Evaluation of DNA damage in white blood cells of healthy human volunteers using the alkaline comet assay and the chromosome aberration test

• Authors: Nevenka Kopjar , Davor Želježić , Verica Garaj-Vrhovac
• Languages of publication: EN

Abstracts

EN

The present study was undertaken to contribute to the characterization of the degree of variability in baseline damage in white blood cells from control population, and to investigate how this variability is associated with external and internal factors. Altogether 170 healthy volunteers, randomly selected from the general population of the Republic of Croatia, participated in the study. Two sensitive tests: the alkaline comet assay and the chromosome aberration test were applied to study the background levels of DNA damage in their white blood cells. The results point to inter-individual differences, indicating different genome sensitivity. As revealed by both assays, the background levels of DNA damage were mostly influenced by smoking habit as well as medical exposure (especially to diagnostic X-rays). Sex and age of subjects did not significantly influence the values of DNA damage recorded in the white blood cells. Although higher levels of DNA damage were recorded in blood samples collected during winter and autumn, they were mostly influenced by medicinal exposure and smoking habit. Statistical evaluation of the data confirmed that a positive correlation exists between DNA migration and the number of long-tailed nuclei found with the comet assay and the total number of chromosome aberrations. The data obtained can serve as control values in forthcoming biomonitoring studies.

Keywords

EN

peripheral blood; chromosome aberration test; white blood cells; DNA damage; alkaline comet assay; lymphocytes

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2006
• Volume: 53
• Issue: 2
• Pages: 321-336

Dates

published

2006

received

2006-01-16

revised

2006-02-16

accepted

2006-02-23

(unknown)

