Subcellular localization of superoxide dismutases, glutatione peroxidase and catalase in developing rat cerebral cortex

Mechanisms of Ageing and Development, 48 (1989) 15--31

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Elsevier Scientific Publishers Ireland Ltd.

SUBCELLULAR LOCALIZATION OF SUPEROXIDE DISMUTASES, GLUTATHIONE PEROXIDASE AND CATALASE IN DEVELOPING RAT CEREBRAL CORTEX

ROLANDO DEL MAESTRO and WARREN McDONALD Brain Research Laboratories, Experimental Research Unit, Department of ClinicalNeurological Sciences, VictoriaHospital, University of Western Ontario, London, Ontario N6A 4G5 (Canada) (Received May 3rd, 1988) (Revision received November 9th, 1988)

SUMMARY

The levels of copper- and zinc-containing superoxide dismutase (CuZnSOD), manganese-containing superoxide dismutase (MnSOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activity have been assessed in a nuclear fraction (NF), a mitochondrial fraction (MF) and a non-mitochondrial, non-nuclear fraction (NMNNF) isolated from developing rat cerebral cortex. The NF showed increasing CuZn and MnSOD activities and static, low activities of GSH-Px and CAT during development. The MF had increased levels of MnSOD and GSH-Px activities and a rapid decrease in CAT activity associated with development. Histochemical methods have localized greater CAT activity in mitochondria isolated from 2-day-old rat brain when compared to 77-day-old animals. Development was associated with increasing CuZnSOD activity, a decrease in CAT activity and, after an initial fall at 19 days, increasing GSH-Px activity in the NMNNF. Measurable activity of MnSOD were found in the NMNNF and appeared to be static during the time period assessed. A distinct ontogenetic pattern of oxidative enzyme activities and subcellular locations is associated with development in rat cerebral cortex.

Key words: Neonatal cerebral development; CuZn- and Mn-superoxide dismutase; Catalase; Glutathione peroxidase; Cytochrome c oxidase; Mitochondria INTRODUCTION

The cerebral ontogenetic pattern of oxidative enzymes appears to be quite complex. Neonatal cerebral development in rats is characterized by an increase in copAddress correspondence and reprint requests to: Dr. Rolando Del Maestro, Brain Research Laboratory, Department of Clinical Neurological Sciences, Victoria Hospital, South Street Campus, London, Ontario, Canada N6A 4G5. 0047-6374/89/$03.50 Printed and Published in Ireland

© 1989 Elsevier Scientific Publishers Ireland Ltd.

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per- and zinc-containing superoxide dismutase (CuZnSOD, superoxide : superoxide oxidoreductase EC 1.15.1.1) activity, static levels of manganese-containing superoxide dismutase (MnSOD), a fall in catalase (CAT, hydrogen peroxide : hydrogen peroxide oxidoreductase EC 1.11.1.6) activity and after an inital fall increasing glutathione peroxidase (GSH-Px, glutathione : hydrogen peroxide oxidoreductase EC 1.11.1.9) activity [1]. Similar and comparable changes were seen in homogenates from the four brain regions assessed: cerebral cortex, striatum, cerebellum and brain stem. Developmental changes seen in these enzymes in brain tissue homogenates may not reflect changes that may occur in their subcellular localization. Rat neonatal cerebral development is associated with increases in a large number of enzymes involved in oxidative respiration [2,3], increased mitochondria and alterations in other organelles [4]. Changes in the intracellular localization of scavenging enzymes may provide information on the development of subcellular enzymatic scavenging mechanisms and provide some insight into the site of origin of superoxide anion radical (02-) and H202 during brain development. Mavelli et al. [5] have shown increased mitochondrial MnSOD and cytoplasmic CuZnSOD during development in rat brain but concluded that independent regulation of the expression of scavenging enzyme activities occurred during brain differentiation. Data obtained from brain homogenates from four different cerebral regions demonstrate a consistent ontogenetic pattern of scavenging enzyme activity during development [I]. It is hypothesized that intracellularly these enzymes function as a scavenging system and that their subcellular concentration and location is linked both to the generation of the appropriate substrate and the presence of other components of the scavenging enzyme system. The levels of CuZnSOD, MnSOD, GSH-Px and CAT in a nuclear fraction (NF), a mitochondrial fracture (MF) and a non-mitochondrial, non-nuclear fraction (NMNNF) have been assessed in developing rat cerebral cortex. A concept of oxidative enzyme ontogenesis at a subcellular level is presented. M A T E R I A L S AND M E T H O D S

