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Activities of monoamine oxidase-A and -B in adult rat cerebellum following neonatal X-irradiation
NEUROSCIENCE RESEARCH ELSEVIER
Neuroscience Research 25 (1996) 97-100
Rapid communication
Activities of monoamine oxidase-A and -B in adult rat cerebellum following neonatal X-irradiation L a u r a R . G u e l m a n a, L u i s M . Z i e h e r a, M a r i a A. Z o r r i l l a Z u b i l e t e a, A l e j a n d r o M . D o p i c o b'* al-~ C~tedra de Farmacologla, Facultad de Medicina, Universidad de Buenos Aires. Paraguay 2155 P. 15, 1121 Buenos Aires, Argentina bDepartment of Pharmacology and Molecular Toxicology, University of Massachusetts Medical School 55 Lake Av. North, Worcester MA 01655, USA
Received 4 December 1995; revised 12 January 1996; accepted 1 February 1996
Abstract
The activities of monoetmine oxidases, MAO-A and MAO-B, were separately determined in the cerebellum (CE) from adult rats neonatally exposed to 5 Gy X-irradiation. They were found to be markedly reduced: 58% and 66% of values from nonirradiated, littermate controls. Since the specific activities of both isoenzymes (per mg tissue weight) were not significantly different from controls, the reduction of activity per CE is basically explained by the irradiation-induced cerebellar atrophy. The unmodified MAO-A specific activity makes it highly improbable that the increase in the cerebellar noradrenaline content, characteristic of neonatally X-irradiated rats, could be due to a decreased neuronal metabolism of noradrenaline by this enzyme. Keywords: MAO, monamine: oxygen oxidoreductase (deaminating) (ravin containing); MAO-A; MAO-B; Neonatal X-irradiation;
Cerebellum; Noradrenaline
The exposure of neonatal rats to a single dose of X-irradiation (5 Gy) produces permanent changes in the neurochemistry and cytoarchitecture of cerebellum (CE), accompanied by characteristic motor abnormalities (Dopico et al., 198!)). Adult rats that were X-irradiated within the first 48 h after birth show a motor syndrome which consists of dystonia-like movements, fine tremor and atax~c gait. Their CE is markedly atrophic, with an agranular cortex in which Purkinje cells are randomly scattered at all depths (Dopico et al., 1989; Guelman et al., 1993). The neurochemical alterations include a mild but significant increase in the noradrenaline (NA) content of CE ( + 20-36%) which, due to the X-ray-induced cerebellar atrophy, results in a marked increase in NA concentration (+225%) (Dopico et al., 1989; Dopico and Zieher, 1993; Guelman et al., 1993). * Corresponding author. Tel.: + 1 508 856 6989; fax: + 1 508 856 5080; e-mail: [email protected]
The biochemical mechanism(s) that leads to the increase in the NA content of X-irradiated CE is largely unknown. One factor that determines the endogenous level of a neurotransmitter is its degradation rate. Monoamine oxidase [monoamine: oxidase oxidoreductase (deaminating), EC 1.4.3.4, MAO] is a primary catabolic enzyme for NA in mammalian brain (Berry et al., 1994). In addition, it is well known that brain MAO activity is highly sensitive to radiation, being either decreased or increased depending upon the type and dose of radiation (Catravas and McHale, 1975; Bod6 and Benk6, 1987). MAO exists in two forms, MAO-A and MAO-B, which can be differentiated based on their substrate specificities and inhibitor sensitivities. Whereas MAO-A is inhibited by low concentrations of clorgyline, MAOB is selectively inhibited by L-deprenyl (Johnston, 1968; Berry et al., 1994). NA is a preferential substrate for MAO-A (Youdim and Riederer, 1993), which is the main type present in the noradrenergic terminals (Berry et al., 1994). The primary route for the inactivation of
0168-0102/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PH SO168-0102(96)01022-X
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L.R. Guelman et al. / Neuroscience Research 25 (1996) 97-100
Table 1 In vitro activities of monoamine oxidase-A and -B in the cerebellum of adult rats exposed to a single dose (5 Gy) of neonatal X-irradiation pM 4-OHQ/mg wet tissue/h
#mol 4-OHQ/CE/h
Total MAO MAO-A MAO-B MAO-A/MAO-B
Control
X-irradiated
Control
X-irradiated
1.17 + 0.22 0.36 _+0.04 0.86 _+0.16 0.42 _+0.05
0.69 _+0.18a (58.9 _+15.4) 0.21 + 0.01a (58.3 _+ 4.2) 0.57 _ 0 . 0 9 b (66.2 _+15.1) 0.37 _+0.03 (88.1 _+_ 3.6)
3.92 _ 0.46 1.48 _+0.15 2.80 _+0.06 0.52 _+0.05
3.50 _+0.38 (89.3 + 9.7) 1.48 _ 0.10 (100.0_ 6.7) 2.90 _+0.05 (103.6_+1.8) 0.51 + 0.03 (98.1 + 3.8)
