Developmental profiles of antioxidant enzymes and trace metals in chick embryo

Mechanisms of Ageing and Development, 65 (1992) 51-64 Elsevier Scientific Publishers Ireland Ltd.

51

DEVELOPMENTAL PROFILES OF ANTIOXIDANT ENZYMES AND TRACE METALS IN CHICK EMBRYO

JOHN X. WILSON a, EDMUND M.K. LUI b and ROLANDO F. DEL MAESTRO c aDepartment of Physiology and bDepartment of Pharmacology and Toxicology, The University of Western Ontario, London, Ontario N6A 5C1 and CBrain Research Laboratory, Experimental Research Unit, Victoria Hospital, London, Ontario N6A 4G5 (Canada.) (Received August 21st, 1991) (Revision received February 19th, 1992)

SUMMARY

It has been previously well documented that partial pressure of oxygen (P02) and weight-specific rate of 02 consumption in chick embryo (Gallus gallus domesticus) transiently increase midway through the 21-day in ovo incubation period. The present study found that these oxidative changes were paralleled by the concentrations of glutathione (GSH) and Zn in liver and by the specific activity of superoxide dismutase (SOD) in brain. Levels of antioxidant enzymes and their trace metal cofactors were markedly higher in liver than in brain. Hepatic catalase activity changed in parallel with the concentration of its cofactor, Fe. However, the relative abundance of metal cofactors did not appear to be the determining influence on other antioxidant enzyme activities. Rates of extra-mitochondrial hydrogen peroxide release were also much greater in liver than in brain. Taken together, the results of this initial study of embryonic chick antioxidant systems suggest that certain antioxidants may be regulated by P02 and rate of oxidative metabolism during fetal development.

Key words: Antioxidant enzymes; Trace metals; Glutathione; Avian embryo; Gallus gallus domesticus Correspondence to: John X. Wilson, Department of Physiology, Medical Sciences Building, University of Western Ontario, London, Canada N6A 5C1. Abbreviations: GSH, reduced glutathione; GSH-Px, glutathione peroxidase; GSSG, oxidized glutathione; Po 2, partial pressure of oxygen; SOD, superoxide dismutase. 0047-6374/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

52 INTRODUCTION Univalent reduction of 02 yields superoxide anion radical, hydrogen peroxide and hydroxyl radical. These metabolites are capable of reacting with a variety of cellular components, including nucleic acids, lipids, proteins, free amino acids and carbohydrates. The resulting oxidative free radicals are obligate intermediates of many metabolic reactions but may also cause pathological damage [1,2]. Thus, control of redox balance is critical to cellular development, differentiation and homeostasis [3]. Antioxidant enzymes counteract excessive formation and deleterious effects of reactive oxygen metabolites [4]. For example, superoxide dismutase (SOD) catalyzes the conversion of superoxide anion radical to H202, catalase reduces H202 to water, while glutathione peroxidase (GSH-Px) acts in conjunction with other enzymes to reduce H202 and to terminate lipid peroxidation. Changes in antioxidant activities may occur under conditions that alter the rates of formation of reactive oxygen radicals. It has been demonstrated that, in at least some experimental animal preparations, the production of superoxide anion radical is facilitated by elevated P02 and augmented oxidative metabolism [5] and, in turn, this radical can induce MnSOD [6]. Expression of antioxidant metalloenzyme activities in developing tissues may be limited by the availability of their trace metal cofactors. For example, catalase activity in perinatal rat lung is highly influenced by the pregnant dam's intake of the enzyme cofactor, Fe [7]. Relevant observations also have been made in postnatal chicks. On the one hand, a decrease in Mn concentration is associated with a fall in MnSOD activity in chick liver during the first week after hatching [8]. On the other hand, the CuZnSOD activity of chick liver more than doubles during this postnatal week in spite of decreasing hepatic Cu and Zn concentrations [8]. Dietary experiments with postnatal chicks have shown that erythrocyte CuZn SOD activity is increased by feeding Cu-enriched diets but is not affected by Zn-enriched diets [9]. As for prenatal development, antioxidant enzyme activities have been me~;sured during late stages of gestation in mammals [10-14] but have not been related to trace metal concentrations. Studies of trace metal concentrations in avian embryos [15] did not make comparisons with antioxidant defenses. Thus, it is not known if the availability of metal cofactors limits antioxidant enzyme activity during the prenatal period. The present report examines an extensive period of fetal development in a nonmammalian vertebrate, the chicken (Gallus gallus domesticus). Many aspects of developmental biology which may be relevant to the expression of antioxidant enzymes are known for this species. It has been established that growth of the chick in ovo involves transient but profound changes in the P02 of oxygenated blood and tissues [16-18], the oxygenation of hemoglobin [19] and the weight-specific rate of 02 consumption [20]. The changes in these factors indicate that widely fluctuating

