Effects of prenatal X-irradiation on the ontogenesis of acetylcholinesterase and carbonic anhydrase in rat central nervous system

BRAIN RESEARCH

383

EFFECTS OF P R E N A T A L X - I R R A D I A T I O N ON T H E O N T O G E N E S I S OF A C E T Y L C H O L I N E S T E R A S E A N D C A R B O N I C A N H Y D R A S E IN R A T C E N T R A L N E R V O U S SYSTEM*

V. NAIR AND D. BAU

Neuropharmacology and Biochemistry Laboratories, Michael Reese Hospital Psychiatric Institute and Department of Pharmacology, Chicago Medical School, Chicago, HI. 60616 (U.S.A.) (Accepted May 23rd, 1969)

INTRODUCTION

Numerous experimental and clinical studies of recent years have indicated that exposure to X-irradiation during specific stages of pregnancy can result in various morphological and behavioral changes in the offspringS,8,23, 26. Equally important and less well understood are the subtler effects on biochemical systems. Unlike the gross malformations and abnormalities, these are not readily evident and may be revealed only by special tests. A molecular or biochemical lesion may remain dormant and may manifest itself later in life as a functional impairment, behavioral disorder or enhanced adverse reaction to drugs. Presently, we have little information about these events. With these considerations in mind, we have initiated an investigation on the effects of exposure in pregnancy to low doses of X-rays on the biochemical and functional development of the offspring. An earlier report has dealt with the effects on the development of drug detoxicating systems in liver 21. This report describes our findings on the ontogenesis of carbonic anhydrase (CA)** and acetylcholinesterase (ACHE)** in the central nervous system (CNS) of prenatally irradiated rats. The determinations were done with respect to sex and age.

* A part of this work has been presented at the Third International Congress of Radiation Research, Cortina, Italy, July 1966. ** The Commission on Enzymes of the International Union of Biochemistry has recommended that acetylcholine hydrolase (System No. 3.1.1.7) be the formal name and acetylcholinesterase be the trivial name for the enzyme having the higher affinity for acetylcholine than for other esters. Similarly, carbonate hydro-lyase (System No. 4.2.1.1) is the term recommended for the enzyme catalyzing the hydration of CO2, and carbonic anhydrase, the trivial name.

Brain Research, 16 (1969) 383-394

384

V, NAIR A N D D. BAU

MATERIALS A N D METHODS

Animals

Sprague-Dawley rats were used. They were housed under controlled environmental conditions (temp. 22.2 °C, light 5 a.m. to 7 p.m.). Food and water were given ad libitum. Female rats in estrus were housed overnight with males ( 2 : 1 ) . The following morning vaginal smears were taken and examined. If the smears were positive for sperm, that day was taken as day t of pregnancy. The pregnant animals were exposed to X-irradiation on the fourteenth day of gestation. X-Irradiation

Animals received a single exposure of 25 or 50 R to the pelvic region 111-12 R/min) with the remainder of the body shielded. The physical factors of radiation were: 250 kV, 30 mA, filter 0.5 mm Cu and 1 m m AI. half-value layer t.45 mm Cu. During irradiation, the unanesthetized animals were held in lucite tubes provided with a large number of air holes. For shielding, a sheet of lead 3.5 mm thick, shaped to cover the lucite tubes, was used. Control animals were sham irradiated. They were subjected to all the procedures used for the irradiated animals except radiation exposure. The animals were allowed to deliver and raise their offspring. The offspring were examined daily, from birth until weaning, for gross malformations; they were also weighed periodically during this time. They were weaned at 21 days of age and separated according to sex. At 10, 20, 40. and 80 days of age 6-8 animals (or less in certain cases) of each sex were killed by decapitation, and the brain was removed rapidly. In brain, enzyme activity was determined on a regional basis. This was accomplished by the use of the frozen state dissection method reported earlier ls,tg. Briefly, this procedure consisted of transferring the decapitated head to a cold room I 10°), rapid removal of the brain and freezing on a bed of dry ice to the appropriate consistency for secuoning. Serial coronal sections, 2-3 m m in thickness, were then obtained by means of freehand sectioning with a razor blade. The selected anatomical regions were dissected out from the frozen sections with the aid of a dissecting lens and scalpel blades; macroscopically visible blood was scraped off the samples before they were weighed and stored. The whole operation was performed in the cold room. thereby minimizing the various errors which may result from leaching of the blood, condensation of moisture (when dissection is performed at room temperature on dry ice), water shift, etc. For ACHE, the brain regions examined were: frontal cortex, occipital cortex, paleocortex, caudate nucleus, hypothalamus and medulla. For CA. the regions of the central nervous systems studied were: frontal cortex, caudate nucleus, hippocampus, cerebellum, medulla and spinal cord. Brain Research, 16 (1969) 383-394

