TNT . J . RADIAT . BIOL ., 1979, VOL . 36, NO . 1, 65-73
Radiation-induced DNA strand breaks and their repair in the developing rat brain
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HUMBERTO CERDA, KARL J . JOHANSON and KERSTIN ROSANDER The Gustaf Werner Institute, Department of Physical Biology, Box 531, S-751 21 Uppsala, Sweden (Received 6 July 1978 ; accepted 27 September 1978)
Rats, 5, 10 or 25 days old, were 60 Co gamma irradiated . The induction of DNA strand breaks was studied after killing the rats within 1 min after irradiation, and the repair of the induced breaks after various intervals up to 180 min . Cell suspensions were prepared from the brain and samples were transferred into alkaline solutions . The fraction of DNA remaining double-stranded after 30 min alkali treatment was estimated after separation of single- and double-stranded DNA on hydroxylapatite . The amount of DNA strand breaks induced per Gray (1-8 Gray) was found to be in accordance with earlier in vivo studies of the mouse small intestine and mouse spleen . The DNA strand breaks in the rat brain induced by 4 Gray 60 Co gamma irradiation were repaired 30 min after irradiation in all age groups studied . 1.
Introduction The brain develops through rapid cell proliferation and successive differentiation . Proliferation of neurons in the cerebrum of the rat occurs mainly in utero, while glial cells continue to divide after birth (Haas, Werner and Fliedner 1970) . The neurons of the cerebellum, on the other hand, develop mostly postnatally . Proliferative activity in the cerebellum is rather low at birth, increases very rapidly to a maximum at 10 days, and then decreases (Fish and Winick 1969, Altman and Nicholson 1971) . The adult rat brain consists mainly of neurons, which are nonproliferative, and glial cells, which have the ability to divide . The developing brain is known to be much more radio-sensitive than the adult brain . Various anomalies can be induced by prenatal irradiation (Hicks and D'Amato 1966) . At the cellular level there are alterations in the development of neurons in rats, after 0 . 1-0•4 Gray (Hicks and D'Amato 1963), and necrosis of proliferative and migrating cells of the cerebellar cortex of rats after 2 Gray (Altman, Anderson and Wright 1968) . It is well known that irradiation introduces DNA strand breaks in mammalian cells in vitro . These breaks are usually rejoined with rather high efficiency (cf . Kihlman 1977) . Some efforts have been made to determine DNA strand breaks in the canine brain after irradiation, with the sucrose density gradient technique (Wheeler and Lett 1972 and 1974, Wang and Wheeler 1978) . DNA strand breaks and their repair can now be studied with a simple and more sensitive technique using the process of DNA unwinding in alkaline solution, followed by separation of singlestranded and double-stranded DNA on hydroxylapatite (Ahnstrom and Erixon 1973, Rydberg 1975) . The result is expressed as the percentage of the labelled DNA found in the double-stranded fraction . This is interpreted as a measure of the mean R .B .
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length between two breaks, or alkali-labile sites, in DNA (Rydberg 1975) . The introduction of DNA strand breaks thus causes a decrease in the percentage of double-stranded DNA . This method has been applied to in vivo studies of radiation-induced DNA strand breaks in the proliferative crypt and the non-proliferative villous cells of the small intestine of the mouse (Rydberg and Johanson 1975), and in the spleen of the mouse (Ostling and Johanson 1978) . The aim of the present work was to study the radiation-induced DNA strand breaks and their repair in the cerebrum and cerebellum of young rats at different ages . 2. Material and method 2 .1 . Animals Rats of the Sprague-Dawley strain were used . They were given food and water ad libitum . The newborn rats were weighed within 24 hours after birth, and only those weighing more than 6 . 0 g were taken for the experiments . The size of the litters was always reduced to eight animals . No sex distinction was made . Growing rats were separated from their mother at 22 days of age . 2.2 . Injections 3 H-thymidine (1 . 85 x 1011 Bq/mmol (5 Ci/mmol), Radiochemical Centre, Amersham) was diluted to 1 . 85 x 10' Bq/ml (0.5 mCi/ml) in 0 . 9 per cent NaCl . All animals were injected twice, each time with 1 . 11 x 10 4 Bq/g (0. 3 pCi/g) body weight . The rats used for the experiments at five days of age were injected on days 3 and 4 . For other ages (10 and 25 days), the animals were injected on days 3 and 5 . 2 .3 . Irradiation Rats 5, 10 or 25 days old, were placed in a plastic cage and whole-body irradiated with a freely radiating 60 Co gamma source at the Gustaf Werner Institute (Kinell and Larsson 1960) . The dose rate was 2 Gray/min (200 rad/min), as determined by Fricke dosimetry (Spinks and Woods 1976) . Control rats were sham-irradiated under similar conditions . A whole litter was used for each age and experiment in order to reduce the variations . 