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The influence of anoxia or oxygenation on the induction of chromosome aberrations in human lymphocytes by 15-MeV neutrons
Mutation Research, 84 (I 981) 365-373 Elsevier/North-Holland Biomedical Press
365
The influence of anoxia or oxygenation on the induction of chromosome aberrations in human lymphocytes by 15-MeV neutrons J.S. P r o s s e r a n d L . D . S t i m p s o n National Radiological Protection Board, Chilton, Didcot, Oxon OX11 ORQ (Great Britain)
(Received 20 November 1980) (Resubmitted 1 July 1981) (Accepted 13 July 1981)
Summary Dicentric and total aberration yields induced in human lymphocytes by 15-MeV neutrons under conditions of oxygenation and of anoxia have been fitted to a dose-response curve using the function Y = aD + flD 2. An oxygen-enhancement ratio (OER) ranging from 3.7 at low yields to 1.6 at high yields was calculated from the co-efficients of the dicentric yield curves and evidence is presented which suggests that oxygen does not act as a dose-modifying agent in this system. High dose RBE values of 1.2 and 2.1 with respect to 250 kVp X-rays for oxygenated and anoxic conditions were also obtained. Coefficients for total aberration yield gave similar values to dicentrics for both OER and RBE
The increase in the radio-resistance of tumours induced by the presence of even a very small proportion of hypoxic cells is well-documented [10,17,26] and one method of overcoming this problem may be by using radiations of high LET. The relationship between LET and OER has been studied in detail [2] since Thoday and Read [25] first demonstrated a low OER with high LET radiation. The decrease in OER with increasing LET is influenced by the type of experimental system used [1,3,4] and, in the case of neutrons, by the energy [2,27]. Unstable chromosome-aberration induction in human lymphocytes has been used to examine the difference in OER at high and low doses of 250 kVp X-rays [18] and in this study the effects of 15-MeV neutron irradiation on the same system under conditions of oxygenation and anoxia have been examined.
0027-5107/81/0000-0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
366 Materials and methods
14.9-MeV neutrons were generated by the 3H(d,n)He4 reaction using a 300-keV deuteron accelerator and monitored by two U-238 fission counters calibrated by measurements of absorbed dose at various distances at 0 ° to the deuteron beam using a pair of ionisation chambers [24]. The accompanying photon dose was determined to be 396 of the total and the absolute uncertainty in the specification of neutron dose at a point was about _.+496 (standard error). Samples of whole blood, equilibrated with humidified oxygen or 596 CO 2 in nitrogen in an apparatus described previously [18] were irradiated at about 20 cm from the target in a perspex phantom maintained at 37 __+0.2°C by means of a warm-air supply controlled by a thermistor mounted in the sample holder. The dose-rate was typically 0.25 Gy rain -l. The sample container was in the form of a hollow disc 5 cm in diameter, the thickness of the blood in the direction of the beam being 3 mm. Charged-particle equilibrium was established in the 2 mm thick front face of the container. The maximum difference in neutron-fluence rate within the blood was about 9%. To prevent settling of the cellular fraction with resultant uneven distribution of kerma and to encourage mixing thus reducing variation in absorbed dose per cell, the perspex container was rotated about its central axis during irradiation. Doses specified were kerma in blood reduced by a calculated 2.6% to account for the mis-match between persex and blood. It was estimated that an effective uncertainty of -+-296 (standard error) in expected mean dose per cell was attributable to the size and nature of the sample. Separated lymphocyte cultures were set up according to a standard procedure [19], incubated at 37 ° C and fixed onto microscope slides 48 h later. Colemid was added 3 h before fixation. Complete orcein-stained cells were analyzed for unstable chromosome aberrations, i.e. dicentrics, centric rings and excess fragments. Over a period of several months, blood sampled from the same donor was sham-irradiated and control cultures containing 7 #M BrdU were set up as before. After harlequin staining by a modification of the method of Perry and Wolff [16] these were analyzed for the numbers of first and second division cells.
Results
The numbers of first and second cycle metaphases observed in 48-h control cultures on different occasions are given in Table 1 and show no difference between each of the pretreatment conditions. The average value for second division metaphase contamination is 3.1%. Neutron doses of 0.08-2.40 Gray to oxygenated blood and 0 . 0 8 - 3.20 Gray to anoxic blood were given and the numbers of aberrations observed for each of these doses is shown in Table 2. Aberration distributions between cells were tested for goodness of fit to a Poisson by the dispersion-index test [22] and as expected from previously published results from neutron irradiation [8, 9, 23] significant overdispersion was observed. The negative binomial distribution was
367 TABLE I T H E N U M B E R S OF S E C O N D M E T A P H A S E CELLS OBSERVED PER 100 CELLS A N A L Y Z E D F R O M 48-h C U L T U R E S OF B L O O D P R E T R E A T E D F O R I h AS I N D I C A T E D The experiment was repeated 4 times with replicates analyzed on each occasion. Expt.