Wistar rats (Woodland Laboratories, Guelph, Canada) of both sexes and 2, 19 and 77 days of age were used. Animals from the same litters were used for each time period studied. Animals were sacrificed by decapitation, a heparinized blood sample obtained, the brain immediately removed and rinsed in iced saline and the cerebral cortex from the frontal and parietal lobes was obtained bilaterally at 0°C using magnified vision [ I]. For studies using 2-day-old rats, blood and brain samples were pooled from 3 to 6 animals for each data point. In animals used from other time periods studied, all tissue fractions and each animal's corresponding blood sample was available for study.

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Tissue fractionation Nuclear fraction (NF). Samples were placed in 2 - - 3 ml of 0°C 0.25 M sucrose and thoroughly homogenized using 10 strokes in a Potter-Elvehjem tissue homogenizer (clearance 125 ta). The homogenate was centrifuged at 1200 g for 10 min and the supernatant saved. The grey layer directly above the erythrocyte layer was carefully removed and placed in 4 vols. o f 0.004 M MgC12, 0.4 M sucrose and 0.25°70 Triton X-100 for 30 rain at 20°C. This solution was then centrifuged at 1500 g for 10 min, the supernatant discarded, and the pellet washed with 4 vols. o f 0.25 M sucrose and centrifuged at 1200 g for 5 rain. This was repeated two more times using 1000 g for 5 min each. The final pellet was resuspended in 0.25 M sucrose (4 vols.) and centrifuged at 12 000 g for 1 min, the supernatant was discarded and the pellet (NF) stored at - 80 °C until assayed.

Mitochondrial fraction (MF) and non-mitochondrial non-nuclear fracture (NMNNF). The initial supernatant was centrifuged for 7.5 min at 12 000 g and the supernatant frozen and stored at - 80°C (NMNNF). The white layer was removed from the pellet and the pellet resuspended in 0.25 M sucrose and centrifuged at 12 000 g for 7.5 min. The white layer was again carefully removed and the procedure repeated once more. The final pellet was stored at - 80 °C until assayed (MF). Isolation fraction purity. All three fractions from 77-day-old animals were assayed biochemically using marker enzymes and the NF and MF were assessed using light and electron microscopy. Light microscopy. The NF was stained using methylene blue and examined microscopically. Electron microscopy. The MF pellets were fixed for 1.5 h in a solution of 3070 glutaraldehyde in Sorenson phosphate buffer (pH 7.2). Following fixation, the pellet was washed a number of times with Sorenson buffer, post-fixed for 1 h in 1070 osmium tetroxide in Sorenson buffer and dehydrated through a series of ethanol solutions (50--100070). After washing twice with 10007o propylene oxide, the sample was put through increasing grades of a varient of Mollenhauer Epon-araldite in propylene oxide. The sections were cut, then stained with Reynoid's lead citrate and a saturated solution of uranyl acetate in 70°-/o ethanol and view on a Philips EM 201 electron microscope. Histochemical localization of CA T in the mitochondrial fraction. The measurement of C A T activity in the MF fraction could be due to C A T activity in mitochondria, lysosomal and other contamination or all of these. To help determine the localization of C A T in the MF, a histochemical method was employed [6]. The MF pellets were fixed in 3070 glutaraldehyde in 0.1 M bicarbonate buffer (pH 10.5) containing 0.05070 CaCI 2. The pellet was consistently agitated for 2 h and then 10 -2 M 3,3'-diaminobenzidine tetrahydrochloride (DAB) was added in 0.I M bicarbonate buffer (ph 10.5). After 1 h o f preincubation, 10 -1 M HzO 2 was added and followed by 2h of incubation in the complete medium. The pellet was then fixed with osmium and processed as described for electron microscopy.