2006-04-03

Contributors

author

Nevenka Kopjar

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

author

Davor Želježić

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

author

Verica Garaj-Vrhovac

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

References

• Albertini RJ, Anderson D, Douglas GR, Hagmar L, Hemminki K, Merlo F, Natarajan AT, Norppa H, Shuker DEG, Tice R, Waters MD, Aitio A (2000) ICPS guidelines for the monitoring of genotoxic effects of carcinogens in humans. Mutat Res 463: 111-172.
• Anderson D, Jenkinson PC, Dewdney RS, Francis AJ, Godbert P, Butterworth KR (1988) Chromosome aberrations, mitogen-induced blastogenesis and proliferative rate index in peripheral lymphocytes from 106 control individuals of the U.K. population. Mutat Res 204: 407-420.
• Anderson D, Francis AJ, Godbert P, Jenkinson PC, Butterworth KR (1993) Variability in chromosome aberrations, sister chromatid exchanges, and mitogen-induced blastogenesis in peripheral lymphocytes from control individuals. Environ Health Perspect 101: (suppl 3) 83-88.
• Andreoli C, Rossi S, Leopardi P, Crebelli R (1999) DNA damage by hydroquinone in human white blood cells: analysis by alkaline single-cell gel electrophoresis. Mutat Res 438: 37-45.
• Awara WM, El-Nabi SH, El-Gohary M (1998) Assessment of vinyl chloride-induced DNA damage in lymphocytes of plastic industry workers using a single cell gel electrophoresis technique. Toxicology 128: 9-16.
• Bajpayee M, Dhawan A, Parmar D, Kumar Pendey A, Mathur N, Seth PK (2002) Gender-related differences in basal DNA damage in lymphocytes of a healthy Indian population using the alkaline comet assay. Mutat Res 520: 83-91.
• Bender MA, Awa AA, Brooks AL, Evans HJ, Groer PO, Littlefield LG, Pereira C, Preston J, Wachholz BW (1988a) Current status of cytogenetic procedures to detect and quantify previous exposure to radiation. Mutat Res 196: 103-159.
• Bender MA, Preston J, Leonard RC, Pyatt BE, Gooch PC, Shelby MD (1988b) Chromosomal aberration and sister chromatid exchange frequencies in peripheral lymphocytes of a large human population sample. Mutat Res 204: 421-433.
• Benke KK (1995) Biological effects of low-level radiation and diagnostic medical X-rays J Aust Coll Nutr Environ Med 14: 17-20.
• Berrington de González A, Darby S (2004) Risk of cancer from diagnostic X-rays: estimates for the UK and 14 other countries. Lancet 363: 345-351.
• Betti C, Davini T, Giannessi L, Loprieno N, Barale R (1994) Microgel electrophoresis assay (comet test) and SCE analysis in human lymphocytes from 100 normal subjects. Mutat Res 307: 323-333.
• Betti C, Davini T, Giannessi L, Loprieno N, Barale R (1995) Comparative studies by comet assay and SCE analysis in human lymphocytes from 200 healthy subjects. Mutat Res 343: 201-207.
• Bolognesi C, Abbondandolo A, Barale R, Casalone R, Dalprà L, De Ferrari M, Degrassi F, Forni A, Lamberti L, Lando C, Migliore L, Padovani P, Pasquini R, Puntoni R, Sbrana I, Stella M, Bonassi S (1997) Age-related increase of baseline frequencies of sister chromatid exchanges, chromosome aberrations and micronuclei in human lymphocytes. Cancer Epidemiol Biomarkers Prev 6: 249-256.
• Bonassi S, Bolognesi C, Abbondandolo A, Barale R, Bigatti P, Camurri L, Dalprà L, De Ferrari M, Forni A, Lando C, Padovani P, Pasquini R, Stella M, Puntoni R (1995) Influence of sex on cytogenetic end points: evidence from a large human sample and review of the literature. Cancer Epidemiol Biomarkers Prev 4: 671-679.
• Butler PF, Conway BJ, Suleiman OH, Koustenis GH, Showalter CK (1985) Chest radiography: a survey of techniques and exposure levels currently used. Radiology 156: 533-536.
• Chuan C-H, Hu M-L (2004) Use of whole blood directly for single-cell gel electrophoresis (comet) assay in vivo and white blood cells for in vitro assay. Mutat Res 564: 75-82.
• Collins AR (2004) The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol 26: 249-261.
• Frenzilli G, Betti C, Davini T, Desideri M, Fornai E, Giannessi L, Maggiorelli F, Paoletti P, Barale R (1997) Evaluation of DNA damage in leukocytes of ex-smokers by single cell gel electrophoresis. Mutat Res 375: 117-123.
• Garaj-Vrhovac V (1999) Micronucleus assay and lymphocyte mitotic activity in risk assessment of occupational exposure to microwave radiation. Chemosphere 39: 2301-2312.
• Garaj-Vrhovac V, Kopjar N (2000) Cytogenetic monitoring of cardiology unit hospital workers exposed to ultrasound. J Appl Toxicol 20: 259-264.
• Garaj-Vrhovac V, Kopjar N (2003) The alkaline comet assay as biomarker in assessment of DNA damage in medical personnel occupationally exposed to ionizing radiation. Mutagenesis 18: 265-271.
• Garaj-Vrhovac V, Fučić A, Kubelka D, Hebrang A (1997) Assessment of genome damage in occupational exposure to ionizing radiation and ultrasound. Mutat Res 395: 101-105.
