Brain peroxidative and glutathione status after moderate hypoxia in normal weight and intra-uterine growth-restricted newborn piglets

Exp Toxic Patho11995; 47: 139-147 Gustav Fischer Verlag Jena

Institute of Pharmacology and Toxicology*, Institute of Pathological Physiology**, Neurochemical Lab. Neurological Clinic***. Friedrich Schiller University Jena, Germany

Brain peroxidative and glutathione status after moderate hypoxia in normal weight and intra-uterine growth-restricted newborn piglets A. BARTH*, R. BAUER**, H. KLUGE***, T. GEDRANGE*, B. WALTER**, W. KLINGER*, and U. ZWIENER** With 5 figures and 2 tables Received: November 8, 1994; Accepted: December 15, 1994 Address for correspondence: PD Dr. A. BARTH, Institute of Pharmacology and Toxicology, L6bderstr. 1, D - 07743 Jena, Germany. Key words: Newborn piglets; Brain, hypoxia; Oxygen free radicals; Lipid peroxidation; Glutathione; Chemiluminescence; Hypoxia, brain; Radicals, oxygen free; Peroxidation, lipid.

Abbreviations TBAR: NW: IUGR: LPO:

thiobarbituric acid reactants normal weight newborn piglets intra-uterine growth-restricted newborn piglets iron stimulated lipid peroxidation

Summary In order to investigate the pathogenetic factors causing the relatively frequent occurrence of brain injury in intrauterine growth-restricted newborns, lipid peroxidation products (TBAR), glutathione (GSH, GSSG) and in vitro production of reactive oxygen species (chemiluminescence, stimulated lipid peroxidation, HP2 formation) were studied in the brain of normal weight (NW) and intra-uterine growthrestricted newborn piglets (lUGR) after 1 hour of hypoxia (Fi0 2 11 %) and 90 min reoxygenation. Cardiocirculatory parameters and catecholamine release into the blood were also measured. In the cerebellum, higher GSH content, but also higher in vitro production of lucigenin amplified chemiluminescence were found in comparison to other brain regions, independent of growth restriction and hypoxia. Moderate hypoxia without acidosis and hypercapnia resulted in GSH depletion especially in the brain ofIUGR, but no changes in GSSG concentrations were measured. Though TBAR decreased after hypoxialreoxygenation, in some brain areas of IUGR higher TBAR values were found in comparison to NW. HP2 formation, stimulated lipid peroxidation and lucigenin and luminol amplified chemiluminescence in the 9000 x g supernatant of brain tissue did not reveal special response of IUGR to hypoxialreoxygenation. Hypoxiainduced circulatory centralisation due to increased release of catecholamines into the plasma prevented oxygen deficiency also in the brain of IUGR. The role of brain monoamine metabolism in the production of reactive oxygen spe-

cies, followed by greater GSH depletion and higher in vivo formation of lipid peroxides in IUGR is discussed.

Introduction Asymmetric intra-uterine growth retardation is caused predominantly by chronic placental insufficiency. A feature of intra-uterine growth retardation is a preservation of brain development at the expense of other organs. Intrauterine growth-restricted newborn piglets (IUGR) show similar morphometric features including changes in brain! liver ratio as known from human babies, as well as similarities in cerebral oxidative metabolism (WIDDOWSON 1971, FLECKNELL et al. 1982, BAUER et al. 1989a). Because intra-uterine growth retardation is associated with increased frequency of severe motor disturbances and mental retardation (KYLLERMAN 1982), the question arises what could be the reasons for a higher risk of brain injury. One hypothesis is that growth-restricted newborns suffer more often from cardiopulmonary disorders during the transition from intra- to extra-uterine life (LEVENE et al. 1985). In previous studies we have shown a tendency towards reduced cardiocirculatory compensation in IUGR during moderate hypoxia (BAUER et al. 1989b). However, the amount of changes cannot elucidate putative brain disorders due to brain oxygen deficits, because a preservation of spontaneous brain electrical activity was shown during the hypoxia period. The brain of fetuses and newborns has a high concentration of polyunsaturated fatty acids increasing its susceptibility to oxidative injury (OGIHARA et al. 1991, KAPLAN 1991). Moreover, polyunsaturated fatty acids, i.e. prostanoids, essentially involved in the brain vessel resExp Toxic Pathol47 (1995) 2-3

139

Table 1. Arterial blood pressure, gases, and pH. Baseline Sham normal weight piglets (n = 4) Mean arterial pressure, mmHg 63 ± 3 Arterial blood 112 ± 13 POz,mmHg PCOz,mmHg 40 ± 0.4 pH 7.47 ± 0.026 Sham IUGR piglets (n = 6) Mean arterial pressure, mmHg Arterial blood POz,mmHg PCOz,mmHg pH