2.4 . Preparation of cell suspension and alkali treatment The rats were decapitated within 1 min or after various intervals after irradiation . The brain was rapidly resected and divided mid-sagittally . The anterior parts of the cerebrum, except the olfactory lobes, were excised and transferred to an ice-chilled glass dish . The cerebellar halves were also transferred to an ice-chilled dish . Each half of the cerebrum and cerebellum was used as a separate sample, in order to evaluate variations due to the experimental procedure . In the 25-day-old rats the anterior part of the cerebellum was discarded, in order to obtain an adequate number of cells (see below) . The cell preparation was performed in the cold, using an ice-bath and pre-chilled solutions, instruments and glassware . The tissue was minced with a scalpel and suspended in 2 ml Earle-Eagle's medium by gentle drawing 2-3 times through a 19 gauge needle (cerebrum) or 3-5 times through a 18 gauge needle (cerebellum) . The cell suspension was filtered through a nylon net of 250 ym mesh size . A sample of 0 . 1 ml (about 10 6 cells) was transferred to 1 ml of the alkaline solution (0 . 03 M NaOH, 0 . 01 M Na,HPO 4i 0. 9 M
Radiation induced DNA strand breaks in the developing rat brain
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NaCI) and left in the dark at 20 ° C . After 30 min the solution was neutralized by addition of I ml 0 . 036 M HC1 and sonicated (Branson Sonifier B-12 with microtip), at level 2 for 15 s (Rydberg 1975) . Two millilitres of 2 per cent sodium dodecyl sulphate solution were added, and the sample was stored at - 20°C until chromatography was performed .
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2 .5 . Hydroxylapatite chromatography
The procedure described by Rydberg (1975) was essentially followed . Chromatography was performed at 60°C, single-stranded DNA being eluted with 3 ml 0-125M phosphate buffer, and double-stranded DNA with 1 . 5 ml 0-250M phosphate buffer and then diluted with water to 3 ml . Seven millilitres of scintillator solution (PCS, Nuclear Chicago Instrument Co) was added to each DNA fraction . The samples were heated to 60 ° C, and thoroughly shaken . The 3 H-activities were determined in a liquid scintillation counter (Nuclear Chicago, Mark I I) operating at 4°C .
3 . Results 3 .1 . Preparation of the cell suspension The major technical problem in developing the present method was to obtain cells of acceptable quality in suspension . The mechanical treatment had to be reduced to a minimum, otherwise DNA strand breaks were introduced, seen as a lower percentage of double -stranded DNA . Tissue disruption by repeated drawing through a needle gave a suspension of cell nuclei, single cells and cell aggregates . The larger aggregates were excluded by filtration through a 250 ,um nylon net . When analysing DNA strand breaks in control rats, values of 70-80 per cent double-stranded DNA were obtained as a rule . Variations within the range of 20 per
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Figure 1 . Induction of DNA strand breaks in cells from cerebrum and cerebellum of 5-dayold rats irradiated in vivo : the percentage double-stranded DNA immediately after irradiation as a function of absorbed dose . E2
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cent of this control value could be seen from one experiment to another, but for one litter (eight rats) used during 1 day, the variations were smaller, within a range of about 10 per cent . The use of needles of smaller diameter caused a significant decrease in the percentage of double-stranded DNA . In the ultimate method EarleEagle medium was used . When other solutions, such as 0 . 9 per cent NACI, KrebsRinger phosphate buffer pH 7 . 4, or tris-buffer (0 . 114 M tris, 0.077 NaCl, pH 7 . 4) were used, a lower percentage of double-stranded DNA was found . Addition of detergents, in low concentrations, to the solutions seemed to have no effect . 3 .2 . Induction of DNA strand breaks Rats of different ages were irradiated with various doses and killed within 1 min after irradiation . Each of figures 1, 2 and 3 represents a complete experiment with one litter (see Irradiation § 2 .3) . As both halves of the anterior part of the cerebrum and the cerebellum were analysed, one rat thus gave rise to two values at a given dose . The difference between these points gives the variation in the results caused by preparation of the cell suspension and the following treatment . Only minor differences in the slopes of the curves for different ages can be seen . There seem also to be only minor differences in dose response between cerebrum and cerebellum . Similar results were obtained in repeated experiments . 3 .3 . Rejoining of DNA strand breaks Rats from one litter were irradiated at 4 Gray and killed after various periods . As can be seen in figures 4, 5 and 6, the percentage of double-stranded DNA increased to a maximum, or a plateau, within 30 min of irradiation . One exception was the cerebrum in 5-day-old rats where a maximum was found after 60 min . This increase is here interpreted as a measure of the repair of DNA strand breaks and alkali-labile sites in DNA .
