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Adaptive response to 2 low doses of X-rays in human blood lymphocytes
Mutation Research, 243 (1990) 53-56 Elsevier
53
MUTLET 0300
Adaptive response to 2 low doses of X-rays in human blood lymphocytes S. Fan, Vijayalaxmi, G. Mindek I and W. Burkart Radiation Biology Unit, Paul Scherrer Institute, CH-5232 Villigen PSI (Switzerland) and l Institute of Radiation Biology, University of Ziirich, CH-8029 Ziirich (Switzerland) (Accepted 14 September 1989)
Keywords: Adaptive response; Pre-exposure, single-dose, 2-dose
Summary Human peripheral blood lymphocytes exposed to a single adaptive dose of 1 cGy X-rays or 2 adaptive doses, each of 1 cGy, were found to be equally resistant to the induction of chromosome damage by subsequent challenge with a high dose of 1 Gy X-rays, as compared to cells that were not pre-exposed. They responded with a significantly reduced incidence of chromatid and isochromatid breaks. These results indicate the presence of an inducible chromosomal repair mechanism in human blood lymphocytes and confirm the observations made by earlier investigators. The incidence of chromosome damage was found to be similar in the lymphocytes pre-exposed to a single or 2 adaptive doses, suggesting that, under the conditions tested, the second adaptive dose did not offer any additional protection against the chromosome damage induced by the challenge dose.
There is growing evidence for the existence of an inducible 'adaptive response' in human blood lymphocytes similar to that found in Escherichia coli (Samson and Cairns, 1977) and rodent cells (Samson and Schwartz, 1980). Olivieri et al. (1984) first reported that pre-exposure of human lymphocytes to low levels of radioactive thymidine led to a significant reduction in the chromosome damage induced by subsequent challenge with a high dose of X-rays. This was followed by a series of investigations which suggested some of the condi-
Correspondence: S. Fan, Radiation Biology Unit, Paul Scherrer Institute, CH-5232 Villigen PSI (Switzerland).
tions required for eliciting such an inducible process. It was found that the exposure of lymphocytes to an X-ray dose as low as 0.5 cGy could significantly reduce the cytogenetic damage induced by subsequent challenge with a high dose of 1.5 Gy X-rays (Shadley and Wolff, 1987). Cells irradiated as early as 4 h after phytohemagglutinin (PHA) stimulation exhibited the adaptive response and once induced, it persisted for 3 cell cycles (Shadley et al., 1987). The radiation dose used for the adaptation seemed to have an effect, as did the time interval between the adaptive and the challenge doses, the dose rate and the quality of radiation (Shadley et al., 1987; Wiencke et al., 1987). Lymphocytes exposed to an adaptive dose
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
54 of 1 cGy X-rays were observed to express some novel proteins which could be considered potential candidates for DNA repair enzymes (Wolff and Wiencke, 1988). The adaptive response induced by low doses of radiation offered protection against chromosome damage induced by subsequent challenge with not only radiation but also with chemical mutagens which induce similar kinds of lesions in DNA and vice versa (Wolff et al., 1988; Vijayalaxmi and Burkart, 1989a). The magnitude of the adaptive response seemed to vary between the blood samples from different donors (Sankaranarayanan et al., 1989; Bosi and Olivieri, 1989) and also on the sampling time at which the cells were fixed after the challenge dose (Fan and Mindek, 1989). The adaptive response was also found to be negated when 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, was added to the lymphocyte cultures immediately following the challenge dose (Wiencke et al., 1986; Shadley et al., 1987; Vijayalaxmi and Burkart, 1989b), thus implicating a possible role of repair enzyme(s) in this inducible process. In all studies mentioned above, a single adaptive dose was given to the lymphocytes to study the adaptive response. It might be possible that the exposure of lymphocytes to 2 adaptive doses, instead of one, would induce higher levels of repair enzymes or protective substances resulting in an enhanced reduction in the chromosome damage induced by the challenge dose. The results of such a study are reported in this paper. Materials and methods
