"On est donc passé, en une quarantaine d’années, de la sûreté absolue, à l’accident possible, puis à l’accident certain, tellement certain qu’il faut travailler, non pas la sûreté pour l’éviter, mais l’acceptabilité de son occurrence par les populations".
Coup de gueule
par Raymond SENÉ
De l’évolution de la doctrine de l’industrie nucléaire par Raymond SENÉ, physicien nucléaire
*Les allégations lors des années 50 étaient : “L’énergie nucléaire permettra de produire de l’énergie en quantité illimitée et quasiment
gratuite”.*
Puis “Atom for peace” annonce le bonheur pour l’humanité et ce … sans risques.
Puis, c’est l’accident de Three Mile Island (1) en 1979 : une grosse peur … mais les Américains sont des ânes et … c’est dû à l’embonpoint du chef de quart dont la bedaine empêchait la lecture des indications sur le panneau de conduite.
Par contre, chez nous, le Francatome est d’une sûreté inébranlable.
Néanmoins, on va remplacer les soupapes Fischer, responsables de l’accident de Three Mile Island (car en cas de décharge, elles se coincent en position ouverte). J’avais oublié de vous dire que notre palier des centrales de 900 Mw est purement du Westinghouse, construit sous licence américaine, donc après avoir été conçu par les ânes cités plus haut.
Arrive Tchernobyl en 1986 : la grosse frayeur : un réacteur à neutrons lents peut devenir surcritique prompt et vous sauter à la figure comme un vulgaire surgénérateur. Quel manque de savoir vivre !
Vite il faut expliquer que les Soviétiques sont des nuls, que leurs réacteurs sont mal conçus …. etc … même si la veille de l’accident, on vous les donnait encore en exemple. Je me souviens d’une réunion contradictoire tenue à Saclay, où un physicien du Commissariat à l’Energié Atomique, un communiste pur et dur nous expliquait, sans sourire, qu’en URSS le rendement de Carnot était plus favorable que dans les pays capitalistes. Ce qu’il voulait nous dire, c’était que la construction des centrales à proximité des villes, permettait d’utiliser les rejets d’eaux chaudes pour faire du chauffage urbain, ce qui améliorait le rendement global de l’installation. Pauvre Carnot !!! et pauvres habitants de Pripiat …
Mais après un moment de stupeur, et la décision de hâter la fermeture des Graphites – Gaz (Chinon 2 et 3, St Laurent 1 et 2 et Bugey 1) qui n’avaient guère plus d’enceinte de confinement que les réacteurs RBMK soviétiques (telle que la centrale de Tchernobyl), notre cher M. Tanguy (ancien directeur de l'Institut de protection et de sûreté nucléaire) se hâta d’expliquer que la probabilité pour qu’un accident grave survienne sur un de nos réacteurs du type PWR était … peanuts !!!
*Sûr et archi-sûr*
Donc, depuis le début du Francatome, on nous ressasse que le nucléaire est sûr, archi-sûr et que tout est prévu pour éviter, pour empêcher qu’un accident grave puisse se produire.
D’ailleurs, en France, nous avons une solution pour obtenir ce résultat
: il suffit de publier au journal officiel un arrêté fixant les modalités de qualité de fabrication, de construction, permettant d’obtenir cette sûreté absolue. D’accord, on est en France, donc un dernier article de cet arrêté donne la possibilité de dérogations (2).
Puis arrive l’EPR (European Pressurized Reactor) (3). La vague de libéralisme submerge la sûreté. Il faut que cette machine produise des KWh moins chers, pas pour le client, mais permettant plus de profits pour les futurs actionnaires de la future boite privée que va devenir EDF. Donc on étudie des astuces permettant de gagner sur la disponibilité de la machine. Que certaines de ces options mettent en péril la sûreté, c’est certain. Les cycles longs avec des hauts taux de combustion exigent des combustibles ayant une charge fissile au démarrage à la limite des zones dangereuses, les puissances résiduelles plus importantes rendent inopérants les dispositifs d’évacuation de la chaleur en cas de gros pépin … (4) .
Qu’à cela ne tienne, les dogmes des barrières (souvenez vous : 1e barrière : la gaine du combustible, 2e barrière : le circuit primaire avec la cuve, 3e barrière : l’enceinte de confinement) en prennent un sacré coup.
- Les gaines … boff … avec des taux de combustions de 80 à 90 GWjour/tonne ne sont garanties que grâce à une aide divine.
- Donc si le cœur fond, la cuve … fond aussi. D’où l’apparition, tel Zorro, du récupérateur de corium, dispositif destiné, d’après ses concepteurs à rassembler tout le corium fondu dans une zone où il serait possible de le refroidir. Il va falloir prévoir dans les procédures, une procession annuelle pour essayer de mettre les Dieux dans de bonnes dispositions … (5)
Mais, je pense que vous avez remarqué qu’on est passé subrepticement du zéro accident grave à un dispositif destiné à confiner le résultat d’un accident grave programmé.
*C’est cela le progrès technique.*
La phase suivante consiste, désormais puisque l’accident grave est envisagé comme étant quasi certain, à étudier le post-accidentel. Pour cela on dispose, grâce à Tchernobyl, d’un retour d’expérience … pas très encourageant !!!
De nombreuses réunions de groupes de travail, en France (CODIRPA (6) ), et au niveau européen (European Nuclear Energy Forum), ont lieu depuis début 2008. Un volet particulier y est étudié : l’acceptabilité par les populations (… du nucléaire ? : vous rigolez, Non bien sûr !) d’un accident et de ses conséquences.
Ces groupes de travail, composés en quasi-totalité de représentants des constructeurs et des autorités administratives, débattent doctement des astuces psychologiques qu’il faudra mettre en œuvre en cas d’accident.
Ce n’est pas surprenant que les citoyens de base n’y soient pas représentés. Ils pourraient avoir leur mot à dire car, en fait, après une première phase relativement courte où ce seront les agents du site qui seront en première ligne, ce seront eux, les voisins plus ou moins proches de l’installation, qui auront à subir pendant des dizaines d’années, voire beaucoup plus – mais là il s’agit de générations, les nuisances et les effets sur leur santé et sur l’environnement.
On est donc passé, en une quarantaine d’années, de la sûreté absolue, à l’accident possible, puis à l’accident certain, tellement certain qu’il faut travailler, non pas la sûreté pour l’éviter, mais l’acceptabilité de son occurrence par les populations.
Et si on arrêtait le nucléaire …
Raymond SENÉ
Physicien nucléaire
Mail : m-r.sene@wanadoo.fr
Groupement de scientifiques pour l’information sur l’énergie nucléaire
(GSIEN)
(1) Premier accident sur un 1000 MW, mais auparavant des réacteurs
expérimentaux eurent des états d’âme destructifs, et en particulier un
suisse construit à Lucens (1969), qui divergea puis ne s’arrêta qu’une
fois fondu.
(2) Voir décret 99-1046 du 13-12-1999 article 27
(3) L’EPR n’est, par rapport aux réacteurs des paliers 900 et 1300 Mwe
(y compris N4), qu’une petite évolution du même style que celle qui fit
passer des réacteurs graphite gaz de Chinon 2 et 3 et St Laurent 1 et 2,
à celui de Bugey 1. En fait de troisième génération, c’est une resucée
de la seconde, en beaucoup plus dangereux ! ! !
(4) D’ailleurs, nous avons appris, à l’occasion des réunions du débat
public, qu’au dessus d’une puissance nominale de 600 MWe, les
dispositifs de refroidissement destinés à sauver la cuve seraient
insuffisants, voire inopérants.
(5) Le puits de cuve est d’ailleurs revêtu d’une couche de "béton
sacrificiel". Quand on vous dit qu’il y a un recours aux dieux ! ! !
(6) CODIRPA : COmité DIRecteur pour la gestion de la phase
Post-Accidentelle d’un accident nucléaire ou d’une situation d’urgence
radiologique.
dimanche 14 septembre 2008
A 1,4-dihydropyridine derivative reduces DNA damage and stimulates DNA repair in human cells in vitro
Mutation Research 587 (2005) 52–58
A 1,4-dihydropyridine derivative reduces DNA damage and
stimulates DNA repair in human cells in vitro
Nadezhda I. Ryabokon a,1, Rose I. Goncharova b, Gunars Duburs c,
Joanna Rzeszowska-Wolny a,∗
a Department of Experimental and Clinical Radiobiology, Centre of Oncology, M. Sklodowska-Curie Memorial Institute,
Wybrzeze Armii Krajowej 15, 44-101 Gliwice, Poland
b Laboratory of Genetic Safety, Institute of Genetics and Cytology, National Academy of Sciences of Belarus,
Akademichnaya 27, 220070 Minsk, Republic of Belarus
c Latvian Institute of Organic Synthesis, Aizkaukles 21, LV-1006 Riga, Latvia
Received 29 April 2005; received in revised form 18 July 2005; accepted 30 July 2005
Available online 3 October 2005
Abstract
Compounds of the 1,4-dihydropyridine (1,4-DHP) series have been shown to reduce spontaneous, alkylation- and radiationinduced
mutation rates in animal test systems. Here we report studies using AV-153, the 1,4-DHP derivative that showed the highest
antimutagenic activity in those tests, to examine if it modulates DNA repair in human peripheral blood lymphocytes and in two
human lymphoblastoid cell lines, Raji and HL-60. AV-153 caused a 50% inhibition of growth (IC50) of Raji and HL-60 cells at
14.9±1.2 and 10.3±0.8 mM, respectively, but did not show a cytotoxic effect at concentrations <100>3 g/kg when administered
intravenously or >10 g/kg with oral administration) [2]
this group of compounds appears to offer promise for
1383-5718/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2005.07.009
N.I. Ryabokon et al. / Mutation Research 587 (2005) 52–58 53
medical applications. Among 12 screened 1,4-DHPs
that differed in chemical structure, six _-carbonyl-1,4-
dihydropyridines that are analogues of dihydronicotinamide,
the hydrogen- and electron-transferring moiety
of the redox coenzymes NADH and NADPH, showed
antimutagenic activity and significantly reduced spontaneous
and alkylation-induced point mutations and chromosome
breaks in germ cells of Drosophila [13,14],
alkylation-induced micronuclei in mouse bone-marrow
cells [15], and radiation-induced chromosome aberrations
and other cytogenetic end-points in fish [16]. The
reduction of mutation frequency reached 85% in some
cases [14]. Studies on Drosophila suggested that the 1,4-
DHPsinhibit chemical mutagenesis due to modulation of
DNA repair [13–15], and in the present study we examined
the effects of AV-153, a 1,4-DHP that showed the
highest antimutagenic activity in animals [13,14], on
spontaneous, chemically- and radiation-induced DNA
damage and its repair in human cells in vitro.
2. Materials and methods
2.1. AV-153
Sodium 3,5-bis-ethoxycarbonyl-2,6,dimethyl-1,4-dihydropyridine-
4-carboxylate (AV-153), synthesized in the Latvian
Institute of Organic Synthesis, is an analogue of the active center
of the reduced form of nicotinamide adenine dinucleotide
(NADH) or its phosphate (NADPH) (Fig. 1). It is a yellow
crystalline powder, resistant to temperature fluctuations, soluble
in water and it passes the cell membrane. Stock solutions
were prepared in phosphate-buffered saline (PBS) or culture
medium and kept in the dark at 4 ◦C during a period of one
month.
2.2. Cells and media
Venous blood from a young non-smoking healthy female
was collected in heparinized tubes. Histopaque-1077 (ICN)
was used to separate mononuclear lymphocytes, which were
washed in RPMI 1640 (Sigma–Aldrich) at 4 ◦C and incubated
in RPMI 1640 with 10% fetal bovine serum (FBS; Gibco), lglutamine
and 0.1% gentamicine at 37 ◦Cin a humidified atmosphere
with 5% CO2. Human HL-60 (promyelocytic leukemia)
and Raji (B-lymphoblastic leukemia) cells were cultured in the
same conditions and used during exponential growth.
Fig. 1. Structure of AV-153. Positions R3 and R5 bear a OC2H5 group
and position R4 a COONa group.
2.3. Assessment of cell viability
We used the colorimetric methyl-thiazol-tetrazolium
(MTT) assay [17] to study cell survival after incubation with
AV-153. Briefly, cells in RPMI 1640 without phenol red
(Sigma–Aldrich) were seeded in 96-well plates (NUNC, Denmark)
using 1.5×104 cells/100_l/well. Equal aliquots of PBS
containing AV-153 at different concentrations were added 24 h
later. Wells without cells or without AV-153 were used to
determine baseline values. After further incubation for 24 h
at 37 ◦C, MTT (Sigma–Aldrich) was added to all wells to
a final concentration of 0.5 mg/ml and the plates were incubated
for 3 h in the same conditions. The formazan crystals
formed were dissolved by addition of 150_l of DMSO and
the optical density (OD) at 570 nm was measured with an
ELx800 microplate reader (Bio-Tek Instruments, USA). The
percentage cell survival was calculated by the equation [viable
cells = (ODt −ODtb)/(ODc −ODcb)×100], where ODt is for
the cell sample with AV-153, ODtb for AV-153 alone (test
blank), ODc for control cells, and ODcb for the control blank
without cells and AV-153. All experimental series were performed
at least three times with triplicate samples. The IC50,
i.e. the concentration at which 50% of cells showed inhibition
of MTT processing, was calculated from the dose-response
curve. The standard Trypan-blue exclusion test was also used
according to the manufacturer’s indications (Sigma–Aldrich)
and blue (non-viable) cells were scored under the microscope.
2.4. Treatment with genotoxic factors
Cells were used at a concentration of 4×105/ml. Gammairradiation
was performed on ice using a 60Co radiotherapy
source (Gammatron, Siemens) at a dose rate of 0.8 Gy/min
to a total dose of 2Gy. In other experiments cells suspended
in ice-cold PBS were incubated with ethylmethane sulfonate
(EMS, Sigma–Aldrich) or hydrogen peroxide (H2O2) at final
concentrations of 100 _Mfor 1–3 min,washed in ice-cold PBS,
and suspended in complete medium at 37 ◦C. Equal aliquots
of AV-153 at different concentrations were added immediately
after irradiation or after washing off the chemical agents.
2.5. Alkaline single-cell gel electrophoresis (comet) assay
DNA breaks were assessed by comet assays as described
in [18–20] with all steps of preparation performed on ice.
Images of 100 randomly selected cells were analyzed per experimental
point. In initial experiments an image-analysis system
(Lucia version 4.60, Laboratory Imaging Ltd.) was compared
with visual scoring and a statistically significant correlation
(r≥0.72, p < d="A1" y ="−a×ln(x)">0.92 and p <>100_Mand the IC50 differed slightly
for the different cell types tested, HL-60 cells being more
sensitive than Raji cells.HL60cells are p53-deficient due
to deletions in the gene coding for this protein [24], but
nevertheless they undergo apoptosis readily and show a
G2 checkpoint [25]. They have been found to be more
sensitive than other cell lines in studies of anticancer
drugs [26,27].
In the lower range of concentrations tested, AV-153
caused a decrease in the level of DNA SSBs as measured
by alkaline comet assays, which detect single and
double strand breaks including those associated with
replication, incomplete excision repair of DNA damage,
or alkali-labile (mostly apurinic and apyrimidinic,
AP) sites [19]. The levels of both endogenous SSBs,
which are common lesions caused byDNAoxidation and
AP sites formed during metabolic processes (up to 104
depurinizations occur in human cells per day [28]), and
of SSBs caused by ionizing radiation (oxidative damage
to bases and cross-links) or alkylating agents like
EMS (N-alkylation of purines and AP sites) were significantly
reduced. These effects suggest that AV-153
may directly stimulate DNA repair, which is consistent
with its influence on DNA-repair kinetics seen here and
also with the results of Goncharova and Kuzhir [13,14]
who showed that it reduced the frequency of spontaneous
and EMS-induced point mutations in germ cells of
Drosophila by 85% and 40%, respectively.AV-153 could
influence cellular redox equilibria, because it may possess
antioxidant activity [14] like several other 1,4-DHP
derivatives [29–31], but this mechanism is improbable,
N.I. Ryabokon et al. / Mutation Research 587 (2005) 52–58 57
Table 3
Effect of AV-153 on DNA-repair rate after irradiation or exposure to H2O2 or EMS
AV-153, M Parameter τ
100_MH2O2 2Gy _-radiation 100_M EMS
Raji Lymphocytes HL-60 Lymphocytes
Mean±S.E. p Mean±S.E. p Mean±S.E. p Mean±S.E. p
0 27.0±2.8 40.6±3.3 12.7 ± 1.2 48.9±14.5
10−5 21.1±2.1 0.07 – – 15.5 ± 2.2 0.24 39.2±13.2 0.06
10−6 – – 40.3±1.7 0.94 8.3 ± 2.3 0.11 – –
10−7 19.2±6.3 0.07 25.8±4.3 0.05 7.7 ± 0.9 0.021 26.9±12.1 0.31
10−8 – – 22.8±2.0 0.04 – – – –
10−9 15.7±1.4 0.0002 – – – – 26.2±2.6 0.2
The experimental points were fitted to the exponential equation y = a×exp(t/−τ) + c, where τ is a time constant inversely related to the rate of DNA
repair. Values in bold show p < 0.05.
because it influencesDNAdamage induced by alkylating
agents.
The structure of AV-153 resembles that of dihydronicotinamide,
the hydrogen- and electron-transferring
moiety of NADH and NADPH, suggesting two possible
mechanisms for its protective effect against DNA
damage. The oxidized form of NADH is a substrate
for ADP-ribosyl cyclases, and cyclic ADP-ribose mobilizes
calcium (reviewed in [32]). We therefore considered
that AV-153 could influence the cell cycle through
calcium signaling, which in turn could influence the
SSB level. However, we observed that AV-153 had no
influence on the cell cycle parameters of HL-60 cells
at concentrations from 10−9 to 10−5M as assessed by
cytofluorometry (data not shown). A second hypothesis
is that since NADH and NaDPH are substrates for
poly(ADP-ribosyl)polymerase, which modifies proteins
involved in DNA repair [32], a modulating effect of AV-
153 on poly(ADP)ribosylation reactions could underlie
its effects on DNA-repair kinetics. Elucidation of the
details of the mechanism of action of AV-153 requires
further studies.
