Radiol Oncol 2005; 39(2): 153-8. Cytogenetic analysis of peripheral blood lymphocytes after arteriography (exposure to x-rays and contrast medium) Ljubomira Popova , Valeria Hadjidekova , Georgy Karadjov , Savina Agova , Danail Traskov , Vassil Hadjidekov National Center of Radiobiology and Radiation Protection, Sofia 1756, Bulgaria 2Department of Medical Genetics, Medical University, Sofia 1431, Bulgaria 3Coordinated Science Laboratory, University of Illinois, IL 61801, USA 4University Hospital Alexandrovska, Department of Radiology, Sofia 1431, Bulgaria Backgrounds. The purpose of our study is to investigate the cytogenetic analysis findings in peripheral blood lymphocytes of 29 patients who had undergone diagnostic radiography. Methods. Peripheral blood samples were taken from 22 patients submitted to renal arteriography and 7 patients submitted to cerebral arteriography (17 male and 12 female, aged between 13-68 years). Cytogenetic analyses of peripheral lymphocytes were performed before the procedure, immediately after and 24 hours later. The entrance skin dose obtained during the whole diagnostic X-ray exposure was measured by thermoluminescent dosimeters and varied between 0.03-0.30 Gy. Both low and high osmolarity contrast media were used. Chromosomal aberrations and micronuclei frequency were used as biomarkers of genotoxicity. Results. The estimated frequency of chromosomal aberrations and micronuclei in the peripheral blood lymphocytes of patients after arteriography examination was significantly higher than the level before the diagnostic exposure. The mean frequency of cells with chromosomal aberrations was nearly double after examination and proved to be constant in the analysis after 24 hours. Conclusions. Radiological diagnostic procedures involving iodinated contrast media as arteriography may cause a significant increase in cytogenetic damage in peripheral blood lymphocytes. Key words: angiography - adverse effects; lymphocytes; chromosome aberrations; micronucleus tests Introduction Iodinated contrast media are largely needed in diagnostic radiology. In angiography and in-Received 17 October 2004 Accepted 11 November 2004 Correspondence to: Vassil Hadjidekov, MD, PhD, University Hospital Alexandrovska, Department of Radiology, Sofia 1431, Bulgaria; Phone: +359 888940801; E-mail: hadjidekov@yahoo.com terventional radiology, especially high diagnostic doses are obtained - relatively long fluoroscopy time plus serial radiography (several frames per second). Cytogenetic analysis findings of diagnostic doses of x-rays and contrast media were investigated in experimental studies on cell cultures in vitro. , Parallel clinical investigations showed an increased genotoxicity in the peripheral blood lymphocytes of the patients undergoing angiography. The results indicate that some contrast media can 154 Popova L et al / Lymphocytes after arteriography induce genotoxic effects alone, and when applied in combination with X-rays, can increase, even double the radiation induced genetic damage. Radiological contrast media do not only increase the absorbed dose, but may also enhance the sensitivity of blood cells to the radiation induced cell damage. Cytogenetic analysis results are of great concern as they are involved in the mechanism of cancer genesis. It is generally accepted that chromosomal mutations are causal events in the development of neoplasia and it has been postulated that an increased cytogenetic damage may be an indication of an enhanced cancer risk. The aim of the present study is to investigate the effects of contrast media and diagnostic radiation on cytogenesis of the peripheral blood lymphocytes of the patients undergoing arteriography. Chromosomal aberrations (CA) and micronuclei (MN) in the peripheral blood lymphocytes are used as cytogenetic biomarkers. Methods Subjects investigated Twenty-nine patients with limited history of previous