Data from X-irradiated CE expressed as percentages of their respective controls are given in parentheses. One enzyme determination was performed per CE. Data are mean + S.E.M. from 3-6 determinations. 4-OHQ, 4-hydroxyquinoline.CE, cerebellum. aSignificantly different from controls (P<0.01). bSignificantlydifferent from controls (P < 0.05). released N A is the reuptake into the nerve terminal (Iversen, 1974), and MAO-A is considered the form responsible for presynaptic metabolism of NA (Fagervall and Ross, 1986). On the other hand, MAO-B is mainly localized in non-neuronal elements, being the astrocytes critical contributors of its activity in both normal and injured CNS (Levitt et al., 1982; Berry et al., 1994). An increase in MAO-B activity is found when astrogliosis occurs (Ekblom et al., 1993), and astrogliosis may be induced by X-irradiation in the early postnatal period (Sykovfi et al., 1992). Therefore, in this study we evaluated separately the activities of MAO-A and MAO-B in CE from adult rats exposed to X-irradiation in the neonatal period using an in vitro assay. Newborn littermate Wistar rats of both sexes (8-10 pups/litter) were separated into experimental and control (nonirradiated) groups. Rats from each group were housed 4-5/cage, given rat chow and water ad libitum, and maintained on a 12 h light/dark cycle at 25°C. The radiation source was a 225 kV, 17 mA, X-ray unit (Philips 220/25 for profound radiotherapy, Philips Groeilmpen Fabrieken, Eindhoven, The Netherlands). A 1 mm copper filter equivalent to 1.3 mm copper half-value level (HVL) was used for filtration. Exposure time was fixed by previous dosimetry, using a Simplex probe (Simplex Universal Dosimeter Physikalisch Technische Werkstatten, Freiburg, Germany). Animals were not anesthetized. Rats from the experimental group were exposed simultaneously to a single dose of X-irradiation (5 Gy; IR/m = 195 R/min), within the first 48 h after birth. Only the head was exposed, the rest of the body being protected with a 4 mm thick lead sheet. Control rats were handled similarly to their respective X-irradiated littermates. Animals were killed by decapitation at PN day 90. The brain was exposed, and the CE was isolated on ice. After determining its weight, the CE was homogenized in water (diluted 1:10, wt./ vol.). MAO activity in the homogenate was determined using kynuramine as the substrate, according to Weiss-
bach et al. (1980), with slight modifications (Dopico and Zieher, 1993). Activities of MAO-A and -B were measured in separate homogenates by incubating the homogenate with either 1 p M L-deprenyl or 1 p M clorgyline at 0°C, 30 min before the addition of the substrate. One homogenate was obtained from each CE, and one determination was performed per homogenate. The protein content was determined using the Follin phenol reagent method of Lowry et al. (1951), with bovine serum albumin as standard. Data were analyzed using analysis of variance. The post hoc analysis to test the significance of the difference between individual means was performed according to Bonferroni (Hochberg and Tamhane, 1987). Data are expressed as mean _+ S.E.M. Kynuramine dihydrobromide, 4-hydroxyquinoline, and clorgyline were purchased from Sigma Chemical Co., St. Louis, MO. L-deprenyl was a generous gift from Dr. Daniel Livio, Lab. Armstrong, Argentina. Total MAO activity in adult rat CE following a single dose of neonatal X-irradiation was significantly reduced (59% of nonirradiated littermate controls, P < 0.01; Table 1), in agreement with a previous finding (Dopico and Zieher, 1993). The comparison of data from X-irradiated animals with those from agematched (littermate) controls rules out any contribution of age, a particular modifier of MAO activity (Oreland et al., 1990), to the observed difference. In addition, X-irradiated animals showed a marked decrease in cerebellar weight when compared to controls: 148.8 _+ 6.5 mg vs. 238.5___4.4 mg, P < 0 . 0 1 . This reduction in weight ( - 37.6%) is related to the action o f the noxa on developing cerebellar neurons in the early postnatal period, which results in an agranular cerebellar cortex (Dopico et al., 1989). When MAO activity was expressed taking into account the reduction in cerebellar weight, the whole MAO specific activity was not significantly different from the controls (Table 1). The fact that X-irradiation did not modify the protein content of the organ (11.7_+0.8 vs. 11.3_-4-1.5% of wet tissue weight from irradiated CE and controls, respectively)
L.R. Guelman et aL / Neuroscience Research 25 (1996) 97-100
