Proc. Nati. Acad. Sci. USA Vol. 85, pp. 4020-4024, June 1988 Medical Sciences Central nervous system radiation syndrome in mice from preferential 10B(n,a)7Li irradiation of brain vasculature (lethality/endothelium/x-ray/enhancement) D. N. SLATKIN, R. D. STONER, K. M. ROSANDER*, J. A. KALEF-EZRAt, AND J. A. LAISSUEf Medical Department, Brookhaven National Laboratory, Upton, NY 11973 Communicated by Eugene P. Cronkite, January 4, 1988 ABSTRACT Ionizing radiations were directed at the heads of anesthetized mice in doses that evoked the acute cen- tral nervous system (CNS) radiation syndrome. Irradiations were done using either a predominantly thermal neutron field at a nuclear reactor after intraperitoneal i'jection of '°B-en- riched boric acid or 250-kilovolt-peak x-rays with and without previous intraperitoneal injection of equivalent unenriched bo- ric acid. Since '°B concentrations were =3-fold higher in blood than in cerebral parenchyma during the reactor irradiations, more radiation from a and 7Li particles was absorbed by brain endothelial cells than by brain parenchymal cells. Com- parison of the LDjO dose for CNS radiation lethality from the reactor experiments with the LD50 dose from the x-ray experi- ments gives results compatible with morphologic evidence that endothelial cell damage is a maijor determinant of acute lethali- ty from the CNS radiation syndrome. It was also observed that boric acid is a low linear energy transfer radiation-enhance- ment agent in vivo. Irradiation of the head of a mouse by more than 120 Gy of x- rays usually causes death from the acute central nervous system (CNS) radiation syndrome within 3.5 days after irra- diation (1). Blood vessels are damaged in the acute CNS syn- drome (2). If a 10B-enriched substance is injected rapidly into a mouse and then penetrates the blood-brain barrier slowly, endothelial cells and parenchymal cells of the brain will be irradiated unequally by heavy charged particles (HCP) from the 10B(n,a)7Li nuclear reaction (3) when the head is ex- posed to thermal neutrons before concentrations of borate in the vascular and extravascular fluid compartments of the brain equilibrate. Thus, slow penetration of the blood-brain barrier by borate anions facilitates boron-neutron capture ir- radiation of the brain to induce the CNS radiation syndrome in the mouse under conditions of preferential irradiation of brain endothelial cells. The lethality of such microscopically anisotropic irradiation of mouse brains is compared quantita- tively with comparable lethality 'from exposure of mouse brains to 250-kilovolt-peak (kVp) x-rays to provide dosimet- ric evidence for an "endothelial" pathogenesis of the acute CNS radiation syndrome.§ METHODS Irradiations. Anesthetized mice (8- to 16-week-old, female Swiss albino mice of the Brookhaven National Laboratory Hale-Stoner strain) were confined to plastic tubes for place- ment into body-shielding holders (Fig. 1) for boron-neutron capture irradiation or x-irradiation of their heads (5, 1). Neu- tron exposures were carried out at the Brookhaven National Laboratory Medical Research Reactor (6), operated at 3 MW power. X-ray exposures were carried out with a Maxitron 250 (General Electric) at 250 kVp through copper (0.5 mm) and aluminum (1.0 mm) filters (first half-value layer, 1.77 mm Cu; homogeneity coefficient, 0.49).11 Boric acid [95.0 ± 0.5 atom % °B-enriched H3BO3 (Eagle-Picher, Miami, OK)** in reactor experiments or normal unenriched H3BO3 in some (see Table 3) x-ray experiments] was injected intra- peritoneally 15-35 min before the start of irradiation in an aqueous solution that provided 12.5 umol of boron and 0.02 ml of H20 per g of body weight. Dosimetry. The minor, naturally occurring stable isotope of boron, 10B, has an exceptionally high effective capture cross-section for thermal neutrons, 3.40 x 10-25 m2 at 2482 ms-1, the average neutron speed at 200C (8, 9). In 6% of such captures, the 10B(n,a)7Li reaction leads to the lower 7Li energy state (Ea = 1.777 MeV; ELU = 1.014 MeV; 1 eV = 1.602 x 10-19 J), whereas in 94% of captures, the reaction leads to the first excited