Acta Radiologica: Oncology, Radiation, Physics, Biology
ISSN: 0348-5196 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ionc18
Pharmacologic Effects of Radiation Protective Compounds Related to Their Protective Effect in Mice C.-O. Criborn & C. Rönnbäck To cite this article: C.-O. Criborn & C. Rönnbäck (1979) Pharmacologic Effects of Radiation Protective Compounds Related to Their Protective Effect in Mice, Acta Radiologica: Oncology, Radiation, Physics, Biology, 18:1, 31-44, DOI: 10.3109/02841867909128188 To link to this article: https://doi.org/10.3109/02841867909128188
Published online: 08 Jul 2009.
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FROM THE NATIONAL DEFENCE RESEARCH INSTITUTE, DIVISION OF RADIATION BIOLOGY, S-172 04 SUNDBYBERG, SWEDEN.
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS RELATED TO THEIR PROTECTIVE EFFECT IN MICE C.-0. CRIBORN and C.
RONNBACK
The effects of presumptive radiation protective compounds have mainly been investigated only with respect to their influence on the survival rate (expressed as LD,,,), but seldom as regards physiologic effects before, during and after irradiation. Neither have their negative effects been considered, sometimes due to the absence of suitable methods. CRIBORN (1969, 1970) presented methods concerning the determination of respiratory minute volume, oxygen consumption and respiratory response to acoustic excitation. A number of compounds with proved radiation protective effects were found to depress the oxygen consumption and respiratory response strongly during their active phase. In the present investigation the depressive effect of cysteamine on these parameters was simulated by the use of some psychopharmaca. Attempts were made (1) to decrease the effect of cysteamine on respiration and body temperature by using a central stimulating compound, amphetamine, and (2) to correlate the effects of these compounds to a change in the radiation protective effect of cysteamine. This would give a more differentiated evaluation of the effect of presumptive radiation protective compounds.
Material and Methods Male CBA mice weighing 28.3 ? 4 g, aged 70 to 110 days, were used in the experiments. The different substances were administered intraperitoneally, and the concentration of the solutions was adjusted to give an injection volume of 0.1 to Submitted for publication 3 March 1978. Acta Radiologica Oncology 18 (1979) F a x . 1
31
32
C.-0. CRIBORN AND C. RONNBACK
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Fig. 1. Effects of radiation protective compounds on respiratory minute volume and rectal temperature in mice. Controls. 0 Cysteamine, 210 mg/kg body weight. (3 A 295, 530 mg/kg body weight. 0 A 331, 18 mg/kg body weight. Mercaptoetanole, 180 mg/kg body weight.
4
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30
60 min
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3
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Time after irradiation
0.3 ml. Control animals were at the same time injected with 0.3 ml physiologic saline. A Muller MG300 apparatus gave a dose rate of 11.7 mGy/s (74 R/min) at 260 kV, 1 1 mA (HVL 2 mm Cu). The focal distance was 45 cm.
33
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS
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Fig. 2. a) Effects on oxygen consumption (left, 0 ) and rectal temperature (right, a)of a dose of 50 mg mebumal/kg body weight. b) The mortality related to time interval between administration of mebumal and start of irradiation 6.41 Gy (675 R); H indicate irradiation time.
The animals were irradiated in groups of 10 in a wheel of plastic, rotating 1 rpm to eliminate irregularities in the irradiation field. Following the irradiation, 5 animals were kept per cage. The body weight of the animals was measured with an accuracy of 0.1 g from the day before irradiation until some of the animals in that group had died. The weighing procedure was performed at 8 a.m. The rectal temperature was measured with a termistor thermometer. The oxygen consumption, respiratory minute volume and acoustic reaction were measured by means of a specially constructed apparatus (CRIBORN1969). This gave an estimate of the breathing and the acoustic reaction with a simultaneous measurement of the oxygen concentration in the tank surrounding the animals at examination. The oxygen consumption was obtained by pumping the air into the tank through a paramagnetic oxygen meter, which was read off before the animal was placed in the tank and also 3 min later. The oxygen consumption could then be calculated with a knowledge of the volume of the tank.
Results The respiratory minute volume as well as the rectal temperature decreased following the administration of the three radiation protective substances cysteamine, A295 or A331 (AKERFELDT et coll. 1967).(Fig. 1). The former decreased rapidly whereas the temperature reached its minimum point after about one hour. A recovery of the values started within one hour for all three substances. 3 - 795845
c.-0.CRIBORN AND c .
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Fig. 3. Effects on rectal temperature (a) and respiratory volume ( 0 ) by: Cysteamine, 210 mg/kg body weight (top); morphine chloride, 4.4 mg/kg body weight (middle); morphine chloride, 14.0 mg/kg body weight (bottom).
3020mon
Fig. 4. Effect on the oxygen consumption by: Cysteamine, 210 mg/kg body weight (top); morphinechloride, 4.4 mg/kg body weight (middle); morphine chloride, 14.0 mg/kg body weight (bottom).
On the other hand, the effect of a fourth substance, mercaptoethanol, had a long duration. It reached its minimum values first after about 6 h but was still evident after 24 h. When the animals were anaesthetized with mebumalnatrium before irradiation, the effect on oxygen consumption and rectal temperature lasted for a shorter time. The two minimum values of the mortality curve coincided with the minimum points for oxygen consumption and rectal temperature (Fig. 2). A low dose of morphine chloride decreased the respiratory minute volume with a cycle similar to that of cysteamine but without any effect on the rectal temperature (Fig. 3). The oxygen consumption (Fig. 4) was depressed to about 75 per cent during the irradiation compared to about 35 per cent when cysteamine was used. Morphine in higher doses (60 mg/kg body weight) gives a dosz reduction factor 1962, ANDREWS & LILJEGREN 1954). Dose in mice of the order of 1.4 (THOMSON response determinations with morphine chloride showed that 14 mg/kg was enough to reveal effects on breathing, body temperature and oxygen consumption similar to those from cysteamine (Fig. 4).