Oxygenated
Venous
Anoxic
1
a b
4 1
0 2
4 2
2
a b
9 5
2 I
5 3
3
a b
4 4
2 4
6 6
4
a b
2 0
1 4
0 3
3.6
2.0
3.6
Mean (%)
found to provide a good fit to these data ( P = 0.97). Dicentric and total aberration yields have been fitted to the linear-quadratic dose-response equation Y = aD + BD 2 using a least-squares method and weighting each value of aberrations per cell by the inverse of the sample variance ( n i / s 2, where n i is the number of cells). Results are shown in eqns. (1) to (4) and curves for TABLE 2 C H R O M O S O M E A B E R R A T I O N S I N D U C E D BY 15-MeV N E U T R O N S IN O X Y G E N A T E D A N D ANOXIC BLOOD Dose (Gy)
Cells scored
Normal ceils
Dicentrics
Total aberrations
200 1000 1000 1000 695 657 258 199
198 973 901 845 497 431 83 29
0 17 74 113 174 197 207 311
2 35 131 214 299 360 394 495
200 500 500 500 329 500 500 226 100
200 494 493 465 307 421 331 109 35
0 5 6 19 20 64 147 113 93
0 7 10 48 39 117 282 199 133
Oxygenated 0 0.08 0.20 0.40 0.60 0.80 1.60 2.40 Anox~ 0 0.08 0.20 0.40 0.60 0.80 1.60 2.40 3.20
368
dicentric yield are plotted in Fig. 1. YN = (6.81 ___ 1.34) × 10 -2 D + (6.61 ___0.84) X 10 --2
( P = 0.55)
(1)
Yo = (25.50 __+3.07) × 10-2 D + (16.15 ___2.82) × 10-2 D 2 ( p = 0.10)
(2)
D2
YN and Yo are dicentric yields observed after radiation dose D in anoxic and fully oxygenated conditions respectively. AN =(16.23_3.39)×
10-2 D + (9.17___ 2.02) × 1 0 - 2 D 2
( P = 0.06)
A o=(49.50__+4.60)>(lO-2D+(24.25___4.02)XlO-2D2 ( P = 0 . 1 0 )
(3) (4)
A N and A o are yields of total aberrations observed after radiation dose D in anoxic and fully oxygenated conditions respectively. Included are multicentrics, centric rings, and acentric fragments, all of which can prevent a cell from undergoing repeated cycles of division [6,30]. The a and fl coefficients from eqns. 1-4 have been used to derive limiting values for the OER which are given in Table 3 with comparable results obtained previously by X-irradiation. At low neutron doses below about 1 Gray, the linear (aD) component of yield predominates and an OER of 3.7 _+ 0.9 is obtained from the a
1.6
1.4 OXYGENATED 1.2
_-
o=
1.0
== .~ 0.8
¢3 0.6
ANOXIC
0.4
0.2
0m.a"~-
t
I
I
1.0
2.0
3.0
Dose, Gy
Fig. !. The yield of dicentrics per cell -4-SE plotted against dose for lymphocytes irradiated under oxygenated or anoxic conditions.
369 TABLE 3 A COMPARISON OF THE a AND ~ COEFFICIENTS FROM THE LINEAR-QUADRATIC EQUATIONS FITTED TO ABERRATION YIELDS INDUCED BY 15-MeV NEUTRONS AND 250 kVp X-RAYS UNDER OXYGENATED (O) AND A N O X I C (N) CONDITIONS
Neutron
~o
~o
~#o
Dicentrics SE
3.7 0.9
2.4 0,5
1.6 0.2
Total aberrations SE
3.1 0.7
2.6 0.7
t .6 0.2
X-Ray
Dicentrics
7.2
[ 18]
SE
4.6
7.1 0.9
2.7 0.2
Total aberrations SE
3.6 1.9
7.4 1.3
2.7 0.2
coefficient ratio for dicentric production. This compares with a ratio of 7.2 for 250 kVp X-rays [18]. As the neutron dose is increased and the_flD 2 component of yield becomes more important, the OER tends towards fflOflN, which is 1.6 ± 0.2 for dicentrics (Table 3). Table 4 gives values for the low dose RBE [15] for 15-MeV neutrons with respect to 250-kVp X-radiation for dicentric and total aberration induction [18]. With increasing dose of neutrons the flD 2 term becomes more important and the RBE
TABLE 4 MAXIMUM RBE VALUES CALCULATED FROM aNEUTRON/aX.RAY DERIVED FROM Eqns. 1-4 AND X-IRRADIATION [18] Dicentrics Oxygenated SE Anoxic SE
Total aberrations
5.4 2.1
5.5 1.9
10.3 5.5
6.5 2.8
TABLE 5 MINIMUM RBE VALUES CALCULATED FROM V/flNEUTRON/flX.RAV DERIVED FROM Eqns. 1-4 AND X-IRRADIATION [18]. Dicentrics
Total aberrations
Oxygenated SE
1.3 0.1
1.2 0.1
Anoxic SE
2.2 0.2
2.0 0.3
370
~/(flneutron/flX-ray)"
tends t o w a r d s High dose RBE values are given in Table 5 and it is evident that ratios obtained from both dicentric and total aberration yields are significantly greater in the case of blood irradiated under anoxic conditions than with oxygenated blood. Discussion
