1975, British Journal of Radiology, 48, 312-314
Diurnal variations in radiosensitivity of mouse intestine B y J . H . Hendry, Ph.D. Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX, England. {Received March, 1974 and in revised form July, 1974) ABSTRACT
Crypts in the mouse small intestine show a diurnal rhythm in radiosensitivity and are most sensitive in the middle of the dark period, when mitotic activity in the crypt is high. Analysis of crypt survival on a cellular basis shows that the changes in cryptogenic cell radiosensitivity are dose-modifying between 1,000 and 1,450 rads y rays such that the Do value varies between 85 rads and 140 rads.
Circadian variations are a feature of most biological systems. In the mouse, such rhythms have been reported, among others, in the rate of cell turnover in the intestine and the bone marrow (Sigdestad and Lesher, 1970; Pizzarello and Witcofski, 1970), and the survival of mice to whole-body irradiation (Pizzarello et ah, 1964; Concannon et ah, 1973). Data are presented here which show the magnitude of the variations in radiosensitivity of the progenitor cells of the intestine, using the microcolony assay technique (Withers and Elkind, 1970).
The data at the maximum and minimum in whole crypt survival were transformed to values of crypt cell survival (Fig. 2) as described by Hendry and Potten (1974). The left ordinate is a logarithmic scale, and each unit represents a cell depopulation of 63 per cent i.e. that caused by a dose Do rads. The transformed data for three radiation doses at each of the times used, supported further by data at neighbouring times (Fig. 1), are linear with dose and thus fit the model described by Gilbert (1974). The maximum and minimum D o values are 140 rads and 85 rads respectively. Also, there is a large variation in the point of extrapolation (—No. of cells X CRYPT SURVIVING FRACTION i
1
1
1
i
1
1
i
i
MATERIALS AND METHODS
Three-month-old (C57BL/6?X DBA2^) F t hybrid mice were used throughout, and maintained on a 12 h light (07-00 h to 19.00 h) 12 h dark cycle from birth. At each dose level and each time of day, four mice were whole-body irradiated in a Cs-137 yirradiator at 540 rads min"1. The intestinal microcolony assay technique was performed as described previously (Hendry and Potten, 1974). The crypt survival data were corrected for the increase in the size of the regenerating crypts, which gives rise to an increased scoring rate (Hendry and Potten, 1974) and analysed on a cellular basis using the method described by Gilbert (1974). An estimate of the cell turnover in the crypt was made by scoring mitotic figures per crypt section (X 1,000 magnification).
0.1-
0.01
RESULTS
In these experiments, the average transverse diameter of control crypts was measured to be smaller (2-83 arbitary units) than that of regenerating crypts (3-91 units) by a factor 0-725. The crypt survival data at all doses and times were reduced by this factor. The variations in crypt radiosensitivity with time of day are shown in Fig. 1 for four radiation doses.
0.001 11.00
19.00
3.00 11.00 TIME of DAY
19.00
FIG. 1. Survival of whole crypts at four radiation dose levels, with time of day. Solid symbols—repeat data from first part of curve. Shaded area—dark period. Representative standard errors shown in Fig. 2.
312
APRIL 1975
Diurnal variations in radiosensitivity of mouse intestine PER CENT CRYPT SURVIVAL (S)
-In(-lnd-S))
99 95 80 -63
0-
40 20 10 5
extrapolation number) of the multicell survival curve to zero dose, from about 970 (13.00 h) to about 69,600 (01.00 h) in these experiments. Fig. 3 shows the diurnal variation in crypt survival after 1,300 rads (top curve), taken from Fig. 1, together with the results from a repeat experiment (circles) in which the average number of mitotic figures was measured in sections of normal crypts from mice killed at different times of day (lower curve, Fig. 3). A repeat measurement of this parameter is also shown (squared symbols). DISCUSSION
Ol.OOhrs 0.5 0.2 [000
flOO 12^0 DOSE RADS
1300"
FIG. 2. Whole crypt survival (right ordinate) expressed as cryptogenic cell survival (left ordinate) at maxima and minima of crypt survivals shown in Fig. 1. Standard errors shown. Each left ordinate unit represents a cell depopulation of 63 per cent, as caused by a dose Do rads.
PER CENT CRYPt SURVIVAL I MITOTIC FIGURES/CRYPT SECTION
•
• 8
0.5 11.00
19.00
03.00 11.00 19.00 TIME of DAY FIG. 3. Upper curve—whole crypt survival after 1,300 rads from Fig. 1 (triangles) and a repeat experiment (circles). Lower curve—mean mitotic figures per crypt section in two experiments (circles, squares). Solid symbols and shaded area as for Fig. 1.