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Enzyme assay Lactic dehydrogenase (LDH, L-lactate : NAD oxidorectase EC I. 1.1.27) was assayed using the method of Wroblewski and LeDue [7] as supplied by Sigma Chemical Co. Ltd. Cytochrome c oxidase (cytochrome c : oxygen oxidoreductase EC 1.9.3. l) activity was assessed by the method of Smith [8]. Monoamine oxidase (MAO, amine : oxygen oxidoreductase EC 1.4.3.4) was assayed by the method of Weissbach et al. [10]. Succinate dehydrogenase (SDH, succinate : tetrazolium oxidoreductase EC 1.3.99.1) was measured by the method of Pennington [11]. Acid phosphate (orthophosphoric monoester phorphohydrolase EC 3.1.3.2) was assayed using a Sigma kit (Sigma Chemical Co., St. Louis, No. 104). Scavenging enzymes The levels of CuZnSOD, MnSOD, GSH-Px and CAT were measured in all samples as previously described by Del Maestro and McDonald [1,12]. The pyrogallol autooxidation method was used to determine CuZn and MnSOD [13]. Cyanide insensitive SOD which was considered to represent MnSOD was assayed by adding l mM sodium cyanide to the supernatant [12]. The method of Gunzler et al. [14] was used to measure GSH-Px. The measurement of CAT was carried out immediately after obtaining the supernatant using a Clark electrode [ 12,15]. The hemoglobin [16] and enzyme content of each fraction and each animal's corresponding blood sample was determined and appropriate corrections made [1,12]. Protein The method of Waddell and Hill [17] was used to determine protein and bovine serum albumin was used as a standard. Chemicals The chemicals used and their sources were as follows. All the buffer salts, pyrogaUol and sodium cyanide were obtained from Fischer Scientific Co., NJ. Catalase was obtained from Boehringer Mannheim, F.R.G. and all other chemicals and enzymes were obtained from Sigma Chemical Co. Ltd., St. Louis, MO. Statistics All results are expressed as means - standard error of the mean (S.E.M.). Statistical significance was calculated using the student unpaired t-test with P < 0.05 being considered significant. RESULTS

The Fractionation Procedure Light and electron microscopy. The NF when stained and examined microscopically demonstrated sheets of densely blue staining nuclei and very few had any evi-

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dence of surrounding cytoplasm. Electron microscopy of the NF demonstrated no evidence of small organelles. The electron micrograph of typical MF specimens demonstrated that the majority of the mitochondria appeared intact and contain two membranes. Swollen mitochondria, mitochondria in synaptosomes and occasional lysosomes were seen. Biochemical studies. Table I gives the biochemical assessment of the three fractions used. The NF fracture was characterized by having less than 1 070 of the L D H of the N M N N F and no measurable MAO or SDH. The NF contains some acid phosphatase; however, this was 1.6070 of the total acid phosphatase recovered in the three fractions (data not shown). These results would suggest that little cytoplasmic, mitochondrial or lysosomal contamination is present in the NF. The MF fraction also had very little measurable L D H and was about 30 times richer in mitochondrial enzymes than the NMNNF, Acid phosphatase was clearly present in this fraction and occasional lysosomes were seen on electron microscopy; 9070 of the total acid phosphate recovered was in this fraction and 90070 recovered in the NMNNF (data not shown). The MF has very little cytoplasmic contamination (as measured by LDH) is rich in mitochondria but contaminated with some lysosomes. The NMNNF is very rich in LDH, poor in mitochondria and contains lysosomal enzymes. Developmental changes in NF. The levels of SOD activity for the 3 ages assessed can be seen in Table II while the levels o f GSH-Px and CAT activity in the same subcellular fractions can be seen in Table III. Total SOD activity increased progressively in the NF with 77-day-old animals having significantly more CuZn, Mn and total SOD than either the 2- or 19-day groups. Although some variability is seen in the data, low levels of GSH-Px and CAT activity were measured at the 3 ages assessed. At 19 days, a significant increase in GSH-Px and a significant fall in CAT was seen. Developmental changes in MF. Total SOD activity was not significantly different between 2 and 19 days in the MF; however, a significant fall in CuZnSOD activity and a significant increase in MnSOD was seen between these two time periods. No TABLE I B I O C H E M I C A L ASSESSMENT OF S U B C E L L U L A R F R A C T I O N S Values are expressed as means ± S.E.M. in units of the individual e n z y m e / m g protein (see Material and Methods). N = 3 in each case and 77-day-old rats were used and ND means that no measurable enzyme activity was detected.

