1977, British Journal of Radiology, 50, 658-662
Some long-term effects of negative pions in mice exposed to partial body irradiation By J . E. Coggle, Ph.D. Department of Radiobiology, Medical College of St. Bartholomew's Hospital, Charterhouse Square, London EC1M6BQ {Received February, 1977 and in revised form June, 1977) ABSTRACT
of the dose at the peak (Ellis et al., 1976). The absorption of pions in tissue is the result of nuclear interactions with carbon, nitrogen and oxygen causing nuclear disintegration with the release of neutrons and "stars" of protons, alpha particles and heavy recoils which are locally absorbed. The LET distribution in the plateau is lower than in the peak region because of the smaller contribution from stars produced by pions interacting in flight. In the peak, pion stars contribute about 50% of the dose; based Negative pion beams of biologically significant in- on calculations of Alsmiller et al. (1974) 25% of the tensity and momentum spread are now available and peak dose is due to particles with LET greater than much of the recent work has been reviewed by Raju 10 KeV fim-1 and 10% from those greater than (1974). The 70 MeV pion beam at the Rutherford 100 KeV [xmr1. In contrast, in the plateau these high High Energy Laboratory, England, which delivers LET contributions are reduced by a factor of five. about 150 rad h"1, has been used by a number of Prior to all mouse irradiations a depth dose curve groups to study pion radiobiology. This work has of the pion beam was determined and the position of recently been comprehensively reviewed by Ellis the Bragg peak scanned in both vertical and horiet al. (1976). In 1976 Coggle et al. reported on the induction of lens opacities in one-day-old mice zontal planes to facilitate positioning of the mice. given irradiation to the head and upper part of the Since the irradiations, which took place in 1973/74, body with either 60Co y rays at 1.5 rad min^1 or were designed to study the induction of lens opacipions at 1 rad min^1. These mice have now been ties, and since the2 field size at the 80% isodose was systematically studied to determine both their life- only 2.5 X 2.5 cm , it was decided to irradiate only span and the main causes of death and the results are the head and upper part of the body of one-day-old mice. This enabled eight mice to be irradiated simulgiven here. taneously, four in the plateau and four in the peak region of the beam. The mice were restrained in MATERIALS AND METHODS The pion beam from the proton cyclotron ventilated Perspex containers and arranged 3 so that NIMROD is obtained from the interaction of a their heads were within the 2.5 X 2.5 X 3 cm irradiaThe containers were placed in a 10 X 7 GeV proton beam with a copper or tungsten tion volume. 3 target. The pions produced are focused along a 6 m 10x18 cm Perspex phantom at the depths required The pion dose rate beam and brought to the shielded experimental for plateau and peak irradiation. 1 was about 1 rad min. For comparison, groups of room. Ionization measurements show a peak to plateau dose ratio of 1.6 for particles of 160 MeV/c four one-day-old mice were placed in ventilated 60 with a mean momentum spread of 15%. The current Perspex containers and positioned 2 m from a Co beam line using a tungsten target gives a dose rate of y-ray source. Their heads were1 directly in the beam 150 rad h"1 over a volume of 2.5 X 2.5 X 3 cm 3 at the and irradiated at 1.5 rad min" , whilst their bodies 80% isodose contour. The electron, muon and were shielded by lead wedges designed to reproduce neutron contamination of the beam is reasonably the field size and distribution of the pion beam. The small. Muons contribute about 1 % of the total peak dose to the body was not more than 10% of that to ionization and are of low LET. Low LET electrons the head. The doses and numbers of mice used are contribute about 5% of the total ionization at the given in Table I. pion peak. High LET neutron contamination is All mice were caged under identical conditions, reduced by shielding and contributes less than 1 % fed MRC diet 41B and given water ad lib. They were The long-term effects of partial body exposure of one-dayold mice given either 60Co y rays or negative pions have been studied. Both radiations produced considerable lifeshortening; for pions 6.8 ±1.5% of life is lost per 100 rad and for y rays the value is 5.7 ±0.5% per 100 rad. The RBE of pions for 60ten weeks of life-shortening is about 1.3 compared with Co y rays, although at lower doses the RBE may be higher reaching about two for six weeks of lifeshortening. The incidence rate of tumours at any particular age was greater in mice irradiated with pions at the peak and in those given higher doses of y rays than in the controls.