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS
35
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Fig. 5. Changes in body weight after treatment with different substances before irradiation with 6.65 Gy (700 R). o Physiologic saline. 0 Morphine chloride 4.4 mg/kg body weight. (3 Morphine chloride 14.0 mg/ kg body weight. Cysteamine. (No. of animals in each group =
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C.-0. CRIBORN AND C. RGNNBACK Phys. saline t Amphetamine Cysteamine Irradiation
Fiz. 6. Compensation of decreased respiratory minute volume and depressed rectal temperature caused by cysteamine. Interval between administration of the two compounds: 60 min.
(TOP) Respiratory minute volume (TOP) Rectal temperature (Bottom) 0,-consumption
Physiologic saline+ cysteamine
Amphetamine+ cysteamine
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and oxygen consumption (Fig. 6). Also the lethal effect was thus strongly reduced. Lower doses than 12 mg/kg gave a minute effect only. The relation between the rectal temperature during an irradiation with 8.55 Gy (900 R) and the decrease of body weight after 1, 4, 8 and 12 days is presented in Fig. 7. Of these animals all cysteamine treated survived; 4 per cent of those given the combined treatment died, which has to be compared to a mortality of 100 per cent within 12 days in the unprotected group. In Fig. 8 the body temperature during irradiation is compared to the subsequent decrease of body weight 8 days later in animals to which both cysteamine and amphetamine was administered. Cysteine belongs to the substances with a weak radiation protective effect (GOLDIE et coll. 1951), but because of its SH-group it was of interest to estimate its effect on rectal temperature, breathing and oxygen consumption.
37
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS
a
1
0
During irradiation
Days after lrradlation
a
b
12
Fig. 7. Reduction of depressive effect from cysteamine by administration of amphetamine. IrradiaCysteamine (210 mg/kg body weight) and amtion dose 8.55 Gy (900 R). 0 Physiologic saline. phetamine. (12 mg/kg body weight); two parallel groups. IIIIII)Cysteamine (210 mg/kg body weight). a) Rectal temperature during irradiation. b) Decrease of body weight (in per cent).
Following the administration of 210 mg cysteine/kg body weight to unirradiated mice, the rectal temperature decreased slightly during a short interval, the respiratory minute volume strongly, and the oxygen consumption moderately (Fig. 9). The radiation protective effect of cysteine administered 15 min before irradiation seems insignificant; after 6.65 Gy (700 R) all 5 animals survived, but after 8.55 Gy (900 R) all animals died. (1970) has shown that the respiratory activity in mice Previously CRIBORN increases during the influence of sound. The reaction occurs practically instantly when a sound of definite frequency and strength is switched on. The acoustic reaction is very individual. One prerequisite for its registration is that the normal respiratory minute volume in the animal is sufficiently low. Every tone pulse (Fig. 10, bottom curve) corresponds to an increase of altitude of the respiratory activity, and this acoustic reaction does not decrease during an experiment with an untreated animal lasting nearly 10 min. The time necessary for the tested substances to influence the nervous system can be estimated by alterations in the acoustic reaction. Cysteamine caused this reaction to cease after 3 to 4 min. At the same time the respiratory activity increased temporarily. Cysteine (Fig. 10 c) gave slight indications to the acoustic effects, and only mice with an initially high acoustic reaction gave a clear response. The reaction began to decrease about 5 min after the administration of cysteine. Still 45 s later the respiratory activity increased temporarily and then returned to an ordinary acoustic reaction. Repeated experiments showed a short disturbance of the acoustic reaction between 4 and 5 min after injection with a duration of about 14 min.
38
c.-0.CRIBORN AND c. R ~ N N B A C K
Fig. 8. Correlation between rectal temperature during irradiation and decrease in body weight on the 8th day post irradiation. 8.55 Gy (900 R) Amphetamine (12 mg/kg) + 0 9.50 Gy (1 OOO R) cysteamine (210 mg/kg)
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The lower dose of morphine caused a continually increasing acoustic reaction without any possibility of estimating the maximum effect within a 7 min interval (Fig. 10 d), most probably due to the depressive effect of the morphine on respiratory activity. The high dose of morphine gave rise to a more marked acoustic reaction of approximately the same kind (Fig. 10e). Hibernal (Fig. 10 f) gave a continuously decreasing response. The acoustic reaction suddenly ceased 3 to 4 min after the injection. At the same time the respiratory activity transiently increased. A characteristic record of the acoustic reaction was obtained with Mebumal (Fig. 10 g). Already after one minute a more defined reaction had ceased whereas the respiratory minute volume increased for another minute. About 3 min after the injection the acoustic response had ceased totally, and the respiratory activity also decreased rapidly. At that time the animal had fallen asleep and the curve showed the respiratory activity in that condition.
Discussion Though cysteamine is one of the best known radiation protective substances, few investigations of its pharmacologic effects have been performed. The effect of that substance as well as of other radiation protective compounds has mainly been described as an increased survival (LDbO).Cysteamine has a protective effect of short duration (NELSON 1954). He found the best protection if the mice were injected up to 15 min before irradiation. The protective effect was very low if the substance was administrated 30 to 60 min before irradiation, and it vanished rapidly if the interval increased.
39
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS
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Fig. 9. Effect of cysteamine on: Oxygen consumption.