state of 7Li, from which a 478-keV photon is emitted (Ea = 1.471 MeV; ELI = 0.839 MeV). For uniform distribution of boron, the kinetic energy released in matter (kerma) rate due to these HCP is, therefore, 7.68 x 10-12 FBq Gy-s-', where FB is the 10B mass fraction in the tissue and 4 is the thermal neutron fluence rate (s-1.m-2).tt For endothelial cells, the usual assumption of equivalence between the kerma rate and the absorbed dose rate is inap- plicable. Since endothelial cells demarcate the physiological blood-brain barrier anatomically (10, 11), and since blood 10B concentrations were about 3 times greater than paren- chymal 10B concentrations during exposure to neutrons (Fig. 2, Table 1), there was then a significant difference between the average 10B mass fractions in the blood, Fb,B, and in the parenchyma on the extraluminal side of endothelial cells, FpB- Almost all kinetic energy is imparted to small cylindri- cal volumes of tissue [:14 ,um long (a 9 um, 7Li 5 ,um; ref. 13) and :0.1 ,um in diameter] that envelop the colinear paths of the two mutually recoiling HCP. The radial gradi- Abbreviations: CNS, central nervous system; GyE, Gy equivalent; HCP, heavy charged particle; kerma, kinetic energy released in mat- ter; kVp, kilovolt peak; RBE, relative biological effectiveness. *Research Collaborator from the Gustaf Werner Institute, Uppsala University, S-75121 Uppsala, Sweden and the National Defence Research Institute, S-90182 Umei, Sweden. tResearch Collaborator from the Department of Medical Physics, Faculty of Medicine, University of Ioannina, Ioannina, 45332 Greece. tResearch Collaborator from the Institute of Pathology, Cantonal Hospital, CH-6004 Lucerne, Switzerland. §Preliminary results of this study were presented at the 1986 Winter Meeting of the American Nuclear Society (4). IThe upper surface of the plastic mouse holder (Fig. 1 c and d), not the upper surface of its Pb shield (5), was in a horizontal plane 9.5 cm below the focal point of the anode of the x-ray generator. Ther- moluminescent dosimetry shows that mouse heads were irradiated within the penumbra of a cone of radiation from the x-ray generator and that backscattered radiation was less than estimated previously (5). 1The homogeneity coefficient is the ratio of the first to the second half-value layer after penetration of the beam into a target. **In some experiments, 95.0 ± 0.5 atom % 10B-enriched boric acid prepared by P. C. Tompkins and A. D. Conger was used (7). ttThe kerma rates (Gy s-1) due to a and 7Li HCP are 4.89 x 10-12 FBO and 2.79 X 10-12 FB4, respectively. 4020 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Transcript
Proc. Nati. Acad. Sci. USAVol. 85, pp. 4020-4024, June 1988Medical Sciences
Central nervous system radiation syndrome in mice frompreferential 10B(n,a)7Li irradiation of brain vasculature
(lethality/endothelium/x-ray/enhancement)
D. N. SLATKIN, R. D. STONER, K. M. ROSANDER*, J. A. KALEF-EZRAt, AND J. A. LAISSUEfMedical Department, Brookhaven National Laboratory, Upton, NY 11973
Communicated by Eugene P. Cronkite, January 4, 1988
ABSTRACT Ionizing radiations were directed at theheads of anesthetized mice in doses that evoked the acute cen-tral nervous system (CNS) radiation syndrome. Irradiationswere done using either a predominantly thermal neutron fieldat a nuclear reactor after intraperitoneal i'jection of '°B-en-riched boric acid or 250-kilovolt-peak x-rays with and withoutprevious intraperitoneal injection of equivalent unenriched bo-ric acid. Since '°B concentrations were =3-fold higher in bloodthan in cerebral parenchyma during the reactor irradiations,more radiation from a and 7Li particles was absorbed bybrain endothelial cells than by brain parenchymal cells. Com-parison of the LDjO dose for CNS radiation lethality from thereactor experiments with the LD50 dose from the x-ray experi-ments gives results compatible with morphologic evidence thatendothelial cell damage is a maijor determinant of acute lethali-ty from the CNS radiation syndrome. It was also observed thatboric acid is a low linear energy transfer radiation-enhance-ment agent in vivo.