658
SEPTEMBER
1977
Some long-term effects of negative pions in mice exposed to partial body irradiation TABLE I TABLE II AGES AT WHICH MICE OF BOTH SEXES REACH 50% SURVIVAL (MEDIAN AGE)
RADIATION SCHEME FOR DAY-OLD MICE (EQUAL NUMBERS OF MALES AND FEMALES) 60
Median
Pions
C y rays
Dose (rad)
No. of mice
0 40 60 100 200 300
120 48 48 48 60 60
Dose (rad)
No. of mice
40 (plateau) 60(peak) 136 (plateau) 200 (peak)
160 60 60 60 60
0
Dose (rad) 60
No. of
(weeks)
Standard deviation
Standard error
mice
111.5 107.3 106.5 102.0 97.6 92.5
24.9 22.8 24.5 20.8 22.3 23.0
2.1 3.4 3.6 3.1 3.0 3.0
120 48 48 48 59 60
110.9 105.5 102.1 99.8 95.8
23.0 21.3 17.6 27.5 25.3
2.1 3.0 2.3 3.8 3.8
146 54 60 54 46
age
Co y rays 0
40
oU 1 nn ^nn 300 I VU
Pions
examined daily for deaths or signs of morbidity, moribund mice being killed before death. A full post mortem on each mouse gave data on gross pathological lesions, many samples being studied histopathologically. This enabled a probable cause of death to be ascribed to 80% of the mice. However, difficulty remained in mice that were eaten, autolysed or which showed no significant pathological changes. The classification of pathological findings used in this study follows that of Lindop and Rotblat (1961b). RESULTS
The age at death of every mouse was recorded and the percentage survival plotted against age in weekly intervals for each dose group. The difference between the sexes was so small that the analysis of lifeshortening has been made for both sexes combined. Figure 1 shows the survival curves for control mice and those given 60 rad and 200 rad of pions at the peak to the head region alone. Figure 2 shows TT
MESON
0
40 (plateau) 60 (peak) 136 (plateau) 200 (peak)
similar curves for controls and those given 60 rad and 300 rad of 60Co y irradiation. As can be seen the effect of such irradiations is to shift the survival curves towards a younger age. For the sake of clarity the survival curves for the other dose groups are not included in Figs. 1 and 2. The amount of life-shortening for a given dose can be determined by comparing the ages at which mice, from different dose groups, reach a given percentage survival. Table II lists the median age (age for 50% survival) for mice of both sexes as a function of dose together with the standard deviation and error. The errors are obtained from a linear transformation of the survival curves. A plot of the probit of the percentage survival against age gave very good CO-60
AGE ( WEEKS ) FIG.1.
Survival curves of mice of both sexes for controls and mice given 60 and 200 rad of peak pions, at the peak at 1 rad min"1 to the head region only. 659
FIG. 2. Survival curves of mice of60 both sexes for controls and mice given 60 and 300 rad of Co at 1.5 rad min"1 to the head region only.
VOL.
