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 ( : ) 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 One-way analysis of variance (ANOVA) followed by was . per metaphases, while the means for pair-wise comparisons revealed that the Hadžići group chromosome- and chromatid-type aberrations were had a signifi cantly higher frequency of total aberrations , and , (Table , summarized in Table ). than the other two groups (p = ,). There were For the Hadžići group, chromosome-type aber- no significant differences between the aberration fre- rations accounted for of all aberrations and quencies detected for the Sarajevo and Posušje groups. chromatid-type aberrations, . Three individual The frequency of chromosome-type aberrations samples from this population had dicentric chromo- was significantly higher for the Hadžići group than somes and one of these had two dicentric chromo- either of the two other groups (p < ,), while
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 Aberrations/100 metaphases Sample Sample (a) Chromatid (b) Chromosome a+b (a) Chromatid (b) Chromosome a+b 1066 1000 2077 2000 3022 3011 4112 4000 5044 5000 6044 6011 7033 7000 8066 8123 9 0 10 10 9011 10112 10000 11000 11000 12099 12101 13000 13213 14000 14101 15044 15156 16000 16011 17000 17044 18000 18224 19055 19000 20033 20101 21112 21123 22022 22101 23123 23101 24088 24101 25000 25022 26033 26303 27303 TABLE 3. Standard chromosome aberration analysis of lymphocytes 28022 from the Hadžići study group TABLE 4. Standard chromosome aberration analysis of lymphocytes from the Posušje study group the frequency of chromatid-type aberrations was significantly higher for the Sarajevo and Posušje groups than the group from Hadžići (p = ,). that there was no statistically significant correlation between any of the cytogenetic endpoints and either Stratifi ed analysis revealed that cigarette smoking could the sex or the smoking habits of the subjects. Individu- be a confounder for association between depleted als from the Hadžići group were older than the sub- uranium exposure and chromatid-type aberrations as jects in the other two groups, however, and there was well as total aberrations, while smoking was not a con- a strong positive linear association between age and founder for chromosome-type aberrations in the three the frequency of chromosome aberrations (p < ,). experimental groups. Stratifi ed analysis also indicated that age could have a confounding effect on total ab- Discussion errations and on chromosome-type aberrations in the individual groups as well as for the entire samples. Industrial development is closely related with increased environmental pollution. Various mutagens present Analysis of point bi-serial coeffi cients for the entire in the environment increase the genetic burden of all samples pooled from the three study groups indicated living populations, including humans. Environmen-
Aberrations/100 metaphases (a) Chromatid (b) Chromosome a+b Sarajevo Hadžići Posušje Sarajevo Hadžići Posušje Sarajevo Hadžići Posušje
Xav 0,633 0,154 0,679 0,967 3,115 0,857 1,600 3,269 1,536 s 0,718 0,368 0,905 1,188 3,024 1,297 1,453 2,947 1,551 sX av 0,131 0,072 0,171 0,217 0,593 0,245 0,265 0,578 0,293 V 113,428 238,961 133,284 122,854 97,079 151,342 90,812 90,15 100,976
TABLE 5. Arithmetic means and variability measures for chromosome aberration data from the three study groups
BOSNIAN JOURNAL OF BASIC MEDICAL SCIENCES 2007; 7 (4): 311-316 SANIN HAVERIĆ ET AL.: CHROMOSOME ABERRATIONS AS BIOINDICATORS OF ENVIRONMENTAL GENOTOXICITY
tal pollution is a contributing factor to the approxi- tion is consistent with our previous results demonstrat- mately of inhabitants of developed countries who ing a signifi cant increase in micronucleus frequency in die from malignant disease (). Th e frequency of cells the group exposed to depleted uranium (). Positive with structural chromosomal aberrations in peripheral correlations have been reported previously between blood lymphocytes is the fi rst biomarker for which an micronucleus frequency and the frequency of specifi c association has been established with cancer risk (). chromosomal aberrations, such as acentric fragments In the research conducted by Liou et al. (), the cy- and dicentric chromosomes (). In addition, a cytoge- togenetic analysis of a reference group and a group netic investigation on peripheral blood lymphocytes of of individuals who had developed cancer and who three groups of residents from areas of south and cen- inhabited an area with a known high cancer risk, indi- tral Serbia contaminated with depleted uranium and a cates that the frequency of chromosome aberration group of workers occupationally exposed to X-rays de- in groups at high risk for developing cancer is signifi - tected an increased frequency of chromosome aberra- cantly higher in comparison with control populations. tions among the subjects exposed to depleted uranium. Elevated lung cancer rates have been detected in Gulf This increase, however, was bellow the frequency of War and Balkan confl ict veterans (). We hypothesize chromosome aberrations in individuals occupationally that exposure to war-related carcinogens and mutagens, exposed to ionizing radiation (). In vitro studies with among which depleted uranium has been identifi ed as human osteoblast cells suggest that depleted uranium an oncogene-inducing factor both in vitro and in vivo results in genomic instability manifested as delayed (), may be involved in this increased cancer incidence. reproductive death and micronucleus formation ().
One-way ANOVA revealed that the Hadžići group Uranium was released into the environment of Bos- had a signifi cantly higher frequency of total aberrations nia and Herzegovina at least seven years before the than the other two groups. As the Hadžići group was sampling of peripheral blood conducted for this re- made up of individuals who work in an area known search. As mentioned earlier, however, uranium iso- to be contaminated with depleted uranium, these topes have a relatively long half-life and similar studies data support our hypothesis that depleted uranium conducted years after exposure to radioactivity suggest is a risk factor for cancer in Bosnia and Herzegovina. that the genome of peripheral blood lymphocytes is a reliable biological system for cytogenetic analysis even Our research revealed that the blood samples of work- long after exposure to genotoxins. For instance, Ner- ers in the depleted-uranium-contaminated tank re- onova et al. () performed cytogenetic analysis – pair facility in Hadžići had an increased frequency of years after the Chernobyl accident and showed an el- structural chromosome aberrations, including several evated frequency of acentrics, chromatid exchanges, samples with dicentric chromosomes. This observa- 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 de- pleted 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.
BOSNIAN JOURNAL OF BASIC MEDICAL SCIENCES 2007; 7 (4): 311-316 SANIN HAVERIĆ ET AL.: CHROMOSOME ABERRATIONS AS BIOINDICATORS OF ENVIRONMENTAL GENOTOXICITY
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