022-0222148-2137 Genotoxicity of chemical and physical agents of natural and anthropogenic origin



Principal Investigator: Vilena Kašuba

Aleksandra Fučić Davor Želježić
Nevenka Kopjar Vesna Pavlica
Ivan Milas Marin Mladinić
Mirta Milić Ružica Rozgaj
Feodora Stipoljev


Exposure to physical and chemical agents cause damage to cellular structures and macromolecules, which can lead to various pathological changes, impaired body function and development of various diseases, including malignant ones. Mutagens from  living and working environment can indicate clastogenic and / or aneugenic effect. The cellular response to genotoxic and cytotoxic agents is still not fully understood.

The project ˝Genotoxicity of chemical and physical agents of natural and anthropogenic origin˝ is  investigating  mechanisms of action of ionizing radiation and various chemical agents. The aim is to clarify the effects on cells and macromolecules as well as to contribute to the development of ecotoxicological profiles of individual agents and their mixtures.

The study involves in vitro (mammalian cell culture) and in vivo models (mice and rats).The project purpose is to provide the most appropriate experimental model that will indicate the optimal biomarkers of sensitivity and exposure that would have application in assessing exposure and health effects in human epidemiological studies. The international cooperation activities are carried out around the standardization of methods of micronucleus-test and its application in the investigation of possible correlation between the frequency of genomic damage and cancer.
Special attention is on investigations of  to DNA damage and  repair processes. We areInvestigating the interindividual differences in susceptibility to cancer, especially in developing organisms. The intention is to set up cellular and genetic framework for better understanding of the adaptive response and evaluation of its impact on genotoxicity. We are also exploring the dynamics of the frequency and the ratio of stable and unstable aberrations in different professional groups as set forth in the general population of Croatian. Research has been focused on assessing the inter-individual differences in chemo / radio-sensitivity based on polymorphism of genes responsible for the maintenance of genome integrity. It is expected that the results obtained can find application in programs dealing with human and environmental exposure to low doses of ionizing radiation and chemical agents and the possible adverse health effects that may result from these exposures.

The level of  damage in DNA and cellular macromolecules is carried out using molecular, biochemical and biological markers of exposure, effect and sensitivity to lymphocyte cultures  in vitro, cell lines of human origin, and in vivo in experimental animals (adult individuals and entities in development). The project estimates the specificity and determine the extent of genome damage caused by the exposure to low doses of mutagens (ionizing radiation, anesthetic, antineoplastic drugs, acetylcholinesterase inhibitors, pesticides, mycotoxins, the materials used in dentistry, heavy metals, etc.).The dynamics of DNA damage formation and its repair are also explored, and the interdependence of unstable types of damage and translocation with respect to the type of mutagen.

The project is partly focused on basic biological research leading to new insights into the mechanisms of damage, their repair and examined the interaction of agents with genome and their possible effects on enzyme systems of cells. Data on direct and indirect effects of mutagens may indicate critical levels of exposure to low doses of physical and chemical agents that may be the basis for further research on the level of cells, organisms and populations.

Potential applications of this project “Genotoxicity of chemical and physical agents of natural and anthropogenic origin”:

  • Identification of the target sites of action of individual agents at the level of the organism and cells.
  • Finding the most suitable biomarkers to assess exposure, effect and sensitivity in biomonitoring of populations exposed to low doses of physical and chemical mutagens.
  • The results of studies of interactions with cytotoxic drugs and acetylcholinesterase genome can contribute to effective implementation of drug and reduce the level of toxicity and ease the side effects of chemotherapy treated patients.
  • Defining the threshold of sensitivity to the activity of the genome of physical and chemical agents with respect to the age of the children.
  • Comparison of cyto-and genotoxicity of various dental materials to choose those who have the highest biocompatibility in order to protect the health of patients.
  • Most of the research results obtained within the proposed project serves as a basis for introducing and / or re-evaluation of existing guidelines in the legislation in the field of occupational, environmental, residential and natural exposure to ionizing radiation and chemicals.

