- Email: [email protected]
Chromosome aberrations in lymphocytes and clastogenic factors in plasma detected in Belarus children 10 years after Chernobyl accident
Mutation Research 446 Ž1999. 245–253 www.elsevier.comrlocatergentox Community address: www.elsevier.comrlocatermutres
Chromosome aberrations in lymphocytes and clastogenic factors in plasma detected in Belarus children 10 years after Chernobyl accident Federica Gemignani a , Michela Ballardin a , Francesca Maggiani a , Anna M. Rossi a , A. Antonelli b, Roberto Barale a,) a
Dipartimento di Scienze dell’Uomo e dell’Ambiente UniÕersita` di Pisa, Õia S. Giuseppe 22, 56100 Pisa, Italy b Dipartimento di Medicina Interna, Õia Roma 67, UniÕersita` di Pisa, 56100 Pisa, Italy Received 18 June 1999; received in revised form 28 September 1999; accepted 29 September 1999
Abstract In 1996, 10 years after Chernobyl accident, a cytogenetic analysis was carried out to assess whether chromosome aberrant cells ŽCA. were still detectable in the lymphocytes and clastogenic factors ŽCFs. were present in the plasma of children coming from Gomel ŽBelarus., one of the most heavily contaminated regions. Furthermore, the possible contribution of plasmatic CFs to the amount of CA was investigated. The presence of CA was examined in the lymphocytes from 29 thyroid tumour-affected children and 41 healthy children Žlocal controls.. Thirty healthy children living in Pisa ŽItaly. were enrolled in the study as additional controls from an uncontaminated area. No significant difference was observed between the two control groups, whereas a significantly increased frequency of CA was found in the tumour-affected children, as compared with Gomel and Pisa controls Ž x 2-test, p - 0.001.. However, when soil contamination level was taken into account, the chromosome type CA frequency observed in tumour-affected children coming from the more contaminated areas Ž) 4 Cirkm2 . resulted significantly higher than that in other children, either affected or not Ž p s 0.003.. The presence of CFs was analyzed on the plasma ultrafiltrate from 41 children. 7r10 Ž70%. plasma samples from tumour-affected children and 17r23 Ž74%. Gomel controls resulted to possess clastogenic activity irrespective of soil contamination levels. No activity was detected in the plasma of eight Pisa controls Ž0%.. The difference between both Gomel groups and Pisa controls was highly significant Ž p s 0.002.. A borderline, but not statistically significant correlation Ž p s 0.08. was observed between basal CA frequency and CF potency, which became significant Ž p s 0.03. when only chromosome type of aberrations was considered. We conclude that, although the presence of CFs in the plasma of these children might be partly responsible of the cytogenetic effects observed, the main source of damage has considered to be do to the previous andror continuous exposure to environmental radiocontaminants. Tumour-affected children may represent a subset of the population either more sensitive to clastogenic damage or exposed to higher levels of contaminants. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Chromosome aberrant cells; Lymphocytes; Clastogenic factors; Belarus; Chernobyl accident
)
Corresponding author. Tel.: q0039-50-551-217; fax: q0039-50-551-290; e-mail: [email protected]
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 1 9 4 - 1
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
246
1. Introduction After the Chernobyl accident, several cytogenetic analyses have been carried out on human populations living in the different regions of the former USSR, in order to assess the possible clastogenic effects following the exposure to radioactive fallout since the first years w1x. Increased chromosome aberrant ŽCA. frequencies have been observed in subjects directly involved in the recovery immediately after the nuclear power plant explosion Žclean-up workersr liquidators. and also in groups of residents in areas contaminated at different levels w2–6x. In some cases, relationships between CA frequencies and radioactive environmental contamination levels have been described w7x. In 1994 we examined a group of children living in the Gomel region ŽBelarus. and an excess of CA and micronucleus ŽMN. frequencies was found to be significantly correlated with the amount of 137Cs excreted in their urine w8,9x. These findings led to the assumption that the observed cytogenetic abnormalities were due to the continuous exposure to contaminating radionuclides in the environment. However, another hypothesis concerning the presence of clastogenic factors ŽCFs. in the plasma of these children exposed to radioactive fallout is also worth of consideration, since it has been demonstrated that CFs are still detectable in the plasma of people many years after different types of radiation exposure w3x. In particular, CFs have been shown to be present in the plasma of children coming from radiocontaminated areas of the former USSR up to 6–8 years after the Chernobyl accident w10x, raising the question whether CFs might actually contribute to the total cytogenetic damage observable in vivo. To our knowledge, such an theory has yet to be
investigated. Therefore, in 1996, 10 years after the Chernobyl accident, we undertook a cytogenetic study with the aim of assessing the possible correlation between the presence of CFs in the plasma and the basal frequency of CA in the lymphocytes of 70 children coming from Gomel, one of the most contaminated areas during the event.
2. Material and methods 2.1. Donor selection, sample collection and metaphase analysis In the framework of a protocol for medical assistance, settled between Italian and Belarus governments in 1995, every year several thyroid tumour-affected children coming from Belarus are treated at Pisa hospital. Additional care is also provided to hundreds of healthy children coming from the same region ŽGomel. by private and public organizations to allow them to spend a vacation period in an uncontaminated area. Within this framework, we have analyzed 70 Gomel children who arrived in Pisa between June and August 1996. Out of these children, 29 had been diagnosed as thyroid tumouraffected children, whilst 41 exhibited no clinical sign attributable to ionising radiation exposure. In addition, 30 healthy children from Pisa ŽItaly. were enrolled in the study as controls. The demography of all the children enrolled in the study is shown in Table 1. Pisa controls resulted younger Žabout 4 years. than Gomel children and age was therefore accounted for in multivariate analysis. According to the ‘‘1995 Report on Chernobyl Sasakawa Project
Table 1 Demography of Gomel and Pisa children Gomel children
Males Age " S.D. Females Age " S.D. Total Age " S.D. S.D.s Standard deviation.