Acknowledgements
The authors thank Ronald Hancock for critical reading
of the manuscript and discussions. This work was
carried out at the Department of Experimental and Clinical
Radiobiology, Centre of Oncology,M. Sklodowska-
Curie Memorial Institute, Gliwice in the framework of
the scientific agreement between this institute and the
Institute of Genetics and Cytology, National Academy of
Sciences of Belarus, Minsk. The studies were supported
by fellowship funds from the Association for the Support
of Cancer Research, UNESCO (Polish Committee), and
the National Cancer Institute (Bethesda, USA) and by a
grant 4T11F01824 from the Polish State Committee for
Scientific Research (KBN).
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hypertension, Clin. Chim. Acta 299 (2000) 1–10.
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1550–1564.
A 1,4-dihydropyridine derivative reduces DNA damage and
stimulates DNA repair in human cells in vitro
Nadezhda I. Ryabokon a,1, Rose I. Goncharova b, Gunars Duburs c,
Joanna Rzeszowska-Wolny a,∗
a Department of Experimental and Clinical Radiobiology, Centre of Oncology, M. Sklodowska-Curie Memorial Institute,
Wybrzeze Armii Krajowej 15, 44-101 Gliwice, Poland
b Laboratory of Genetic Safety, Institute of Genetics and Cytology, National Academy of Sciences of Belarus,
Akademichnaya 27, 220070 Minsk, Republic of Belarus
c Latvian Institute of Organic Synthesis, Aizkaukles 21, LV-1006 Riga, Latvia
Received 29 April 2005; received in revised form 18 July 2005; accepted 30 July 2005
Available online 3 October 2005
Abstract
Compounds of the 1,4-dihydropyridine (1,4-DHP) series have been shown to reduce spontaneous, alkylation- and radiationinduced
mutation rates in animal test systems. Here we report studies using AV-153, the 1,4-DHP derivative that showed the highest
antimutagenic activity in those tests, to examine if it modulates DNA repair in human peripheral blood lymphocytes and in two
human lymphoblastoid cell lines, Raji and HL-60. AV-153 caused a 50% inhibition of growth (IC50) of Raji and HL-60 cells at
14.9±1.2 and 10.3±0.8 mM, respectively, but did not show a cytotoxic effect at concentrations <100>3 g/kg when administered
intravenously or >10 g/kg with oral administration) [2]
this group of compounds appears to offer promise for
1383-5718/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2005.07.009
N.I. Ryabokon et al. / Mutation Research 587 (2005) 52–58 53
medical applications. Among 12 screened 1,4-DHPs
that differed in chemical structure, six _-carbonyl-1,4-
dihydropyridines that are analogues of dihydronicotinamide,
the hydrogen- and electron-transferring moiety
of the redox coenzymes NADH and NADPH, showed
antimutagenic activity and significantly reduced spontaneous
and alkylation-induced point mutations and chromosome
breaks in germ cells of Drosophila [13,14],
alkylation-induced micronuclei in mouse bone-marrow
cells [15], and radiation-induced chromosome aberrations
and other cytogenetic end-points in fish [16]. The
reduction of mutation frequency reached 85% in some
cases [14]. Studies on Drosophila suggested that the 1,4-
DHPsinhibit chemical mutagenesis due to modulation of
DNA repair [13–15], and in the present study we examined
the effects of AV-153, a 1,4-DHP that showed the
highest antimutagenic activity in animals [13,14], on
spontaneous, chemically- and radiation-induced DNA
damage and its repair in human cells in vitro.
2. Materials and methods
2.1. AV-153
Sodium 3,5-bis-ethoxycarbonyl-2,6,dimethyl-1,4-dihydropyridine-
4-carboxylate (AV-153), synthesized in the Latvian
Institute of Organic Synthesis, is an analogue of the active center
of the reduced form of nicotinamide adenine dinucleotide
(NADH) or its phosphate (NADPH) (Fig. 1). It is a yellow
crystalline powder, resistant to temperature fluctuations, soluble
in water and it passes the cell membrane. Stock solutions
were prepared in phosphate-buffered saline (PBS) or culture
medium and kept in the dark at 4 ◦C during a period of one
month.
2.2. Cells and media
Venous blood from a young non-smoking healthy female
was collected in heparinized tubes. Histopaque-1077 (ICN)
was used to separate mononuclear lymphocytes, which were
washed in RPMI 1640 (Sigma–Aldrich) at 4 ◦C and incubated
in RPMI 1640 with 10% fetal bovine serum (FBS; Gibco), lglutamine
and 0.1% gentamicine at 37 ◦Cin a humidified atmosphere
with 5% CO2. Human HL-60 (promyelocytic leukemia)
and Raji (B-lymphoblastic leukemia) cells were cultured in the
same conditions and used during exponential growth.
Fig. 1. Structure of AV-153. Positions R3 and R5 bear a OC2H5 group
and position R4 a COONa group.
2.3. Assessment of cell viability
We used the colorimetric methyl-thiazol-tetrazolium
(MTT) assay [17] to study cell survival after incubation with
AV-153. Briefly, cells in RPMI 1640 without phenol red
(Sigma–Aldrich) were seeded in 96-well plates (NUNC, Denmark)
using 1.5×104 cells/100_l/well. Equal aliquots of PBS
containing AV-153 at different concentrations were added 24 h
later. Wells without cells or without AV-153 were used to
determine baseline values. After further incubation for 24 h
at 37 ◦C, MTT (Sigma–Aldrich) was added to all wells to
a final concentration of 0.5 mg/ml and the plates were incubated
for 3 h in the same conditions. The formazan crystals
formed were dissolved by addition of 150_l of DMSO and
the optical density (OD) at 570 nm was measured with an
ELx800 microplate reader (Bio-Tek Instruments, USA). The
percentage cell survival was calculated by the equation [viable
cells = (ODt −ODtb)/(ODc −ODcb)×100], where ODt is for
the cell sample with AV-153, ODtb for AV-153 alone (test
blank), ODc for control cells, and ODcb for the control blank
without cells and AV-153. All experimental series were performed
at least three times with triplicate samples. The IC50,
i.e. the concentration at which 50% of cells showed inhibition
of MTT processing, was calculated from the dose-response
curve. The standard Trypan-blue exclusion test was also used
according to the manufacturer’s indications (Sigma–Aldrich)
and blue (non-viable) cells were scored under the microscope.
2.4. Treatment with genotoxic factors
Cells were used at a concentration of 4×105/ml. Gammairradiation
was performed on ice using a 60Co radiotherapy
source (Gammatron, Siemens) at a dose rate of 0.8 Gy/min
to a total dose of 2Gy. In other experiments cells suspended
in ice-cold PBS were incubated with ethylmethane sulfonate
(EMS, Sigma–Aldrich) or hydrogen peroxide (H2O2) at final
concentrations of 100 _Mfor 1–3 min,washed in ice-cold PBS,
and suspended in complete medium at 37 ◦C. Equal aliquots
of AV-153 at different concentrations were added immediately
after irradiation or after washing off the chemical agents.
2.5. Alkaline single-cell gel electrophoresis (comet) assay
DNA breaks were assessed by comet assays as described
in [18–20] with all steps of preparation performed on ice.
Images of 100 randomly selected cells were analyzed per experimental
point. In initial experiments an image-analysis system
(Lucia version 4.60, Laboratory Imaging Ltd.) was compared
with visual scoring and a statistically significant correlation
(r≥0.72, p < d="A1" y ="−a×ln(x)">0.92 and p <>100_Mand the IC50 differed slightly
for the different cell types tested, HL-60 cells being more
sensitive than Raji cells.HL60cells are p53-deficient due
to deletions in the gene coding for this protein [24], but
nevertheless they undergo apoptosis readily and show a
G2 checkpoint [25]. They have been found to be more
sensitive than other cell lines in studies of anticancer
drugs [26,27].
In the lower range of concentrations tested, AV-153
caused a decrease in the level of DNA SSBs as measured
by alkaline comet assays, which detect single and
double strand breaks including those associated with
replication, incomplete excision repair of DNA damage,
or alkali-labile (mostly apurinic and apyrimidinic,
AP) sites [19]. The levels of both endogenous SSBs,
which are common lesions caused byDNAoxidation and
AP sites formed during metabolic processes (up to 104
depurinizations occur in human cells per day [28]), and
of SSBs caused by ionizing radiation (oxidative damage
to bases and cross-links) or alkylating agents like
EMS (N-alkylation of purines and AP sites) were significantly
reduced. These effects suggest that AV-153
may directly stimulate DNA repair, which is consistent
with its influence on DNA-repair kinetics seen here and
also with the results of Goncharova and Kuzhir [13,14]
who showed that it reduced the frequency of spontaneous
and EMS-induced point mutations in germ cells of
Drosophila by 85% and 40%, respectively.AV-153 could
influence cellular redox equilibria, because it may possess
antioxidant activity [14] like several other 1,4-DHP
derivatives [29–31], but this mechanism is improbable,
N.I. Ryabokon et al. / Mutation Research 587 (2005) 52–58 57
Table 3
Effect of AV-153 on DNA-repair rate after irradiation or exposure to H2O2 or EMS
AV-153, M Parameter τ
100_MH2O2 2Gy _-radiation 100_M EMS
Raji Lymphocytes HL-60 Lymphocytes
Mean±S.E. p Mean±S.E. p Mean±S.E. p Mean±S.E. p
0 27.0±2.8 40.6±3.3 12.7 ± 1.2 48.9±14.5
10−5 21.1±2.1 0.07 – – 15.5 ± 2.2 0.24 39.2±13.2 0.06
10−6 – – 40.3±1.7 0.94 8.3 ± 2.3 0.11 – –
10−7 19.2±6.3 0.07 25.8±4.3 0.05 7.7 ± 0.9 0.021 26.9±12.1 0.31
10−8 – – 22.8±2.0 0.04 – – – –
10−9 15.7±1.4 0.0002 – – – – 26.2±2.6 0.2
The experimental points were fitted to the exponential equation y = a×exp(t/−τ) + c, where τ is a time constant inversely related to the rate of DNA
repair. Values in bold show p < 0.05.
because it influencesDNAdamage induced by alkylating
agents.
The structure of AV-153 resembles that of dihydronicotinamide,
the hydrogen- and electron-transferring
moiety of NADH and NADPH, suggesting two possible
mechanisms for its protective effect against DNA
damage. The oxidized form of NADH is a substrate
for ADP-ribosyl cyclases, and cyclic ADP-ribose mobilizes
calcium (reviewed in [32]). We therefore considered
that AV-153 could influence the cell cycle through
calcium signaling, which in turn could influence the
SSB level. However, we observed that AV-153 had no
influence on the cell cycle parameters of HL-60 cells
at concentrations from 10−9 to 10−5M as assessed by
cytofluorometry (data not shown). A second hypothesis
is that since NADH and NaDPH are substrates for
poly(ADP-ribosyl)polymerase, which modifies proteins
involved in DNA repair [32], a modulating effect of AV-
153 on poly(ADP)ribosylation reactions could underlie
its effects on DNA-repair kinetics. Elucidation of the
details of the mechanism of action of AV-153 requires
further studies.
Acknowledgements
The authors thank Ronald Hancock for critical reading
of the manuscript and discussions. This work was
carried out at the Department of Experimental and Clinical
Radiobiology, Centre of Oncology,M. Sklodowska-
Curie Memorial Institute, Gliwice in the framework of
the scientific agreement between this institute and the
Institute of Genetics and Cytology, National Academy of
Sciences of Belarus, Minsk. The studies were supported
by fellowship funds from the Association for the Support
of Cancer Research, UNESCO (Polish Committee), and
the National Cancer Institute (Bethesda, USA) and by a
grant 4T11F01824 from the Polish State Committee for
Scientific Research (KBN).
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R. I. Goncharova: Transgenerational accumulation of radiation damage
Radiat Environ Biophys (2006) 45: 167–177
DOI 10.1007/s00411-006-0054-3
ORIGINAL PAPER
Nadezhda I. Ryabokon · R. I. Goncharova
Transgenerational accumulation of radiation damage
in small mammals chronically exposed to Chernobyl fallout
Received: 5 March 2006 / Accepted: 17 June 2006 / Published online: 22 July 2006
© Springer-Verlag 2006
Abstract The purpose of this investigation has been the
analysis of the long-term development of biological damage
in natural populations of a model mammalian species,
the bank vole (Clethrionomys glareolus, Schreber),
which were chronically exposed to low doses of ionizing
radiation over 22 animal generations within 10 years following
the Chernobyl accident. The time course of the
biological end-points (chromosome aberrations in bone
marrow cells and embryonic lethality) was compared
with the time course of the whole-body absorbed dose
rate from external and internal exposure in the studied
populations inhabiting monitoring sites in Belarus with
diVerent ground deposition of radionuclides. The yield of
chromosome aberrations and, in lesser degree, embryonic
lethality was associated with the radionuclide contamination
of the monitoring areas in a dose-dependent
manner. As a main feature of the long-term development
of biological damage under low dose rate irradiation,
permanently elevated levels of chromosome aberrations
and an increasing frequency of embryonic lethality have
developed over 22 animal generations. This contrasts
with the assumption that the biological damage would
gradually disappear since in the same period of time the
whole-body absorbed dose rate decreased exponentially
with a half-value time of about 2.5–3 years. Furthermore,
gravid females were captured, and their oVspring, born
and grown up under contamination-free laboratory conditions,
showed the same enhanced level of chromosome
aberrations. Therefore the authors suggest that, along
with the biological damage attributable to the individual
exposure of each animal, the observed cellular and systemic
eVects reXect the transgenerational transmission
and accumulation, via genetic and/or epigenetic pathways,
of damage attributable to the chronic low-dose
rate exposure of the preceding generations of animals.
They also suggest that the level of the accumulated transmissible
damage in the investigated populations will
decrease in future due to the further recession of the
chronic exposure and as a consequence of selection processes.
Introduction
The Chernobyl accident in April 1986 caused the deposition
of radionuclides across Europe, followed by a longterm
artiWcial increase of the radiation background [1].
In addition to the classical subject of mutagenesis after
acute radiation exposure [2], the study of the time course
of biological damage associated with chronic low-dose
radiation exposure of mammals and the endeavors to
predict biological damage in consecutive generations
have become a relevant issue. Since there is little information
on this topic [3, 4], the present work addresses
these important questions.
Starting with 1986, we were engaged in studying biological
eVects of chronic low dose radiation in natural
populations of bank vole (Clethrionomys glareolus, Schreber)
in a series of many animal generations. The bank
vole is a widespread rodent species that is used as indicator
of environmental quality. It is a convenient object for
many genetic tests, which originally have been devised for
the laboratory mouse [5]. Comparison of own and literature
data on doubling doses of acute irradiation for chromosome
injuries had shown that the sensitivity of somatic
cells of the bank vole to ionizing radiation is very similar
to the sensitivity of human lymphocytes and germ cells of
laboratory mice [6]. This Wnding conWrms that the bank
vole is a suitable model species for assessment of genetic
radiation risks in mammals.
According to the Atlas of Caesium Deposition in
Europe [1], practically the whole Belarusan territory was
contaminated by 137Cs above the level of global fallout.
A ground deposition of 137Cs equal to 37 kBq/m2 (1 Ci/
km2) was chosen to discriminate between so-called
“clean” regions and contaminated regions. We selected
monitoring sites representing large areas of Belarus with
a strong gradient of radionuclide ground deposition,
from 8 to 8,500 kBq/m2 of 137Cs, i.e., from light contamination
in “clean” regions to high contamination in the
evacuated zone. In comparison with the pre-Chernobyl
data, increased frequencies of chromosome aberrations
and genomic mutations in somatic cells, abnormal sperm
heads and embryonic losses were observed in bank vole
populations at the monitoring sites [7, 8].
The aim of the present study was to analyze the longterm
development of chromosome aberration frequency
and embryonic lethality in bank vole populations over
22 animal generations living in 1986–1996 and to compare
it with the time course of the whole-body absorbed
dose rate.
Materials and methods
Monitoring sites
The animals were collected at Wve monitoring sites in
large forestry areas with limited human activities, far
from possible sources of industrial or domestic pollution.
These sites are located at diVerent distances and directions
from the Chernobyl nuclear power plant (Fig. 1)
and represent diVerent levels of radionuclide contamination:
site 1—the Priluksky Reserve, Minsk region,
330 km NW, site 2—the Berezinsky Biosphere Reserve,
Vitebsk region, 400 km NNW, site 3—the vicinity of
Majsk village, Bragin district, Gomel region, 60 km N,
site 4—the vicinity of the evacuated Babchin village,
Khoiniki district, Gomel region, 40 km NNW and site
5—the vicinity of the evacuated Radin village, Khoiniki
district, Gomel region, 18 km N. The initial levels of
radionuclide contamination of soil at these sites were
determined earlier [9] and are shown in Table 1. The animals
were captured using live traps with bait. The Wrst
animals were captured at sites 1, 3, and 4 in September
1986, i.e., about 5 months after the period of acute irradiation
by short- and long-lived radionuclides. In the subsequent
years, the animals were as well usually collected
in the period of August–September in order to exclude
the inXuence on the data of seasonal changes in the age
structure of populations. Data obtained outside this collection
period (e.g., the data from site 1 in 1991 and 1996
[7, 8]) were not included in this study. Investigations at
sites 2 and 5 started in 1991 and 1996, respectively, i.e., 5
and 10 years after the accident.
Assessment of animal age and change of animal
generations
For age determination, root, cusp, and height of the Wrst
mandibular molar (M2) were measured according to
Bashenina [10]. The specimens were divided into seven
age groups, which corresponded to ages of approximately
2 weeks, 1, 2, 3, and 4 months and 1 and 1.5 years.
