cai Y., Patel D.J., Geacintov N.E., Broyde S. (2007) Dynamics of benzo[a]pyren-derived

guanine DNA lesion in TGT and CGC sequence contexts: Enhanced mobility in

TGT explains conformational heterogeneity, flexible bending, and greater

susceptibility to nucleotide excision repair. Journal of Molecular Biology 374, 292-305.

Cecchini S., Girouard S., Huels M.A., Sanche L., Hunting D.J. (2004) Single strand specific

radiosensitization of DNA by Bromodeoxyuridine. Radiation Research 162, 604-615.

Cecchini S., Girouard S., Huels M.A., Sanche L., Hunting D.J. (2005a) Interstrand crosslinks:

a new type of gamma-ray damage in bromodeoxyuridine-substituted DNA.

Biochemistry 44, 1932–1940.

Cecchini S., Masson C., La Madeleine C., Huels M.A., Sanche L., Wagner J.R., Hunting D.J.

(2005b) Interstrand cross-link induction by UV radiation in bromodeoxyuridine-

substituted DNA: Dependence on DNA conformation. Biochemistry, 44, 16957-

16966.

Chen J., Stubbe JA. (2004) Synthesis and Characterization of Oligonucleotides Containing a

4‘-Keto Abasic Site. Biochemistry 43, 5278–5286.

Chen T., Cook G.P., Koppisch A.T., Greenberg M.M. (2000) Investigation of the Origin of the

Sequence Selectivity for the 5-Halo-2’-deoxyuridine Sensitization of DNA to

Damage by UV-Irradiation. Journal of the American Chemical Society 122, 3861-3866.

Cutrona G., Carpaneto E.M., Ulivi M., Roncella S., Landt O., Ferrarini M., Boffa L.C. (2000)

Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal.

Nature Biotechnology 18, 300-303.

D’Andrea A.D., Grompe M. (2003) The Fanconi anaemia/BRCA pathway. Nature Reviews

Cancer 3, 23-34.

Dedon P.C. (2008) The Chemical Toxycology of 2-Deoxyribose Oxidation in DNA. Chemical

Research in Toxicology 21, 206-219.

Selected Topics in DNA Repair

432

Demidov V.V., Yavnilovich M.V., Belotserkovskii B.P., Frank-Kamenetskii M.D., Nielsen

P.E. (1995) Kinetics and mechanism of polyamide ("peptide") nucleic acid binding

to duplex DNA. Proceedings of the National Academy of Sciences USA 92, 2637-2641.

Dextraze M.-E., Wagner J.R., Hunting D.J. (2007) 5-Bromodeoxyuridine radiosensitization:

conformation-dependent DNA damage. Biochemistry 46, 9089–9097.

Dextraze M.-E., Cecchini S., Bergeron F., Girouard S., Turcotte K., Wagner J.R., Hunting D.J.

(2009) Reaching for the other side: Generating sequence-dependent interstrand

crosslinks with 5-bromodeoxyuridine and -rays. Biochemistry 48, 2005-2011.

Ding H., Greenberg M.M. (2007) -Radiolysis and Hydroxyl Radical Produce Interstrand

Cross-Links in DNA Involving Thymidine. Chemical Research in Toxicology 20, 1623–

1628.

Ding H., Majumdar A., Tolman J.R., Greenberg M.M. (2008) Multinuclear NMR and Kinetic

Analysis of DNA Interstrand Cross-Link Formation. Journal of the American Chemical

Society, 130, 17981–17987.

Ding H., Greenberg M.M. (2010) DNA Damage and Interstrand Cross-Link Formation upon

Irradiation of Aryl Iodide C-Nucleotide Analogues. Journal of Organic Chemistry 75,

535–544.

Doyle D.F., Braasch D.A., Simmons C.G., Janowski B.A., Corey D.R. (2001) Inhibition of gene

expression inside cells by peptide nucleic acids: Effect of mRNA target sequence,

mismatched bases, and PNA length. Biochemistry 40, 53-64.

Egholm M., Buchardt O., Christensen L., Behrens C., Freir S.M., Driver D.A., Berg R.H., Kim

S.K., Norden B., Nielsen P.E. (1993) PNA hybridizes to complementary

oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365,

566-568.

Faruqi F.A., Egholm M., Glazer P.M. (1998) Peptide nucleic acid-targeted mutagenesis of a

chromosomal gene in mouse cells. Proceedings of the National Academy of Sciences

U.S.A. 95, 1398-1403.