0
Rectal temperature (left). o Respiratory minute volume (right).
The correlation between the protective effect and the rectal temperature and the oxygen consumption, respectively, was discussed by LANGENDORFF & KOCH(1955), BACQ et coll. (1965), BETZet coll. (1967). Neither LANGENDORFF & KOCHnor BACQ noted the early and relatively short phase of increased radiation protection most probably caused by decreased oxygen consumption. They postulated that the protective effect, correlated to a later decreased rectal temperature, was very low. BETZet coll. in experiments with radiation protective substances in rats, did not find any simple correlation between oxygen consumption and postirradiation survival. In mice anaesthetized with the sodium salt of Mebumal, CRIBORN (1967) found a radiation protective effect of the anaesthetic limited to certain intervals after the administration. He found a correlation between the minimum points for oxygen consumption and rectal temperature, respectively, and the minima for the mortality curve (Fig. 2). The importance of hypoxia has been discussed by several authors (LIMPEROS 1950, DOWDY et coll. 1950, BRUES& PATT 1953). LANGENDORFF & K o c ~have shown that a decrease of body temperature of about 6°C gives some protection against radiation. Variations in the body weight after treatment with radiation protective substances have been presented by many authors (NELSON 1965, BURNETTJR et coll. 1953, LIMPEROS) . Because of technical reasons only small groups of animals have been used in the present investigation. The radiation injury has been expressed as well as LD,, as a decrease of body weight in per cent of the weight before the irradiation. The in-
40
C.-0. CRIBORN AND C. RONNBACK
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.
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Fig. 10. Acoustic reaction after administration of: a) Physiologic saline. b) Cysteamine (240 mg/kg body weight). c) Cysteine (240 mg/kg body weight). d) Morphine (4.4 mg/kg body weight). e) Morphine (14.0 mg/kg body weight). f) Hibernal (8 mg/kg body weight). g) Mebumal (50 mg/kg body weight).
fluence on body temperature and the respiratory conditions of the animals produced by the substances tested have also been recorded. In two groups of cystearnine-treated animals, one irradiated with 6.65 Gy (700 R), the other unirradiated, the body weight decreased in both irradiated and unirradiated animals during the first 24 hours. The unirradiated animals recovered rapidly and on the 8th day after irradiation the decrease had changed to an increase of the same order of magnitude (about 7%). The irradiated, cysteamine-treated animals recovered more slowly (Fig. 12). The changes in body weight after administration of other compounds were less (Fig. 11). These measurements during the irradiation have suggested that the protection is correlated to the decrease of the oxygen consumption and rectal temperature of the animals during the irradiation. Compounds with a low influence on the oxygen consumption also appeared to have a low protective effect (cf. the low dose of morphine). However, the higher dose .of morphine giving the same decrease of oxygen consumption as cysteamine, did not cause a protection corresponding to that of cysteamine (Fig. 5). The radiation protective effect of the latter is therefore
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS
41
Values during irradiation
I
100
a
1
8
1
12
Days after irradiation
b Fig. 11. a) Rectal temperature and oxygen consumption during irradiation. b) Loss of body weight (in per cent). 0 Physiologic saline Morphine (4 mg/kg body weight) FA Morphine (14 mg/kg body weight) Cysteamine (210 mg/kg body weight) Cysteamine Unirradiated
not only to be ascribed to its decreasing effect on the oxygen consumption, but also to specific qualities of that substance. It should be observed that the oxygen consumption measured is not likely to be equivalent with the intercellular oxygen pressure in different organs.
42
c . - 0 . CRIBORN AND
c. RONNBACK
Using autoradiography with 35S-labeled cysteamine, NELSON & ULLBERG (1960) showed that the uptake of cysteamine, when administered intravenously, varies in different organs. This would confirm the ideas of JACOBSONet coll. (1949), suggesting that especially some critical organs ought to be protected during irradiation. By means of a polarographic method VAN DER MEERet coll. (1961) did not find any significant decrease of the oxygen tension in the spleeen and femoral bone marrow in CBA mice after an administration of 3 mg cysteamine per animal. They therefore rejected the theory of the radiation protective effect of hypoxia in these organs. However, no information was given on the intervals between the injections and the time for measurement. This is important as the protective effect is of relatively short duration. In the present investigation the oxygen consumption was measured every 10 min during the hour following the intraperitoneal administration of 6 mg cysteamineHCl per animal (Fig. 4a). Measurements of the acoustic reaction demonstrated that a change occurred 4 to 5 min after the administration of cysteamine and cysteine; with Hibernal and Mebumal the change occurred still earlier. The present experiments were intended to elucidate the optimum conditions for the protective substances, such as the dose and the duration of the effective period. The results of these experiments may in some cases lead to an adjustment of the irradiation technique (CRIBORN & RONNBACK 1969) with the exposure time better fitted to the effective period of the protective substance in question. In the light of the results its seems necessary to extend the evaluation of the general effect on the organism caused by presumptive protective substances in every special case. The strong decrease of the oxygen consumption giving a decrease of the physiologic capacity together with a decrease of the body temperature in the treated individual must be looked upon as seriously limiting the use of some of the substances in question.
SUMMARY Effects of presumptive radiation protective substances on physiologic criteria such as oxygen consumption, rectal temperature, respiratory and acoustic response are described. The rate of oxygen consumption and rectal temperature cannot entirely be considered a reliable measure of radiation protection. Some substances cause a decrease in the investigated parameters, strong enough to incapacitate the treated individuals severely. This limits the use of such substances. It is suggested that not only the effect on the survival but also the metabolic effects on the organism caused by the substance have to be investigated.