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Enzyme measurements Carbonic anhydrase. Brain was homogenized in cold (5 °) isotonic sodium chloride solution (1 in 20). The enzyme activity in the homogenate was determined according to the method of Maren 13 utilizing phenolsulfonphthalein as the indicator. This micromethod permitted measurement of enzyme activity in different brain regions using only one rat brain. A single determination could be performed with a sample size as low as 1 mg (0.02 ml of homogenate). In brief, the assay consists of measuring, with the aid o f a pH indicator, the time required for the change o f p H during the formation of carbonic acid from carbon dioxide and water. Foaming and loss of solution may occur with brain homogenates during the reaction, but this can be prevented by the prior application of silicone (Dow Coming Antifoam Spray) to the reaction vessel. This is best done by applying the silicone to a piece of gauze and wiping the lip of the reaction vessel with it. The validity of the method was first established using purified carbonic anhydrase (Nutritional Biochemicals, Cleveland) and hemolyzed blood. The enzyme activity is expressed in units, where 1 unit = (TB T~)/TE. TB and TE represent the reaction times measured in the absence and presence of enzyme respectively. Brain Research, 16 (1969) 383-394

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Acetylcholinesterase activity was determined by the colorimetric method of Hestrin 7. Each sample was assayed in duplicate. The enzyme activity is expressed in #moles of acetylcholine hydrolyzed per gram wet tissue per hour. RESULTS

Gross effects. There were no significant differences between the control and irradiated groups with respect to the duration of pregnancy, average litter size or 50 of early death in the offspring (until 21 days of age). No visible malformations were noted in the offspring of irradiated mothers. Body weight changes were similar to those in the control rats. Acetylcholinesterase. In control animals, AChE activity increased with age up to 80 days in all the brain regions examined except in the hypothalamus nnd medulla (Figs. 1 and 2). In the latter 2 regions, levels as high as those noted at 80 days were present even at 20 days of age. The highest activity was noted in the caudate nucleus in both sexes. There was no sex difference in the activity of the enzyme m the different regions studied. In the caudate nucleus and medulla, prenatal irradiation (50 R) produced a suppression of the development of the AChE in both sexes (Figs. 3 and4). A similar suppression but of lesser magnitude was noted in the frontal and occipital cortices in the male and in the paleo- and occipital cortices in the female. Brain Research, t6 (1969) 383-394

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Fig. 3. Effect o f prenatal X-irradiation on postnatal development o f A C h E in rat C N S (males). E a c h point is the m e a n f r o m 4 - 6 animals. In the caudate nucleus, the values for 50 R are significantly different f r o m controls ( P < 0.01); for medulla P < 0.05.