Fazio D., Trindler C., Heil K., Chatgilialoglu C., Carell T. (2011) Investigation of Excess-

Electron Transfer in DNA Double-Duplex Systems Allows Estimation of Absolute

Excess-Electron Transfer and CPD Cleavage Rates. Chemistry A European Journal 17,

206-212.

Gaballah S.T., Collier G., Netzel T.L. (2005) Charge Transfer Excited-State Dynamics in DNA

Duplexes Substituted with an Ethynylpyrenyldeoxyuridine Electron Source and a

Fluorodeoxyuridine Electron Trap. Journal of Physical Chemistry B, 109, 12175–12181.

Gaillard H., Fitzgerald D.J., Smith C.L., Peterson C.L., Richmond T.J., Thoma F. (2003)

Chromatin remodeling activities act on UV-damaged nucleosomes and modulate

DNA damage accessibility to photolyase. Journal of Biological Chemistry 278, 17655-

17663.

Gantchev T.G., Cecchini S., Hunting D.J. (2005) Dynamic conformational states of DNA

containing T•T or BrdU•T mispaired bases: wobble H-bond pairing versus cross-

strand inter-atomic contacts. Journal of Molecular Modeling 11, 141–159.

Gantchev T.G., Hunting D.J. (2008) Probing the interactions of the solvated electron with

DNA by molecular dynamics simulations: bromodeoxyuridine substituted DNA.

Journal of Molecular Modeling 14, 451–464.

DNA Radiosensitization: The Search for Repair Refractive

Lesions Including Double Strand Breaks and Interstrand Crosslinks

433

Gantchev T.G., Hunting D.J. (2009) Probing the interactions of the solvated electron with

DNA by molecular dynamics simulations: II. Bromodeoxyuridine - thymidine

mismatched. Journal of Molecular Modeling 15, 9 –23.

Gantchev T.G., Girouard S., Dodd D.W., Wojciechowski F., Hudson R.H.E., Hunting D.J.

(2009) γ-Radiation Induced Interstrand Cross-Links in PNA:DNA Heteroduplexes.

Biochemistry 48, 7032–7044.

Gospodinov A., Herceg Z. (2011) Chromatin: The entry to and exit from DNA repair. Post-

translational Modifications in Health and Disease, C. Vidal (ed.), Protein Reviews,

Springer, v. 13, pp. 387-409.

Guan L., Greenberg M.M. (2009) DNA Interstrand Cross-Link Formation by the 1,4-

Dioxobutane Abasic Lesion. Journal of the American Chemical Society, 131, 15225–

15231.

Hanada K., Budzowska M., Modesti M., Maas A., Wyman C., Essers J., Kanaar R. (2006) The

structure-specific endonuclease Mus81–Eme1 promotes conversion of interstrand

DNA crosslinks into double-strands breaks. The EMBO Journal 25, 4921–4932.

Hara R., Mo, J. Sancar, A. (2000) DNA damage in the nucleosome core is refractory to repair

by human excision nuclease. Molecular Cell Biology 20, 9173-9181.

Haranczyk M., Gutowski M., Li X., Bowen K.H. (2007) Bound anionic states of adenine.

Proceedings of the National Academy of Sciences 104, 4804–4807.

Hempel K., Mildenberger E. (1987) Determination of G-values for single and double strand

break induction in plasmid DNA using agarose gel electrophoresis and a curve-

fitting procedure. International Journal of Radiation Biology 52, 125-138.

Higgins G.S., Prevo R., Lee Y.-F., Helleday T., Muschel R.J., Taylor S., Yoshimura M.,

Hickson I.D., Bernhard E.J., McKenna W.G. (2010) A Small Interfering RNA Screen

of Genes Involved in DNA Repair Identifies Tumor-Specific Radiosensitization by

POLQ Knockdown. Cancer Research 70, 2984–2993.

Hlavin E.M., Smeaton M.B., Noronha A.M., Wilds C.J., Miller P.S. (2010) Cross-Link

Structure Affects Replication-Independent DNA Interstrand Cross-Link Repair in

Mammalian Cells. Biochemistry 49, 3977–3988.

Hong I.S., Ding H., Greenberg M.M. (2006) Radiosensitization by a Modified Nucleotide that

Produces DNA Interstrand Cross-Links under Hypoxic Conditions. Journal of the

American Chemical Society 128, 2230–2231.