66±5 122 ± 13 36 ± 2.1 7.49 ± 0.029

Hypoxia normal weight piglets (n = 6) Mean arterial pressure, mmHg 57 ±2 Arterial blood 115 ± 15 POz,mmHg 41 ± 2.2 PCOz,mmHg pH 7.42 ± 0.023 Hypoxia IUGR piglets (n = 7) Mean arterial pressure, mmHg Arterial blood POz,mmHg PCOz,mmHg pH

63 ±4 116±9 41 ± 2.6 7.50 ±0.030

10 min hypoxia 45 min hypoxia 30 min recovery 90 min recovery

60±2 113±11 40 ±0.5 7.48 ± 0.028 60±4 115 ± 13 37 ± 1.1 7.50 ± 0.021 60± 3 37 ± 6* 41 ± 2.9 7.44 ± 0.014 64±5

63 ±5 111 ± 13 38 ± 3.7 7.46 ± 0.043 60± 3 117 ± 14 39 ± 1.1 7.45 ± 0.016 65 ±4 39 ±3* 38 ± 3.3 7.39 ± 0.018 57 ±4

34± 3* 44 ± 3.1 7.49 ± 0.039

39 ± 1* 37 ± 1.9 7.49 ± 0.016+

66 ±6 106 ± 15 40 ± 1.9 7.45 ± 0.030 63 ±4 119 ± 16 40 ± 2.5 7.42 ± 0.025 61 ±4 118 ± 11 37 ± 2.5 7.41 ± 0.018 57 ±4 106±5 40 ± 1.4 7.46 ± 0.024

66 ±5 112 ± 12 41 ± 1.4 7.44 ± 0.029 60 ± 8 126 ± 17 40 ± 1.3 7.43 ± 0.021 60±4 104 ± 10 37 ± 2.7 7.40 ± 0.025 53 ±8 115 ± 6 39 ± 1.1 7.47 ± 0.019

Values are means ± S.E.M., n = no. of pigs. * P < 0.05 compared with sham groups, +P < 0.05 compared between NW and IUGR groups.

ponse to hypercapnia (LEFFLER et al. 1990), cerebral perfusion pressure decrease (LEFFLER et al. 1986), and seizures, have a more important influence on brain blood flow regulation as known from adult brain blood regulation (LEFFLER and BUSHA 1987, ARMSTEAD et al. 1989). Furthermore, it has been shown that in newborn piglets levels of lipid peroxidation products (conjugated dienes and fluorescent compounds) increased significantly due to systemic hypoxia (DiGIACOMO et al. 1992). The involvement of reactive oxygen species and lipid peroxidation in brain hypoxiaJreperfusion injury is assumed (TRA YSTMAN et al. 1991, GOPLERUD et al. 1992, EVANS 1993, GRAMMAS et al. 1993, HALL and BRAUGHLER 1993, ANDERSON and THOMAS 1994, TRUELOVE et al. 1994). The high content of unsaturated lipids in the membranes and the localisation of glutathione as antioxidant system primarily in glia makes neurons particularly susceptible to oxidative stress (RAPS et al. 1989, KAPLAN 1991, MAKAR et al. 1994). According to VERITY (1994), neuronal survival and differentiation are dependent upon the intracellular glutathione redox potential. In previous investigations we found no influence of intra-uterine growth retardation on basic data of peroxidative and glutathione status nor on the production of reactive oxygen species in 140

Exp Toxic Patho147 (1995) 2-3

the brain (BARTH et al. 1994). However, during moderate hypoxia a marked redistribution of circulating blood occurred with concomitant increase of circulating catecholarnines (BAUER et al. 1993). It was the aim of these investigations to find out whether the hypoxia-induced changes in cardiovascular and monoaminergic response have any relations to the peroxidative and glutathione status in the brain of NW and IUGR.