53

levels of oxidative stress occur spontaneously. It has also been demonstrated that 02 supply influences the rate of embryonic development. For example, electroencephalograms of 2-week-old chick embryos under hypoxic experimental conditions resemble normal electroencephalograms of younger embryos [21]. Furthermore, it may be anticipated that the intensity of oxidative stress varies between tissues because of differing rates of maturation and metabolism and that this variation would require tissue-specific responses of the developing antioxidant defense systems. We hypothesize that prenatal development of antioxidant defenses is related to spontaneous changes in the supply of 02, the maturational state of tissues and the concentrations of metalloenzyme cofactors. The present study appears to be the first to elucidate the ontogenetic profiles of antioxidant enzymes in chick embryo. Identification of these profiles allows their comparison with the known pattern of changes in incident PO2 and 02 consumption. Additionally, liver and brain were compared to evaluate the relative importance of metalloenzyme cofactors and metabolic activity for the expression of antioxidant activities. MATERIALS AND METHODS

Materials Fertilized White Leghorn chicken eggs (Gallus gallus domesticus) were purchased from McKinley Hatcheries (St. Mary's, Ontario). Catalase (EC 1.11.1.6) was obtained from Boehringer Mannheim. Nagase was purchased from the Enzyme Development Corporation. Bovine CuZnSOD (EC 1.15.1.1), glucose oxidase (EC 1.1.3.4), GSH, homovanillic acid and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) were obtained from Sigma Chemical Company.

Tissue collection Eggs were incubated at 39°C in an automatic forced-air incubator. Ambient 02 concentration was held at 21%. At the times indicated, the embryos were removed from their shells and decapitated. Allantoic blood was collected by syringe while the heart was still beating. Brain and liver were either used immediately for studies of mitochondrial function or stored at -80°C for subsequent assays.

Mitochondrial function Mitochondria were isolated by the method of Clark and Nicklas [22], modified by deleting the Ficoll gradient step. Tissues were minced in cold MSE solution (225 mM mannitol, 75 mM sucrose and 1 mM EGTA). Nagase was then added and the samples were homogenized in a Dounce homogenizer with a loose pestle for 1 min, followed by two strokes of the tight pestle. Homogenates were centrifuged (2000 x g, 3 min), the pellet discarded and the supernatant centrifuged again (10 000 x g, 8 min). The resulting pellet was resuspended in cold MSE solution.

54 Samples of the pellet were used for the H202 assay, which was performed by the method of Ruch et al. [23] with the following modifications. The assay buffer contained 145 mM KC1, 5 mM KH2PO4, 3 mM MgCI2, 0.1 mM EGTA and 30 mM Hepes (pH 7.4). At the time of assay, 1 unit of horse radish peroxidase, 100 #mol homovanillic acid and 50/zl of mitochondrial extract were added to 1 ml of buffer, followed by 100/~1 of 1 M succinate to start the reaction. Half of the samples also contained 50 units of CuZn SOD to trap 02-. After 15 min, the reaction was terminated with 250 #1 of 0.1 mM glycine-NaOH (pH 12.2). The samples were then centrifuged (12 000 x g, 5 min) and the supernatant measured in a Perkin-Elmer LS-5 spectrofluorimeter. The H202 assay was standardized using glucose-glucose oxidase. Cytochrome oxidase Cytochrome oxidase activity was assayed by the method of Wharton and Tzagoloff [24]. Results were expressed as units/mg mitochondrial protein, where 1 unit = 1 #mol oxidized cytochrome c. Antioxidant enzymes Brain samples were pooled on day 6 (three per pool) and day 8 (two per pool), whereas older brains and all livers were analyzed individually. Tissues and blood were homogenized in 10 mM potassium phosphate buffer (4°C, pH 7.4) supplemented with 30 mM KCI [25]. An aliquot of tissue homogenate was taken for determination of protein content [26]. Remaining homogenates were centrifuged (12 000 x g, 5 min) and the resulting supernatants were assayed for catalase, SOD, GSH-Px and hemoglobin according to the procedures of Del Maestro and McDonald [25]. The hemoglobin concentration in tissue samples was used to estimate the extent of blood contamination. Correction for this contamination was made for each tissue sample by subtracting the value obtained for blood in terms of amount of enzyme per mg hemoglobin. Glutathione and trace metals Concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) in cerebral and hepatic cytosols were measured as described by Kuo and Hook [27]. Estimation of metal concentrations was performed following tissue digestion [28]. To 0.25-0.5 ml of liver or brain homogenate (20%) 1 ml of a nitric acidsulphuric acid (1:2) solution was added. This mixture was heated at 60°C for 1 h and, after being cooled to room temperature, 200 #1 of 30% H202 solution was added. The resulting solution was diluted with 1 ml of double-distilled and deionized water prior to measurement of Zn, Cu and Fe by atomic absorption spectrophotometry. Zn, Cu and Fe analyses were performed on a Varian AA-475 absorption spectrophotometer using an acetylene-air flame. Recovery was more than 95% by standard additions [28].