List of relevant publications  (2007-2010)


1. Brozović G, Oršolić N, Rozgaj R, Kašuba V, Knežević F, Knežević AH, Benković V, Lisičić D, Borojević N, Dikić D.DNA damage and repair after exposure to sevoflurane in vivo, evaluated in Swiss albino mice by the alkaline comet assay and micronucleus test. J Appl Genet 2010; 51(1):1-8

2. Šegvić-Klarić M, Daraboš D, Rozgaj R, Kašuba V, Pepeljnjak S.Beauvericin and ochratoxin A genotoxicity evaluated using the alkaline comet assay: single and combined genotoxic action. Arch Toxicol DOI 10.1007/s00204-010-0535-7

3. Kopjar N, Kašuba V, Milić M, Rozgaj R, Želježić D, Gajski G, Mladinić M, Garaj-Vrhovac V.Normalne i granične vrijednosti mikronukleus-testa na limfocitima periferne krvi u ispitanika opće populacije Republike Hrvatske. Arh Hig Rada Toksikol 2010;61:219-234

4. Kašuba V, Rozgaj R, Gamulin M, Trošić I. Assessment of cyto/genotoxicity of irinotecan in V79 cells using the comet, micronucleus, and chromosome aberration assay. Arh Hig Rada Toksikol 2010;61:1-9

5. Kopjar N, Želježić D, Kašuba V, Rozgaj R. Antineoplastični lijekovi kao čimbenik rizika u radnom okolišu: mehanizmi djelovanja na razini stanice i pregled metoda za otkrivanje njihovih genotoksičnih učinaka. Arh Hig Rada Toksikol 2010;61:121-146. (Review)

6. Kašuba V, Rozgaj R, Milić M, Želježić D, Kopjar N, Pizent A, Kljaković-Gašpić Z.Evaluation of lead exposure in battery-manufactoring workers with focus on different biomarkers. J Appl Toxicol 2010; 30: 321-328.

7. Hrenović J, Želježić D, Kopjar N, Sarpola A, Bronić J, Sekovanić L. Antimicrobial activity of commercial zeolite A on Acinetobacter junii and Saccharomyces cerevisiae. Journal of Hazardous Materials 2010;183(1-3):655-663.

8. Rusak G, Piantanida I, Mašić L, Kapuralin K, Durgo K, Kopjar N. Spectrophotometric analysis of flavonoid-DNA interactions and DNA damaging/protecting and cytotoxic potential of flavonoids in human peripheral blood lymphocytes. Chemico-Biological Interactions 2010;188(1):181-189.



1. Benković V, Horvat Knežević A, Oršolić N, Bašić I, Ramić S, Viculin T, Knežević F, Kopjar N. Evaluation of radioprotective effects of propolis and its flavanoid constituents:in vitro study on human white blood cells. Phytother Res 2009; 23(8):1159-68.

2. Benković V, Horvat Knežević A, Đikić D, Lisičić D, Oršolić N, Bašić I, Kopjar N. Evaluation of the radioprotective effects of propolis and quercetin in gamma irradiated mice: comet assay study. Arh Hig Rada Toksikol 2009;60(2):1-9.

3. Brzović V, Miletić I, Želježić D, Mladinić M, Ramić S, Anić I. In vitro genotoxicity of root canal sealers. Int Endodont J 2009;42(3):253-63.

4. Durgo, K, Oreščanin V,  Lulić S, Kopjar N,  Želježić D,  Franekić Čolić J. The assessment of genotoxic effects of wastewater from a fertilizer factory. J Appl Toxicol 2009;29(1):42-51.

5. Knežević A, Želježić D, Kopjar N, Tarle Z. Influence of curing mode intensities on cell culture cytotoxicity/genotoxicity. Am J Dent 2009;22(1):43-8.

6. Kopjar N, Garaj-Vrhovac V, Kašuba V, Rozgaj R, Ramić S, Pavlica V, Želježić D.Assessment of genotoxic risks in Croatian health care workers occupationally exposed to cytotoxic drugs: a multi biomarker approach. Int J Hyg Environ Health; 2009;212(4):414-31.

7. Kopjar N, Kašuba V, Rozgaj R, Želježić D, Milić M, Ramić S, Pavlica V, Milković-Kraus S. The genotoxic risk in health care workers occupationally exposed to cytotoxic drugs. A comprehensive evaluation by the SCE assay. J Environ Sci Health A 2009;44(5):462-79. (Review)

8. Lucić Vrdoljak A,  Berend S, Želježić D,  Piljac-Žegarac J, Pleština S,  Kuča K,  Radić B,  Mladinić M,  Kopjar N. Irinotecan side effects relieved by the use of HI-6 oxime: in vivo experimental approach. Basic Clin Pharmacol Toxicol 2009;105:401-9

9. Mihaljević Z, Ternjej I, Stanković I, Kerovec M, Kopjar N. Application of the comet assay and detection of DNA damage in haematocytes of medical leech affected by aluminium pollution: A case study. Environ Poll 2009;157(5):1565-72.

10. Mladinić M, Perković P, Želježić D. Characterization of chromatin instabilities induced by glyphosate, terbuthylazine and carbofuran using cytome FISH assay. Toxicol Lett 2009;189:130-7.