Irradiation of the head of a mouse by more than 120 Gy of x-rays usually causes death from the acute central nervoussystem (CNS) radiation syndrome within 3.5 days after irra-diation (1). Blood vessels are damaged in the acute CNS syn-drome (2). If a 10B-enriched substance is injected rapidly intoa mouse and then penetrates the blood-brain barrier slowly,endothelial cells and parenchymal cells of the brain will beirradiated unequally by heavy charged particles (HCP) fromthe 10B(n,a)7Li nuclear reaction (3) when the head is ex-posed to thermal neutrons before concentrations of borate inthe vascular and extravascular fluid compartments of thebrain equilibrate. Thus, slow penetration of the blood-brainbarrier by borate anions facilitates boron-neutron capture ir-radiation of the brain to induce the CNS radiation syndromein the mouse under conditions of preferential irradiation ofbrain endothelial cells. The lethality of such microscopicallyanisotropic irradiation of mouse brains is compared quantita-tively with comparable lethality 'from exposure of mousebrains to 250-kilovolt-peak (kVp) x-rays to provide dosimet-ric evidence for an "endothelial" pathogenesis of the acuteCNS radiation syndrome.§
METHODSIrradiations. Anesthetized mice (8- to 16-week-old, female
Swiss albino mice of the Brookhaven National LaboratoryHale-Stoner strain) were confined to plastic tubes for place-ment into body-shielding holders (Fig. 1) for boron-neutroncapture irradiation or x-irradiation of their heads (5, 1). Neu-tron exposures were carried out at the Brookhaven NationalLaboratory Medical Research Reactor (6), operated at 3 MWpower. X-ray exposures were carried out with a Maxitron250 (General Electric) at 250 kVp through copper (0.5 mm)and aluminum (1.0 mm) filters (first half-value layer, 1.77
mm Cu; homogeneity coefficient, 0.49).11 Boric acid [95.0 ±0.5 atom % °B-enriched H3BO3 (Eagle-Picher, Miami,OK)** in reactor experiments or normal unenriched H3BO3in some (see Table 3) x-ray experiments] was injected intra-peritoneally 15-35 min before the start of irradiation in anaqueous solution that provided 12.5 umol of boron and 0.02ml of H20 per g of body weight.