The variations in crypt survival are in agreement with the difference (1313R to 1426R) in the LD 5o / 6 values (Concannon et al., 1973) of mice irradiated at the peak (seven mitotic figures and 120 labelled nuclei per crypt at 02.00 h) and nadir (two mitotic figures and 50 labelled nuclei at 14.00 h) of crypt cell mitotic activity and DNA-synthetic activity (Sigdestad and Lesher, 1970). The above times of the peak and nadir are similar to those shown in Fig. 3. The radiosensitivity differences could be explained by the oxygen status of the cells or by the age responses through the cell cycle of a diurnallysynchronized cell population. The former is unlikely, as it has been shown that when mice breathed air during irradiations, performed presumably during the day, the crypt cell Do value was unaffected (Hornsey, 1970) or increased by a factor of only 1-06 (Withers and Elkind, 1970) over the value for mice breathing oxygen. Crypt cells show marked variations in radiosensitivity through the cell cycle (Gillette, Withers, and Tannock, 1970; Hagemann and Lesher, 1971) and so do cells in vitro, e.g., Terasima and Tolmach (1963) reported a 2-5-fold Do difference for Chinese hamster cells. With values of about 44 for the number of cryptogenic cells per crypt and about 130 for their extrapolation number (Hendry and Potten, 1974), measured in the middle of the light period, variations in the product of these two parameters by a factor of 70 during the day are possible. It is curious that the crypts are most radiosensitive at a time when there are more cells in mitosis (Fig. 3). This is because the peaks in mitotic index and labelling index occur at the same time (Sigdestad and Lesher, 1970) and in an artificially-synchronized crypt, S is the most resistant phase in the cycle (Hagemann and Lesher, 1971; second peak in labelled cells only—Gillette et al., 1970). The anomaly is expected if cryptogenic cells and proliferative cells are indeed different populations (Hendry and Potten, 1974).
313
VOL.
48, No. 568 J. H. Hendry
Mice are nocturnally active and the present GILLETTE, E. L., WITHERS, H. R. and TANNOCK, I. F., 1970. The age sensitivity of epithelial cells of mouse small experiments imply that for animals active during the intestine. Radiology, 96, 639-643. day {e.g., humans), less gut damage would be in- HAGEMANN, R. F., and LESHER, S., 1971. Intestinal crypt survival and total and per crypt levels of proliferative curred when irradiations are performed in the middle cellularity following irradiation: age response and animal of the dark period (with single doses from 1,000 to lethality. Radiation Research, 47, 159-167. 1,450 rads). This is under further investigation HENDRY, J. H., and POTTEN, C. S., 1974. Cryptogenic cells and proliferative cells in intestinal epithelium. Internausing lower dose fractions in view of its therapeutic tional Journal of Radiation Biology, 25, 583-588. interest. HORNSEY, S., 1970. The effect of hypoxia on the sensitivity
of the epithelial cells of the jejunum. International Journal of Radiation Biology, 18, 539-546.
ACKNOWLEDGMENTS
I thank Dr. C. S. Potten for helpful discussions, Miss Janice Grainger for technical assistance, and the Pathology Department for preparing the tissue sections. This work was supported by grants from the Cancer Research Campaign and the Medical Research Council.
PIZZARELLO, D. J., ISAAK, D., CHUA, K. E., and RHYNE, A.
L., 1964. Circadian rhythmicity in the sensitivity of two strains of mice to whole-body irradiation. Science, 145, 286-291. PIZZARELLO, D. J., and WITCOFSKI, R. L., 1970. A possible
link between diurnal variations in radiation sensitivity and cell division in bone marrow of male mice. Radiology, 97, 165-167. SIGDESTAD, C. P., and LESHER, S., 1970. Further studies on
REFERENCES
the circadian rhythm in the proliferative activity of mouse intestinal epithelium. Experientia, 26, 1321-1322.
CONCANNON, J. P., DALBOW, M. H., WEIL, C , and HODG-
SON, S. E., 1973. Radiation and actinomycin D studies: TERASIMA, T., and TOLMACH, L. J., 1963. Variations in Circadian variations in lethality due to independent several responses of HeLa cells to X-irradiation during effects of either agent. International Journal of Radiation the division cycle. Biophysics Journal, 3, 11—33. Biology, 24,405-411. WITHERS, H. R., and ELKIND, M. M., 1970. Microcolony GILBERT, C. W., 1974. A double minus log transformation survival assay for cells of mouse intestinal mucosa exof mortality probabilities. International Journal of Radiaposed to radiation. International Journal of Radiation tion Biology, 25, 633-634. Biology, 17,261-267.