55

Statistics

Results are presented as the mean 4- standard error of the mean (SEM) of a number (n) of independent determinations. Differences between means were evaluated at a 5% level of significance using repeated measures analysis of variance and the Tukey-Kramer method for multiple comparisons [29]. RESULTS

Examination of embryonic chick blood showed that hemoglobin concentration increased 5-fold during days 6-18, then doubled again by day 20 (Table I). The specific activity of catalase in blood remained constant, whereas GSH-Px increased and SOD decreased during days 8-10 before reaching stable values (Fig. 1). In liver, the concentration of total protein (mg/g liver) increased from 10.9 ± 0.47 on day 8 to 14.5 ± 0.36 on day 18. The major fraction of hepatic Zn was acquired between days 8 and 12 (data not shown), with the result that Zn concentration peaked on day 12 (Fig. 2). In contrast, most of the Cu deposition in liver occurred after day 14 (data not shown) and Cu concentration increased rapidly between days 14 and 16 (Fig. 2). Hepatic Fe concentration increased just prior to hatching (Fig. 2). Hepatic concentrations of GSH and of GSH + GSSG combined (#mol/g liver, n = 7-10) were maximal at day 12 and fell at a later stage of incubation (GSH 2.87 ± 0.12 and GSSG 0.25 ± 0.06 on day 8; GSH 3.31 4-0.06 and GSSG 0.04 4- 0.01 on day 12; GSH 1.68 4- 0.03 and GSSG 0.37 4- 0.02 on day 18). The specific activities of GSH-Px and catalase in liver rose during the second half of the incubation period (Fig. 3), with catalase activity paralleling the concentration of its cofactor, Fe. While there were no significant changes in the specific activities of total, CuZn-dependent or Mn-dependent SOD in developing liver, the highest mean values for these enzymes were observed on day 12 (Fig. 4).

TABLE I CONCENTRATION OF HEMOGLOBIN IN BLOOD OF CHICK EMBRYOS INCUBATED WITH 21% AMBIENT 02

Days of incubation

Hemoglobin (mg/ml blood)

6 8 10 12 14 16 18 20

4.9 5.9 5.5 8.8 8.1 15.3 25.9 49.6

± ± ± + ± ± ± ±

0.4 1.0 1.0 0.7 0.6 1.6 2.2 4.1

Values are mean ± SEM for 20-40 independent determinations.

56

Liver 48

I 0

O

On;O

I\,

~q

BD

30

o~

A

18 6

*

-

D ,~ ABD ' ~-~,£~ A AB a--a

/

A

-

8

10

!2

14

"6

"8

20 N

~ - I ~ 0

800

i O~.~

A/l

[~

l

\6~O---O

AB AB

~

60

O ~ © L-

o~

0J

20

0

n:

i

0 B

o-, .,o

sT

1'6 ~

6o

~" 6ooi

×!

,.~ 200i

1+2-1'4

A

i

A

A

A

IB A A/ ? ~-- 0 /

sJ[]----[]J@ 8

10

~2

~,

18

I\

n

Wo

1T-,'8

D r

D

j

9~-o

D

12

•t ~

B

a

S

B

B

I

"~c~

8 8

10

!'2 [] : v ~

16

"8

20

A 8

ABC/9 AB~o ~ o ~o

~2

~¢

Days

~6

1'8

2o

Fig. 1. Ontogenetic profiles of catalase (top panel), glutathione peroxidase (GSH-Px, middle panel) and superoxide dismutase (SOD, bottom panel) in allantoic blood. Enzyme activities are normalized to 1 mg blood hemoglobin (Hb) content• Each data point plotted is the mean + SEM of 20 independent determinations. Within each panel, groups with different superscripts are significantly different (P < 0.05). Fig. 2. Developmental changes in the concentrations of Zn (top panel), Fe (middle panel) and Cu (bottom panel) in liver. Each data point plotted is the mean + SEM of 7-11 independent determinations. Within each panel, groups with different superscripts are significantly different (P < 0.05).