ZUSAMMENFASSUNG Die Wirkungen von moglichen Strahlenschutzsubstanzen auf physiologische Kriterien wie Sauerstoffverbrauch, Rektaltemperatur, Atmung und akustische Respons werden beschrieben. Der Umfang des Sauerstoffverbrauchs und die Rektaltemperatur konnen nicht
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS
43
ausschliesslich als zuverlassiges Mass fur einen Strahlenschutz angesehen werden. Einige Substanzen verursachen ein Absinken in den untersuchten Parametern, stark genug, um die behandelten Mausen schwer zu beeintrachtigen. Dieses begrenzt den Gebrauch dieser Substanzen. Nicht nur der Effekt auf das Uberleben sondern auch metabolische Effekte auf den Organismus, hervorrufen durch diese Substanzen, mhssen untersucht werden.
RESUME Les auteurs decrivent les effets de substances prksumees radioprotectrices sur des critkes physiologiques tels que la consommation d’oxygbne, la temperature rectale, la rkponse respiratoire et acoustique. Le taux de consommation d’dxygbne et la temperature rectale ne peuvent pas Ctre consideres comme une mesure entikrement fiable de I’effet radioprotecteur. Certaines substances diminuent certains des parametres etudies au point d‘invalider grafement les individus trait&. Ceci limite I’emploi de ces substances. Les auteurs pensent qu’il ne faut pas seulement etudier l’effet de ces substances sur la survie mais Btudier aussi leur effet metabolique dans l’organisme.
REFERENCES
AKERFELDTS., RONNBACKC. and NELSONA.: Radioprotective agents: Results with S-(3-amino-2-hydroxypropyl)phosphorothioate, amidophosphorothioate and some related compounds. Radiat. Res. 31 (1967), 850. ANDREWS H. L. and LILJEGREN E. J.: Effect of morphine and L-allylnormorphine on radiation mortality. Radiat. Res. l (1954), 487. BACQZ.M., BEAUMARIAGE M. L. et LIBBECQ-HUTTER S.: Relation entre la radioprotection et l’hypothermie induite par certaines substances chimiques. Int. J. Radiat. Biol. 9 (1965),175. BETZE. H., LELIEVRE P. and SMOLIAR V.: Protective effectiveness of some sulphur-containing substances and oxygen uptake in the rat. Int. J. Radiat. Biol. 12 (1967), 163. BORELLA L. E.,PAQUETTE R. and HERRF.: The effect of some CNS depressants on the hypermofility and anorexia induced by amphetamine in rats. Canad. J. Physiol. Pharmacol. 47 (1969), 841. BRUESA. M. and PATTH. M.: Mechanisms of protection against mammalian radiation injury. Physiol. Rev. 33 (1953), 85. BURNETT JR W. T., BURKEJR A. W. and UPTONA. C.: Protective effect of acetyl-betamethylcholine, carbamylcholine and atropine on X-irradiated mice. Amer. J. Physiol. 174 (1953), 254. CRIBORN C.-0.: Undersokning av somnmedels inverkan pa CBA-moss i avseende pi%rektaltemperatur, andningsfrekvens, minutvolym och tonreaktion. (In Swedish.) FOA 1 intern rapport C 1250-27 (31), 1967. - Anvandbarheten av vissa fysiologiska kriterier for bestamning av prognosen hos bestr8lade moss. (In Swedish.) FOA 1 intern rapport C 1262-27, 1967. - Recording of the respiratory process in mice without strain on the respiratory organs. Life Sciences 8, Part I (1969), 1351. - Respiratory response to acoustic excitation of mice. Life Sciences 9, Part I (1970), 13. - and RONNBACK C.: Inverkan av olika strhingsintensiteter vid superletala straldoser pa moss (In Swedish.) FOA 1 rapport A 1481-27,1969. L. R. and CHASTAIN S. M.: Protective action of anoxic anoxia against DOWDY A. H., BENNETT total body roentgen irradiation of mammals. Radiology 55 (1950), 879.
44
C.-0. CRIBORN AND C. RONNBACK
GOLDIEH., GADSON J., TARLETON JR G. J. and HAHNP. F.: Effect of pretreatment with cysteine on survival of mice exposed to external and internal irradiation. Proc. SOC.exp. Biol. 77 (1951), 790. JACOBSON L. O., MARKSE. K., GASTON E. O., ROBSON M. and ZIRKLER. E.: The role of the spleen in radiation injury. Proc. SOC.exp. Biol. 70 (1949), 740. LANGENDORFF H. und KOCH R.: Strahlenschaden und Narkose. Arzneimittel-Forsch. 5 (1955), 677.
LIMPEROS G. J.: Effects of varying oxygen tensions on mortality of X-rayed mice. J. Franklin Institute 249 (1950), 513. VAN DER MEERC., VALKENBURG P. W. and REMMELTS M: Effect of radioprotective sulphydryl compounds on the oxygen tension in the spleen of mice. Nature 189 (1961), 588.
NELSONA.: The protective effect of cystearnine on young mice exposed to roentgen rays. Acta radiol. 42 (1954), 485. - and ULLBERG S.: Distribution of Sas in mice after injection of Sa6-cysteamine.An autoradiographic investigation. Acta radiol. 53 (1960), 305. THOMSON J. F.: Radiation protection in mammals. Reinhold Publishing Corp., New York, 1962.
