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In vivo somatic cell mutations at the hprt locus in atomic bomb survivors
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Bradley, W.E.C., and Karen Messing, Department of Biological Sciences, University of Quebec at Montreal, Montreal H3C 3P8 (Canada) Studies of mutation at the
hprt locus
The hprt T-cell cloning assay - - establishment of characteristics among control individuals, and its application to workplace situations We wish eventually to establish the T-cell cloning technique as a tool for screening for genotoxic agents in the workplace. To this end we have first analysed mutant frequencies among many (110) individuals not exposed to known agents with the exception of cigarette smoke. Mutant frequencies did not vary significantly between the groups of smokers and non-smokers, males and females and also did not vary with age. This latter observation is not in agreement with that of some other groups, but may be explained by the lack of inclusion of individuals under 20 years of age in our study. Several groups of individuals exposed to a known genotoxic agent, ionising radiation, have been analysed. We have previously reported that radiotherapy patients, with a 50-Gy acute dose, undergo an increase in mutant frequency, and we have now shown that over the following 3 years, the mutant frequency stays high. We have also reported increases in 3 groups exposed to low doses of 10-80 mSv (radiotherapy technicians, nuclear medicine technicians and nuclear medicine patients). In the first group we were initially unable to show a dose-response relationship, but we have recently found that such a relationship does exist if only the recent dose (previous 6 months) is considered. A programme has recently been initiated to determine mutant frequencies among workers and controls (unexposed) at a nuclear power plant in Qu6bec. Initial results show no difference between the groups unless smokers only are considered. It is therefore possible that a synergistic effect of smoking and occupational exposure to the type of radiation involved exists. Structural changes in the hprt gene have been examined by Southern blot analysis of DNA from a total of more than 100 mutants from 63 control individuals. In addition to previously published
results we have found 11 alterations, of which 10 are deletions and 1 a complex rearrangement.
Mutant frequencies at hemizygous vs. heterozygous loci - - a potential limitation of the hprt locus in measuring large deletion frequencies CHO cells which are hemizygous or heterozygous for the" aprt locus were mutagenised with soft X-rays. A difference of 30-100-fold was found in aprt- mutant frequencies, those from the heterozygotes being greater. Hprt- mutant frequencies were the same in both types of cell lines, being intermediate between the above values. These results are consistent with the hypothesis that essential sequences lie in the flanking regions of both genes, and that the mutations caused by the X-irradiation, probably being mostly large deletions, were not all viable in the hemizygous systems (at hprt, and at aprt in the aprt hemizygotes), because these sequences tended to be deleted. Hprt, being about 20 times bigger than aprt, would tolerate a larger proportion of these events since a bigger deletion would be required to involve the flanking regions. Southern blot analysis supported the hypothesis. One important consequence of these observations is that the possibility is now raised that the hemizygosity of the hprt locus may not allow us to detect all the large deletion events which affect this gene. It may therefore be appropriate to develop a mutational assay system employing an autosomal marker.