allowed us to expres,~ the specific activity on a wet tissue basis. The unchanged total MAO activity referred to weight tissue indicates that the marked reduction in MAO activity/CE is basically due to the permanent atrophy of the organ produced by the ionizing treatment. The activity of total MAO provides a very limited insight into a possible reduction of NA metabolism by the enzyme in X-irradiated CE. For example, a hypothetical permanent reduction in presynaptic MAO (essentially MAO-A) induced by neonatal X-irradiation, which could explain tile increase in the NA content of adult CE characteristic', of this model, would be masked if X-irradiation also induces an increase of MAO activity from nonmonoarninergic cell populations (essentially MAO-B). Thi,; situation would occur, for example, with astrocytic gliosis reactive to X-rays. Astrocytes are known to contain high amounts of MAO-B (Berry et al., 1994) and proliferate in response to neonatal X-irradiation (Sykov~i et al., 1992). Conversely, as the intraneuronal portion of MAO is a small proportion of the total amount (Oreland et al., 1990), our technique, as others that use brain tissue homogenates, primarily estimates extra-monoaminesynaptosomal MAO. Then, the reduction in total MAO/CE reported here could be related to the impairment of development of nonmonoaminergic cell populations by the noxa. According to this interpretation, the unchanged specific activity of total MAO found in the X-rays-induced atrophic CE might reflect a relative greater contribution of presynaptic MAO (assuming an unmodified number of NA terminals) to the total MAO activity. In consequence, it is essential to consider separately the effects of X-irradiation on MAO-A and MAO-B activities. We evaluated the separate activities of MAO-A and MAO-B through an in vitro assay, blocking each isoenzyme with either clorgyline (1 /zM) or L-deprenyl (1 p M), and measuring the amount of activity left toward a nonspecific substrate. These in vitro concentrations of the blockers are known to selectively block MAO-B and MAO-A, respectively (Tsang et al., 1986; Thakkar and Mallick, 1993). The activity of MAO-A/CE was significantly decreased by the ionizing treatment (58.3% of controls, P < 0.01, Table 1). This finding, together with the fact that two other indicators of biochemical activity of the noradrenergic system, such as the net 3H retention by cerebellar cortex slices labeled with [3H]NA (which reflects the number of synaptic NA terminals) and the activity of tyrosine hydroxylase under optimal conditions (the rate-limiting enzyme in catecholamine biosynthesis) were not increased in X-irradiated CE (Dopico and Zieher, 1993), indicates that a single dose of X-rays at birth does not induce a longterm sprouting of biochemically functional NA terminals. Then, the permanent increase in cerebellar NA
99
content may be alternatively interpreted as an increase in the amount of neurotransmitter per NA terminal. In spite of the well known and widely documented sensitivity of rodent brain MAO to different forms of radiation (Catravas and McHale, 1975; Bod6 and Benk6, 1987), the specific activity of MAO-A in adult CE after a single dose of X-irradiation applied in the neonatal period remained unchanged (Table 1). This unchanged activity, together with an unaltered number of biochemically functional NA terminals in X-irradiated CE (see above) makes it highly improbable that the permanent increase in NA content of adult CE following neonatal X-irradiation could be due to a decreased neuronal metabolism of the neurotransmitter by this enzyme. Whether NA in X-irradiated CE is preferentially stored in a vesicular compartment, resulting in a diminished availability of the amine for mitochondrial MAO-A, is a possibility to be considered, although unlikely. We have previously found that the vesicular release of NA by either reserpine or RO-1284, a reserpine-like agent, remains unmodified in adult rat CE after neonatal X-irradiation (Dopico and Zieher, 1993). Changes in the activities of both isoenzymes were reflected on the MAO-A/MAO-B ratio. A MAO-A/ MAO-B ratio of 0.42 in the controls (Table 1) confirms previous reports which indicated that MAO-B seems to be the predominant type in the rat cerebellar cortex (Berry et al., 1994). The differential decrease in the isoenzyme activities produced by the treatment (MAOB, - 34% vs. MAO-A, - 41.7%) results in a reduction of the ratio in irradiated animals (88.1% of controls). However, the MAO-A/MAO-B ratio was unmodified when data were expressed per mg tissue (98.1% of controls). Thus, the decreased ratio when activities were expressed on a CE basis possibly indicates that the cell populations that largely contain MAO-A and MAO-B in rat CE (NA axons and astrocytes, respectively) are, in the