The bank vole, like other rodent species, is known to
have a short generation time with a complete change of
about 2–3 generations per year [10]. According to data of
Rozhdestvenskaya [11] and our own estimates of animal
participation in reproduction, this holds also for the
Fig. 1 Map of Belarus with localization of the monitoring sites. The
position of the Chernobyl nuclear power plant (ChNPP) in the
neighboring Ukraine is also indicated
Table 1 Densities of radionuclide contamination of soil (kBq/m2) at Wve monitoring sites (data of April–May 1986 for 137Cs, 134Cs, 106Ru,
144Ce and data of August 1996 for 90Sr and transuranic radionuclides) according to data [9]
Site April–May 1986 August 1996
137Cs 134Cs 106Ru 144Ce 90Sr 238Pu 239,240Pu 241Pu 241Am
1 8 4 5 0 4 0.04 0.10 2.98 0.14
2 18 9 12 0 5 0.07 0.14 5.10 0.19
3 220 140 150 440 39 0.62 1.28 48.80 1.81
4 1,530 1,020 1,090 3,050 117 1.17 2.35 86.70 3.21
5 8,500 5,650 5,790 1,7200 1,200 4.90 11.00 420.00 15.00
169
populations studied in the present work. Due to seasonal
reproduction and short life-span, in the trapping periods
of late summer and early autumn only few animals born
in the preceding year were observed at the monitoring
sites, showing that the populations were almost completely
renewed every subsequent year by the time of
observation.
The populations living at the time of the accident, in
early spring of 1986, consisted of adult animals born in
the previous year. Due to the turnover of generations,
our investigations, beginning in September 1986, started
with the Wrst and second post-accidental generations of
bank vole. In the period from 1986 to 1996, at least 22
generations were studied.
Determination of radionuclide concentration
and estimation of absorbed dose rate
Concentrations of radionuclides in the soil and the
whole-body animal samples were assessed as described in
[9]. BrieXy, the _-spectrometry of samples was performed
in the Hydrometeorological Centre of Belarus in Minsk
using the _-spectrometer ADCAM-300 equipped with a
high-purity germanium detector GEM-30185 (EG&G
Ortec, USA). SpeciWc activities of 90Sr and transuranic
elements were determined by the staV of the Institute of
Radiobiology, National Academy of Sciences of Belarus,
using radiochemical methods and the _-spectrometer
Ortec 576A with the silicon surface barrier detector
(EG&G Ortec, USA), the liquid scintillation counter Tricarb
2700TR (Packard Company) and a gas-Xow
counter (Tesla Automat, Slovakia).
Dose rates due to internal and external exposure were
assessed in the studied specimens as described previously
[9]. The whole-body absorbed dose rate from incorporated
_-emitting radionuclides was calculated according
to the absorbed fraction model. For the dose rate attributable
to incorporated _- and _-emitters, we used the
local absorption model. The whole-body absorbed dose
was calculated as the product of the individual wholebody
absorbed dose rate and the age of the animal at the
time of capture.
Metaphase analysis of chromosome aberrations
Cytogenetic eVects in bank vole somatic cells were studied
by metaphase analysis of chromosome aberrations in
red bone marrow cells according to a standard protocol
[12] as described [7, 8]. BrieXy, visually healthy animals of
diVerent age, sex, and state of maturation were randomly
chosen for this test from numerous groups of animals
captured shortly after trapping. Colchicine at concentration
of 0.1 mg per 10 g animal weight was injected intraperitoneally
for 1.5 h to accumulate metaphases.
Animals were sacriWced by ethyl ether and cervical dislocation,
and the marrow was aspirated from the femurs
using inactivated fetal bovine serum. The marrow suspension
was incubated at 37°C for 20 min, treated with
0.56% potassium chloride for the next 20 min and Wxed
in methanol:acetic acid (3:1 v/v). Fixed cells were spread
on clean slides, Xame-dried and stained with Giemsa.
Coded slides were screened for chromatid- and chromosome-
type aberrations in approximately 100 well-spread
metaphases per specimen using standard criteria [12, 13].
Achromatic lesions (gaps) were not included in the statistical
analysis.
Selected age groups
The chromosome aberrations and embryonic losses were
also recorded from selected cohorts of mature animals
with age from 2 to 4 months in order to exclude any agedependent
bias of the biological eVects.
Furthermore, two gravid females were taken to a laboratory
in Minsk, where they gave litters shortly after
capture. Mother animals and their oVspring were fed
with uncontaminated food and were studied for chromosome
aberrations in red bone marrow cells when the age
of the oVspring reached 1.5 months.
Analysis of embryonic losses
Assessment of embryonic mortality was performed in all
captured and visually healthy females at 7–22 days of
pregnancy using the conventional approaches [14]. The
content of the uteri was examined to determine the number
of implants including live and dead embryos. The
early (pre-implantation) losses were calculated as the
ratio (number of corpora lutea minus number of
implants)/(number of corpora lutea). The late (postimplantation)
losses were calculated as the ratio of the
number of dead embryos to the number of implants. The
total losses were determined as the ratio (number of corpora
lutea minus number of live embryos)/(number of
corpora lutea). All ratios are expressed in percent.
Statistics
Chi-square and U tests, regression and correlation analysis
were employed as a part of the Statistica software
package (StatSoft Inc., USA). Figure 2 gives an example
of the scatter of the individual aberration frequency and
whole-body dose rate data at a given site and given season
of the study (site 4, analyzed 5 years after the accident).
In the following, these Xuctuations are represented
by the mean value and the standard deviation of the
mean.
Results
Time course of the absorbed dose rate
The time course of the absorbed dose rates in populations
of bank vole at the monitoring sites was determined
previously [9] using numerous groups of captured
animals. It was shown that external _-irradiation and
170
internal _-irradiation by incorporated 137Cs and 134Cs
delivered the most prominent contributions to the
whole-body dose rate over the monitoring period, which
started 5 months after the accident with the Wrst and second
post-accident animal generations and ended 1996
after about 22 animal generations [9]. Here, we analyzed
the time course of the mean whole-body absorbed dose
rate due to internal _-irradiation and to total irradiation
speciWcally for those animals which, out of the whole
cohorts, were randomly chosen for assessment of chromosome
aberrations (Table 2; Fig. 3a, b) and embryonic
mortality (Table 3; Fig. 3c, d). The values of the wholebody
dose rates and their temporal development in these
sub-cohorts of animals were similar to those in the larger
groups of bank vole used in the earlier dosimetrical
investigations of monitored populations [9].
Fig. 2 Correlation of the chromosome aberration frequency in
bone marrow cells of bank voles inhabiting site 4, with the individual
whole-body absorbed dose rates 5 years after the Chernobyl accident.
The dose rates are due to external _- and internal _ + _-radiation
of incorporated 137Cs and 134Cs
Mean value
0
2
4
6
8
10
12
60 80 100 120 140
Whole-body dose rate, μGy/d
Chromosome aberrations per 100 cells
Fig. 3 Time course of the mean whole-body absorbed dose rates in
bank vole specimens captured at sites 1–5 and studied for chromosome
aberrations (a, b) and for embryonic mortality (c, d). The contributions
by internal _-irradiation from incorporated 137Cs and
134Cs are presented in a and c, the total whole-body dose rates by
external _-irradiation and internal _ + _-irradiation from incorporated
137Cs and 134Cs are shown, together with exponential approximation
curves, in b and d. Error bars are not shown for clarity
Internal â-irradiation Total irradiation
Site 1 Site 2
Site 3
Site 4 Site 5
1
10
100
1000
0 6 10
Years since accident
Site 1
Site 2
Site 3
Site 5
Site 4
0.1
1
10
100
1000
Years since accident
Whole-body dose rate, μGy/d
Site 2
Site 3
Site 4
1
10
100
1000
Years since accident
Site 2
Site 3
Site 4
0.1
1
10
100
1000
Years since accident
0.1
0.1
2 4 8 0 2 4 6 8 10
0 2 4 6 8 10 0 2 4 6 8 10
(c)
(a) (b)
(d)
Table 2 Chromosome aberrations in bone marrow cells of bank vole at Wve monitoring sites
a Pre-accident data according to [15]
b All data on total frequencies of aberrations and aberrant cells in animals at sites 2–5 in 1986–1996 were signiWcantly higher in comparison
with pre-accident data by Chi-square test
Site Year Animal generation
since the accident
Whole-body
absorbed dose
rate (_Gy/day)
Number of analyzed Aberrations per 100 cells § SD Aberrant
cells, %
Animals Cells Chromatid
type
Chromosome
type
Total
1 1986 1–2 6.44 § 0.14 10 992 0.30 § 0.14 0.10 § 0.09 0.40 § 0.22 0.40 § 0.22
1988 5–6 3.36 § 0.02 3 310 0.64 § 0.32 0 0.64 § 0.32 0.64 § 0.32
2 1981–1983a Pre-accident – 24 2,437 0.41 § 0.12 0 0.41 § 0.12 0.41 § 0.12
1991 11–12 4.43 § 0.38 19 1,945 1.19 § 0.37 0.03 § 0.03 1.22 § 0.37b 1.22 § 0.37
1992 13–14 – 17 1,962 0.90 § 0.27 0.27 § 0.17 1.17 § 0.13 1.09 § 0.24
1996 21–22 2.41 § 0.03 8 585 1.06 § 0.54 0.11 § 0.11 1.17 § 0.53 1.17 § 0.53
3 1986 1–2 87.52 § 4.50 18 1,987 1.36 § 0.45 0.76 § 0.28 2.12 § 0.59 1.99 § 0.53
1988 5–6 76.06 § 8.90 16 1,630 1.22 § 0.49 0.52 § 0.18 1.74 § 0.57 1.51 § 0.03
1991 11–12 16.83 § 2.27 16 1,655 2.67 § 0.81 0.20 § 0.13 2.87 § 0.83 2.49 § 0.76
1996 21–22 7.02 § 0.27 11 1,121 1.86 § 0.41 0.18 § 0.10 2.04 § 0.39 1.95 § 0.38
4 1986 1–2 605.46 § 7.75 16 1,739 1.45 § 0.43 0.06 § 0.06 1.51 § 0.45 1.21 § 0.10
1987 3–4 258.57 § 11.33 36 3,675 0.79 § 0.18 0.44 § 0.15 1.24 § 0.24 1.10 § 0.22
1988 5–6 321.64 § 33.12 21 1,942 1.75 § 0.39 0.17 § 0.09 1.92 § 0.40 1.86 § 0.04
1991 11–12 95.93 § 3.69 31 3,780 1.63 § 0.34 0.31 § 0.10 1.94 § 0.40 1.84 § 0.36
1996 21–22 41.80 § 0.96 14 1,821 1.90 § 0.49 0.24 § 0.15 2.14 § 0.42 2.04 § 0.38
5 1996 21–22 274.52 § 7.27 11 492 4.74 § 0.74 1.47 § 0.61 6.21 § 0.85 5.10 § 0.78
171
The maxima of internal _-irradiation due to incorporated
137Cs and 134Cs were observed in the second year
after the accident (Fig. 3a, c) due to the increasing biological
availability of cesium isotopes from the biota [7-9], followed
by a decrease by one order of magnitude in the
subsequent period. In [9] it was shown that external _-irradiation
was the essential determinant for the time course of
the total whole-body dose rate; this was also true for the
animals analyzed for chromosome aberrations (Fig. 3b) or
embryonic losses (Fig. 3d). The highest values of total dose
rate were observed in the year of the accident, followed by
an approximately exponential decrease over the next
10 years (r2=0.95, P<0.05 and r2=0.91, P<0.05 for
sites 3 and 4, respectively, in Fig. 3b). The reduction factor
since the accident was about 15, corresponding to a halflife
of 2.5–3 years. These data show that after the primary
insult in the year of the accident, every successive generation
of animals was exposed to lower whole-body dose
rates of ionizing radiation than the preceding one.
The mean whole-body absorbed doses in the studied
cohorts of animals were also maximal in the year of radionuclide
deposition. The highest individual value, determined
in the Wrst and second post-accidental generations
of animals at site 4, was 73 mGy. By the end of the monitoring
period, the mean whole-body doses were about 0.3,
0.7, 12, and 25 mGy for sites 2, 3, 4, and 5, respectively [9].
At any given time of the observation period, absorbed
dose rates (Fig. 3) and absorbed doses at the diVerent sites
diVered by about two orders of magnitude, thereby representing
a strong gradient in the level of animal exposure.
Temporal development of the chromosome aberration
frequency
The time course of chromosome aberration frequencies
in the studied animals is presented in Table 2. The frequency
of chromosome aberrations in bone marrow cells
of the bank vole inhabiting the Berezinsky Biosphere
Reserve, i.e., site 2 in our study, was Wrst recorded 3–5 years
before the accident [15] and can be taken as a pre-accident
(historical) control for our data. The data of this
pre-accident control are similar to those observed at the
less contaminated site 1 (Minsk region) in the years 1986
and 1988. Before the accident, only chromatid-type aberrations
were observed, and the cells never contained multiple
aberrations. In contrast, in the post-accident period,
the chromosome anomalies consisted of both chromatidand
chromosome-type aberrations, containing single and
paired fragments in the majority of cases, but also rare
Robertsonian translocations (fusion of acrocentric chromosomes
at the centromeres). In animals inhabiting
contaminated sites, the mean frequencies of both chromosome
aberrations and aberrant cells were signiWcantly
higher (P<0.01, Chi-square test) than the pre-accident
value, and were increased in a dose-dependent manner,
by a factor of 3–7 at sites 2, 3, and 4, and of about 15 at
site 5. Also Pearson correlation analysis of the total aberration
frequencies and those of the Robertsonian translocations
at sites 2, 3 4, and 5 in 1996 (Fig. 4a, b) hints at
a relationship between mean aberration frequencies and
the whole-body dose rates at the time of capture. The
increased frequency of chromosome aberrations
observed in animals inhabiting contaminated areas
appears to remain relatively constant over the years of
the investigation (Table 2).
The data in Table 2 comprise the level of chromosome
aberrations in animals varying by age, sex and
maturity. These animals represent the studied populations,
from where they were chosen at random. Generally,
the mean age of the animals in each group did not
diVer from the usual mean age of about 3–4 months at
the season of capture. There was a single exception at site
Table 3 Embryonic lethality in bank vole populations at three monitoring sites
a Pre-accident level according to [16]
* P< 0.05 and ** P< 0.01 in comparison with pre-accident data (Chi-square test)
Site Year Animal
generation
since the
accident
Whole-body
absorbed dose
rate (_Gy/day)
Number of Mean embryonic lethality,
% (95% binomial conWdence limits)
Animals
analyzed
Yellow
bodies
Embryos
analyzed/
dead
embryos
Before
implantation
After
implantation
Total
2 1981–1983a Pre-accident – 45 249 235/4 5.62 (3.11–9.25) 1.70 (0.47–4.30) 7.23 (4.34–11.18)
1991 11–12 5.5 § 0 12 56 56/3 0 (0.00–5.21) 5.36 (1.12–14.87) 5.36 (1.10–14.87)
1992m 13–14 – 19 92 90/2 2.17 (0.26–7.68) 2.22 (0.27–7.80) 4.35 (1.20–10.76)
1996 21–22 2.51 § 0.13 8 44 39/0 11.36 (3.79–24.56) 0 (0.00–9.03) 11.36 (3.79–24.56)
3 1988 5–6 58.01 § 5.40 7 36 36/0 0 (0.00–7.98) 0 (0.00–7.98) 0 (0.00–7.98)
1989 7–8 44.38 § 3.71 30 138 130/1 5.80 (2.50–11.10) 0.77 (0.02–4.21) 6.52 (3.03–12.08)
1991 11–12 17.95 § 2.53 14 71 65/1 8.45 (3.16–17.49) 1.54 (0.04–8.28) 9.86 (4.06–19.26)
1996 21–22 6.58 § 0.07 3 15 12/1 20.00* (4.33–48.09) 8.33 (0.21–38.48) 26.67* (7.74–55.10)
4 1988 5–6 245.6 § 20.21 14 63 61/1 3.17 (0.39–11.00) 1.64 (0.04–8.80) 4.76 (0.99–13.29)
1989 7–8 265.63 § 34.53 40 201 192/3 4.48 (2.07–8.33) 1.56 (0.32–4.50) 5.97 (3.12–10.20)
1991 11–12 103.78 § 5.96 21 103 91/4 11.65* (6.27–19.76) 4.40 (1.21–10.87) 15.53* (9.15–24.00)
1996 21–22 43.47 § 0.87 11 51 44/4 13.73* (5.70–26.26) 9.09* (2.53–21.67) 21.57** (11.29–35.32)
172
4, 10 years after the accident, when by chance 10 of 14
animals randomly sampled for cytogenetic analysis were
at the age of one year or older, whereas in the more
numerous group of specimens captured at this site for
dosimetrical investigations [9] there was no detectable
change in age distribution. In order to exclude any possible
inXuence of variations in the age distribution on the
observed frequencies of chromosome aberration, analysis
of the time course of the chromosome aberration frequency
was limited to mature 2–4 months old animals,
whose mean values and temporal development of age
over the period of this study is shown in Fig. 5a. The
temporal pattern of the frequency of chromosome aberrations
in the 2–4 months old animals (Fig. 5b) was
almost identical with the pattern observed in all animals
(Table 2).
As is evident from Fig. 5, the frequencies of chromosome
aberrations observed in 2–4 months old bank voles
remain fairly constant over time, in spite of the approximately
exponential decrease of the whole-body dose rate.
This suggests that the induction of aberrations depends
not only on the actual exposure level. To test this, aberration
frequencies were investigated in the oVspring of
mice captured in 1988 at sites 3 and 4, which were born
and brought up in the laboratory and fed with uncontaminated
food. The chromosome aberration frequency
observed in the oVspring animals showed no signiWcant
diVerence to that observed in animals from the same sites
that had grown up in the contaminated environment,
and to the aberration frequency of their mother animals
(Fig. 6).
Embryonic mortality
Studies on embryonic losses in the monitored populations
were started 2 years after the accident. Since at sites
1 and 5 no gravid females after the stage of embryonic
implantation were captured, the analysis on embryonic
mortality is limited to sites 2, 3, and 4. Figure 7a shows
that there was no increase over the monitoring period in
the age of gravid females, which could have aVected the
values of embryonic lethality. Rather, the mean age of
Fig. 4 Correlation between mean frequency values of all types of
chromosome aberrations (a) and of Robertsonian translocation (b)
with the mean values of the whole-body absorbed dose rate in bank
vole populations inhabiting sites 2–5, 10 years after the accident.