Pisa children
Patients
Healthy
Total
Healthy
11 13.36 " 2.61 18 13.16 " 1.94 29 13.24 " 2.18
13 12.76 " 2.24 28 13.07 " 2.53 41 12.97 " 2.42
24 13.04 " 2.38 46 13.10 " 2.30 70 13.08 " 2.31
12 9.25 " 3.64 18 8.66 " 3.23 30 8.9 " 3.35
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
Workshop’’ w11x, Gomel children lived in districts characterized by soil contamination with different levels of 137Cs Ž0.8–8.2 Cirkm2 .. A venous blood sample Ž10 ml. collected in sodium-heparinized tubes was obtained from each donor, at the arrival and prior to start of any therapy Žpatients.. A total 0.3 ml of whole blood was added to 4.7 ml of RPMI medium ŽGibco., supplemented with 10% fetal calf serum ŽICN Biomedicals., 1.5% phytohemagglutinin Ž Wellcome Diagnostics . and antibiotics Ž100 IU penicillin and 100 mg streptomycinrml, Sigma Aldrich.. The cultures were incubated at 378C for 48 h. A 4 mgrml colchicine ŽSigma Aldrich. was added 2 h before cell harvesting and fixing, according to standard procedures. Following standard Giemsa staining of coded slides, 150–200 well spread metaphases per donor were scored, for the analysis of the presence of cytogenetic abnormalities, that is gaps, breaks, fragments, exchanges, rings, dicentrics and other morphologically abnormal chromosomes, according to Savage’s classification criteria w12x. 2.2. CFs isolation and testing for clastogenic actiÕity Plasma fraction was separated from whole blood Ž4 mlrdonor. by centrifugation at 3000 rpm for 15 min and immediately frozen at y208C until further manipulation. For isolation of CFs, plasma was ultrafiltrated at 3000 rpm for 2 h through a 10-kDa cut-off filter ŽCentriplus concentrators, Amicon. to remove any high molecular mass component which might interfere with the cell culture, due to blood group incompatibilities w13x. Ultrafiltrate was immediately frozen at y808C and tested within few days. Aliquots of these ultrafiltrates Ž0.4 ml. were added to test cultures settled with 0.3 ml whole blood obtained from a reference donor in 4.7 ml TCM 199 ŽFlow Laboratories., a tissue culture medium poor in L-cysteine, which is a free radical scavenger, 1.5% of phytohemagglutinin Ž Wellcome Diagnostics . and antibiotics Ž100 IU penicillin and 100 mg Streptomycinrml, Sigma Aldrich.. The cultures were incubated at 378C for 48 h. A 4 mgrml colchicine ŽSigma Aldrich. was added 2 h before cell harvesting and fixing, according to standard procedures. Microscope slides were prepared as above and 150– 200 well spread metaphases were scored for each
247
control and CF-treated culture. Six replicas of each experiment were performed on blood samples obtained from one healthy person. For each experiment, the basal level of CA of the same healthy person Žcontrol culture., as well as the effect of his own ultrafiltrate on his own cells has been assessed. 2.3. Statistical analysis of the data Among the structural aberrations scored — either of chromosome or chromatid type — were registered, while gaps, although reported, were not included in the statistical analysis. The distribution of CA frequency Ž p ., either spontaneously occurring or induced by the CFs, approached a gaussian when p was transformed as arcsin 6p, thus allowing the use of parametric statistical analysis. The significance of the differences observed among children groups was assessed by means of analysis of variance ŽANOVA. or by multifactor analysis of variance ŽMANOVA., when several factors such as gender, health status Žtumour-affected vs. healthy., soil contamination level and age as covariate were accounted for. Multiple regression analysis ŽMRA. was used to assess the possible correlation between baseline and CF-induced CA, taking into account the above mentioned factors.
3. Results 3.1. Basal CA frequency Both Gomel and Pisa healthy children showed similar frequencies of total aberrant cells even considering separately the different types of aberrations Ž x 2-test, with Yates correction, p ) 0.05, in all cases, Table 2.. However, by considering chromosome type aberrations only, higher frequencies of aberrant cells were observed among tumour-affected children compared to both Pisa and Gomel controls Ž x 2-test, with Yates correction, p - 0.001 for both comparisons, Table 2.. Also the MANOVA, taking into account age and sex of the donors, did not reveal any significant difference between the frequencies of aberrant cells of the two groups of healthy children, whereas a significant difference Ž p s 0.05. was found compar-
248
Group of children
Gomel controls Gomel patients Pisa controls
No. of subjects
No. of metaphases
41 29 30
7605 5800 5863
Chromatid aberrations
Chromosome aberrations
Gaps
brs
ace
Total aberrations excluding gaps
Gaps
brs
ace
dic
rqt
Total aberrations excluding gaps
105 106 106
32 16 18
11 7 11
43 23 29
7 7 7
3 5 3
24 30 a 16
3 20b 1
1 6 2
31 61c 22
x 2-test, a p- 0.05, b p- 0.01, c p- 0.001, for comparisons with Pisa controls.
Total CA cells Ž%. excluding gaps
0.97 1.44 0.87
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
Table 2 Structural CA in lymphocytes of Gomel children as compared to Pisa controls brs s breaks; acesacentric fragments; dic sdicentrics; r q t s centric and acentric rings and translocated chromosomes.
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
ing tumour-affected children with the both Pisa or Gomel controls. Age and gender had no significant effect Ž p ) 0.05.. By restricting the MANOVA to unstable chromosome rearrangements Žrings and translocationss r q t . plus dicentrics, the significance of the difference between tumour-affected children and healthy ones increased considerably Ž p s 0.0006.. The finding that CA cells are more frequent in tumour-affected children raises the question whether such an increase of CA frequency might be due to the presence of the tumour, or other reasons, possibly related to genetic andror environmental factors, might account for both cytogenetic damage and cancer proneness. Therefore, the effect of the levels of exposure was examined and the distribution frequency in children according to 137Cs levels in the soil resulted bimodal. Gomel children living in districts with soil contamination - 4 Cirkm2 were referred as group 1 Ž n s 50., while those living in more contaminated districts Ž) 4 Cirkm2 . were referred as group 2 Ž n s 20.. This classification has proven to be a significant Ž p s 0.0034. factor in the ANOVA of the frequency of chromosome type aberrant cells observed in Belarus children Žeither affected or not.. When the health status was also considered in MANOVA, a complex picture was obtained. That is, both soil contamination and presence of tumour showed high statistically significant correlations with the cytogenetic damage Ž p s 0.006 and p - 0.001, respectively., and a significant Ž p s 0.05. interaction between the two factors emerged ŽTable 3.: no significant difference was found between healthy and affected children living in lower contaminated areas Ž- 4 Cirkm2 ., nor between the two groups of Table 3 ANOVA for arcsin
249
healthy children, whilst an increased frequency of CA was observed in tumour-affected children respect to healthy ones living in more contaminated areas Ž) 4 Cirkm2 . or tumour-affected living in less contaminated areas Ž p s 0.05.. 3.2. Assessment of clastogenic actiÕity of children plasma ultrafiltrates Due to the limited amount of blood available from each donor, plasma ultrafiltrates from 10 tumour-affected children, 23 Gomel healthy children and 8 Pisa healthy controls were tested for their possible clastogenic activity against the reference donor’s lymphocytes. In six untreated cultures of the reference donor’s blood, 2r810 Ž0.25%. aberrant metaphases have been observed, while 7r863 Ž0.81%. aberrant cells were found in six cultures of the same blood treated with its own plasma ultrafiltrate Ž x 2 s 0.21; p ) 0.05... The clastogenic effect of plasma ultrafiltrates, evaluated as CA induced on donor’s cells, is reported in Table 4. Ultrafiltrates obtained from Pisa children proved to be devoid of any clastogenic effect Ž x 2 s 0.14; p ) 0.05 as compared with untreated cultures.. On the contrary, plasma ultrafiltrates obtained from Gomel children, either tumour-affected or not, induced a statistically significant increase of both chromatid and chromosome type aberrations as compared to Pisa control values Žsee Table 4 for significance.. The frequency of aberrant cells induced by plasma ultrafiltrates obtained from Gomel healthy children compared to Pisa controls by ANOVA are significantly higher Ž p s 0.002.. When only Gomel children were considered, MANOVA revealed a significant effect of
'Ždic q rear. rcell
Source
Sum of squares
Main effects A: Soil contamination B: Tumour presence
0.321036 0.746154
Interactions AB Residual Total Žcorrected.