long-term, differentially affected by X-irradiation. One possibility is that the development of coming NA terminals to CE is more seriously impaired than that of microneurons and astrocytes by X-rays. This conclusion is not supported by the marked increase in the concentration of different biochemical indicators of presynaptic noradrenergic activity in the atrophic CE of X-irradiated rats previously reported (Dopico and Zieher, 1993). Alternatively, the reduction in MAO-B, associated with the neuronal loss of nonnoradrenergic populations, could be partially masked by an increase of MAO-B activity due to reactive gliosis of astrocytes in response to X-irradiation. This phenomenon would possibly be reflected in a smaller reduction of MAO-B activity/CE compared to that of MAO-A. A permanent gliosis of the astrocytic population after a single dose of X-irradiation at birth would be consistent with previous
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L.R. Guelman et al. / Neuroscience Research 25 (1996) 97-100
findings from others, showing astrogliosis in the rat spinal cord reactive to early postnatal irradiation (Sykovfi et al., 1992), and ourselves, demonstrating a disarrangement of the glial fibrillary acidic protein, a specific marker for differentiated astrocytes, in adult rat CE following the same irradiation protocol as the one used here (Dopico et al., 1989). Finally, we should mention that the generation of free radicals by MAO-B activity is thought to be involved in some models of neuronal degeneration (Cohen, 1984). Free radicals, in turn, are known to be responsible for neuronal injury caused by ionizing radiation (Verity, 1994). The net reduction of MAO-B activity in adult CE from neonatally X-irradiated rats reported here does not totally rule out a pathogenic role for this isoenzyme in the generation of the permanent changes in the cytoarchitecture of CE by X-rays, as a consequence of the action of the noxa on developing cell populations in the early postnatal period. To obtain some insight into this issue, a time-course of MAO-B activity including determinations close to the time of exposure would be needed. In conclusion, we reported here a marked reduction of MAO-A and MAO-B activities in adult rat CE after exposure to a single dose of neonatal X-irradiation. These decreased activities are largely explained by the permanent atrophy of the organ produced by the action of the ionizing noxa on cerebellar developing neurons. The lack of change in the specific activity of MAO-A suggests that an impaired presynaptic metabolism of NA by this enzyme is not responsible for the permanent increase in cerebellar NA content characteristic of this model.
Acknowledgements We thank Dr. C. Olivieri and Dr. D. Feld for making available the X-irradiation facility of the Hospital Municipal de Oncologia, Buenos Aires. We also thank Diana Prahic, Alicia Petragali, and A. Karpluk for their excellent technical assistance.
References Berry, M.D., Juorio, A.V. and Paterson, I.A. (1994) The functional role of monoamine oxidases A and B in the mammalian central nervous system. Prog. Neurobiol., 42: 375-391. Bodr, K. and Benk6, Gy. (1987) Alterations in monoamine oxidase activity of the mouse brain and liver after mixed neutron-gamma irradiation. Acta Physiol. Hung., 69: 181-188.
Catravas, G.N. and McHale, C.G. (1975) Changed activities of brain enzymes involved in neurotransmitter metabolism in rats exposed to different qualities of ionizing radiation. J. Neurochem., 24: 673-676. Cohen, G. (1984) Oxy-radical toxicity in catecholamine neurons. Neurotoxicology, 5: 77-82. Dopico, A.M. and Zieher, L.M. (1993) Neurochemical characterization of the alterations in the noradrenergic afferents to the cerebellum of adult rats exposed to X-irradiation at birth. J. Neurochem, 61: 481-489. Dopico, A.M., Zieher, L.M., Mayo, J., Altschiiller, N., L6pez J.J. and Jaim-Etcheverry, G. (1989) Long-term changes in noradrenaline (NA) and dopamine (DA) contents of rat cerebellum following neonatal X-irradiation. Neurochem. Int., 15: 97-105. Ekblom, J., Aquilonius, S.