Similar cytogenetic effects of sodium-meglumine diatrizoate and sodium-meglumine ioxithalamate in lymphocytes of patients undergoing brain CT scan

Toxicology Letters 98 (1998) 25 – 30

Similar cytogenetic effects of sodium-meglumine diatrizoate and sodium-meglumine ioxithalamate in lymphocytes of patients undergoing brain CT scan Hossein Mozdarani *, Shahram Fadaei Department of Radiology, School of Medical Sciences, Tarbiat Modarres Uni6ersity, P.O. Box: 14155 -4838, Tehran, Iran Received 9 March 1998; received in revised form 5 May 1998; accepted 7 May 1998

Abstract Cytogenetic effects of two ionic contrast media (CM), Urografin 76% a sodium-meglumine diatrizoate, and Telebrix 38, a sodium-meglumine ioxythalamate, were tested on lymphocytes of patients undergoing brain CT Scan. Both compounds have approximately similar iodine concentrations. Chromosomal aberrations were scored in peripheral lymphocytes obtained from 15 patients undergoing brain CT with either urografin 76% or telebrix 38 before and after examination. Results showed no difference in aberration frequency for patients who underwent brain CT without contrast materials compared to controls. However, injection of CM resulted in a high frequency of chromosomal aberrations which significantly differed from controls (PB 0.05). The effect of urografin 76% appeared to be similar to telebrix 38. Therefore, both CM exhibited clastogenic effects on peripheral lymphocytes in vivo. An increase in chromosomal aberrations due to CM used in this study were similar to that reported for other ionic and non-ionic compounds. © 1998 Elsevier Science Ireland Ltd. All rights reserved.

1. Introduction Apart from the systemic reactions, chromosomal damage in lymphocytes of patients undergoing radiologic examinations with contrast media has been demonstrated. Clastogenic effects of X* Corresponding author. Present address: St. Andrews University, School of Biomedical Sciences, Bute Medical Buildings St Andrews, Scotland KY16 9TS, UK; fax: + 44 1334 46300; [email protected]

ray CM was reported by various investigators for both ionic and non-ionic CM using metaphase analysis or micronuclei scoring (Adams et al., 1977; Stentrand and Edgren, 1981; Parvez et al., 1987; Cochran and Norman, 1994a,b). Study of newly developed low osmolarity CM has also shown similar effects on lymphocytes of patients who underwent excretory urography (Cochran and Norman, 1994b). Although cytogenetic effects of CM was shown by in vitro lymphocyte culture (Stentrand and Edgren, 1981), most of the

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H. Mozdarani, S. Fadaei / Toxicology Letters 98 (1998) 25–30

in vivo studies were associated with relatively large doses of radiation such as in excretory urography (Cochran and Norman, 1994b), angiocardiography (Adams et al., 1977) and digital subtraction angiography (DSA) (Parvez et al., 1987). In all these examinations the in vivo effect of radiation on lymphocytes cannot be excluded, so the synergistic effects of radiation with CM cannot be ignored. On the other hand, the amount of CM injected was mainly based on iodine concentration (Parvez et al., 1987). Studies with non-ionic CM showed that iodine might not be the major clastogenic cause of CM. Therefore in the present study we employed brain CT examination for evaluation of two commonly used contrast media in Iran; namely Urografin 76%, a sodium-meglumine diatrizoate and Telebrix 38, a sodium-meglumine ioxythalamate which have relatively similar iodine concentration and osmolality. The main conceptual basis for using cytogenetic assays for biological monitoring is that genetic damage in a non-target tissue, most often peripheral blood lymphocytes, reflects similar events in the carcinogenic process (Sorsa et al., 1992). Therefore, chromosomal damage in human somatic cells may represent an event in a process that will eventually lead to the manifestation of ill health such as cancer. Cytogenetic surveillance which can be routinely used and has been previously, may serve as an early indicator of hazards of CM, thereby preventing adverse effects.

2. Materials and methods

2.1. Subjects Three groups consisting of 35 relatively young patients who had not undergone radiography in the previous 1 year were established. They were free of viral infections, neoplastic disease and toxic habits (alcohol and tobacco) and had not received medication in the previous 3 months. These patients were referred for brain CT scan mainly for headache and blurred vision. They were 20 women and 15 men ranging in age from 15 to 55 years (average 30 years) and

weight from 50 to 85 kg (mean= 65 kg). All patients undergoing CT with contrast materials recieved 1 ml/kg CM. This was a case-control study, i.e. each patient served as his or her own control too. Five patients (two women and three men) who underwent brain CT scan without CM injection were used as radiation controls. The contrast agents employed were sodium meglumine diatrizoate (Urografin 76%, Schering AG; iodine concentration= 370 mg/ml, viscosity= 8.9 mPa.s and osmolality = 2.10 Osm/ kgH2O) and sodium meglumine ioxythalamate (Telebrix 38, Guerbet Paris; iodine concentration= 380 mg/ml; viscosity= 8.3 mPa.s and osmolality= 2.10 Osm/kgH2O). Blood samples were collected in sterile heparinised tubes shortly before CM injection and 30 min towards the end of the examination. For brain CT, GE 9800 Advantage X-ray machine was used. Exposure condition was 120 kVp and average MA of 100. Axial sections of 13–16 was taken for each patient; scanning time for each section was 3 s. All patients were exposed to X-rays under similar conditions.

