anti-oxidant enzymes in fetal guinea pig brain during development and the effect of maternal hypoxia

Developmental Brain Research, 42 (1988) 173-179 173 Elsevier

BRD 50778

Anti-oxidant enzymes in fetal guinea pig brain during development and the effect of maternal hypoxia

Om Prakash Mishra and Maria Delivoria-Papadopoulos Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 (U.S.A.)

(Accepted 8 March 1988)

Key words: Anti-oxidant enzyme; Hypoxia; Brain; Superoxide dismutase; Catalase; Glutathione peroxidase

The development of the anti-oxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase was studied in the fetal guinea pig brain at 30, 35, 40, 45, 50, 55, and 60 days of gestation. The activities of these enzymes remained constant during 30-45 days of gestation and increased significantly during the 45-60 day period, with the exception of superoxide dismutase, which remained unchanged throughout the gestational period. The enzyme activities in fetal brain tissue at every gestational age were unaffected by maternal hypoxia (inspired oxygen, 7% for 40 min). The concurrent development of glucose-6-phosphate dehydrogenase, glutathione reductase, and glutathione peroxidase during 45-60 days of gestation indicates an increased ability of the fetal brain to detoxify lipid peroxidation products by reinforcing the glutathione system. The results of this study indicate that the anti-oxidant enzymatic defense mechanisms in the guinea pig brain are fairly mature at birth. How- ever, these mechanisms are underdeveloped during the early stages of gestation and, therefore, during this period the brain might be at potential risk for lipid peroxidative damage under conditions leading to increased formation of oxygen free radicals.

INTRODUCTION

Brain tissue is potentially predisposed to lipid peroxidation due to its high metabol ic rate and its high concentration of polyunsaturated fatty acids 33, which are susceptible to oxidation 34. Lipid peroxidat ion has been proposed as a major mechanism of cellular membrane damage during brain ageing 17, cerebral hypoxia t9 and ischemia 1°'2°'32. Evidence suggesting

an increase in the product ion of superoxide anions and hydrogen peroxide under hypoxic conditions has been adequately discussed lz, and mechanisms for these reactions have been proposed 15. Anti-oxidant

enzymes, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase play an important role in protecting tissues f rom oxidative free radical reactions. While it has been documented that the guinea pig brain is morphologically, biochemically, and electrophysiologically more mature at birth than other animal species 1'2'9"11, thus far the enzymatic

mechanisms of anti-oxidant defenses in the fetal brain have not been investigated. In order to assess

the susceptibility of the fetal brain to oxidative reactions, the present study examines the activities of the anti-oxidant enzymes superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), glutathione peroxidase (EC 1.11.1.9), glutathione reductase (EC 1.6.4.2), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) in the brains of fetal guinea pigs during development and the effect of maternal hypoxia on these activities.

MATERIALS AND METHODS

All the reagents and chemicals used in the experiments were of analytical grade of the highest purity. Xanthine oxidase, glutathione reductase, reduced glutathione, cytochrome C, hydrogen peroxide, cumene hydroperoxide, and bovine serum albumin were purchased f rom Sigma. Pregnant , Dunkin Hart- ley guinea pigs of 30, 35, 40, 45, 50, 55, and 60 days of

Correspondence: O.P. Mishra, Department of Physiology, Room A201, Richards Bldg., G/4, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, U.S.A.

0165-3806/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

174

gestation were purchased from Hazleton Research

Animals.

l:or each gestational age, fetuses were obtained

from 6 normoxic (control group) and 6 hypoxic (ex-

perimental group) pregnant guinea pigs. To obtain control group fetuses, the mother was carefully re-

strained and the abdomen was anesthetized by intra- dermal infiltration of 5.0 ml lidocaine (1%). An inci-

sion was made in the lower abdomen and the fetuses

were removed and quickly decapitated. Their brains

were frozen in liquid nitrogen within 4-10 s of deca- pitation and samples were stored until the enzyme ac-

tivities could be assayed. For experimental group fe-

tuses, hypoxia was induced individually in guinea pig

mothers by allowing them to breathe 7% O, for 40

min in a specially designed chamber fitted with a

probe to monitor oxygen tension. The bottom of the

chamber was layered with Baralyme to remove CO~.

Arterial pO 2 of the mother was determined at 10-min intervals during the exposure. Maternal p~,O: de- creased to approximately 20 mm Hg within 10 min of

exposure and was maintained at that level for the du-

ration of the experiment. Following hypoxia, fetuses

were removed and treated as previously described.