Hakoda, M. 1.3, y . Hirai l, H. Shimba 2 and M. Akiyama 1, Departments of 1 Radiobiology and 2 Genetics, Radiation Effects Research Foundation, 5-2, Hijiyama Park, Minami-Ku, Hiroshima 732, and 3 Institute of Rheumatology, Tokyo Women's Medical College, Box 116, NS BLD., 2-4-1, Hishi-Shinjuku, Shinjuku-ku, Tokyo, 163 (Japan) In vivo somatic cell mutations at the atomic bomb survivors
hprt locus in
The most prominent late effect of atomic bomb radiation is an increase in the incidence and mortality of malignant tumors. To investigate the
76
somatic cell gene damage caused by atomic b o m b radiation, 6-thioguanine-resistant (TG r) mutant T-lymphocytes have been cloned from the peripheral blood of atomic bomb survivors. The mean frequency of these mutant cells for 30 randomly selected survivors was 5 . 2 × 10 6 significantly higher than that for 17 controls (3.4 × 10 6). There was a slight but significant positive correlation between mutant T-cell frequency and radiation exposure dose. Mutant frequency also positively correlated with the frequency of lymphocytes carrying chromosome aberrations. These results suggest that the effects of atomic b o m b radiation on somatic cell genes persist for more than 40 years. Molecular analyses of mutant T-cells were carried out by Southern blotting, using a full-length hprt c D N A as a probe. Of 105 mutant colonies isolated from 3 non-irradiated controls, 14 (13%) had various gross changes of the hprt gene. On the other hand, a significantly higher frequency of mutant colonies with gross hprt changes (16/61, 26%) was observed in one survivor (radiation dose: 245 rad, mutant frequency: 9.3 × 10-6). However, another survivor (radiation dose: 215 rad, mutant frequency: 14.4 × 10 6) did not show a higher frequency of gross hprt changes (8/59, 14%). This might suggest a difference in the sensitivity to induction of certain types of mutations among individuals. Of 225 mutant colonies analyzed, 5 sets of mutants (each set consisted of 2 6 mutants) showed the same hprt changes. Of these 5 sets, 2 had different rearrangements of T-cell receptor genes, indicating that the mutation occurred in undifferentiated stem cells. The other 3 sets of mutants had the same rearrangements of T-cell receptor genes, indicating that the mutation occurred in mature peripheral T-cells. Thus, the effect of atomic b o m b radiation detected at the hprt locus in peripheral T-cells is not so striking. This is probably due to the disappearance of induced mutant T-cells during the 40 years after exposure to the bomb. This disappearance of mutant T-cells from the survivors seems to be attributable to the in vivo kinetics of T-cells rather than to the presumable selection against hprt cells. This is suggested by the following findings. Although not included in the population study, one male survivor was found to have an extremely
high mutant frequency (2 × 10 4) for at least 6 months. All the mutant T-cell colonies examined had the same chromosome aberration (20q - ) and the same alteration pattern of the hprt gene, indicating that all these cells were derived from a single progenitor cell. However, rearrangement patterns of T-cell receptor genes were different between the colonies. Furthermore, T G r B-cells, carrying the same chromosome aberration and the same hprt gene changes as were found in the TG" T-cells, were also cloned from the same person. Thus, a single mutational event, occurring in a stem cell before differentiation into T- and B-cells, produced an extremely high mutant frequency. These results suggest that some hprt mutations occurring in certain populations in the development of T-cell lineage can persist for a long time. The number of cells belonging to such populations may be too small for the mutations to be frequently induced. In order to detect mutations in the cells with different in vivo kinetics from that of T-cells, we have developed a method for cloning T G r B-cells using Epstein Barr virus transformation. B-cells were enriched from a large number of peripheral blood mononuclear cells prepared by a blood cell separator. The frequencies of the T G r B-cells for 4 healthy adults were around 1 × 10 5. Molecular analyses of these mutant B-cells are now conducted in our laboratory. Measurement of the frequency of T G ~ B-cells in the peripheral blood of atomic b o m b survivors is also under way. References Hakoda, M.. M. Akiyama, S. Kyoizumi, K. Kobuke, A.A. Awa and M. Yamakido (1988) Measurement of in vivo HGPRT-deficient mutant cell frequency using a modified method for cloning h u m a n peripheral blood T-lymphocytes, Mutation Res., 197, 161-169. Hakoda, M., M. Akiyama, S. Kyoizumi, A.A. Awa, M. Yamakido and M. Otake (1988a) Increased somatic cell mutant frequency in atomic bomb survivors, Mutation Res., in press. Hakoda, M., M. Akiyama, Y. Hirai, S. Kyoizumi and A.A. Awa (1988b) In vivo mutant T cell frequency in atomic bomb survivors carrying outlying values of chromosome aberration frequency, Mutation Res., in press. Hakoda, M., Y. Hirai, Y. Kusunoki and M. Akiyama (1988c) Cloning of in vivo-derived thioguanine-resistant h u m a n B cells, Mutation Res., in press.