Hunting D.J. et al., unpublished.

Ito T., Kondo A., Kamashita, T., Tanabe K., Yamada H., Nishimoto S. (2009) Pathways of

excess electron transfer in phenothiazine-tethered DNA containing single-base

mismatches, Organic & Biomolecular Chemistry 7, 2077–2081.

Jackson S.P. (2002) Sensing and repairing DNA double-strand breaks. Carcinogenesis 23,

687-696.

Kaihatsu K., Jonowski B.A., Corey D.R. (2004) Recognition of chromosomal DNA by PNAs.

Chemistry & Biology 11, 749-758.

Kim Y., Hong I.S. (2008) PNA/DNA interstrand cross-links from a modified PNA base upon

photolysis or oxidative conditions. Bioorganic and Medicinal Chemistry Letters 18,

5054-5057.

Kim K.-H. Nielsen P.E., Glazer P.M. (2006) Site-specific gene modification by PNAs

conjugated to psoralen. Biochemistry, 45, 314-323.

Selected Topics in DNA Repair

434

Lin G., Zhang J., Zeng Y., Luo H., Wang Y. (2010) Conformation-Dependent Formation of

the G[8-5]U Intrastrand Cross-Link in 5-Bromouracil-Containing G-Quadruplex

DNA Induced by UVA Irradiation. Biochemistry 49, 2346–2350.

Liu T., Ghosal G., Yuan J., Chen J., Huang J. (2010) FAN1 Acts with FANCI-FANCD2 to

Promote DNA Interstrand Cross-Link Repair. Science 329, 693- 696.

Mazumder A., Neamati N., Pilon A.A., Sunder S., Pommier Y. (1996) Chemical trapping of

ternary complexes of human immunodeficiency virus type I integrase, divalent

metal, and DNA substrates containing an abasic site. Journal of Biological Chemistry

271, 27330-27338.

Menchise V., De Simone G., Tedeschi T., Corradini R., Sforza S., Marchelli R., Capasso D.,

Saviano M., Pedone C. (2003) Insights into peptide nucleic acid (PNA) structural

features: The crystal structure of a D-lysine-based chiral PNA-DNA duplex.

Proceedings of the National Academy of Sciences USA 100, 12021-12026.

Méndez-Acuña, L., Di Tomaso M.V., Palitti F., Martínez-López W. (2010) Histone post-

translational modifications in DNA damage response. Cytogenetic and Genome

Research 128, 28-36.

Michaels H.B., Hunt J.W. (1978) A model for radiation damage in cells by direct effect and

by indirect effect: A radiation chemistry approach. Radiation Research 74, 23-34.

Moldovan G.L., D’Andrea A.D. (2009a) FANCD2 Hurdles the DNA Interstrand Crosslink.

Cell 139, 1222.

Moldovan G.-L., D’Andrea A.D. (2009b) How the Fanconi Anemia Pathway Guards the

Genome. Annual Review of Genetics 43, 223.

Nabben F.J., Karman J.Pl., Loman H. (1982) Inactivation of biologically active DNA by

hydrated electrons. International Journal of Radiation Biology 42, 23-30.

Nag R., Smerdon M.J. (2009) Altering the chromatin landscape for nucleotide excision

repair. Mutation Research: Reviews in Mutation Research 682, 13-20.

Nese C., Yuan Z., Schuchmann M.N., von Sonntag C. (1992) Electron transfer from

nucleobase electron adducts to 5-bromouracil. Is guanine an ultimate sink for the

electron in irradiated DNA? International Journal of Radiation Biology 62, 527-541.

Nielsen P.E. (1995) DNA analogues with nonphosphodiester backbones. Annual Review of

Biophysics and Biomolecular Structure 24, 167-183.

Nielsen P.E., Egholm M., Buchardt O. (1994) Sequence-specific transcription arrest by

peptide nucleic acid bound to the DNA template strand. Gene 149, 139-145.

Op de Beeck M., Madder A. (2011) Unprecedented C-Selective Interstrand Cross-Linking

through in Situ Oxidation of Furan-Modified Oligodeoxynucleotides. Journal of the

American Chemical Society 133, 796–807.

Pehrson, J.R. (1995) Probing the conformation of nucleosome linker DNA in situ with

pyrimidine dimer formation. Journal of Biological Chemistry 270, 22440-22444.