ISSN: 0348-5196 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ionc18
Pharmacologic Effects of Radiation Protective Compounds Related to Their Protective Effect in Mice C.-O. Criborn & C. Rönnbäck To cite this article: C.-O. Criborn & C. Rönnbäck (1979) Pharmacologic Effects of Radiation Protective Compounds Related to Their Protective Effect in Mice, Acta Radiologica: Oncology, Radiation, Physics, Biology, 18:1, 31-44, DOI: 10.3109/02841867909128188 To link to this article: https://doi.org/10.3109/02841867909128188
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 12
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ionc18
FROM THE NATIONAL DEFENCE RESEARCH INSTITUTE, DIVISION OF RADIATION BIOLOGY, S-172 04 SUNDBYBERG, SWEDEN.
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS RELATED TO THEIR PROTECTIVE EFFECT IN MICE C.-0. CRIBORN and C.
RONNBACK
The effects of presumptive radiation protective compounds have mainly been investigated only with respect to their influence on the survival rate (expressed as LD,,,), but seldom as regards physiologic effects before, during and after irradiation. Neither have their negative effects been considered, sometimes due to the absence of suitable methods. CRIBORN (1969, 1970) presented methods concerning the determination of respiratory minute volume, oxygen consumption and respiratory response to acoustic excitation. A number of compounds with proved radiation protective effects were found to depress the oxygen consumption and respiratory response strongly during their active phase. In the present investigation the depressive effect of cysteamine on these parameters was simulated by the use of some psychopharmaca. Attempts were made (1) to decrease the effect of cysteamine on respiration and body temperature by using a central stimulating compound, amphetamine, and (2) to correlate the effects of these compounds to a change in the radiation protective effect of cysteamine. This would give a more differentiated evaluation of the effect of presumptive radiation protective compounds.
Material and Methods Male CBA mice weighing 28.3 ? 4 g, aged 70 to 110 days, were used in the experiments. The different substances were administered intraperitoneally, and the concentration of the solutions was adjusted to give an injection volume of 0.1 to Submitted for publication 3 March 1978. Acta Radiologica Oncology 18 (1979) F a x . 1
31
32
C.-0. CRIBORN AND C. RONNBACK
2 1 L
I 01
I1
I
I
I
5
15
30
60
2
3
L
39
5
6
hour
niin
4
Fig. 1. Effects of radiation protective compounds on respiratory minute volume and rectal temperature in mice. Controls. 0 Cysteamine, 210 mg/kg body weight. (3 A 295, 530 mg/kg body weight. 0 A 331, 18 mg/kg body weight. Mercaptoetanole, 180 mg/kg body weight.
4
1’5
30
60 min
2
3
L
5
6
hour
Time after irradiation
0.3 ml. Control animals were at the same time injected with 0.3 ml physiologic saline. A Muller MG300 apparatus gave a dose rate of 11.7 mGy/s (74 R/min) at 260 kV, 1 1 mA (HVL 2 mm Cu). The focal distance was 45 cm.
33
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS
-0
L 60
30
niin
90
do
' 3 0 "
a
'
In:,
do
b
Fig. 2. a) Effects on oxygen consumption (left, 0 ) and rectal temperature (right, a)of a dose of 50 mg mebumal/kg body weight. b) The mortality related to time interval between administration of mebumal and start of irradiation 6.41 Gy (675 R); H indicate irradiation time.
The animals were irradiated in groups of 10 in a wheel of plastic, rotating 1 rpm to eliminate irregularities in the irradiation field. Following the irradiation, 5 animals were kept per cage. The body weight of the animals was measured with an accuracy of 0.1 g from the day before irradiation until some of the animals in that group had died. The weighing procedure was performed at 8 a.m. The rectal temperature was measured with a termistor thermometer. The oxygen consumption, respiratory minute volume and acoustic reaction were measured by means of a specially constructed apparatus (CRIBORN1969). This gave an estimate of the breathing and the acoustic reaction with a simultaneous measurement of the oxygen concentration in the tank surrounding the animals at examination. The oxygen consumption was obtained by pumping the air into the tank through a paramagnetic oxygen meter, which was read off before the animal was placed in the tank and also 3 min later. The oxygen consumption could then be calculated with a knowledge of the volume of the tank.
Results The respiratory minute volume as well as the rectal temperature decreased following the administration of the three radiation protective substances cysteamine, A295 or A331 (AKERFELDT et coll. 1967).(Fig. 1). The former decreased rapidly whereas the temperature reached its minimum point after about one hour. A recovery of the values started within one hour for all three substances. 3 - 795845
c.-0.CRIBORN AND c .
34
R~NNBACK
100-
Irradiation
9080-
7 0b0-
50Lou)-
a!
5 0
C
0
100-
f
90-
g
80-
0 0
10-
E
f >
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391
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37-
12
70-
36-
10
60-
35-
B
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6
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-
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Fig. 3. Effects on rectal temperature (a) and respiratory volume ( 0 ) by: Cysteamine, 210 mg/kg body weight (top); morphine chloride, 4.4 mg/kg body weight (middle); morphine chloride, 14.0 mg/kg body weight (bottom).
3020mon
Fig. 4. Effect on the oxygen consumption by: Cysteamine, 210 mg/kg body weight (top); morphinechloride, 4.4 mg/kg body weight (middle); morphine chloride, 14.0 mg/kg body weight (bottom).