Standard deviations of the mean values are indicated as error bars
Site 2
Site 3
Site 4
Site 5
r = 0.99
P = 0.011
0
2
4
6
8
0 100 200 300
Whole-body dose rate (μGy/d)
Chromosome aberrations per 100 cells
Site 5
Site 4
Site 3
Site 2
r = 0.95
P = 0.048
0
0 100 200 300
Whole-body dose rate (μGy/d)
Robertsonian translocations per 100 cells
0.8
0.6
0.4
0.2
(a)
(b)
Fig. 5 Time course of animal age (a) and chromosome aberration
frequency (b) in a sub-cohort of adult, 2–4 months old bank voles at
four sites. Mean data and standard deviation are shown. Pre-accident
data according to [15]
(a)
(b)
0
1
2
3
4
5
6
0 2 4 6 8 10
Years since accident
Age of animals (months)
Site 1
Site 2
Site 3
Site 4
Site 1
preaccident
Site 2
Site 3
Site 4
0
1
2
3
4
0 2 4 6 8 10
Years since accident
Chromosome aberrations per 100 cells
173
the gravid females tended to decrease over the period of
monitoring. The frequencies of embryonic losses in the
population inhabiting the Berezinsky Biosphere Reserve
3–5 years prior to the accident [16] were used as pre-accident
control (Table 3; Fig. 7b). A comparison of pre- and
post-accident data shows that both pre- and postimplantation
embryonic mortality in populations in the
moderately contaminated site 2 and in the more highly
contaminated sites 3 and 4 remained on the level of preaccident
mortality frequencies during the Wrst years of
observation. After 5–10 years, however, a tendency
towards increasing embryonic lethality was found in all
populations studied, reaching a statistically signiWcant
increase in samples from sites 3 and 4 after 10 years (by a
factor 2–5 in comparison with pre-accident data).
Discussion
In this study free-living small mammals, the bank voles,
were used as indicator organisms for the monitoring of
environmental eVects. The studied populations lived at
diVerent distances from the Chernobyl nuclear power
plant. The investigations were started 5 months after the
accident, from the 1 and 2 post-accident generations of
animals that were irradiated at doses one order of magnitude
less than animals in the acute period after the
accident. The particular subject of this study was to compare
the time course of biological damage in animals,
representing up to 22 generations living during 10 years
following the Chernobyl accident, with the time course
of the radiation exposure experienced by these animals.
The main results of this study are (a) a dose-dependent
increase in the frequencies of chromosome aberrations
and embryonic losses in animals living in contaminated
areas as compared to historic controls and to animals
living in an area with the lowest level of contamination,
(b) the fact that the chromosome aberration frequencies
remained on elevated levels (Fig. 5) and the frequencies
of embryonic losses even increased (Fig. 7) over 10 years
after the accident, although the whole-body absorbed
dose rates experienced by the animals declined almost
exponentially (Fig. 3).
Historic controls raise the concern that they might
not directly be comparable to the samples under study.
In this study the frequencies of chromosome aberrations
observed in 1986 and 1988 in animals living at the almost
uncontaminated site 1 can be used as controls, and they
were similar to those assessed in the historic control
(Table 2; Fig. 3). Thereby we can exclude that the
increases in chromosome aberration frequency seen in
the contaminated areas have reasons other than the
higher radiation exposure. Unfortunately, we have no
data from site 1 for the years 1991 and 1996 collected in
the period August–September, but the fact that all investigations
at the contaminated sites were done by the
same team guarantees that the methods of investigation
Fig. 6 Chromosome aberration frequencies in bank vole females
captured at sites 3 and 4 in 1988, and of their oVspring, born, and
raised under laboratory conditions for about 1.5 months before
analysis. Data are compared in bank vole populations living at sites
3 and 4 at the same period. Means, standard deviations of the means
and the number of analyzed animals are shown
n = 2
n = 9
n = 37
Pre-accident
level
0
1
2
3
Mothers Offspring Populations
Chromosome aberrations per 100 cells
Fig. 7 a: Time course of the age of gravid bank vole females analyzed
for embryonic losses. Means and standard deviation of the
means are show. b: Time course of embryonic lethality in animals at
site 2 and at sites 3 and 4 (pooled). Means and binomial 95% conWdence
limits are indicated. Pre-accident data according to [16]
(a)
0
2
4
6
8
10
0 2 4 6 8 10
Years since accident
Age of animals (months)
Site 2
Site 3
Site 4
(b)
**
0
20
40
60
0 2 4 6 8 10
Years since accident
Embryonic lethality (%)
Site 2 Site 3+4
preaccident
174
have not changed in these years. Therefore, and in line
with the unequivocal dose-rate dependency displayed in
Figs. 4, 5 and 7, we propose a radiogenic etiology of the
observed biological eVects.
Steady decrease of the whole-body dose rate over
the monitoring period of 1986-1996
At all sites investigated, whole-body absorbed dose rates
were found to decrease over about one order of magnitude
in 10 years, which is attributable to the gradual disappearance
of the radioactive fall-out material from the
biota [9]. More speciWcally, we show that the whole-body
dose rate decreased approximately according to an exponential
function with a half-life of 2.5–3 years (Fig. 3b,
d). As the observed biological damage did not decrease
in the same animals over the studied period, it is important
to exclude that the exposure levels in the later years
of the investigation, due to long-lived _-emitting transuranic
radionuclides and/or increasing 90Sr concentrations
in the studied populations, were underestimated.
Our analyses of radionuclide concentrations in animals
showed that 10 years after the accident the contribution
of transuranic elements and 90Sr to the whole-body dose
rate in the studied specimens of bank vole did not exceed
3 and 8%, respectively [9]. This contribution was estimated
to be two orders of magnitude lower than the
whole-body dose rates during the year of the accident [9].
The contribution of 90Sr to local doses in bone tissue in
the later years of the investigation was about 2–10£ lower than that of external _-irradiation. The absorbed
dose rate in the red bone marrow due to 90Sr _-radiation,
which is approximately equal to the absorbed dose rates
averaged over the bone tissues, was of the same small
magnitude, comparable to that of 137+134Cs _-radiation
in the later years and an order of magnitude lower than
_-radiation of 137+134Cs in early period after the accident.
We conclude that doses at late time points were not
underestimated by our approach.
Dose dependence of the observed eVects
Data presented in Tables 2 and 3, as well as in Figs. 5
and 7, hint at a strong dependence of the observed biological
eVects on radiation exposure. This is manifested
by relatively low levels of aberrations and, where present,
embryonic losses at the less contaminated sites 1 and 2,
the medium levels at sites 3 and 4, and the relatively high
level at the most contaminated site 5. Furthermore, there
is a statistically signiWcant linear correlation between
mean values of the chromosome aberration frequency
observed 10 years post-accident and the mean wholebody
dose rates prevailing in the studied animals at the
same time (Fig. 4). A similar association of mean
micronuclei frequencies with exposure at low dose rates
(4.22–39.4 _Gy/day) has been shown in bank vole populations
studied 2 years after the accident at four Swedish
regions contaminated by Chernobyl fallout at deposition
levels of 1.8, 22, 90, and 145 kBq/m2 [17], i.e., levels similar
to those at our monitoring sites 1–3 (Table 1). Mean
levels of chromosome aberrations were also increased in
a dose-dependent manner in laboratory mice, exposed in
our monitoring areas during 4 months to whole-body
dose rates from external and internal irradiation of 3–
145 _Gy/day [8], i.e., at exposure levels close to those in
free-living animals.
Dose dependence of the biological damage is a basic
requirement for conclusions concerning the causal role
of radiation exposure for the observed eVects. This
includes the possibility of direct as well as indirect modes
of action, in other words, the induction of the eVects seen
in a given generation by radiation exposure of the same
or of the preceding generations of animals. In such a
way, a linear correlation observed between mean values
of chromosome aberration frequencies and the dose
rates in late generations of animals 10 years after the
accident can as well demonstrate a linear correlation of
the aberration frequencies with the dose rates in all previous
years, because the dose rates at the various sites
remained in constant proportions to each other, while
they decreased in an exponential fashion over the monitoring
period (Fig. 4). On the other hand, the scatter
plots of the individual frequency of chromosome aberrations
versus the individual level of the whole-body
absorbed dose rate or whole-body dose may hint at a
dose dependence of the biological eVect even on the
exposure of the individual animal (Fig. 2 and data not
shown). An analogous correlation has been observed
between the individual frequencies of micronuclei and
the individual radiation exposures in the same population
of animals [6]. These dose-eVect relationships should
be studied in future.
Long-term persistence of biological eVects and possible
mechanism of their transgenerational transmission
Our data (Tables 2, 3; Figs. 5, 7) suggest that biological
eVects in bank voles living in regions contaminated by
the Chernobyl accident persisted on a stable level (chromosome
aberrations) or even increased (embryonic
loses) over 10 years, corresponding to at least 22 animal
generations, although the dose rates have substantially
decreased during this period. This observation is in fundamental
contrast to the assumption that the biological
injury would gradually disappear in direct connection
with the exponential reduction of the whole-body dose
rate. Thus, the biological eVects cannot be explained
alone by the exposure experienced by the individual animals.
Rather, our observations call for a diVerent interpretation,
namely that the biological eVects reXect a
transgenerational transmission of radiation damage that
occurred in early generations, especially in the generations
living at the time of the accident and in the early
post-accidental generations, and was further accumulated
in the course of the chronic exposure of the following
generations. The fact that oVspring from captured
females, irradiated in utero at contaminated areas, but
born in the laboratory and fed with uncontaminated
175
food, did not show a signiWcant reduction in aberration
frequencies in comparison with animals born and grown
up in the contaminated environment (Fig. 6) corroborates
the hypothesis that some mechanism of transgenerational
transmission of radiation damage has been
manifested.
The occurrence of radiation damage in cells and tissues
that have not been directly exposed to radiation is
not without precedence. For example, genomic instability
is a long-term radiation eVect that has been intensively
investigated. The term “genomic instability” refers
to the de novo production of delayed responses of the
genome in the progeny of the exposed cells in a cell culture
or in an irradiated animal [18–22]. Genomic instability,
including chromosomal instability in hemopoietic
stem cells in vitro and in vivo, has been shown to be activated
by high- and low-LET ionizing radiations at relatively
low doses [21–25] and to exhibit a dose-dependent
increase at higher doses [24]. Dose dependence was also
observed for chromosomal instability in mice fetuses
irradiated as zygotes [26]. Other authors did, however,
not Wnd a dose relationship for genomic instability over
a wide range of radiation doses [27–29]. The expression
of genomic instability was described to persist for up to
or even more than 70–80 population doublings at higher
doses [29, 30]. In the progeny of long-term repopulating
haemopoietic stem cells of mice, chromosomal instability
was observed over 24 months after cell irradiation and
transplantation [22]. There is evidence that the genetic
background or genotype can inXuence radiation-induced
instability [25]. The mechanisms of genomic instability
are largely unknown. At least part of them may rely on
epigenetic transmission of information. For instance,
genomic instability was suggested to reXect altered signal
transduction pathways and changes in the DNA microenvironment
[31, 32]. Since DNA methylation is linked
to chromosome condensation [33], chromosome and
chromatid fragments as well as Robertsonian translocations
may be due explained by the radiation-induced
increased fragility of the chromatin during chromosome
condensation [34]. These types of chromosome aberrations
have been suggested as hallmarks of induced genomic
instability, although other types of chromosome
aberration can also occur [24, 26, 27, 30].
However, in contrast to studies where long-term
eVects are investigated in the same animals, which had
been exposed to radiation at an earlier stage of individual
development, the interpretation of the eVects seen in
our study must deal with damage persisting over many
generations of animals. In the literature, the term “transgenerational
transmission of radiation damage” has
mainly been used in a descriptive sense, including genetic
and epigenetic as well as hitherto uncharacterized mechanisms
of transmission damage to the progeny [21, 35–
42]. Delayed radiation-induced responses attributable to
genomic instability were revealed in the Wrst few animal
generations after irradiation of male germ cells [43] or
zygotes [37]. Heritable tumors and anomalies were
observed in descendants of exposed mice [35], and physiological
or developmental disorders were seen in the
progenies of irradiated parents, which resulted in embryonic
and early postnatal death, fertility disturbances,
congenital abnormalities or malformations [44]. Genomic
instability in the Wrst-generation oVspring (F1) of
irradiated mice or rats covers diverse endpoints such as
chromosome aberrations, micronuclei, point mutations
and reversions in somatic cells (reviewed in [39]) as well
as early (pre-implantation) and late (post-implantation)
embryonic resorption [37], reduced fertilization rate [45],
increased number of sterile females [37] and reduction in
the proliferative ability of both F1 and F2 embryonic
cells [36] and F1 liver cells [38]. Transgenerational germline
instability was also demonstrated in the oVspring of
irradiated mice (reviewed in [39]), as well as in children
born from irradiated parents [46] and in barn swallows
breeding not far (25–50 km) from the Chernobyl nuclear
power plant [47]. In addition, transgenerational transmission
of genomic and developmental eVects was
reported after parental exposure to certain chemicals
[e.g., 35, 41].
In conclusion, we suggest that in the studied bank vole
populations the radiation exposure of the parental generations
has lead to an accumulated pool of germline mutations
and/or of epigenetic changes, which resulted in the
observed, persistently elevated levels of chromosome aberrations
in somatic cells and in increased embryonic losses
in later generations. With regard to the continuous buildup
of transgenerationally-transmitted damage in the
course of chronic radiation exposure of the parental generations,
we will shortly speak of the transgenerational
accumulation of transmitted biological damage.
The present report is the Wrst in which the time course
of biological damage in mammals chronically exposed to
ionizing radiation over a series of generations has been
studied. So far, the long-term development of transgenerationally
transmitted radiation damage has been studied
in a restricted number of consecutive generations of animals,
generally in the Wrst 1–2 generations after irradiation
of parents [21, 31, 35–39, 43, 46]. Increased
frequencies of chromosome aberrations were also
observed in bone marrow cells after 25–30 generations of
red vole [3] and after 75–80 generations of common vole
[48] chronically irradiated in regions of heavy radioactive
contamination in the Urals as well as in bordering areas.
The latter observation was explained by hereditary chromosome
instability [48], but there is no data on the quantitative
time course of these eVects. The question arises
whether the levels of chromosomal aberrations and
embryonic lethality that in our study were reached in
1996 after 22 animal generations, will be maintained permanently,
or whether a phase of reduction will follow in
subsequent animal generations. Our study indicates that
a further reduction of chronic radiation exposure has to
be anticipated, and that there exists an eVective selection
mechanism, the dose-dependent increased embryonic
lethality in animal populations in radiocontaminated
areas. Further research will be required to clarify this
genetically and ecologically interesting point.
176
Acknowledgments This work was performed within the framework
of the State Program of the Republic of Belarus for minimizing and
overcoming consequences of the Chernobyl Accident (1986–2002).
The authors wish to thank the administrations of the Chernobyl
Exclusion Zone and the Berezinsky Biosphere Reserve for access to
the zone and the reserve. We are indebted to former and present staV
of the Antimutagenesis Laboratory, the Institute of Genetics and
Cytology, NAS of Belarus, for enthusiastic Weld and laboratory
assistance. We are grateful to Dr. M. Malko, the Institute of Physical
and Chemical Radiation Problems, NAS of Belarus, for the recommendations
in dose rate assessment. The authors especially
acknowledge Prof. D. Harder, University of Göttingen, for critically
reviewing the manuscript and helpful discussions that signiWcantly
improved this work.
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DOI 10.1007/s00411-006-0054-3
ORIGINAL PAPER
Nadezhda I. Ryabokon · R. I. Goncharova
Transgenerational accumulation of radiation damage
in small mammals chronically exposed to Chernobyl fallout
Received: 5 March 2006 / Accepted: 17 June 2006 / Published online: 22 July 2006
© Springer-Verlag 2006
Abstract The purpose of this investigation has been the
analysis of the long-term development of biological damage
in natural populations of a model mammalian species,
the bank vole (Clethrionomys glareolus, Schreber),
which were chronically exposed to low doses of ionizing
radiation over 22 animal generations within 10 years following
the Chernobyl accident. The time course of the
biological end-points (chromosome aberrations in bone
marrow cells and embryonic lethality) was compared
with the time course of the whole-body absorbed dose
rate from external and internal exposure in the studied
populations inhabiting monitoring sites in Belarus with
diVerent ground deposition of radionuclides. The yield of
chromosome aberrations and, in lesser degree, embryonic
lethality was associated with the radionuclide contamination
of the monitoring areas in a dose-dependent
manner. As a main feature of the long-term development
of biological damage under low dose rate irradiation,
permanently elevated levels of chromosome aberrations
and an increasing frequency of embryonic lethality have
developed over 22 animal generations. This contrasts
with the assumption that the biological damage would
gradually disappear since in the same period of time the
whole-body absorbed dose rate decreased exponentially
with a half-value time of about 2.5–3 years. Furthermore,
gravid females were captured, and their oVspring, born
and grown up under contamination-free laboratory conditions,
showed the same enhanced level of chromosome
aberrations. Therefore the authors suggest that, along
with the biological damage attributable to the individual
exposure of each animal, the observed cellular and systemic
eVects reXect the transgenerational transmission
and accumulation, via genetic and/or epigenetic pathways,
of damage attributable to the chronic low-dose
rate exposure of the preceding generations of animals.
They also suggest that the level of the accumulated transmissible
damage in the investigated populations will
decrease in future due to the further recession of the
chronic exposure and as a consequence of selection processes.
Introduction
The Chernobyl accident in April 1986 caused the deposition
of radionuclides across Europe, followed by a longterm
artiWcial increase of the radiation background [1].
In addition to the classical subject of mutagenesis after
acute radiation exposure [2], the study of the time course
of biological damage associated with chronic low-dose
radiation exposure of mammals and the endeavors to
predict biological damage in consecutive generations
have become a relevant issue. Since there is little information
on this topic [3, 4], the present work addresses
these important questions.