Contrary to some observations [5] but not others [20], the average frequency of second-division metaphases observed in 48-h cultures of venous blood (Table 1) was not significant, amounting to only 2%. A low yield of second cycle metaphases (less than 14%) under the culture conditions used here has been noted previously [20] and further experiments [21; Purrott, personal communication] have indicated that the use of an anhydric incubator and hence an effectively shortened time at 37°C and also the use of the relatively impoverished Eagle's Minimal Essential Medium leads to a significant reduction in the frequency of second-cycle metaphases at 48 h in all donors examined so far. There is good evidence that this frequency is further reduced with increasing radiation dose due to mitotic delay [20]: None of the values in Table 1 is significantly different from the overall average of 3.1% showing that the pretreatment did not influence the rate of response of the lymphocytes to PHA. This indicates that the variation in aberration yield with oxygen concentration is due entirely to a change in radiosensitivity caused by the presence of oxygen and not to any effect on the cell-cycle kinetics. In the radiation experiments described here, irradiated blood was cultured without the addition of BrdU to enable direct comparison with previously published data for X-rays [18] and to avoid any complicating effects resulting from the incorporation of this mutagenic base analogue into chromosomal DNA. The numbers of second-cycle metaphases analyzed, with consequent underestimation of aberration yields are considered insignificant, particularly at the higher doses. For neutrons with an energy high enough to be clinically useful, the OER is usually in the range 1.5 to 1.8 compared with about 2.5 for X-rays [7]. Watson [29], in a study of the cytogenetic effect of 15-MeV neutrons on human lymphocytes irradiated in saline suspension, observed OER values which were dependent upon the frequency of dicentrics and ranged from 3.7 __+1.5 at 0. I dicentrics per cell to 1.2-+-0.3 at 0.4 dicentrics pet cell. Corresponding values calculated from eqns. (1) and (2) are 2.3 _ 0.6 at 0.1 dicentrics per cell and 2.1 ___0.5 at 0.4 dicentrics per cell. Watson's results were obtained at a culture time of 60 h when the maximum aberration frequency was observed and her dose-response curves were complex, some even showing a decrease in yield at the highest dose. In contrast, results in Table 2 were obtained after 48 h in culture and particularly for dicentric aberrations fitted satisfactorily to the linear-quadratic expression. As argued previously [18], for an "effect-modifying" factor where the ratio of aberration yields is constant for any given dose: O/O__ flO __ (XO
~N
constant
371
From Table 3 it can be seen that a o / a N and flo//BN are not significantly different. For a dose=modifying factor where the ratio of doses is constant for any given aberration yield: a_..~o_ ~
= constant
aN--
Columns 2 and 4 of Table 3 were compared by calculating the value of y and its standard error using the equation
aN
and the standard formula for the variance of a function of several random variables [12]. For a dose-modifying factor the value of y will not be significantly different from zero. From the coefficients for dicentric yield the value of y/s.e..r~_~-f_)~ascalculated to be 2.1, indicating that a o / a N is significantly different from ~/Bo/BN at the 5% level and therefore the value of OER is not constant with dose. Using coefficients for total aberration yield from Eqns. 3 and 4 a value of 1.6 is obtained for y/s.e.(y) indicating that a o / a ~ is not significantly different from ~/Bo/Bs even at the 10% level. Acentric aberrations are generally less reliable as an indicator of radiation damage and their inclusion in the total aberration data means that it becomes impossible to discriminate between the effect-modifying and dose-modifying hypotheses. For dicentric induction, however, it can be concluded that oxygen is not acting as a dose-modifying agent. Results from animal experiments suggests that an increase in the RBE of neutrons under hypoxic conditions would be expected. The actual values differ with the tissue examined and may not be relevant to man [7]. Tables 4 and 5 give values for the RBE of 15-MeV neutrons calculated from Eqns. 1-4 and data obtained using blood From the same donor irradiated with 250 kVp X-rays [18]. Because of the large errors in Table 4 due mainly to the small number of aberrations observed after low doses of X-rays, there appears to be no difference at low doses between the RBE under oxygenated or anoxic conditions. However, high dose values calculated from ~/Bneutron/flx-,,y (Table 5) do show a marked increase in RBE for anoxic irradiations giving approximately twice the oxygenated value for both dicentric and total aberrations.
