CHROMOSOME ABERRATIONS AS BIOINDICATORS OF ENVIRONMENTAL &GENOTOXICITY Slavica Ibrulj¹,² , Sanin Haverić²*, Anja Haverić² ¹ Faculty of Medicine, University of Sarajevo, Čekaluša , Sarajevo, Bosnia and Herzegovina Institute for Genetic Engineering and Biotechnology, Gajev trg , Sarajevo, Bosnia and Herzegovina * Corresponding author Abstract Due to the exposure to various potentially genotoxic xenobiotics, derived from recent war activities such as NATO air strikes with antitank ammunition contain- ing depleted uranium, we have evaluated chromosome aberrations in periph- eral blood samples from three local populations. One population sample included individuals who lived in the Sarajevo area during and after the war (exposed to potential genotoxins), second population was presented with employees of the tank repair facility in Hadžići (target of NATO air strikes), and inhabitants of Posušje (not exposed to war-related activities) were treated as sample of control population. Th e mean of chromosome aberration frequencies for the population from Hadžići was signifi cantly higher than the frequencies for the two other popu- lations. Point bi-serial coeffi cient analysis did not reveal any relationship between the frequencies of chromosome aberrations and smoking habits or gender. Results suggest that depleted uranium could be a risk factor for human health. KEY WORDS: environmental genotoxins, depleted uranium, human lymphocytes BOSNIAN JOURNAL OF BASIC MEDICAL SCIENCES 2007; 7 (4): 311-316 SANIN HAVERIĆ ET AL.: CHROMOSOME ABERRATIONS AS BIOINDICATORS OF ENVIRONMENTAL GENOTOXICITY Introduction dicated necessity to perform additional studies (). In the present study, we have conducted chromosome Environmental contamination presents serious threats aberration analysis on peripheral blood lymphocytes for human health. From to , the citizens of from three local human populations from Bosnia and Bosnia and Herzegovina were exposed to weapons, Herzegovina. One of these populations works at a loca- used ammunition and waste products of ammunition tion known to be contaminated with depleted uranium. decay, and they were forced to use expired pharmaceu- ticals and potentially contaminated food. According to Materials and Methods Th e Study of the Battle and Siege of Sarajevo, the city absorbed approximately shell impacts per day (). Subjects were recruited from three local populations Depleted uranium, used in NATO air strikes in and provided their informed consent prior to experi- and , is among the potential war-related genotox- mentation. Volunteers who were previously exposed ins present at certain sites in Bosnia and Herzegovina. to radiotherapy or chemotherapy, who were exposed In their report, the United Nations Environment to diagnostic X-rays, and those who were using pre- Programme confirmed higher levels of radioactivity scription were excluded from the study. One of the at localities that were targets of these strikes (). De- study groups was composed of individuals who pleted uranium contamination and higher radioac- inhibited Sarajevo during the war and the postwar pe- tivity were detected in samples of soil, water, air, and riod and were directly exposed to war activities. Th e even lichens from three of sites, including the tank second group included workers from the tank re- repair and ammunition storage facility in Hadžići. pair facility in Hadžići, who are suspected of exposure An additional concern is that six NATO targets sur- to depleted uranium contamination as a consequence rounding Sarajevo have not been examined, as their of NATO air strikes. Th e third group served as a refer- coordinates are not available (). Although there have ence population, and included inhabitants of the west been several nongovernmental and SFOR (Stabilisa- Herzegovina region (Posušje). Th is locality was chosen tion Force) decontamination efforts, much of the ra- due to a lack of military activities during the war and dioactive ammunition has not yet been removed and the absence of known environmental contamination. the amount of potential contaminants remains high. Th e proportions of males and females, the average age, Depleted uranium is a waste product of the atomic en- and the percentage of smokers in the three study groups ergy industry (). During decay, uranium isotopes emit are presented in Table . α particles, which possess high energy but are poorly Number of Gender penetrating. Th us, uranium poses primarily an internal Average Population analyzed SD ratio Smokers radiation hazard to tissue in close proximity. Uranium age (years) individuals ( : V) isotopes have relatively long half-lives of approximately Sarajevo 30 32,37 9,90 33% : 67% 47% - years (), and are, therefore, a long-lasting threat. Posušje 