Material and methods Piglets of both sexes (race "Deutsches Landschwein") were transported from a farm in an infant incubator. The piglets were 12-23 h old. Two animal groups with different body weights were studied: NW (n = 10, body weight: 1533 ± 60 g) and IUGR (n = 13, body weight: 847 ± 13 g). The piglets were anesthetized with 0.50 % halothane in a gas mixture of 70 % nitrous oxide and 30 % oxygen. The piglets were intubated with an endotracheal tube (14-16 French Charrier), relaxed with pancuronium (2 mg/kg x h i.v.) and artificially ventilated (volume controlled; Servo Ventilator 900 C, Siemens-Elema Uppsala). Catheters were inserted into the abdominal aorta via the right femoral artery, into the sagittal sinus after a skull trepanation (diameter 3 mm; in the mid-

GSH

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Fig.t. GSH (Ilg/g wet weight) in different brain regions: temporoparietal (TP), frontal (F), occipital (0), basal ganglia (B), cerebellum (C), midbrain (M) of normal weight (NW) and intra-uterine growth-restricted newborn piglets (IUGR). Arithmetic means ± S.E.M., n = 4-8. + significant differences from other brain regions within the same animal group, Student's t-test pairwise comparison, * significant differences between the same regions of NW and IUGR, Student's t-test, p::; 0.05.

line, 2 mm caudal from the coronal suture) and into the upper caval vein via the left external jugular vein for measurement of the blood gases, pH (ABL 50, Radiometer Copenhagen), arterial blood pressure (Statham transducer), and for collecting blood samples to measure glutathione and lipid peroxides in the blood. Blood volume obtained was replaced by an equal volume of donor blood, obtained from a piglet of the same litter. After ending the surgical procedure, the animals were allowed to recover for at least one hour. In this time halothane concentration was lowered to 0.25 %. Then baseline values were obtained. Afterwards, both groups were divided into a hypoxic and a sham hypoxic group. The hypoxia groups got a ventilatory gas mixture with a lowered fractional inspired oxygen concentration (Fi02) of 11 % in exchange to nitrogen for one hour. The hypoxia period was followed by a recovery period with the same gas mixture as before hypoxia. Sham hypoxic animals obtained the same procedures without hypoxia. After 90 min recovery the brains were frozen in situ using liquid nitrogen [funnel technique (PONTEN et al. 1993)]. The head was stored at -80°C. The frozen brains were sliced under continuous irrigation with liquid nitrogen and divided into ten portions: frontal (F),

temporo-parietal (TP), occipital (0), basal ganglia (B), cerebellum (C) and midbrain (M), sometimes right and left areas. In sham hypoxic NW and IUGR only TP areas and C were investigated. After subdivision the brain portions were stored for no longer than one week at - 20°C up to the preparation of homogenates and supernatants. In the homogenates of different brain regions reduced glutathione (GSH) was measured according to ELLMAN (1959) and oxidized glutathione (GSSG) according to HISS IN and HILF (1976), lipid peroxides were determined as TBAR with the method ofYAGI (1987). To investigate the possibility of in vitro peroxidation and radical production, the 9000 g supernatant of brain tissue was used to estimate LPO (BUEGE and AUST 1978), HP2 formation (HILDEBRANDT et al. 1978) as well as luminol (lum) and lucigenin (Juc) amplified chemiluminescence (CI). CL to measure reactive oxygen mediated photoemission was determined according to MULLERPEDDINGHAUS and WURL (1987) with a BERTHOLD-Auto Lumat LB 953 after starting the reduction of oxygen in the 9000 g supernatant with NADPH (KLINGER et al. 1994). The measurement of plasma catecholamines was performed by high performance liquid chromatography with an electrochemical detector according to the Instruction Exp Toxic Pathol47 (1995) 2-3

141

LI PI D PEROXI DES [nmol TBAR/g brain]

control

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Fig. 2. Lipid peroxides as thiobarbituric acid reactants (nmol TBARlg wet weight) measured as malondialdehyde equivalents in different brain areas (see legend Fig. 1). Arithmetic means ± S.E.M. * significant differences between the same regions ofNW and IUGR, Student's t-test, p::; 0.05.

Manual (Catalog number 195-6050/51) of Bio-Rad Laboratories. The results are given as arithmetic means ± S. E. M. For statistical analysis Student's t-test was conducted to compare the results between NW and IUGR, in the same animal group pairwise comparison was used. Comparisons among groups concerning physiological parameters summarized in tab. 1 and 2 were made using analysis of variance with Duncan new multiple range post hoc test. A significance level of p < 0.05 was used.