2.2. Cytogenetic methods 2.2.1. Cell culture Venous blood was drawn into heparinised tubes and the samples coded and cultures established the same day. To culture lymphocytes, 0.4 ml whole blood were added to 4.5 ml of RPMI-1640 (Sigma) supplemented with 15% heat inactivated fetal calf serum (Sigma) and 0.1 ml phytohemaglutinin (PHA-M). All cultures were incubated at 37°C for 48–52 h. Two hours prior to harvesting, colchicine was added at a final concentration of 0.2 mg/ml. After hypotonic treatment with 0.075 mol/l KCl for 10 min the lymphocytes were fixed in a mixture of methanol and acetic acid with a ratio of 3:1 and then transferred onto glass slides (IAEA, 1986). Three slides were made for each pre-CM and post-CM blood samples. After staining with 5% Giemsa (Merck) solution, 100 mitotic cells were analysed for each sample. Lesions were classified according to the International System of Cytogenetic Nomenclature for acquired chromosome aberrations (ISCN, 1985). Chromosome aberrations were divided into

H. Mozdarani, S. Fadaei / Toxicology Letters 98 (1998) 25–30

chromosome and chromatid types. Chromosomal lesions including chromosomal breaks, interstitial deletions (min) and exchange figures were analysed. Chromatid gaps were defined as achromatic lesions if less than the width of the chromatid, whereas chromatid deletions were scored if the separation was greater than the width of the chromatid and if there was a displacement of the chromatid arm (Buckton and Evans, 1973; Savage, 1976).

2.3. Statistical methods Kolmograv–Smirnov test showed that the cytogenetic data did not fit a normal distribution. The statistical significance of the differences between each sample in each group was established using Wilcoxon rank sum non-parametric test for paired data. The difference between the two CM in the groups were evaluated using the same test.

3. Results and discussion On the basis of cytogenetic analysis of lymphocytes, it can be concluded that CM used in this investigation are capable of chromosome aberration induction. Results are summarised in Table 1 and shown in Figs. 1 and 2. As observed, diagnostic X-ray alone did not produce a significant number of chromosomal aberrations (Table 1, Fig. 2). This might be due to the amount of whole body radiation dose received in a brain CT scan which is too low for aberration induction. UNSCEAR Report in 1988 showed that in a brain CT a whole body dose of 0.111 cSv (111 mrem) is received (Bushberg et al., 1994). It is also shown that 5 cGy of acute X-ray exposure did not change the frequency of chromosomal aberrations in lymphocytes (Pohl-Ruling et al., 1993) and consequently the lower limit for micronuclei formation was 5 cGy (Fenech and Morley, 1985). Our results are in agreement with other reports and is indicative that X-ray dose received by peripheral lymphocytes in brain CT is too low for chromosomal aberration induction. A relatively high number of aberrant cells calculated for radiation control group before and after CT scan

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was due to the observation of high frequency of gaps (achromatic lesions) which are supposed to arise from DNA single strand break. Similar frequency of gaps and hence aberrant cells before and after CT scan in same individuals indicate that radiation had almost no effect in chromosomal aberration induction (Table 1). Therefore the frequency of aberrations observed in this study could be attributed to the effect of contrast materials. In our study the radiation dose was not significantly different within the two groups in which contrast media was used (Table 1). In principle, this would appear to emphasis the importance of the direct action of chemical media in aberration induction. In the diatrizoate group, there was a significant increase in chromosomal aberrations of both chromosome and chromatid type breaks and exchanges (Table 1 and Figs. 1 and 2). Mutation frequency and chromosome aberration induction have been studied by using various diatrizoate compounds. Using Renografin, (a Na-meg. diatrizoate) and Hypaque (a Na-diatrizoate), Adams et

Fig. 1. Frequency of various types of chromosomal aberrations (Chtd =chromatid type and Chrom =chromosome type) observed in lymphocytes of patients undergoing brain CT scan before and after injection of contrast media (Tele = telebrix 38; Uro= urografin 76%).

5

15 15 15 15

After scan

Urografin 76% Before scan After scan

Telebrix 38 Before scan After scan 1500 1500

1500 1500

500

500

No. of cells analysed

Errors are standard error of mean values.