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Figure 2 . Induction of DNA strand breaks in cells from cerebrum and cerebellum of 10day-old rats irradiated in vivo : the percentage double-stranded DNA immediately after irradiation as a function of absorbed dose .
Radiation induced DNA strand breaks in the developing rat brain
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Figure 3 . Induction of DNA strand breaks in cells from cerebrum and cerebellum of 25 dayold-rats irradiated in vivo : the percentage double-stranded DNA immediately after irradiation as a function of absorbed dose .
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Figure 4 . Rejoining of DNA strand breaks in cells from cerebellum and cerebrum of 5-dayold rats after irradiation in vivo : the percentage double-stranded DNA plotted against time after irradiation at 4 Gray .
In the youngest rats a decrease in the percentage of double-stranded DNA was observed between 30 and 180 min (figure 4) . It was also found, although less pronounced, in the 10-day-old rats (figure 5), but not in the rats aged 25 days, where a plateau was reached after 30 min (figure 6) . Similar results were obtained in repeated experiments .
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Figure 5 . Rejoining of DNA strand breaks in cells from cerebellum and cerebrum of 10-dayold rats after irradiation in vivo : the percentage double-stranded DNA plotted against time after irradiation at 4 Gray .
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Figure 6 . Rejoining of DNA strand breaks in cells from cerebellum and cerebrum of 25-dayold rats after irradiation in vivo : the percentage double-stranded DNA plotted against time after irradiation at 4 Gray .
4.
Discussion
In this work cells of the developing rat brain were labelled by injection of 3 Hthymidine 3-5 days after birth . Consequently,, the radiation-induced DNA strand breaks were studied in the cell population proliferating at this time . The cell population labelled in the cerebrum were mostly glial cells, but in the cerebellum
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mainly neurons were labelled as seen after autoradiography (Haas et al . 1970, Altman and Nicholson 1971) . No major differences in the number of DNA strand breaks induced per Gray could be observed between the different age groups . In rats aged 10 days slightly more DNA strand breaks seem to be introduced than in the other age groups . The interpretation of these results, however, is very difficult, because of the heterogeneous pattern of proliferation and differentiation of the neuronal and glial cell populations . As estimated by the slope of the dose-effect curve (figures 1-3), the DNA strand breaks induced per Gray in rats aged 5 and 25 days were in accordance with earlier results found in the crypt and villous cells of the mouse small intestine . Only minor differences were observed between the proliferative and radiosensitive crypt cells and the non-proliferative and rather radio-resistant villous cells (Rydberg and Johanson 1975) . A similar number of DNA strand breaks per Gray seem to be introduced also in the splenic cells of mouse in vivo (Ostling and Johanson 1978) . The kinetics of rejoining of DNA strand breaks (and repair of alkali-labile sites in DNA) after irradiation at 4 Gray were similar in the cerebrum and cerebellum of rats at 10 and 25 days of age . The DNA strand breaks seem to be repaired, as control values were obtained within 30 min after irradiation . Similar rates of repair of DNA strand breaks were found in crypt and villous cells of the small intestine, and in the splenic cells of the mouse (Rydberg and Johanson 1975, Ostling and Johanson 1978) . Since the method is very sensitive small variations in the mechanical treatment could give rise to differences in the percentage of double-stranded DNA from day to day . As mentioned earlier the position of a curve can differ from experiment to experiment (cf. §3 .1) . However, the shapes of the curves were always similar in repeated experiments . Gutin, Hilton, Fein, Allen and Walker (1977), estimated the rate of strand separation of DNA in alkali combined with S 1 nuclease treatment instead of hydroxylapatite chromatography . They found that the damaged DNA seemed to be repaired within 30 min after irradiation of intracerebral rat gliosarcoma in vivo at 600 rad . The observed decrease in the percentage of double-stranded DNA 2 hours after irradiation in the 5-day-old rats (less pronounced in cerebellum at 10 days) may be due to the occurrence of cell death . Histological studies of radiation damage in the cerebellum of the rat show that the nuclei of the damaged cells begin to be pycnotic about 2 hours after irradiation (Altman and Nicholson 1971, Cerda 