In two additional experiments, we determined that

under the above conditions of maternal hypoxia, fe-

tal brain ATP level was reduced by approximately 50%, indicating that severe hypoxic insult had been

induced in the fetal brain. In addition, 6 control

guinea pigs of 14 days postnatal age were decapitated and their brains frozen in liquid nitrogen for the anal-

ysis of enzyme activity.

Brain tissue frozen in liquid nitrogen was homog- enized in 50 mM potassium phosphate buffer (pH

7.4) containing 0.5 mM EDTA to yield a 10% (w/v) homogenate, which was centrifuged at 2000 g for 15 rain at 0-4 °C. The resulting supernatant was used

for enzymatic analysis. Catalase activity was determined according to the

method of Homes and Masters tS. The activity was assayed at 25 °C by determining the rate of degradation

of H20 2 at 240 nm in the presence of 25/xl tissue ex- tract in a 1-ml assay medium containing 6.6 mM H20: in 10 mM potassium phosphate buffer (pH 7.0). E H20 2 = 43.6 cm2/mmol was used for calculation. The enzyme activity was expressed as/~mol of H202 degraded/min/mg protein.

Glutathione peroxidase activity was determined

according to the method of Paglia and Valentine ~

The enzyme activity was assayed at 25 ':'( by dctcr-

mining the rate of oxidation of NADPH a[ 340 nm in

a 1-ml assay medium containing 50 mM K-phosphate

buffer (pH 7.0), 3.6 mM sodium azide. ~J.3 mM

NADPH, and 5 mM reduced glutathione GSH.

Tubes were incubated for 5 min after addition of 10!,1

(l:1) diluted glutathione reductase. The reaction was

initiated by the addition of 25 ul of cumene hydroperoxide (1.5 mM). E NADPH = 6.22 cme/,umol was

used for calculations. The enzyme activity was ex-

pressed as nmol of NADPH oxidized/min/mg protein.

Glutathione reductase activity was determined as described by Ray and Prescott xg. Enzyme activity

was assayed at 25 °C by measuring the rate of oxida-

tion of NADPH at 340 nm in a loml assay medium

containing 50 mM K-phosphate buffer (pH 7.0), 0.5 mM EDTA-mercaptoethanol, and 0.125 mM

NADPH. The reaction was initiated by the addition of 0.1 ml oxidized glutathione GSSG (I0 mM). The

activity was expressed as nmol of NADPH oxi-

dized/min/mg protein.

Glucose-6-phosphate dehydrogenase was determined according to the method of Lohr and Wal- ters 22. The reaction was carried out at 25 °C in 1-ml

assay medium containing 50 mM Tris-HCl buffer (pH 7.5), 0.6 mM NADP and 0.2 ml tissue sample. The

reaction was initiated by the addition of 20 ¢xl of 40

mM glucose-6-phosphate. The enzyme activity was

assayed by measuring the rate of reduction of NADP at 340 nm and expressed as nmol of NADPH formed/

min/mg protein. Superoxide dismutase activity was determined ac-

cording to the method of McCord and Fridovich >, with increased sensitivity by measuring at 25 °C the

decrease in the reduction of cytochrome C at pH 10,0 in a xanthine-xanthine oxidase system containing: l(/ /xM cytochrome C, 100/,M xanthine, 16/xM NaCN, 50 mM sodium carbonate buffer (pH 10.0), and approximately 20 lxl of xanthine oxidase (diluted 1:300) to give a slope of 0.025 absorbance units per min at 550 nm. Approximately 10 jxl of diluted tissue supernatant was used to provide a 50% reduction in the slope. The method determines both cytosolic Cu-Zn superoxide dismutase and mitochondrial Mn- superoxide dismutase. To completely inhibit cytochrome oxidase and thereby prevent the reoxidation of cyto-

chrome C which mimics superoxide dismutase activi-

ty, 16#M NaCN was used. A similar concentration of

10 / tM NaCN, or slightly higher, is recommended while using tissue homogenates 6. The activity of the

enzyme was expressed as units per mg protein, where

one unit of superoxide dismutase activity was defined

as a 50% reduction in the slope of the standard reaction of this system. Protein was determined accord-

ing to the method of Lowry et al. 23.