Dosimetry. The minor, naturally occurring stable isotopeof boron, 10B, has an exceptionally high effective capturecross-section for thermal neutrons, 3.40 x 10-25 m2 at 2482ms-1, the average neutron speed at 200C (8, 9). In 6% ofsuch captures, the 10B(n,a)7Li reaction leads to the lower 7Lienergy state (Ea = 1.777 MeV; ELU = 1.014 MeV; 1 eV =
1.602 x 10-19 J), whereas in 94% of captures, the reactionleads to the first excited state of 7Li, from which a 478-keVphoton is emitted (Ea = 1.471 MeV; ELI = 0.839 MeV). Foruniform distribution of boron, the kinetic energy released inmatter (kerma) rate due to these HCP is, therefore, 7.68 x10-12 FBq Gy-s-', where FB is the 10B mass fraction in thetissue and 4 is the thermal neutron fluence rate (s-1.m-2).ttFor endothelial cells, the usual assumption of equivalencebetween the kerma rate and the absorbed dose rate is inap-plicable. Since endothelial cells demarcate the physiologicalblood-brain barrier anatomically (10, 11), and since blood10B concentrations were about 3 times greater than paren-chymal 10B concentrations during exposure to neutrons (Fig.2, Table 1), there was then a significant difference betweenthe average 10B mass fractions in the blood, Fb,B, and in theparenchyma on the extraluminal side of endothelial cells,FpB- Almost all kinetic energy is imparted to small cylindri-cal volumes of tissue [:14 ,um long (a 9 um, 7Li 5 ,um;ref. 13) and :0.1 ,um in diameter] that envelop the colinearpaths of the two mutually recoiling HCP. The radial gradi-
Abbreviations: CNS, central nervous system; GyE, Gy equivalent;HCP, heavy charged particle; kerma, kinetic energy released in mat-ter; kVp, kilovolt peak; RBE, relative biological effectiveness.*Research Collaborator from the Gustaf Werner Institute, UppsalaUniversity, S-75121 Uppsala, Sweden and the National DefenceResearch Institute, S-90182 Umei, Sweden.tResearch Collaborator from the Department of Medical Physics,Faculty of Medicine, University of Ioannina, Ioannina, 45332Greece.tResearch Collaborator from the Institute of Pathology, CantonalHospital, CH-6004 Lucerne, Switzerland.§Preliminary results of this study were presented at the 1986 WinterMeeting of the American Nuclear Society (4).IThe upper surface of the plastic mouse holder (Fig. 1 c and d), notthe upper surface of its Pb shield (5), was in a horizontal plane 9.5cm below the focal point of the anode of the x-ray generator. Ther-moluminescent dosimetry shows that mouse heads were irradiatedwithin the penumbra of a cone of radiation from the x-ray generatorand that backscattered radiation was less than estimated previously(5).1The homogeneity coefficient is the ratio of the first to the secondhalf-value layer after penetration of the beam into a target.
**In some experiments, 95.0 ± 0.5 atom % 10B-enriched boric acidprepared by P. C. Tompkins and A. D. Conger was used (7).
ttThe kerma rates (Gy s-1) due to a and 7Li HCP are 4.89 x 10-12FBO and 2.79 X 10-12 FB4, respectively.
4020
The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Proc. Natl. Acad. Sci. USA 85 (1988) 4021
FIG. 1. (a and b) Reactor irradiation mouse holder; front (a) andrear (b) views. There is a 12-mm-wide air gap between the front faceof the holder and the Bi metal face plate of the reactor port, intowhich mouse heads project downward. A plastic plate is used hereas a transparent phantom for the Bi face plate. (c and d) Rotating x-
irradiation mouse holder without its 1/4-inch Pb shield; general (c)and close-up (d) views. Mouse heads are irradiated in an unshielded12-mm-wide air gap. Scale is indicated by the 1-inch diameter mousetubes.
ents of absorbed dose in these slender cylinders are verysteep (14, 15) and therefore are neglected because most
10 20 30 40 50 60 70
TIME AFTER HO0B0 INJECTION (min)3 3
FIG. 2. Concentrations of 10B in blood and cerebrum after a sin-gle intraperitoneal injection of boric acid [12.5 ,umol of boron (12.0jamol of 10B) per g of body weight]. Each point in a vertical barrepresents mean and range of 10B concentrations in two to fourmice. At 50 and 60 min after injection, cerebral concentrations arefrom one mouse each.