Results As shown in table 1, change in Fi02 from 30 to 11 % resulted in a strong reduction of arterial p02' This was the only significant change in all parameters controlled during the moderate hypoxia and the recovery period. The moderate hypoxia failed to produce metabolic acidosis and hypercapnia as well as changes in blood glutathione and lipid peroxides (BARTH, unpub!.) . In the brain, however, different responses were found in NW and IUGR. After hypoxialreoxygenation the total GSH content de142

Exp Toxic Patho147 (1995) 2- 3

creased in the brain of both NW and IUGR, more obviously in IUGR. Compared to NW, GSH concentration was found to be significantly lower in all regions investigated (fig. 1). In sham hypoxic control, NW and IUGR GSH did not differ, but in the cerebellum a significantly higher GSH concentration was seen in comparison to TP. This GSH difference between C and other brain regions remained also after hypoxialreoxygenation, at least in NW (fig. 1). GSSG was evenly distributed throughout the brain without any differences between NW and IUGR after hypoxia and reoxygenation (NW, C: 211.5 ± 10.7 ug GSSG/g wet weight; IUGR, C: 233 .0 ± 17.5 ug GSSG/g wet weight). Fig. 2 shows the lipid peroxides measured as TBAR in the brain of sham hypoxic and hypoxic NW and IUGR. Surprisingly, IUGR showed lower TBAR levels in both regions. But after hypoxia and reoxygenation there was an inverse situation, TBAR levels had generally decreased, but IUGR showed more lipid peroxides in the brain than NW (fig. 2). Measuring LPO in the 9000 g brain supernatant, significantly more TBAR were found in the TP region and cerebellum of IUGR (fig. 3). After hypoxialreoxygenation these differences disappeared, moreover in some regions

LIPIDPEROXIDATION 9000xg supernatant [nmol TBARjmgxmin]

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Fig. 3. Iron stimulated lipid peroxidation (LPO) in the 9000 g brain supernatant, incubated 10 minutes at 37 °C and pH 7.4, measured as production of thiobarbituric acid reactants (TBAR) per mg protein and minute. Brain areas see legend Fig. 1. Arithmetic means ± S.E.M. * significant differences between the same regions of NW and IUGR, Student' s t-test, p ~ 0.05 .

of the brain in IUGR the production of TBAR was significanty lower compared to NW (fig. 3). Hydrogen peroxide formation in the 9000 g brain supernatant, reflecting the oxidase function of cyt P-450, was not changed at all after hypoxialreoxygenation in NW and IUGR (not shown). There was only a tendency towards enhanced lum CI after hypoxialreoxygenation in the brain of NW, but nevertheless no significant differences between NW and IUGR were found (fig. 4). Fig. 5 demonstrates the luc CL in the brain of sham and hypoxic NW and IUGR. It is of interest that in the cerebellum luc CL was higher than in other regions, more pronounced in NW. But even Iuc CL did not reveal special responses of IUGR to hypoxialreoxygenation. Under baseline conditions, plasma catecholamine release was similar in both groups (tab. 2). NW showed an increase of catecholamine content due to moderate hypoxia, but the amount returned to prehypoxic values only for dopamine. Plasma epinephrine and norepinephrine remained on moderately elevated levels. IUGR (sham and hypoxia group) showed a continuous increase of plasma

epinephrine and norepinephrine content throughout the whole experiment up to high values at the end of the observation. Plasma dopamine showed a similar time course as found in NW with a moderate increase during hypoxia and further return to prehypoxic values (tab. 2).

Discussion Apart from lipid soluble alpha-tocopherol, GSH as watersoluble substrate of GSH-peroxidases and GSH-Stransferases is one of the most important antioxidants. Also reactivation of alpha-tocopherol after radicalisation requires ascorbate and/or GSH (CHAN 1993). Loss of GSH and increase of GSSG are used to indicate oxidative stress (ADAMS et al. 1983, SASTRE et al. 1992, GIBSON et al. 1993). In accordance with previous results, intra-uterine growth retardation did not significantly influence GSH and GSSG content in the brain as seen in TP and cerebellum of sham hypoxic IUGR in comparison to NW (fig. I). The higher GSH content of cerebellum in contrast to other brain regions is a recurrent finding indepenExp Toxic Pathol47 (\995) 2-3

143

CHEMILUMINESCENCE 9000xg supernatant LU (E +4 CPM/mg prot.] luminol

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Fig. 4. Luminol amplified chemiluminescence in the 9000 g brain supernatant, given as light units (LU) in counts per minute (CPM) and mg protein, measured over 3.5 minutes. Brain regions see legend Fig. 1.