1978) . A slight decrease in the percentage of double-stranded DNA was also observed in studies of the repair of DNA strand breaks in splenic cells in vivo (Ostling and Johanson 1978) . Limited ability to repair DNA damage may result in accumulation of DNA breaks in non-proliferative, terminally differentiated cells . Price, Modak and Makinodan (1971), studied the DNA template activity with calf thymus DNA polymerase in fixed sections of various tissues from young and senescent mice . They found indications of an age-associated increase in DNA strand scission in neurons and astrocytes of the brain, and in Kupfer cells of the liver . DNA in microglia of the brain and hepatocytes seems to be either more resistant to damage to strand scission, or is more effectively repaired when damaged . Ageing thus shows a similar effect to irradiation . Karron and Ormerod (1973), studied the repair of DNA strand breaks, using sedimentation in gradients of alkaline sucrose and estimating DNA by fluorimetry . They found that DNA from non-dividing cells, such as rat muscle cells
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and chicken erythrocytes, lacked the ability to repair X-ray induced DNA singlestrand breaks . Cells from the thymus and spleen of rats could repair such breaks . Lieberman and Forbes (1973), found little or no unscheduled DNA-synthesis in cerebellar neurons or striated myocytes after U . V .-irradiation or treatment with chemical carcinogens, indicating a decreased ability to repair DNA damage . Wheeler and Lett using density gradient centrifugation to study the induction and repair of DNA strand breaks in neurons from canine cerebellum, found that the efficiency in producing DNA strand breaks was similar in neurons in vitro or in vivo and in normal cells in culture . The radiation-induced DNA strand breaks were repaired after irradiation, although the molecular events which accompany strand break repair seemed to be different from those observed in cultured dividing cells (Wheeler and Lett 1972) . The results show that radiation-induced DNA strand breaks in the rat brain can be repaired up to at least 25 days of age . Since mixed cell populations from the cerebrum or cerebellum labelled at days 3 to 5 after birth were used, we cannot exclude the possibility that some cell populations are unable to repair the DNA strand breaks . Here a complicated problem arises, i .e . how to devise a sensitive and rapid method for studying the induction and repair of DNA strand breaks in different cell types, as in the brain . A new approach combines the principle of studying DNA strand separation in alkaline solution with the use of fluorochromes binding to DNA . This permits microscope fluorimetry of individual cells as a means of estimating the percentage of double-stranded DNA in these cells (Rydberg and Johanson 1978) .
Acknowledgments We wish to thank Mrs . U . Johanson for skilful technical assistance . This work has been financially supported by grants from the Swedish Atomic Research Council and the Swedish Cancer Society .
Des ratons a l'age de 5, 10, ou 25 jours, ont ete irradi&s par 60 Co gamma . L'induction des ruptures de chaines d'ADN a &te determinee apres des intervalles diff&rents jusqu'a 180 min . Des suspensions de cellules on et& preparees du cerveau et des echantillons ont et& transferes dans des solutions alkalines . La fraction d'ADN rest& a 1'&tat de double-chaine apres le traitement par de 1'alkali pendant 30 minutes a ete estim&e apres la separation d'ADN en simple- et double-chaine au moyen de chromatographie a l'hydroxylapatite . Il s'est trouve que la quantite de ruptures des chaines d'ADN provoquees par Gray (1-8 Gray) a ete en conformite avec des etudes in vivo de l'intestin grele et de la rate de souris . Les ruptures des chaines d'ADN dans le cerveau des ratons, provoquees par irradiation avec 4 Gray 60Co gamma, ont et& reparees 30 min apres irradiation dans toutes les categories d'age &tudi&es . Ratten, 5, 10 oder 25 Tage alt, wurden mit 60 Co-Gamma bestrahit . Die Induktion von DNS-Strangbriichen wurde nach Toten der Ratten innerhalb 1 Min nach der Bestrahlung studiert, and ebenso die Reparatur der induzierten Bruche nach verschiedenen Intervallen bis 180 Min . Zellsuspensionen vom Gehirn wurden prapariert, and Proben wurden in alkalische Losungen iibergefiihrt . Der Anteil von doppelstrangiger DNS, nach alkalischer Behandlung, 30 Min, wurde nach Trennung von einfach- and doppel-strangiger DNS auf Hydroxylapatite abgeschatzt . Die Zahl der DNS-Strangbruche, per Gray (1-8 Gray) war ahnlich wie in friiheren Studien in vivo im Dunndarm and in der Milz in Mausen . DNS-Strangbriiche im Rattengehirn nach 4 Gray 60 Co-Gamma-strahlung, waren in 30 Min nach der Bestrahlung in alien Altersgruppen, repariert .