In brain, p r o t e i n c o n c e n t r a t i o n (mg/g b r a i n ) increased f r o m 3.7 ± 0.15 on d a y 6 to 6.9 ± 0.32 on d a y 20. C e r e b r a l c o n t e n t s o f Fe, C u a n d Z n increased p r o p o r tionately with b r a i n weight as a function o f age ( d a t a n o t shown). Thus, the concent r a t i o n s (/~g/g b r a i n ) o f these metals r e m a i n e d c o n s t a n t at 6.8 for Zn, 4.0 for C u a n d 7.5 for F e d u r i n g d a y s 8 - 2 0 . Both the c o n c e n t r a t i o n o f G S H a n d the specific activity o f G S H - P x were severalfold lower in b r a i n t h a n in liver. M o r e o v e r , unlike the p a t t e r n occurring in liver

57 1.6 (~

@

Liver

[

1.4.

~ 0f

o2

O0 0_

t

1.2

~E D 0.8 120

B 1(~ o

'Live~

C

b £

c)

q

8

1'0

12

~+4

1~6

2~0

,o ~"

t

CT) 0 o.8

o_

8E8o

118

C N ~ c~ ~ 0.7

A ~ o

(-') " ~ 0.6 60

~--~o--~'~

~'6

5oo

~'~

/o

o~

C

&× , •_ 20o~

110

1~2

ll4

116

0.6

~13/

c ~ ' ~ 0.5.

q- CT, 150

1 O0

0*

1~8 70

[

[]

8

~

1'0 ~ - - ~ 4 - 4

Days

1'6

I'8

~0

0.4.

Z~

0.3

8

1~0

1~2

1~4

116

118

210

Days

Fig. 3. Ontogenetic profiles of catalase (top panel) and glutathione peroxidase (GSH-Px, bottom panel) in liver. The enzyme activities were corrected for blood contamination and normalized to 1 mg total protein content. Each data point plotted is the mean ± SEM of 20 independent determinations. Within each panel, groups with different superscripts are significantly different (P < 0.05). Fig. 4. Ontogenetic profiles of total superoxide dismutase (total SOD, top panel), CuZnSOD (middle panel) and MnSOD (bottom panel) in liver. The enzyme activities were corrected for blood contamination and normalized to I mg total protein content. Each data point plotted is the mean ± SEM of 20 independent determinations.

during the period studied, concentrations of GSH and GSSG in brain (#mol/g brain, n = 9-10) did not fluctuate significantly (GSH 0.88 ± 0.03 and GSSG 0.24 ± 0.02 on day 8; GSH 1.01 ± 0.0~i and GSSG 0.21 4- 0.01 on day 12; GSH 0.84 ± 0.08 and GSSG 0.20 ± 0.02 on ,day 18). Cerebral specific activity of GSH-Px doubled during the final 2 weeks in ovo (Fig. 5), paralleling but remaining consistently lower than the hepatic specific activity of GSH-Px. In an even more striking contrast with

58 16T

~3rgir

%

~.~

?

.....

8

lO

(~

L~

I

(3_ 10

B 0.6 G4 6

Brc~ A O"

s~

A -(),

01 O

\

£3

A

A

12

i ~-

16

18

20

AB .]~

1

18

2C

A

A,

18

20

0

0 ~ c4~ c,

D," L,L,

'<\ B,C CD

©

"4

o61

@

\

12

'/ '

/

O

,, }

0

I

2~

&

S.

.

+

(}

8

/

4

G

~°i'

.c:

1~

6

~){21

10

12 T

•

j

8

.J

i

-

c AB AB A_~

Q

A

,,

A

A

~"\

A/I [

J ',) ;

16

•

~

- - - -

:

a

....

~

2

:

/

1,1

~ 16

i

i E

i

20

04

;

£

8

10

"2

"4

"6

}(3/

Fig. 5. Ontogenetic profiles of catalase (top panel) and glutathione peroxidase (GSH-Px, bottom panel) in brain. The enzymeactivities were corrected for blood contamination and normalized to 1 mg total protein content. Each data point plotted is the mean ± SEM of 20 independent determinations. Within each panel, groups with different superscripts are significantly different (P < 0.05). Fig. 6. Ontogenetic profiles of total superoxide dismutase (total SOD, top panel), CuZnSOD (middle panel) and MnSOD (bottom panel) in brain. The enzyme activities were corrected for blood contamination and normalized to 1 mg total protein content. Each data point plotted is the mean + SEM of 20 independent determinations. Within each panel, groups with different superscripts are significantly different (P < 0.05).