Heparinized blood was collected from healthy non-smoking adults, one male and one female, aged 25-30 years. Separate cultures were set up from each sample, using 0.5 ml blood in 5 ml of RPMI 1640 medium (with 25 mM Hepes buffer and L-glutamine, Gibco) containing 20070 fetal calf serum (Seromed, Seromed GmbH) and 20/0 PHAM (Seromed, Biochrom KG). The cultures were kept at 37°C in a 5070COz incubator. They were exposed to the adaptive dose(s) of 1 cGy X-rays at 4 and/or 24 h and a challenge dose of 1 Gy at 48 h
(Isovolt X-ray machine, 250 kV, 0.25-mm Cu filter, half-value layer of 1.05 mm Cu, dose rates of 0.2 Gy/min for adaptive dose and 1 Gy/min for challenge dose; during irradiation, cultures were kept in a water-filled plexiglass phantom preheated at 37°C) and the cultures returned to the inCubator. Colchicine (0.05 #g/ml) was added 52 h after culture initiation. Lymphocytes were collected 2 h later, treated with 0.075 M KCI for 8 min and fixed in methanol:acetic acid mixture (3:1 v/v). Fixed cells were dropped onto clean glass slides, air-dried and stained with Giemsa. Coded slides were used for microscopic analysis. From each culture, 200 well-spread metaphases were examined for chromosome damage. Gaps and achromatic lesions less than the width of a chromatid were not included in the scoring. Very few chromatid exchanges were observed, but not included in the analysis. Dicentric chromosomes were not found. The results on the chromatid and isochromatid breaks were analyzed using a 1-tailed Student's t-test. Results and discussion
There were no significant differences in the response of the lymphocytes from the 2 blood samples. Therefore the data were pooled. They are summarized in Table 1. The incidence of chromatid and isochromatid breaks in the lymphocytes exposed to 1 cGy X-rays 4 and 24 h after PHA stimulation was found to be 0.75070 and 1.0070 above the background level, respectively. When the cells were exposed to 2 adaptive doses of 1 cGy each, the effect was slightly less than additive (1.5 070above the control value). The challenge dose alone, given 48 h after culture initiation, had induced a significant increase in chromosomal damage (30070). However, cells pre-exposed to a single adaptive dose of 1 cGy (either at 4 or at 24 h) exhibited at 41-45070 reduction (P<0.005) in the incidence of chromatid and isochromatid breaks when challenged with I Gy X-rays, while the decrease in the cells treated with 2 adaptive doses was 41070. The former observation confirms the results of earlier investigations mentioned in the In-
55 TABLE 1 CHROMOSOME DAMAGE INDUCED BY A CHALLENGE DOSE OF 1 Gy X-RAYS IN HUMAN BLOOD LYMPHOCYTES PRE-EXPOSED TO 1 OR 2 ADAPTIVE DOSES OF 1 cGy X-RAYS Adaptive dose 1 (cGy)
1
1 1 1
Adaptive dose 2 (cGy)
Challenge dose (Gy)
Chromatid and isochromatid breaks in 200 cells Blood 1
Blood 2
Total breaks observed in 400 cells (%)
-
-
1
2
3 (0.75)
-
-
2
4
6
1
-
2
5
7
1 1 1
1 1 1 1
4 62 34 33 39
5 55 32 38 36
9 117 66 71 75
Expected
Percent decrease
(%)
(1.50) (1.75)
(2.25) (29.25) (16.50)* (17.75)* (18.75)*
120 (30.00) 121 (30.25) 127 (31.75)
45 41 41
Expected values are the sum of the 2-3 individual treatments minus the control. *Significantly different from expected values (P<0.005, l-tailed Student's t-test).
troduction, while the latter result indicates that the second adaptive dose, given to the lymphocytes during the same cell cycle, did not offer any additional protection against the chromosome damage induced by the challenge dose. It should be mentioned that in this study we used only 1 fixation time of 6 h after the challenge dose. The results of our previous investigation, where the adapted lymphocytes were fixed over a period of 2.5-8.5 h after the challenge dose, indicated that the reduction in aberration frequencies observed in G2 lymphocytes varied with fixation time. These results point to a differential sensitivity of cells within the G2 phase of the cell cycle, and probably reflect the state of condensation of the chromatin which was accessible to the repair enzymes to reach the damaged sites (Fan and Mindek, 1989). It is possible that once the repair enzymes were induced by the first adaptive dose, the second had little effect, i.e., that there exists a saturation for the induction of repair enzymes involved in the adaptive response. If the second dose were given at later cell cycle(s), it might have a beneficial effect in eliciting such a response. References
Bosi, A., and G. Olivieri (1989) Variability of the adaptive response to ionizing radiation in humans, Mutation ges., 211, 13-17.