Starting with 1986, we were engaged in studying biological
eVects of chronic low dose radiation in natural
populations of bank vole (Clethrionomys glareolus, Schreber)
in a series of many animal generations. The bank
vole is a widespread rodent species that is used as indicator
of environmental quality. It is a convenient object for
many genetic tests, which originally have been devised for
the laboratory mouse [5]. Comparison of own and literature
data on doubling doses of acute irradiation for chromosome
injuries had shown that the sensitivity of somatic
cells of the bank vole to ionizing radiation is very similar
to the sensitivity of human lymphocytes and germ cells of
laboratory mice [6]. This Wnding conWrms that the bank
vole is a suitable model species for assessment of genetic
radiation risks in mammals.
According to the Atlas of Caesium Deposition in
Europe [1], practically the whole Belarusan territory was
contaminated by 137Cs above the level of global fallout.
A ground deposition of 137Cs equal to 37 kBq/m2 (1 Ci/
km2) was chosen to discriminate between so-called
“clean” regions and contaminated regions. We selected
monitoring sites representing large areas of Belarus with
a strong gradient of radionuclide ground deposition,
from 8 to 8,500 kBq/m2 of 137Cs, i.e., from light contamination
in “clean” regions to high contamination in the
evacuated zone. In comparison with the pre-Chernobyl
data, increased frequencies of chromosome aberrations
and genomic mutations in somatic cells, abnormal sperm
heads and embryonic losses were observed in bank vole
populations at the monitoring sites [7, 8].
The aim of the present study was to analyze the longterm
development of chromosome aberration frequency
and embryonic lethality in bank vole populations over
22 animal generations living in 1986–1996 and to compare
it with the time course of the whole-body absorbed
dose rate.
Materials and methods
Monitoring sites
The animals were collected at Wve monitoring sites in
large forestry areas with limited human activities, far
from possible sources of industrial or domestic pollution.
These sites are located at diVerent distances and directions
from the Chernobyl nuclear power plant (Fig. 1)
and represent diVerent levels of radionuclide contamination:
site 1—the Priluksky Reserve, Minsk region,
330 km NW, site 2—the Berezinsky Biosphere Reserve,
Vitebsk region, 400 km NNW, site 3—the vicinity of
Majsk village, Bragin district, Gomel region, 60 km N,
site 4—the vicinity of the evacuated Babchin village,
Khoiniki district, Gomel region, 40 km NNW and site
5—the vicinity of the evacuated Radin village, Khoiniki
district, Gomel region, 18 km N. The initial levels of
radionuclide contamination of soil at these sites were
determined earlier [9] and are shown in Table 1. The animals
were captured using live traps with bait. The Wrst
animals were captured at sites 1, 3, and 4 in September
1986, i.e., about 5 months after the period of acute irradiation
by short- and long-lived radionuclides. In the subsequent
years, the animals were as well usually collected
in the period of August–September in order to exclude
the inXuence on the data of seasonal changes in the age
structure of populations. Data obtained outside this collection
period (e.g., the data from site 1 in 1991 and 1996
[7, 8]) were not included in this study. Investigations at
sites 2 and 5 started in 1991 and 1996, respectively, i.e., 5
and 10 years after the accident.
Assessment of animal age and change of animal
generations
For age determination, root, cusp, and height of the Wrst
mandibular molar (M2) were measured according to
Bashenina [10]. The specimens were divided into seven
age groups, which corresponded to ages of approximately
2 weeks, 1, 2, 3, and 4 months and 1 and 1.5 years.
The bank vole, like other rodent species, is known to
have a short generation time with a complete change of
about 2–3 generations per year [10]. According to data of
Rozhdestvenskaya [11] and our own estimates of animal
participation in reproduction, this holds also for the
Fig. 1 Map of Belarus with localization of the monitoring sites. The
position of the Chernobyl nuclear power plant (ChNPP) in the
neighboring Ukraine is also indicated
Table 1 Densities of radionuclide contamination of soil (kBq/m2) at Wve monitoring sites (data of April–May 1986 for 137Cs, 134Cs, 106Ru,
144Ce and data of August 1996 for 90Sr and transuranic radionuclides) according to data [9]
Site April–May 1986 August 1996
137Cs 134Cs 106Ru 144Ce 90Sr 238Pu 239,240Pu 241Pu 241Am
1 8 4 5 0 4 0.04 0.10 2.98 0.14
2 18 9 12 0 5 0.07 0.14 5.10 0.19
3 220 140 150 440 39 0.62 1.28 48.80 1.81
4 1,530 1,020 1,090 3,050 117 1.17 2.35 86.70 3.21
5 8,500 5,650 5,790 1,7200 1,200 4.90 11.00 420.00 15.00
169
populations studied in the present work. Due to seasonal
reproduction and short life-span, in the trapping periods
of late summer and early autumn only few animals born
in the preceding year were observed at the monitoring
sites, showing that the populations were almost completely
renewed every subsequent year by the time of
observation.
The populations living at the time of the accident, in
early spring of 1986, consisted of adult animals born in
the previous year. Due to the turnover of generations,
our investigations, beginning in September 1986, started
with the Wrst and second post-accidental generations of
bank vole. In the period from 1986 to 1996, at least 22
generations were studied.
Determination of radionuclide concentration
and estimation of absorbed dose rate
Concentrations of radionuclides in the soil and the
whole-body animal samples were assessed as described in
[9]. BrieXy, the _-spectrometry of samples was performed
in the Hydrometeorological Centre of Belarus in Minsk
using the _-spectrometer ADCAM-300 equipped with a
high-purity germanium detector GEM-30185 (EG&G
Ortec, USA). SpeciWc activities of 90Sr and transuranic
elements were determined by the staV of the Institute of
Radiobiology, National Academy of Sciences of Belarus,
using radiochemical methods and the _-spectrometer
Ortec 576A with the silicon surface barrier detector
(EG&G Ortec, USA), the liquid scintillation counter Tricarb
2700TR (Packard Company) and a gas-Xow
counter (Tesla Automat, Slovakia).
Dose rates due to internal and external exposure were
assessed in the studied specimens as described previously
[9]. The whole-body absorbed dose rate from incorporated
_-emitting radionuclides was calculated according
to the absorbed fraction model. For the dose rate attributable
to incorporated _- and _-emitters, we used the
local absorption model. The whole-body absorbed dose
was calculated as the product of the individual wholebody
absorbed dose rate and the age of the animal at the
time of capture.
Metaphase analysis of chromosome aberrations
Cytogenetic eVects in bank vole somatic cells were studied
by metaphase analysis of chromosome aberrations in
red bone marrow cells according to a standard protocol
[12] as described [7, 8]. BrieXy, visually healthy animals of
diVerent age, sex, and state of maturation were randomly
chosen for this test from numerous groups of animals
captured shortly after trapping. Colchicine at concentration
of 0.1 mg per 10 g animal weight was injected intraperitoneally
for 1.5 h to accumulate metaphases.
Animals were sacriWced by ethyl ether and cervical dislocation,
and the marrow was aspirated from the femurs
using inactivated fetal bovine serum. The marrow suspension
was incubated at 37°C for 20 min, treated with
0.56% potassium chloride for the next 20 min and Wxed
in methanol:acetic acid (3:1 v/v). Fixed cells were spread
on clean slides, Xame-dried and stained with Giemsa.
Coded slides were screened for chromatid- and chromosome-
type aberrations in approximately 100 well-spread
metaphases per specimen using standard criteria [12, 13].
Achromatic lesions (gaps) were not included in the statistical
analysis.
Selected age groups
The chromosome aberrations and embryonic losses were
also recorded from selected cohorts of mature animals
with age from 2 to 4 months in order to exclude any agedependent
bias of the biological eVects.
Furthermore, two gravid females were taken to a laboratory
in Minsk, where they gave litters shortly after
capture. Mother animals and their oVspring were fed
with uncontaminated food and were studied for chromosome
aberrations in red bone marrow cells when the age
of the oVspring reached 1.5 months.
Analysis of embryonic losses
Assessment of embryonic mortality was performed in all
captured and visually healthy females at 7–22 days of
pregnancy using the conventional approaches [14]. The
content of the uteri was examined to determine the number
of implants including live and dead embryos. The
early (pre-implantation) losses were calculated as the
ratio (number of corpora lutea minus number of
implants)/(number of corpora lutea). The late (postimplantation)
losses were calculated as the ratio of the
number of dead embryos to the number of implants. The
total losses were determined as the ratio (number of corpora
lutea minus number of live embryos)/(number of
corpora lutea). All ratios are expressed in percent.
Statistics
Chi-square and U tests, regression and correlation analysis
were employed as a part of the Statistica software
package (StatSoft Inc., USA). Figure 2 gives an example
of the scatter of the individual aberration frequency and
whole-body dose rate data at a given site and given season
of the study (site 4, analyzed 5 years after the accident).
In the following, these Xuctuations are represented
by the mean value and the standard deviation of the
mean.
Results
Time course of the absorbed dose rate
The time course of the absorbed dose rates in populations
of bank vole at the monitoring sites was determined
previously [9] using numerous groups of captured
animals. It was shown that external _-irradiation and
170
internal _-irradiation by incorporated 137Cs and 134Cs
delivered the most prominent contributions to the
whole-body dose rate over the monitoring period, which
started 5 months after the accident with the Wrst and second
post-accident animal generations and ended 1996
after about 22 animal generations [9]. Here, we analyzed
the time course of the mean whole-body absorbed dose
rate due to internal _-irradiation and to total irradiation
speciWcally for those animals which, out of the whole
cohorts, were randomly chosen for assessment of chromosome
aberrations (Table 2; Fig. 3a, b) and embryonic
mortality (Table 3; Fig. 3c, d). The values of the wholebody
dose rates and their temporal development in these
sub-cohorts of animals were similar to those in the larger
groups of bank vole used in the earlier dosimetrical
investigations of monitored populations [9].
Fig. 2 Correlation of the chromosome aberration frequency in
bone marrow cells of bank voles inhabiting site 4, with the individual
whole-body absorbed dose rates 5 years after the Chernobyl accident.
The dose rates are due to external _- and internal _ + _-radiation
of incorporated 137Cs and 134Cs
Mean value
0
2
4
6
8
10
12
60 80 100 120 140
Whole-body dose rate, μGy/d
Chromosome aberrations per 100 cells
Fig. 3 Time course of the mean whole-body absorbed dose rates in
bank vole specimens captured at sites 1–5 and studied for chromosome
aberrations (a, b) and for embryonic mortality (c, d). The contributions
by internal _-irradiation from incorporated 137Cs and
134Cs are presented in a and c, the total whole-body dose rates by
external _-irradiation and internal _ + _-irradiation from incorporated
137Cs and 134Cs are shown, together with exponential approximation
curves, in b and d. Error bars are not shown for clarity
Internal â-irradiation Total irradiation
Site 1 Site 2
Site 3
Site 4 Site 5
1
10
100
1000
0 6 10
Years since accident
Site 1
Site 2
Site 3
Site 5
Site 4
0.1
1
10
100
1000
Years since accident
Whole-body dose rate, μGy/d
Site 2
Site 3
Site 4
1
10
100
1000
Years since accident
Site 2
Site 3
Site 4
0.1
1
10
100
1000
Years since accident
0.1
0.1
2 4 8 0 2 4 6 8 10
0 2 4 6 8 10 0 2 4 6 8 10
(c)
(a) (b)
(d)
Table 2 Chromosome aberrations in bone marrow cells of bank vole at Wve monitoring sites
a Pre-accident data according to [15]
b All data on total frequencies of aberrations and aberrant cells in animals at sites 2–5 in 1986–1996 were signiWcantly higher in comparison
with pre-accident data by Chi-square test
Site Year Animal generation
since the accident
Whole-body
absorbed dose
rate (_Gy/day)
Number of analyzed Aberrations per 100 cells § SD Aberrant
cells, %
Animals Cells Chromatid
type
Chromosome
type
Total
1 1986 1–2 6.44 § 0.14 10 992 0.30 § 0.14 0.10 § 0.09 0.40 § 0.22 0.40 § 0.22
1988 5–6 3.36 § 0.02 3 310 0.64 § 0.32 0 0.64 § 0.32 0.64 § 0.32
2 1981–1983a Pre-accident – 24 2,437 0.41 § 0.12 0 0.41 § 0.12 0.41 § 0.12
1991 11–12 4.43 § 0.38 19 1,945 1.19 § 0.37 0.03 § 0.03 1.22 § 0.37b 1.22 § 0.37
1992 13–14 – 17 1,962 0.90 § 0.27 0.27 § 0.17 1.17 § 0.13 1.09 § 0.24
1996 21–22 2.41 § 0.03 8 585 1.06 § 0.54 0.11 § 0.11 1.17 § 0.53 1.17 § 0.53
3 1986 1–2 87.52 § 4.50 18 1,987 1.36 § 0.45 0.76 § 0.28 2.12 § 0.59 1.99 § 0.53
1988 5–6 76.06 § 8.90 16 1,630 1.22 § 0.49 0.52 § 0.18 1.74 § 0.57 1.51 § 0.03
1991 11–12 16.83 § 2.27 16 1,655 2.67 § 0.81 0.20 § 0.13 2.87 § 0.83 2.49 § 0.76
1996 21–22 7.02 § 0.27 11 1,121 1.86 § 0.41 0.18 § 0.10 2.04 § 0.39 1.95 § 0.38
4 1986 1–2 605.46 § 7.75 16 1,739 1.45 § 0.43 0.06 § 0.06 1.51 § 0.45 1.21 § 0.10
1987 3–4 258.57 § 11.33 36 3,675 0.79 § 0.18 0.44 § 0.15 1.24 § 0.24 1.10 § 0.22
1988 5–6 321.64 § 33.12 21 1,942 1.75 § 0.39 0.17 § 0.09 1.92 § 0.40 1.86 § 0.04
1991 11–12 95.93 § 3.69 31 3,780 1.63 § 0.34 0.31 § 0.10 1.94 § 0.40 1.84 § 0.36
1996 21–22 41.80 § 0.96 14 1,821 1.90 § 0.49 0.24 § 0.15 2.14 § 0.42 2.04 § 0.38
5 1996 21–22 274.52 § 7.27 11 492 4.74 § 0.74 1.47 § 0.61 6.21 § 0.85 5.10 § 0.78
171
The maxima of internal _-irradiation due to incorporated
137Cs and 134Cs were observed in the second year
after the accident (Fig. 3a, c) due to the increasing biological
availability of cesium isotopes from the biota [7-9], followed
by a decrease by one order of magnitude in the
subsequent period. In [9] it was shown that external _-irradiation
was the essential determinant for the time course of
the total whole-body dose rate; this was also true for the
animals analyzed for chromosome aberrations (Fig. 3b) or
embryonic losses (Fig. 3d). The highest values of total dose
rate were observed in the year of the accident, followed by
an approximately exponential decrease over the next
10 years (r2=0.95, P<0.05 and r2=0.91, P<0.05 for
sites 3 and 4, respectively, in Fig. 3b). The reduction factor
since the accident was about 15, corresponding to a halflife
of 2.5–3 years. These data show that after the primary
insult in the year of the accident, every successive generation
of animals was exposed to lower whole-body dose
rates of ionizing radiation than the preceding one.
The mean whole-body absorbed doses in the studied
cohorts of animals were also maximal in the year of radionuclide
deposition. The highest individual value, determined
in the Wrst and second post-accidental generations
of animals at site 4, was 73 mGy. By the end of the monitoring
period, the mean whole-body doses were about 0.3,
0.7, 12, and 25 mGy for sites 2, 3, 4, and 5, respectively [9].
At any given time of the observation period, absorbed
dose rates (Fig. 3) and absorbed doses at the diVerent sites
diVered by about two orders of magnitude, thereby representing
a strong gradient in the level of animal exposure.
Temporal development of the chromosome aberration
frequency
The time course of chromosome aberration frequencies
in the studied animals is presented in Table 2. The frequency
of chromosome aberrations in bone marrow cells
of the bank vole inhabiting the Berezinsky Biosphere
Reserve, i.e., site 2 in our study, was Wrst recorded 3–5 years
before the accident [15] and can be taken as a pre-accident
(historical) control for our data. The data of this
pre-accident control are similar to those observed at the
less contaminated site 1 (Minsk region) in the years 1986
and 1988. Before the accident, only chromatid-type aberrations
were observed, and the cells never contained multiple
aberrations. In contrast, in the post-accident period,
the chromosome anomalies consisted of both chromatidand
chromosome-type aberrations, containing single and
paired fragments in the majority of cases, but also rare
Robertsonian translocations (fusion of acrocentric chromosomes
at the centromeres). In animals inhabiting
contaminated sites, the mean frequencies of both chromosome
aberrations and aberrant cells were signiWcantly
higher (P<0.01, Chi-square test) than the pre-accident
value, and were increased in a dose-dependent manner,
by a factor of 3–7 at sites 2, 3, and 4, and of about 15 at
site 5. Also Pearson correlation analysis of the total aberration
frequencies and those of the Robertsonian translocations
at sites 2, 3 4, and 5 in 1996 (Fig. 4a, b) hints at
a relationship between mean aberration frequencies and
the whole-body dose rates at the time of capture. The
increased frequency of chromosome aberrations
observed in animals inhabiting contaminated areas
appears to remain relatively constant over the years of
the investigation (Table 2).