-M. and Jossan, S.S. (1993) Differential increases in catecholamine metabolizing enzymes in amyotrophic lateral sclerosis. Exp. Neurol., 123: 289-294. Fagervall, I. and Ross, S.B. (1986) A and B forms of monoamine oxidase within the monoaminergic neurons of the rat brain. J. Neurochem., 47: 569-576. Guelman, L.R., Zieher, L.M., Rios, H., Mayo, J. and Dopico, A.M. (1993) Motor abnormalities and changes in the noradrenaline content and the cytoarchitecture of developing cerebellum following X-irradiation at birth. Mol. Chem. Neuropathol., 20: 45-57. Hochberg, Y. and Tamhane, A. (1987) Multiple Comparison Procedures, J. Wiley & Sons, Inc., New York. Iversen, L.L. (1974) Uptake mechanisms for neurotransmitter amines. Biochem. Pharmacol., 23: 1927-1935. Johnston, J.P. (1968) Some observations upon a new inhibitor of monoamine oxidase in brain. Biochem. Pharmacol., 17:1285 1297. Levitt, P., Pintar, J.E. and Breakfield, X.O. (1982) Immunocytochemical demonstration of monoamine oxidase B in brain astrocytes and serotonergic neurons. Proc. Natl. Acad. Sci. USA, 79: 63856389. Lowry, O.H., Rosebrough, N.J., Farrand, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem,, 193: 265-275. Oreland, L., Hiraga, Y., Jossan, S.S., Regland, B. and Gottfries, C.G. (1990) Increased monoamine oxidase activity and vitamin B-12 deficiency in dementia disorders. In: P. Dostert et al. (Eds.), Early Markers in Parkinson's and Alzheimer's Diseases, Springer-Verlag, Austria, pp. 267-286. Sykov/t, E., Jendelov~i P., Simonovfi, Z. and Chvfital, A. (1992) K + and pH homeostasis in the developing rat spinal cord is impaired by early postnatal X-irradiation. Brain Res., 594: 19-30. Thakkar, M. and Mallick, B.N. (1993) Effect of rapid eye movement sleep deprivation on rat brain monoamine oxidases. Neuroscience, 55: 677-683. Tsang, D., Ho, K.P. and Wen, H.L. (1986) Ontogenesis of multiple forms of monoamine oxidase in rat brain regions and liver. Dev. Neurosci., 8: 243-250. Verity, M.A. (1994) Oxidative damage and repair in the developing nervous system. Neurotoxicology, 15: 81-92. Weissbach, H., Smith, T.E., Daly, J.W., Witkop, B. and Underfriend, S. (1980) A rapid spectrophotometric assay of MAO based on the rate of disappearance of kynuramine. J. Biol. Chem., 235:11601163. Youdim, M.B.H. and Riederer, P. (1993) The relevance of glial monoamine oxidase-B and polyamines to the action of selegiline in Parkinson's disease. Mov. Dis., 8 (Suppl. I): $8-S13.
Neuroscience Research 25 (1996) 97-100
Rapid communication
Activities of monoamine oxidase-A and -B in adult rat cerebellum following neonatal X-irradiation L a u r a R . G u e l m a n a, L u i s M . Z i e h e r a, M a r i a A. Z o r r i l l a Z u b i l e t e a, A l e j a n d r o M . D o p i c o b'* al-~ C~tedra de Farmacologla, Facultad de Medicina, Universidad de Buenos Aires. Paraguay 2155 P. 15, 1121 Buenos Aires, Argentina bDepartment of Pharmacology and Molecular Toxicology, University of Massachusetts Medical School 55 Lake Av. North, Worcester MA 01655, USA
Received 4 December 1995; revised 12 January 1996; accepted 1 February 1996
Abstract
The activities of monoetmine oxidases, MAO-A and MAO-B, were separately determined in the cerebellum (CE) from adult rats neonatally exposed to 5 Gy X-irradiation. They were found to be markedly reduced: 58% and 66% of values from nonirradiated, littermate controls. Since the specific activities of both isoenzymes (per mg tissue weight) were not significantly different from controls, the reduction of activity per CE is basically explained by the irradiation-induced cerebellar atrophy. The unmodified MAO-A specific activity makes it highly improbable that the increase in the cerebellar noradrenaline content, characteristic of neonatally X-irradiated rats, could be due to a decreased neuronal metabolism of noradrenaline by this enzyme. Keywords: MAO, monamine: oxygen oxidoreductase (deaminating) (ravin containing); MAO-A; MAO-B; Neonatal X-irradiation;
Cerebellum; Noradrenaline
The exposure of neonatal rats to a single dose of X-irradiation (5 Gy) produces permanent changes in the neurochemistry and cytoarchitecture of cerebellum (CE), accompanied by characteristic motor abnormalities (Dopico et al., 198!)). Adult rats that were X-irradiated within the first 48 h after birth show a motor syndrome which consists of dystonia-like movements, fine tremor and atax~c gait. Their CE is markedly atrophic, with an agranular cortex in which Purkinje cells are randomly scattered at all depths (Dopico et al., 1989; Guelman et al., 1993). The neurochemical alterations include a mild but significant increase in the noradrenaline (NA) content of CE ( + 20-36%) which, due to the X-ray-induced cerebellar atrophy, results in a marked increase in NA concentration (+225%) (Dopico et al., 1989; Dopico and Zieher, 1993; Guelman et al., 1993). * Corresponding author. Tel.: + 1 508 856 6989; fax: + 1 508 856 5080; e-mail: [email protected]