On the other hand, the effect of a fourth substance, mercaptoethanol, had a long duration. It reached its minimum values first after about 6 h but was still evident after 24 h. When the animals were anaesthetized with mebumalnatrium before irradiation, the effect on oxygen consumption and rectal temperature lasted for a shorter time. The two minimum values of the mortality curve coincided with the minimum points for oxygen consumption and rectal temperature (Fig. 2). A low dose of morphine chloride decreased the respiratory minute volume with a cycle similar to that of cysteamine but without any effect on the rectal temperature (Fig. 3). The oxygen consumption (Fig. 4) was depressed to about 75 per cent during the irradiation compared to about 35 per cent when cysteamine was used. Morphine in higher doses (60 mg/kg body weight) gives a dosz reduction factor 1962, ANDREWS & LILJEGREN 1954). Dose in mice of the order of 1.4 (THOMSON response determinations with morphine chloride showed that 14 mg/kg was enough to reveal effects on breathing, body temperature and oxygen consumption similar to those from cysteamine (Fig. 4).
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS
35
29
28
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Fig. 5. Changes in body weight after treatment with different substances before irradiation with 6.65 Gy (700 R). o Physiologic saline. 0 Morphine chloride 4.4 mg/kg body weight. (3 Morphine chloride 14.0 mg/ kg body weight. Cysteamine. (No. of animals in each group =
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36
C.-0. CRIBORN AND C. RGNNBACK Phys. saline t Amphetamine Cysteamine Irradiation
Fiz. 6. Compensation of decreased respiratory minute volume and depressed rectal temperature caused by cysteamine. Interval between administration of the two compounds: 60 min.
(TOP) Respiratory minute volume (TOP) Rectal temperature (Bottom) 0,-consumption
Physiologic saline+ cysteamine
Amphetamine+ cysteamine
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and oxygen consumption (Fig. 6). Also the lethal effect was thus strongly reduced. Lower doses than 12 mg/kg gave a minute effect only. The relation between the rectal temperature during an irradiation with 8.55 Gy (900 R) and the decrease of body weight after 1, 4, 8 and 12 days is presented in Fig. 7. Of these animals all cysteamine treated survived; 4 per cent of those given the combined treatment died, which has to be compared to a mortality of 100 per cent within 12 days in the unprotected group. In Fig. 8 the body temperature during irradiation is compared to the subsequent decrease of body weight 8 days later in animals to which both cysteamine and amphetamine was administered. Cysteine belongs to the substances with a weak radiation protective effect (GOLDIE et coll. 1951), but because of its SH-group it was of interest to estimate its effect on rectal temperature, breathing and oxygen consumption.
37
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS
a
1
0
During irradiation
Days after lrradlation
a
b
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Fig. 7. Reduction of depressive effect from cysteamine by administration of amphetamine. IrradiaCysteamine (210 mg/kg body weight) and amtion dose 8.55 Gy (900 R). 0 Physiologic saline. phetamine. (12 mg/kg body weight); two parallel groups. IIIIII)Cysteamine (210 mg/kg body weight). a) Rectal temperature during irradiation. b) Decrease of body weight (in per cent).
Following the administration of 210 mg cysteine/kg body weight to unirradiated mice, the rectal temperature decreased slightly during a short interval, the respiratory minute volume strongly, and the oxygen consumption moderately (Fig. 9). The radiation protective effect of cysteine administered 15 min before irradiation seems insignificant; after 6.65 Gy (700 R) all 5 animals survived, but after 8.55 Gy (900 R) all animals died. (1970) has shown that the respiratory activity in mice Previously CRIBORN increases during the influence of sound. The reaction occurs practically instantly when a sound of definite frequency and strength is switched on. The acoustic reaction is very individual. One prerequisite for its registration is that the normal respiratory minute volume in the animal is sufficiently low. Every tone pulse (Fig. 10, bottom curve) corresponds to an increase of altitude of the respiratory activity, and this acoustic reaction does not decrease during an experiment with an untreated animal lasting nearly 10 min. The time necessary for the tested substances to influence the nervous system can be estimated by alterations in the acoustic reaction. Cysteamine caused this reaction to cease after 3 to 4 min. At the same time the respiratory activity increased temporarily. Cysteine (Fig. 10 c) gave slight indications to the acoustic effects, and only mice with an initially high acoustic reaction gave a clear response. The reaction began to decrease about 5 min after the administration of cysteine. Still 45 s later the respiratory activity increased temporarily and then returned to an ordinary acoustic reaction. Repeated experiments showed a short disturbance of the acoustic reaction between 4 and 5 min after injection with a duration of about 14 min.
38
c.-0.CRIBORN AND c. R ~ N N B A C K
Fig. 8. Correlation between rectal temperature during irradiation and decrease in body weight on the 8th day post irradiation. 8.55 Gy (900 R) Amphetamine (12 mg/kg) + 0 9.50 Gy (1 OOO R) cysteamine (210 mg/kg)
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The lower dose of morphine caused a continually increasing acoustic reaction without any possibility of estimating the maximum effect within a 7 min interval (Fig. 10 d), most probably due to the depressive effect of the morphine on respiratory activity. The high dose of morphine gave rise to a more marked acoustic reaction of approximately the same kind (Fig. 10e). Hibernal (Fig. 10 f) gave a continuously decreasing response. The acoustic reaction suddenly ceased 3 to 4 min after the injection. At the same time the respiratory activity transiently increased. A characteristic record of the acoustic reaction was obtained with Mebumal (Fig. 10 g). Already after one minute a more defined reaction had ceased whereas the respiratory minute volume increased for another minute. About 3 min after the injection the acoustic response had ceased totally, and the respiratory activity also decreased rapidly. At that time the animal had fallen asleep and the curve showed the respiratory activity in that condition.
Discussion Though cysteamine is one of the best known radiation protective substances, few investigations of its pharmacologic effects have been performed. The effect of that substance as well as of other radiation protective compounds has mainly been described as an increased survival (LDbO).Cysteamine has a protective effect of short duration (NELSON 1954). He found the best protection if the mice were injected up to 15 min before irradiation. The protective effect was very low if the substance was administrated 30 to 60 min before irradiation, and it vanished rapidly if the interval increased.