Fan, S., and G. Mindek (1989) In vitro cytogenetic investigations of X-ray induced adaptive response of human lymphocytes and tumor cells at different times in the G2 phase, Mutation Res., in press. Olivieri, G., J. Bodycote and S. Wolff (1984) Adaptive response of human lymphocytes to low concentrations of radioactive thymidine, Science, 223, 594-597. Samson, L., and J. Cairns (1977) A new pathway for DNA repair in Escherichia coli, Nature (London), 267, 281-283. Samson, L., and J.L. Schwartz (1980) Evidence for an adaptive DNA repair pathway in CHO and human skin fibroblast cell lines, Nature (London), 287, 861-863. Sankaranarayanan, K., A. van Duyn, M.J. Loos and A.T. Natarajan (1989) Adaptive response of human lymphocytes to low concentrations of radio-isotopes, Mutation Res., 211, 7-12. Shadley, J.D., and S. Wolff (1987) Very low doses of X-rays can cause human lymphocytes to become less susceptible to ionizing radiation, Mutagenesis, 2, 95-96. Shadley, J.D., V. Afzai and S. Wolff (1987) Characterization of the adaptive response to ionizing radiation induced by low doses of X-rays to human lymphocytes, Radiat. Res., 111, 511-517. Vijayalaxmi, and W. Burkart (1989a) Resistance and crossresistance to chromosome damage in human blood lymphocytes adapted to bleomycin, Mutation Res., 211, 1-5. Vijayalaxmi, and W. Burkart (1989b) Effect of 3-aminobenzamide on chromosome damage in human blood lymphocytes adapted to bleomycin, Mutagenesis, 4, 187-189. Wiencke, J.A., V. Afzal, G. Olivieri and S. Wolff (1986) Evidence that the [3H]thymidine-induced adaptive response of human lymphocytes to subsequent doses of X-rays involves the induction of a chromosomal repair mechanism, Mutagenesis, 1,375-380. Wiencke, J.A., J.D. Shadley, K.T. Kelsey, A. Kronenberg and
56 J.B. Little (1987) Failure of high intensity X-ray treatments or densely ionizing fast neutrons to induce the adaptive response in human lymphocytes, in: E.M. Fielden, J.F. Fowler, J.H. Hendry and D. Scott (Eds.), Proceedings of the VIII International Congress on Radiation Research, 1, Taylor and Francis, London, p. 212. Wolff, S., and J.K. Wiencke (1988) The induction of' chromosomal repair enzymes by lcGy (1 rad) of X-rays to
human lymphocytes, Environ. Mol. Mutagen., 2 (Suppl. 11), 114 (Abstract). Wolff, S., V. Afzal, J.K. Wiencke, G. Olivieri and A. Michaeli (1988) Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA, Int. J. Radiat. Biol., 53, 39-48. Communicated by F.H. Sobels
53
MUTLET 0300
Adaptive response to 2 low doses of X-rays in human blood lymphocytes S. Fan, Vijayalaxmi, G. Mindek I and W. Burkart Radiation Biology Unit, Paul Scherrer Institute, CH-5232 Villigen PSI (Switzerland) and l Institute of Radiation Biology, University of Ziirich, CH-8029 Ziirich (Switzerland) (Accepted 14 September 1989)
Keywords: Adaptive response; Pre-exposure, single-dose, 2-dose
Summary Human peripheral blood lymphocytes exposed to a single adaptive dose of 1 cGy X-rays or 2 adaptive doses, each of 1 cGy, were found to be equally resistant to the induction of chromosome damage by subsequent challenge with a high dose of 1 Gy X-rays, as compared to cells that were not pre-exposed. They responded with a significantly reduced incidence of chromatid and isochromatid breaks. These results indicate the presence of an inducible chromosomal repair mechanism in human blood lymphocytes and confirm the observations made by earlier investigators. The incidence of chromosome damage was found to be similar in the lymphocytes pre-exposed to a single or 2 adaptive doses, suggesting that, under the conditions tested, the second adaptive dose did not offer any additional protection against the chromosome damage induced by the challenge dose.
There is growing evidence for the existence of an inducible 'adaptive response' in human blood lymphocytes similar to that found in Escherichia coli (Samson and Cairns, 1977) and rodent cells (Samson and Schwartz, 1980). Olivieri et al. (1984) first reported that pre-exposure of human lymphocytes to low levels of radioactive thymidine led to a significant reduction in the chromosome damage induced by subsequent challenge with a high dose of X-rays. This was followed by a series of investigations which suggested some of the condi-
Correspondence: S. Fan, Radiation Biology Unit, Paul Scherrer Institute, CH-5232 Villigen PSI (Switzerland).