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REFERENCES AHNSTROM, G ., and ERIxoN, K ., 1973, Int . J. Radiat . Biol ., 23, 285 . ALTMAN, J ., ANDERSON, W . J ., and WRIGHT, K . A ., 1968, Expl . Neural., 21, 69 . ALTMAN, J ., and NICHOLSON, J . L ., 1971, Radiat . Res ., 46, 476 . CERDA, H ., 1978, Manuscript, GWI . FISH, I ., and WINICK, M ., 1969, Pediat . Res ., 3, 407 . GUTIN, P . H ., HILTON, J ., FEIN, V . J ., ALLEN, A . E ., and WALKER, M . D ., 1977, Radiat . Res .,
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72,100 . HAAS, R . J ., WERNER, J ., and FLIEDNER, T . M ., 1970, J. Anat ., 107, 421 . HICKS, S . P ., and D'AMATO, C . J ., 1963, Science, N. Y ., 141, 903 ; 1966, Advances in Teratology, edited by D . H . M . WOOLLAM (London : Loges Press), p . 195 . KARRON, P ., and ORMEROD, M . G ., 1973, Biochim . biophys . Acta, 299, 64 . KIHLMAN, B . A ., 1977, Caffeine and chromosomes (Oxford : Elsevier Sci . Publ . Co .), pp . 153-
246 . KINELL, P . 0 ., and LARSSON, B ., 1960, Riso Rep ., 16, 9 . LIEBERMAN, M . N ., and FORBES, P . D ., 1973, Nature, New Biol ., 241, 199 . OSTLING, 0 ., and JOHANSON, K. J ., 1978, Manuscript, GWI . PRICE, G . B ., MODAK, S . P ., and MAKINODAN, T ., 1971, Science, N .Y., 171, 917 . RYDBERG, B ., 1975, Radiat . Res ., 61, 274 . RYDBERG, B ., and JOHANSON, K . J ., 1975, Radiat . Res ., 64, 281 ; 1978, DNA Repair
edited by P . C . Hanawalt, E . C . Friedberg and C . F . Fox . (London : Academic Press), p . 465 . SPINKS, J . W . T ., and WOODS, R . J ., 1976, An Introduction to Radiation Chemistry (New York : Wiley-Interscience), p . 93 . WANG, T . S ., and WHEELER, K . T ., 1978, Radiat . Res ., 73, 464 . WHEELER, K . T ., and LETT, J . T ., 1972, Radiat . Res ., 52, 59 ; 1974, Proc . natn . Acad . Sci . Mechanism,
U .S . A ., 71, 1862 .
Radiation-induced DNA strand breaks and their repair in the developing rat brain
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HUMBERTO CERDA, KARL J . JOHANSON and KERSTIN ROSANDER The Gustaf Werner Institute, Department of Physical Biology, Box 531, S-751 21 Uppsala, Sweden (Received 6 July 1978 ; accepted 27 September 1978)
Rats, 5, 10 or 25 days old, were 60 Co gamma irradiated . The induction of DNA strand breaks was studied after killing the rats within 1 min after irradiation, and the repair of the induced breaks after various intervals up to 180 min . Cell suspensions were prepared from the brain and samples were transferred into alkaline solutions . The fraction of DNA remaining double-stranded after 30 min alkali treatment was estimated after separation of single- and double-stranded DNA on hydroxylapatite . The amount of DNA strand breaks induced per Gray (1-8 Gray) was found to be in accordance with earlier in vivo studies of the mouse small intestine and mouse spleen . The DNA strand breaks in the rat brain induced by 4 Gray 60 Co gamma irradiation were repaired 30 min after irradiation in all age groups studied . 1.
Introduction The brain develops through rapid cell proliferation and successive differentiation . Proliferation of neurons in the cerebrum of the rat occurs mainly in utero, while glial cells continue to divide after birth (Haas, Werner and Fliedner 1970) . The neurons of the cerebellum, on the other hand, develop mostly postnatally . Proliferative activity in the cerebellum is rather low at birth, increases very rapidly to a maximum at 10 days, and then decreases (Fish and Winick 1969, Altman and Nicholson 1971) . The adult rat brain consists mainly of neurons, which are nonproliferative, and glial cells, which have the ability to divide . The developing brain is known to be much more radio-sensitive than the adult brain . Various anomalies can be induced by prenatal irradiation (Hicks and D'Amato 1966) . At the cellular level there are alterations in the development of neurons in rats, after 0 . 1-0•4 Gray (Hicks and D'Amato 1963), and necrosis of proliferative and migrating cells of the cerebellar cortex of rats after 2 Gray (Altman, Anderson and Wright 1968) . It is well known that irradiation introduces DNA strand breaks in mammalian cells in vitro . These breaks are usually rejoined with rather high efficiency (cf . Kihlman 1977) . Some efforts have been made to determine DNA strand breaks in the canine brain after irradiation, with the sucrose density gradient technique (Wheeler and Lett 1972 and 1974, Wang and Wheeler 1978) . DNA strand breaks and their repair can now be studied with a simple and more sensitive technique using the process of DNA unwinding in alkaline solution, followed by separation of singlestranded and double-stranded DNA on hydroxylapatite (Ahnstrom and Erixon 1973, Rydberg 1975) . The result is expressed as the percentage of the labelled DNA found in the double-stranded fraction . This is interpreted as a measure of the mean R .B .
E
66
H . Cerda et al.