50, No. 597 J. E. Goggle
straight lines, six of t h e 11 lines having coefficients of correlations greater t h a n 0.99, four w i t h coefficients greater t h a n 0.98 and one with a coefficient of 0.975. T h e derivation of confidence limits from statistical analysis of life-shortening is described b y L i n d o p a n d Rotblat (1961a). F i g u r e s 3 and 4 are o b t a i n e d from data in T a b l e I I a n d show life-shortening in weeks as a function of dose for pion a n d 6 0 C o y irradiation respectively. T h e a t t e m p t to fit a straight line by regression analysis to t h e p i o n data (Fig. 3) p r o v e d r a t h e r unsatisfactory, w i t h a coefficient of correlation of 0.93. I n contrast, t h e 6 0 C o data (Fig. 4) fit a straight line very well with a coefficient of 0.99 a n d an intersection at t h e origin of 0 . 9 ^ 0 . 7 weeks, s h o w i n g t h a t t h e r e is p r o b a b l y n o t h r e s h o l d for lifes h o r t e n i n g in y o u n g mice. I n Fig. 3 t h e d o t t e d line, d r a w n b y eye t h r o u g h t h e pion points, curves steeply from t h e origin suggesting a greater relative effectiveness c o m p a r e d w i t h y rays at low doses. F o r example, t h e relative biological effect of pions for six weeks of life-shortening is a b o u t t w o c o m p a r e d w i t h 6 0 C o y rays. F r o m t h e linear regression lines it can be calculated t h a t for pions 6.83 ± 1 . 5 1 % of life is lost p e r 100 rad a n d for 6 0 C o t h e value is 5 . 6 7 ± 0 . 4 6 % life lost p e r 100 rad. T h e relative biological efficiency of pions for t e n weeks life-shortening is 1.27 c o m p a r e d w i t h 6 0 C o y rays.
causes of d e a t h did not really vary w i t h dose, a finding in a g r e e m e n t with t h e m u c h larger s t u d y of L i n d o p a n d Rotblat (1961b). However, the n u m b e r s of mice in t h e experiment (column 11) are too small to allow a meaningful analysis of m e a n age at death for each cause of death. However, by c o m b i n i n g mice dying from all t u m o u r s (columns 1-5 in T a b l e I I I ) it is possible to analyse incidence rates of t u m o u r s using t h e m e t h o d s of Peto (1974; 1976). Such an analysis shows t h a t t h e incidence rate of t u m o u r s at any particular age is greater in the irradiated t h a n in t h e control g r o u p for doses of 200 rad (p=0.0\) and 60 rad (p
Some long-term effects of negative pions in mice exposed to partial body irradiation By J . E. Coggle, Ph.D. Department of Radiobiology, Medical College of St. Bartholomew's Hospital, Charterhouse Square, London EC1M6BQ {Received February, 1977 and in revised form June, 1977) ABSTRACT
of the dose at the peak (Ellis et al., 1976). The absorption of pions in tissue is the result of nuclear interactions with carbon, nitrogen and oxygen causing nuclear disintegration with the release of neutrons and "stars" of protons, alpha particles and heavy recoils which are locally absorbed. The LET distribution in the plateau is lower than in the peak region because of the smaller contribution from stars produced by pions interacting in flight. In the peak, pion stars contribute about 50% of the dose; based Negative pion beams of biologically significant in- on calculations of Alsmiller et al. (1974) 25% of the tensity and momentum spread are now available and peak dose is due to particles with LET greater than much of the recent work has been reviewed by Raju 10 KeV fim-1 and 10% from those greater than (1974). The 70 MeV pion beam at the Rutherford 100 KeV [xmr1. In contrast, in the plateau these high High Energy Laboratory, England, which delivers LET contributions are reduced by a factor of five. about 150 rad h"1, has been used by a number of Prior to all mouse irradiations a depth dose curve groups to study pion radiobiology. This work has of the pion beam was determined and the position of recently been comprehensively reviewed by Ellis the Bragg peak scanned in both vertical and horiet al. (1976). In 1976 Coggle et al. reported on the induction of lens opacities in one-day-old mice zontal planes to facilitate positioning of the mice. given irradiation to the head and upper part of the Since the irradiations, which took place in 1973/74, body with either 60Co y rays at 1.5 rad min^1 or were designed to study the induction of lens opacipions at 1 rad min^1. These mice have now been ties, and since the2 field size at the 80% isodose was systematically studied to determine both their life- only 2.5 X 2.5 cm , it was decided to irradiate only span and the main causes of death and the results are the head and upper part of the body of one-day-old mice. This enabled eight mice to be irradiated simulgiven here. taneously, four in the plateau and four in the peak region of the beam. The mice were restrained in MATERIALS AND METHODS The pion beam from the proton cyclotron ventilated Perspex containers and arranged 3 so that NIMROD is obtained from the interaction of a their heads were within the 2.5 X 2.5 X 3 cm irradiaThe containers were placed in a 10 X 7 GeV proton beam with a copper or tungsten tion volume. 