Bradley, W.E.C., and Karen Messing, Department of Biological Sciences, University of Quebec at Montreal, Montreal H3C 3P8 (Canada) Studies of mutation at the
hprt locus
The hprt T-cell cloning assay - - establishment of characteristics among control individuals, and its application to workplace situations We wish eventually to establish the T-cell cloning technique as a tool for screening for genotoxic agents in the workplace. To this end we have first analysed mutant frequencies among many (110) individuals not exposed to known agents with the exception of cigarette smoke. Mutant frequencies did not vary significantly between the groups of smokers and non-smokers, males and females and also did not vary with age. This latter observation is not in agreement with that of some other groups, but may be explained by the lack of inclusion of individuals under 20 years of age in our study. Several groups of individuals exposed to a known genotoxic agent, ionising radiation, have been analysed. We have previously reported that radiotherapy patients, with a 50-Gy acute dose, undergo an increase in mutant frequency, and we have now shown that over the following 3 years, the mutant frequency stays high. We have also reported increases in 3 groups exposed to low doses of 10-80 mSv (radiotherapy technicians, nuclear medicine technicians and nuclear medicine patients). In the first group we were initially unable to show a dose-response relationship, but we have recently found that such a relationship does exist if only the recent dose (previous 6 months) is considered. A programme has recently been initiated to determine mutant frequencies among workers and controls (unexposed) at a nuclear power plant in Qu6bec. Initial results show no difference between the groups unless smokers only are considered. It is therefore possible that a synergistic effect of smoking and occupational exposure to the type of radiation involved exists. Structural changes in the hprt gene have been examined by Southern blot analysis of DNA from a total of more than 100 mutants from 63 control individuals. In addition to previously published
results we have found 11 alterations, of which 10 are deletions and 1 a complex rearrangement.
Mutant frequencies at hemizygous vs. heterozygous loci - - a potential limitation of the hprt locus in measuring large deletion frequencies CHO cells which are hemizygous or heterozygous for the" aprt locus were mutagenised with soft X-rays. A difference of 30-100-fold was found in aprt- mutant frequencies, those from the heterozygotes being greater. Hprt- mutant frequencies were the same in both types of cell lines, being intermediate between the above values. These results are consistent with the hypothesis that essential sequences lie in the flanking regions of both genes, and that the mutations caused by the X-irradiation, probably being mostly large deletions, were not all viable in the hemizygous systems (at hprt, and at aprt in the aprt hemizygotes), because these sequences tended to be deleted. Hprt, being about 20 times bigger than aprt, would tolerate a larger proportion of these events since a bigger deletion would be required to involve the flanking regions. Southern blot analysis supported the hypothesis. One important consequence of these observations is that the possibility is now raised that the hemizygosity of the hprt locus may not allow us to detect all the large deletion events which affect this gene. It may therefore be appropriate to develop a mutational assay system employing an autosomal marker.