39
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS
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Fig. 9. Effect of cysteamine on: Oxygen consumption.
0
Rectal temperature (left). o Respiratory minute volume (right).
The correlation between the protective effect and the rectal temperature and the oxygen consumption, respectively, was discussed by LANGENDORFF & KOCH(1955), BACQ et coll. (1965), BETZet coll. (1967). Neither LANGENDORFF & KOCHnor BACQ noted the early and relatively short phase of increased radiation protection most probably caused by decreased oxygen consumption. They postulated that the protective effect, correlated to a later decreased rectal temperature, was very low. BETZet coll. in experiments with radiation protective substances in rats, did not find any simple correlation between oxygen consumption and postirradiation survival. In mice anaesthetized with the sodium salt of Mebumal, CRIBORN (1967) found a radiation protective effect of the anaesthetic limited to certain intervals after the administration. He found a correlation between the minimum points for oxygen consumption and rectal temperature, respectively, and the minima for the mortality curve (Fig. 2). The importance of hypoxia has been discussed by several authors (LIMPEROS 1950, DOWDY et coll. 1950, BRUES& PATT 1953). LANGENDORFF & K o c ~have shown that a decrease of body temperature of about 6°C gives some protection against radiation. Variations in the body weight after treatment with radiation protective substances have been presented by many authors (NELSON 1965, BURNETTJR et coll. 1953, LIMPEROS) . Because of technical reasons only small groups of animals have been used in the present investigation. The radiation injury has been expressed as well as LD,, as a decrease of body weight in per cent of the weight before the irradiation. The in-
40
C.-0. CRIBORN AND C. RONNBACK
.
.
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Fig. 10. Acoustic reaction after administration of: a) Physiologic saline. b) Cysteamine (240 mg/kg body weight). c) Cysteine (240 mg/kg body weight). d) Morphine (4.4 mg/kg body weight). e) Morphine (14.0 mg/kg body weight). f) Hibernal (8 mg/kg body weight). g) Mebumal (50 mg/kg body weight).
fluence on body temperature and the respiratory conditions of the animals produced by the substances tested have also been recorded. In two groups of cystearnine-treated animals, one irradiated with 6.65 Gy (700 R), the other unirradiated, the body weight decreased in both irradiated and unirradiated animals during the first 24 hours. The unirradiated animals recovered rapidly and on the 8th day after irradiation the decrease had changed to an increase of the same order of magnitude (about 7%). The irradiated, cysteamine-treated animals recovered more slowly (Fig. 12). The changes in body weight after administration of other compounds were less (Fig. 11). These measurements during the irradiation have suggested that the protection is correlated to the decrease of the oxygen consumption and rectal temperature of the animals during the irradiation. Compounds with a low influence on the oxygen consumption also appeared to have a low protective effect (cf. the low dose of morphine). However, the higher dose .of morphine giving the same decrease of oxygen consumption as cysteamine, did not cause a protection corresponding to that of cysteamine (Fig. 5). The radiation protective effect of the latter is therefore
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS
41
Values during irradiation
I
100
a
1
8
1
12
Days after irradiation
b Fig. 11. a) Rectal temperature and oxygen consumption during irradiation. b) Loss of body weight (in per cent). 0 Physiologic saline Morphine (4 mg/kg body weight) FA Morphine (14 mg/kg body weight) Cysteamine (210 mg/kg body weight) Cysteamine Unirradiated
not only to be ascribed to its decreasing effect on the oxygen consumption, but also to specific qualities of that substance. It should be observed that the oxygen consumption measured is not likely to be equivalent with the intercellular oxygen pressure in different organs.
42
c . - 0 . CRIBORN AND
c. RONNBACK
Using autoradiography with 35S-labeled cysteamine, NELSON & ULLBERG (1960) showed that the uptake of cysteamine, when administered intravenously, varies in different organs. This would confirm the ideas of JACOBSONet coll. (1949), suggesting that especially some critical organs ought to be protected during irradiation. By means of a polarographic method VAN DER MEERet coll. (1961) did not find any significant decrease of the oxygen tension in the spleeen and femoral bone marrow in CBA mice after an administration of 3 mg cysteamine per animal. They therefore rejected the theory of the radiation protective effect of hypoxia in these organs. However, no information was given on the intervals between the injections and the time for measurement. This is important as the protective effect is of relatively short duration. In the present investigation the oxygen consumption was measured every 10 min during the hour following the intraperitoneal administration of 6 mg cysteamineHCl per animal (Fig. 4a). Measurements of the acoustic reaction demonstrated that a change occurred 4 to 5 min after the administration of cysteamine and cysteine; with Hibernal and Mebumal the change occurred still earlier. The present experiments were intended to elucidate the optimum conditions for the protective substances, such as the dose and the duration of the effective period. The results of these experiments may in some cases lead to an adjustment of the irradiation technique (CRIBORN & RONNBACK 1969) with the exposure time better fitted to the effective period of the protective substance in question. In the light of the results its seems necessary to extend the evaluation of the general effect on the organism caused by presumptive protective substances in every special case. The strong decrease of the oxygen consumption giving a decrease of the physiologic capacity together with a decrease of the body temperature in the treated individual must be looked upon as seriously limiting the use of some of the substances in question.
SUMMARY Effects of presumptive radiation protective substances on physiologic criteria such as oxygen consumption, rectal temperature, respiratory and acoustic response are described. The rate of oxygen consumption and rectal temperature cannot entirely be considered a reliable measure of radiation protection. Some substances cause a decrease in the investigated parameters, strong enough to incapacitate the treated individuals severely. This limits the use of such substances. It is suggested that not only the effect on the survival but also the metabolic effects on the organism caused by the substance have to be investigated.