blood vessels have dimensions >0.5 ,um.Using either bare or Cd-shielded Au foils and wires with a
polymethylmethacrylate mouse dosimetry phantom, it wasdetermined that the thermal neutron fluence rate at the Bimetal face plate of the reactor port, against which mouseheads were buttressed (Fig. 1), was 6.9 x 1014 s-1m-2 (Cdratio, 13), and that the fluence rate decreased almost expo-nentially by the factor e-l07d with distance d (in meters) fromthe vertical plane of the face plate to a parallel plane in themouse phantom. This result was verified by inserting Auwires transversely at several positions in the brains of re-cently killed mice through a hypodermic needle and then ir-radiating the dead mice at the reactor as the experimentalmice and the mouse dosimetry phantom were irradiated.Thus, the absorbed dose rate, DHCPB (Gy's-), to endotheli-
Table 1. Reactor irradiations of 10B-enriched boric acid-injected miceAverage 10B
Duration of Average H3BO3 injection concentration duringirradiation, to midirradiation interval irradiation, gg/g Mortality <4 days after
Mouse group s (± SD), min Blood Cerebrum irradiation, fraction
Summary of acute (<4 days postirradiation) mortality data from 321 anesthetized, H310B03-injected (see text) miceexposed to a shutter-controlled irradiation port ofthe Brookhaven National Laboratory Medical Research Reactor (operatedat 3 MW) with their heads unshielded and their bodies substantially shielded from thermal neutrons. The duration ofirradiation that corresponds to LD50 was extrapolated from these data by normalizing the average concentration of '0B inthe blood during reactor irradiations (column 3) to a standard 10B concentration (105 jzg/g). The corresponding 10Bconcentration in the cerebrum is 37 Ag/g (see Fig. 2), a value that differs only slightly from the weighted average ofobservedvalues (35 Aug/g). The extrapolation to LD" was performed by probit analysis using Bliss's weighting coefficients (12).
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Table 2. Significant sources of reactor-induced radiations in the brains of '0B-injected miceSource Tissue dose rate, Gy s'lno., j Type of radiation RBE, Rj (SI units)
1 585-keV protons and 42-keV 14C particles from 14N(n,p)14C 2.0 [RN] 7.06 x 10-16 FNOOe- 107d2 478-keV y photons from '0B(n,a)7Li 1.0 1. x 10-14FB4O3 2.22-MeV y photons from 1H(ny)2H due to the hydrogen content of the mouse head 1.0 4 x 10-18 4o4 /3 particles from decay of radionuclides created by slow neutrons in tissue 1.0 1 x lo-18 4,5 y photons from the mouse holder and from the reactor 1.0 0.84 x 10-26 Fast and epithermal neutrons from the reactor 2.0 1.17 x 10-27 7Li and a particles from 10B(n,a)7Li - [RB] 7.68 x 10-12 FB4oe-l07dSignificant sources of radiation to brain cells of 10B-treated mice, the heads of which were exposed to a port of the Brookhaven Medical
Research Reactor operated at 3 MW. Indices j = 1-6 correspond to the most important radiations that accompany the radiations from the10B(n,a)7Li reaction. RB denotes the RBE of the HCP from the 10B(n,a)7Li reaction (relative to a standard RBE of 1.0 for 250-kVp x-rays) withrespect to lethality from the acute CNS syndrome before 4 days after irradiation.