Carbonic anhydrase. A caudo-rostral gradient in the appearance of this enzyme is suggested from Figs. 5 and 6. At 10 days of age, medulla and spinal cord had significantly higher levels of enzyme activity than the other parts. In contrast to ACHE, a sex difference in the levels and distribution was noted with this enzyme. In general, activity was higher in the females. In females, for all the regions except spinal cord, enzyme activity increased with age up to 80 days. Only medulla and spinal cord showed this increase in the males. Furthermore, hippocampus in the females showed more than two-fold activity in comparison with that in the male rats. Irradiation at both doses inhibited enzyme development in the medulla and spinal cord in the male rats while a slight stimulation of activity was noted in the females (Figs. 7 and 8). In spinal cord, enzyme activity was significantly increased at 40 and 80 days of age (P < 0.01), in medulla at 20 and 40 days of age (P < 0.05) and in caudate and hippocampus at 40 days (P < 0.05). Brain Research, 16 (1969) 383-394

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Fig. 4. Effect of prenatal X-irradiation on postnatal development of AChE in rat CNS (females). Each point is the mean from 4-6 animals. For eaudate nucleus, the values are significanttydifferent from controls (P < 0.01) in both 25 and 50 R groups; for paleocortex, occipital cortex and medulla the difference is barely significant (P < 0.05).

DISCUSSION

The pattern of regional development of AChE in the brain of control rats noted in this study is in general agreement with the observations of previous investigators 2,4,9,1°,17. The maximal activity is attained first in the medulla, midbrain and cerebellum and later in the cerebral hemispheres. By 20 days of age and thereafter, the region richest in enzyme is the caudate nucleus. This remains so in the irradiated animal, even though the enzyme development was suppressed by prenatal radiation. Sex-linked differences in butyrylcholinesterase distribution have been reported by Woolley 25 but not for ACHE. In this study, no sex differences in the development and distribution of AChE were observed. The development of CA in the CNS of many species has been studied by Ashby and Schuster 1. However, their investigations did not include a regional study of sex Brain Research, 16 (1969) 383-394

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differences, as was done in the present study. The caudo-rostral gradient in the appearance of the enzyme within the CNS is consistent with their observations; that is, the enzyme appeared first in the medulla and spinal cord and later in the cerebral cortex. In addition, our studies have revealed a sex difference in the distribution and level of CA in the CNS, the enzyme activity generally being higher in the female than in the male. Furthermore, the hippocampus in the female stands out in this respect, being considerably higher in CA activity (more than two-fold) than that of the male. The implications of this difference are not presently understood. In prenatally irradiated animals, the development of CA was suppressed in medulla and spinal cord of male offspring while no inhibition was noted in the females. There was an initial rise in CA activity in the subcortical regions and spinal cord in the females, the rise in spinal cord being maintained beyond 80 days of age. Giacobini 6 has demonstrated that CA is concentrated in the glial cells. Electron microscopic studies have shown that acetylcholinesterase activity is largely confined to the neurons, where it is associated chiefly with portions of endoplasmic reticulum and synapses3,1~. Thus, the changes in these two enzymes resulting from irradiation may also be taken as a reflection of changes in the glial and neuronal compartments. The effects of neonatal radiation on the development of acetyl- and butyrylcholinesterases have been studied by Maletta and TimiraslL They found that after exposure to 450 R in 2-day-old rats, brain acetylcholinesterase activity was signifiBrain Research, 16 (1969) 383-394

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Fig. 6. Postnatal development of CA in the different regions of rat CNS (females). Each point is the mean from 4-6 animals. cantly decreased in the ontogenetically newer structures at day 10 but not at 64 days. Butyrylcholinesterase activity was decreased in some areas 1 week after irradiation, but returned to normal at 24 days. They found no gross behavioral alterations when the enzyme was maximally inhibited, but suggested the possibility of disturbances in functional brain maturation. It ~s not surprising to see that suppression Of enzyme development (caudate) resulted at a much lower radiation dose level prenatally than when administered postnatally considering that day 14 is a period of great metabolic and cellular activity. Mechanisms underlying the impairment a r e n o t clear. Our initial efforts were directed towards measurement of total protein in the tissue homogenate and various cell fractions. To date, no alterations in protein content were detected in the irradiated brain. In interpreting this, it should be noted that changes restricted to isolated enzymes, even if present, may not be reflected in the cell fractions. We are also studying the effects of radiation when given at other periods in gestation. It is hoped that analysis of these results in the light of the morphological data available on the development of rat CNS s should provide further insight into the mechanisms. Functional correlates of these two enzymes have been the objects of many investigations. However, there is no unanimity of opinion on the correlation. The expectation that there should be a relationship between function and the activity of enzymes taking part in the function is not surprising. The problems and pitfalls Brain Research, 16 (1969) 383-394