Pellestor F., Paulasova P., Hamamah S. (2008) Peptide nucleic acids (PNAs) as diagnostic

devices for genetic and cytogenetic analysis. Current Pharmaceutical Design 14, 2439-

2444.

Peng X., Ghosh A.K., Van Houten B., Greenberg M.M. (2010) Nucleotide Excision Repair of

a DNA Interstrand Cross-Link Produces Single- and Double-Strand Breaks.

Biochemistry 49, 11–19.

DNA Radiosensitization: The Search for Repair Refractive

Lesions Including Double Strand Breaks and Interstrand Crosslinks

435

Peng X., Hong I.S., Li H., Seidman M.M., Greenberg M.M. (2008) Interstrand Cross-Link

Formation in Duplex and Triplex DNA by Modified Pyrimidines. Journal of the

American Chemical Society 130, 10299–10306.

Peterson C.L., Côte J. (2004) Cellular machineries for chromosomal DNA repair. Genes &

Development 18, 602-616.

Pooga M., Land T., Bartfai T., Langel Ü. (2001) PNA oligomers as tools for specific

modulation of gene expression. Biomolecular Engineering 17, 183-192.

Porcheddu A. and Giacomelli G. (1996) PNA-nucleic acid complexes. Structure, stability and

dynamics. Quarterly Reviews of Biophysics 29, 369-394.

Porcheddu A., Giacomelli G. (2005) Peptide nucleic acids (PNAs), A chemical Overview.

Current Medicinal Chemistry 12, 2561-2599.

Quanz M., Berthault N., Roulin C., Roy M., Herbette A., Agrario C., Alberti C., Josserand V.,

Coll J.-L., Sastre-Garau X., Cosset J.-M., Larue L., Sun J.-S., Dutreix M. (2009) Small-

molecule drugs mimicking DNA damage: A new strategy for sensitizing tumors to

radiotherapy. Clinical Cancer Research 15, 1308-1316.

Ray A., Norden B. (2000) Peptide nucleic acid (PNA): its medical and biotechnical

applications and promise for the future, FASEB Journal 14, 1041-1060.

Regulus P., Duroux B., Bayle P.-A., Favier, A., Cadet J., Ravanat J.-L. (2007) Oxidation of the

sugar moiety of DNA by ionizing radiation or bleomycin could induce the

formation of a cluster DNA lesion. Proceedings of the National Academy of Sciences

104, 14032–14037.

Richardson N.A., Gu J., Wang S., Xie Y., Schaefer H.F. III. (2004) DNA Nucleosides and

Their Radical Anions: Molecular Structures and Electron Affinities. Journal of the

American Chemical Society, 126, 4404–4411.

Romanelli A., Pedone C., Saviano M., Bianchi N., Borgatti M., Mischiati C., Gambari R.

(2001) Molecular interactions between nuclear factor B (NF-B) transcription

factors and a PNA-DNA chimera mimicking NF-B binding sites. European Journal

of Biochemistry 268, 6066-6075.

Sawada S., Okada S. (1972) Effects of BUdR-labelling on radiation-induced DNA breakage

and subsequent rejoining in cultured mammalian cells. International Journal of

Radiation Biology 21, 599-602.

Sczepanski J.T., Jacobs A.C., Greenberg M.M. (2008) Self-Promoted DNA Interstrand Cross-

Link Formation by an Abasic Site. Journal of the American Chemical Society, 130, 9646–

9647.

Sczepanski J.T., Jacobs A.C., Majumdar A., Greenberg M.M. (2009a) Scope and Mechanism

of Interstrand Cross-Link Formation by the C4′-Oxidized Abasic Site. Journal of the

American Chemical Society, 131, 11132–11139.

Sczepanski J., Jacobs A.C., Van Houten B., Greenberg M.M. (2009b) Double-Strand Break

Formation during Nucleotide Excision Repair of a DNA Interstrand Cross-Link.

Biochemistry 48, 7565–7567.

Sherer C., Cramer C.J. (2004) Structural and dynamic variations in DNA hexamers

containing T-T and F-F single and tandem internal mispairs. Theoretical Chemistry

Accounts 111, 311-327.

Smerdon M.J., Lieberman, M.W. (1978) Nucleosome rearrangement in human chromatin

during UV-induced DNA-repair synthesis.

Proceedings of the National Academy of

Sciences U.S.A. 75, 4238-4241.