tions required for eliciting such an inducible process. It was found that the exposure of lymphocytes to an X-ray dose as low as 0.5 cGy could significantly reduce the cytogenetic damage induced by subsequent challenge with a high dose of 1.5 Gy X-rays (Shadley and Wolff, 1987). Cells irradiated as early as 4 h after phytohemagglutinin (PHA) stimulation exhibited the adaptive response and once induced, it persisted for 3 cell cycles (Shadley et al., 1987). The radiation dose used for the adaptation seemed to have an effect, as did the time interval between the adaptive and the challenge doses, the dose rate and the quality of radiation (Shadley et al., 1987; Wiencke et al., 1987). Lymphocytes exposed to an adaptive dose
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
54 of 1 cGy X-rays were observed to express some novel proteins which could be considered potential candidates for DNA repair enzymes (Wolff and Wiencke, 1988). The adaptive response induced by low doses of radiation offered protection against chromosome damage induced by subsequent challenge with not only radiation but also with chemical mutagens which induce similar kinds of lesions in DNA and vice versa (Wolff et al., 1988; Vijayalaxmi and Burkart, 1989a). The magnitude of the adaptive response seemed to vary between the blood samples from different donors (Sankaranarayanan et al., 1989; Bosi and Olivieri, 1989) and also on the sampling time at which the cells were fixed after the challenge dose (Fan and Mindek, 1989). The adaptive response was also found to be negated when 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, was added to the lymphocyte cultures immediately following the challenge dose (Wiencke et al., 1986; Shadley et al., 1987; Vijayalaxmi and Burkart, 1989b), thus implicating a possible role of repair enzyme(s) in this inducible process. In all studies mentioned above, a single adaptive dose was given to the lymphocytes to study the adaptive response. It might be possible that the exposure of lymphocytes to 2 adaptive doses, instead of one, would induce higher levels of repair enzymes or protective substances resulting in an enhanced reduction in the chromosome damage induced by the challenge dose. The results of such a study are reported in this paper. Materials and methods
Heparinized blood was collected from healthy non-smoking adults, one male and one female, aged 25-30 years. Separate cultures were set up from each sample, using 0.5 ml blood in 5 ml of RPMI 1640 medium (with 25 mM Hepes buffer and L-glutamine, Gibco) containing 20070 fetal calf serum (Seromed, Seromed GmbH) and 20/0 PHAM (Seromed, Biochrom KG). The cultures were kept at 37°C in a 5070COz incubator. They were exposed to the adaptive dose(s) of 1 cGy X-rays at 4 and/or 24 h and a challenge dose of 1 Gy at 48 h
(Isovolt X-ray machine, 250 kV, 0.25-mm Cu filter, half-value layer of 1.05 mm Cu, dose rates of 0.2 Gy/min for adaptive dose and 1 Gy/min for challenge dose; during irradiation, cultures were kept in a water-filled plexiglass phantom preheated at 37°C) and the cultures returned to the inCubator. Colchicine (0.05 #g/ml) was added 52 h after culture initiation. Lymphocytes were collected 2 h later, treated with 0.075 M KCI for 8 min and fixed in methanol:acetic acid mixture (3:1 v/v). Fixed cells were dropped onto clean glass slides, air-dried and stained with Giemsa. Coded slides were used for microscopic analysis. From each culture, 200 well-spread metaphases were examined for chromosome damage. Gaps and achromatic lesions less than the width of a chromatid were not included in the scoring. Very few chromatid exchanges were observed, but not included in the analysis. Dicentric chromosomes were not found. The results on the chromatid and isochromatid breaks were analyzed using a 1-tailed Student's t-test. Results and discussion
There were no significant differences in the response of the lymphocytes from the 2 blood samples. Therefore the data were pooled. They are summarized in Table 1. The incidence of chromatid and isochromatid breaks in the lymphocytes exposed to 1 cGy X-rays 4 and 24 h after PHA stimulation was found to be 0.75070 and 1.0070 above the background level, respectively. When the cells were exposed to 2 adaptive doses of 1 cGy each, the effect was slightly less than additive (1.5 070above the control value). The challenge dose alone, given 48 h after culture initiation, had induced a significant increase in chromosomal damage (30070). However, cells pre-exposed to a single adaptive dose of 1 cGy (either at 4 or at 24 h) exhibited at 41-45070 reduction (P<0.005) in the incidence of chromatid and isochromatid breaks when challenged with I Gy X-rays, while the decrease in the cells treated with 2 adaptive doses was 41070. The former observation confirms the results of earlier investigations mentioned in the In-
55 TABLE 1 CHROMOSOME DAMAGE INDUCED BY A CHALLENGE DOSE OF 1 Gy X-RAYS IN HUMAN BLOOD LYMPHOCYTES PRE-EXPOSED TO 1 OR 2 ADAPTIVE DOSES OF 1 cGy X-RAYS Adaptive dose 1 (cGy)
1
1 1 1
Adaptive dose 2 (cGy)
Challenge dose (Gy)
Chromatid and isochromatid breaks in 200 cells Blood 1
Blood 2
Total breaks observed in 400 cells (%)
-
-
1
2
3 (0.75)
-
-
2
4
6
1
-
2
5
7
1 1 1
1 1 1 1
4 62 34 33 39
5 55 32 38 36
9 117 66 71 75
Expected
Percent decrease
(%)
(1.50) (1.75)
(2.25) (29.25) (16.50)* (17.75)* (18.75)*
120 (30.00) 121 (30.25) 127 (31.75)
45 41 41
Expected values are the sum of the 2-3 individual treatments minus the control. *Significantly different from expected values (P<0.005, l-tailed Student's t-test).