the hepatic profile of a n t i o x i d a n t development, cerebral catalase fell 4-fold d u r i n g this prenatal period (Fig. 5). Differences in enzyme specific activities between liver a n d b r a i n were of much smaller m a g n i t u d e s for S O D t h a n for G S H - P x or catalase. However, divergent patterns were observed in b r a i n for C u Z n - d e p e n d e n t a n d M n - d e p e n d e n t S O D (Fig. 6). Cerebral C u Z n S O D showed a transient peak on day 8. I n contrast, cerebral

59

TABLE 11 EXTRAMITOCHONDRIAL RELEASE OF H202 IN DAY 18 CHICK EMBRYO Control

SOD

Liver

1.62

1.67

Brain

0.013 4- 0.003

± 0.33

± 0.34

0.013 4- 0.003

Embryos were incubated until day 18 with 21% ambient 02. Subsequently mitochondria were isolated from liver and brain and extra-mitochondrial release of H202 was measured. Half of the samples received 50 units of CuZn-superoxide dismutase (SOD) to trap 02-. H202 release is expressed as nmol O2/min/mg mitochondrial protein. Values are mean ± SEM for 10 determinations in liver and 8 determinations in brain.

Mn SOD underwent a gradual increase during days 8-14 before returning to the low levels typical of the first week of incubation. Because MnSOD activity always exceeded that of CuZn SOD, the specific activity of total SOD was sustained at an elevated level in brain during days 8-14 (Fig. 6). Parameters of oxidative metabolism in brain and liver were compared on day 18. Cytochrome oxidase activity, expressed as units/mg mitochondrial protein, was 1.03 ± 0.11 in liver and 0.86 • 0.11 in brain (n = 8). However, rate of extramitochondrial release of H202 was two orders of magnitude greater in liver than in brain (Table II). DISCUSSION

The ontogenetic profiles of antioxidant enzymes differ markedly between blood (Fig. 1), liver (Figs. 3 and 4) and brain (Figs. 5 and 6). This variation between tissues indicates that the development of antioxidant systems cannot be determined solely by the level of incident 02. Nevertheless, changes in certain antioxidants do appear to be related to changes in 02 supply. In chicken eggs incubated with 21% ambient 02 concentration, embryonic P02 rises from minimal values at day 4 [18] to much higher levels by day 10 [16,17]. From day 10 or 12 onwards, P02 and weightspecific rate of 02 consumption gradually decline until pipping allows breathing to begin [16,17,20]. For example, P02 in chorioallantoic venous blood, expressed in mmHg, is 80 at day 10, 79 at day 12, 74 at day 14, 65 at day 16 and 57 at day 18 [17]. While P02 and rate of oxidative metabolism obtain high levels during the second week of incubation and subsequently fall, there is a similar pattern for concentrations of GSH (present experiments) and Zn (Fig. 2) in liver, concentration of the antioxidant ascorbic acid in brain [301 and specific activity of SOD in brain (Fig. 6). The antioxidant metalloenzyme cofactors Fe, Cu and Zn were investigated in the present experiments. It is evident that these metals are essential for maturational

60

processes in the chick embryo, since deficiencies in their supply lead to severe structural and functional abnormalities [31]. As for the normal pattern of trace metal distribution, Dewar et al. [32] reported that concentrations of Fe, Cu and Zn in whole chick embryo decrease between days 5 and 18 in ovo, with the most rapid declines occurring from day 5 to day 10. Upon examining individual organs of the chick, we have found that concentrations of Fe, Cu and Zn are markedly greater in liver than in brain and are similar to those previously reported for the liver and brain of neonatal mice [33]. Catalase activity in perinatal rodent lung is highly influenced by the pregnant dam's intake of Fe [7]. In the fetal chick, mobilization of Fe from the yolk [34] is accompanied by simultaneous increases in hepatic levels of Fe (Fig. 2) and the hemecontaining enzyme, catalase (Fig. 3). The levels of catalase and Fe both are consistently higher in liver than in brain (Fig. 5). Taken together, these relationships are consistent with the hypothesis that expression of catalase activity in certain fetal tissues is modulated by the availability of its metal cofactor, Fe. Contrastingly, chick CuZn SOD specific activity does not correlate with either Cu or Zn concentrations during either the prenatal period (Figs. 2, 4 and 6) or the week following hatching [8]. Therefore, factors other than the availability of metal cofactors appear to determine the expression of CuZn SOD in chick. The transient peaking of hepatic Zn concentration midway through the fetal period may be related to antioxidant functions of this metal which are independent of CuZn SOD. In this respect it is noteworthy that injection of Zn during the final week of chick embryonic development increases hepatic metallothionein mRNA levels [35]. Metallothionein, a cysteine-rich, cytosolic, metal-binding protein, has been considered an antioxidant by virtue of its ability to scavenge reactive oxygen species [36,37]. The protective role of Zn-metallothionein in oxidative stress is believed to be mediated by the Zn ions that are released upon the inactivation of metal binding sites following oxidation of the cysteinyl thiolate groups by reactive oxygen metabolites. Temporal changes in antioxidants within a tissue may reflect proliferation and differentiation of particular cell types. In embryonic chick brain, for example, both GSH-Px specific activity (Fig. 5) and glial cell number [38,39] increase markedly during the second half of the in ovo incubation period. This is consistent with previous findings that GSH-Px specific activity is higher in glial cells than in neurons [40,41]. Furthermore, it suggests that the late induction of GSH-Px in brain may result from glial proliferation and differentiation. Similarly, SOD activities may vary because of changes in the relative abundance of particular cell types. For example, among adult rat brain cells, the specific activity of total SOD is approximately 10-fold higher in glial cells than in neurons [40]. Furthermore, immunolocalization methods indicate that CuZn SOD occurs in neurons and oligodendroglia, but not in astroglia [42]. Cultures of chick embryonic neurons also have been shown to express CuZn SOD [43]. Analysis of the chick embryonic