0.158948 2.61587 3.48907
All F ratios are based on the residual mean square error.
df
Mean square
F ratio
p Value
1 1
0.321 0.746
8.10 17.32
0.0059 0.0001
1 66 69
0.159 0.040
4.01
0.0490
250
Group of children Gomel controls Gomel patients Pisa controls
No. of subjects
No. of metaphases
Chromatid aberrations Gaps brs Ace
Tot-gap
Chromosome aberrations Gaps brs Ace
23 10 8
3994 1452 1184
136c 79c 15
24a 81c 0
13 6 2
20a 77c 0
4 4 0
0 15c 0
41b 10a 1
dic
rqt
Tot-gap
Total CA cells Ž%. excluding gaps
1 5 1
1 6 1
43a 36c 3
1.67 8.05 0.25
x 2-test, a p- 0.05, b p- 0.01, c p- 0.001, for comparisons with Pisa controls. brs s breaks; acesacentric fragments; dic sdicentrics; r q t s centric and acentric rings and translocated chromosomes.
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
Table 4 Structural CA induced by FCs of Pisa and Gomel’s children
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
the presence of tumour pathology on the frequency of aberrant cells induced by plasma CFs Ž p s 0.004., but not age, gender and soil contamination level. The distribution of clastogenic scores obtained by ranking the frequencies of aberrant cells induced in the reference donor’s lymphocytes by plasma ultrafiltrates in seven classes are shown in Table 5. While all ultrafiltrates from Pisa controls fell in the first class Ž- 1% of induced CA., several samples from Gomel children, either tumour-affected or not, fell in higher ranks. In this context, it is noteworthy to observe that half of tumour-affected children’s ultrafiltrates fell in the highest clastogenic rank Ži.e., ) 10% of induced CA., suggesting that highly active CFs are present in the plasma of tumour-affected children. Considering the effects of plasma ultrafiltrates from all Belarus children, we observed that CFs were able to induce either chromatid Ž57%. or chromosome type of aberrations Ž43%. in the 48-h culture. Then we tested whether the clastogenic potency of CFs, i.e., the frequency of CA induced in vitro by a plasma unit, might affect the basal frequency of overall CA. No significant correlation, although the results suggest a possible relationship, was found Ž p s 0.08. probably due to the limited number of donors assayed. However, by considering only the basal frequency of chromosome type of aberrations, a significant correlation ŽMRA, p s 0.03. with CFs clastogenic potency was found.
Table 5 Clastogenic scores for Pisa and Gomel children according to the clastogenic activity of serum ultrafiltrates Clastogenic score a
Number of children Pisa Gomel controls controls
Gomel patients
0 -1% 1- 2% 2 - 3% 3- 4% 4 -6% 6 -10% )10% Total children
8 0 0 0 0 0 0 8
3 0 1 0 1 0 5 10
a
6 7 7 2 1 0 0 23
Percentage of chromosome aberrant cells obtained from reference donor cultures treated with children plasma ultrafiltrates. Kruskal–Wallis test showed a highly significant difference Ž p0.002. between the groups of children in clastogenic score.
251
4. Discussion Several cytogenetic studies reported the presence, detectable even several years after the disaster, of significant increases of chromosomal aberrations or DNA damages in the peripheral lymphocytes of children exposed to Chernobyl radioactive fall-out w8,14–17x. The adverse effects were attributed to the current exposure to environmental radionuclides since some positive correlation with soil contamination w9x or internal radioactivity w8x was observed. In the present study we have shown that, 10 years after the nuclear power plant explosion, healthy children living in contaminated areas of Belarus did not present increased CA frequencies in the lymphocytes, with respect to a control group of children living in Pisa, an uncontaminated area. On the other hand, chromosome type aberrations occurred more frequently in tumour-affected children, raising the questions: whether the presence of the tumour itself might affect the CA formation process, e.g., through the impairment of cell cycle checkpoints or DNA repair systems, or that these children are constitutively more sensitive to chromosome damage or might be or have been exposed to higher levels of radionuclides. Stratifying tumour-affected children in low- and high-soil contamination groups, children living in more contaminated areas, presented significantly higher frequencies of chromosome type aberrant cells, compared either to affected children living in low-contamination areas or compared to both Pisa and Gomel healthy children. In addition, it should be mentioned that the highest frequencies of dicentrics plus rearrangements, i.e., 2.7% and 4.0%, were detected in two tumour-affected children living in the most contaminated areas, i.e., 7.4 and 8.2 Cirkm2 , respectively, thus confirming the correlation between soil contamination and cytogenetic damage. These findings are in agreement with our previous results, which showed that 19 tumour-affected children had higher CA than 17 healthy controls who arrived from Belarus 2 years earlier w8x. Differences in CA frequency have been reported to occur between healthy and tumour-affected children living in another contaminated province ŽBryansk: contamination level up to 5 Cirkm2 ., whereas no increase in CA have been observed in tumour-affected children living in uncontaminated area such as Moscow w18x. This latter
252
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
finding seems to exclude a possible role of the tumour in determining increases of CA. Moreover, in the present study no difference in CA was found between healthy and affected children living in lower contaminated areas Ž- 4 Cirkm2 ., further supporting the hypothesis that the tumour itself may not have a causative role on CA increase. Therefore, the increased frequency of CA observed in tumour-affected children living in areas exposed to higher contamination Ž) 4 Cirkm2 . supports the hypothesis that these children might be more susceptible to radiation damage, and perhaps to carcinogenic outcome, or that they have been more heavily exposed than healthy children living in the same districts. We have also explored the possibility that the higher occurrence of CA in tumour-affected children might be determined by high levels of CFs in their plasma, being possibly correlated with environmental exposure. Clastogenic activity of plasma ultrafiltrates has been reported for subjects therapeutically or accidentally exposed to X-rays and for A-bomb survivors many years afterwards w19x. Moreover, people exposed to Chernobyl fallout have been found positive for the presence of CFs in their plasma w3,10x. We assayed the plasma ultrafiltrates of several children coming from Gomel, either tumour-affected or not, and roughly 70% of them showed positive for CFs, whilst no clastogenic activity was detected in the plasma of Pisa controls. It should, therefore, be concluded that the presence of CFs in the plasma of Gomel children might represent the consequence of a biological reaction to the exposure to a stressor w20x, possibly associated with the Gomel environment itself. Furthermore, since a much higher clastogenic activity was detected in the plasma ultrafiltrate of one half of the tumour-affected children Žnot related to the soil contamination level of their living places., it can be suggested that the presence of tumour might enhance CF activity, although it is not the main determining factor. These findings are in agreement with those obtained from other groups of children coming from radiocontaminated areas of Gomel and Kiev w10x. Since many children still showed positive 2–4 years after immigration in an uncontaminated country, the presence of CFs is more likely to be attributable to a self-sustaining mechanism rather than to a current exposure to a stressor such as environmental radiation. Accordingly, we did not
find any correlation between the presence of CFs and the soil contamination level, thus suggesting that the extent of this biological response is based more likely on individual susceptibility rather than to the amount of environmental exposure. However, it should be stressed that, even if no clear evidence of biological or pathological consequences has been so far reported for the presence of CFs, it is very remarkable that 10 years after the Chernobyl disaster, these children continue to retain relevant amounts of CFs in their plasma and able to induce ‘‘in vitro’’ significant increases of CA in the lymphocytes. In contrast with other studies which reported a prevalence of chromatid type aberrations induced by CFs w10x, the CFs analyzed in this study were able to induce either chromatid and chromosome type aberrations. This discrepancy may be due to the fact that other authors tested frozen ultrafiltrates up to several weeks after blood collection, whereas we tested them within few days. Different storage conditions might have determined the observed differences in clastogenic activity of ultrafiltrates. Therefore, in our case, the basal frequency of both types of aberration were expected to be enhanced by CFs if they have some activity when naturally present in culture and not when added after concentration. Although we could not demonstrate a statistically significant correlation between basal CA frequencies and clastogenic potency of plasma ultrafiltrates, an indication of a possible relationship was indeed obtained Ž p s 0.08.. Reasonably, CFs amounts naturally present in plasma are not large enough to induce significantly detectable effects. However, by considering only chromosome type of aberrations, such an effect was indeed observed. In addition, it should be mentioned that we have observed among adult cancer patients undergoing 131 I therapy a significant correlation between micronuclei frequency in peripheral blood lymphocytes and the clastogenic activity elicited by their plasma ultrafiltrates w21x. This can further suggest that part of the cytogenetic damage induced by ionizing radiation may be the consequence of indirect mechanisms, such as the induction of CF formation. Because it has been shown that ginko biloba extracts have an effective antioxidant power — able to reduce or to eliminate the presence of CFs in vivo w22x — if it would be definitely clarified that CFs play a role in maintaining elevated levels of CA ‘‘in
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
vivo’’, it might be worth considering to establish preventive programs by administering antioxidant agents to exposed children.
