CHROMOSOME ABERRATIONS AS BIOINDICATORS OF ENVIRONMENTAL GENOTOXICITY

Due to the exposure to various potentially genotoxic xenobiotics, derived from recent war activities such as NATO air strikes with antitank ammunition containing depleted uranium, we have evaluated chromosome aberrations in  peripheral 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 populations. 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.


Introduction
Environmental contamination presents serious threats for human health.From  to , the citizens of Bosnia and Herzegovina were exposed to weapons, used ammunition and waste products of ammunition decay, and they were forced to use expired pharmaceuticals and potentially contaminated food.According to Th e Study of the Battle and Siege of Sarajevo, the city absorbed approximately  shell impacts per day ().Depleted uranium, used in NATO air strikes in  and , is among the potential war-related genotoxins present at certain sites in Bosnia and Herzegovina.In their  report, the United Nations Environment Programme confirmed higher levels of radioactivity at localities that were targets of these strikes ().Depleted uranium contamination and higher radioactivity were detected in samples of soil, water, air, and even lichens from three of  sites, including the tank repair and ammunition storage facility in Hadžići.An additional concern is that six NATO targets surrounding Sarajevo have not been examined, as their coordinates are not available ().Although there have been several nongovernmental and SFOR (Stabilisation Force) decontamination efforts, much of the radioactive ammunition has not yet been removed and the amount of potential contaminants remains high.Depleted uranium is a waste product of the atomic energy industry ().During decay, uranium isotopes emit α particles, which possess high energy but are poorly penetrating.Th us, uranium poses primarily an internal radiation hazard to tissue in close proximity.Uranium isotopes have relatively long half-lives of approximately   -  years (), and are, therefore, a long-lasting threat.Depleted uranium has been identifi ed as an oncogeneinducing factor both in vitro and in vivo ().Also, depleted uranium neoplastically transforms cultured human osteoblasts (), further suggesting that depleted uranium may be involved in cancer induction.An investigation of sixteen British Gulf War and Balkans' War veterans (two females and  males), who were suspected of being exposed to depleted uranium in  and later on, demonstrated a statistically signifi cant over-dispersion of dicentric and centric ring chromosomes in comparison with Bremen laboratory controls ().An increased frequency of malignant tumors was detected in Sarajevo Canton in  and a signifi cant increase in individuals with malignant diseases, the most common being lung and breast cancers, was found after  ().Our previous research of micronuclei frequencies in Sarajevo population due to the war related environmental contamination, in-dicated necessity to perform additional studies ().
In the present study, we have conducted chromosome aberration analysis on peripheral blood lymphocytes from three local human populations from Bosnia and Herzegovina.One of these populations works at a location known to be contaminated with depleted uranium.

Materials and Methods
Subjects were recruited from three local populations and provided their informed consent prior to experimentation.Volunteers who were previously exposed to radiotherapy or chemotherapy, who were exposed to diagnostic X-rays, and those who were using prescription were excluded from the study.One of the study groups was composed of  individuals who inhibited Sarajevo during the war and the postwar period and were directly exposed to war activities.Th e second group included  workers from the tank repair facility in Hadžići, who are suspected of exposure to depleted uranium contamination as a consequence of NATO air strikes.Th e third group served as a reference population, and included  inhabitants of the west Herzegovina region (Posušje).Th is locality was chosen due to a lack of military activities during the war and the absence of known environmental contamination.Th e proportions of males and females, the average age, and the percentage of smokers in the three study groups are presented in Table .
Peripheral blood samples for chromosome aberration analysis were collected in LH vacutainers (BD Vacutainer Systems, Plymonth, UK) during  and .Th e blood was cultivated according to the standardized procedure described by Moorhead et al. (). μl of peripheral blood were added to  ml of  RPMI medium supplemented with L-glutamine,  of fetal bovine serum, PHA and antibiotics (GIBCO-Invitrogen, Carlsbad, CA).Th e cultures were incubated in sterile, plastic, conical -ml tubes (NUNC, Rochester, NY) at °C for  hr.Cell division was blocked by the addition of colcemid (GIBCO-Invitrogen) , hr prior to the end of the cultivation period.For each blood sam-