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of correlating behavior or functional changes with alterations of cholinergic systems have been discussed in a recent symposium by Russell 24. With CA, Millichap 14 has related the changes in susceptibility to electroshock seizure occurring with age in rats to changes in the level of brain enzyme activity. In rats younger than 20 days, the maximal electroshock seizure pattern is less severe than in older rats, and the level of brain CA is also correspondingly lower in the younger animals. This relationship has been extended further through the use of acetazolamide 15, where Millichap has demonstrated a parallelism between the anticonvulsant effect of the drug and the degree of inhibition of the enzyme. In another study, reported earlier, we have confirmed Millichap's findings, and in addition shown that the maximal electroshock seizure response was more specifically related to the degree of enzyme activity in the caudate nucleus 20. Environmental manipulations of various sorts in early life are known to alter the development of brain ACHE. For instance, Rosenzweig et al. 22 have shown that enriched environment of postweaning rats lowered AChE activity in the cortex and Brain Research, 16 (1969) 383-394

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increased it in the subcortical structures• We found in an earlier study that handling or light stimulation during the first 10 days of postnatal life suppressed the enzymic development in the cortical structures while accelerating the enzymic ontogenes~s in the subcortical regions 1l. None of these findings seems to offer adequate evidence for a causal relationship between function and enzyme levels. Additional associated changes must be demonstrated before any valid relationships can be formulated. Studies are under way in our laboratories to assess the functional and behavioral changes in the prenatally irradiated animals. SUMMARY

Effects of prenatal X-irradiation on the ontogenesis of carbonic anhydrase and acetylcholinesterase in rat CNS have been studied. In both male and female offspring of irradiated mothers, development of AChE was impaired in the caudate nucleus. Brain Research, 16 (1969) 383- 394

t 80

RADIATION EFFECTS ON BRAIN ENZYME DEVELOPMENT

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Ontogenesis of CA, on the other hand, was suppressed in the medulla and spinal cord of male rats while a slight stimulation was noted in the females. In the spinal cord, the stimulation was noted beyond 80 days of age. Approaches towards understanding the mechanisms underlying these effects and their possible functional significance are discussed. ACKNOWLEDGEMENTS