3 target. The pions produced are focused along a 6 m 10x18 cm Perspex phantom at the depths required The pion dose rate beam and brought to the shielded experimental for plateau and peak irradiation. 1 was about 1 rad min. For comparison, groups of room. Ionization measurements show a peak to plateau dose ratio of 1.6 for particles of 160 MeV/c four one-day-old mice were placed in ventilated 60 with a mean momentum spread of 15%. The current Perspex containers and positioned 2 m from a Co beam line using a tungsten target gives a dose rate of y-ray source. Their heads were1 directly in the beam 150 rad h"1 over a volume of 2.5 X 2.5 X 3 cm 3 at the and irradiated at 1.5 rad min" , whilst their bodies 80% isodose contour. The electron, muon and were shielded by lead wedges designed to reproduce neutron contamination of the beam is reasonably the field size and distribution of the pion beam. The small. Muons contribute about 1 % of the total peak dose to the body was not more than 10% of that to ionization and are of low LET. Low LET electrons the head. The doses and numbers of mice used are contribute about 5% of the total ionization at the given in Table I. pion peak. High LET neutron contamination is All mice were caged under identical conditions, reduced by shielding and contributes less than 1 % fed MRC diet 41B and given water ad lib. They were The long-term effects of partial body exposure of one-dayold mice given either 60Co y rays or negative pions have been studied. Both radiations produced considerable lifeshortening; for pions 6.8 ±1.5% of life is lost per 100 rad and for y rays the value is 5.7 ±0.5% per 100 rad. The RBE of pions for 60ten weeks of life-shortening is about 1.3 compared with Co y rays, although at lower doses the RBE may be higher reaching about two for six weeks of lifeshortening. The incidence rate of tumours at any particular age was greater in mice irradiated with pions at the peak and in those given higher doses of y rays than in the controls.
658
SEPTEMBER
1977
Some long-term effects of negative pions in mice exposed to partial body irradiation TABLE I TABLE II AGES AT WHICH MICE OF BOTH SEXES REACH 50% SURVIVAL (MEDIAN AGE)
RADIATION SCHEME FOR DAY-OLD MICE (EQUAL NUMBERS OF MALES AND FEMALES) 60
Median
Pions
C y rays
Dose (rad)
No. of mice
0 40 60 100 200 300
120 48 48 48 60 60
Dose (rad)
No. of mice
40 (plateau) 60(peak) 136 (plateau) 200 (peak)
160 60 60 60 60
0
Dose (rad) 60
No. of
(weeks)
Standard deviation
Standard error
mice
111.5 107.3 106.5 102.0 97.6 92.5
24.9 22.8 24.5 20.8 22.3 23.0
2.1 3.4 3.6 3.1 3.0 3.0
120 48 48 48 59 60
110.9 105.5 102.1 99.8 95.8
23.0 21.3 17.6 27.5 25.3
2.1 3.0 2.3 3.8 3.8
146 54 60 54 46
age
Co y rays 0
40
oU 1 nn ^nn 300 I VU
Pions
examined daily for deaths or signs of morbidity, moribund mice being killed before death. A full post mortem on each mouse gave data on gross pathological lesions, many samples being studied histopathologically. This enabled a probable cause of death to be ascribed to 80% of the mice. However, difficulty remained in mice that were eaten, autolysed or which showed no significant pathological changes. The classification of pathological findings used in this study follows that of Lindop and Rotblat (1961b). RESULTS
The age at death of every mouse was recorded and the percentage survival plotted against age in weekly intervals for each dose group. The difference between the sexes was so small that the analysis of lifeshortening has been made for both sexes combined. Figure 1 shows the survival curves for control mice and those given 60 rad and 200 rad of pions at the peak to the head region alone. Figure 2 shows TT
MESON
0
40 (plateau) 60 (peak) 136 (plateau) 200 (peak)
similar curves for controls and those given 60 rad and 300 rad of 60Co y irradiation. As can be seen the effect of such irradiations is to shift the survival curves towards a younger age. For the sake of clarity the survival curves for the other dose groups are not included in Figs. 1 and 2. The amount of life-shortening for a given dose can be determined by comparing the ages at which mice, from different dose groups, reach a given percentage survival. Table II lists the median age (age for 50% survival) for mice of both sexes as a function of dose together with the standard deviation and error. The errors are obtained from a linear transformation of the survival curves. A plot of the probit of the percentage survival against age gave very good CO-60
AGE ( WEEKS ) FIG.1.