5

No. of subjects studied

Control Before scan

Treatment

30.46 93.14

30.13 9 3.23

29.2 92.08

Mean age 9 S.E.

1.06 9 0.3 2.0 9 0.47

0.8 90.24 2.2 9 0.35

1.2 9 0.36 2.66 90.58

1.2 9 0.40

2.8 9 0.37

1.06 9 0.37 2.26 9 0.57

1.4 90.51

Chromatid type

0.53 9 0.21 0.86 9 0.21

0.139 0.09 0.939 0.2

0.2 9 0.2

0

Chromosome type

Mean No. deletions and exchanges

2.8 9 0.37

Mean No. gaps and isogaps

1.339 0.27 3.069 0.31

1.339 0.36 3.609 0.52

1.49 0.51

1.49 0.51

Mean total breaks

2.399 0.13 5.069 0.59

2.399 0.55 5.859 0.61

4.29 0.8

4.29 0.4

13.290.52

13.490.25

15 91.41

Mean aberrant Mean No. of cells scan

Table 1 Frequency of chromosomal aberrations observed in patients before and after brain CT examination in the presence or absence of contrast media

28 H. Mozdarani, S. Fadaei / Toxicology Letters 98 (1998) 25–30

H. Mozdarani, S. Fadaei / Toxicology Letters 98 (1998) 25–30

Fig. 2. Comparison of frequency of total chromosomal aberrations (breaks and exchanges) induced by radiation alone and contrast media before and after bratin CT Scan. Gaps are not included.

al. (1977) showed a higher frequency of chromosome aberrations and micronuclei both, in vitro and in vivo. Norman et al. (1978) had reported that the diatrizoate group of contrast agents were capable of producing chromosomal damage in lymphocytes of patients undergoing angiocardiography. Other reports indicate that CM contributed in chromosomal damage only in the presence of radiation where an enhanced yield of aberrations in lymphocytes were obtained (Matsubara et al., 1982). It is also shown that various contrast media are capable of micronuclei induction in peripheral blood lymphocytes (Parvez et al., 1987). Micronuclei arise due to clastogenic or aneugenic activity of chemical agents. These results indicate that irrespective of ionic and osmolarity differences, CM agents are capable of producing chromosomal damage in peripheral lymphocytes. Patients who underwent excretory urography exhibit an increase in micronuclei formation with diatrizoate (Sinues et al., 1991). Although chromosomal aberration and micronuclei formation are consequences of mutagenic activity of chemicals, Ames test failed to show a mutagenic effect on Renografin 76% and Hypaque (Wheeler et al., 1980). Similarly Nelson et al.

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(1984) did not find an increase in SCE (Sister Chromatid Exchange) frequency in CHO cells following Na-meg. diatrizoate treatment. Up to now cytogenetic effects of ioxithalamate CM have not been studied as extensively as other CM agents. Telebrix 38 used in this study belongs to this group and is a structurally ionic monomer. Inspite of extensive studies on the clastogenic effects of diatrizoate contrast media group, fewer studies have been done on the effects of ioxithalamate CM. (As shown in Table 1 and Figs. 1 and 2) Telebrix 38 is as potent as Urografin in chromosomal aberration induction, of both chromosome and chromatid types. Studies with other ionic monomers such as metrizoate (Stentrand and Edgren, 1981) and iothalamate, showed that they can cause chromosome damage (Norman et al., 1978; Matsubara et al., 1982). Urografin 76% and telebrix 38 are ionic monomer compounds with similar ionic concentration and osmolality, but they are structurally different, therefore, the clastogenic effects of these agents might not be due to their chemical structures as proposed by Sinues et al. (1991). The mechanisms which CM use to cause chromosome damage are not well understood and established. It is shown that CM can enter into cells (Nordby et al., 1989) and might inhibit mitochondrial respiration resulting in the production of superoxide and hydroxyl radicals (Hodnick et al., 1986). However, based on the results obtained in this study, the cytogenetic damage is not molecule specific but may be caused by iodine, ionicity or osmolality of contrast materials. An increased frequency of chromosomal aberrations in a population may be considered to indicate an increased risk of cancer. Structural chromosome changes can lead to the activation of proto-oncogenes and elimination of tumour-suppressor genes and therefore represent an important mechanism of tumorigenesis (Heim and Mitelman, 1996). Numerous reports indicate that most neoplasms are associated with chromosomal rearrangements (Mitelman, 1988; Trent et al., 1989; Mitelman, 1990). It is also known that genetic predisposition to cancer is associated with certain chromosomal instability syndrome such as ataxia-telangiectasia, suggesting the possible

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health significance of chromosomal breakage at the individual level (Heim et al., 1989). Therefore it would be valuable if routine clinical trials used contrast materials with minimum genotoxicity.

Acknowledgements This work was partly supported by Tarbiat Modarres University Research Council.

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