Selected Topics in DNA Repair

436

Špačková N., Berger I., Šponer J. (2000) Nanosecond Molecular Dynamics of Zipper-like

DNA Duplex Structures Containing Sheared G·A Mismatch Pairs. Journal of the

American Chemical Society 122, 7564–7572.

Stokes S.T., Li X., Grubisic A., Ko Y.J., Bowen K.H. (2007) Intrinsic electrophilic properties of

nucleosides: Photoelectron spectroscopy of their parent anions. Journal of Chemical

Physics 127, 084321.

Takata M., Sasaki M.S., Sonoda E., Morrison C., Hashimoto M., Utsumi H., Yamaguchi-Iwai

Y., Shinohara A., Takeda S. (1998) Homologous recombination and non-

homologous end-joining pathways of DNA double-strand break repair have

overlapping roles in the maintenance of chromosomal integrity in vertebrate cells.

EMBO Journal 17, 5497-5508.

Thoma F. (2005) Repair of UV lesions in nucleosomes – intrinsic properties and remodeling.

DNA Repair 4, 855-869.

Van Attikum H., Gasser, S.M. (2005) The histone code at DNA breaks: A guide to repair?

Nature Reviews Molecular Cell Biology. 6, 757-765.

Wang C.-R., Nguyen J., Lu Q.-B. (2009) Bond Breaks of Nucleotides by Dissociative Electron

Transfer of Nonequilibrium Prehydrated Electrons: A New Molecular Mechanism

for Reductive DNA Damage. Journal of the American Chemical Society 131, 11320-

11322.

Wang C.-R., Lu Q.-B. (2010) Molecular Mechanism of the DNA Sequence Selectivity of 5-

Halo-2′-Deoxyuridines as Potential Radiosensitizers. Journal of the American

Chemical Society 132, 14710–14713.

Wang W. (2007) Emergence of a DNA-damage response network consisting of Fanconi

anaemia and BRCA proteins. Nature Reviews Genetics 8, 735-748.

Weng X., Ren L., Weng L., Huang J., Zhu S., Zhou X., Weng L. (2007) Synthesis and

Biological Studies of Inducible DNA Cross-Linking Agents. Angewandte Chemie 119,

8166–8169.

Yoshioka Y., Kitagawa Y., Takano Y., Yamaguchi K., Nakamura T., Saito I.J. (1999)

Experimental and Theoretical Studies on the Selectivity of GGG Triplets toward

One-Electron Oxidation in B-Form DNA. American Chemical Society 121, 8712-8719.

Zeng Y., Wang Y. (2006) Sequence-dependent formation of intrastrand crosslink products

from the UVB irradiation of duplex DNA containing a 5-bromo-20-deoxyuridine or

5-bromo-20-deoxycytidine. Nucleic Acids Research 34, 6521–6529.

Zeng Y., Wang Y. (2007) UVB-induced formation of intrastrand cross-link products of DNA

in MCF-7 cells treated with 5-bromo-20-deoxyuridine. Biochemistry 46, 8189–8195.

Zimbrick J.D., Ward J.F., Myers L.S. Jr. (1969a) Studies on the chemical basis of cellular

radiosensitization by 5-bromouracil substitution in DNA. I. Pulse- and steady-state

radiolysis of 5-bromouracil and thymine", International Journal of Radiation Biology

16, 505-523.

Zimbrick J.D., Ward J.F., Myers L.S. Jr. (1969b) Studies on the chemical basis of cellular

radiosensitization by 5-bromouracil substitution in DNA. II. Pulse- and steady-state

radiolysis of bromouracil-substituted and unsubstituted DNA, International Journal

of Radiation Biology 16, 524-534.

The Influence of Individual Genome Sensitivity

in DNA Damage Repair Assessment in

Chronic Professional Exposure to

Low Doses of Ionizing Radiation

Mirta Milić

, Ružica Rozgaj

, Vilena Kašuba

, Ana Marija Jazbec

Patrizia Hrelia

and Sabrina Angelini

Institute for Medical Research and Occupational Health, Mutagenesis Unit, Zagreb

Faculty of Forestry, University of Zagreb, Zagreb,

Department of Pharmacology,University of Bologna, Bologna,

1,2

Croatia

Italy

1. Introduction

During their working and living activities, humans are constantly exposed to different

environmental genotoxic agents. Biological consequences of the exposure are accumulation

of different mutations and DNA damage that can lead to disruption of genetic material.