medical radiation exposures and undergoing angiography examination [22 renal arteriographies (RAr) and 7 cerebral arteriographies (CAr)] were selected for this study. In the selected group of patients, 17 were males and 12 females, ranging in age from 13 to 68 years (average age 41.6 years). A Philips Medical Systems angiographic equipment »PolyDiagnost C« was used with DSI viewing console and Easy Vision workstation. The unit was operated at 60 - 90 kV range and up to 250 mA with a filtration of 2 mm Al. Blood samples were collected in sterile vacationers with Li-heparin. Three samples were taken: (1) before angiographic run, (2) immediately after, and (3) 24 hours after the examination. The radiation exposure assessment was made by thermoluminescent Radiol Oncol 2005; 39(2): 153-8. dosimeters. The radiation exposure varied from 0.03 to 0.30 Gy (Table 1) and was estimated as skin entrance dose. The type and the volume of contrast material used are given in Table 1. For all subjects, a questionnaire was completed to assess their general physical condition, life style, previous x-ray examinations, diets, use of medications. Cytogenetic endpoints Lymphocyte cultures were prepared in 5 ml RPMI-1640 medium supplemented with 10% fetal calf serum and phytochaemaglutinin P. For chromosomal aberration analysis, Colchicine 0.5 mkg/ml was added to the cultures 48 hours after incubation. The cells were harvested two hours later. Twenty-eight subjects were analyzed for chromosomal aberrations (CA). The cells scored per sample for structural chromosomal aberrations after staining with 10% Giemsa ranged from 100 to 400. For cytokinesis blocked micronucleus test, Cytochalasin B was added 44 hours after incubation. The cells were harvested after 72 hours (7). Ten patients were analyzed for the presence of micronuclei (MN) in binucleated lymphocytes immediately before (1) and after (2) radiodiagnostic examination. Two thousand cells per each sample were analyzed. Ethics Informed consent was obtained from all investigated subjects after they had received an explanation of the study. The reports were reviewed and approved by the local ethics committee. The volume of the samples (1) and (2) is the blood collected during the air trapping prevention and catheters flushing. Statistical analysis Student t- test and x - test was applied before and after arteriography of patients to analyze Popova L et al / Lymphocytes after arteriography 155 statistical significance of the difference between the frequencies of chromosomal aberrations and micronuclei formation, respectively. Results A total of 29 subjects submitted to angiography were investigated cytogenetically. Chromosomal aberrations were analyzed in 28 of them, and in 10 subjects, micronuclei forma- tion in binucleated lymphocytes was investigated (Table 2). The frequency of chromosomal aberrations was increased in most of the patients immediately after the examination and remained constant at the sampling after 24 hours (Table 2). Dicentric chromosomes, which are the most sensitive indicators of radiation exposure, were found in 7 cases. It must be noted that, despite selection, some of the patients underwent some kind of radiodi- Table 1. Characteristics of the investigated patients undergoing arteriography No Case Age Sex Smoker Type* Contrast Total of agent volume examination (mg J/ml) (ml) Entrance skin dose (Gy) Ro-exam. Sampling in last time** 1 year CA MN 1. GI 28 M yes CAr iodixanol 320 80 Head CT 1; 2 1; 2 2. KG 45 F CAr iodixanol 320 80 Head CT 1; 2 1; 2 3. DG 56 F no CAr iopromide 300 50 Head CT 1; 2 1; 2 4. RF 44 F RAr iopromide 370 40 IVU 1; 2 1; 2 5. DS 39 M yes RAr iohexol 350 40 no 1; 2 1; 2 6. SM 49 M yes CAr iohexol 350 50 Head CT 1; 2 1; 2 7. DK 36 M yes CAr iohexol 350 50 Head CT 1; 2 1; 2 8. ED64 F no CAr iohexol 350 50 Head CT 1; 2 9. MP 50 M CAr iopromide 300 80 Head CT 1; 2 1; 2 10. PD35 M yes RAr diatrizoate 370 