References w1x G. Stephan, U. Oestreicher, An increased frequency of structural chromosome aberrations in persons present in the vicinity of Chernobyl during and after the reactor accident. Is this effect caused by radiation exposure, Mutat. Res. 223 Ž1989. 7–12. w2x L. Verschaeve, E.V. Domracheva, S.A. Kuznetsov, V.V. Nechai, Chromosome aberrations in inhabitants of Byelorussia: consequence of the Chernobyl accident, Mutat. Res. 287 Ž1993. 253–259. w3x I. Emerit, A. Levy, L. Cernjavski, R. Arutyunyan, N. Oganesian, A. Pogosian, H. Mejlumian, T. Sarkisian, M. Gulkandanian, M. Quastel, J. Goldsmith, E. Riklis, E. Kordysh, S. Poliak, L. Merklin, Transferable clastogenic activity in plasma from persons exposed as salvage personnel of the Chernobyl reactor, J. Cancer Res. Clin. Oncol. 120 Ž1994. 558–561. w4x M.A. Pilinskaya, A.M. Shemetun, Yu. Bondars, ´ S.S. Dybskii, Cytogenetic effect in the somatic cells of persons exposed to radiation after the Chernobyl accident Žin Russian., Vestn. Akad. Med. Nauk. SSSR 8 Ž1991. 40–43. w5x V.A. Schevchenko, E.A. Akayeva, I.M. Veliseyeva, T.V. Yelisova, E.L. Yofa, I.N. Nicova, A.B. Symov, W. Burkat, Human cytogenetic consequences of the Chernobyl accident, Mutat. Res. 361 Ž1996. 29–34. w6x N. Slozina, E. Neronova, T. Kharchenko, A. Nikiforov, Increased level of chromosomal aberrations in lymphocytes of Chernobyl liquidators 6–10 years after the accident, Mutat. Res. 379 Ž1997. 121–125. w7x L. Padovani, D. Caporossi, B. Tedeschi, P. Vernole, B. Nicoletti, F. Mauro, Cytogenetic study in lymphocytes from children exposed to ionizing radiation after the Chernobyl accident, Mutat. Res. 319 Ž1993. 55–60. w8x R. Barale, F. Gemignani, C. Morizzo, A. Lori, A. Rossi, A. Antonelli, G. Di Pretoro, G. Panasiuk, M. Ballardin, Cytogenetic damage in lymphocytes of healthy and tumour-affected children from the Gomel region ŽBelarus., Mutat. Res. 405 Ž1998. 89–95. w9x L. Zotti Martelli, L. Migliore, G. Panasiuk, R. Barale, Micronucleus frequency in Gomel ŽBelarus. children affected and not affected by thyroid cancer, Mutat. Res. 440 Ž1999. 35–43. w10x I. Emerit, M. Quastel, J. Goldsmith, L. Merkin, A. Levy, L. Cernjavski, A. Alaoul-Youssefi, A. Pogossian, E. Riklis, Clastogenic factors in the plasma of children exposed at Chernobyl, Mutat. Res. 373 Ž1997. 47–54.
253
w11x V.E. Derzhitskii, G.D. Panasyuk, N.K. Derzhitskana, A.N. Demidenko, V.A. Kalimulin, I.V. Anikina, V.A. Cot, V.B. Masyakin, Results of the Examination of the Health Status of Children in Gomel Oblast 1991–1994 Chernobyl Sasakawa Project, A Report on the Chernobyl Sasakawa Project Workshop, Published by: Sasakawa Memorial Health Foundation, Japan 1996. w12x J.R.K. Savage, Classification and relationships of induced chromosomal structural changes, J. Med. Genet. 13 Ž1976. 103–122. w13x I. Emerit, Radiation induced transferable clastogenic factors, in: E. Riklis ŽEd.., Frontiers in Radiation Biology, Balaban Publishers, Weinheim, 1990, pp. 679–684. w14x L. Padovani, L. Stronati, F. Mauro, A. Testa, M. Appolloni, P. Anzidei, D. Caporossi, B. Tedeschi, P. Vernole, Cytogenetic effects in lymphocytes from children exposed to radiation fall-out after the Chernobyl accident, Mutat. Res. 395 Ž1997. 249–254. w15x S. Salomaa, A. Koivistoinen, W. Paile, T. Rahola, T. Ritomaa, Chromosomal aberrations of Byelorussian children exposed to Chernobyl fall-out, Proceedings IRPA 8 Congress, International Radiation Protection Association, Montreal, Vol. 1, 1992, pp. 801–804. w16x G. Frenzilli, A. Lori, G. Panasiuk, M. Ferdeghini, R. Barale, Comet assay on children’s leukocytes 8 years after the Chernobyl disaster, Mutat. Res. 415 Ž1998. 151–158. w17x M.G. Grollino, P. Eleuteri, H.F. Mark, S. Bazzarri, D. Cavallo, G.S. Crescanzi, R. De Vita, Children exposed to chronic contamination after the Chernobyl accident: cytogenetic and radiotoxicological analyses, Arch. Environ. Health 53 Ž5. Ž1998. 344–346. w18x E.K. Khandogina, V.A. Ageikin, S.V. Zvereva, L.F. Marchenko, G.R. Mutovin, G.P. Snigireva, R.V. Lenskaia, V.M. Buiankin, V.V. Shakhtarin, A.P. Akif’ev, The cytogenetic examination of different groups of children living in regions of Bryansk Province contaminated as a result of the Chernobyl accident, Radiat. Biol. Radioecol. 35 Ž5. Ž1995. 618–625. w19x I. Emerit, Chromosome breakage factors: origin and possible significance, Prog. Mutat. Res. 4 Ž1982. 61–74. w20x I. Emerit, Reactive oxigen species, chromosome mutation, and cancer: possible role of clastogenic factors in carcinogenesis, Free Radical Biol. Med. 16 Ž1994. 99–109. w21x M. Ballardin, R. Barale, M. Ferdeghini, L. Bodei, F. Gemignani, Micronuclei in lymphocytes of patients therapeutically treated with 131-Iodine, in preparation. w22x I. Emerit, N. Oganesian, T. Sarkisian, R. Arutyunyan, A. Pogosian, K. Asrian, A. Levy, L. Cernjavski, Clastogenic factors in the plasma of chernobyl accident recovery workers: anticlastogenic effect of ginkgo biloba extract, Radiat. Res. 144 Ž1995. 198–205.