Hakoda, M. 1.3, y . Hirai l, H. Shimba 2 and M. Akiyama 1, Departments of 1 Radiobiology and 2 Genetics, Radiation Effects Research Foundation, 5-2, Hijiyama Park, Minami-Ku, Hiroshima 732, and 3 Institute of Rheumatology, Tokyo Women's Medical College, Box 116, NS BLD., 2-4-1, Hishi-Shinjuku, Shinjuku-ku, Tokyo, 163 (Japan) In vivo somatic cell mutations at the atomic bomb survivors
hprt locus in
The most prominent late effect of atomic bomb radiation is an increase in the incidence and mortality of malignant tumors. To investigate the
76
somatic cell gene damage caused by atomic b o m b radiation, 6-thioguanine-resistant (TG r) mutant T-lymphocytes have been cloned from the peripheral blood of atomic bomb survivors. The mean frequency of these mutant cells for 30 randomly selected survivors was 5 . 2 × 10 6 significantly higher than that for 17 controls (3.4 × 10 6). There was a slight but significant positive correlation between mutant T-cell frequency and radiation exposure dose. Mutant frequency also positively correlated with the frequency of lymphocytes carrying chromosome aberrations. These results suggest that the effects of atomic b o m b radiation on somatic cell genes persist for more than 40 years. Molecular analyses of mutant T-cells were carried out by Southern blotting, using a full-length hprt c D N A as a probe. Of 105 mutant colonies isolated from 3 non-irradiated controls, 14 (13%) had various gross changes of the hprt gene. On the other hand, a significantly higher frequency of mutant colonies with gross hprt changes (16/61, 26%) was observed in one survivor (radiation dose: 245 rad, mutant frequency: 9.3 × 10-6). However, another survivor (radiation dose: 215 rad, mutant frequency: 14.4 × 10 6) did not show a higher frequency of gross hprt changes (8/59, 14%). This might suggest a difference in the sensitivity to induction of certain types of mutations among individuals. Of 225 mutant colonies analyzed, 5 sets of mutants (each set consisted of 2 6 mutants) showed the same hprt changes. Of these 5 sets, 2 had different rearrangements of T-cell receptor genes, indicating that the mutation occurred in undifferentiated stem cells. The other 3 sets of mutants had the same rearrangements of T-cell receptor genes, indicating that the mutation occurred in mature peripheral T-cells. Thus, the effect of atomic b o m b radiation detected at the hprt locus in peripheral T-cells is not so striking. This is probably due to the disappearance of induced mutant T-cells during the 40 years after exposure to the bomb. This disappearance of mutant T-cells from the survivors seems to be attributable to the in vivo kinetics of T-cells rather than to the presumable selection against hprt cells. This is suggested by the following findings. Although not included in the population study, one male survivor was found to have an extremely
high mutant frequency (2 × 10 4) for at least 6 months. All the mutant T-cell colonies examined had the same chromosome aberration (20q - ) and the same alteration pattern of the hprt gene, indicating that all these cells were derived from a single progenitor cell. However, rearrangement patterns of T-cell receptor genes were different between the colonies. Furthermore, T G r B-cells, carrying the same chromosome aberration and the same hprt gene changes as were found in the TG" T-cells, were also cloned from the same person. Thus, a single mutational event, occurring in a stem cell before differentiation into T- and B-cells, produced an extremely high mutant frequency. These results suggest that some hprt mutations occurring in certain populations in the development of T-cell lineage can persist for a long time. The number of cells belonging to such populations may be too small for the mutations to be frequently induced. In order to detect mutations in the cells with different in vivo kinetics from that of T-cells, we have developed a method for cloning T G r B-cells using Epstein Barr virus transformation. B-cells were enriched from a large number of peripheral blood mononuclear cells prepared by a blood cell separator. The frequencies of the T G r B-cells for 4 healthy adults were around 1 × 10 5. Molecular analyses of these mutant B-cells are now conducted in our laboratory. Measurement of the frequency of T G ~ B-cells in the peripheral blood of atomic b o m b survivors is also under way. References Hakoda, M.. M. Akiyama, S. Kyoizumi, K. Kobuke, A.A. Awa and M. Yamakido (1988) Measurement of in vivo HGPRT-deficient mutant cell frequency using a modified method for cloning h u m a n peripheral blood T-lymphocytes, Mutation Res., 197, 161-169. Hakoda, M., M. Akiyama, S. Kyoizumi, A.A. Awa, M. Yamakido and M. Otake (1988a) Increased somatic cell mutant frequency in atomic bomb survivors, Mutation Res., in press. Hakoda, M., M. Akiyama, Y. Hirai, S. Kyoizumi and A.A. Awa (1988b) In vivo mutant T cell frequency in atomic bomb survivors carrying outlying values of chromosome aberration frequency, Mutation Res., in press. Hakoda, M., Y. Hirai, Y. Kusunoki and M. Akiyama (1988c) Cloning of in vivo-derived thioguanine-resistant h u m a n B cells, Mutation Res., in press.