al cells from HCP due to 10B in blood and parenchyma canbe expressed as
DHCP,B = (7.68 X 10-12)LfBFb B + (1 - fB)Fp,B1]oe107d, [1]
where: fB is the fraction of the HCP dose to endothelial cellsthat would be due to 10B in the adjacent blood if the endothe-lial cells were surrounded completely by a tissue with thesame concentration of 10B and the same HCP stopping pow-er as the blood; Fb,B and FP,B are the 10B mass fractions inblood and parenchyma, respectively; 40 is the thermal neu-tron fluence rate (s-1.m-2) at the face plate of the reactorport. Although the dose from 10B within endothelial cells isneglected in these calculations because of our ignorance ofthe gradients of borate concentrations across these cells dur-ing irradiations, it is assumed that borate diffuses in aqueoustissue compartments and that 10B concentrations in endothe-lial cells were intermediate between blood and parenchymalconcentrations. The neglect introduces some error into thesecalculations (16), but if endothelium does not accumulate bo-rate selectively this source of error must be small because ofthe thinness of endothelial cells relative to the range of alphaparticles.Other nuclides in brain tissue, most importantly 1H, '4N,
23Na, 31P, 38C1, and 41K, also capture thermal neutrons andirradiate the brain by liberating HCP and photons and byforming ,/emitting radionuclides. The non-1OB-related radia-tion dose rates to the brain (Table 2) are based on averageconcentrations of elements in the brain (17) and on generallyaccepted relative biological effectiveness (RBE) values (Ta-ble 2). As in the case of 10B, the nitrogen mass fraction inmammalian blood is higher than that in brain: Fb,N = 0.029;FP,N= 0.019 (17). Thus, the absorbed dose rate (Gy s-1) inendothelial cells due to 0.585 MeV protons and 0.042 MeV14C particles from the 14N(np)14C reaction (effective ther-mal neutron capture cross-section, 1.64 x 10-28 M2) iS:#
To a first approximation, fN = fB = f because a particlesfrom the 10B(n,a)7Li reaction and protons from the 4N(n,p)-14C reaction have similar ranges in tissue (=9 ,m) and ac-quire a major portion of the energy liberated by their respec-tive reactions of origin and because, in this study, the ab-sorbed dose of HCP radiation to endothelial cells from 14Ndisintegrations was <4% of that from 10B disintegrations.A tissue-equivalent ionization chamber (IC-17A, Far West
Technology) was used to determine doses from exogenousgamma photons and from fast and epithermal neutrons whilethe reactor port was shielded with 2-mm-thick 6Li metal toabsorb thermal neutrons. In separate exposures, 7Li-en-riched thermoluminescent dosimeters (TLD-700, Harshaw/Filtrol Partnership) were irradiated at the center of small vi-als containing 6Li-enriched LiF powder. The shapes of glowcurves (18) and the activation of Au wires confirmed ade-quate shielding from thermal neutrons by 6Li metal. The ex-trinsic 'y (j = 5) and nonthermal neutron (j = 6) dose rateswere determined by combining the thermoluminescent do-simeter and ionization chamber measurements. The doserate from induced ,B radioactivity (j = 4) in the head wascalculated by assuming that the average /8 particle tracklength within the head was half the average chord length in a1-cm-diameter sphere (-3 mm) (19). The dose rates to themouse head from y photons generated by neutron capturereactions in the mouse (j = 2, 3) were calculated (20).The total biologically effective dose rate [Gy equivalent
(GyE) s-] to brain endothelial cells in a vertical plane d me-ters from the face plate of the reactor port is then
(7.68 x 10'12)RB[fFb, B + (1 - f)FpB1Oe 10
+ (7.06 x 10-16)RN[fFbN + (1 - f)Fp,N]Oe -107d
6
+ I RjDj, [3]j=2
where Dj (j = 2, . . ., 6) are absorbed dose rates from thefive minor radiations (Table 2) that are assumed to irradiatethe brain uniformly and Rj are the corresponding RBE withrespect to death from the CNS radiation syndrome before 4days after irradiation. RB and RN are the RBE for HCP fromneutron capture by 10B and 14N, respectively. It is assumedthat the arithmetic sum of GyE measures of biologicallyeffective radiation from the individual components of themixed reactor radiation field is an appropriate measure ofthe effectiveness of the combined radiations for acute CNSlethality.
X-ray dosimetry was performed with LiF thermolumines-cent dosimeters. The absorbed x-ray dose rate, Dx, at theanterior, buttressed surfaces of mouse heads was 153 ± 10mGy s-1. The absorbed dose of x-rays in mice decreasedradially by the factor e-21d, where d (in meters) is the dis-tance from the plane that is tangent to the vertical, cylindri-cal plastic head buttress of the x-irradiation mouse holder toa parallel plane of interest in the mouse or in the mouse do-simetry phantom. Because the width of the gaps between theanterior rims of the cylindrical tubes in which mouse bodieswere confined and the vertical buttresses to which mouseheads were apposed was the same (12 mm) for x-ray andreactor irradiations (Fig. 1), structures in mice that were intransverse, vertical planes at the same distance d from these
#For uniform distribution of nitrogen in tissue (mass fraction FN'99.63 atom % 14N) the total kerma rate, 7.06 x 1016 FNO Gy*s,comprises 6.59 x 10-16 FNq5 from proton and 0.47 x 10-16 FNofrom 14C HCP, respectively.