The data in Table 2 comprise the level of chromosome
aberrations in animals varying by age, sex and
maturity. These animals represent the studied populations,
from where they were chosen at random. Generally,
the mean age of the animals in each group did not
diVer from the usual mean age of about 3–4 months at
the season of capture. There was a single exception at site
Table 3 Embryonic lethality in bank vole populations at three monitoring sites
a Pre-accident level according to [16]
* P< 0.05 and ** P< 0.01 in comparison with pre-accident data (Chi-square test)
Site Year Animal
generation
since the
accident
Whole-body
absorbed dose
rate (_Gy/day)
Number of Mean embryonic lethality,
% (95% binomial conWdence limits)
Animals
analyzed
Yellow
bodies
Embryos
analyzed/
dead
embryos
Before
implantation
After
implantation
Total
2 1981–1983a Pre-accident – 45 249 235/4 5.62 (3.11–9.25) 1.70 (0.47–4.30) 7.23 (4.34–11.18)
1991 11–12 5.5 § 0 12 56 56/3 0 (0.00–5.21) 5.36 (1.12–14.87) 5.36 (1.10–14.87)
1992m 13–14 – 19 92 90/2 2.17 (0.26–7.68) 2.22 (0.27–7.80) 4.35 (1.20–10.76)
1996 21–22 2.51 § 0.13 8 44 39/0 11.36 (3.79–24.56) 0 (0.00–9.03) 11.36 (3.79–24.56)
3 1988 5–6 58.01 § 5.40 7 36 36/0 0 (0.00–7.98) 0 (0.00–7.98) 0 (0.00–7.98)
1989 7–8 44.38 § 3.71 30 138 130/1 5.80 (2.50–11.10) 0.77 (0.02–4.21) 6.52 (3.03–12.08)
1991 11–12 17.95 § 2.53 14 71 65/1 8.45 (3.16–17.49) 1.54 (0.04–8.28) 9.86 (4.06–19.26)
1996 21–22 6.58 § 0.07 3 15 12/1 20.00* (4.33–48.09) 8.33 (0.21–38.48) 26.67* (7.74–55.10)
4 1988 5–6 245.6 § 20.21 14 63 61/1 3.17 (0.39–11.00) 1.64 (0.04–8.80) 4.76 (0.99–13.29)
1989 7–8 265.63 § 34.53 40 201 192/3 4.48 (2.07–8.33) 1.56 (0.32–4.50) 5.97 (3.12–10.20)
1991 11–12 103.78 § 5.96 21 103 91/4 11.65* (6.27–19.76) 4.40 (1.21–10.87) 15.53* (9.15–24.00)
1996 21–22 43.47 § 0.87 11 51 44/4 13.73* (5.70–26.26) 9.09* (2.53–21.67) 21.57** (11.29–35.32)
172
4, 10 years after the accident, when by chance 10 of 14
animals randomly sampled for cytogenetic analysis were
at the age of one year or older, whereas in the more
numerous group of specimens captured at this site for
dosimetrical investigations [9] there was no detectable
change in age distribution. In order to exclude any possible
inXuence of variations in the age distribution on the
observed frequencies of chromosome aberration, analysis
of the time course of the chromosome aberration frequency
was limited to mature 2–4 months old animals,
whose mean values and temporal development of age
over the period of this study is shown in Fig. 5a. The
temporal pattern of the frequency of chromosome aberrations
in the 2–4 months old animals (Fig. 5b) was
almost identical with the pattern observed in all animals
(Table 2).
As is evident from Fig. 5, the frequencies of chromosome
aberrations observed in 2–4 months old bank voles
remain fairly constant over time, in spite of the approximately
exponential decrease of the whole-body dose rate.
This suggests that the induction of aberrations depends
not only on the actual exposure level. To test this, aberration
frequencies were investigated in the oVspring of
mice captured in 1988 at sites 3 and 4, which were born
and brought up in the laboratory and fed with uncontaminated
food. The chromosome aberration frequency
observed in the oVspring animals showed no signiWcant
diVerence to that observed in animals from the same sites
that had grown up in the contaminated environment,
and to the aberration frequency of their mother animals
(Fig. 6).
Embryonic mortality
Studies on embryonic losses in the monitored populations
were started 2 years after the accident. Since at sites
1 and 5 no gravid females after the stage of embryonic
implantation were captured, the analysis on embryonic
mortality is limited to sites 2, 3, and 4. Figure 7a shows
that there was no increase over the monitoring period in
the age of gravid females, which could have aVected the
values of embryonic lethality. Rather, the mean age of
Fig. 4 Correlation between mean frequency values of all types of
chromosome aberrations (a) and of Robertsonian translocation (b)
with the mean values of the whole-body absorbed dose rate in bank
vole populations inhabiting sites 2–5, 10 years after the accident.
Standard deviations of the mean values are indicated as error bars
Site 2
Site 3
Site 4
Site 5
r = 0.99
P = 0.011
0
2
4
6
8
0 100 200 300
Whole-body dose rate (μGy/d)
Chromosome aberrations per 100 cells
Site 5
Site 4
Site 3
Site 2
r = 0.95
P = 0.048
0
0 100 200 300
Whole-body dose rate (μGy/d)
Robertsonian translocations per 100 cells
0.8
0.6
0.4
0.2
(a)
(b)
Fig. 5 Time course of animal age (a) and chromosome aberration
frequency (b) in a sub-cohort of adult, 2–4 months old bank voles at
four sites. Mean data and standard deviation are shown. Pre-accident
data according to [15]
(a)
(b)
0
1
2
3
4
5
6
0 2 4 6 8 10
Years since accident
Age of animals (months)
Site 1
Site 2
Site 3
Site 4
Site 1
preaccident
Site 2
Site 3
Site 4
0
1
2
3
4
0 2 4 6 8 10
Years since accident
Chromosome aberrations per 100 cells
173
the gravid females tended to decrease over the period of
monitoring. The frequencies of embryonic losses in the
population inhabiting the Berezinsky Biosphere Reserve
3–5 years prior to the accident [16] were used as pre-accident
control (Table 3; Fig. 7b). A comparison of pre- and
post-accident data shows that both pre- and postimplantation
embryonic mortality in populations in the
moderately contaminated site 2 and in the more highly
contaminated sites 3 and 4 remained on the level of preaccident
mortality frequencies during the Wrst years of
observation. After 5–10 years, however, a tendency
towards increasing embryonic lethality was found in all
populations studied, reaching a statistically signiWcant
increase in samples from sites 3 and 4 after 10 years (by a
factor 2–5 in comparison with pre-accident data).
Discussion
In this study free-living small mammals, the bank voles,
were used as indicator organisms for the monitoring of
environmental eVects. The studied populations lived at
diVerent distances from the Chernobyl nuclear power
plant. The investigations were started 5 months after the
accident, from the 1 and 2 post-accident generations of
animals that were irradiated at doses one order of magnitude
less than animals in the acute period after the
accident. The particular subject of this study was to compare
the time course of biological damage in animals,
representing up to 22 generations living during 10 years
following the Chernobyl accident, with the time course
of the radiation exposure experienced by these animals.
The main results of this study are (a) a dose-dependent
increase in the frequencies of chromosome aberrations
and embryonic losses in animals living in contaminated
areas as compared to historic controls and to animals
living in an area with the lowest level of contamination,
(b) the fact that the chromosome aberration frequencies
remained on elevated levels (Fig. 5) and the frequencies
of embryonic losses even increased (Fig. 7) over 10 years
after the accident, although the whole-body absorbed
dose rates experienced by the animals declined almost
exponentially (Fig. 3).
Historic controls raise the concern that they might
not directly be comparable to the samples under study.
In this study the frequencies of chromosome aberrations
observed in 1986 and 1988 in animals living at the almost
uncontaminated site 1 can be used as controls, and they
were similar to those assessed in the historic control
(Table 2; Fig. 3). Thereby we can exclude that the
increases in chromosome aberration frequency seen in
the contaminated areas have reasons other than the
higher radiation exposure. Unfortunately, we have no
data from site 1 for the years 1991 and 1996 collected in
the period August–September, but the fact that all investigations
at the contaminated sites were done by the
same team guarantees that the methods of investigation
Fig. 6 Chromosome aberration frequencies in bank vole females
captured at sites 3 and 4 in 1988, and of their oVspring, born, and
raised under laboratory conditions for about 1.5 months before
analysis. Data are compared in bank vole populations living at sites
3 and 4 at the same period. Means, standard deviations of the means
and the number of analyzed animals are shown
n = 2
n = 9
n = 37
Pre-accident
level
0
1
2
3
Mothers Offspring Populations
Chromosome aberrations per 100 cells
Fig. 7 a: Time course of the age of gravid bank vole females analyzed
for embryonic losses. Means and standard deviation of the
means are show. b: Time course of embryonic lethality in animals at
site 2 and at sites 3 and 4 (pooled). Means and binomial 95% conWdence
limits are indicated. Pre-accident data according to [16]
(a)
0
2
4
6
8
10
0 2 4 6 8 10
Years since accident
Age of animals (months)
Site 2
Site 3
Site 4
(b)
**
0
20
40
60
0 2 4 6 8 10
Years since accident
Embryonic lethality (%)
Site 2 Site 3+4
preaccident
174
have not changed in these years. Therefore, and in line
with the unequivocal dose-rate dependency displayed in
Figs. 4, 5 and 7, we propose a radiogenic etiology of the
observed biological eVects.
Steady decrease of the whole-body dose rate over
the monitoring period of 1986-1996
At all sites investigated, whole-body absorbed dose rates
were found to decrease over about one order of magnitude
in 10 years, which is attributable to the gradual disappearance
of the radioactive fall-out material from the
biota [9]. More speciWcally, we show that the whole-body
dose rate decreased approximately according to an exponential
function with a half-life of 2.5–3 years (Fig. 3b,
d). As the observed biological damage did not decrease
in the same animals over the studied period, it is important
to exclude that the exposure levels in the later years
of the investigation, due to long-lived _-emitting transuranic
radionuclides and/or increasing 90Sr concentrations
in the studied populations, were underestimated.
Our analyses of radionuclide concentrations in animals
showed that 10 years after the accident the contribution
of transuranic elements and 90Sr to the whole-body dose
rate in the studied specimens of bank vole did not exceed
3 and 8%, respectively [9]. This contribution was estimated
to be two orders of magnitude lower than the
whole-body dose rates during the year of the accident [9].
The contribution of 90Sr to local doses in bone tissue in
the later years of the investigation was about 2–10£ lower than that of external _-irradiation. The absorbed
dose rate in the red bone marrow due to 90Sr _-radiation,
which is approximately equal to the absorbed dose rates
averaged over the bone tissues, was of the same small
magnitude, comparable to that of 137+134Cs _-radiation
in the later years and an order of magnitude lower than
_-radiation of 137+134Cs in early period after the accident.
We conclude that doses at late time points were not
underestimated by our approach.
Dose dependence of the observed eVects
Data presented in Tables 2 and 3, as well as in Figs. 5
and 7, hint at a strong dependence of the observed biological
eVects on radiation exposure. This is manifested
by relatively low levels of aberrations and, where present,
embryonic losses at the less contaminated sites 1 and 2,
the medium levels at sites 3 and 4, and the relatively high
level at the most contaminated site 5. Furthermore, there
is a statistically signiWcant linear correlation between
mean values of the chromosome aberration frequency
observed 10 years post-accident and the mean wholebody
dose rates prevailing in the studied animals at the
same time (Fig. 4). A similar association of mean
micronuclei frequencies with exposure at low dose rates
(4.22–39.4 _Gy/day) has been shown in bank vole populations
studied 2 years after the accident at four Swedish
regions contaminated by Chernobyl fallout at deposition
levels of 1.8, 22, 90, and 145 kBq/m2 [17], i.e., levels similar
to those at our monitoring sites 1–3 (Table 1). Mean
levels of chromosome aberrations were also increased in
a dose-dependent manner in laboratory mice, exposed in
our monitoring areas during 4 months to whole-body
dose rates from external and internal irradiation of 3–
145 _Gy/day [8], i.e., at exposure levels close to those in
free-living animals.
Dose dependence of the biological damage is a basic
requirement for conclusions concerning the causal role
of radiation exposure for the observed eVects. This
includes the possibility of direct as well as indirect modes
of action, in other words, the induction of the eVects seen
in a given generation by radiation exposure of the same
or of the preceding generations of animals. In such a
way, a linear correlation observed between mean values
of chromosome aberration frequencies and the dose
rates in late generations of animals 10 years after the
accident can as well demonstrate a linear correlation of
the aberration frequencies with the dose rates in all previous
years, because the dose rates at the various sites
remained in constant proportions to each other, while
they decreased in an exponential fashion over the monitoring
period (Fig. 4). On the other hand, the scatter
plots of the individual frequency of chromosome aberrations
versus the individual level of the whole-body
absorbed dose rate or whole-body dose may hint at a
dose dependence of the biological eVect even on the
exposure of the individual animal (Fig. 2 and data not
shown). An analogous correlation has been observed
between the individual frequencies of micronuclei and
the individual radiation exposures in the same population
of animals [6]. These dose-eVect relationships should
be studied in future.
Long-term persistence of biological eVects and possible
mechanism of their transgenerational transmission
Our data (Tables 2, 3; Figs. 5, 7) suggest that biological
eVects in bank voles living in regions contaminated by
the Chernobyl accident persisted on a stable level (chromosome
aberrations) or even increased (embryonic
loses) over 10 years, corresponding to at least 22 animal
generations, although the dose rates have substantially
decreased during this period. This observation is in fundamental
contrast to the assumption that the biological
injury would gradually disappear in direct connection
with the exponential reduction of the whole-body dose
rate. Thus, the biological eVects cannot be explained
alone by the exposure experienced by the individual animals.
Rather, our observations call for a diVerent interpretation,
namely that the biological eVects reXect a
transgenerational transmission of radiation damage that
occurred in early generations, especially in the generations
living at the time of the accident and in the early
post-accidental generations, and was further accumulated
in the course of the chronic exposure of the following
generations. The fact that oVspring from captured
females, irradiated in utero at contaminated areas, but
born in the laboratory and fed with uncontaminated
175
food, did not show a signiWcant reduction in aberration
frequencies in comparison with animals born and grown
up in the contaminated environment (Fig. 6) corroborates
the hypothesis that some mechanism of transgenerational
transmission of radiation damage has been
manifested.
The occurrence of radiation damage in cells and tissues
that have not been directly exposed to radiation is
not without precedence. For example, genomic instability
is a long-term radiation eVect that has been intensively
investigated. The term “genomic instability” refers
to the de novo production of delayed responses of the
genome in the progeny of the exposed cells in a cell culture
or in an irradiated animal [18–22]. Genomic instability,
including chromosomal instability in hemopoietic
stem cells in vitro and in vivo, has been shown to be activated
by high- and low-LET ionizing radiations at relatively
low doses [21–25] and to exhibit a dose-dependent
increase at higher doses [24]. Dose dependence was also
observed for chromosomal instability in mice fetuses
irradiated as zygotes [26]. Other authors did, however,
not Wnd a dose relationship for genomic instability over
a wide range of radiation doses [27–29]. The expression
of genomic instability was described to persist for up to
or even more than 70–80 population doublings at higher
doses [29, 30]. In the progeny of long-term repopulating
haemopoietic stem cells of mice, chromosomal instability
was observed over 24 months after cell irradiation and
transplantation [22]. There is evidence that the genetic
background or genotype can inXuence radiation-induced
instability [25]. The mechanisms of genomic instability
are largely unknown. At least part of them may rely on
epigenetic transmission of information. For instance,
genomic instability was suggested to reXect altered signal
transduction pathways and changes in the DNA microenvironment
[31, 32]. Since DNA methylation is linked
to chromosome condensation [33], chromosome and
chromatid fragments as well as Robertsonian translocations
may be due explained by the radiation-induced
increased fragility of the chromatin during chromosome
condensation [34]. These types of chromosome aberrations
have been suggested as hallmarks of induced genomic
instability, although other types of chromosome
aberration can also occur [24, 26, 27, 30].
However, in contrast to studies where long-term
eVects are investigated in the same animals, which had
been exposed to radiation at an earlier stage of individual
development, the interpretation of the eVects seen in
our study must deal with damage persisting over many
generations of animals. In the literature, the term “transgenerational
transmission of radiation damage” has
mainly been used in a descriptive sense, including genetic
and epigenetic as well as hitherto uncharacterized mechanisms
of transmission damage to the progeny [21, 35–
42]. Delayed radiation-induced responses attributable to
genomic instability were revealed in the Wrst few animal
generations after irradiation of male germ cells [43] or
zygotes [37]. Heritable tumors and anomalies were
observed in descendants of exposed mice [35], and physiological
or developmental disorders were seen in the
progenies of irradiated parents, which resulted in embryonic
and early postnatal death, fertility disturbances,
congenital abnormalities or malformations [44]. Genomic
instability in the Wrst-generation oVspring (F1) of
irradiated mice or rats covers diverse endpoints such as
chromosome aberrations, micronuclei, point mutations
and reversions in somatic cells (reviewed in [39]) as well
as early (pre-implantation) and late (post-implantation)
embryonic resorption [37], reduced fertilization rate [45],
increased number of sterile females [37] and reduction in
the proliferative ability of both F1 and F2 embryonic
cells [36] and F1 liver cells [38]. Transgenerational germline
instability was also demonstrated in the oVspring of
irradiated mice (reviewed in [39]), as well as in children
born from irradiated parents [46] and in barn swallows
breeding not far (25–50 km) from the Chernobyl nuclear
power plant [47]. In addition, transgenerational transmission
of genomic and developmental eVects was
reported after parental exposure to certain chemicals
[e.g., 35, 41].
In conclusion, we suggest that in the studied bank vole
populations the radiation exposure of the parental generations
has lead to an accumulated pool of germline mutations
and/or of epigenetic changes, which resulted in the
observed, persistently elevated levels of chromosome aberrations
in somatic cells and in increased embryonic losses
in later generations. With regard to the continuous buildup
of transgenerationally-transmitted damage in the
course of chronic radiation exposure of the parental generations,
we will shortly speak of the transgenerational
accumulation of transmitted biological damage.
The present report is the Wrst in which the time course
of biological damage in mammals chronically exposed to
ionizing radiation over a series of generations has been
studied. So far, the long-term development of transgenerationally
transmitted radiation damage has been studied
in a restricted number of consecutive generations of animals,
generally in the Wrst 1–2 generations after irradiation
of parents [21, 31, 35–39, 43, 46]. Increased
frequencies of chromosome aberrations were also
observed in bone marrow cells after 25–30 generations of
red vole [3] and after 75–80 generations of common vole
[48] chronically irradiated in regions of heavy radioactive
contamination in the Urals as well as in bordering areas.
The latter observation was explained by hereditary chromosome
instability [48], but there is no data on the quantitative
time course of these eVects. The question arises
whether the levels of chromosomal aberrations and
embryonic lethality that in our study were reached in
1996 after 22 animal generations, will be maintained permanently,
or whether a phase of reduction will follow in
subsequent animal generations. Our study indicates that
a further reduction of chronic radiation exposure has to
be anticipated, and that there exists an eVective selection
mechanism, the dose-dependent increased embryonic
lethality in animal populations in radiocontaminated
areas. Further research will be required to clarify this
genetically and ecologically interesting point.