314
Diurnal variations in radiosensitivity of mouse intestine B y J . H . Hendry, Ph.D. Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX, England. {Received March, 1974 and in revised form July, 1974) ABSTRACT
Crypts in the mouse small intestine show a diurnal rhythm in radiosensitivity and are most sensitive in the middle of the dark period, when mitotic activity in the crypt is high. Analysis of crypt survival on a cellular basis shows that the changes in cryptogenic cell radiosensitivity are dose-modifying between 1,000 and 1,450 rads y rays such that the Do value varies between 85 rads and 140 rads.
Circadian variations are a feature of most biological systems. In the mouse, such rhythms have been reported, among others, in the rate of cell turnover in the intestine and the bone marrow (Sigdestad and Lesher, 1970; Pizzarello and Witcofski, 1970), and the survival of mice to whole-body irradiation (Pizzarello et ah, 1964; Concannon et ah, 1973). Data are presented here which show the magnitude of the variations in radiosensitivity of the progenitor cells of the intestine, using the microcolony assay technique (Withers and Elkind, 1970).
The data at the maximum and minimum in whole crypt survival were transformed to values of crypt cell survival (Fig. 2) as described by Hendry and Potten (1974). The left ordinate is a logarithmic scale, and each unit represents a cell depopulation of 63 per cent i.e. that caused by a dose Do rads. The transformed data for three radiation doses at each of the times used, supported further by data at neighbouring times (Fig. 1), are linear with dose and thus fit the model described by Gilbert (1974). The maximum and minimum D o values are 140 rads and 85 rads respectively. Also, there is a large variation in the point of extrapolation (—No. of cells X CRYPT SURVIVING FRACTION i
1
1
1
i
1
1
i
i
MATERIALS AND METHODS
Three-month-old (C57BL/6?X DBA2^) F t hybrid mice were used throughout, and maintained on a 12 h light (07-00 h to 19.00 h) 12 h dark cycle from birth. At each dose level and each time of day, four mice were whole-body irradiated in a Cs-137 yirradiator at 540 rads min"1. The intestinal microcolony assay technique was performed as described previously (Hendry and Potten, 1974). The crypt survival data were corrected for the increase in the size of the regenerating crypts, which gives rise to an increased scoring rate (Hendry and Potten, 1974) and analysed on a cellular basis using the method described by Gilbert (1974). An estimate of the cell turnover in the crypt was made by scoring mitotic figures per crypt section (X 1,000 magnification).
0.1-
0.01
RESULTS
In these experiments, the average transverse diameter of control crypts was measured to be smaller (2-83 arbitary units) than that of regenerating crypts (3-91 units) by a factor 0-725. The crypt survival data at all doses and times were reduced by this factor. The variations in crypt radiosensitivity with time of day are shown in Fig. 1 for four radiation doses.
0.001 11.00
19.00
3.00 11.00 TIME of DAY
19.00
FIG. 1. Survival of whole crypts at four radiation dose levels, with time of day. Solid symbols—repeat data from first part of curve. Shaded area—dark period. Representative standard errors shown in Fig. 2.
312
APRIL 1975
Diurnal variations in radiosensitivity of mouse intestine PER CENT CRYPT SURVIVAL (S)
-In(-lnd-S))
99 95 80 -63
0-
40 20 10 5
extrapolation number) of the multicell survival curve to zero dose, from about 970 (13.00 h) to about 69,600 (01.00 h) in these experiments. Fig. 3 shows the diurnal variation in crypt survival after 1,300 rads (top curve), taken from Fig. 1, together with the results from a repeat experiment (circles) in which the average number of mitotic figures was measured in sections of normal crypts from mice killed at different times of day (lower curve, Fig. 3). A repeat measurement of this parameter is also shown (squared symbols). DISCUSSION
Ol.OOhrs 0.5 0.2 [000
flOO 12^0 DOSE RADS
1300"
FIG. 2. Whole crypt survival (right ordinate) expressed as cryptogenic cell survival (left ordinate) at maxima and minima of crypt survivals shown in Fig. 1. Standard errors shown. Each left ordinate unit represents a cell depopulation of 63 per cent, as caused by a dose Do rads.
PER CENT CRYPt SURVIVAL I MITOTIC FIGURES/CRYPT SECTION
•
• 8
0.5 11.00
19.00
03.00 11.00 19.00 TIME of DAY FIG. 3. Upper curve—whole crypt survival after 1,300 rads from Fig. 1 (triangles) and a repeat experiment (circles). Lower curve—mean mitotic figures per crypt section in two experiments (circles, squares). Solid symbols and shaded area as for Fig. 1.