Enzymes

Subcellular Fractions Nuclear fraction

Lactic dehydrogenase Monoamineoxidase Succinatedehydrogenase Cytochromecoxidase Acid phosphate

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Non-mitochondrial non-nuclear fraction 2056 2.2 3.3 0.3 1.01

± 209 ± 2.2 __. 2.4 ± 0.07 ± 0.09

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22 significant change was seen in C u Z n S O D between 19 and 77 days while MnSOD more than doubled and made up 9007o of the SOD activity measured in the MF at 77 days. There was a significant and marked fall (10-fold) in C A T concentration of the MF fraction between 2 and 19 days of life and no further significant changes were seen at 77 days. The concentration of G S H - P x significantly increased doubling between 2 and 19 days but no significant change was seen between 19 and 77 days.

Histochemical localization o f CA T Figure 1 has examples of electron micrographs of mitochondria isolated from 2- and 77-day-old rats. At 2 days, mitochondrial reaction product can be seen associated with both the inner and outer membranes. Only about 1°70 of the mitochondria isolated f r o m 77-day-old animals demonstrated evidence of reaction product and the occasional lysosome was positive. Developmental changes in NMNNF. At two days of age, 8207o of the total SOD activity measured in this fraction was MnSOD. However, C u Z n S O D increased significantly both between 2 and 19 days and 19 and 77 days. There was no significant difference in M n S O D between 2 and 77 days at which time MnSOD made up 4007o of the total SOD measured in the N M N N F . These results suggest that some MnSOD is not associated with the mitochondria during development of the cerebral cortex in rat. Two-day-old rats contained significantly more C A T in this fraction than either 19- or 77-day-old animals. A relatively small but significant increase in C A T activity was seen between 19 and 77 days. Seventy-seven-day-old animals had significantly higher levels of G S H - P x than the other two ages studied. Significantly lower levels of G S H - P x were found at 19 days when compared to either 2- or 77-day-old animals. Developmental changes in cytochrome c oxidase. In Table IV, the changes seen in cytochrome c oxidase activity in whole brain homogenates from cerebral cortex and the MF fraction from cerebral cortex for the 3 ages studied can be seen. Significant and progressive increases (approx. 3-fold) in cytochrome c oxidase activity was seen in the homogenate specimens. No significant change in MF cytochrome c oxidase was seen between 2 and 19 days, but a significant increase (50070) was seen at 77 days. These results suggest that an increase in both mitochondrial numbers and cytochrome c oxidase activity per mitochondria occurs during cerebral development. DISCUSSION The assessment of the enzymatic scavenging system in brain tissue has a number of limitations. The brain is a heterogenous organ composed of a variety of anatomically distinct regions, each undergoing dynamic change during development and aging. A complex histological architecture composed of varying numbers of glial and neuronal cells are seen in each region while vascular elements and their entrapped

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24 TABLE IV EFFECT OF AGE ON THE CYTOCHROME c OXIDASE ACTIVITY OF WHOLE BRAIN TISSUE HOMOGENATES AND OF THE MITOCHONDRIAL FRACTION Values represent means _+ S.E.M. and values in brackets represent the number of animals. Cytochrome t' oxidase activity is expressed as/~mol cytochrome c units/mg oxidized/min/mg protein. Statistical significance was calculated using the unpaired student's t-test the " P " value between each group indicated and P >i 0.05 was considered non-significant (NS).

Age (days) 2 19 77

Brain homogenate 1 . 4 _ 0.2(6) P < 0.01' 2.2 --- 0.2(6)-

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I P < 0.001 P < 0-001 l 3.7__. 0.2(6, - I