RESULTS

Catalase activity in developing fetal guinea pig brain, as shown in Fig. 1, remained at a constant level

during 30-45 days of gestation and increased 1.6-fold

from 3.31 + 0.67 to 5.17 + 0.75/~mol o f H 2 0 2 degra-

ded/min/mg protein towards the end of the gestatio-

nal period. Maternal hypoxia (inspired oxygen, 7%

for 40 min) at 30, 35, 40, 45, 50, 55 and 60 days of gestation did not affect the activity of the fetal brain en-

zyme. The activity at 60 days was significantly higher

(P < 0.005) than at 45 days. The developmental pattern of the glutathione per-

oxidase activity in the fetal guinea pig brain during gestation is presented in Fig. 2. The activity re-

mained constant during the 30-50 days of gestation

but showed almost a two-fold increase from

27.70 + 3.03 to 53.19 + 4.40 nmol of N A D P H oxi-

~ 6.0

i 5.0

i; , o

3.0 11 # 2.0

' * 1.0

~ o 45 50 55 6.0 Days of Gestation

Fig. 1. Catalase activity in guinea pig brain during gestation. The enzyme activity is expressed as /~mol H202 degraded/ min/mg protein. Each point represents mean + S.D. of 12 fetuses; 12 control and 12 hypoxic fetuses were used at each age. Hypoxia samples were not significantly different from their corresponding controls (P values were > 0.50).

175

i i 60.0 i ~ 50.0

40.0 r T/~/ ' / (

"" 30.0 I --'~-

"** / I / . - . coo,to, ~ 10.0 . . . . . Hypox~a

" " I i o "~'o 3; 4.0 4; 5.0 5; 6'0 Days of Gestation

Fig. 2. Glutathione peroxidase activity in fetal guinea pig brain during gestation. The enzyme activity is expressed as nmol NADPH oxidized/min/mg protein. Each point represents mean + S.D. of 12 fetuses; 12 control and 12 hypoxic fetuses were used at each age. Hypoxia samples were not significantly different from their corresponding controls (P values were > O.50).

dized/min/mg protein during 50-60 days of gestation

(P < 0.005). The activity was unaffected by maternal hypoxia. It was slightly lower at 40 and 45 days in hy-

poxic brain samples; however, it was not statistically

significant (P values were > 0.05 at both ages).

As shown in Fig. 3, glutathione reductase activity

in the developing fetal guinea pig brain remained un-

changed during 30-45 days of gestation. However, a 1.7-fold increase from 18.9 _+ 2.30 to 32.54 + 4.83

50.0

40.0 / I ~ ~"t

~ 30.0 / ~ ~ 20.0 I ' "T !

,~ ~ . - . Oon,ro,

~ 10 .0 • . . . . Hy,~oxia

~ 30 35 4'0 4'5 50 5'5 6'0 ~ Days of Gestation

Fig. 3. Glutathione reductase activity in fetal guinea pig brain during gestation. The activity is expressed as nmol NADPH oxidized/min/mg protein. Each point represents mean + S.D. of 12 fetuses; 12 control and 12 hypoxic fetuses were used at each age. Hypoxia samples were not significantly different from their corresponding controls (P values were > 0.50).

176

nmol of NADPH oxidized/min/mg protein (P < 0.005) during 45-60 days of gestation. The activity leveled off after 5(1 days of gestation. Maternal hypoxia did not affect the enzyme activity.

Glucose-6-phosphate dehydrogenase activity in fetal guinea pig brain during gestation is presented in Fig. 4. The activity remained at a constant level during 35-50 days of gestation but increased 1.6-fold from 24.70 + 0.50 to 38.69 + 1.39 nmol of NADPH formed/min/mg protein (P < 0.05) during 50-60 days of gestation. The activity achieved its maximum steady-state level at 55 days and remained unchanged thereafter. Maternal hypoxia did not affect

the activity. The activity of superoxide dismutase (cytosolic

and mitochondrial) in fetal guinea pig brain during

gestation is shown in Fig. 5. The enzyme activity was unchanged during the entire period of gestation. Un- like other enzymes, superoxide dismutase activity did not show a significant increase (25.46 _+ 4.57 to 32.11 + 2.85 units/mg protein; P > 0.50) during 45-60 days of gestation. The activity was not affected by maternal hypoxia.

At 14 days postnatal age the activities of catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase were: catalase: 6.00 _+ 1.18/~mol HzO 2 degraded/min/mg protein; glutathione peroxidase: 53.57 + 3.83 nmol NADPH

~ 50.0

I 1 I • ® 30 .0 ~ <

~ 2 0 0

~= .--. Co~tro,

d. ~ 10.0 . . . . Hypoxia

COo

o 3'5 4? 4; 5'0 5; 6'o Days of Gestation

Fig. 4. Glucose-6-phosphate dehydrogenase activity in fetal guinea pig brain during gestation. The activity is expressed as nmol NADPH formed/min/mg protein. Each point represents mean + S.D. of 12 fetuses; 12 control and 12 hypoxic fetuses were used at each age. Hypoxia samples were not significantly different from their corresponding controls (P values were > 0.50).