61 telencephalon revealed that essentially all postmitotic neurons are generated between days 4 and 9 but most astroglial cells originate after day 10 [38]. Similarly, glial cells are not detectable in chick embryonic optic tectum before day 9 [39]. These observations suggest that, on the one hand, the brain's CuZn SOD peak on day 8 is associated with a relative abundance of neurons and, on the other hand, the subsequent increase in Mn SOD coincides with augmented numbers of astroglia. A striking finding of the present study is how markedly fetal liver and brain differ with respect to the developmental profiles of their antioxidant systems. Our data are consistent with the hypothesis that the expression of antioxidant systems is influenced by the rate at which oxygen radicals are formed by oxidative metabolism. Thus, the relatively greater GSH concentration, GSH-Px specific activity and catalase specific activity in liver correspond with the extensive metabolizing and detoxifying functions of this organ, processes that lead to the formation of reactive oxygen species. Rate of extra-mitochondrial release of H202 is approximately two orders of magnitude greater in liver than in brain (Table II), and this may account for the higher levels of H202-scavenging enzymes GSH-Px and catalase in liver (Figs. 3 and 5). The importance of rate of oxidative metabolism for tissue-specific expression of GSH-Px and catalase has previously been suggested by the observation that the activities of these two enzymes are higher in liver than in lung of fetal guinea pig [14]. Therefore, data from both avian and mammalian studies suggest that rate of oxidative metabolism is more important than incident Po2 for induction of GSH-Px and catalase. Comparisons between birds and mammals may reveal developmental patterns that have been conserved during evolution. For example, hepatic GSH-Px and catalase specific activities increase in both birds (Fig. 3) and mammals [14,44] during the final week before birth. This process may have adaptive significance because a progressive rise in the rate of lipid catabolism [45] may accentuate the role of these antioxidant enzymes in terminating lipid peroxidation. Aside from hepatic GSH-PX and catalase, however, the expression of antioxidant enzymes differs between chick and mammalian embryos in a number of ways. We have suggested that the changes in SOD and GSH-Px which occur in chick brain during the final 2 weeks of embryonic development may result from the particular timing of neuronal and glial proliferation and differentiation in this species. Although this explanation remains speculative, it is clearly evident that the developmental patterns of chick cerebral GSH-Px and catalase differ from those of mammalian species. In the brain of the guinea pig embryo, for example, the activities of GSH-Px and catalase increase during days 45-60 of gestation [12]. In the rat brain, on the other hand, the neonatal period from 19 days gestational age through 2 days after birth is marked by decreases in the specific activities of both enzymes [11]. Contrastingly, in embryonic chick brain during the final 2 weeks in ovo the specific activity of GSH-Px doubles and that of catalase falls 4-fold (Fig. 5). The ontogeny of SOD also varies between vertebrate species. CuZn SOD is the

62

predominant isozyme in late gestational and neonatal rat brain [11] but Mn SOD predominates in embryonic chick brain (Fig. 6). Additionally, the specific activities of SOD enzymes in brain vary markedly during the development of embryonic chick (Fig. 6), whereas they are maintained at constant levels in guinea pig during days 45-60 of gestation [12] and in rat from day 19 of gestation through 2 days after birth [11]. Taken together, the data indicate that many tissue-specific ontogenetic profiles of antioxidant enzymes were not highly conserved during vertebrate evolution. ACKNOWLEDGEMENTS