Numerous endogenous genotoxic agents can also cause DNA damage. Loss of normal cell

function can cause cell death or can result in different health disorders, including

teratogenic and cancerogenic effects (Jeggo & Lavin, 2009). There is an increasing concern

about mutagenic and cancerogenic effects of genotoxic agents and their influence on

individuals who are exposed to them by accident, or by living/working lifestyle (Au, 1991;

Carrano & Natarajan, 1988; Kassie et al., 2000).

Exposure to ionising radiation, as a well known genotoxic agents (Balasem & Ali, 1991;

Erexon et al., 1991; Fenech et al., 1990; He et al., 2000; Jeggo & Lavin, 2009), can leed to

different DNA damage, such as oxidative damage, base and sugar modification in DNA,

apurinic/apirimidinic places, single/double strand chromosomal breaks, adduct creation,

inter/intra DNA cross linking and other types of damage (Hall & Giaccia, 2006). Numerous

studies deal with ionising radiation exposure of individuals who are chronically

professionally exposed to low doses (Andreassi, 2009; Au, 1991; Carrano & Natarajan, 1988;

Kassie et al., 2000). The results showed the increase in chromosomal damage during

chronically low dose exposure (Barquinero et al., 1993; Boutcher, 1985; Cardoso et al., 2001;

Jha, 1991; Nowak & Jankowski, 1991), but without confirmed relationship between the

received dose and the intensity of DNA damage (Bolus, 2001; Coates et al., 2004; Morgan,

2003; Mothersill et al., 2000, 2001; Seymour & Mothersill, 2000). The influence of chronically

low dose exposure has been considered mutagenic and cancerogenic (Fachini et al., 2009).

Although it is possible to estimate the influence of absorbed dose and the effect on the

Selected Topics in DNA Repair

438

exposed individuals, consequences of continuous low dose exposure are still a great topic of

researches.

The medical stuff represents the best investigated group of professionals exposed to low

levels of ionising radiation (UNSCEAR, 2000). During their work, all the employees wear

dosimeters (film, TLD or electronic ones) that are analysed on monthly bases. In addition,

they are under regular medical control.

Duration and the intensity of exposure to low doses of ionising radiation have significantly

decreased over the last decades. The results of analysed dosimeters showed that the

received doses are significantly below the regulatory threshold of 20 mSv per year,

sometimes even under the dosimeters detection abilities (Kubelka et al., 2011).

Persons employed in emergency medical units and nuclear medicine workers are exposed to

higher doses and show higher DNA damage than workers in diagnostic radiology

departments (Sari-Minodier et al., 2007).

Andreassi et al. (2009) reported higher amount of DNA damage among interventional

cardiologists when compared to clinical cardiologists. Epidemiological researches have shown

the connection between the amounts of accumulated doses and the risk of tumour developing

(Berrington et al., 2001; Maitre et al., 2003; Wang et al., 2002; Yoshinaga et al., 1999). Cardisi et

al. (2005) reported the relationship between exposure to low doses of ionising radiation and

cancer in study that involved 400 000 nuclear industry workers.

Today’s general opinion is that initial event in radiation carcinogenesis is un/ missrepaired

double strand break of DNA molecule, which is a major lesion that leads to developing of

chromosomal abnormalities and genetic mutations (Little, 2000).

Accidental or therapeutical acute exposure to ionising radiation can cause different

cytogenetic damage, including higher amount of micronuclei (small amount of chromatin in

the shape of small nuclei in cytoplasm that was not divided into two new nuclei after first

cell division) and chromosomal aberrations (single /double strand breaks of chromosome,

inter/intra chromosomal exchange, dicentrics, acentrics, etc.). Recently, different biomarkers

are used for monitoring of people occupationally exposed to chronical low doses of ionising

radiation. The use of combined biomarkers can offer better assessment and health care of

those individuals. With them a phenomenon of adaptive response has been observed. This

response can be seen after the first adapting dose of ionising radiation is received (mostly

under 10 cGy). After exposure to higher, so called challenging dose, above 100 cGy, those

individuals show lower amount of DNA damage when compared to those who did not

receive the first, adapting dose. There is also evidence that the amount of dose received can

influence the adaptive response (Gourabi & Mozdarani, 1998). A great variability in DNA

damage response to ionising radiation exposure of cell lines in vitro and individuals in vivo

have been reported (Bosi & Olivieri, 1989; Shadley & Wiencke, 1989). Adaptive response has

been shown also in persons after clinical, environmental or working exposure (Barquinero et

al., 1993, 1995, 1996; Monsieurs et al., 2000; Padovani et al., 1995; Szumiel, 1998; Tedeschi et

al., 1995, 1996). Individual differences in DNA repair genes can also influence on this

response (Milić, 2010).