Chromosome aberrations in lymphocytes and clastogenic factors in plasma detected in Belarus children 10 years after Chernobyl accident Federica Gemignani a , Michela Ballardin a , Francesca Maggiani a , Anna M. Rossi a , A. Antonelli b, Roberto Barale a,) a
Dipartimento di Scienze dell’Uomo e dell’Ambiente UniÕersita` di Pisa, Õia S. Giuseppe 22, 56100 Pisa, Italy b Dipartimento di Medicina Interna, Õia Roma 67, UniÕersita` di Pisa, 56100 Pisa, Italy Received 18 June 1999; received in revised form 28 September 1999; accepted 29 September 1999
Abstract In 1996, 10 years after Chernobyl accident, a cytogenetic analysis was carried out to assess whether chromosome aberrant cells ŽCA. were still detectable in the lymphocytes and clastogenic factors ŽCFs. were present in the plasma of children coming from Gomel ŽBelarus., one of the most heavily contaminated regions. Furthermore, the possible contribution of plasmatic CFs to the amount of CA was investigated. The presence of CA was examined in the lymphocytes from 29 thyroid tumour-affected children and 41 healthy children Žlocal controls.. Thirty healthy children living in Pisa ŽItaly. were enrolled in the study as additional controls from an uncontaminated area. No significant difference was observed between the two control groups, whereas a significantly increased frequency of CA was found in the tumour-affected children, as compared with Gomel and Pisa controls Ž x 2-test, p - 0.001.. However, when soil contamination level was taken into account, the chromosome type CA frequency observed in tumour-affected children coming from the more contaminated areas Ž) 4 Cirkm2 . resulted significantly higher than that in other children, either affected or not Ž p s 0.003.. The presence of CFs was analyzed on the plasma ultrafiltrate from 41 children. 7r10 Ž70%. plasma samples from tumour-affected children and 17r23 Ž74%. Gomel controls resulted to possess clastogenic activity irrespective of soil contamination levels. No activity was detected in the plasma of eight Pisa controls Ž0%.. The difference between both Gomel groups and Pisa controls was highly significant Ž p s 0.002.. A borderline, but not statistically significant correlation Ž p s 0.08. was observed between basal CA frequency and CF potency, which became significant Ž p s 0.03. when only chromosome type of aberrations was considered. We conclude that, although the presence of CFs in the plasma of these children might be partly responsible of the cytogenetic effects observed, the main source of damage has considered to be do to the previous andror continuous exposure to environmental radiocontaminants. Tumour-affected children may represent a subset of the population either more sensitive to clastogenic damage or exposed to higher levels of contaminants. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Chromosome aberrant cells; Lymphocytes; Clastogenic factors; Belarus; Chernobyl accident
)
Corresponding author. Tel.: q0039-50-551-217; fax: q0039-50-551-290; e-mail: [email protected]
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 1 9 4 - 1
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
246
1. Introduction After the Chernobyl accident, several cytogenetic analyses have been carried out on human populations living in the different regions of the former USSR, in order to assess the possible clastogenic effects following the exposure to radioactive fallout since the first years w1x. Increased chromosome aberrant ŽCA. frequencies have been observed in subjects directly involved in the recovery immediately after the nuclear power plant explosion Žclean-up workersr liquidators. and also in groups of residents in areas contaminated at different levels w2–6x. In some cases, relationships between CA frequencies and radioactive environmental contamination levels have been described w7x. In 1994 we examined a group of children living in the Gomel region ŽBelarus. and an excess of CA and micronucleus ŽMN. frequencies was found to be significantly correlated with the amount of 137Cs excreted in their urine w8,9x. These findings led to the assumption that the observed cytogenetic abnormalities were due to the continuous exposure to contaminating radionuclides in the environment. However, another hypothesis concerning the presence of clastogenic factors ŽCFs. in the plasma of these children exposed to radioactive fallout is also worth of consideration, since it has been demonstrated that CFs are still detectable in the plasma of people many years after different types of radiation exposure w3x. In particular, CFs have been shown to be present in the plasma of children coming from radiocontaminated areas of the former USSR up to 6–8 years after the Chernobyl accident w10x, raising the question whether CFs might actually contribute to the total cytogenetic damage observable in vivo. To our knowledge, such an theory has yet to be
investigated. Therefore, in 1996, 10 years after the Chernobyl accident, we undertook a cytogenetic study with the aim of assessing the possible correlation between the presence of CFs in the plasma and the basal frequency of CA in the lymphocytes of 70 children coming from Gomel, one of the most contaminated areas during the event.
2. Material and methods 2.1. Donor selection, sample collection and metaphase analysis In the framework of a protocol for medical assistance, settled between Italian and Belarus governments in 1995, every year several thyroid tumour-affected children coming from Belarus are treated at Pisa hospital. Additional care is also provided to hundreds of healthy children coming from the same region ŽGomel. by private and public organizations to allow them to spend a vacation period in an uncontaminated area. Within this framework, we have analyzed 70 Gomel children who arrived in Pisa between June and August 1996. Out of these children, 29 had been diagnosed as thyroid tumouraffected children, whilst 41 exhibited no clinical sign attributable to ionising radiation exposure. In addition, 30 healthy children from Pisa ŽItaly. were enrolled in the study as controls. The demography of all the children enrolled in the study is shown in Table 1. Pisa controls resulted younger Žabout 4 years. than Gomel children and age was therefore accounted for in multivariate analysis. According to the ‘‘1995 Report on Chernobyl Sasakawa Project
Table 1 Demography of Gomel and Pisa children Gomel children
Males Age " S.D. Females Age " S.D. Total Age " S.D. S.D.s Standard deviation.