troduction, while the latter result indicates that the second adaptive dose, given to the lymphocytes during the same cell cycle, did not offer any additional protection against the chromosome damage induced by the challenge dose. It should be mentioned that in this study we used only 1 fixation time of 6 h after the challenge dose. The results of our previous investigation, where the adapted lymphocytes were fixed over a period of 2.5-8.5 h after the challenge dose, indicated that the reduction in aberration frequencies observed in G2 lymphocytes varied with fixation time. These results point to a differential sensitivity of cells within the G2 phase of the cell cycle, and probably reflect the state of condensation of the chromatin which was accessible to the repair enzymes to reach the damaged sites (Fan and Mindek, 1989). It is possible that once the repair enzymes were induced by the first adaptive dose, the second had little effect, i.e., that there exists a saturation for the induction of repair enzymes involved in the adaptive response. If the second dose were given at later cell cycle(s), it might have a beneficial effect in eliciting such a response. References
Bosi, A., and G. Olivieri (1989) Variability of the adaptive response to ionizing radiation in humans, Mutation ges., 211, 13-17.
Fan, S., and G. Mindek (1989) In vitro cytogenetic investigations of X-ray induced adaptive response of human lymphocytes and tumor cells at different times in the G2 phase, Mutation Res., in press. Olivieri, G., J. Bodycote and S. Wolff (1984) Adaptive response of human lymphocytes to low concentrations of radioactive thymidine, Science, 223, 594-597. Samson, L., and J. Cairns (1977) A new pathway for DNA repair in Escherichia coli, Nature (London), 267, 281-283. Samson, L., and J.L. Schwartz (1980) Evidence for an adaptive DNA repair pathway in CHO and human skin fibroblast cell lines, Nature (London), 287, 861-863. Sankaranarayanan, K., A. van Duyn, M.J. Loos and A.T. Natarajan (1989) Adaptive response of human lymphocytes to low concentrations of radio-isotopes, Mutation Res., 211, 7-12. Shadley, J.D., and S. Wolff (1987) Very low doses of X-rays can cause human lymphocytes to become less susceptible to ionizing radiation, Mutagenesis, 2, 95-96. Shadley, J.D., V. Afzai and S. Wolff (1987) Characterization of the adaptive response to ionizing radiation induced by low doses of X-rays to human lymphocytes, Radiat. Res., 111, 511-517. Vijayalaxmi, and W. Burkart (1989a) Resistance and crossresistance to chromosome damage in human blood lymphocytes adapted to bleomycin, Mutation Res., 211, 1-5. Vijayalaxmi, and W. Burkart (1989b) Effect of 3-aminobenzamide on chromosome damage in human blood lymphocytes adapted to bleomycin, Mutagenesis, 4, 187-189. Wiencke, J.A., V. Afzal, G. Olivieri and S. Wolff (1986) Evidence that the [3H]thymidine-induced adaptive response of human lymphocytes to subsequent doses of X-rays involves the induction of a chromosomal repair mechanism, Mutagenesis, 1,375-380. Wiencke, J.A., J.D. Shadley, K.T. Kelsey, A. Kronenberg and
56 J.B. Little (1987) Failure of high intensity X-ray treatments or densely ionizing fast neutrons to induce the adaptive response in human lymphocytes, in: E.M. Fielden, J.F. Fowler, J.H. Hendry and D. Scott (Eds.), Proceedings of the VIII International Congress on Radiation Research, 1, Taylor and Francis, London, p. 212. Wolff, S., and J.K. Wiencke (1988) The induction of' chromosomal repair enzymes by lcGy (1 rad) of X-rays to
human lymphocytes, Environ. Mol. Mutagen., 2 (Suppl. 11), 114 (Abstract). Wolff, S., V. Afzal, J.K. Wiencke, G. Olivieri and A. Michaeli (1988) Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA, Int. J. Radiat. Biol., 53, 39-48. Communicated by F.H. Sobels