Proc. NatL Acad Sci. USA 85 (1988)
Proc. Natl. Acad Sci. USA 85 (1988) 4023
two buttresses were anatomically comparable targets of irra-diation.
This "endothelial" model of the pathogenesis of acuteCNS radiation deaths implies the following equality for bio-logically equivalent doses of radiation (GyE) to endothelialcells in anatomically comparable planes of the brain fromreactor irradiations (left side of equation) and from 250-kVpx-rays (right side of equation):
tr[(7.68 X 10 12)oeO 107dRB[FP,B + f(Fb,B -FP,B)]
+ (7.06 x 10 16)4Ooe107dRN[FPN + f(FbN - FP,N)I6
+ 2> RjDj] = txbxe-21d,j=2
[4]
where tr and t,, are the extrapolated LD50 irradiation timesfor the reactor exposures (649 s; Table 1) and for the x-rayexposures (890 s; Table 3), respectively. The RBE of x-rayswas assumed to be 1.0. Solving Eq. 4 for RB, one derives an
where K1, K2 . .. K5 are constants calculated from the dataof Tables 1, 2, and 3. These constants are 1.070, 0.0943,0.526, 0.181, and 1.838, respectively. The relationships ofTable 4 are computed from Eq. 5.Boron Concentrations. Mice were exsanguinated under
deep ether anesthesia before removing cerebra for '0B analy-sis. Concentrations of 10B in whole blood and cerebra weremeasured by prompt neutron activation analysis (21). The10B uptake in blood and cerebrum is shown in Fig. 2. Supple-mentary experiments (data not shown) indicate that the rateof transport of borate into brain parenchyma was not alteredby approximate LD50 doses of reactor radiations or of x-radi-ation to the heads of mice during the first hour after injectionof similar doses of boric acid.
RESULTS
The physical conditions of irradiations and the fractions ofmice that died before 4 days after irradiation (day of irradia-tion = day 0) are summarized in Tables 1, 3, and 5. Charac-teristic signs of the acute CNS syndrome without diarrhea(1, 5) were observed during the first 3 days after irradiation.Those mice that lived 4 days or more after head irradiationdied within 10 days after irradiation, apparently from com-bined gastrointestinal (2, 22, 23) and CNS radiation damage.Thus, the fraction of mice that died before 4 days after headirradiation was a convenient measure of the lethality of radi-
Table 3. X-ray irradiations of non-'0B-enriched boricacid-injected mice
Average H3BO3Duration X-ray dose injection to Mortality <4
of at head midirradiation days afterMouse irradiation, buttress, interval (± SD), irradiation,group s Gy min fraction
Summary of acute (<4 days postirradiation) mortality data from320 anesthetized mice exposed to 250-kVp x-rays. The heads wereunshielded and the bodies were substantially shielded in a slowlyrotating holder (Fig. 1) during exposure. Extrapolation to LD50 wasby probit analysis using Bliss's weighting coefficients (12). Mice wereinjected with boric acid before irradiation (see text). cum, Cumula-tive.
ation damage to the brain (1). The brains of mice killed 2days after an approximate LD50 dose of predominantly 10B-(n,a)7Li irradiation of the head show dilatation of capillariesand of pericapillary spaces with swelling of endothelial cellcytoplasm. The extravascular structures of the cerebrum(but not of the cerebellum) appear to be histologically normal2 days after such reactor irradiation (J.A.L., unpublisheddata).