Pisa children
Patients
Healthy
Total
Healthy
11 13.36 " 2.61 18 13.16 " 1.94 29 13.24 " 2.18
13 12.76 " 2.24 28 13.07 " 2.53 41 12.97 " 2.42
24 13.04 " 2.38 46 13.10 " 2.30 70 13.08 " 2.31
12 9.25 " 3.64 18 8.66 " 3.23 30 8.9 " 3.35
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
Workshop’’ w11x, Gomel children lived in districts characterized by soil contamination with different levels of 137Cs Ž0.8–8.2 Cirkm2 .. A venous blood sample Ž10 ml. collected in sodium-heparinized tubes was obtained from each donor, at the arrival and prior to start of any therapy Žpatients.. A total 0.3 ml of whole blood was added to 4.7 ml of RPMI medium ŽGibco., supplemented with 10% fetal calf serum ŽICN Biomedicals., 1.5% phytohemagglutinin Ž Wellcome Diagnostics . and antibiotics Ž100 IU penicillin and 100 mg streptomycinrml, Sigma Aldrich.. The cultures were incubated at 378C for 48 h. A 4 mgrml colchicine ŽSigma Aldrich. was added 2 h before cell harvesting and fixing, according to standard procedures. Following standard Giemsa staining of coded slides, 150–200 well spread metaphases per donor were scored, for the analysis of the presence of cytogenetic abnormalities, that is gaps, breaks, fragments, exchanges, rings, dicentrics and other morphologically abnormal chromosomes, according to Savage’s classification criteria w12x. 2.2. CFs isolation and testing for clastogenic actiÕity Plasma fraction was separated from whole blood Ž4 mlrdonor. by centrifugation at 3000 rpm for 15 min and immediately frozen at y208C until further manipulation. For isolation of CFs, plasma was ultrafiltrated at 3000 rpm for 2 h through a 10-kDa cut-off filter ŽCentriplus concentrators, Amicon. to remove any high molecular mass component which might interfere with the cell culture, due to blood group incompatibilities w13x. Ultrafiltrate was immediately frozen at y808C and tested within few days. Aliquots of these ultrafiltrates Ž0.4 ml. were added to test cultures settled with 0.3 ml whole blood obtained from a reference donor in 4.7 ml TCM 199 ŽFlow Laboratories., a tissue culture medium poor in L-cysteine, which is a free radical scavenger, 1.5% of phytohemagglutinin Ž Wellcome Diagnostics . and antibiotics Ž100 IU penicillin and 100 mg Streptomycinrml, Sigma Aldrich.. The cultures were incubated at 378C for 48 h. A 4 mgrml colchicine ŽSigma Aldrich. was added 2 h before cell harvesting and fixing, according to standard procedures. Microscope slides were prepared as above and 150– 200 well spread metaphases were scored for each
247
control and CF-treated culture. Six replicas of each experiment were performed on blood samples obtained from one healthy person. For each experiment, the basal level of CA of the same healthy person Žcontrol culture., as well as the effect of his own ultrafiltrate on his own cells has been assessed. 2.3. Statistical analysis of the data Among the structural aberrations scored — either of chromosome or chromatid type — were registered, while gaps, although reported, were not included in the statistical analysis. The distribution of CA frequency Ž p ., either spontaneously occurring or induced by the CFs, approached a gaussian when p was transformed as arcsin 6p, thus allowing the use of parametric statistical analysis. The significance of the differences observed among children groups was assessed by means of analysis of variance ŽANOVA. or by multifactor analysis of variance ŽMANOVA., when several factors such as gender, health status Žtumour-affected vs. healthy., soil contamination level and age as covariate were accounted for. Multiple regression analysis ŽMRA. was used to assess the possible correlation between baseline and CF-induced CA, taking into account the above mentioned factors.
3. Results 3.1. Basal CA frequency Both Gomel and Pisa healthy children showed similar frequencies of total aberrant cells even considering separately the different types of aberrations Ž x 2-test, with Yates correction, p ) 0.05, in all cases, Table 2.. However, by considering chromosome type aberrations only, higher frequencies of aberrant cells were observed among tumour-affected children compared to both Pisa and Gomel controls Ž x 2-test, with Yates correction, p - 0.001 for both comparisons, Table 2.. Also the MANOVA, taking into account age and sex of the donors, did not reveal any significant difference between the frequencies of aberrant cells of the two groups of healthy children, whereas a significant difference Ž p s 0.05. was found compar-
248
Group of children
Gomel controls Gomel patients Pisa controls
No. of subjects
No. of metaphases
41 29 30
7605 5800 5863
Chromatid aberrations
Chromosome aberrations
Gaps
brs
ace
Total aberrations excluding gaps
Gaps
brs
ace
dic
rqt
Total aberrations excluding gaps
105 106 106
32 16 18
11 7 11
43 23 29
7 7 7
3 5 3
24 30 a 16
3 20b 1
1 6 2
31 61c 22
x 2-test, a p- 0.05, b p- 0.01, c p- 0.001, for comparisons with Pisa controls.
Total CA cells Ž%. excluding gaps
0.97 1.44 0.87
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
Table 2 Structural CA in lymphocytes of Gomel children as compared to Pisa controls brs s breaks; acesacentric fragments; dic sdicentrics; r q t s centric and acentric rings and translocated chromosomes.
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
ing tumour-affected children with the both Pisa or Gomel controls. Age and gender had no significant effect Ž p ) 0.05.. By restricting the MANOVA to unstable chromosome rearrangements Žrings and translocationss r q t . plus dicentrics, the significance of the difference between tumour-affected children and healthy ones increased considerably Ž p s 0.0006.. The finding that CA cells are more frequent in tumour-affected children raises the question whether such an increase of CA frequency might be due to the presence of the tumour, or other reasons, possibly related to genetic andror environmental factors, might account for both cytogenetic damage and cancer proneness. Therefore, the effect of the levels of exposure was examined and the distribution frequency in children according to 137Cs levels in the soil resulted bimodal. Gomel children living in districts with soil contamination - 4 Cirkm2 were referred as group 1 Ž n s 50., while those living in more contaminated districts Ž) 4 Cirkm2 . were referred as group 2 Ž n s 20.. This classification has proven to be a significant Ž p s 0.0034. factor in the ANOVA of the frequency of chromosome type aberrant cells observed in Belarus children Žeither affected or not.. When the health status was also considered in MANOVA, a complex picture was obtained. That is, both soil contamination and presence of tumour showed high statistically significant correlations with the cytogenetic damage Ž p s 0.006 and p - 0.001, respectively., and a significant Ž p s 0.05. interaction between the two factors emerged ŽTable 3.: no significant difference was found between healthy and affected children living in lower contaminated areas Ž- 4 Cirkm2 ., nor between the two groups of Table 3 ANOVA for arcsin
249
healthy children, whilst an increased frequency of CA was observed in tumour-affected children respect to healthy ones living in more contaminated areas Ž) 4 Cirkm2 . or tumour-affected living in less contaminated areas Ž p s 0.05.. 3.2. Assessment of clastogenic actiÕity of children plasma ultrafiltrates Due to the limited amount of blood available from each donor, plasma ultrafiltrates from 10 tumour-affected children, 23 Gomel healthy children and 8 Pisa healthy controls were tested for their possible clastogenic activity against the reference donor’s lymphocytes. In six untreated cultures of the reference donor’s blood, 2r810 Ž0.25%. aberrant metaphases have been observed, while 7r863 Ž0.81%. aberrant cells were found in six cultures of the same blood treated with its own plasma ultrafiltrate Ž x 2 s 0.21; p ) 0.05... The clastogenic effect of plasma ultrafiltrates, evaluated as CA induced on donor’s cells, is reported in Table 4. Ultrafiltrates obtained from Pisa children proved to be devoid of any clastogenic effect Ž x 2 s 0.14; p ) 0.05 as compared with untreated cultures.. On the contrary, plasma ultrafiltrates obtained from Gomel children, either tumour-affected or not, induced a statistically significant increase of both chromatid and chromosome type aberrations as compared to Pisa control values Žsee Table 4 for significance.. The frequency of aberrant cells induced by plasma ultrafiltrates obtained from Gomel healthy children compared to Pisa controls by ANOVA are significantly higher Ž p s 0.002.. When only Gomel children were considered, MANOVA revealed a significant effect of
'Ždic q rear. rcell
Source
Sum of squares
Main effects A: Soil contamination B: Tumour presence
0.321036 0.746154
Interactions AB Residual Total Žcorrected.
0.158948 2.61587 3.48907
All F ratios are based on the residual mean square error.
df
Mean square
F ratio
p Value
1 1
0.321 0.746
8.10 17.32
0.0059 0.0001
1 66 69
0.159 0.040
4.01
0.0490
250
Group of children Gomel controls Gomel patients Pisa controls
No. of subjects
No. of metaphases
Chromatid aberrations Gaps brs Ace
Tot-gap
Chromosome aberrations Gaps brs Ace
23 10 8
3994 1452 1184
136c 79c 15
24a 81c 0
13 6 2
20a 77c 0
4 4 0
0 15c 0
41b 10a 1
dic
rqt
Tot-gap
Total CA cells Ž%. excluding gaps
1 5 1
1 6 1
43a 36c 3
1.67 8.05 0.25
x 2-test, a p- 0.05, b p- 0.01, c p- 0.001, for comparisons with Pisa controls. brs s breaks; acesacentric fragments; dic sdicentrics; r q t s centric and acentric rings and translocated chromosomes.