The biochemical mechanism(s) that leads to the increase in the NA content of X-irradiated CE is largely unknown. One factor that determines the endogenous level of a neurotransmitter is its degradation rate. Monoamine oxidase [monoamine: oxidase oxidoreductase (deaminating), EC 1.4.3.4, MAO] is a primary catabolic enzyme for NA in mammalian brain (Berry et al., 1994). In addition, it is well known that brain MAO activity is highly sensitive to radiation, being either decreased or increased depending upon the type and dose of radiation (Catravas and McHale, 1975; Bod6 and Benk6, 1987). MAO exists in two forms, MAO-A and MAO-B, which can be differentiated based on their substrate specificities and inhibitor sensitivities. Whereas MAO-A is inhibited by low concentrations of clorgyline, MAOB is selectively inhibited by L-deprenyl (Johnston, 1968; Berry et al., 1994). NA is a preferential substrate for MAO-A (Youdim and Riederer, 1993), which is the main type present in the noradrenergic terminals (Berry et al., 1994). The primary route for the inactivation of
0168-0102/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PH SO168-0102(96)01022-X
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Table 1 In vitro activities of monoamine oxidase-A and -B in the cerebellum of adult rats exposed to a single dose (5 Gy) of neonatal X-irradiation pM 4-OHQ/mg wet tissue/h
#mol 4-OHQ/CE/h
Total MAO MAO-A MAO-B MAO-A/MAO-B
Control
X-irradiated
Control
X-irradiated
1.17 + 0.22 0.36 _+0.04 0.86 _+0.16 0.42 _+0.05
0.69 _+0.18a (58.9 _+15.4) 0.21 + 0.01a (58.3 _+ 4.2) 0.57 _ 0 . 0 9 b (66.2 _+15.1) 0.37 _+0.03 (88.1 _+_ 3.6)
3.92 _ 0.46 1.48 _+0.15 2.80 _+0.06 0.52 _+0.05
3.50 _+0.38 (89.3 + 9.7) 1.48 _ 0.10 (100.0_ 6.7) 2.90 _+0.05 (103.6_+1.8) 0.51 + 0.03 (98.1 + 3.8)
Data from X-irradiated CE expressed as percentages of their respective controls are given in parentheses. One enzyme determination was performed per CE. Data are mean + S.E.M. from 3-6 determinations. 4-OHQ, 4-hydroxyquinoline.CE, cerebellum. aSignificantly different from controls (P<0.01). bSignificantlydifferent from controls (P < 0.05). released N A is the reuptake into the nerve terminal (Iversen, 1974), and MAO-A is considered the form responsible for presynaptic metabolism of NA (Fagervall and Ross, 1986). On the other hand, MAO-B is mainly localized in non-neuronal elements, being the astrocytes critical contributors of its activity in both normal and injured CNS (Levitt et al., 1982; Berry et al., 1994). An increase in MAO-B activity is found when astrogliosis occurs (Ekblom et al., 1993), and astrogliosis may be induced by X-irradiation in the early postnatal period (Sykovfi et al., 1992). Therefore, in this study we evaluated separately the activities of MAO-A and MAO-B in CE from adult rats exposed to X-irradiation in the neonatal period using an in vitro assay. Newborn littermate Wistar rats of both sexes (8-10 pups/litter) were separated into experimental and control (nonirradiated) groups. Rats from each group were housed 4-5/cage, given rat chow and water ad libitum, and maintained on a 12 h light/dark cycle at 25°C. The radiation source was a 225 kV, 17 mA, X-ray unit (Philips 220/25 for profound radiotherapy, Philips Groeilmpen Fabrieken, Eindhoven, The Netherlands). A 1 mm copper filter equivalent to 1.3 mm copper half-value level (HVL) was used for filtration. Exposure time was fixed by previous dosimetry, using a Simplex probe (Simplex Universal Dosimeter Physikalisch Technische Werkstatten, Freiburg, Germany). Animals were not anesthetized. Rats from the experimental group were exposed simultaneously to a single dose of X-irradiation (5 Gy; IR/m = 195 R/min), within the first 48 h after birth. Only the head was exposed, the rest of the body being protected with a 4 mm thick lead sheet. Control rats were handled similarly to their respective X-irradiated littermates. Animals were killed by decapitation at PN day 90. The brain was exposed, and the CE was isolated on ice. After determining its weight, the CE was homogenized in water (diluted 1:10, wt./ vol.). MAO activity in the homogenate was determined using kynuramine as the substrate, according to Weiss-