• Garaj-Vrhovac V, Kopjar N, Besendorfer V, Papeš D (1999) Induction of micronuclei in human lymphocytes after occupational exposure to ultrasound. Chemosphere 38: 3541-3553.
• Hagelström AH, Gorla NB, Larippa IB (1995) Chromosomal damage in workers occupationally exposed to chronic low level ionizing radiation. Toxicol Lett 76: 113-117.
• Hagmar L, Bonassi S, Strömberg U, Mikoczy Z, Lando C, Hansten I-L, Huici Montagud A, Knudsen L, Norppa H, Reuterwall C, Tinnerberg H, Brøgger A, Forni A, Högstedt B, Lambert B, Mitelman F, Nordenson I, Salomaa S, Skerfving S (1998) Cancer predictive value of cytogenetic markers used in occupational health surrveilance programs: a report from an ongoing study by the European Study Group on Cytogenetic Biomarkers and Health. Mutat Res 405: 171-178.
• Hellman B, Vaghef H, Friis L, Edling C (1997) Alkaline single cell gel electrophoresis of DNA fragments in biomonitoring for genotoxicity: an introductory study on healthy human volunteers. Int Arch Occup Environ Health 69: 185-192.
• Hellman B, Friis L, Vaghef H, Edling C (1999) Alkaline single cell gel electrophoresis and human biomonitoring for genotoxicity: a study on subjects with residential exposure to radon. Mutat Res 442: 121-132.
• Hininger I, Chollat-Namy A, Sauvagio S, Osman M, Faure H, Cadet J, Favier A, Rousell A-M (2004) Assessment of DNA damage by comet assay on frozen total blood: method and evaluation in smokers and non-smokers. Mutat Res 558: 75-80.
• Hoffman H, Speit G (2005) Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test. Mutat Res 581: 105-114.
• International Atomic Energy Agency (2001) International Atomic Agency Technical Report Series 405, Cytogenetic Analysis for Radiation Dose Assessment (Vienna: IAEA).
• Kassie F, Parzefall W, Knasmüller S (2000) Single cell gel electrophoresis assay: a new technique for human biomonitoring studies. Mutat Res 463: 13-31.
• Kašuba V, Šentija K, Garaj-Vrhovac V, Fučić A (1995) Chromosome aberrations in peripheral blood lymphocytes from control individuals. Mutat Res 346: 187-193.
• Kašuba V, Rozgaj R, Kopjar N, Želježić D, Jazbec A (2002) Sister chromatid exchange analysis in peripheral blood lymphocytes of healthy individuals. Biologia 57: 401-407.
• Kopjar N, Garaj-Vrhovac V (2001) Application of the alkaline comet assay in human biomonitoring for genotoxicity: a study on Croatian medical personnel handling antineoplastic drugs. Mutagenesis 16: 71-78.
• Landi S, Frenzilli G, Milillo PC, Cocchi L, Sbrana I, Scapoli C, Barale R (1999) Spontaneous sister chromatid exchange and chromosome aberration frequency in humans: the familial effect. Mutat Res 444: 337-345.
• Major J, Jakab MG, Tompa A (1998) Genotoxicological monitoring of 175 subjects living in the green belts, inner town of near chemical industrial estates in Greater Budapest agglomeration, Hungary. Mutat Res 412: 9-16.
• Maluf SW, Ferreira Passos D, Bacelar A, Speit G, Erdtman B (2001) Assessment of DNA damage in lymphocytes of workers exposed to X-radiation using the micronucleus test and the comet assay. Environ Mol Mutagen 38: 311-315.
• Mastaloudis A, Yu T-W, O’Donnel RP, Frei B, Dashwood RH, Traber MG (2004) Endurance exercise results in DNA damage as detected by the comet assay. Free Radic Biol Med 36: 966-795.
• Mayer C, Popanda O, Zelezny O, von Brevern MC, Bach A, Bartsch H, Schmetzer P (2002) DNA repair capacity after γ-irradiation and expression profiles of DNA repair genes in resting and proliferating human peripheral blood lymphocytes. DNA Repair 1: 237-250.
• Mendoza-Núñez VM, Sánchez-Rodríguez MA, Retana-Ugalde R, Vargas-Guadarrama LA, Altamirano-Lozano MA (2001) Total antioxidant levels, gender, and age as risk factors for DNA damage in lymphocytes of the elderly. Mech Ageing Dev 122: 835-847.
• Møller P, Knudsen LE, Frentz G, Dybdahl M, Wallin H, Nexø BA (1998) Seasonal variation of DNA damage and repair in patients with non-melanoma skin cancer and referents with and without psoriasis. Mutat Res 407: 25-34.
• Møller P, Knudsen LE, Loft S, Wallin H (2000) The comet assay as rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors. Cancer Epidemiol Biomarkers Prev 9: 1005-1015.
• Møller P, Wallin H, Holst E, Knudsen LE (2002) Sunlight-induced DNA damage in human mononuclear cells. FASEB J 16: 45-53.
• Moretti M, Villarini M, Scassellati Sforzilini G, Pasquini R (2000) Pesticide-induced primary DNA damage in peripheral blood leukocytes of farm workers evaluated by the computerized “comet” assay. Biomarkers 5: 192-204.
• Morillas MJ, Guillamet E, Surralles J, Creus A, Marcos R (2002) Spontaneous and induced genetic damage in T lymphocyte subsets evaluated by the comet assay. Mutat Res 514: 39-48.