DISCUSSIONCalculations of HCP doses to endothelial cells (24-26) showthat f is within the range 0.1-0.5 for radiation targets that are<2 Am from a blood vessel lumen. Since vital structures ofthe CNS occupied the zone 8 mm < d < 16 mm in the cranialcavity and vertebral canal, it seems likely from the data ofTable 4 that RB - 2.6. The compatibility of this result with invivo measurements ofRB (27-29) lends credence to an endo-thelial pathogenesis of the CNS radiation lethality syndromeand thereby underlines the significance of 10B in the bloodduring boron neutron capture therapy of brain tumors (30,31).When the CNS radiation syndrome was evoked with x-
RB is the in vivo RBE of particle radiation from the 10B(na)7Li reaction, distances d (meters) aremeasured horizontally from the mouse head buttress to radiation targets in the mouse (see text), andf is the average fraction of heavy charged particle radiation to endothelial cells that would be derivedfrom disintegration of 10B in the lumens of blood vessels if 10B were distributed uniformly around thosecells at the same concentration as in the blood (see text).
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4024 Medical Sciences: Slatkin et aL.
Table 5. X-ray irradiations of non-boric acid-injected mice
Duration X-ray doseof at head
Mouse irradiation, buttress, Mortality <4 days aftergroup s Gy irradiation, fraction
Summary of acute (<4 days postirradiation) mortality data from221 anesthetized mice, the heads of which were exposed to 250-kVpx-rays (Fig. 1). Extrapolation to LD5o was by probit analysis usingBliss's weighting coefficients (12). Boric acid was not administeredto these mice. cum., Cumulative.
rays after injection of boric acid (Table 3), the LD50 was 136Gy. Without preinjection of boric acid (Table 5), the LD50was 221 Gy. Thus, boric acid is a low linear energy transferradiation-enhancement agent with a radiation-enhancementfactor of :1.6. Whether this enhancement is primarily bio-chemical or primarily radiochemical (32), is unknown. Nev-ertheless, it should be considered appropriate for any pro-spective boron neutron capture therapy boron carrier sub-stance to be tested for its radiation-enhancement orradiation-protective characteristics.A RBE value of 2.3 for 10B-neutron capture radiation, de-
termined from an in vitro V79 Chinese hamster cell radiationlethality experiment (33), is within the RBE s2.6 limit indi-cated by this in vivo study.§§ Whether this in vitro-in vivocorrespondence reflects radiation damage to similar struc-tures in V79 cells and in brain endothelial cells may bedoubted because of the 34-fold difference between the radia-tion doses (-4 Gy and =136 Gy, respectively, from 250-kVpx-rays) required to observe 50% death in the two disparateexperimental systems. A RBE of 3.7 reported for the lethali-ty of 10B(n,a)7Li radiation in vitro (34) is apparently inappli-cable to acute CNS lethality, in vivo because such a largeRBE would place the most vital targets of brain irradiationoutside the cranial cavity, at least 18 mm (Table 4) from thehead buttress.
§§Atypical mortality fractions were observed from only 72 of the 617mice used to estimate the range of RBE for HCP from the 10B-(n,a)7Li reaction in vivo [in groups RiB and R1C (Table 1) andX2B (Table 2)]. Since the number of mice in each group is a multi-plicative factor for statistical weight of the group in this probitanalysis (12), elimination of the atypical data would not apprecia-bly affect the results of the study.
We thank C. N. Bachelet, E. J. Caiazza, E. N. Carter, A. B. Chi-tuk, M. Eskinatji, D. D. Greenberg, P. L. Micca, S. Sajnacki, N. R.Tempel, R. J. Walton, and M. Wigger for technical assistance. Wethank J. 0. Archambeau, A. L. Aronson, V. P. Bond, E. P. Cron-kite, R. G. Fairchild, D. D. Joel, B. Larsson, D. C. Rorer, L. Wie-lopolski, and anonymous reviewers for helpful comments. K.M.R.acknowledges support from the Swedish Medical Research Counciland the Magnus Bergvalls Stiftelse. This study was performed undercontract DE-AC02-76CH00016 with the U.S. Department of Ener-gy.
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