28 39,57 12,54 43% : 57% 43% Depleted uranium has been identifi ed as an oncogene- Hadžići 26 46,46 5,97 92% : 8% 46% inducing factor both in vitro and in vivo (). Also, deplet- Total 84 39,13 11,38 55% : 45% 46% ed uranium neoplastically transforms cultured human TABLE 1. Details of study groups osteoblasts (), further suggesting that depleted uranium may be involved in cancer induction. An investigation of Peripheral blood samples for chromosome aberration sixteen British Gulf War and Balkans’ War veterans (two analysis were collected in LH vacutainers (BD Vacu- females and males), who were suspected of being ex- tainer Systems, Plymonth, UK) during and . posed to depleted uranium in and later on, demon- Th e blood was cultivated according to the standardized strated a statistically signifi cant over-dispersion of dicen- procedure described by Moorhead et al. (). μl tric and centric ring chromosomes in comparison with of peripheral blood were added to ml of RPMI Bremen laboratory controls (). An increased frequency medium supplemented with L-glutamine, of fetal of malignant tumors was detected in Sarajevo Canton in bovine serum, PHA and antibiotics (GIBCO-Invitrogen, and a signifi cant increase in individuals with malig- Carlsbad, CA). Th e cultures were incubated in sterile, nant diseases, the most common being lung and breast plastic, conical -ml tubes (NUNC, Rochester, NY) cancers, was found after (). Our previous research at °C for hr. Cell division was blocked by the ad- of micronuclei frequencies in Sarajevo population due dition of colcemid (GIBCO-Invitrogen) , hr prior to to the war related environmental contamination, in- the end of the cultivation period. For each blood sam- BOSNIAN JOURNAL OF BASIC MEDICAL SCIENCES 2007; 7 (4): 311-316 SANIN HAVERIĆ ET AL.: CHROMOSOME ABERRATIONS AS BIOINDICATORS OF ENVIRONMENTAL GENOTOXICITY Aberrations/ 100 metaphases ple, metaphase spreads containing μ chromo- Sample (a) Chromatid (b) Chromosome a+b somes were analyzed microscopically. Aberrations were 1011 scored according to International System for Human 2112 Cytogenetic Nomenclature (). Verifi ed aberrations 3213 were subclassifi ed as chromosome-type (chromosome 4101 breaks, dicentric chromosomes, acentric and minute 5000 6022 fragments) and chromatid-type aberrations (chromatid 7202 breaks). Gaps were not scored as aberrations (, ). 8123 The results for each study group were expressed as 9022 10112 arithmetic means (Xav) and variability measures (stan- 11000 dard deviation - s, standard error of the mean - sX , and av 12000 coeffi cient of variation - V). Statistical analysis included 13000 analysis of variance (ANOVA), point bi-serial correla- 14033 tion coeffi cients, simple linear regression and stratifi ed 15224 analysis conducted with Winks . Professional software 16123 17000 (TexaSoft, Cedar Hill, TX). 18202 The significance of differences between arithmetic 19123 means of the cytogenetic parameters for the study 20112 groups was determined by ANOVA followed by pair- 21112 wise comparisons with Newman-Keuls multiple com- 22011 23156 parison test. Correlations between the cytogenetical pa- 24000 rameters and gender and cigarette smoking were tested 25101 by the point bi-serial correlation coefficient. Simple 26022 linear regression was applied in order to determine the 27101 association between age and the frequency of chromo- 28000 29000 some aberration. Stratifi ed analysis was used to assess 30000 the possible confounding eff ects () of cigarette smok- TABLE 2. Standard chromosome aberration analysis of lymphocytes ing and age on chromosome aberrations in the three from the Sarajevo study group study groups. Th e research was approved and fi nancially supported by somes in metaphases. The arithmetic mean for the Federal Ministry of Science and Education, Federa- chromatid-type aberrations was , and the mean tion of Bosnia and Herzegovina (grant number: -- of chromosome-type of aberrations was ,; the -/). Th e Scientifi c Council of the Institute for arithmetic mean for all aberrations was , per Genetic Engineering and Biotechnology approved real- metaphases (Table , summarized in Table ). ization of this research. For the Posušje reference group, chromosome-type aberrations accounted for of all aberrations, and Results chromatid-type aberrations, . The arithmetic mean for chromatid aberration was , and for In the present study, we found that approximately chromosome-type aberrations ,; thus, the arith- of the chromosome aberrations in samples from metic mean for both types of aberrations was , the Sarajevo group were chromosome-type aberra- per metaphases (Table , summarized in Table ). tions, while were chromatid-type aberrations. Th e arithmetic mean of total chromosome aberrations