Conclusions The analysis of lymphocytes for unstable chromosome aberrations which are lethal lesions in dividing cell systems is a useful radiobiological method for investigating the relationship between RBE, OER and LET. Whilst the absolute values derived depend very much on the experimental system used, the data presented here conform with other radiobiological end points in that values of OER which ranged
372 f r o m 3.7 to 1.6, are m a r k e d l y lower t h a n values o b t a i n e d previously for 250-kVp X-rays (7.2 to 2.7). T h e results do, however, suggest that oxygen does n o t act as a d o s e - m o d i f y i n g agent i n this system. Values derived for R B E were greatest at low n e u t r o n dose where the presence of oxygen did n o t i n f l u e n c e the value obtained. A t high doses RBEs were greater for a b e r r a t i o n s i n d u c e d u n d e r anoxic conditions. This is p r o b a b l y due to the greater c o n t r i b u t i o n of single-track d a m a g e i n d u c t i o n b y n e u t r o n s u n d e r these c o n d i t i o n s c o m p a r e d with X-rays.
Acknowledgements T h e e x p e r i m e n t a l a p p a r a t u s was designed b y A.G. Sherwin who together with C.L. H a r v e y a n d J.B. O ' H a g a n provided n e u t r o n i r r a d i a t i o n a n d dosimetry. Dr. S.C. D a r b y was responsible for statistical analyses a n d curve-fitting. This work was partly s u p p o r t e d b y E u r a t o m c o n t r a c t 171-76-1 B 1 0 - U K .
References 1 Alper, T., J.L. Moore and D.K. Bewley, LET as a determinant of bacterial radiosensitivity and its modification by anoxia and glycerol, Radiat. Res., 32 (1967) 277-293. 2 Barendsen, G.W., C.J. Koot, G.R. van Kerson, D.K. Bewley, S.B. Field and C.J. Parnell, The effect of oxygen on impairment of the proliferative capacity of human cells in culture by ionizing radiations of different LET, Int. J. Radiat. Biol., 10 (1966) 317-327. 3 Berry, R.J., Hypoxic protection against fast neutrons of different energies--a review. Eur. J. Cancer, 7 (1971) 145-152. 4 Bryant, P.E., LET as a determinant of oxygen enhancement ratio and shape of survival curve for chlamydomonas, Int. J. Radiat. Biol., 23 (1973) 217-226. 5 Crossen, P.E., and W.F. Morgan, Analysis of human lymphocyte cell cycle time in culture measured by sister chromatid differential staining, Exp. Cell Res., 104 (1977) 453-457. 6 Dewey, W.C., H.H Miller and D.B. Leeper, Chromosomal aberrations and mortality of X-irradiated mammalian cells: emphasis.on repair, Proc. Natl. Acad. Sci. (U.S.A.), 68 (1971) 667-671. 7 Field, S.B., An historical survey of radiobiology and radiotherapy with fast neutrons, Curr. Top. Radiat. Res., 11 (1976) 1-86. 8 Haag, J., J. Brenot and N. Parmentier, Chromosomal aberrations in pig lymphocytes after neutron irradiation in vitro, Radiat. Res., 70 (1977) 187-197. 9 Holmberg, M., The effects of recoiling oxygen nuclei on the frequency of chromosome breakage in human lymphocytes after fast neutron irradiation, in: H.J. Evans and D.C. Lloyd (Eds.), Mutageninduced Chromosome Damage in Man, University of Edinburgh Press, Edinburgh 1978, pp. 14-21. 10 Hewitt, H.B., Studies of the dissemination and quantitative transplantation of a lymphocytic leukaemia of CBA mice, Br. J. Cancer, 12 (1958) 378-400. 11 Kallman, R.F., The phenomenon of reoxygenation and its implications for fractionated radiotherapy, Radiology, 105 (1972) 135-142. 12 Kendall, M.G., and A. Stuart, The Advanced Theory of Statistics, Vol. 1, Distribution Theory, Griffin, London, 1969, pp. 231-232. 13 Lloyd, D.C., R.J. Purrott, G.W. Dolphin and A.A. Edwards, Chromosome aberrations induced in human lymphocytes by neutron irradiation, Int. J. Radiat. Biol., 29 (1976) 169- 182. 14 Lloyd, D.C., R.J. Purrott, J.S. Prosser, G.W. Dolphin, P.A. Tipper, E.J. Reeder, C.M. White, S.J. Cooper and B.D. Stephenson, The study of chromosome aberration yield in human lymphocytes as an