dent of growth retardation or hypoxialreoxygenation (fig. 1) (BARTH et al. 1994). On the other hand luc CI was found to be higher in the cerebellum, indicating increased ability of free radical production in the cerebellum compared to other regions, more pronounced in NW independent of hypoxialreoxygenation (fig. 5). Literature data confirm the special position of the cerebellum concerning the peroxidative and glutathione status. Whereas JOHNSON et al. (1993) and VILLALOBOS et al. (1994) found higher protective capacity in the cerebellum against oxidative stress, other authors described a special susceptibility of the cerebellum to oxidative damage induced by organometals, ethanol or asphyxia (KAPLAN 1991, ALI et al. 1992, BONDY and PEARSON 1993, DEHAAN et al. 1993). Selective GSH depletion in the cerebellum may contribute to drug induced cerebellar damage (DURUIBE et al. 1991). The most important result after hypoxialreoxygenation is the more marked GSH depletion in IUGR in all brain regions investigated (fig. 1). This GSH consumption was accompanied by higher contents of lipid peroxides in some areas of the brain of IUGR (fig. 2), but not with higher GSSG concentrations. GIBSON et al. (1993) demonstrated that the ratio GSSG/GSH as an indicator of 144

Exp Toxic Pathol47 (1995) 2-3

oxidative stress after ischemialreperfusion increased due to GSH disappearance rather than due to an increase in GSSG. Although GSSG is released from the cells by active process (SIES and AKERBOOM 1984), we did not measure higher GSSG concentrations in the cerebral venous blood after hypoxialreoxygenation in IUGR (BARTH, unpubl.). The reason for a higher GSH depletion is unknown. Because lum CL predominantly measures hydrogen superoxide (AHKREM et al. 1985, MDLLER-PEDDINGHAUS and WURL 1987) and luc CI is selective and sensitive in detecting superoxide anion radicals (ISCHIROPOULOS et al. 1989, FAULKNER and FRIDOVICH 1993) apparently these two reactive oxygen species may not be responsible for GSH depletion and higher contents of lipid peroxides in the brain of IUGR after hypoxialreoxygenation. According to TRA YSTMAN et al. (1991), KLINGER et al. (1994) and OHOI et al. (1993) CI with lum and luc is not specific for one radical generated, but measuring oxygen-derived free radicals generally produced in oxygenated tissues. Nevertheless in vitro LPO, HP2' and radical production measured as lum and luc CI did not reveal special responses of IUGR to hypoxialreoxygenation (fig. 3,4,5).

CHEMILUMINESCENCE 9000xg supernatant LU [E +5 CPM/mg prot.] lucigenin

Irzn right ~ left D

hypoxia

control

total

25 r-------------------------,

+

+

2

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Fig.s. Lucigenin amplified chemiluminescence in the 9000 g brain supernatant, given as light units (LU) in counts per minute (CPM) and mg protein, measured over 3.5 minutes. Brain regions see legend Fig. 1. + significantly different from TP, F, 0 (right and left) of NW, Student's t-test pairwise comparison, p ~ 0 .05.

Table 2. Plasma catecholamine content in normal weight (NW) and intrauterine growth restricted (IUGR) newborn piglets. Baseline

10 min hypoxia

45 min hypoxia

30 min recovery

90 min recovery

Sham NW piglets (n =4) epinephrine, nMol1L norepinephrine, nMol1L dopamine, nMol1L

368 ± 52 702 ± 114 326± 98

404± 64 644± 71 259 ± 64

477 ± 55 662 ± 79 218 ± 63

609 ± 54 882 ± 98 220± 33

711 ± 107 1024 ± 182 231 ± 58

Sham IUGR (n =6) epinephrine, nMollL norepinephrine, nMol1L dopamine, nMollL

780 ± 100 1156 ± 93 147 ± 13

794 ± 141 1114± 97 178 ± 35

1441 ± 328+ 1851±321 233 ± 38

2311 ± 741 2800 ± 686+ 232 ± 46

3818 ± 1153+ 5555 ± 1306+ 326 ± 73

Hypoxia NW (n =6) epinephrine, nMol1L norepinephrine, nMol1L dopamine, nMol1L

616 ± 154 774 ± 188 286 ± 107

1674 ± 357* 2497 ± 808 725 ± 203*

1888 ± 393* 2272 ±600* 491 ± 73*

1456 ± 339 1292 ± 230 376± 60

1852 ± 557 1673 ± 243 293 ± 80

Hypoxia IUGR (n =7) epinephrine, nMol1L norepinephrine, nMol1L dopamine, nMol1L

426± 75 1007 ± 142 341 ± 51

1568 ± 434 2414 ± 872 401 ± 60

1512 ± 214 1895 ± 379 421 ± 60*

2815 ± 516 2791 ± 521 398 ± 53

4810 ± 1349 3780 ± 970 364± 67

Values are means ± S.E.M., n =no. of pigs. *p < 0.05 compared with sham groups. +P < 0.05 compared between NW and IUGR groups. Exp Toxic Pathol47 (1995) 2-3