bach et al. (1980), with slight modifications (Dopico and Zieher, 1993). Activities of MAO-A and -B were measured in separate homogenates by incubating the homogenate with either 1 p M L-deprenyl or 1 p M clorgyline at 0°C, 30 min before the addition of the substrate. One homogenate was obtained from each CE, and one determination was performed per homogenate. The protein content was determined using the Follin phenol reagent method of Lowry et al. (1951), with bovine serum albumin as standard. Data were analyzed using analysis of variance. The post hoc analysis to test the significance of the difference between individual means was performed according to Bonferroni (Hochberg and Tamhane, 1987). Data are expressed as mean _+ S.E.M. Kynuramine dihydrobromide, 4-hydroxyquinoline, and clorgyline were purchased from Sigma Chemical Co., St. Louis, MO. L-deprenyl was a generous gift from Dr. Daniel Livio, Lab. Armstrong, Argentina. Total MAO activity in adult rat CE following a single dose of neonatal X-irradiation was significantly reduced (59% of nonirradiated littermate controls, P < 0.01; Table 1), in agreement with a previous finding (Dopico and Zieher, 1993). The comparison of data from X-irradiated animals with those from agematched (littermate) controls rules out any contribution of age, a particular modifier of MAO activity (Oreland et al., 1990), to the observed difference. In addition, X-irradiated animals showed a marked decrease in cerebellar weight when compared to controls: 148.8 _+ 6.5 mg vs. 238.5___4.4 mg, P < 0 . 0 1 . This reduction in weight ( - 37.6%) is related to the action o f the noxa on developing cerebellar neurons in the early postnatal period, which results in an agranular cerebellar cortex (Dopico et al., 1989). When MAO activity was expressed taking into account the reduction in cerebellar weight, the whole MAO specific activity was not significantly different from the controls (Table 1). The fact that X-irradiation did not modify the protein content of the organ (11.7_+0.8 vs. 11.3_-4-1.5% of wet tissue weight from irradiated CE and controls, respectively)
L.R. Guelman et aL / Neuroscience Research 25 (1996) 97-100
allowed us to expres,~ the specific activity on a wet tissue basis. The unchanged total MAO activity referred to weight tissue indicates that the marked reduction in MAO activity/CE is basically due to the permanent atrophy of the organ produced by the ionizing treatment. The activity of total MAO provides a very limited insight into a possible reduction of NA metabolism by the enzyme in X-irradiated CE. For example, a hypothetical permanent reduction in presynaptic MAO (essentially MAO-A) induced by neonatal X-irradiation, which could explain tile increase in the NA content of adult CE characteristic', of this model, would be masked if X-irradiation also induces an increase of MAO activity from nonmonoarninergic cell populations (essentially MAO-B). Thi,; situation would occur, for example, with astrocytic gliosis reactive to X-rays. Astrocytes are known to contain high amounts of MAO-B (Berry et al., 1994) and proliferate in response to neonatal X-irradiation (Sykov~i et al., 1992). Conversely, as the intraneuronal portion of MAO is a small proportion of the total amount (Oreland et al., 1990), our technique, as others that use brain tissue homogenates, primarily estimates extra-monoaminesynaptosomal MAO. Then, the reduction in total MAO/CE reported here could be related to the impairment of development of nonmonoaminergic cell populations by the noxa. According to this interpretation, the unchanged specific activity of total MAO found in the X-rays-induced atrophic CE might reflect a relative greater contribution of presynaptic MAO (assuming an unmodified number of NA terminals) to the total MAO activity. In consequence, it is essential to consider separately the effects of X-irradiation on MAO-A and MAO-B activities. We evaluated the separate activities of MAO-A and MAO-B through an in vitro assay, blocking each isoenzyme with either clorgyline (1 /zM) or L-deprenyl (1 p M), and measuring the amount of activity left toward a nonspecific substrate. These in vitro concentrations of the blockers are known to selectively block MAO-B and MAO-A, respectively (Tsang et al., 1986; Thakkar and Mallick, 1993). The activity of MAO-A/CE was significantly decreased by the ionizing treatment (58.3% of controls, P < 0.01, Table 1). This finding, together with the fact that two other indicators of biochemical activity of the noradrenergic system, such as the net 3H retention by cerebellar cortex slices labeled with [3H]NA (which reflects the number of synaptic NA terminals) and the activity of tyrosine hydroxylase under optimal conditions (the rate-limiting enzyme in catecholamine biosynthesis) were not increased in X-irradiated CE (Dopico and Zieher, 1993), indicates that a single dose of X-rays at birth does not induce a longterm sprouting of biochemically functional NA terminals. Then, the permanent increase in cerebellar NA
99