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length between two breaks, or alkali-labile sites, in DNA (Rydberg 1975) . The introduction of DNA strand breaks thus causes a decrease in the percentage of double-stranded DNA . This method has been applied to in vivo studies of radiation-induced DNA strand breaks in the proliferative crypt and the non-proliferative villous cells of the small intestine of the mouse (Rydberg and Johanson 1975), and in the spleen of the mouse (Ostling and Johanson 1978) . The aim of the present work was to study the radiation-induced DNA strand breaks and their repair in the cerebrum and cerebellum of young rats at different ages . 2. Material and method 2 .1 . Animals Rats of the Sprague-Dawley strain were used . They were given food and water ad libitum . The newborn rats were weighed within 24 hours after birth, and only those weighing more than 6 . 0 g were taken for the experiments . The size of the litters was always reduced to eight animals . No sex distinction was made . Growing rats were separated from their mother at 22 days of age . 2.2 . Injections 3 H-thymidine (1 . 85 x 1011 Bq/mmol (5 Ci/mmol), Radiochemical Centre, Amersham) was diluted to 1 . 85 x 10' Bq/ml (0.5 mCi/ml) in 0 . 9 per cent NaCl . All animals were injected twice, each time with 1 . 11 x 10 4 Bq/g (0. 3 pCi/g) body weight . The rats used for the experiments at five days of age were injected on days 3 and 4 . For other ages (10 and 25 days), the animals were injected on days 3 and 5 . 2 .3 . Irradiation Rats 5, 10 or 25 days old, were placed in a plastic cage and whole-body irradiated with a freely radiating 60 Co gamma source at the Gustaf Werner Institute (Kinell and Larsson 1960) . The dose rate was 2 Gray/min (200 rad/min), as determined by Fricke dosimetry (Spinks and Woods 1976) . Control rats were sham-irradiated under similar conditions . A whole litter was used for each age and experiment in order to reduce the variations . 2.4 . Preparation of cell suspension and alkali treatment The rats were decapitated within 1 min or after various intervals after irradiation . The brain was rapidly resected and divided mid-sagittally . The anterior parts of the cerebrum, except the olfactory lobes, were excised and transferred to an ice-chilled glass dish . The cerebellar halves were also transferred to an ice-chilled dish . Each half of the cerebrum and cerebellum was used as a separate sample, in order to evaluate variations due to the experimental procedure . In the 25-day-old rats the anterior part of the cerebellum was discarded, in order to obtain an adequate number of cells (see below) . The cell preparation was performed in the cold, using an ice-bath and pre-chilled solutions, instruments and glassware . The tissue was minced with a scalpel and suspended in 2 ml Earle-Eagle's medium by gentle drawing 2-3 times through a 19 gauge needle (cerebrum) or 3-5 times through a 18 gauge needle (cerebellum) . The cell suspension was filtered through a nylon net of 250 ym mesh size . A sample of 0 . 1 ml (about 10 6 cells) was transferred to 1 ml of the alkaline solution (0 . 03 M NaOH, 0 . 01 M Na,HPO 4i 0. 9 M
Radiation induced DNA strand breaks in the developing rat brain
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NaCI) and left in the dark at 20 ° C . After 30 min the solution was neutralized by addition of I ml 0 . 036 M HC1 and sonicated (Branson Sonifier B-12 with microtip), at level 2 for 15 s (Rydberg 1975) . Two millilitres of 2 per cent sodium dodecyl sulphate solution were added, and the sample was stored at - 20°C until chromatography was performed .
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2 .5 . Hydroxylapatite chromatography
The procedure described by Rydberg (1975) was essentially followed . Chromatography was performed at 60°C, single-stranded DNA being eluted with 3 ml 0-125M phosphate buffer, and double-stranded DNA with 1 . 5 ml 0-250M phosphate buffer and then diluted with water to 3 ml . Seven millilitres of scintillator solution (PCS, Nuclear Chicago Instrument Co) was added to each DNA fraction . The samples were heated to 60 ° C, and thoroughly shaken . The 3 H-activities were determined in a liquid scintillation counter (Nuclear Chicago, Mark I I) operating at 4°C .