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
Table 4 Structural CA induced by FCs of Pisa and Gomel’s children
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
the presence of tumour pathology on the frequency of aberrant cells induced by plasma CFs Ž p s 0.004., but not age, gender and soil contamination level. The distribution of clastogenic scores obtained by ranking the frequencies of aberrant cells induced in the reference donor’s lymphocytes by plasma ultrafiltrates in seven classes are shown in Table 5. While all ultrafiltrates from Pisa controls fell in the first class Ž- 1% of induced CA., several samples from Gomel children, either tumour-affected or not, fell in higher ranks. In this context, it is noteworthy to observe that half of tumour-affected children’s ultrafiltrates fell in the highest clastogenic rank Ži.e., ) 10% of induced CA., suggesting that highly active CFs are present in the plasma of tumour-affected children. Considering the effects of plasma ultrafiltrates from all Belarus children, we observed that CFs were able to induce either chromatid Ž57%. or chromosome type of aberrations Ž43%. in the 48-h culture. Then we tested whether the clastogenic potency of CFs, i.e., the frequency of CA induced in vitro by a plasma unit, might affect the basal frequency of overall CA. No significant correlation, although the results suggest a possible relationship, was found Ž p s 0.08. probably due to the limited number of donors assayed. However, by considering only the basal frequency of chromosome type of aberrations, a significant correlation ŽMRA, p s 0.03. with CFs clastogenic potency was found.
Table 5 Clastogenic scores for Pisa and Gomel children according to the clastogenic activity of serum ultrafiltrates Clastogenic score a
Number of children Pisa Gomel controls controls
Gomel patients
0 -1% 1- 2% 2 - 3% 3- 4% 4 -6% 6 -10% )10% Total children
8 0 0 0 0 0 0 8
3 0 1 0 1 0 5 10
a
6 7 7 2 1 0 0 23
Percentage of chromosome aberrant cells obtained from reference donor cultures treated with children plasma ultrafiltrates. Kruskal–Wallis test showed a highly significant difference Ž p0.002. between the groups of children in clastogenic score.
251
4. Discussion Several cytogenetic studies reported the presence, detectable even several years after the disaster, of significant increases of chromosomal aberrations or DNA damages in the peripheral lymphocytes of children exposed to Chernobyl radioactive fall-out w8,14–17x. The adverse effects were attributed to the current exposure to environmental radionuclides since some positive correlation with soil contamination w9x or internal radioactivity w8x was observed. In the present study we have shown that, 10 years after the nuclear power plant explosion, healthy children living in contaminated areas of Belarus did not present increased CA frequencies in the lymphocytes, with respect to a control group of children living in Pisa, an uncontaminated area. On the other hand, chromosome type aberrations occurred more frequently in tumour-affected children, raising the questions: whether the presence of the tumour itself might affect the CA formation process, e.g., through the impairment of cell cycle checkpoints or DNA repair systems, or that these children are constitutively more sensitive to chromosome damage or might be or have been exposed to higher levels of radionuclides. Stratifying tumour-affected children in low- and high-soil contamination groups, children living in more contaminated areas, presented significantly higher frequencies of chromosome type aberrant cells, compared either to affected children living in low-contamination areas or compared to both Pisa and Gomel healthy children. In addition, it should be mentioned that the highest frequencies of dicentrics plus rearrangements, i.e., 2.7% and 4.0%, were detected in two tumour-affected children living in the most contaminated areas, i.e., 7.4 and 8.2 Cirkm2 , respectively, thus confirming the correlation between soil contamination and cytogenetic damage. These findings are in agreement with our previous results, which showed that 19 tumour-affected children had higher CA than 17 healthy controls who arrived from Belarus 2 years earlier w8x. Differences in CA frequency have been reported to occur between healthy and tumour-affected children living in another contaminated province ŽBryansk: contamination level up to 5 Cirkm2 ., whereas no increase in CA have been observed in tumour-affected children living in uncontaminated area such as Moscow w18x. This latter
252
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
finding seems to exclude a possible role of the tumour in determining increases of CA. Moreover, in the present study no difference in CA was found between healthy and affected children living in lower contaminated areas Ž- 4 Cirkm2 ., further supporting the hypothesis that the tumour itself may not have a causative role on CA increase. Therefore, the increased frequency of CA observed in tumour-affected children living in areas exposed to higher contamination Ž) 4 Cirkm2 . supports the hypothesis that these children might be more susceptible to radiation damage, and perhaps to carcinogenic outcome, or that they have been more heavily exposed than healthy children living in the same districts. We have also explored the possibility that the higher occurrence of CA in tumour-affected children might be determined by high levels of CFs in their plasma, being possibly correlated with environmental exposure. Clastogenic activity of plasma ultrafiltrates has been reported for subjects therapeutically or accidentally exposed to X-rays and for A-bomb survivors many years afterwards w19x. Moreover, people exposed to Chernobyl fallout have been found positive for the presence of CFs in their plasma w3,10x. We assayed the plasma ultrafiltrates of several children coming from Gomel, either tumour-affected or not, and roughly 70% of them showed positive for CFs, whilst no clastogenic activity was detected in the plasma of Pisa controls. It should, therefore, be concluded that the presence of CFs in the plasma of Gomel children might represent the consequence of a biological reaction to the exposure to a stressor w20x, possibly associated with the Gomel environment itself. Furthermore, since a much higher clastogenic activity was detected in the plasma ultrafiltrate of one half of the tumour-affected children Žnot related to the soil contamination level of their living places., it can be suggested that the presence of tumour might enhance CF activity, although it is not the main determining factor. These findings are in agreement with those obtained from other groups of children coming from radiocontaminated areas of Gomel and Kiev w10x. Since many children still showed positive 2–4 years after immigration in an uncontaminated country, the presence of CFs is more likely to be attributable to a self-sustaining mechanism rather than to a current exposure to a stressor such as environmental radiation. Accordingly, we did not
find any correlation between the presence of CFs and the soil contamination level, thus suggesting that the extent of this biological response is based more likely on individual susceptibility rather than to the amount of environmental exposure. However, it should be stressed that, even if no clear evidence of biological or pathological consequences has been so far reported for the presence of CFs, it is very remarkable that 10 years after the Chernobyl disaster, these children continue to retain relevant amounts of CFs in their plasma and able to induce ‘‘in vitro’’ significant increases of CA in the lymphocytes. In contrast with other studies which reported a prevalence of chromatid type aberrations induced by CFs w10x, the CFs analyzed in this study were able to induce either chromatid and chromosome type aberrations. This discrepancy may be due to the fact that other authors tested frozen ultrafiltrates up to several weeks after blood collection, whereas we tested them within few days. Different storage conditions might have determined the observed differences in clastogenic activity of ultrafiltrates. Therefore, in our case, the basal frequency of both types of aberration were expected to be enhanced by CFs if they have some activity when naturally present in culture and not when added after concentration. Although we could not demonstrate a statistically significant correlation between basal CA frequencies and clastogenic potency of plasma ultrafiltrates, an indication of a possible relationship was indeed obtained Ž p s 0.08.. Reasonably, CFs amounts naturally present in plasma are not large enough to induce significantly detectable effects. However, by considering only chromosome type of aberrations, such an effect was indeed observed. In addition, it should be mentioned that we have observed among adult cancer patients undergoing 131 I therapy a significant correlation between micronuclei frequency in peripheral blood lymphocytes and the clastogenic activity elicited by their plasma ultrafiltrates w21x. This can further suggest that part of the cytogenetic damage induced by ionizing radiation may be the consequence of indirect mechanisms, such as the induction of CF formation. Because it has been shown that ginko biloba extracts have an effective antioxidant power — able to reduce or to eliminate the presence of CFs in vivo w22x — if it would be definitely clarified that CFs play a role in maintaining elevated levels of CA ‘‘in
F. Gemignani et al.r Mutation Research 446 (1999) 245–253
vivo’’, it might be worth considering to establish preventive programs by administering antioxidant agents to exposed children.