• Natarajan, A. T (1993) Mechanisms for induction of mutations and chromosome alterations. Environ Health Perspect 101: (suppl 3) 225-229.
• Natarajan AT, Boei JJ, Darroudi F, Van Diemen PCM, Dulout F, Hande MP, Ramalho AT (1996) Current cytogenetic methods for detecting exposure and effects of mutagens and carcinogens. Environ Health Perspect 104: (suppl 3) 445-248.
• Palus J, Dziubałtowska E, Rydzynski K (1999) DNA damage detected by the comet assay in the white blood cells of workers in a wooden furniture plant. Mutat Res 444: 61-74.
• Pfeiffer P, Goedecke W, Obe G (2000) Mechanisms of DNA double strand break repair and their potential to induce chromosomal aberrations. Mutagenesis 15: 289-302.
• Piperakis SM, Visvardis EE, Sagnou M, Tassiou AM (1998) Effects of smoking and aging on oxidative DNA damage of human lymphocytes. Carcinogenesis 19: 695-698.
• Pitarque M, Vaglenov A, Nosko M, Hirvonen A, Norppa H, Creus A, Marcos R (1999) Evaluation of DBA damage by the comet assay in shoe workers exposed to toluene and other organic solvents. Mutat Res 441: 115-127.
• Rehani MM (2000) CT: caution on radiation dose. Ind J Radiol Imaging 10: 19-20.
• Rojas E, Lopez MC, Valverde M (1999) Single cell gel electrophoresis assay: methodology and applications. J Chromatogr B Biomed Sci Appl 722: 225-254.
• Rozgaj R, Kašuba V, Šentija K, Prlić I (1999) Radiation-induced chromosomal aberrations and haematological alterations in hospital workers. Occup Med 49: 353-360.
• Rozgaj R, KašubaV, Jazbec A (2001) Preliminary study of cytogenetic damage in personnel exposed to anesthetic gases. Mutagenesis 16: 139-143.
• Şardaş S, Aygün N, Gamli M, Ünal Y, Berk N, Karakaya AE (1998) Use of alkaline comet assay (single cell gel electrophoresis technique) to detect DNA damages in lymphocytes of operating room personnel occupationally exposed to anaestethic gases. Mutat Res 418: 93-100.
• Singh NP (2000) Microgels for estimation of DNA strand breaks, DNA protein crosslinks and apoptosis. Mutat Res 455: 111-127.
• Singh NP, Mc Coy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175: 184-191.
• Singh NP, Danner DB, Tice RR, Pearson JD, Brant LJ, Morrell CH, Schneider EL (1991) Basal DNA damage in individual human lymphocytes with age. Mutat Res 256: 1-6.
• Somorovská M, Szabová E, Vodička P, Tulinská J, Barančoková M, Fábry R, Liškova A, Riegerová Z, Petrovská H, Kubová J, Rausová K, Dušinská M, Collins AR (1999) Biomonitoring of genotoxic risk in workers in a rubber factory: comparison of the comet assay with cytogenetic methods and immunology. Mutat Res 445: 181-192.
• Speit G, Witton-Davies T, Heepchantree W, Trenz K, Hoffman H (2003) Investigations on the effect of cigarette smoking in the comet assay. Mutat Res 542: 33-42.
• Stephan G, Pressl S (1999) Chromosomal aberration in peripheral lymphocytes from healthy subjects as detected in first cell division. Mutat Res 446: 231-237.
• Šrám RJ, Podrazilová K, Dejmek J, Mračková G, Pilčík T (1998) Single cell gel electrophoresis assay: sensitivity of peripheral white blood cells in human population studies. Mutagenesis 13: 99-103.
• Tice RR (1995) The single cell gel / comet assay: a microgel electrophoretic technique for the detection of DNA damage and repair in individual cells. In Environmental Mutagenesis (Phillips DH, Vennit S, eds), pp 315-339 Oxford, Bioscientific.
• Tice RR, Agurell E, Anderson D, Burlinson B, Hartmenn A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35: 206-121.
• Valverde M, Ostrosky-Wegman P, Rojas E, Fortoul E, Meneses MNF, Ramírez M, Díaz-Barriga F, Cebrian M (1999) The application of single cell gel electrophoresis or comet assay to human monitoring studies. Salud Pública Méx 41: 109-113.
• Wojewódzka M, Kruszewski M, Iwanenko T, Collins AR, Szumiel I (1998) Application of the comet assay for monitoring DNA damage in workers exposed to chronic low-dose irradiation. I. Strand breakage. Mutat Res 416: 21-35.
• Wojewódzka M, Kruszewski M, Iwanenko T, Collins AR, Szumiel I (1999) Lack of adverse effect of smoking habit on DNA strand breakage and base damage, as revealed by the alkaline comet assay. Mutat Res 440: 19-25.
• Zhu CQ, Lam TH, Jiang CQ, Wei BX, Lou X, Liu WW, Lao XQ, Chen YH (1999) Lymphocyte DNA damage in cigarette factory workers measured by the comet assay. Mutat Res 444: 1-6.
• Zhu CQ, Lam TH, Jiang CQ (2001) Lymphocyte DNA damage in bus manufacturing workers. Mutat Res 491: 173-181.
• Želježić D, Garaj-Vrhovac V (2001) Chromosomal aberration and single-cell gel electrophoresis (comet) assay in the longitudinal risk assessment of occupational exposure to pesticides. Mutagenesis 16: 359-363.
• Želježić D, Garaj-Vrhovac V (2002) Sister chromatid exchange and proliferative rate index in the longitudinal risk assessment of occupational exposure to pesticides. Chemosphere 46: 295-303.