3 . Results 3 .1 . Preparation of the cell suspension The major technical problem in developing the present method was to obtain cells of acceptable quality in suspension . The mechanical treatment had to be reduced to a minimum, otherwise DNA strand breaks were introduced, seen as a lower percentage of double -stranded DNA . Tissue disruption by repeated drawing through a needle gave a suspension of cell nuclei, single cells and cell aggregates . The larger aggregates were excluded by filtration through a 250 ,um nylon net . When analysing DNA strand breaks in control rats, values of 70-80 per cent double-stranded DNA were obtained as a rule . Variations within the range of 20 per
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Figure 1 . Induction of DNA strand breaks in cells from cerebrum and cerebellum of 5-dayold rats irradiated in vivo : the percentage double-stranded DNA immediately after irradiation as a function of absorbed dose . E2
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cent of this control value could be seen from one experiment to another, but for one litter (eight rats) used during 1 day, the variations were smaller, within a range of about 10 per cent . The use of needles of smaller diameter caused a significant decrease in the percentage of double-stranded DNA . In the ultimate method EarleEagle medium was used . When other solutions, such as 0 . 9 per cent NACI, KrebsRinger phosphate buffer pH 7 . 4, or tris-buffer (0 . 114 M tris, 0.077 NaCl, pH 7 . 4) were used, a lower percentage of double-stranded DNA was found . Addition of detergents, in low concentrations, to the solutions seemed to have no effect . 3 .2 . Induction of DNA strand breaks Rats of different ages were irradiated with various doses and killed within 1 min after irradiation . Each of figures 1, 2 and 3 represents a complete experiment with one litter (see Irradiation § 2 .3) . As both halves of the anterior part of the cerebrum and the cerebellum were analysed, one rat thus gave rise to two values at a given dose . The difference between these points gives the variation in the results caused by preparation of the cell suspension and the following treatment . Only minor differences in the slopes of the curves for different ages can be seen . There seem also to be only minor differences in dose response between cerebrum and cerebellum . Similar results were obtained in repeated experiments . 3 .3 . Rejoining of DNA strand breaks Rats from one litter were irradiated at 4 Gray and killed after various periods . As can be seen in figures 4, 5 and 6, the percentage of double-stranded DNA increased to a maximum, or a plateau, within 30 min of irradiation . One exception was the cerebrum in 5-day-old rats where a maximum was found after 60 min . This increase is here interpreted as a measure of the repair of DNA strand breaks and alkali-labile sites in DNA .
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Radiation induced DNA strand breaks in the developing rat brain
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Figure 4 . Rejoining of DNA strand breaks in cells from cerebellum and cerebrum of 5-dayold rats after irradiation in vivo : the percentage double-stranded DNA plotted against time after irradiation at 4 Gray .
In the youngest rats a decrease in the percentage of double-stranded DNA was observed between 30 and 180 min (figure 4) . It was also found, although less pronounced, in the 10-day-old rats (figure 5), but not in the rats aged 25 days, where a plateau was reached after 30 min (figure 6) . Similar results were obtained in repeated experiments .
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Figure 6 . Rejoining of DNA strand breaks in cells from cerebellum and cerebrum of 25-dayold rats after irradiation in vivo : the percentage double-stranded DNA plotted against time after irradiation at 4 Gray .
4.
Discussion
In this work cells of the developing rat brain were labelled by injection of 3 Hthymidine 3-5 days after birth . Consequently,, the radiation-induced DNA strand breaks were studied in the cell population proliferating at this time . The cell population labelled in the cerebrum were mostly glial cells, but in the cerebellum
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mainly neurons were labelled as seen after autoradiography (Haas et al . 1970, Altman and Nicholson 1971) . No major differences in the number of DNA strand breaks induced per Gray could be observed between the different age groups . In rats aged 10 days slightly more DNA strand breaks seem to be introduced than in the other age groups . The interpretation of these results, however, is very difficult, because of the heterogeneous pattern of proliferation and differentiation of the neuronal and glial cell populations . As estimated by the slope of the dose-effect curve (figures 1-3), the DNA strand breaks induced per Gray in rats aged 5 and 25 days were in accordance with earlier results found in the crypt and villous cells of the mouse small intestine . Only minor differences were observed between the proliferative and radiosensitive crypt cells and the non-proliferative and rather radio-resistant villous cells (Rydberg and Johanson 1975) . A similar number of DNA strand breaks per Gray seem to be introduced also in the splenic cells of mouse in vivo (Ostling and Johanson 1978) . The kinetics of rejoining of DNA strand breaks (and repair of alkali-labile sites in DNA) after irradiation at 4 Gray were similar in the cerebrum and cerebellum of rats at 10 and 25 days of age . The DNA strand breaks seem to be repaired, as control values were obtained within 30 min after irradiation . Similar rates of repair of DNA strand breaks were found in crypt and villous cells of the small intestine, and in the splenic cells of the mouse (Rydberg and Johanson 1975, Ostling and Johanson 1978) . Since the method is very