145

In connection with these results, it is of interest that hypoxemia in the brain was certainly well compensated with regard to brain oxygen supply due to an increased cerebral blood flow as a component of the hypoxia-induced redistribution of the circulatory blood volume to "vital" organs (myocardium, brain, adrenals) at the expense of other (abdominal) organs. This cardiovascular response to hypoxia is well established in newborns (HEYMANN et al. 1981) and also existent in IUGR (BAUER et al. 1991). The sustained brain oxygen supply is shown by unchanged neurophysiological parameters such as the quantified electrocorticogram (BAUER et al. 1993). The increased release of catecholamines into the plasma is certainly involved in the hypoxia-induced circulatory centralization, but in IUGR a gradual increase occurred throughout the hypoxia and reoxigenation period (tab. 2). Apparently, IUGR are able to keep stable systemic cardiovascular functions, such as arterial blood pressure by gradual improvement of sympathetic activity. However, the relevance of this for the findings on brain peroxidative and glutathione status remained uncertain. Immediately before brain freezing, there were no correlations between brain GSH, GSSG, TBAR and plasma catecholamine levels. Indeed, these levels need not be closely correlated to cerebral catecholamine release, because there are two different mechanism to release catecholamines: First, the early (fetal) established directly stimulated output from adrenals by hypoxia and other stress factors. Second, the neural control of plasma catecholamine release (PADBURY and MARTINEZ 1988). The maturity of neural control of plasma catecholamine release is in line with other features of brain development. So, species with prenatal spurts in brain development, such as sheep, show a mature neural control of plasma catecholamine release at birth, but rats as postnatal brain developers have established this function not before the second week after birth. Data from piglets are not available, but other comparable parameters of brain development suggest that pigs as "perinatal brain developers" at birth should be in the transition of maturity of neurally controlled plasma catecholamine release. But the role of monoamines for the generation of free oxygen radicals remained ambivalent: monoamine oxidases in the brain are able to produce reactive oxygen species, and oxygen dependent catecholamine metabolism was found to take part in CNS oxygen toxicity (ZHANG et al. 1993). On the contrary, monoamines as norepinephrine, dopamine and their metabolites have been found to inhibit lipid peroxidation and to scavenge free oxygen radicals in vitro in the brain (Lm and MORI 1993). Possibly brain monoamine metabolism in vivo, especially monoamine oxidase activity, is responsible for the production of reactive oxygen species followed by greater GSH depletion and higher in vivo formation of lipid peroxides in IUGR, making them more susceptible to hypoxic events. Summarizing the results we found higher GSH concentrations and higher in vitro production of reactive oxygen species in the cerebellum, independent of growth retardation or hypoxia, demon146

Exp Toxic Patho147 (1995) 2-3

strating the special position of the cerebellum concerning the peroxidative and glutathione status. The moderate hypoxia without acidosis and hypercapnia resulted in GSH depletion and in vivo lipid peroxidation especially in the brain of IUGR. These results may indicate possible involvement of free radicals in disturbed adaptation to extra-uterine life in IUGR. Acknowledgements: We gratefully acknowledge the skilled technical assistance of ELKE KARGE and HElKE STADLER. Supported by BMFT grant num. 01ZZ9104/8; 5.