Supported by the Natural Sciences and Engineering Research Council (Canada). We thank W. McDonald, G. Osborne and E. Jaworski for technical assistance. REFERENCES 1 C.E. Cross, B. Halliwell, E,T. Boush, W.A. Pryor, B.N. Ames, R.C. Saul, J. McCord and D. Harmon, Oxygen radicals and human disease. Ann. Int. Med, 107 (1987) 526-545. 2 R.A. Floyd, Role of oxygen free radicals in carcinogenesis and brain ischemia. FASEB J.. 4 (1990) 2587-2597. 3 R.G. Allen and A.K. Balin, Oxidative influence on development and differentiation: an overview of a free radical theory of development. Free Rad. Biol. Med., 6 (1989) 631-661. 4 I.A. Cotgreave, P. Moldeus and S. Arrenius, Host biochemical defense mechanisms against prooxidants. Annu. Rev. PharmacoL Toxicol., 28 (1988) 189-212. 5 L. Barthelemy, A. Belaud and C. Chastel, A comparative study of oxygen toxicity in vertebrates. Respir. Physiol., 4 (1981) 261-268. 6 I. Fridovich, Superoxide dismutases. An adaptation to a paramagnetic gas. J 3iol. Chem.. 264 (1989) 7761-7764. 7 A.K. Tanswell and B.A. Freeman, Pulmonary antioxidant enzyme maturation in the fetal and neonatal rat. II. The influence of maternal iron supplements upon fetal lung catalase activity. Pediatr. Res., 18 (1984) 871-874. 8 G. DeRosa, C.L. Keen, R.M. Leach and L.S. Hurley, Regulation of superoxide dismutase activity by dietary manganese. Z Nutr., 110 (1980) 795-804. 9 W.J. Bettger, J.E. Savage and B.L. O'Dell, Effects of dietary copper and zinc on erythrocyte superoxide dismutase activity in the chick. Nutr. Rep. Int., 19 (1979) 893-900. 10 A.K. Tanswell and B.A. Freeman, Pulmonary antioxidant enzyme maturation in the fetal and neonatal rat. I. Developmental profiles. Pediatr. Res., 18 (1984) 584-587. 11 R.F. Del Maestro and W. McDonald, Distribution of superoxide dismutase, glutathione peroxidase and catalase in developing rat brain. Mech. Ageing Dev., 41 (1987) 29-38. 12 O.P. Mishra and M. Delivoria-Papadropoulos, Anti-oxidant enzymes in fetal guinea pig brain tissue during development and the effect of maternal hypoxia. Dev. Brain Res., 42 (1988) 173-179. 13 R.F. Del Maestro and W. McDonald, Subcellular localization of superoxide dismutases, glutathione peroxidase and catalase in developing rat cerebral cortex. Mech. Ageing Dev., 48 (1989) 15-31. 14 G.M. Rickett and F.J. Kelly, Developmental expression of antioxidant enzymes in guinea pig lung and liver. Development, 108 (1990) 331-336. 15 M.P. Richards and N.C. Steele, Trace element metabolism in the developing avian embryo: a review. J. Exp. Zool., Suppl. 1, (1987) 39-51. 16 B.M. Freeman and B.H. Misson, B.H. pH, Po: and Pco 2 of blood from the foetus and neonate of Gallus gallus domesticus. Comp. Biochem. Physiol., 33 (1970) 763-772. 17 H. Tazawa, T, Mikami and C. Yoshimoto, Effect of reducing the shell area on the respiratory properties of chicken embryonic blood. Respir. Physiol., 13 (1971) 352-360.