176
Acknowledgments This work was performed within the framework
of the State Program of the Republic of Belarus for minimizing and
overcoming consequences of the Chernobyl Accident (1986–2002).
The authors wish to thank the administrations of the Chernobyl
Exclusion Zone and the Berezinsky Biosphere Reserve for access to
the zone and the reserve. We are indebted to former and present staV
of the Antimutagenesis Laboratory, the Institute of Genetics and
Cytology, NAS of Belarus, for enthusiastic Weld and laboratory
assistance. We are grateful to Dr. M. Malko, the Institute of Physical
and Chemical Radiation Problems, NAS of Belarus, for the recommendations
in dose rate assessment. The authors especially
acknowledge Prof. D. Harder, University of Göttingen, for critically
reviewing the manuscript and helpful discussions that signiWcantly
improved this work.
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Goncharova R. I.: Health Effects of the Chernobyl Catastrophe
Health Effects of the Chernobyl Catastrophe
Goncharova R. I.
Institute of Genetics and Cytology, National Academy of Sciences
Akademichnaya St., 27, Minsk, 220072, REPUBLIC OF Belarus
E-mail: R.Goncharova@igc.bas-net.by
Slide 2
My report is devoted to the memory of Solange Fernex (1934-2006), a prominent French pacifist activist and politician, the former member of the European Parliament,
for her stubborn struggle for the full information on the health and other effects of the Chernobyl catastrophe in all the affected countries, including France, and for development of the adequate health assistance to affected populations.
Doctor Rosalie Bertell and Solange Fernex were initiators of the organization of Permanent People’s Tribunal, Session on Chernobyl in Vienna in 1996.
Solange Fernex actively participated in the work of Permanent People’s Tribunal, Session on Chernobyl.
Solange Fernex was the final editor of the book: Chernobyl Environmental, Health and Human Rights Implications
Solange Fernex has made the report “Health effects of Chernobyl: a dogma or a quest of truth?” at 3td International Conference: “ Health Effects of the Chernobyl Accident, Results of the 15-years Follow-up Studies” in Kiev in 2001
Slide 3
The Chernobyl accident has caused the deposition of radioisotopes over very wide areas of the Northern Hemisphere, in particular in Europe (Atlas of Caesium Deposition on Europe after the Chernobyl Accident, 1998), followed by chronic exposure of many millions of people to a mixture of external and internal radiation. You can see the map of radioactive contamination of Europe after the Chernobyl accident.
Slide 4
However, the Republic of Belarus was affected by the accident more than any other country of the world. According to the Atlas practically the whole territory of Belarus was contaminated with different radioisotopes above the level of global fall-out. The ground deposition density equal to 37 kBq/m2 is accepted as a limited value for distinguishing the so-called “clean” regions from the contaminated ones making up 23% of the country area. So, 22 years after the world’s worst nuclear accident the entire population of Belarus is involuntary taking part in a decades-long experiment on how low doses of radiation affects human health.
Clear understanding of this fact is of great importance for interpreting the effects recorded.
Since 1986 we have been studying the biological effects of chronic low dose radiation in natural populations of wild small mammals named bank vole, in laboratory mice and evaluating remote consequences of the Chernobyl accident.
Here, on the map of Belarus, you can also see monitoring sites. These sites are located at different distances from the Chernobyl nuclear power plant and represent different levels of radioisotope contamination:
Priluksky reserve (near Minsk, 330 km north-west from the Chernobyl power station, 8 kBq/m2);
Berezinsky Biosphere reserve (Vitebsk region, 400 km NNW, 18 kBq/m2);
The vicinity of Majsk village (Bragin district of Gomel region, 60 km N, 220 kBq/m2);
The vicinity of Babchin village, exclusion zone (Khoiniki district of Gomel region, 40 km NNW, 1530 kBq/m2). Now it is Polessky Radiation reserve.
The vicinity of the Radin village, exclusion zone (Khoiniki district of Gomel region, about 18 km N, 8500 kBq/m2). Now it is Polessky Radiation reserve.
Slide 5
Bank voles are collected at 5 trapping sites different in radionuclide ground deposition. These data is given on this slide.
The data on 137Cs, 134Cs, 106Ru, 144Ce contamination were obtained in the year of accident (1986). The concentrations of 90Sr and transuranic elements in samples of soils were measured in 1996.
It is necessary to note that after the primary insult in the year of the accident, every successive generation of animals under investigation was exposed to permanently decreasing whole-body dose rates of ionizing radiation.
United Nations Chernobyl Forum under the aegis of WHO issued the massive report on the health and environmental consequences of the accident. The WHO report “Health Effects of the Chernobyl Accident and Special Health Care Programmes”(2006) confirmed radiation induced huge increase of the thyroid cancer in those exposed in childhood and adolescence and declared that there has been no significant increase so far in the incidence of other cancers and congenital malformations that can be attributed to radiation exposure. It is intriguing to note that prognosis estimate of future deaths due to the Chernobyl is much less in Joint News Release WHO/IAEA/UNDP “Chernobyl: the true scale of the accident; 20 years later a UN report provides definitive answers and ways to repair lives” and the report’s 50-page summary in Health effect of Chernobyl fallout that firmly established
The established health consequences of the Chernobyl
Slide 6
Reconstruction of iodine–131 fallout has shown that the whole territory of the Republic of Belarus was contaminated this isotope (National report, 2006).
On this slide you can see calculated collective thyroid doses for two age groups, namely for children and adolescents and for adult at the time of accident, for all regions of Belarus.
For the time being scientific society has accepted only significant increase of thyroid cancer incidence in children and adolescents. Growth of thyroid cancer in adult population has not been reported in official scientific conferences as a proved scientific fact.
Slide 7
As for concerning whole body absorbed doses, the data on collective effective dose you can see on this slide. According to the National report of Belarus, 2006, table 3.11) up to 90% of collective cumulative effective dose was formed in the first decade after the accident.
Annual collective dose to population residing on the territory of radioactive contamination is about 21 person-Sv, average individual dose is 0,15 mSv.
For all of exposed groups, estimates of numbers of fatal cancers can be derived from collective doses. Such estimates depend on the assumed risk coefficient
Slide 8
The incidence of thyroid cancer in those who were adults at the time of exposure is reported to have increased in the many exposed populations, including Poland (Mahoney, Ostapenko, 2004), although the relationship to radiation is not so clear. Time course of thyroid cancer incidence among childhood and adult populations of Belarus up to 2001 have been shown on this slide.
According National Report of Belarus, 2006 thyroid cancer incidence continues growing steadily among adult population of Belarus.
The concentration of effort on the major increase in those exposed as children has meant that the smaller risk to adults has not been adequately investigated.
As for concerning future estimates of thyroid cancer for Belarus it is necessary to note.
Currently, individuals exposed as children are now adolescents or young adults but continue to carry an increased risk of developing thyroid carcinoma during all lifespan.
For predicting, we have to know risk coefficients obtained on the basis of studying suffered populations. Depending of the calculated values of risk coefficient, the predicted values of the future incidence of thyroid cancer differ greatly. The latest estimate for the year 2056 ranges from 3,400 to 72,000 according Cardis et al., 2006 (Estimates of the cancer burden in Europe from radioactive fallout from the Chernobyl accident, Int J Cancer).
According estimates of Malko, in Belarus alone, approximately 31,400 additional thyroid cancers (15,400 to 47,400) are expected. For France this estimate is 678 –1,153 additional cases, for Germany –1,479-2,514, for Romania –2,239-3,976.
Slide 9 standardized incidence rate
It is very important to assess an incidence of other cancers. I already mention that the fact of additional increase of other tumors among the population and liquidators is considered as not being evident.
It is relevant to the current view of Chernobyl impact on human health to reflect on the understanding in 1965 of the health effects of radiation from the atomic bombs in Japan in 1945 year. The only significant consequences observed in survivors 20 years after the atoms bombs were increases in leukemia and thyroid cancer, and the general view of the future was reassuring. But in 1974, a significant increase in solid cancers was detected from survivors with dose estimates in excess of 1Gy. But they comprise less than 3% of the cohort. In the full LSS cohort of 120,321 individuals about 48% were still alive at the end of 1998.
The analyses of the mortality from cancer diseases in the atomic bomb survivors over the period of 1950–1990, i. e. over 40 years of follow up, has revealed the statistically significant radiation effects beginning from 0.2Sv (200 mSv or 20 rem) (Pierse et al., 1996).
In 2000, Pierce and Preston evaluated solid cancer incidence for the period of1958 through 1994 focusing on the subcohort of about 50,000 lSS survivors who had dose estimate of less than 0.5 Gy to clarify cancer risks at low doses. They concluded there was a statistically significant dose response in the range of 0–0.15 Gy. This conclusion was confirmed by new publication Preston et al., 2007 on solid cancer incidence in atomic bomb survivor: 1958–1998. The authors write “ there is a statistically significant dose response when analyses were limited to cohort members with doses of 0.15Gy or less”.
Today, leukemia and thyroid cancer form only a small fraction of the accepted total radiation related health detriment of atomic bombs.
That is why it is too early to assess the overall impact of Chernobyl fallout on human health and animal populations.
According prof. Alexej Okeanov, 2007 the data on Incidence of malignant tumors among different groups of Belarusian population are as follows;
Among population living on the territories with ground deposition of 37–555 kBq/m2, an increase of relative risk of colon cancer, breast and thyroid cancer was found. There is statistically significant increase of breast cancer incidence in women, the most intensive in the age of 20–40 years. Population of this territory presents the most numerous of all follow up groups and includes 1,245 individuals.
Slide 10
You can see data on a relative risk of malignant tumors incidence among the population living in regions with density contamination of 37-555 kBq/m2. Population of this territory presents the most numerous of all follow up groups and includes 1,245 individuals.
Relative risk (RR) was calculated as ratio of standardized rate (TASR) of incidence among different exposed groups to the incidence of the control group (non-exposed group). Vitebsk region was accepted as a control region as far as its population was exposed to Chernobyl fallout much less. Vitebsk region is considered as so-called clean region.
Comparative analysis of relative risk of cancer incidence showed the considerable growth in 1997-2003 as compared with the previous period 1993-1996.
The period 1997-2003 was characterized by significant increase of cancer incidence of all sites including colon, mammary gland, skin and thyroid.
Slide 11
For the period of 1990–2003 there is a statistically significant increase of breast cancer incidence among women of Gomel region in comparison with appropriate value among women living in the less contaminated areas. Particularly, dose dependent three fold increase of breast cancer incidence was shown from women of Gomel region (National report, 2006).
At the same time the report of the Chernobyl Forum in Vienna, 2005 stated that the group of international experts did not reveal any evidences of leukemia and cancer growth among the inhabitants of the affected regions that can be attributed to radiation exposure.
However, a large body of data amassed by the present makes it possible to draw other conclusions concerning the health effects of low doses of radiation.
There are some reasons for such disagreement.
First of all, it is evident that a few attentions have been paid to assessment of whole body absorbed doses in comparison with the reconstruction of thyroid doses.
Therefore, we should admit non-availability of reliable data of individual and group doses appropriate for long-term cohort studies of dose dependence of the cancer incidence (mortality). For the time being, such investigations were not conducted. Only a cohort study of thyroid cancer and other thyroid diseases after the Chernobyl accident is conducting in Belarus and Ukraine, that includes 25,161 subjects under the age of 18 years in 1986.
Second, an assessment of the remote consequences of the Chernobyl fallout was reduced to the estimation of the health effects of low doses of radiation delivering with low dose rates. One of the specific features of the Chernobyl accident is long term exposure of numerous groups of population to low dose radiation. In more details, relatively high dose rate exposure (days to weeks) was followed by prolong (some decades) exposure to a low dose rate.
Before the Chernobyl disaster a reality of genetic effects of very low doses of IR remained unclear.
Slide 11 Our data
A number of studies conducted during the past two decades give reliable data about serious biological and medical effects of the Chernobyl accident and about harmful impact of irradiation at low doses and low dose rates. Such results were established for cell, animals and human.
So, we established genetic effects of low doses (less than 100 mSv) in somatic and germ cells of a model mammalian species, the bank vole, which was chronically exposed to low doses of ionizing radiation over 22 animal generations within 10 years following the Chernobyl accident (Ryabokon, Goncharova, 2006).
For example on this slide you can see the dose response curve for total chromosome aberrations frequencies and those of the Robertsonean translocation in bone marrow of bank voles inhabiting fives sites with different ground deposition of radionuclides in 1996 year. The accumulated doses in five natural mammalian populations living in the contaminated areas range were 0.3, 0.7, 12 and 25 mGy.
The analysis of our and literature data shows that the doubling dose estimates for acute irradiation of somatic cells in bank vole and human lymphocytes as well as for germ cells in laboratory mice are close to each other (Goncharova, Smolich, 2002). Therefore, the choice of bank voles as a model species for assessing radiation genetic risk is justified.
Slide 13
Our data on statistically significant genetic effects of very low doses are in line with data about radiation-related cancer risks at low doses among atomic bomb survivors.
The recent general report on mortality in the cohort of atomic bomb survivors followed up by the Radiation Research Foundation give strong evidence that there is direct, statistically significant evidence of risk in the dose range of approximately 0-0.10 Sv. You can see the dose-effect curve on this slide. Pierce and Preston used Life Span Study (LSS) solid cancer incidence data for the period from 1958 through 1994 in an assessment of low-dose risks.
It is important to note that radiation-related cancer risks at low doses among atomic survivors are well established in 45 years after bombardment. It is known the lower radiation doses the longer latent periods are.
On the other hand, the recent general report on solid cancer incidence in atomic bomb survivors over 1958—1998 years (Preston et al., 2007) and publication (Pierce, Preston, 2000) have presented direct, statistically significant evidence of risk in the dose range of 0–0.15 Gy.
Whole body doses received by exposed populations of the three most affected countries Republic of Belarus, Ukraine are estimated to be in this range, i. e. within the range that led to a significant increase in cancer incidence after nuclear explosion.
Although the Radiation Research Foundation investigations are often considered the high-dose research, in reality, approximately 30% of the exposed individuals of the cohort received doses from 5 to 200 mGy (Preston et al., 2007).
Such doses of the whole body irradiation were delivered as a result of the Chernobyl accident to inhabitants of high-contaminated areas in Belarus, Ukraine and Russia too. This indicates the possibility of radiation-induced cancers caused by the Chernobyl accident.
According to the present knowledge (BEIR VII, 2006) there is no threshold for the carcinogenic effect of IR. Therefore any additional irradiation will induce additional cancers in exposed populations, not only in the three most contaminated countries but all around Europe, including France, Swiss, Germany and other countries.
It is important to note that radiation-related cancers at low doses among atomic survivors were established later than in the range of high doses. The longer latency period in case of low doses of irradiation is responsible for this effect. Thus, one needs to expect more pronounced manifestation of additional cancers from the Chernobyl accident in the future time. Particularly, dose dependent three fold increase of breast cancer incidence was recently shown for women of Gomel region (National report, 2006).
According to Malko’s estimates, in Belarus alone, approximately 28,300 solid cancers other than thyroid and non-melanoma skin cancers (11,800 to 44,800) are expected. It is nearly 6-times more (130,400 for all Europe) than predicted by Cardis et al., 2006 (22,800).
For France –1,220, for Germany – 9,280 cases.
Further, recent cellular and molecular studies have increased our understanding of low dose radiation effects, first of all induced genome instability and bystander effects.
Transgenerational accumulation of radiation damage over 22 animal generations was found by the levels of chromosome aberrations in bone marrow cells and embryonic lethality in bank vole populations chronically exposed to low doses delivered with very low dose-rates (Ryabokon, Goncharova, 2006).
Evidently, non-targeted effects of ionizing radiation such as genomic instability, bystander effects and other new phenomena have to contribute to short-term and long-term overall outcomes for human health after low dose ionizing radiation. In this connection we study a genomic instability of different risk groups in Belarusian population (Goncharova et al., 2008).
I suppose that increased thyroid cancer incidence of children from irradiated parents chronically exposed due to Chernobyl accident might be a manifestation of the induced genomic instability (Goncharova, 2005).
For the elucidation of the causal role of low dose radiation exposure due to Chernobyl fallout for the observed increases of many types of cancer and congenital malformations in Belarus (National report, 2006), long-term radiation-epidemiological studies with reconstruction of whole body absorbed doses must be carried out in the future. So far, all declaration on the absence of radiation-linked increases in the incidence of other types of cancer and congenital malformations simply means the absence of an adequate research.
Slide 14
There is a set of data on increased sensitivity of somatic and germ cells of animals and human to low radiation doses (Vilenchik, Knudson, 2000).
There is also similar data concerning cancer risks attributable to low doses. In this connection I would like to give you the data published by Radiation Effects Research Foundation on Excess Relative Risk for cancer mortality.
The estimated excess relative risk (ERR) per Sv for the selected dose ranges of Life Span Study cohort was the highest for the lowest dose category, namely from 0 to 20 mSv in comparison with dose range between 0 and 3 Sv.
Recently, the scientists of the RERF gave also reliable evidences of radiation effects on noncancer mortality. Statistically significant increases are seen for heart diseases, stroke, digestive, respiratory and other diseases (Preston et al., 2003).
Slide 15 conclusions
The whole body doses received by exposed populations of the Republic of Belarus, Ukraine and contaminated regions of the Russian Federation are estimated to be in the dose range of 0–0.15 Gy, i. e. within the range that led to a significant increase in cancer incidence in atomic bomb survivors.
Thyroid cancer incidence continues growing steadily among adult population of Belarus (National report, 2006)
For the period of 1990–2003 there is a statistically significant increase of breast cancer incidence among women of Gomel region in comparison with appropriate value among women living in the less contaminated areas. Dose dependence between accumulated radiation dose and realized relative risk of breast cancer was shown.
According A. Okeanow data among population living in the regions of 37–555 kBq/m2 considerable growth of relative risk of cancer incidence occurred in 1997-2003 in comparison with the previous period 1993-1996.
Summing up all these data allows us to conclude that the accident at the Chernobyl nuclear power plant will result in a number of unfavorable health consequences for both affected people and coming generations.
Particularly, an increased thyroid cancer incidence of children born from irradiated parents chronically exposed due to Chernobyl accident might be a manifestation of the induced genomic instability.