50.0

¢J

'~ 40.0 o

~ 30.0 Q.

E = ~'2 ~ 20.0

10.0 Q

0

e - - e Control

o - - - - o Hypoxia

~; 3'5 4; 4'5 5'0 5; 6'0 Days of Gestation

Fig. 5. Superoxide dismutase activity in fetal guinea pig brain during gestation. The activity is expressed as units/mg protein. Each point represents mean + S.D. of 12 fetuses; 12 control and 12 hypoxic fetuses were used at each age. Hypoxia samples were not significantly different from their corresponding con- trois (P values were > 0.50).

oxidized/min/mg protein; glutathione reductase: 34.24 + 6.72 nmol NADPH oxidized/min/mg protein; glucose-6-phosphate dehydrogenase: 38.59 _+ 4.38 nmol NADPH formed/min/mg protein, which are similar to the levels at 60 days of gestation. The activity of superoxide dismutase increased to 44.78 + 2.65 units.

DISCUSSION

It is well established that potentially toxic oxygen species, superoxide radicals and hydrogen peroxide are formed under normal aerobic metabolism. Hy- drogen peroxide is a known cytotoxin and is produced in brain tissue during the oxidation of mono- amines 35 and glycine 16. Hydrogen peroxide is re-

duced by catalase and therefore it is possible that the activity of this enzyme plays a major role in protecting brain tissue from hydrogen peroxide-dependent oxidative damage. The activity of catalase in the developing fetal guinea pig brain, as presented in Fig. 1, did not increase during 30-45 days of gestation, but did increase during 50-60 days of gestation. The activity at 60 days of gestation is similar to that at two weeks of postnatal age, indicating that the guinea pig brain is mature at birth with regard to catalase activity. In addition to the normal developmental process, it is possible that the increase in activity during later periods of gestation is an adaptive response to the in-

crease in mitochondrial function during this period 2. However, the activity of monoamine oxidase, which generates hydrogen peroxide, attains its adult level as early as 30 days of gestation in the guinea pig brain ~. The presence of low levels of catalase activity in the fetal guinea pig brain during the early periods of gestation might result in increased accumulation of hydrogen peroxide in the brain tissue. However, in

addition to catalase, the activity of glutathione peroxidase should also be considered, as this enzyme too detoxifies H20 2.

Glutathione peroxidase detoxifies lipid peroxides by converting them to their corresponding monohydroxy unsaturated fatty acids 4'5"2~ and has been proposed as a major protective mechanism against peroxidative damage in brain tissue s . Low activity of this enzyme has been reported in the brains of different animal species, including guinea pig 8. However, later studies have shown considerably higher activities 3'26'3°. The activity of glutathione peroxidase in

the developing fetal guinea pig brain as presented in Fig. 2 shows a constant low level during 30-50 days of gestation, followed by a significant increase over the final 10 days. The activity at 60 days of gestation was found to be similar to that of two weeks of postnatal age, suggesting the maturation of guinea pig brain at birth with regard to this anti-oxidant enzyme. The lower glutathione peroxidase activity in the brain of guinea pig fetuses during the early days of gestation suggests the possibility of increased accumulation of lipid peroxides during this period as compared to later prenatal ages. However, it should be realized that the level of enzyme activity need not be considered as an absolute index of the susceptibility of an animal to the toxic effects of oxygen 12.

Glutathione reductase catalyzes the reduction of oxidized glutathione to reduced glutathione in a reaction coupled with the oxidation of NADPH. The reduced glutathione is further used as a substrate for the reduction of lipid peroxides by glutathione peroxidase. Therefore, the activity of glutathione reductase is important for maintaining a constant high level of cellular reduced glutathione. Results showed that the guinea pig brain is fairly mature in terms of glutathione reductase activity before birth (Fig. 3). A low level of glutathione reductase activity in the developing fetal guinea pig brain during 30-45 days of gestation again indicates an underdeveloped anti-oxidant

177

mechanism in the fetal brain. In a recent study 28, it

was shown that the inhibition of brain glutathione reductase by carmustine increased the susceptibility of the animal to hyperbaric oxygen-induced convul- sions.