This work was supported in part by grants from the State of Illinois Department of Mental Health (17, 337 and 1711). The authors wish to thank the University of Chicago, Argonne Cancer Hospital and Department of Radiology for the use of the radiation facilities, and Mr. James Bland for technical assistance with the radiation procedures. Grateful acknowledgement is also due to Mr. Terry Rothstein for assistance in part of this work. REFERENCES 1 ASHBY, W., AND SCHUSTER, E. M., Carbonic anhydrase in the brain of the newborn in relation to functional maturity, J. biol. Chem., 184 (1950) 109-116. 2 BENNETT, E. L., ROSENZWEIG, M. R., KRECH, D., KARLSSON, H., DYE, N., AND OHLANDER, A., Individual, strain, and age differences in cholinesterase activity of the rat brain, J. Neurochern., 3 (1958) 144-152. 3 DE LORENZO, A. J. D., Electron microscopy of the cerebral cortex. I. The ultrastructure and histochemistry of synaptic functions, Bull. Johns Hopk. Hosp., 108 (1961) 258 279. 4 ELKES,J., EAYKS,J. T., AND TODRICK, A., On the effect and lack of effect of some drugs on postnatal development in the rat. In H. WAELSH (Ed.), Biochemistry o f the Developing Nervous System, Academic Press, New York, 1955, pp. 499-509. 5 FURCHTGOTT, E., Behavioural effects of ionizing radiations: 1955-1961, Psychol. Bull., 60 (1963) 157 199. 6 GIACOBIN], E., A cytochemical study of the localization of carbonic anhydrase in the nervous system, J. Neurochem., 9 (1962) 169-177. 7 HESTRIN, S., The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine and its analytical application, J. biol. Chem., 180 (1949) 249-261. 8 HICKS, S. P., D'AMATO, C. J. AND LOWE, M. J., The development of the mammalian nervous system, J. cornp. Neurol., 113 (1959)435 469. 9 lsml, Y., The histochemical studies of cholinesterase in the central nervous system, l and II, Arch. histol, jap., 12 (1957) 587-637. 10 KAP.CZMAR, A. G., Ontogenesis of cholinesterases. In G. B. KOELLE (Ed.), Handbuch der EAperimentellen Pharmakologie, Springer, Berlin, 1963, pp. 129-186. I 1 KLING, A., FINER. S., AND NAIR, V., Effects of early handling and light stimulation on the acetylcholinesterase activity of the developing rat brain, Int. J. Neuropharmacol., 4 (1965) 353-357. 12 MALETTA, G. J., AND TIM1RAS, P. S., Acetyl- and butyrylcholinesterase activity of selected brain areas in developing rats after neonatal X-irradiation, J. Neurochem., 13 (1966) 75-84. 13 MAREN, T. H., A simplified micromethod for the determination of carbonic anhydrase and its inhibitors, J. Pharmacol. exp. Ther., 130 (1960) 26-29. 14 MILLICHAP, J. G., WOODBURY, D. M., AND GOODMAN, L. S., Mechanism of the anticonvulsant action of acetazolamide, a carbonic anhydrase inhibitor, J. Pharmacol. exp. Ther., 115 (1955) 251-258. 15 MILLICHAP, J. G., Seizure patterns in young animals. Significance of brain carbonic anhydrase, !I, Proc. Soc. exp. Biol. ( N. Y.) , 97 (1958) 606-611. 16 MORI, S. T. M., AND SHIMIZU, N., Electron-microscopic histochemistry of cholinesterases in the rat brain, Histochemie, 4 (1964) 65-72.

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17 NACHMANSOHN,D., Chemical and Molecular Basis of Nerve Activity, Academic Press. New York. 1959. 18 NA1R, V., LAL, H., AND ROTH, L. J., A demonstration of the early entry of iproniazid into the central nervous system, Int. J. Neuropharmacol., 1 (1962) 361-369. 19 NAIR, V., AND ROTH, L. J.. Penetration of substances into the brain. In L. J. RO'IH ~Ed.l. Isotopes in Experimental Pharmacology, Univ. Chicago Press, 1965, pp. 219-228. 20 NAIR, V., AND BAU, D., Region specific inhibition of brain carbonic anhydrase (CA) and anticonvulsant action, Fed. Proc., 26 (1967) 764. 21 NAIR, V., BAD, D., AND SIEGEL. S.. Effects of prenatal X-irradiation: Studies on the mechanism of X-irradiation induced inhibition of microsomal enzyme development in rat liver. Radiat. Res., 36 (1968) 493-507. 22 ROSENZWEIG, M. R., KRECH. D.. BENNETT. E. L., AND DIAMOND. M. C.. Effects of environmental complexity and training on brain chemistry and anatomy: a replication and extension. J. ~vmp. physiol. PsychoL, 55 (1962) 429-437. 23 RUGH, R., AND WOHLFROMM. M.. Previous reproductive history and the susceptibility to X-ray induced congenital anomalies, Nature (Lond.), 210 (1966) 969-970. 24 RUSSELL, R. W., Behavioural aspects of cholinergic transmission. Fed. Proc.. 28. Suppl. (1969) 121-131. 25 WOOLLEY,D. E., Sex differences in brain pseudo-cholinesterase activity in the rat, J. Neurochem., 10 (1963) 447-452. 26 YAMAZAKI,J. N., A review of the literature on the radiation dosage required to cause manifest central nervous system disturbances from in utero and postnatal exposure, Pediatrics, 37, Suppl. (1966) 877-903.

Brain Research, 16 (1969) 383-394