The variations in crypt survival are in agreement with the difference (1313R to 1426R) in the LD 5o / 6 values (Concannon et al., 1973) of mice irradiated at the peak (seven mitotic figures and 120 labelled nuclei per crypt at 02.00 h) and nadir (two mitotic figures and 50 labelled nuclei at 14.00 h) of crypt cell mitotic activity and DNA-synthetic activity (Sigdestad and Lesher, 1970). The above times of the peak and nadir are similar to those shown in Fig. 3. The radiosensitivity differences could be explained by the oxygen status of the cells or by the age responses through the cell cycle of a diurnallysynchronized cell population. The former is unlikely, as it has been shown that when mice breathed air during irradiations, performed presumably during the day, the crypt cell Do value was unaffected (Hornsey, 1970) or increased by a factor of only 1-06 (Withers and Elkind, 1970) over the value for mice breathing oxygen. Crypt cells show marked variations in radiosensitivity through the cell cycle (Gillette, Withers, and Tannock, 1970; Hagemann and Lesher, 1971) and so do cells in vitro, e.g., Terasima and Tolmach (1963) reported a 2-5-fold Do difference for Chinese hamster cells. With values of about 44 for the number of cryptogenic cells per crypt and about 130 for their extrapolation number (Hendry and Potten, 1974), measured in the middle of the light period, variations in the product of these two parameters by a factor of 70 during the day are possible. It is curious that the crypts are most radiosensitive at a time when there are more cells in mitosis (Fig. 3). This is because the peaks in mitotic index and labelling index occur at the same time (Sigdestad and Lesher, 1970) and in an artificially-synchronized crypt, S is the most resistant phase in the cycle (Hagemann and Lesher, 1971; second peak in labelled cells only—Gillette et al., 1970). The anomaly is expected if cryptogenic cells and proliferative cells are indeed different populations (Hendry and Potten, 1974).
313
VOL.
48, No. 568 J. H. Hendry
Mice are nocturnally active and the present GILLETTE, E. L., WITHERS, H. R. and TANNOCK, I. F., 1970. The age sensitivity of epithelial cells of mouse small experiments imply that for animals active during the intestine. Radiology, 96, 639-643. day {e.g., humans), less gut damage would be in- HAGEMANN, R. F., and LESHER, S., 1971. Intestinal crypt survival and total and per crypt levels of proliferative curred when irradiations are performed in the middle cellularity following irradiation: age response and animal of the dark period (with single doses from 1,000 to lethality. Radiation Research, 47, 159-167. 1,450 rads). This is under further investigation HENDRY, J. H., and POTTEN, C. S., 1974. Cryptogenic cells and proliferative cells in intestinal epithelium. Internausing lower dose fractions in view of its therapeutic tional Journal of Radiation Biology, 25, 583-588. interest. HORNSEY, S., 1970. The effect of hypoxia on the sensitivity
of the epithelial cells of the jejunum. International Journal of Radiation Biology, 18, 539-546.
ACKNOWLEDGMENTS
I thank Dr. C. S. Potten for helpful discussions, Miss Janice Grainger for technical assistance, and the Pathology Department for preparing the tissue sections. This work was supported by grants from the Cancer Research Campaign and the Medical Research Council.
PIZZARELLO, D. J., ISAAK, D., CHUA, K. E., and RHYNE, A.
L., 1964. Circadian rhythmicity in the sensitivity of two strains of mice to whole-body irradiation. Science, 145, 286-291. PIZZARELLO, D. J., and WITCOFSKI, R. L., 1970. A possible
link between diurnal variations in radiation sensitivity and cell division in bone marrow of male mice. Radiology, 97, 165-167. SIGDESTAD, C. P., and LESHER, S., 1970. Further studies on
REFERENCES
the circadian rhythm in the proliferative activity of mouse intestinal epithelium. Experientia, 26, 1321-1322.
CONCANNON, J. P., DALBOW, M. H., WEIL, C , and HODG-
SON, S. E., 1973. Radiation and actinomycin D studies: TERASIMA, T., and TOLMACH, L. J., 1963. Variations in Circadian variations in lethality due to independent several responses of HeLa cells to X-irradiation during effects of either agent. International Journal of Radiation the division cycle. Biophysics Journal, 3, 11—33. Biology, 24,405-411. WITHERS, H. R., and ELKIND, M. M., 1970. Microcolony GILBERT, C. W., 1974. A double minus log transformation survival assay for cells of mouse intestinal mucosa exof mortality probabilities. International Journal of Radiaposed to radiation. International Journal of Radiation tion Biology, 25, 633-634. Biology, 17,261-267.
314