References w1x G. Stephan, U. Oestreicher, An increased frequency of structural chromosome aberrations in persons present in the vicinity of Chernobyl during and after the reactor accident. Is this effect caused by radiation exposure, Mutat. Res. 223 Ž1989. 7–12. w2x L. Verschaeve, E.V. Domracheva, S.A. Kuznetsov, V.V. Nechai, Chromosome aberrations in inhabitants of Byelorussia: consequence of the Chernobyl accident, Mutat. Res. 287 Ž1993. 253–259. w3x I. Emerit, A. Levy, L. Cernjavski, R. Arutyunyan, N. Oganesian, A. Pogosian, H. Mejlumian, T. Sarkisian, M. Gulkandanian, M. Quastel, J. Goldsmith, E. Riklis, E. Kordysh, S. Poliak, L. Merklin, Transferable clastogenic activity in plasma from persons exposed as salvage personnel of the Chernobyl reactor, J. Cancer Res. Clin. Oncol. 120 Ž1994. 558–561. w4x M.A. Pilinskaya, A.M. Shemetun, Yu. Bondars, ´ S.S. Dybskii, Cytogenetic effect in the somatic cells of persons exposed to radiation after the Chernobyl accident Žin Russian., Vestn. Akad. Med. Nauk. SSSR 8 Ž1991. 40–43. w5x V.A. Schevchenko, E.A. Akayeva, I.M. Veliseyeva, T.V. Yelisova, E.L. Yofa, I.N. Nicova, A.B. Symov, W. Burkat, Human cytogenetic consequences of the Chernobyl accident, Mutat. Res. 361 Ž1996. 29–34. w6x N. Slozina, E. Neronova, T. Kharchenko, A. Nikiforov, Increased level of chromosomal aberrations in lymphocytes of Chernobyl liquidators 6–10 years after the accident, Mutat. Res. 379 Ž1997. 121–125. w7x L. Padovani, D. Caporossi, B. Tedeschi, P. Vernole, B. Nicoletti, F. Mauro, Cytogenetic study in lymphocytes from children exposed to ionizing radiation after the Chernobyl accident, Mutat. Res. 319 Ž1993. 55–60. w8x R. Barale, F. Gemignani, C. Morizzo, A. Lori, A. Rossi, A. Antonelli, G. Di Pretoro, G. Panasiuk, M. Ballardin, Cytogenetic damage in lymphocytes of healthy and tumour-affected children from the Gomel region ŽBelarus., Mutat. Res. 405 Ž1998. 89–95. w9x L. Zotti Martelli, L. Migliore, G. Panasiuk, R. Barale, Micronucleus frequency in Gomel ŽBelarus. children affected and not affected by thyroid cancer, Mutat. Res. 440 Ž1999. 35–43. w10x I. Emerit, M. Quastel, J. Goldsmith, L. Merkin, A. Levy, L. Cernjavski, A. Alaoul-Youssefi, A. Pogossian, E. Riklis, Clastogenic factors in the plasma of children exposed at Chernobyl, Mutat. Res. 373 Ž1997. 47–54.
253
w11x V.E. Derzhitskii, G.D. Panasyuk, N.K. Derzhitskana, A.N. Demidenko, V.A. Kalimulin, I.V. Anikina, V.A. Cot, V.B. Masyakin, Results of the Examination of the Health Status of Children in Gomel Oblast 1991–1994 Chernobyl Sasakawa Project, A Report on the Chernobyl Sasakawa Project Workshop, Published by: Sasakawa Memorial Health Foundation, Japan 1996. w12x J.R.K. Savage, Classification and relationships of induced chromosomal structural changes, J. Med. Genet. 13 Ž1976. 103–122. w13x I. Emerit, Radiation induced transferable clastogenic factors, in: E. Riklis ŽEd.., Frontiers in Radiation Biology, Balaban Publishers, Weinheim, 1990, pp. 679–684. w14x L. Padovani, L. Stronati, F. Mauro, A. Testa, M. Appolloni, P. Anzidei, D. Caporossi, B. Tedeschi, P. Vernole, Cytogenetic effects in lymphocytes from children exposed to radiation fall-out after the Chernobyl accident, Mutat. Res. 395 Ž1997. 249–254. w15x S. Salomaa, A. Koivistoinen, W. Paile, T. Rahola, T. Ritomaa, Chromosomal aberrations of Byelorussian children exposed to Chernobyl fall-out, Proceedings IRPA 8 Congress, International Radiation Protection Association, Montreal, Vol. 1, 1992, pp. 801–804. w16x G. Frenzilli, A. Lori, G. Panasiuk, M. Ferdeghini, R. Barale, Comet assay on children’s leukocytes 8 years after the Chernobyl disaster, Mutat. Res. 415 Ž1998. 151–158. w17x M.G. Grollino, P. Eleuteri, H.F. Mark, S. Bazzarri, D. Cavallo, G.S. Crescanzi, R. De Vita, Children exposed to chronic contamination after the Chernobyl accident: cytogenetic and radiotoxicological analyses, Arch. Environ. Health 53 Ž5. Ž1998. 344–346. w18x E.K. Khandogina, V.A. Ageikin, S.V. Zvereva, L.F. Marchenko, G.R. Mutovin, G.P. Snigireva, R.V. Lenskaia, V.M. Buiankin, V.V. Shakhtarin, A.P. Akif’ev, The cytogenetic examination of different groups of children living in regions of Bryansk Province contaminated as a result of the Chernobyl accident, Radiat. Biol. Radioecol. 35 Ž5. Ž1995. 618–625. w19x I. Emerit, Chromosome breakage factors: origin and possible significance, Prog. Mutat. Res. 4 Ž1982. 61–74. w20x I. Emerit, Reactive oxigen species, chromosome mutation, and cancer: possible role of clastogenic factors in carcinogenesis, Free Radical Biol. Med. 16 Ž1994. 99–109. w21x M. Ballardin, R. Barale, M. Ferdeghini, L. Bodei, F. Gemignani, Micronuclei in lymphocytes of patients therapeutically treated with 131-Iodine, in preparation. w22x I. Emerit, N. Oganesian, T. Sarkisian, R. Arutyunyan, A. Pogosian, K. Asrian, A. Levy, L. Cernjavski, Clastogenic factors in the plasma of chernobyl accident recovery workers: anticlastogenic effect of ginkgo biloba extract, Radiat. Res. 144 Ž1995. 198–205.