58 0,09 IVU 1; 2; 3 11. II 65 F no RAr diatrizoate 370 52 0,08 Abdominal CT 1; 2; 3 12. EL 60 F no RAr ioxaglate 320 50 0.20 IVU 1; 2; 3 13. PX 42 M RAr diatrizoate 370 46 no 1; 2; 3 14. HI 33 M no RAr diatrizoate 370 46 0,20 no 1; 2; 3 15. SV 13 F no RAr diatrizoate 370 48 0,19 IVU 1; 2; 3 16. HS 17 F no RAr diatrizoate 370 18 0,05 no 1; 2; 3 17. ML 33 F no RAr diatrizoate 370 50 0,15 no 1; 2; 3 18. VI 18 M no RAr diatrizoate 370 60 0.3 IVU 1; 2; 3 19. TG 38 F no RAr diatrizoate 370 40 0.03 no 1; 3 20. PP 58 M yes RAr diatrizoate 370 30 0.11 RA 1; 2; 3 21. AD29 M no RAr diatrizoate 370 36 0.15 no 1; 2; 3 22. VY 68 M no RAr iopromide 300 50 0.19 no 1; 2 1; 2 23. DZ 46 M yes RAr diatrizoate 370 50 no 1; 2; 3 24. SD44 F RAr diatrizoate 370 35 0.26 IVU 1; 2 25. ID21 M yes RAr diatrizoate 370 40 0.08 no 2; 3 26. ME 68 F RAr diatrizoate 370 14 Abdominal CT; IVU 1; 2 27. GV 64 M RAr diatrizoate 370 45 1; 2 28. HP 52 M RAr diatrizoate 370 45 1; 2 29. TZ 34 M no RAr iopromide 300 50 0.03 no 1; 2 * CAr - Cerebral arteriography, RAr - Renal arteriography; **1 - before arteriography, 2 - after arteriography, 3 - 24 hours after arteriography Radiol Oncol 2005; 39(2): 153-8. 156 Popova L et al / Lymphocytes after arteriography Table 2. Frequency of chromosomal aberrations (CA) and micronuclei (MN) in the peripheral blood lymphocyte of the patients undergoing to arteriography Chromosomal aberrations, % No Case Sampling time* CA, No scored cells Cells with CA, % Chromosome Fragments Dicentrics Chromatide Fragments Total Noof CA, % MN, ? scored cells Cells with MN, %o Total Noof MN,% 1. GI 1. 2. 200 250 2 2,8 1 2,4 0 0 1 0,4 2 2,8 2000 2000 16.00 24.00 19.5 27.00 2. KG 1. 2. 200 200 1 1 1 1 0 0 0 0 1 1 2000 2000 8.00 13.00 8.00 15.00 3. DG 1. 2. 200 200 1 0,5 0,5 0,5 0 0 0,5 0 1 0,5 2000 2000 14.50 14.00 17.00 15.50 4. RF 1. 2. 200 200 0.5 1 0 1 0 0 0,5 0 0,5 1 2000 2000 7.00 9.00 7.00 9.50 5. DS 1. 2. 200 200 1 2 0 1,5 0 0 1 0,5 1 2 2000 2000 7.00 5.50 7.00 6.00 6. SM 1. 2. 200 200 1,5 1,5 1 1 0 0 0,5 0,5 1,5 1,5 2000 2000 7.00 11.50 7.00 11.50 7. DK 1. 2. 200 200 1 1,5 0,5 0,5 0 0,5 0,5 0,5 1 1,5 2000 2000 5.00 8.00 5.00 10.00 8. ED1. 2. 200 200 1 0,5 0 0 0 0 1 0,5 1 0,5 9. MP 1. 2. 200 200 0,5 0,5 0 0,5 0 0 0,5 0 0,5 0,5 2000 2000 7.00 10.00 7.00 11.5 10. PD1. 2. 3. 200 200 100 4.5 3 5 3 1.5 2 0.5 0 1 1 1.5 2 4.5 3 5 11. II 1. 2. 3. 200 400 200 3 6 4.5 3 4.25 6 0 0.25 0.5 0.5 1.75 0 3.5 6.25 6.5 12. EL 1. 2. 3. 200 200 100 2 3 3 1 1.5 2 0 0 0 1 1.5 1 2 3 3 13. PX 1. 2. 3. 100 400 200 1 3.25 3.5 0 1.75 2.5 0 0 0 1 1.5 1 1 3.25 3.5 14. HI 1. 2. 3. 0 0 0 0 0 0 2 2 2 2.5 3 5 2 2 3.5 0 0 0 0.5 1 1.5 2.5 3 5 15. SV 1. 2. 3. 100 300 200 1 2.3 4 1 0.7 1 0 0 0 0 1.6 3 1 2.3 4 16. HS 1. 2. 3. 200 400 200 1.5 4 3.5 1 1.5 3 0 0.5 0 0.5 2 0.5 1.5 4 3.5 17. ML 1. 2. 3. 0 0 0 0 0 0 2 2 2 1 5.5 2 1 4.5 2 0 0 0 0 1 0 1 5.5 2 18. VI 1. 2. 3. 0 0 0 0 0 0 2 2 2 1.5 2 3 0.5 1.5 1.5 0 0 0.5 1 0.5 1 1.5 2 3 19. TG 1. 3. 200 200 1.5 2 1 1 0 0 0.5 1 1.5 2 20. PP 1. 2. 3. 0 0 0 0 0 0 2 2 2 1 1.5 3.5 0.5 1 2.5 0 0 0 0.5 0.5 1 1 1.5 3.5 Radiol Oncol 2005; 39(2): 153-8. Popova L et al / Lymphocytes after arteriography 157 No Case Sampling time* CA, No scored cells Cells with CA, % Chromosome Fragments Dicentrics Chromatide Fragments Total Noof CA, % MN, ? scored cells Cells with MN, %o Total Noof MN,% 21. AD1. 2. 3. 200 200 200 0.5 1 0.5 0.5 0.5 0.5 0 0 0 0 0.5 0 0.5 1 0.5 22. VY 1. 2. 200 400 1.5 3.75 1 3.25 0 0 0.5 2.5 1.5 5.75 2000 2000 9.5 12.5 13.5 15.5 23. DZ 1. 2. 3. 100 200 100 0 3.5 3 0 1.5 2 0 0 0 0 2 1 0 3.5 3 24. SD1. 2. 100 100 5 4 3 2 0 0 2 2 5 4 25. ID2. 3. 200 200 3.5 6.5 2 3.5 0 1 1.5 2.5 3.5 7 26. ME 1. 2. 200 300 3 4.7 1.5 2.7 0.5 0.3 1 1.7 3 4.7 27. GV 1. 2. 200 200 2 2 1 1.5 0 0 1 0.5 2 2 28. HP 1. 2. 200 200 2 1.5 0.5 1 0 0 1.5 0.5 2 1.5 29. TZ 1. 2. 