373 indicator of radiation dose, VI. A review of cases investigated, 1975, NRPB-R41, National Radiological Protection Board, Harwell, 1976. 15 Neary, G.J, J.R.K. Savage, H.J. Evans and J. Whittle, Ultimate maximum values of the RBE of fast neutrons and gamma rays for chromosomal aberrations, Int. J. Radiat. Biol., 6 (1963) 127-136. 16 Perry, P., and S. Wolff, New Giemsa method for the differential staining of sister chromatids, Nature (London), 251 (1974) 156-158. 17 Powers, W.E., and L.J. Tolmach, A multicomponent X-ray survival curve for mouse lymphosarcoma cells irradiated in vivo, Nature (London), 197 (1963) 710-71 I. 18 Prosser, J.S., C.M. White and A.A. Edwards, The effect of oxygen concentration on the X-ray induction of chromosome aberrations in human lymphocytes, Mutation Res., 61 (1979) 287-295. 19 Purrot, R.J., and D.C. Lloyd, The study of chromosome aberration yield in human lymphocytes as an indicator of radiation dose, 1. Techniques, U.K. Natl. Radiol. Protect. Board Rept. (1972) NRPB-R2. 20 Purrot, R.J., N. Vulpis and D.C. Lloyd, The use of harlequin staining to measure delay in the human lymphocyte cell cycle induced by in vitro X-irradiation, Mutation Res., 69 0980) 275-282. 21 Purrot, R.J., N. Vulpis and D.C. Lloyd, The influence of incubation temperature on the rate of human lymphocyte proliferation in vitro, Experientia, 37 (1981) 407-408. 22 Savage, J.R.K., Sites of radiation chromosome exchanges, Curr. Top. Radiat. Res., 6 (1970) 129-194. 23 Schmid, E., and M. Bauchinger, Chromosome aberrations in human lymphocytes after irradiation with 15.0-MeV neutrons in vitro, II. Analysis of the number of absorption events and the interaction distance in the formation of dicentric chromosomes, Mutation Res., 27 (1975) I I l - l 17. 24 Sherwin, A.G., Developments in the use of ionization chambers for mixed field dosimetry, in: G. Burger and H.G. Ebert (Eds.,) Proceedings of the second symposium on Neutron Dosimetry in Biology and Medicine,' Commission of the European Communities, Luxembourg, 1975, EUR5273 d-e-f, pp. 341-358. 25 Thoday, J.M., and J. Read, Effect of oxygen on the frequency of chromosome aberrations produced by alpha rays, Nature (Lond9n), 163 (1949) 133-134. 26 Thomlinson, R.H., and L.H. Gray, The histological structure of some human lung cancers and the possible implications for radiotherapy, Br. J. Cancer, 9 (1955) 539-549. 27 Todd, P., Heavy ion irradiation of cultured human cells, Radiation Res., Suppl. 7 (1967) 196-207. 28 Watson, G.E., 15 MeV neutrons and the oxygen effect on chromosome aberration induction in cultured human lymphocytes (abstract), Br. J. Radiol., 46 (1973) 648. 29 Watson, G.E., and N.E. Gillies, The oxygen enhancement ratio for X-ray induced chromosomal aberrations in cultured human lymphocytes, Int. J. Radiat. Biol., 17 (1970) 279-283. 30 Wolff, S., Genetic effects and radiation induced cell death, in: J.M. Vaeth (Ed.), Frontiers of Radiation Therapy and Oncology, Vol. 16, Karger, Basel, 1972, pp. 459-469.
365
The influence of anoxia or oxygenation on the induction of chromosome aberrations in human lymphocytes by 15-MeV neutrons J.S. P r o s s e r a n d L . D . S t i m p s o n National Radiological Protection Board, Chilton, Didcot, Oxon OX11 ORQ (Great Britain)
(Received 20 November 1980) (Resubmitted 1 July 1981) (Accepted 13 July 1981)
Summary Dicentric and total aberration yields induced in human lymphocytes by 15-MeV neutrons under conditions of oxygenation and of anoxia have been fitted to a dose-response curve using the function Y = aD + flD 2. An oxygen-enhancement ratio (OER) ranging from 3.7 at low yields to 1.6 at high yields was calculated from the co-efficients of the dicentric yield curves and evidence is presented which suggests that oxygen does not act as a dose-modifying agent in this system. High dose RBE values of 1.2 and 2.1 with respect to 250 kVp X-rays for oxygenated and anoxic conditions were also obtained. Coefficients for total aberration yield gave similar values to dicentrics for both OER and RBE
The increase in the radio-resistance of tumours induced by the presence of even a very small proportion of hypoxic cells is well-documented [10,17,26] and one method of overcoming this problem may be by using radiations of high LET. The relationship between LET and OER has been studied in detail [2] since Thoday and Read [25] first demonstrated a low OER with high LET radiation. The decrease in OER with increasing LET is influenced by the type of experimental system used [1,3,4] and, in the case of neutrons, by the energy [2,27]. Unstable chromosome-aberration induction in human lymphocytes has been used to examine the difference in OER at high and low doses of 250 kVp X-rays [18] and in this study the effects of 15-MeV neutron irradiation on the same system under conditions of oxygenation and anoxia have been examined.