Glucose-6-phosphate dehydrogenase, the key enzyme of the hexose monophosphate pathway in glucose metabolism, is primarily responsible for the generation of NADPH which is an essential compo-

nent for the conversion of oxidized glutathione to reduced glutathione by glutathione reductase. There- fore, the effectiveness of the entire glutathione system depends on a continuous supply of NADPH. Similar to other enzymes, the activity of glucose-6- phosphate dehydrogenase was at a constant low level until 50 days of gestation and increased slightly during 50-60 days of gestation. These results are suggestive of an underdeveloped mechanism of tissue lipid peroxide detoxification by glutathione system during early periods of gestation.

Superoxide dismutase can provide defense against superoxide anions which are normally produced in all aerobic cells 14. In brain tissue, aerobic metabolism increases with maturation and therefore an increased level of superoxide dismutase as an anti-oxidant defense is anticipated. The activity of superoxide dismutase in developing guinea pig brain remained at a constant low level during 30-45 days of gestation and did not increase significantly during 45-60 days of gestation. A sharp increase in superoxide dismutase activity (approximately 3-fold) has been reported in rat brain during 40 days of postnatal life 24. The activity of superoxide dismutase in guinea pig brain increased 1.3-fold during the first 14 days of postnatal life, which was similar to the adult level.

In addition to low activities of catalase, glutathione peroxidase, glutathione reductase, and glucose-6- phosphate-dehydrogenase, the lower level of superoxide dismutase in the fetal guinea pig brain is again suggestive of an underdeveloped mechanism for the detoxification of lipid peroxidation products and superoxide radicals and, therefore, probably an increased potential for oxidative damage during the oc- currence of increased oxygen-free radical formation. It appears that, with the exception of superoxide dismutase, the activities of the anti-oxidant enzymes increase sharply in the guinea pig brain during 45-60 days of gestation and attain their maximum level be-

178

fore birth. The term period of gestation in guinea pigs

is approximately 65 days. Since none of these enzymes were affected by ma-

ternal hypoxia, their inhibition cannot be implicated in tissue damage resulting from hypoxia. As under

similar conditions of maternal hypoxia, a significant

decrease (50%) in the activity of Na+,K+-ATPase, a membrane-bound enzyme and an index of mem-

brane function, and a significant increase in lipid per-

oxidation products, was observed in the brain of

guinea pig fetuses. Therefore, the absence of an

adaptive response by anti-oxidant enzymes might

suggest an increased susceptibility of the guinea pig

fetal brain to hypoxia. However, the challenge of hy-

poxia under our condition was only for 40 min. An adaptive response to 95% oxygen exposure was ob-

served in lung anti-oxidant enzymes of rat and rabbit.

whereas in the guinea pig such response was not observed by other investigators l:. A similar response by

lung enzymes to hypoxic exposure (10-11% 02) has been observed in rats 12'3L. However , we do not know

of such adaptive responses of brain tissue anti-oxi-

dant enzymes to hypoxic conditions in any species.

These results suggest that such adaptive response to

hypoxia is absent in brain tissue of guinea pigs. There are several factors that should be considered

in the proposal of the susceptibility of fetal brain to

oxidative free radical reactions at term. As previously stated, the monoamine oxidase system contributes

significantly to the formation of oxygen species, including hydrogen peroxide. Furthermore, there will

most probably be an increased formation of oxygen species as a result of mitochondrial aerobic functions in the last two weeks of gestation :. In addition, the

especially rapid increase in brain pol}unsaturatcd

fatty acids during development and maturation:

should be considered as a major factor for tile suscep-

tibility of brain tissue to oxidative reactions. This is particularly true in the case of unsaturated fatty acids

whose rates of oxidation increase with the degree of unsaturation 3a.

This study has investigated the development of en-

zymatic anti-oxidant mechanisms in the guinea pig

brain during gestation. To our knowledge, it is the

first study to have done so. The results demonstrate

that the activities of catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phos-

phate dehydrogenase remained constant during

30-45 days of gestation, after which they increased,

attaining nearly an adult level of activity before birth.

In contrast, the activity of superoxide dismutase re-

mained constant throughout the gestational period. In addition, the study examined the effects of mater-

nal hypoxia on the activities of these fetal brain tissue

enzymes and found that they were not significantly affected by hypoxia at any gestational age. This ob-

servation, when coupled with the near-adult level of

activity of the majority of these enzymes at birth,

gives evidence of a mature enzymatic anti-oxidant

defense system in the brain of the term guinea pig fe- tus.

ACKNOWLEDGEMENTS

This study was supported by N1H Grant HD- 20337-01 and completed with the technical assistance

of Miss Stephanie Arlis and Mr. Christopher F. Cam-

eron.

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anti-oxidant enzymes in fetal guinea pig brain during development and the effect of maternal hypoxia

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