10.3 ± 1.4(12)=~qNS

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9.8 +- 0.5 ( 1 2 ) ~ - I P < 0.01 P<0.011 15.4 ± 1.4(12) - j

blood elements further complicates enzyme measurement. Attempts have been made to selectively isolate cell populations from brain by size criteria using a variety of meshes and centrifugation techniques [18]. However, the selectivity of these methods is suspect. Isolation methods suggest a predominance of SOD in astrocytes [18] while immunolocalization techniques for CuZnSOD suggest a localization in neurons [19,20]. Cell culture techniques could provide useful information on the content of scavenging enzymes in vivo. However, the activities of scavenging enzymes in glial cell lines are not static responding dynamically to growth of that cell line in a variety of different tissue sites [21]. Malignant glial cell lines derived from human cerebral tumours also do not reflect accurately the level of scavenging enzyme present in the in vivo tumour [21]. These results suggest that the scavenging enzyme content of a tissue is not static, but responds to a number of interacting factors. To accurately assess the enzymatic scavenging system available to specific cerebral ceils, data pertaining to in vivo rates and sites of free radical generation and local enzyme modulation of these reactions would be very pertinent. The application of the techniques of molecular biology may help overcome some of the present problems. However, until then, any information presented must be understood in light of the known limitations of multiple enzyme assays in a complex tissue. The present subcellular fractionation studies were carried out in developing rat cerebral cortex since tissue amounts necessary for the subcellular studies were readily accessible in small animals and homogenates of this tissue accurately reflected changes seen in other brain regions [1]. Nuclear fraction There was no detectable MAO or SDH in the NF suggesting very little mitochondrial contamination in this fraction. The finding of intrinsic cytochrome c

25 oxidase activity in the nucleus is consistent with the results of others [22]. Only small amounts of L D H or acid phosphatase were measured in the NF. At 2 and 19 days, very little CuZn or MnSOD was seen; however, by 77 days, significant amounts of both of these enzymes were measured. With no detectable mitochondrial contamination and only small amounts of cytoplasmic contamination in the NF, it is difficult to suggest that the measured SOD activity is only a contaminant. Cytochrome oxidase preparations have been reported to have weak superoxide dismutase activity which is inhibited by cyanide [23]. However, the SOD activity contributed by this would be small and not explain the non-inhibitable SOD activity measured. Mazeaud et al. [24] have demonstrated the presence of MnSOD in carp erythrocytes which do not contain mitochondria and have suggested a nuclear localization for MnSOD in these cells. Very small amounts of GSH-Px and CAT were measured in the NF fraction at any time period assessed. Our results would be consistent with increasing total SOD, made up of both Mn and CuZnSOD, in the nucleus during development. The presence of low GSH-Px and CAT activities in the nucleus suggest very little intrinsic capacity to reduce H202 in this organelle. Mitoch ondrial fraction The MF was enriched in both outer and inner mitochondrial enzymes and little LDH, as the cytoplasmic marker measured. However, since some acid phosphate was found in the MF fraction, some contamination from lysosomes was present. Mitochondria in rat cerebral cortex undergo a number of changes associated with development [3]. The 3-fold increase in the amount of cytochrome c oxidase/mg protein in the brain homogenate suggests either increasing number of mitochondria, increased cytochrome c oxidase per mitochondria or both. Caley and Maxwell [4], employing quantitative electron microscopic techniques, demonstrated a 4-fold increase in mitochondrial numbers in the rat cerebral cortex during development. An increase was also seen in the amount of cytochrome c oxidase/mg of mitochondrial protein in the MF. Since rat cerebral mitochondria increases 1.5-fold in protein during development [3], it seems reasonable to suggest that the amount of cytochrome c oxidase per mitochondria is also increasing during development. Development in the rat cerebral cortex appears therefore to be associated both with increasing numbers of mitochondria and increases in the components of the respiratory chain of each mitochondria [2--4]. In the MF, total SOD activity increases during development related to a progressive increase in MnSOD activity which is consistent with the results of Mavelli et al. [5]. At 77 days o f age, about 90°-/o o f the total SOD is cyanide insensitive and by definition MnSOD. At 2 days of age, a large amount o f total SOD activity was inhibitable by cyanide. An explanation for the high CnZnSOD measured in 2-day cerebral cortex could be that, in embryonal and early neonatal development, the MnSOD present may be more sensitive to cyanide inhibition than that present in adult mitochondria resulting in a spuriously low MnSOD activity. The measurement of high