0027-5107/81/0000-0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
366 Materials and methods
14.9-MeV neutrons were generated by the 3H(d,n)He4 reaction using a 300-keV deuteron accelerator and monitored by two U-238 fission counters calibrated by measurements of absorbed dose at various distances at 0 ° to the deuteron beam using a pair of ionisation chambers [24]. The accompanying photon dose was determined to be 396 of the total and the absolute uncertainty in the specification of neutron dose at a point was about _.+496 (standard error). Samples of whole blood, equilibrated with humidified oxygen or 596 CO 2 in nitrogen in an apparatus described previously [18] were irradiated at about 20 cm from the target in a perspex phantom maintained at 37 __+0.2°C by means of a warm-air supply controlled by a thermistor mounted in the sample holder. The dose-rate was typically 0.25 Gy rain -l. The sample container was in the form of a hollow disc 5 cm in diameter, the thickness of the blood in the direction of the beam being 3 mm. Charged-particle equilibrium was established in the 2 mm thick front face of the container. The maximum difference in neutron-fluence rate within the blood was about 9%. To prevent settling of the cellular fraction with resultant uneven distribution of kerma and to encourage mixing thus reducing variation in absorbed dose per cell, the perspex container was rotated about its central axis during irradiation. Doses specified were kerma in blood reduced by a calculated 2.6% to account for the mis-match between persex and blood. It was estimated that an effective uncertainty of -+-296 (standard error) in expected mean dose per cell was attributable to the size and nature of the sample. Separated lymphocyte cultures were set up according to a standard procedure [19], incubated at 37 ° C and fixed onto microscope slides 48 h later. Colemid was added 3 h before fixation. Complete orcein-stained cells were analyzed for unstable chromosome aberrations, i.e. dicentrics, centric rings and excess fragments. Over a period of several months, blood sampled from the same donor was sham-irradiated and control cultures containing 7 #M BrdU were set up as before. After harlequin staining by a modification of the method of Perry and Wolff [16] these were analyzed for the numbers of first and second division cells.
Results
The numbers of first and second cycle metaphases observed in 48-h control cultures on different occasions are given in Table 1 and show no difference between each of the pretreatment conditions. The average value for second division metaphase contamination is 3.1%. Neutron doses of 0.08-2.40 Gray to oxygenated blood and 0 . 0 8 - 3.20 Gray to anoxic blood were given and the numbers of aberrations observed for each of these doses is shown in Table 2. Aberration distributions between cells were tested for goodness of fit to a Poisson by the dispersion-index test [22] and as expected from previously published results from neutron irradiation [8, 9, 23] significant overdispersion was observed. The negative binomial distribution was
367 TABLE I T H E N U M B E R S OF S E C O N D M E T A P H A S E CELLS OBSERVED PER 100 CELLS A N A L Y Z E D F R O M 48-h C U L T U R E S OF B L O O D P R E T R E A T E D F O R I h AS I N D I C A T E D The experiment was repeated 4 times with replicates analyzed on each occasion. Expt.
Oxygenated
Venous
Anoxic
1
a b
4 1
0 2
4 2
2
a b
9 5
2 I
5 3
3
a b
4 4
2 4
6 6
4
a b
2 0
1 4
0 3
3.6
2.0
3.6
Mean (%)
found to provide a good fit to these data ( P = 0.97). Dicentric and total aberration yields have been fitted to the linear-quadratic dose-response equation Y = aD + BD 2 using a least-squares method and weighting each value of aberrations per cell by the inverse of the sample variance ( n i / s 2, where n i is the number of cells). Results are shown in eqns. (1) to (4) and curves for TABLE 2 C H R O M O S O M E A B E R R A T I O N S I N D U C E D BY 15-MeV N E U T R O N S IN O X Y G E N A T E D A N D ANOXIC BLOOD Dose (Gy)
Cells scored
Normal ceils
Dicentrics
Total aberrations
200 1000 1000 1000 695 657 258 199
198 973 901 845 497 431 83 29
0 17 74 113 174 197 207 311
2 35 131 214 299 360 394 495
200 500 500 500 329 500 500 226 100
200 494 493 465 307 421 331 109 35
0 5 6 19 20 64 147 113 93
0 7 10 48 39 117 282 199 133
Oxygenated 0 0.08 0.20 0.40 0.60 0.80 1.60 2.40 Anox~ 0 0.08 0.20 0.40 0.60 0.80 1.60 2.40 3.20
368
dicentric yield are plotted in Fig. 1. YN = (6.81 ___ 1.34) × 10 -2 D + (6.61 ___0.84) X 10 --2
( P = 0.55)
(1)
Yo = (25.50 __+3.07) × 10-2 D + (16.15 ___2.82) × 10-2 D 2 ( p = 0.10)