2000 2000 14 17.5 15 17.5 *1 - before arteriography, 2 - after arteriography, 3 - 24 hours after arteriography agnostic examination within the year before entering the study (Table 1). The mean frequency of cells carrying chromosomal aberrations in the group of 28 investigated patients was 1.62% ± 0.18 before angiography, and 2.77% ± 0.21 immediately after diagnostic examination (Figure 1). The difference was statistically significant (t = 3.21; PZZZ0.01). The frequency of cells with aberrations was estimated 24 hours after the diagnostic exposure only in 14 subjects and was found to be 3.61 % ± 0.39. The frequency score for the same subjects immediately after angiography was 3.39 % ± 0.32 and did not differ significantly in the analysis after 24 hours (PVVV0.05). In the group of patients submitted to renal arteriography, the frequency of cells with chromosomal aberrations immediately before and after the exposure was 1.81% ± 0.22, and 3.22% ± 0.25, respectively (Table 2), (PZZZ0.01). No increase in the frequency of chromosomal aberrations was observed in the patients who has undergone cerebral arteriography (PVVV0.05). The yield of micronuclei also increased sig- nificantly after angiography (Table 2). The frequency varied from 5%o to 16%o in subjects before, and from 5.5%o to 24%o in different subjects immediately after the examination. The mean values of micronuclei in peripheral lymphocytes of the investigated subjects was 9.5È ± 0.69 before, and 12.5 ± 0.80 after the examination (Figure2). The difference was statistically significant (x 7.85; PZZZ0.01). Discussion In this study, we found a higher frequency of chromosomal aberrations and micronuclei in the group of patients exposed to the diagnostic x-ray with the application of contrast media during angiography compared to their control values before the exposure. The difference was statistically significant for both cytogenetic biomarkers used: chromosomal aberrations (PZZZ0.01) and micronuclei formation (PZZZ0.01). Micronuclei arose in the cytoplasm of binucleated cells as a result of CA induction and they were proved to be a sensitive bioindicator of genotoxic exposure. Radiol Oncol 2005; 39(2): 153-8. 158 Popova L et al / Lymphocytes after arteriography 1.62 I-----T----- D before D after 12- 12.5 — T —, o 10_ 9.5 i-----1------ °- 8- 4- 0- Zl before D after Figure 1. Mean frequency I SE of chromosomal aberrations (CA) and micronuclei (MN) in the patients immediately before and after arteriography. The use of contrast agent in radiodiagnostic arteriography aimed to increase the absorption of X-rays in blood vessels. This was due to the iodine atom included and resulting effect of high photoelectric absorption. As a consequence, the cells in the vicinity of the contrast agent might have absorbed larger radiation dose and might have been exposed to greater cytotoxic effects. This could explain the observed significant genotoxic damage in the peripheral blood lymphocytes of the investigated patients in our study. Previous in vitro studies found that some contrast agents might possess genotoxic properties by themselves and might have a potential to increase the genotoxicity of X-rays as well. 2,4 Previous studies also proved that certain contrast media could also penetrate the epithelial cells through a transcellu-lar mechanism. 8,9 In conclusion, there is a significant increase in the frequency of chromosome damage in the peripheral blood lymphocytes of the subjects undergoing diagnostic arteriography. These results suggest the need for studying the radiosensitizing property of the contrast media to reduce the patient dose without compromising the image quality. Further in vitro studies are needed to elucidate the mechanism of the combined genotoxic effects of io-dinated contrast agents and radiation. References 1. Parvez Z, Kormano M, Satokari K, Moncada R, Eklund R. Induction of mitotic micronuclei by X-ray contrast media in human peripheral lymphocytes. Mutation Res 1987; 188: 233-9. 2. 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