sensitive small variations in the mechanical treatment could give rise to differences in the percentage of double-stranded DNA from day to day . As mentioned earlier the position of a curve can differ from experiment to experiment (cf. §3 .1) . However, the shapes of the curves were always similar in repeated experiments . Gutin, Hilton, Fein, Allen and Walker (1977), estimated the rate of strand separation of DNA in alkali combined with S 1 nuclease treatment instead of hydroxylapatite chromatography . They found that the damaged DNA seemed to be repaired within 30 min after irradiation of intracerebral rat gliosarcoma in vivo at 600 rad . The observed decrease in the percentage of double-stranded DNA 2 hours after irradiation in the 5-day-old rats (less pronounced in cerebellum at 10 days) may be due to the occurrence of cell death . Histological studies of radiation damage in the cerebellum of the rat show that the nuclei of the damaged cells begin to be pycnotic about 2 hours after irradiation (Altman and Nicholson 1971, Cerda 1978) . A slight decrease in the percentage of double-stranded DNA was also observed in studies of the repair of DNA strand breaks in splenic cells in vivo (Ostling and Johanson 1978) . Limited ability to repair DNA damage may result in accumulation of DNA breaks in non-proliferative, terminally differentiated cells . Price, Modak and Makinodan (1971), studied the DNA template activity with calf thymus DNA polymerase in fixed sections of various tissues from young and senescent mice . They found indications of an age-associated increase in DNA strand scission in neurons and astrocytes of the brain, and in Kupfer cells of the liver . DNA in microglia of the brain and hepatocytes seems to be either more resistant to damage to strand scission, or is more effectively repaired when damaged . Ageing thus shows a similar effect to irradiation . Karron and Ormerod (1973), studied the repair of DNA strand breaks, using sedimentation in gradients of alkaline sucrose and estimating DNA by fluorimetry . They found that DNA from non-dividing cells, such as rat muscle cells
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and chicken erythrocytes, lacked the ability to repair X-ray induced DNA singlestrand breaks . Cells from the thymus and spleen of rats could repair such breaks . Lieberman and Forbes (1973), found little or no unscheduled DNA-synthesis in cerebellar neurons or striated myocytes after U . V .-irradiation or treatment with chemical carcinogens, indicating a decreased ability to repair DNA damage . Wheeler and Lett using density gradient centrifugation to study the induction and repair of DNA strand breaks in neurons from canine cerebellum, found that the efficiency in producing DNA strand breaks was similar in neurons in vitro or in vivo and in normal cells in culture . The radiation-induced DNA strand breaks were repaired after irradiation, although the molecular events which accompany strand break repair seemed to be different from those observed in cultured dividing cells (Wheeler and Lett 1972) . The results show that radiation-induced DNA strand breaks in the rat brain can be repaired up to at least 25 days of age . Since mixed cell populations from the cerebrum or cerebellum labelled at days 3 to 5 after birth were used, we cannot exclude the possibility that some cell populations are unable to repair the DNA strand breaks . Here a complicated problem arises, i .e . how to devise a sensitive and rapid method for studying the induction and repair of DNA strand breaks in different cell types, as in the brain . A new approach combines the principle of studying DNA strand separation in alkaline solution with the use of fluorochromes binding to DNA . This permits microscope fluorimetry of individual cells as a means of estimating the percentage of double-stranded DNA in these cells (Rydberg and Johanson 1978) .
Acknowledgments We wish to thank Mrs . U . Johanson for skilful technical assistance . This work has been financially supported by grants from the Swedish Atomic Research Council and the Swedish Cancer Society .
Des ratons a l'age de 5, 10, ou 25 jours, ont ete irradi&s par 60 Co gamma . L'induction des ruptures de chaines d'ADN a &te determinee apres des intervalles diff&rents jusqu'a 180 min . Des suspensions de cellules on et& preparees du cerveau et des echantillons ont et& transferes dans des solutions alkalines . La fraction d'ADN rest& a 1'&tat de double-chaine apres le traitement par de 1'alkali pendant 30 minutes a ete estim&e apres la separation d'ADN en simple- et double-chaine au moyen de chromatographie a l'hydroxylapatite . Il s'est trouve que la quantite de ruptures des chaines d'ADN provoquees par Gray (1-8 Gray) a ete en conformite avec des etudes in vivo de l'intestin grele et de la rate de souris . Les ruptures des chaines d'ADN dans le cerveau des ratons, provoquees par irradiation avec 4 Gray 60Co gamma, ont et& reparees 30 min apres irradiation dans toutes les categories d'age &tudi&es . Ratten, 5, 10 oder 25 Tage alt, wurden mit 60 Co-Gamma bestrahit . Die Induktion von DNS-Strangbriichen wurde nach Toten der Ratten innerhalb 1 Min nach der Bestrahlung studiert, and ebenso die Reparatur der induzierten Bruche nach verschiedenen Intervallen bis 180 Min . Zellsuspensionen vom Gehirn wurden prapariert, and Proben wurden in alkalische Losungen iibergefiihrt . Der Anteil von doppelstrangiger DNS, nach alkalischer Behandlung, 30 Min, wurde nach Trennung von einfach- and doppel-strangiger DNS auf Hydroxylapatite abgeschatzt . Die Zahl der DNS-Strangbruche, per Gray (1-8 Gray) war ahnlich wie in friiheren Studien in vivo im Dunndarm and in der Milz in Mausen . DNS-Strangbriiche im Rattengehirn nach 4 Gray 60 Co-Gamma-strahlung, waren in 30 Min nach der Bestrahlung in alien Altersgruppen, repariert .
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