(2)
D2
YN and Yo are dicentric yields observed after radiation dose D in anoxic and fully oxygenated conditions respectively. AN =(16.23_3.39)×
10-2 D + (9.17___ 2.02) × 1 0 - 2 D 2
( P = 0.06)
A o=(49.50__+4.60)>(lO-2D+(24.25___4.02)XlO-2D2 ( P = 0 . 1 0 )
(3) (4)
A N and A o are yields of total aberrations observed after radiation dose D in anoxic and fully oxygenated conditions respectively. Included are multicentrics, centric rings, and acentric fragments, all of which can prevent a cell from undergoing repeated cycles of division [6,30]. The a and fl coefficients from eqns. 1-4 have been used to derive limiting values for the OER which are given in Table 3 with comparable results obtained previously by X-irradiation. At low neutron doses below about 1 Gray, the linear (aD) component of yield predominates and an OER of 3.7 _+ 0.9 is obtained from the a
1.6
1.4 OXYGENATED 1.2
_-
o=
1.0
== .~ 0.8
¢3 0.6
ANOXIC
0.4
0.2
0m.a"~-
t
I
I
1.0
2.0
3.0
Dose, Gy
Fig. !. The yield of dicentrics per cell -4-SE plotted against dose for lymphocytes irradiated under oxygenated or anoxic conditions.
369 TABLE 3 A COMPARISON OF THE a AND ~ COEFFICIENTS FROM THE LINEAR-QUADRATIC EQUATIONS FITTED TO ABERRATION YIELDS INDUCED BY 15-MeV NEUTRONS AND 250 kVp X-RAYS UNDER OXYGENATED (O) AND A N O X I C (N) CONDITIONS
Neutron
~o
~o
~#o
Dicentrics SE
3.7 0.9
2.4 0,5
1.6 0.2
Total aberrations SE
3.1 0.7
2.6 0.7
t .6 0.2
X-Ray
Dicentrics
7.2
[ 18]
SE
4.6
7.1 0.9
2.7 0.2
Total aberrations SE
3.6 1.9
7.4 1.3
2.7 0.2
coefficient ratio for dicentric production. This compares with a ratio of 7.2 for 250 kVp X-rays [18]. As the neutron dose is increased and the_flD 2 component of yield becomes more important, the OER tends towards fflOflN, which is 1.6 ± 0.2 for dicentrics (Table 3). Table 4 gives values for the low dose RBE [15] for 15-MeV neutrons with respect to 250-kVp X-radiation for dicentric and total aberration induction [18]. With increasing dose of neutrons the flD 2 term becomes more important and the RBE
TABLE 4 MAXIMUM RBE VALUES CALCULATED FROM aNEUTRON/aX.RAY DERIVED FROM Eqns. 1-4 AND X-IRRADIATION [18] Dicentrics Oxygenated SE Anoxic SE
Total aberrations
5.4 2.1
5.5 1.9
10.3 5.5
6.5 2.8
TABLE 5 MINIMUM RBE VALUES CALCULATED FROM V/flNEUTRON/flX.RAV DERIVED FROM Eqns. 1-4 AND X-IRRADIATION [18]. Dicentrics
Total aberrations
Oxygenated SE
1.3 0.1
1.2 0.1
Anoxic SE
2.2 0.2
2.0 0.3
370
~/(flneutron/flX-ray)"
tends t o w a r d s High dose RBE values are given in Table 5 and it is evident that ratios obtained from both dicentric and total aberration yields are significantly greater in the case of blood irradiated under anoxic conditions than with oxygenated blood. Discussion
Contrary to some observations [5] but not others [20], the average frequency of second-division metaphases observed in 48-h cultures of venous blood (Table 1) was not significant, amounting to only 2%. A low yield of second cycle metaphases (less than 14%) under the culture conditions used here has been noted previously [20] and further experiments [21; Purrott, personal communication] have indicated that the use of an anhydric incubator and hence an effectively shortened time at 37°C and also the use of the relatively impoverished Eagle's Minimal Essential Medium leads to a significant reduction in the frequency of second-cycle metaphases at 48 h in all donors examined so far. There is good evidence that this frequency is further reduced with increasing radiation dose due to mitotic delay [20]: None of the values in Table 1 is significantly different from the overall average of 3.1% showing that the pretreatment did not influence the rate of response of the lymphocytes to PHA. This indicates that the variation in aberration yield with oxygen concentration is due entirely to a change in radiosensitivity caused by the presence of oxygen and not to any effect on the cell-cycle kinetics. In the radiation experiments described here, irradiated blood was cultured without the addition of BrdU to enable direct comparison with previously published data for X-rays [18] and to avoid any complicating effects resulting from the incorporation of this mutagenic base analogue into chromosomal DNA. The numbers of second-cycle metaphases analyzed, with consequent underestimation of aberration yields are considered insignificant, particularly at the higher doses. For neutrons with an energy high enough to be clinically useful, the OER is usually in the range 1.5 to 1.8 compared with about 2.5 for X-rays [7]. Watson [29], in a study of the