Survival curves of mice of both sexes for controls and mice given 60 and 200 rad of peak pions, at the peak at 1 rad min"1 to the head region only. 659
FIG. 2. Survival curves of mice of60 both sexes for controls and mice given 60 and 300 rad of Co at 1.5 rad min"1 to the head region only.
VOL.
50, No. 597 J. E. Goggle
straight lines, six of t h e 11 lines having coefficients of correlations greater t h a n 0.99, four w i t h coefficients greater t h a n 0.98 and one with a coefficient of 0.975. T h e derivation of confidence limits from statistical analysis of life-shortening is described b y L i n d o p a n d Rotblat (1961a). F i g u r e s 3 and 4 are o b t a i n e d from data in T a b l e I I a n d show life-shortening in weeks as a function of dose for pion a n d 6 0 C o y irradiation respectively. T h e a t t e m p t to fit a straight line by regression analysis to t h e p i o n data (Fig. 3) p r o v e d r a t h e r unsatisfactory, w i t h a coefficient of correlation of 0.93. I n contrast, t h e 6 0 C o data (Fig. 4) fit a straight line very well with a coefficient of 0.99 a n d an intersection at t h e origin of 0 . 9 ^ 0 . 7 weeks, s h o w i n g t h a t t h e r e is p r o b a b l y n o t h r e s h o l d for lifes h o r t e n i n g in y o u n g mice. I n Fig. 3 t h e d o t t e d line, d r a w n b y eye t h r o u g h t h e pion points, curves steeply from t h e origin suggesting a greater relative effectiveness c o m p a r e d w i t h y rays at low doses. F o r example, t h e relative biological effect of pions for six weeks of life-shortening is a b o u t t w o c o m p a r e d w i t h 6 0 C o y rays. F r o m t h e linear regression lines it can be calculated t h a t for pions 6.83 ± 1 . 5 1 % of life is lost p e r 100 rad a n d for 6 0 C o t h e value is 5 . 6 7 ± 0 . 4 6 % life lost p e r 100 rad. T h e relative biological efficiency of pions for t e n weeks life-shortening is 1.27 c o m p a r e d w i t h 6 0 C o y rays.
causes of d e a t h did not really vary w i t h dose, a finding in a g r e e m e n t with t h e m u c h larger s t u d y of L i n d o p a n d Rotblat (1961b). However, the n u m b e r s of mice in t h e experiment (column 11) are too small to allow a meaningful analysis of m e a n age at death for each cause of death. However, by c o m b i n i n g mice dying from all t u m o u r s (columns 1-5 in T a b l e I I I ) it is possible to analyse incidence rates of t u m o u r s using t h e m e t h o d s of Peto (1974; 1976). Such an analysis shows t h a t t h e incidence rate of t u m o u r s at any particular age is greater in the irradiated t h a n in t h e control g r o u p for doses of 200 rad (p=0.0\) and 60 rad (p