11. Mladinić M, Berend S, Lucić Vrdoljak A, Kopjar N, Radić B, Želježić D. Evaluation of genome damage and its relation to oxidative stress induced by glyphosate in human lymphocytes in vitro. Environmental and Molecular Mutagenesis 2009;50:800-7.

12. Oreščanin V, Kopjar N, Durgo K, Elez L, Findri Š, Franekić Čolić J. Cytotoxicity status of electroplating wastewater prior/after neutralization/purification with alkaline solid residue of electric arc furnace dust. J Environ Sci Health A 2009;44(3):273-8.

13. Rozgaj R, Kašuba V, Brozović G, Jazbec AM. Genotoxic effects of anaesthetics in operating theatre personnel evaluated by the comet assay and micronucleus test. Int J Hyg Environ Health 2009; 212: 11-7.

14. Ternjej I, Stanković I, Mihaljević Z, Furač L, Želježić D, Kopjar N. Alkaline comet assay as a potentional tool in the assessment of DNA integrity in freshwater zooplankton affected by pollutants from water treatment facility. Water, Air, Soil Poll 2009;204(1-4):299-314.

15. Želježić D, Vrdoljak Lucić A, Lucas JN, Lasan R, Fučić A, Kopjar N, Katić J, Mladinić M, Radić B. Effect of occupational exposure to multiple pesticides on translocation yield and chromosomal aberrations in lymphocytes of plant workers. Environ Sci Technol 2009;43(16):6370-7.



1. Benković V, Horvat-Knežević A, Đikić D, Lisičić D, Bašić N, Kosalec I, Kopjar N.Radioprotective effect of propolis and quercetin gamma-irradiated mice evaluated by the alkaline comet assay. Phytomedicine 2008;15:851-8.

2. Benković V, Kopjar N, Horvat-Knežević A, Đikić D, Bašić I, Ramić S, Viculin T, Knežević F, Oršolić N. Evaluation of radioprotective effects of propolis and quercetin on human white blood cells in vitro. Biol Parm Bull 2008;31:1778-85.

3. Benković V, Oršolić N, Horvat-Knežević A, Ramić S, Đikić D, Bašić I, Kopjar N.Evaluation of radioprotective effects of propolis and flavonoids in gamma-irradiated mice: the alkaline comet assay study. Biol Parm Bull 2008;31:167-72.

4. Gamulin M, Kopjar N, Grgić M, Ramić S, Bišof V, Garaj-Vrhovac V. Genome damage in oropharyngeal cancer patients treated by radiotherapy. Croat Med J 2008;49:515-27.

5. Garaj-Vrhovac V, Đurinec M, Kopjar N, Oreščanin V. A survey on the cytogenetic status of the Croatian general population by use of the cytokinesis-block micronucleus assay. Mutat Res 2008;649:91-100.

6. Kašuba V, Rozgaj R, Jazbec A. Chromosome aberrations in peripheral blood lymphocytes of croatian hospital staff occupationally exposed to low levels of ionising radiation. Arh hig rada toks 2008; 59(4): 251-259.

7. Klarić MS. Pepeljnjak S. Rozgaj R. Genotoxicity of fumonisin B-1, beauvericin and ochratoxin A in porcine kidney PK15 cells: effects of individual and combined treatment. Croat Chem Acta 2008;81:13946.

8. Knežević A, Želježić D, Kopjar N, Tarle Z. Cytotoxicity of composite materials polymerized with LED curing units. Oper Dent 2008;33:23-30.

9. Kopjar N, Mustafić P, Zanella D, Buj I, Ćaleta M, Marčić Z, Milić M, Dolenec Z, Mrakovčić M. Assessment of DNA integrity in erythrocytes of Cobitis elongata affected by water pollution: the alkaline comet assay study. Folia Zool 2008;57:120-30.

10. Kosalec I, Puel O, Delaforge M, Kopjar N, Antolović R, Jelić D, Matica B, Galtier P, Pepeljnjak S. Isolation and cytotoxicity of low-molecular-weight metabolites of Candida albicans. Frontiers Biosci 2008;13:6893-904.

11. Kosalec I, Šafranić A, Pepeljnjak S, Bačun-Družina V, Ramić S, Kopjar N.Genotoxicity of tryptophol in battery of short-term assays on human white blood cells in vitro. Basic Clin Pharmacol Toxicol 2008;102:443-52.

12. Milić M, Kašuba V, Oreščanin V, Želježić D, Kopjar N, Rozgaj R. Chromosome damage in workers in cigarette manufacturing industry. J Appl Toxicol  2008;28:399-404.

13. Peraica, M, Ljubanović, D, Želježić, D, Domijan, AM. The effect of a single dose of fumonisin B1 on rat kidney. Croat Chem Acta 2008;81(1):119-24.