Results
In the present study, we found that approximately  of the chromosome aberrations in samples from the Sarajevo group were chromosome-type aberrations, while  were chromatid-type aberrations.Th e arithmetic mean of total chromosome aberrations was . per  metaphases, while the means for chromosome-and chromatid-type aberrations were , and , (Table , summarized in Table ).
For the Hadžići group, chromosome-type aberrations accounted for  of all aberrations and chromatid-type aberrations, .Three individual samples from this population had dicentric chromosomes and one of these had two dicentric chromo-somes in  metaphases.The arithmetic mean for chromatid-type aberrations was , and the mean of chromosome-type of aberrations was ,; the arithmetic mean for all aberrations was , per  metaphases (Table , summarized in Table ).
For the Posušje reference group, chromosome-type aberrations accounted for  of all aberrations, and chromatid-type aberrations, .The arithmetic mean for chromatid aberration was , and for chromosome-type aberrations ,; thus, the arithmetic mean for both types of aberrations was , per  metaphases (Table , summarized in Table ).
One-way analysis of variance (ANOVA) followed by pair-wise comparisons revealed that the Hadžići group had a signifi cantly higher frequency of total aberrations than the other two groups (p = ,).There were no significant differences between the aberration frequencies detected for the Sarajevo and Posušje groups.
The frequency of chromosome-type aberrations was significantly higher for the Hadžići group than either of the two other groups (p < ,), while the frequency of chromatid-type aberrations was significantly higher for the Sarajevo and Posušje groups than the group from Hadžići (p = ,).
Stratifi ed analysis revealed that cigarette smoking could be a confounder for association between depleted uranium exposure and chromatid-type aberrations as well as total aberrations, while smoking was not a confounder for chromosome-type aberrations in the three experimental groups.Stratifi ed analysis also indicated that age could have a confounding effect on total aberrations and on chromosome-type aberrations in the individual groups as well as for the entire  samples.
Analysis of point bi-serial coeffi cients for the entire  samples pooled from the three study groups indicated that there was no statistically significant correlation between any of the cytogenetic endpoints and either the sex or the smoking habits of the subjects.Individuals from the Hadžići group were older than the subjects in the other two groups, however, and there was a strong positive linear association between age and the frequency of chromosome aberrations (p < ,).

Discussion
Industrial development is closely related with increased environmental pollution.Various mutagens present in the environment increase the genetic burden of all living populations, including humans.Environmen- tal pollution is a contributing factor to the approximately  of inhabitants of developed countries who die from malignant disease ().Th e frequency of cells with structural chromosomal aberrations in peripheral blood lymphocytes is the fi rst biomarker for which an association has been established with cancer risk ().
In the research conducted by Liou et al. (), the cytogenetic analysis of a reference group and a group of individuals who had developed cancer and who inhabited an area with a known high cancer risk, indicates that the frequency of chromosome aberration in groups at high risk for developing cancer is significantly higher in comparison with control populations.Elevated lung cancer rates have been detected in Gulf War and Balkan confl ict veterans ().We hypothesize that exposure to war-related carcinogens and mutagens, among which depleted uranium has been identifi ed as an oncogene-inducing factor both in vitro and in vivo (), may be involved in this increased cancer incidence.
One-way ANOVA revealed that the Hadžići group had a signifi cantly higher frequency of total aberrations than the other two groups.As the Hadžići group was made up of individuals who work in an area known to be contaminated with depleted uranium, these data support our hypothesis that depleted uranium is a risk factor for cancer in Bosnia and Herzegovina.
Our research revealed that the blood samples of workers in the depleted-uranium-contaminated tank repair facility in Hadžići had an increased frequency of structural chromosome aberrations, including several samples with dicentric chromosomes.This observa-tion is consistent with our previous results demonstrating a signifi cant increase in micronucleus frequency in the group exposed to depleted uranium ().Positive correlations have been reported previously between micronucleus frequency and the frequency of specifi c chromosomal aberrations, such as acentric fragments and dicentric chromosomes ().In addition, a cytogenetic investigation on peripheral blood lymphocytes of three groups of residents from areas of south and central Serbia contaminated with depleted uranium and a group of workers occupationally exposed to X-rays detected an increased frequency of chromosome aberrations among the subjects exposed to depleted uranium.This increase, however, was bellow the frequency of chromosome aberrations in individuals occupationally exposed to ionizing radiation ().In vitro studies with human osteoblast cells suggest that depleted uranium results in genomic instability manifested as delayed reproductive death and micronucleus formation ().
Uranium was released into the environment of Bosnia and Herzegovina at least seven years before the sampling of peripheral blood conducted for this research.As mentioned earlier, however, uranium isotopes have a relatively long half-life and similar studies conducted years after exposure to radioactivity suggest that the genome of peripheral blood lymphocytes is a reliable biological system for cytogenetic analysis even long after exposure to genotoxins.For instance, Neronova et al. () performed cytogenetic analysis - years after the Chernobyl accident and showed an elevated frequency of acentrics, chromatid exchanges, dicentrics and rings in Chernobyl cleanup workers.

Conclusion
Results of this research suggest that exposure to depleted uranium could be a risk factor for human genome.More extensive studies, using better matched study groups, are necessary to confi rm the association between exposure to depleted uranium and human health risk.Prudence dictates, however, that continuous screening should be conducted on the environment and on the health of humans in areas potentially impacted by this contamination.

TABLE 1 .
Details of study groups wise comparisons with Newman-Keuls multiple comparison test.Correlations between the cytogenetical parameters and gender and cigarette smoking were tested by the point bi-serial correlation coefficient.Simple linear regression was applied in order to determine the association between age and the frequency of chromosome aberration.Stratifi ed analysis was used to assess the possible confounding eff ects () of cigarette smoking and age on chromosome aberrations in the three study groups.Th e research was approved and fi nancially supported by the Federal Ministry of Science and Education, Federation of Bosnia and Herzegovina (grant number: ---/).Th e Scientifi c Council of the Institute for Genetic Engineering and Biotechnology approved realization of this research.

TABLE 4
. Standard chromosome aberration analysis of lymphocytes from the Posušje study group TABLE 5. Arithmetic means and variability measures for chromosome aberration data from the three study groups

TABLE 3 .
Standard chromosome aberration analysis of lymphocytes from the Hadžići study group