References ADAMS JD, WANG B, KLAIDMAN LK, et a1.: New aspects of brain oxidative stress induced by tertbutylhydroperoxide. Free Rad Bioi Med 1993; 15: 195-202. AKHREM AA, SEMENKOVA GN, CHERENKEVICH SN, et al.: Chemiluminescence of luminol caused by interaction of cytochrome C with cumene hydroperoxide: comparative studies. Biomed Biochim Acta 1985; 44: 1591-1597. ALI SF, LEBEL CP, BONDY SC: Reactive oxygen species formation as a biomarker of methylmercury and trimethyltin neurotoxicity. Neurotoxicology 1992; 13: 637-648. ANDERSON DK, THOMAS CE: Mechanisms and role of oxygen free radicals in CNS pathology. Int Acad Biomed Drug Res. Karger Basel, 1994; 7: 119-124. ARMSTEAD WM, MIRRO R, LEFFLER CW, et a1.: Cerebral superoxide anion generation during seizures in newborn pigs. J Cereb Blood Flow Metab 1989; 9: 175-179. BARTH A, BAUER R, KLINGER W, et al.: Peroxidative status and glutathione content of brain in normal weight and intrauterine growth-retarded newborn piglets. Exp Toxic Patho11993; 45: 519-524. BAUER R, ZWIENER U, BUCHENAU W, et al.: Reduced cardiovascular and cerebral compensatory ability of intrauterine growth-retarded newborn piglets in severe hypoxic load. Int J Feto-Matern Med 1989a; 2: 98-108. BAUER R, ZWIENER U, BUCHENAU W, et al.: Restricted cardiovascular and cerebral performance of intra-uterine growth retarded newborn piglets during severe hypoxia. Biomed Biochim Acta 1989b; 48: 697-705. BAUER R, ZWIENER U, BERGMANN R, et al.: Interaction between systemic circulation and brain injuries in newborns. Exp Patho11991; 42: 197-203. BAUER R, WALTER B, KLUGE H, et a1.: Catecholamine response and cardiovascular reaction to moderate hypoxia in growth retarded (IUGR) newborn piglets. In: COSMI EV, DIRENZO GC (eds): 2nd World Congress of Perinatal Medicine. Proceedings of Communications and Posters 1. Monduzzi Editore S.p.A. Bologna, pp. 423-426. BONDY SC, PEARSON KR: Ethanol-induced oxidative stress and nutritional status. Alcoholism - Clin Exp Res 1993; 17: 651-654. BUEGE JKA, AUST SD: Microsomal lipid peroxidation. Methods Enzymol1978; 52: 302-310. CHAN AC: Partners in defense, vitamin E and vitamin C. Can J Physiol Pharmacol1993; 71: 725-731. DEHAAN HH, V ANREEMPTS JLH, VLES JSH, et a1.: Effects of asphyxia on the fetal lamp brain. Am J Obstet Gynecol 1993; 169: 1493-1501.

DIGIACOMO JE, PANE CR, GWIAZDOWSKI S, et al.: Effect of graded hypoxia on brain cell membrane injury in newborn piglets. BioI Neonate 1992; 61: 25-32. DURumE VA, BLYDEN GT, OKONMAH A: Effect of cyclosporin A on the content of glutathione in the brain of the rat. Neuropharmacology 1991; 30: 353-357. ELLMAN GL: Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-77. EVANS PH: Free radicals in brain metabolism and pathology. Brit Med Bull 1993; 49: 577-587. FAULKNER K, FRIDOVICH I: Luminol and lucigenin as detectors for 02. -. Free Rad BioI Med 1993; 15: 447-451. FLECKNELL PA, WOOTON R, JOHN M: Accurate measurement of cerebral metabolism in the conscious, unrestrained neonatal pig. II. Glucose and oxygen utilization. BioI Neonate 1982; 41: 221-226. GmsoN DD, BRACKETT DJ, SQUIRES RA, et al.: Evidence that the large loss of glutathione observed in ischemialreperfusion of the small intestine is not due to oxidation to glutathione disulfide. Free Rad BioI Med 1993; 14: 427-433. GOPLERUD JM, MISHRA OP, DELIVORIAPAPADOPOULOS M: Brain cell membrane dysfunction following acute asphyxia in newborn piglets. BioI Neonate 1992; 61: 33-41. GRAMMAS P, LIU GJ, WOOD K, et al.: Anoxialreoxygenation induces hydroxyl free radical formation in brain microvessels. Free Rad BioI Med 1993; 14: 553-557. HALL ED, BRAUGHLER JM: Free radicals in CNS injury. Research Publications: Association for Research in Nervous and Mental Disease 1993; 71: 81-105. HEYMANN MA, IWAMOTO HS, RUDOLPH AM: Factors affecting changes in the neonatal systemic circulation. Ann Rev Physiol1981; 43: 371-383. HILDEBRANDT AG, ROOTS I, TJOE M, et al.: Hydrogen peroxide in hepatic microsomes. Methods Enzymol 1978; 52: 342-350. HISSIN PJ, HILF R: A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochern 1976; 74: 214--226. ISCHIROPOULOS H, KUMAE T, KIKKAWA Y: Effect of interferon inducers on superoxide anion generation from liver microsomes detected by lucigenin chemiluminescence. Biochem Biophys Res Commun 1989; 161: 1042-1048. JOHNSON JA, ELBARBARY A, KORNGUTH SE, et al.: Glutathione S-transferase isozymes in rat brain neurons and glia. J Neuroscience 1993; 13: 2013-2023. KAPLAN E: Lipid hydroperoxides in cerebrum and cerebellum obtained from rat brains: effect of freezing in liquid N 2 • Biochem Archs 1991; 7: 227-231. KLINGER W, KARGE E, KRETZSCHMAR M, et al.: Luminoland lucigenin-amplified chemiluminescence with rat liver microsomes: kinetics and influence of iron and copper ions, catalase and superoxide dismutase, glutatione and ascorbic acid, hexobarbital and anilin, and of dimethylsulfoxide. Free Rad Res Commun 1994 (in press). KYLLERMAN M: Dyskinetic cerebral palsy. II. Pathogenetic risk factors and intra-uterine growth. Acta Paediat Scand 1982;71:551-558. LEFFLER CW, BUSIJA DW, BEASLEY DG, et al.: Maintenance of cerebral circulation during hemorrhagic hypotension in newborn pigs: Role of prostanoids. Circulation Res 1986;59: 562-567. LEFFLER CW, BUSIJA DW: Arachnoid acid metabolites and perinatal cerebral hemodynamics. Seminars in Perinatology 1987; 11: 31-42.