14. Petek MJ, Berend S, Kopjar N, Želježić D, Mladinić M, Radić B, Lucić-Vrdoljak A.Tenocyclidine treatment in soman poisoned rats – intriguing results on genotoxicity versus protection. Acta Biochim Polon 2008;55.97-105.

15. Zeljezic D, Lucic Vrdoljak A, Kopjar N, Radic B, Milkovic Kraus S. Cholinesterase-inhibiting and genotoxic effects of acute carbofuran intoxication in man: a case report. Basic Clin Pharmacol Toxicol 2008;103:329-35.



1. Fučić A, Želježić D, Kašuba V, Kopjar N, Rozgaj R, Lasan R, Mijić A, Hitrec V, Lucas JN. Stable and unstable chromosome aberrations measured after occupational exposure to ionizing radiation and ultrasound. Croat Med J 2007; 48:371-7.

2. Kopjar N, Milas I, Garaj-Vrhovac V, Gamulin M. Cytogenetic outcomes of adjuvant chemotherapy in non-target cells of breast cancer patients. Hum Exp Toxicol 2007;26:1-9. (znanstveni rad)

3. Kopjar N,  Želježić D, Lucić Vrdoljak A, Radić B, Milić M, Gamulin M, Pavlica V, Fučić A. Irinotecan toxicity to human blood cells in vitro: relationship between various biomarkers. Basic Clin Pharmacol Toxicol 2007;100(6):403-13.

4. Oršolić N, Benković V, Horvat-Knežević A, Kopjar N, Kosalec I, Bakmaz M, Mihaljević Ž, Bendelja K, Bašić I. Assessment by survival analysis of the radioprotective properties of propolis and its polyphenolic compounds. Biol Pharm Bull 2007;30(5):946-51.

5. Gamulin M, Garaj-Vrhovac V, Kopjar N. Evaluation of DNA damage in radiotherapy-treated cancer patients using the alkaline comet assay. Coll Antropol. 2007;31(3):837-45.

6. Ivičić N, Prejac J, Kopjar N, Momčilović B. The sister chromatid exchange rate is inversely related to the Se, Sr, Mn, Cu, and Be trace element nutritional status. J Trace Elements Med Biol 2007;21(S1):74-76.

7. Radić B, Lucić Vrdoljak A, Želježić D, Fuchs N, Berend S, Kopjar N.  Evaluation of HI-6 oxime: potential use in protection of human acetyl-cholinesterase inhibited by antineoplastic drug irinotecan and its cyto/genotoxicity in vitro. Acta Biochim Pol 2007;54(3):583-93.

8. Rozgaj R, Kašuba V. Genotoxic effects of anaesthetics in operating theatre personnel evaluated by the comet assay and micronucleus test. Int J Hyg  Environ Health 2007.

9. KovačevićG, Želježić D, Horvatin K, Kalafatić M. Morphological features and comet assay of green and brown hydra treated with aluminium. Symbiosis  2007;44(1-3):145-52.

10. Domijan AM, Želježić D, Milić M, Peraica M. Fumonisin B-1: Oxidative status and DNA damage in rats. Toxicology 2007;232(3):163-9.



1. Kopjar N, Želježić D, Garaj-Vrhovac V. Evaluation of DNA damage in the white blood cells of healthy human volunteers using the alkaline comet assay and the chromosome aberration test. Acta Biochim Pol 2006:53(2):321-326.

2. Radić B, Lucić-Vrdoljak A, Petek M J, Kopjar N, Želježić D. In vitro biological efficiency of tenocyclidine – TCP and its adamantane derivative TAMORF. Toxicol in Vitro2006;20:1455-1464.

3. Lucić-Vrdoljak A, Radić B, Garaj-Vrhovac V, Kopjar N, Žlender V. Evaluation of antidotal effects of adamantyl derivative Tamorf in soman poisoning. J Appl Toxicol 2006; 26:56-63.

4. Domijan A-M, Želježić D, Kopjar N, Peraica M. Standard and Fpg-modified comet assay in kidney cells of ochratoxin A- and fumonisin B1-treated cells. Toxicology 2006;222:53-59.

5. Garaj-Vrhovac V, Kopjar N, Poropat M. Evaluation of cytogenetic damage in nuclear medicine personnel occupationally exposed to low-level ionising radiation. Arh Hig Rada Toksikol 2006; 57:31-38.

6. Kopjar N, Miočić S, Ramić S, Milić M, Viculin T. Assessment of the radioprotective effects of amifostine and melatonin on human lymphocytes irradiated with gama-rays in vitro. Arh Hig Rada Toksikol 2006;57:155-163.