26 levels of MnSOD in the N M N N F at 2 days of age does not support this interpretation and no variability in the response of MnSOD in embryonal tissues to cyanide has been reported. Further studies are necessary to clarify this finding. Since mitochondria increase their total protein content [3], a component of the decrease in the activity of CuZnSOD measured at 19 and 77 days may be related to increasing total mitochondrial protein, A number of investigators have suggested that the CuZnSOD measured in liver mitochondria is localized to the intermembrane space while MnSOD is primarily localized in the matrix [22,25,26]. Geller et al. [27] has reported that the CuZnSOD measured in mitochondriat fractions is predominately a result of lysosomal contamination. Histochemical localization studies may be necessary to assess whether any of the CuZnSOD measured in mitochondrial fractions is localized in the intermembrane space or is solely a contaminant. The activity of CAT in the MF drops by 10-fold between 2 and 19 days of age reflecting a similar change which occurs in the NMNNF and in brain homogenates from rat cortical tissue [1]. Nohl and Hegner [28] have shown using a variety of biochemical techniques and histochemically that CAT is present in cardiac mitochondria. Using a similar histochemical technique, it has been shown that mitochondria isolated from 2 day rat cerebral cortex contain more CAT positive staining than mitochondria isolated from 77-day-old rats (Fig. 1). Some of the CAT activity measured in the MF may be related to lysosomal contamination but the histochemical staining supports the concept that CAT is located in cerebral mitochondria and that this appears to decrease during development. The activity of GSH-Px increased in the MF, the NMNNF and in tissue homogenates from rat cerebral cortex [1] during development. Although the cause of the increase in G S H - P x activity during cerebral development is not known, it can be suggested that this increase occurs to cope with both the increased H202 generated by the elevated MnSOD content of mitochondria and their lowered CAT content. Non-mitochondrial non-nuclear fraction This fraction contained large amounts of L D H and acid phosphatase with only small amounts of cytochrome c oxidase activity. This fraction is envisioned as one containing the majority of the cellular cytoplasm and organelles excluding nuclei and most mitochondria. The enzyme changes measured in this fraction mirror the changes reported previously in whole tissue homogenates from rat brain [ 1]. An increase in total SOD is seen in this fraction during development due to increasing CuZnSOD activity. No significant change is seen in MnSOD in this fraction during development. The MnSOD activity measured in the NMNNF cannot be explained only by mitochondrial contamination suggesting that some MnSOD is located in extra-mitochondrial sites. McCord et al. [29] have reported the presence of large amounts of MnSOD in a cytoplasmic fraction of human and baboon liver. The presence of MnSOD in extra mitochondrial sites has also been reported in Mor-

27 ris hepatomas [30]. The human gene for MnSOD is located on the 6 chromosome and the CuZnSOD gene on the 21 chromosome [31]. The synthesis of both MnSOD and CuZnSOD occurs along the endoplasmic reticulum; therefore some of the MnSOD measured in this fraction may be awaiting transport and incorporation into mitochondria. An initial fall occurs in GSH-Px activity which may be related to nutritional availability of selenium during the postnatal period in the rat [1,32] followed by increasing GSH-Px activity related to the abundance of selenium in laboratory food sources. The fall in CAT which occurs in this fraction also occurs in the MF and the total tissue homogenate [1] certainly suggesting that decreasing CAT activity occurs in a number of intracellular sites.

Hypothesis Mcllwain [3] has divided the development of the rat brain into four periods which are: (1) a fetal period of cell proliferation in which over 90% of all cells appear but the brain is only 15°70 of its adult weight; (2) a period of growth of cells, axons and dendrites between birth and day 10; (3) from 10 to 20 days, the majority of blood vessels develop; enzymes involved in oxidative metabolism increase by 3--4-fold and myelination begins; and (4) after 20 days, a period of myelination and further development. The two major events occurring in the early postnatal period in rat cerebral cortex are a significant slowing of cell division associated with differentiation of cells and a switch from predominately anerobic to aerobic metabolism. Figure 2 outlines an hypothesis which relates to these two events. The maturation of the cerebral cortex involves a complex interplay between the developing microcirculatory system to deliver 02 and glucose and the increasing metabolic sophistication of mitochondria. Oxidative metabolism is associated with the quadrivalent reduction of 02 to H20 via the cytochrome oxidase complex and the "univalent leak" of 02- from the respiratory chain [33]. In vitro studies have shown that 1--4070 of the 02 consumption by isolated cardiac mitochondria is involved in this "univalent leak" [34]. In experiments using mitochondria isolated from rat brain similar results have been obtained [35]. The increase in the enzymatic components of the mitochondrial inner membrane and the delivery of substantial amounts of 02 and glucose by the developing microcirculation system result in the release of 02- from a number of sites along the respiratory chain. Increased mitochondrial generation of 02- results in MnSOD induction [36] and incorporation into the mitochondria of cells. This would result in more efficient dismutation of 02- into H202. A portion of this H202 is reduced by intramitochondrial CAT and GSH-Px but some H202 diffuses out of mitochondria [34,35]. One would expect that levels of CAT and GSH-Px would increase with development to cope with increased H20 2 generation and indeed GSH-Px increases.