For the establishment of the causal role of low dose radiation exposure due to Chernobyl fallout for the observed increases of many types of cancer and congenital malformations in Belarus (National report, 2006), health radiation-epidemiological and cohort studies with reconstruction of whole body absorbed doses must be carried out in the future.
So far, all declarations on the absence of radiation-linked increases in the incidence of other types of cancer and congenital malformations simply mean the ignoring of data established in Belarus, Ukraine and the Russian Federation and absence of an adequate research.
Goncharova R. I.
Institute of Genetics and Cytology, National Academy of Sciences
Akademichnaya St., 27, Minsk, 220072, REPUBLIC OF Belarus
E-mail: R.Goncharova@igc.bas-net.by
Slide 2
My report is devoted to the memory of Solange Fernex (1934-2006), a prominent French pacifist activist and politician, the former member of the European Parliament,
for her stubborn struggle for the full information on the health and other effects of the Chernobyl catastrophe in all the affected countries, including France, and for development of the adequate health assistance to affected populations.
Doctor Rosalie Bertell and Solange Fernex were initiators of the organization of Permanent People’s Tribunal, Session on Chernobyl in Vienna in 1996.
Solange Fernex actively participated in the work of Permanent People’s Tribunal, Session on Chernobyl.
Solange Fernex was the final editor of the book: Chernobyl Environmental, Health and Human Rights Implications
Solange Fernex has made the report “Health effects of Chernobyl: a dogma or a quest of truth?” at 3td International Conference: “ Health Effects of the Chernobyl Accident, Results of the 15-years Follow-up Studies” in Kiev in 2001
Slide 3
The Chernobyl accident has caused the deposition of radioisotopes over very wide areas of the Northern Hemisphere, in particular in Europe (Atlas of Caesium Deposition on Europe after the Chernobyl Accident, 1998), followed by chronic exposure of many millions of people to a mixture of external and internal radiation. You can see the map of radioactive contamination of Europe after the Chernobyl accident.
Slide 4
However, the Republic of Belarus was affected by the accident more than any other country of the world. According to the Atlas practically the whole territory of Belarus was contaminated with different radioisotopes above the level of global fall-out. The ground deposition density equal to 37 kBq/m2 is accepted as a limited value for distinguishing the so-called “clean” regions from the contaminated ones making up 23% of the country area. So, 22 years after the world’s worst nuclear accident the entire population of Belarus is involuntary taking part in a decades-long experiment on how low doses of radiation affects human health.
Clear understanding of this fact is of great importance for interpreting the effects recorded.
Since 1986 we have been studying the biological effects of chronic low dose radiation in natural populations of wild small mammals named bank vole, in laboratory mice and evaluating remote consequences of the Chernobyl accident.
Here, on the map of Belarus, you can also see monitoring sites. These sites are located at different distances from the Chernobyl nuclear power plant and represent different levels of radioisotope contamination:
Priluksky reserve (near Minsk, 330 km north-west from the Chernobyl power station, 8 kBq/m2);
Berezinsky Biosphere reserve (Vitebsk region, 400 km NNW, 18 kBq/m2);
The vicinity of Majsk village (Bragin district of Gomel region, 60 km N, 220 kBq/m2);
The vicinity of Babchin village, exclusion zone (Khoiniki district of Gomel region, 40 km NNW, 1530 kBq/m2). Now it is Polessky Radiation reserve.
The vicinity of the Radin village, exclusion zone (Khoiniki district of Gomel region, about 18 km N, 8500 kBq/m2). Now it is Polessky Radiation reserve.
Slide 5
Bank voles are collected at 5 trapping sites different in radionuclide ground deposition. These data is given on this slide.
The data on 137Cs, 134Cs, 106Ru, 144Ce contamination were obtained in the year of accident (1986). The concentrations of 90Sr and transuranic elements in samples of soils were measured in 1996.
It is necessary to note that after the primary insult in the year of the accident, every successive generation of animals under investigation was exposed to permanently decreasing whole-body dose rates of ionizing radiation.
United Nations Chernobyl Forum under the aegis of WHO issued the massive report on the health and environmental consequences of the accident. The WHO report “Health Effects of the Chernobyl Accident and Special Health Care Programmes”(2006) confirmed radiation induced huge increase of the thyroid cancer in those exposed in childhood and adolescence and declared that there has been no significant increase so far in the incidence of other cancers and congenital malformations that can be attributed to radiation exposure. It is intriguing to note that prognosis estimate of future deaths due to the Chernobyl is much less in Joint News Release WHO/IAEA/UNDP “Chernobyl: the true scale of the accident; 20 years later a UN report provides definitive answers and ways to repair lives” and the report’s 50-page summary in Health effect of Chernobyl fallout that firmly established
The established health consequences of the Chernobyl
Slide 6
Reconstruction of iodine–131 fallout has shown that the whole territory of the Republic of Belarus was contaminated this isotope (National report, 2006).
On this slide you can see calculated collective thyroid doses for two age groups, namely for children and adolescents and for adult at the time of accident, for all regions of Belarus.
For the time being scientific society has accepted only significant increase of thyroid cancer incidence in children and adolescents. Growth of thyroid cancer in adult population has not been reported in official scientific conferences as a proved scientific fact.
Slide 7
As for concerning whole body absorbed doses, the data on collective effective dose you can see on this slide. According to the National report of Belarus, 2006, table 3.11) up to 90% of collective cumulative effective dose was formed in the first decade after the accident.
Annual collective dose to population residing on the territory of radioactive contamination is about 21 person-Sv, average individual dose is 0,15 mSv.
For all of exposed groups, estimates of numbers of fatal cancers can be derived from collective doses. Such estimates depend on the assumed risk coefficient
Slide 8
The incidence of thyroid cancer in those who were adults at the time of exposure is reported to have increased in the many exposed populations, including Poland (Mahoney, Ostapenko, 2004), although the relationship to radiation is not so clear. Time course of thyroid cancer incidence among childhood and adult populations of Belarus up to 2001 have been shown on this slide.
According National Report of Belarus, 2006 thyroid cancer incidence continues growing steadily among adult population of Belarus.
The concentration of effort on the major increase in those exposed as children has meant that the smaller risk to adults has not been adequately investigated.
As for concerning future estimates of thyroid cancer for Belarus it is necessary to note.
Currently, individuals exposed as children are now adolescents or young adults but continue to carry an increased risk of developing thyroid carcinoma during all lifespan.
For predicting, we have to know risk coefficients obtained on the basis of studying suffered populations. Depending of the calculated values of risk coefficient, the predicted values of the future incidence of thyroid cancer differ greatly. The latest estimate for the year 2056 ranges from 3,400 to 72,000 according Cardis et al., 2006 (Estimates of the cancer burden in Europe from radioactive fallout from the Chernobyl accident, Int J Cancer).
According estimates of Malko, in Belarus alone, approximately 31,400 additional thyroid cancers (15,400 to 47,400) are expected. For France this estimate is 678 –1,153 additional cases, for Germany –1,479-2,514, for Romania –2,239-3,976.
Slide 9 standardized incidence rate
It is very important to assess an incidence of other cancers. I already mention that the fact of additional increase of other tumors among the population and liquidators is considered as not being evident.
It is relevant to the current view of Chernobyl impact on human health to reflect on the understanding in 1965 of the health effects of radiation from the atomic bombs in Japan in 1945 year. The only significant consequences observed in survivors 20 years after the atoms bombs were increases in leukemia and thyroid cancer, and the general view of the future was reassuring. But in 1974, a significant increase in solid cancers was detected from survivors with dose estimates in excess of 1Gy. But they comprise less than 3% of the cohort. In the full LSS cohort of 120,321 individuals about 48% were still alive at the end of 1998.
The analyses of the mortality from cancer diseases in the atomic bomb survivors over the period of 1950–1990, i. e. over 40 years of follow up, has revealed the statistically significant radiation effects beginning from 0.2Sv (200 mSv or 20 rem) (Pierse et al., 1996).
In 2000, Pierce and Preston evaluated solid cancer incidence for the period of1958 through 1994 focusing on the subcohort of about 50,000 lSS survivors who had dose estimate of less than 0.5 Gy to clarify cancer risks at low doses. They concluded there was a statistically significant dose response in the range of 0–0.15 Gy. This conclusion was confirmed by new publication Preston et al., 2007 on solid cancer incidence in atomic bomb survivor: 1958–1998. The authors write “ there is a statistically significant dose response when analyses were limited to cohort members with doses of 0.15Gy or less”.
Today, leukemia and thyroid cancer form only a small fraction of the accepted total radiation related health detriment of atomic bombs.
That is why it is too early to assess the overall impact of Chernobyl fallout on human health and animal populations.
According prof. Alexej Okeanov, 2007 the data on Incidence of malignant tumors among different groups of Belarusian population are as follows;
Among population living on the territories with ground deposition of 37–555 kBq/m2, an increase of relative risk of colon cancer, breast and thyroid cancer was found. There is statistically significant increase of breast cancer incidence in women, the most intensive in the age of 20–40 years. Population of this territory presents the most numerous of all follow up groups and includes 1,245 individuals.
Slide 10
You can see data on a relative risk of malignant tumors incidence among the population living in regions with density contamination of 37-555 kBq/m2. Population of this territory presents the most numerous of all follow up groups and includes 1,245 individuals.
Relative risk (RR) was calculated as ratio of standardized rate (TASR) of incidence among different exposed groups to the incidence of the control group (non-exposed group). Vitebsk region was accepted as a control region as far as its population was exposed to Chernobyl fallout much less. Vitebsk region is considered as so-called clean region.
Comparative analysis of relative risk of cancer incidence showed the considerable growth in 1997-2003 as compared with the previous period 1993-1996.
The period 1997-2003 was characterized by significant increase of cancer incidence of all sites including colon, mammary gland, skin and thyroid.
Slide 11
For the period of 1990–2003 there is a statistically significant increase of breast cancer incidence among women of Gomel region in comparison with appropriate value among women living in the less contaminated areas. Particularly, dose dependent three fold increase of breast cancer incidence was shown from women of Gomel region (National report, 2006).
At the same time the report of the Chernobyl Forum in Vienna, 2005 stated that the group of international experts did not reveal any evidences of leukemia and cancer growth among the inhabitants of the affected regions that can be attributed to radiation exposure.
However, a large body of data amassed by the present makes it possible to draw other conclusions concerning the health effects of low doses of radiation.
There are some reasons for such disagreement.
First of all, it is evident that a few attentions have been paid to assessment of whole body absorbed doses in comparison with the reconstruction of thyroid doses.
Therefore, we should admit non-availability of reliable data of individual and group doses appropriate for long-term cohort studies of dose dependence of the cancer incidence (mortality). For the time being, such investigations were not conducted. Only a cohort study of thyroid cancer and other thyroid diseases after the Chernobyl accident is conducting in Belarus and Ukraine, that includes 25,161 subjects under the age of 18 years in 1986.
Second, an assessment of the remote consequences of the Chernobyl fallout was reduced to the estimation of the health effects of low doses of radiation delivering with low dose rates. One of the specific features of the Chernobyl accident is long term exposure of numerous groups of population to low dose radiation. In more details, relatively high dose rate exposure (days to weeks) was followed by prolong (some decades) exposure to a low dose rate.
Before the Chernobyl disaster a reality of genetic effects of very low doses of IR remained unclear.
Slide 11 Our data
A number of studies conducted during the past two decades give reliable data about serious biological and medical effects of the Chernobyl accident and about harmful impact of irradiation at low doses and low dose rates. Such results were established for cell, animals and human.
So, we established genetic effects of low doses (less than 100 mSv) in somatic and germ cells of a model mammalian species, the bank vole, which was chronically exposed to low doses of ionizing radiation over 22 animal generations within 10 years following the Chernobyl accident (Ryabokon, Goncharova, 2006).
For example on this slide you can see the dose response curve for total chromosome aberrations frequencies and those of the Robertsonean translocation in bone marrow of bank voles inhabiting fives sites with different ground deposition of radionuclides in 1996 year. The accumulated doses in five natural mammalian populations living in the contaminated areas range were 0.3, 0.7, 12 and 25 mGy.
The analysis of our and literature data shows that the doubling dose estimates for acute irradiation of somatic cells in bank vole and human lymphocytes as well as for germ cells in laboratory mice are close to each other (Goncharova, Smolich, 2002). Therefore, the choice of bank voles as a model species for assessing radiation genetic risk is justified.
Slide 13
Our data on statistically significant genetic effects of very low doses are in line with data about radiation-related cancer risks at low doses among atomic bomb survivors.
The recent general report on mortality in the cohort of atomic bomb survivors followed up by the Radiation Research Foundation give strong evidence that there is direct, statistically significant evidence of risk in the dose range of approximately 0-0.10 Sv. You can see the dose-effect curve on this slide. Pierce and Preston used Life Span Study (LSS) solid cancer incidence data for the period from 1958 through 1994 in an assessment of low-dose risks.
It is important to note that radiation-related cancer risks at low doses among atomic survivors are well established in 45 years after bombardment. It is known the lower radiation doses the longer latent periods are.
On the other hand, the recent general report on solid cancer incidence in atomic bomb survivors over 1958—1998 years (Preston et al., 2007) and publication (Pierce, Preston, 2000) have presented direct, statistically significant evidence of risk in the dose range of 0–0.15 Gy.
Whole body doses received by exposed populations of the three most affected countries Republic of Belarus, Ukraine are estimated to be in this range, i. e. within the range that led to a significant increase in cancer incidence after nuclear explosion.
Although the Radiation Research Foundation investigations are often considered the high-dose research, in reality, approximately 30% of the exposed individuals of the cohort received doses from 5 to 200 mGy (Preston et al., 2007).
Such doses of the whole body irradiation were delivered as a result of the Chernobyl accident to inhabitants of high-contaminated areas in Belarus, Ukraine and Russia too. This indicates the possibility of radiation-induced cancers caused by the Chernobyl accident.
According to the present knowledge (BEIR VII, 2006) there is no threshold for the carcinogenic effect of IR. Therefore any additional irradiation will induce additional cancers in exposed populations, not only in the three most contaminated countries but all around Europe, including France, Swiss, Germany and other countries.
It is important to note that radiation-related cancers at low doses among atomic survivors were established later than in the range of high doses. The longer latency period in case of low doses of irradiation is responsible for this effect. Thus, one needs to expect more pronounced manifestation of additional cancers from the Chernobyl accident in the future time. Particularly, dose dependent three fold increase of breast cancer incidence was recently shown for women of Gomel region (National report, 2006).
According to Malko’s estimates, in Belarus alone, approximately 28,300 solid cancers other than thyroid and non-melanoma skin cancers (11,800 to 44,800) are expected. It is nearly 6-times more (130,400 for all Europe) than predicted by Cardis et al., 2006 (22,800).
For France –1,220, for Germany – 9,280 cases.
Further, recent cellular and molecular studies have increased our understanding of low dose radiation effects, first of all induced genome instability and bystander effects.
Transgenerational accumulation of radiation damage over 22 animal generations was found by the levels of chromosome aberrations in bone marrow cells and embryonic lethality in bank vole populations chronically exposed to low doses delivered with very low dose-rates (Ryabokon, Goncharova, 2006).
Evidently, non-targeted effects of ionizing radiation such as genomic instability, bystander effects and other new phenomena have to contribute to short-term and long-term overall outcomes for human health after low dose ionizing radiation. In this connection we study a genomic instability of different risk groups in Belarusian population (Goncharova et al., 2008).
I suppose that increased thyroid cancer incidence of children from irradiated parents chronically exposed due to Chernobyl accident might be a manifestation of the induced genomic instability (Goncharova, 2005).
For the elucidation of the causal role of low dose radiation exposure due to Chernobyl fallout for the observed increases of many types of cancer and congenital malformations in Belarus (National report, 2006), long-term radiation-epidemiological studies with reconstruction of whole body absorbed doses must be carried out in the future. So far, all declaration on the absence of radiation-linked increases in the incidence of other types of cancer and congenital malformations simply means the absence of an adequate research.
Slide 14
There is a set of data on increased sensitivity of somatic and germ cells of animals and human to low radiation doses (Vilenchik, Knudson, 2000).
There is also similar data concerning cancer risks attributable to low doses. In this connection I would like to give you the data published by Radiation Effects Research Foundation on Excess Relative Risk for cancer mortality.
The estimated excess relative risk (ERR) per Sv for the selected dose ranges of Life Span Study cohort was the highest for the lowest dose category, namely from 0 to 20 mSv in comparison with dose range between 0 and 3 Sv.
Recently, the scientists of the RERF gave also reliable evidences of radiation effects on noncancer mortality. Statistically significant increases are seen for heart diseases, stroke, digestive, respiratory and other diseases (Preston et al., 2003).
Slide 15 conclusions
The whole body doses received by exposed populations of the Republic of Belarus, Ukraine and contaminated regions of the Russian Federation are estimated to be in the dose range of 0–0.15 Gy, i. e. within the range that led to a significant increase in cancer incidence in atomic bomb survivors.
Thyroid cancer incidence continues growing steadily among adult population of Belarus (National report, 2006)
For the period of 1990–2003 there is a statistically significant increase of breast cancer incidence among women of Gomel region in comparison with appropriate value among women living in the less contaminated areas. Dose dependence between accumulated radiation dose and realized relative risk of breast cancer was shown.
According A. Okeanow data among population living in the regions of 37–555 kBq/m2 considerable growth of relative risk of cancer incidence occurred in 1997-2003 in comparison with the previous period 1993-1996.
Summing up all these data allows us to conclude that the accident at the Chernobyl nuclear power plant will result in a number of unfavorable health consequences for both affected people and coming generations.
Particularly, an increased thyroid cancer incidence of children born from irradiated parents chronically exposed due to Chernobyl accident might be a manifestation of the induced genomic instability.
For the establishment of the causal role of low dose radiation exposure due to Chernobyl fallout for the observed increases of many types of cancer and congenital malformations in Belarus (National report, 2006), health radiation-epidemiological and cohort studies with reconstruction of whole body absorbed doses must be carried out in the future.
So far, all declarations on the absence of radiation-linked increases in the incidence of other types of cancer and congenital malformations simply mean the ignoring of data established in Belarus, Ukraine and the Russian Federation and absence of an adequate research.
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