Molecular mechanisms of adaptive response are still not clear. It has been considered that it

depends on the synthesis and/or protein expression, especially those involved in DNA

repair mechanism (Boothman et al., 1989; Ikushima, 1989, 1996; Robson et al., 1999; Wolf et

al., 1989, 1996; Youngblom et al., 1989). Early induction response (Okayasu et al., 2000;

The Influence of Individual Genome Sensitivity in DNA Damage Repair

Assessment in Chronic Professional Exposure to Low Doses of Ionizing Radiation

439

Stecca & Gerber, 1998), changes in gene expression, gene transcription regulation are also

related with adaptive response. Mutations in genes involved in DNA repair can cause DNA

missrepair, chromosome endings fusion, fusion of unprotected telomere ends and double

strand breaks after exposure to ionising radiation (Bailey et al., 2004; Bailey & Goodwin,

2004; Sasaki et al., 2002).

The accuracy of any risk assessment, especially low dose exposures, depends on both the

resolution of the method, and the baseline data obtained in well-selected controls. Alkaline

comet assay (single-cell gel electrophoresis, SCGE) is an easy-to-use, quick and very

sensitive method for detecting primary DNA strand breaks, that is, direct DNA damage

within single cells (Tice et al., 1990; Collins, 2004) and can be applied to proliferating and

non proliferating cells (Kassie et al., 2000) to determine DNA damage as a result of

endogenous factors, lifestyle (Hoffman & Speit, 2005), and occupational or environmental

exposure (Valverde et al., 1999).

After relaxation, DNA can be seen as a comet during electrophoresis due to strand breaks

(Singh, 2000). The advantages of this technique are sensitivity, reproducibility, easy to use,

low expences. It is a rapid method and the amount of sample necessary for the analysis is

very small. Comet assay enables analysis of any sort of cells, whether it is plant, animal or

human origin, no matter are those single cells from the cell culture or from the tissue. Due to

the short-time performance of this technique (it is possible to have results after few hours), it

has become well accepted in investigation of different genotoxic agents both in vitro and in

vivo conditions (Betti et al., 1994; Kassie et al., 2000; Kruszewski et al., 1998; McKelvey-

Martin et al., 1993; Wojewodzka et al., 1998).

In this method, single cells are embedded into so called agarosis sandwich. Cytoplasm and

membranic cell structures are lysed with high concentrated EDTA solution

(ethylenediaminetetraacetic acid) and detergents, causing total cell DNA to be free. There

are two comet assay protocols, whether alkaline or neutral denaturation is used.

Neutral version enables detection of double stranded DNA breaks (Olive et al., 1990). Under

alkaline conditions, single stranded breaks, alkaline labile sites (AP, parts of DNA that can

break easily in alkaline solutions), DNA-DNA and DNA protein crosslinking can be

detected. Alkaline labile sites are apurinic and apirimidinic DNA bases that can break easily

when exposed to alkaline conditions (Singh et al., 1988). After alkaline/neutral

denaturation, the cells undergo electrophoresis. Concerning the high molecular weight of

the entire DNA molecule, DNA can not pass through the agarose pores towards the anode.

Only the small, damaged fragments can pass through. During electrophoresis, the velocity

of those fragments that are caused by single or double chromosome breaks depends on their

molecular weight. The smaller they are, the faster they get (Plappert et al., 1995). After

electrophoresis, the slides are stained with fluorescent dye (usually ethidium bromide).

Epifluorescent microscope is used for single cells analysing. Undamaged cells have round

shape, while damaged one are similar to the shape of the comet. The mostly used

parameters of comet assay are the tail length (TL), percentage of DNA in tail (TI) and the tail

moment (TM). Tail length (usually expressed in micrometers) is measured from the centre of

the comet head till the end of the comet tail (that is the distance of the fragments from the

major DNA that have travelled through the gel during electrophoresis). It is proportional

with DNA damage and with length of the fragments (Singh et al., 1988; Tice et al., 1990). TI

is measured with computer programme for comet assay analysis. The amount of the

Selected Topics in DNA Repair

440

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

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

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, T-

thymine, 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

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

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

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

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