ZUSAMMENFASSUNG Die Wirkungen von moglichen Strahlenschutzsubstanzen auf physiologische Kriterien wie Sauerstoffverbrauch, Rektaltemperatur, Atmung und akustische Respons werden beschrieben. Der Umfang des Sauerstoffverbrauchs und die Rektaltemperatur konnen nicht
PHARMACOLOGIC EFFECTS OF RADIATION PROTECTIVE COMPOUNDS
43
ausschliesslich als zuverlassiges Mass fur einen Strahlenschutz angesehen werden. Einige Substanzen verursachen ein Absinken in den untersuchten Parametern, stark genug, um die behandelten Mausen schwer zu beeintrachtigen. Dieses begrenzt den Gebrauch dieser Substanzen. Nicht nur der Effekt auf das Uberleben sondern auch metabolische Effekte auf den Organismus, hervorrufen durch diese Substanzen, mhssen untersucht werden.
RESUME Les auteurs decrivent les effets de substances prksumees radioprotectrices sur des critkes physiologiques tels que la consommation d’oxygbne, la temperature rectale, la rkponse respiratoire et acoustique. Le taux de consommation d’dxygbne et la temperature rectale ne peuvent pas Ctre consideres comme une mesure entikrement fiable de I’effet radioprotecteur. Certaines substances diminuent certains des parametres etudies au point d‘invalider grafement les individus trait&. Ceci limite I’emploi de ces substances. Les auteurs pensent qu’il ne faut pas seulement etudier l’effet de ces substances sur la survie mais Btudier aussi leur effet metabolique dans l’organisme.
REFERENCES
AKERFELDTS., RONNBACKC. and NELSONA.: Radioprotective agents: Results with S-(3-amino-2-hydroxypropyl)phosphorothioate, amidophosphorothioate and some related compounds. Radiat. Res. 31 (1967), 850. ANDREWS H. L. and LILJEGREN E. J.: Effect of morphine and L-allylnormorphine on radiation mortality. Radiat. Res. l (1954), 487. BACQZ.M., BEAUMARIAGE M. L. et LIBBECQ-HUTTER S.: Relation entre la radioprotection et l’hypothermie induite par certaines substances chimiques. Int. J. Radiat. Biol. 9 (1965),175. BETZE. H., LELIEVRE P. and SMOLIAR V.: Protective effectiveness of some sulphur-containing substances and oxygen uptake in the rat. Int. J. Radiat. Biol. 12 (1967), 163. BORELLA L. E.,PAQUETTE R. and HERRF.: The effect of some CNS depressants on the hypermofility and anorexia induced by amphetamine in rats. Canad. J. Physiol. Pharmacol. 47 (1969), 841. BRUESA. M. and PATTH. M.: Mechanisms of protection against mammalian radiation injury. Physiol. Rev. 33 (1953), 85. BURNETT JR W. T., BURKEJR A. W. and UPTONA. C.: Protective effect of acetyl-betamethylcholine, carbamylcholine and atropine on X-irradiated mice. Amer. J. Physiol. 174 (1953), 254. CRIBORN C.-0.: Undersokning av somnmedels inverkan pa CBA-moss i avseende pi%rektaltemperatur, andningsfrekvens, minutvolym och tonreaktion. (In Swedish.) FOA 1 intern rapport C 1250-27 (31), 1967. - Anvandbarheten av vissa fysiologiska kriterier for bestamning av prognosen hos bestr8lade moss. (In Swedish.) FOA 1 intern rapport C 1262-27, 1967. - Recording of the respiratory process in mice without strain on the respiratory organs. Life Sciences 8, Part I (1969), 1351. - Respiratory response to acoustic excitation of mice. Life Sciences 9, Part I (1970), 13. - and RONNBACK C.: Inverkan av olika strhingsintensiteter vid superletala straldoser pa moss (In Swedish.) FOA 1 rapport A 1481-27,1969. L. R. and CHASTAIN S. M.: Protective action of anoxic anoxia against DOWDY A. H., BENNETT total body roentgen irradiation of mammals. Radiology 55 (1950), 879.
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C.-0. CRIBORN AND C. RONNBACK
GOLDIEH., GADSON J., TARLETON JR G. J. and HAHNP. F.: Effect of pretreatment with cysteine on survival of mice exposed to external and internal irradiation. Proc. SOC.exp. Biol. 77 (1951), 790. JACOBSON L. O., MARKSE. K., GASTON E. O., ROBSON M. and ZIRKLER. E.: The role of the spleen in radiation injury. Proc. SOC.exp. Biol. 70 (1949), 740. LANGENDORFF H. und KOCH R.: Strahlenschaden und Narkose. Arzneimittel-Forsch. 5 (1955), 677.
LIMPEROS G. J.: Effects of varying oxygen tensions on mortality of X-rayed mice. J. Franklin Institute 249 (1950), 513. VAN DER MEERC., VALKENBURG P. W. and REMMELTS M: Effect of radioprotective sulphydryl compounds on the oxygen tension in the spleen of mice. Nature 189 (1961), 588.
NELSONA.: The protective effect of cystearnine on young mice exposed to roentgen rays. Acta radiol. 42 (1954), 485. - and ULLBERG S.: Distribution of Sas in mice after injection of Sa6-cysteamine.An autoradiographic investigation. Acta radiol. 53 (1960), 305. THOMSON J. F.: Radiation protection in mammals. Reinhold Publishing Corp., New York, 1962.