cytogenetic effect of 15-MeV neutrons on human lymphocytes irradiated in saline suspension, observed OER values which were dependent upon the frequency of dicentrics and ranged from 3.7 __+1.5 at 0. I dicentrics per cell to 1.2-+-0.3 at 0.4 dicentrics pet cell. Corresponding values calculated from eqns. (1) and (2) are 2.3 _ 0.6 at 0.1 dicentrics per cell and 2.1 ___0.5 at 0.4 dicentrics per cell. Watson's results were obtained at a culture time of 60 h when the maximum aberration frequency was observed and her dose-response curves were complex, some even showing a decrease in yield at the highest dose. In contrast, results in Table 2 were obtained after 48 h in culture and particularly for dicentric aberrations fitted satisfactorily to the linear-quadratic expression. As argued previously [18], for an "effect-modifying" factor where the ratio of aberration yields is constant for any given dose: O/O__ flO __ (XO
~N
constant
371
From Table 3 it can be seen that a o / a N and flo//BN are not significantly different. For a dose=modifying factor where the ratio of doses is constant for any given aberration yield: a_..~o_ ~
= constant
aN--
Columns 2 and 4 of Table 3 were compared by calculating the value of y and its standard error using the equation
aN
and the standard formula for the variance of a function of several random variables [12]. For a dose-modifying factor the value of y will not be significantly different from zero. From the coefficients for dicentric yield the value of y/s.e..r~_~-f_)~ascalculated to be 2.1, indicating that a o / a N is significantly different from ~/Bo/BN at the 5% level and therefore the value of OER is not constant with dose. Using coefficients for total aberration yield from Eqns. 3 and 4 a value of 1.6 is obtained for y/s.e.(y) indicating that a o / a ~ is not significantly different from ~/Bo/Bs even at the 10% level. Acentric aberrations are generally less reliable as an indicator of radiation damage and their inclusion in the total aberration data means that it becomes impossible to discriminate between the effect-modifying and dose-modifying hypotheses. For dicentric induction, however, it can be concluded that oxygen is not acting as a dose-modifying agent. Results from animal experiments suggests that an increase in the RBE of neutrons under hypoxic conditions would be expected. The actual values differ with the tissue examined and may not be relevant to man [7]. Tables 4 and 5 give values for the RBE of 15-MeV neutrons calculated from Eqns. 1-4 and data obtained using blood From the same donor irradiated with 250 kVp X-rays [18]. Because of the large errors in Table 4 due mainly to the small number of aberrations observed after low doses of X-rays, there appears to be no difference at low doses between the RBE under oxygenated or anoxic conditions. However, high dose values calculated from ~/Bneutron/flx-,,y (Table 5) do show a marked increase in RBE for anoxic irradiations giving approximately twice the oxygenated value for both dicentric and total aberrations.
Conclusions The analysis of lymphocytes for unstable chromosome aberrations which are lethal lesions in dividing cell systems is a useful radiobiological method for investigating the relationship between RBE, OER and LET. Whilst the absolute values derived depend very much on the experimental system used, the data presented here conform with other radiobiological end points in that values of OER which ranged
372 f r o m 3.7 to 1.6, are m a r k e d l y lower t h a n values o b t a i n e d previously for 250-kVp X-rays (7.2 to 2.7). T h e results do, however, suggest that oxygen does n o t act as a d o s e - m o d i f y i n g agent i n this system. Values derived for R B E were greatest at low n e u t r o n dose where the presence of oxygen did n o t i n f l u e n c e the value obtained. A t high doses RBEs were greater for a b e r r a t i o n s i n d u c e d u n d e r anoxic conditions. This is p r o b a b l y due to the greater c o n t r i b u t i o n of single-track d a m a g e i n d u c t i o n b y n e u t r o n s u n d e r these c o n d i t i o n s c o m p a r e d with X-rays.
Acknowledgements T h e e x p e r i m e n t a l a p p a r a t u s was designed b y A.G. Sherwin who together with C.L. H a r v e y a n d J.B. O ' H a g a n provided n e u t r o n i r r a d i a t i o n a n d dosimetry. Dr. S.C. D a r b y was responsible for statistical analyses a n d curve-fitting. This work was partly s u p p o r t e d b y E u r a t o m c o n t r a c t 171-76-1 B 1 0 - U K .
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