content may be alternatively interpreted as an increase in the amount of neurotransmitter per NA terminal. In spite of the well known and widely documented sensitivity of rodent brain MAO to different forms of radiation (Catravas and McHale, 1975; Bod6 and Benk6, 1987), the specific activity of MAO-A in adult CE after a single dose of X-irradiation applied in the neonatal period remained unchanged (Table 1). This unchanged activity, together with an unaltered number of biochemically functional NA terminals in X-irradiated CE (see above) makes it highly improbable that the permanent increase in NA content of adult CE following neonatal X-irradiation could be due to a decreased neuronal metabolism of the neurotransmitter by this enzyme. Whether NA in X-irradiated CE is preferentially stored in a vesicular compartment, resulting in a diminished availability of the amine for mitochondrial MAO-A, is a possibility to be considered, although unlikely. We have previously found that the vesicular release of NA by either reserpine or RO-1284, a reserpine-like agent, remains unmodified in adult rat CE after neonatal X-irradiation (Dopico and Zieher, 1993). Changes in the activities of both isoenzymes were reflected on the MAO-A/MAO-B ratio. A MAO-A/ MAO-B ratio of 0.42 in the controls (Table 1) confirms previous reports which indicated that MAO-B seems to be the predominant type in the rat cerebellar cortex (Berry et al., 1994). The differential decrease in the isoenzyme activities produced by the treatment (MAOB, - 34% vs. MAO-A, - 41.7%) results in a reduction of the ratio in irradiated animals (88.1% of controls). However, the MAO-A/MAO-B ratio was unmodified when data were expressed per mg tissue (98.1% of controls). Thus, the decreased ratio when activities were expressed on a CE basis possibly indicates that the cell populations that largely contain MAO-A and MAO-B in rat CE (NA axons and astrocytes, respectively) are, in the long-term, differentially affected by X-irradiation. One possibility is that the development of coming NA terminals to CE is more seriously impaired than that of microneurons and astrocytes by X-rays. This conclusion is not supported by the marked increase in the concentration of different biochemical indicators of presynaptic noradrenergic activity in the atrophic CE of X-irradiated rats previously reported (Dopico and Zieher, 1993). Alternatively, the reduction in MAO-B, associated with the neuronal loss of nonnoradrenergic populations, could be partially masked by an increase of MAO-B activity due to reactive gliosis of astrocytes in response to X-irradiation. This phenomenon would possibly be reflected in a smaller reduction of MAO-B activity/CE compared to that of MAO-A. A permanent gliosis of the astrocytic population after a single dose of X-irradiation at birth would be consistent with previous
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findings from others, showing astrogliosis in the rat spinal cord reactive to early postnatal irradiation (Sykovfi et al., 1992), and ourselves, demonstrating a disarrangement of the glial fibrillary acidic protein, a specific marker for differentiated astrocytes, in adult rat CE following the same irradiation protocol as the one used here (Dopico et al., 1989). Finally, we should mention that the generation of free radicals by MAO-B activity is thought to be involved in some models of neuronal degeneration (Cohen, 1984). Free radicals, in turn, are known to be responsible for neuronal injury caused by ionizing radiation (Verity, 1994). The net reduction of MAO-B activity in adult CE from neonatally X-irradiated rats reported here does not totally rule out a pathogenic role for this isoenzyme in the generation of the permanent changes in the cytoarchitecture of CE by X-rays, as a consequence of the action of the noxa on developing cell populations in the early postnatal period. To obtain some insight into this issue, a time-course of MAO-B activity including determinations close to the time of exposure would be needed. In conclusion, we reported here a marked reduction of MAO-A and MAO-B activities in adult rat CE after exposure to a single dose of neonatal X-irradiation. These decreased activities are largely explained by the permanent atrophy of the organ produced by the action of the ionizing noxa on cerebellar developing neurons. The lack of change in the specific activity of MAO-A suggests that an impaired presynaptic metabolism of NA by this enzyme is not responsible for the permanent increase in cerebellar NA content characteristic of this model.
Acknowledgements We thank Dr. C. Olivieri and Dr. D. Feld for making available the X-irradiation facility of the Hospital Municipal de Oncologia, Buenos Aires. We also thank Diana Prahic, Alicia Petragali, and A. Karpluk for their excellent technical assistance.
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