LEFFLER CW, BUSIJA DW, ARMSTEAD WM, et al.: Activated oxygen and arachidonate effects on newborn cerebral arterioles. Am J Physiol 1990; 259: H1230-H1238. LEVENE ML, KORNBERG J, WILLIAMS THC: The incidence and severity of post-asphyxial encephalopathy in fullterm infants. Early Hum Develop 1985; 11: 21-26. LIU J, MORI A: Monoamine metabolism provides an antioxidant defense in the brain against oxidant-induced and free radical-induced damage. Arch Biochem Biophys 1993; 302: 118-127. MAKAR TK, NEDERGAARD M, PREUSS A, et al.: Vitamin E, ascorbate, glutathione, glutathione disulfide, and enzymes of glutathione metabolism in cultures of chick astrocytes and neurons - evidence that astrocytes play an important role in anti oxidative processes in the brain. J Neurochem 1994; 62: 45-53. MULLER-PEDDINGHAUS R, WURL M: The amplified chemiluminescence test to characterize antirheumatic drugs as oxygen radical scavengers. Biochem Pharmacol 1987; 36: 1125-1132. OGIHARA T, KITAGAWA M, MIKI M, et al.: Susceptibility of neonatal lipoproteins to oxidative stress. Pediat Res 1991, 29: 39-45. OHOI I, SONE K, TOBARI H, et al.: A simple chemiluminescence method for measuring oxygen-derived free radicals generated in oxygenated rat myocardium. Japanese J Pharmacol 1993;61: 101-107. PADBURY JF, MARTINEZ AM: Sympathoadrenal system activity at birth: Integration of postnatal adaptation. Seminars in Perinatology 1988; 12: 163-172. PONTEN U, RATCHESON A, SALFORD LG, et al.: Optimal freezing conditions for cerebral metabolites in rats. J Neurochem 1973; 21: 1127-1138. RAPS SP, LAI JCK, HERTZ L, et al.: Glutathione is present in high concentrations in cultured astrocytes, but not in cultured neurons. Brain Res 1989; 493: 398-401. SASTRE J, ASENSI M, GASCO E, et al.: Exhaustive physical exercise causes oxidation of glutathione status in blood prevention by antioxidant administration. Am J Physiol 1992; 263: R992-R995. SIES H, ACKERBOOM TPM: Glutathione disulfide (GSSG) efflux from cells and tissues. Methods Enzymol 1984; 105: 445-451. TRAYSTMAN RJ, KIRSCH JR, KOEHLER RC: Oxygen radical mechanisms of brain injury following ischemia and reperfusion. J Appl Physiol1991; 71: 1185-1195. TRUELOVE D, SHuAm A, IJAZ S, et al.: Superoxide dismutase, catalase, and U78517F attenuate neuronal damage in gerbils with repeated brief ischemic insults. Neurochern Res 1994; 19: 665-671. VERITY MA: Oxidative damage and repair in the developing nervous system. Neurotoxicol1994; 15: 81-91. VILLALOBOS MA, DELACRUZ JP, CARRASCO T, et al.: Effects of alpha-tocopherol on lipid peroxidation and mitochondrial reduction of tetraphenyl tetrazolium in the rat brain. Brain Res Bull 1994; 33: 313-318. WIDDOWSON EM: Intra-uterine growth-retardation in the pig. I. Orga!l size and cellular development at birth and after growth to maturity. BioI Neonate 1971; 19: 329-340. Y AGI K: Lipid peroxides and human diseases. Chern Phys Lipids 1987; 45: 337-351. ZHANG J, Su YF, OURY TD, et al.: Cerebral amino acid, norepinephrine and nitric oxide metabolism in CNS oxygen toxicity. Brain Res 1993; 606: 56-62. Exp Toxic Pathol47 (1995) 2-3

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