63 18 19 20 21

22 23

24 25

26 27 28 29 30 31 32 33 34

35

36

37 38

39 40 41

H.-J. Meuer and R. Baumann, Oxygen partial pressure in intra- and extra-embryonic blood vessels of early chick embryo. Respir. Physiol., 71 (1988) 331-343. C. Cirotto and I. Arangi, How do avian embryos breathe? Oxygen transport in the blood of early chick embryos. Comp. Biochem Physiol., 94A (1989) 607-613. M. Hoiby, A. Aulie and O.B. Reite, Oxygen uptake in fowl eggs incubated in air and pure oxygen. Comp. Biochem. Physiol., 74A (1983)315-318. M.A. Corner, J.P. Schade, J. Sedlacek, R. Stoeckart and A.P.C. Bot, Developmental patterns in the central nervous system of birds. 1. Electrical activity in the cerebral hemisphere, optic lobe and cerebellum. Prog. Brain Res., 26 (1967) 145-192. J.B. Clark and W.J. Nicklas, The metabolism of rat brain mitochondria. Preparation and characterization. J. Biol. Chem., 245 (1970) 4724-4731. W. Ruch, P.H. Cooper and M. Baggiolini, Assay of H202 production by macrophages and neutrophils with homovanillic acid and horse-radish peroxidase. J. lmmunol. Methods, 63 (1983) 347-357. D.C. Wharton and A. Tzagoloff, Cytochrome oxidase from beef heart mitochondria. Methods Enzymol., 10 (1967) 245-250. R.F. Del Maestro and W. McDonald, Oxidative enzymes in tissue homogenates. In R.A. Greenwald (ed.), CRC Handbook of Methods for Oxygen Radical Research, CRC Press Inc., Boca Roton, FL, 1985, pp. 291-296. O.H. Lowry, N.J. Roseborough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent. J. Biol. Chem., 192 (1951) 265-275. C. Kuo and J. Hook, Depletion of renal glutathione content and nephrotoxicity of cephaloridine in rabbits, rats and mice. Toxicol. Appl. Pharmacol., 63 (1982) 292-302. I.S. Clarke and E.M.K. Lui, Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepatotoxicity. Can. J. Physiol. Pharmacol., 64 (1986) 1104-1110. R.R. Sokal and R.J. Rohlf, Biometry, W.H. Freeman, San Francisco, 1981. J.X. Wilson, Regulation of ascorbic acid concentration in embryonic chick brain. Dev. Biol., 139 (1990) 292-298. E.J. Butler, Role of trace elements in metabolic processes. In B.M. Freeman (ed.), Physiology and Biochemistry of the Domestic Fowl Academic Press, London, 1983, pp. 175-190. W.A. Dewar, P.W. Teague and J.N. Downie, The transfer of minerals from the egg to the chick embryo from the 5th to the 18th days of incubation. Br. Pouh. Sci., 15 (1974) 119-129. C.L. Keen and L.S. Hurley, Developmental changes in concentrations of iron, copper and zinc in mouse tissues. Mech. Ageing Dev., 13 (1980) 161-176. B.C. Sandrock, S.R. Kern and S.E. Bryan, The movement of zinc and copper from the fertilized egg into metallothionein-like proteins in developing chick hepatic tissue. Biol. Trace Element Res.. 5 (1983) 503-515. D. Wei and G.K. Andrews, Molecular cloning of chicken metallothionein. Deduction of the complete amino acid sequence and analysis of expression using cloned cDNA. NucL Acids Res., 16 (1988) 537-553. P.J. Thornalley and M. Vasak, Possible role for metallothionein in protection against radiationinduced oxidative stress. Kinetics and mechanism of its interaction with superoxide and hydroxyl radicals. Biochim. Biophys. Acta, 827 (1985) 36-44. Z.S. Suntres and E.M.K. Lui, Biochemical mechanism of metallothionein-carbon tetrachloride interaction in vitro. Biochem. PharmacoL, 39 (1990) 833-840. H.M. Tsai, B.B. Garber and L.M.H. Larramendi, [3H]Thymidine autoradiographic analysis of telencephalic histogenesis in the chick embryo: I. Neuronal birthdates of telencephalic compartments in situ. J. Comp. Neurol., 198, (1981) 275-292. P.J. Linser and M. Perkins, Gliogenesis in the embryonic avian optit: tectum: neuronal-glial interactions influence astroglial phenotype maturation. Dev. Brain Res.. 31 (1987) 277-290. H. Savolainen, Superoxide dismutase and glutathione peroxidase activities in rat brain. Res. Commun. Chem. Pathol. Pharmacol., 21 (1978) 173-175. E. Geremia, D. Baratta, S. Zafarana, R. Giordano, M.R. Pinizotto, M.G. La Rosa and A. Garozzo, Antioxidant enzymatic systems in neuronal and glial cell-enriched fractions of rat brain during aging. Neurochem. Res., 15 (1990) 719-723.

64 42 43

44

45

L.G. Thaete, R.K. Crouch and S.S. Spicer, lmmunolocalization of copper-zinc superoxide dismutase. J. Histochem. Cytochem.. 33 (1985) 803-808. G. Tholey, M. Ledig, P. Kopp, L. Sargentini-Maier, M. Leroy, A.A. Grippo and F.C. Wedler, Levels and sub-cellular distribution of physiologically important metal ions in neuronal cells cultured from chick embryo cerebral cortex. Neurochem. Res.. 13 (1988) 1163-1167. S. Stefanini, M.G. Farrace and M.P. Ceru Argento, Differentiation of liver peroxisomes in the foetal and newborn rat. Cytochemistry of catalase and D-aminoacid oxidase. J. Embryol. E_~:p. MorphoL. 88 (1985) 151-163, H.A. Murray, Jr., Physiological ontogeny. A. Chicken embryos. II. Catabolism. Chemical changes in fertile eggs during incubation. Selection of standard conditions. J. Gen. PhysioL, 9 (1925/26) 1-37.