,___-_-_______-______7

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Fig. 2. The hypothesis presented as a diagramatic representation showing the routes for 0, reduction and scavenging enzymes in neonatal and adult mitochondria. Increased levels of H,O, and CAT are represented in boxes and increased levels of a pathway are represented by a solid rather than an interrupted line. A question mark is used to denote the hypothesized pathway for the reaction(s) which result in the divalent reduction of 0: to HZOZ in mitochondria from neonatal brain tissue.

I

-

MITOCHONOKIA

NEONATAL

29

The high neonatal levels of CAT measured in both the MF and NMNNF cannot be explained by this concept and a reaction or group of reactions resulting in the divalent reduction of 02 to H202 has to be proposed to suggest a role for the high levels of CAT measured. The site(s) for this H202 generation are not known but, since CAT is localized to early postnatal mitochondria, an intramitochondrial source(s) of H202 seems probable. Other intracellular sources of H202 may be present. A number of investigators have suggested that the level of intracellular H202 generation influences both rate of cell division and cellular differentiation [37--39]. The mechanisms by which this is felt to occur is by increasing the intracellular cyclic GMP (cGMP), cyclic AMP (cAMP) ratio and/or H202 functioning as a second messenger [37]. The increased levels of H202 released by mitochondria and other intracellular sites during embryonal and prenatal development would result in increased intracellular H202 concentrations, increased cGMP, an increased cGMP/cAMP ratio and increased rates of cellular division. It is interesting to note that a number of malignant murine and human cerebral tumour cell lines and malignant human glial tumours are associated with high CAT [12,40], low MnSOD [12] and low cytochrome oxidase content (Del Maestro and McDonald unpublished results). Associated with late prenatal and postnatal development, sources of this H202 generation alter thus lowering intracellular H202 and cGMP levels which results in a slowing of mitosis and the differentiation of cells. The hypothesis presented in Fig. 2 is speculative. However, a number of components of the hypothesis can be tested and this information may lead to a further understanding of the role of free radical species in both cerebral development and cerebral neoplasia. ACKNOWLEDGEMENTS

The authors would like to thank Jo-Ann Dunn for her secretarial assistance. This work was supported through a grant from the Medical Research Council of Canada Grant MA-8104, the Brain Research Fund Foundation and Revtech Research Ltd. Dr. Del Maestro is a recipient of an Ontario Ministry of Health Career Scientist Award. REFERENCES 1 2 3

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R.F. Del Maestro and W. McDonald, Distribution of superoxide dismutase, glutathione peroxidase and catalase in developing rat brain. Mech. Aging Dev., 41 (1987) 29--38. N.A. Gregson and P.L. Williams, A comparative study of brain and liver mitochondria from newborn and adult rats. J. Neurochem., 16 (1969) 617--626. H. Mcllwain and S. Bachelard, Chemical and enzymatic makeup of the brain during development. In H. Mcllwain and S. Bachelard (eds.), Biochemistry and The Central Nervous System, Churchill Livingstone, London, 1971, pp. 406--444. D.W. Caley and D.S. Maxwell, Ultrastructure of the developing cerebral cortex in the rat. In M.B. Sterman, D.J. McGinty and A.M. Adolf (eds.), Brain Development and Behaviour, Academic Press, New York, 1971, pp. 91--107. I. Mavelli, A. Rigo, R. Federico, M.R. Ciriolo and G. Rotilio, Superoxide dismutase, glutathione peroxidase and catalase in developing rat brain. Biochem. J., 204 (1982) 535--540.

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