National Jewish Medical and Research Center, University of Colorado Health Sciences Center, Denver, Colorado 80206
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ABSTRACT |
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We investigated the effects of gestational age and oxygen exposure on superoxide dismutase (SOD) activities in distal fetal lung tissue in primate models of bronchopulmonary dysplasia. During the final third of fetal life, lung coppper-zinc SOD (Cu,ZnSOD) specific activity decreased, whereas lung manganese SOD (MnSOD) specific activity tended to increase. In the premature newborn (140 days, 78% of term gestation), lung total SOD and Cu,ZnSOD specific activities decreased after 6-10 days of ventilation with as needed [pro re nada (PRN)] or 100% oxygen compared with fetal control animals. Neither Cu,ZnSOD mRNA nor protein expression changed after either oxygen exposure at this gestation (140 days) relative to fetal control animals. At this age (6-10 days), lung MnSOD specific activity did not change in oxygen-exposed relative to fetal control animals, even though lung expression of MnSOD mRNA and protein increased after PRN or 100% oxygen exposure. In the very premature 125-day newborn (69% of term), lung Cu,ZnSOD specific activity and protein decreased, whereas Cu,ZnSOD mRNA increased, after 6-10 days of ventilation with PRN oxygen compared with fetal control animals. In fetal lung explants, hyperoxia also decreased expression of SOD activity acutely (16-h exposure, 125 and 140 days gestation). To conclude, expression of SOD activity in the premature primate lung did not increase in response to elevated oxygen tension, apparently due to effects occurring subsequent to the expression of these mRNAs.
copper-zinc superoxide dismutase; manganese superoxide dismutase; gestation; messenger ribonucleic acid; fetal; explant
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INTRODUCTION |
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SEVERAL ANTIOXIDANT ENZYMES (AOEs) increase in the lung during the final third of gestation in ruminants and rodents, and these parallel the marked increase in surfactant production before birth (19, 41, 44). This increase in antioxidants before birth provides protection from toxic radicals in the transition from the relatively low oxygen fetal environment to the increased oxygen environment of the newborn (18). One classic antioxidant of particular importance in the newborn is the intracellular enzyme superoxide dismutase (SOD). SOD consists of a family of enzymes located in either the cytoplasm [copper-zinc SOD (Cu,ZnSOD)], mitochondrion [manganese SOD (MnSOD)], or extracellular space (endothelial cell SOD, a Cu,Zn-containing enzyme) (5). The close proximity of SOD to intracellular sites of superoxide anion production, such as the nuclear membrane or mitochondrion, protects the cell from the damaging effects of toxic oxygen radicals (17). Although numerous studies (18, 19, 41, 44) have been done in lesser species, very little information is available regarding the changes in the activities of Cu,ZnSOD and MnSOD during fetal lung development in primates or humans. Therefore, we sought to determine the developmental profile of pulmonary SOD activities in the fetal primate.
Previous investigations (7, 16, 24, 29, 47) in both the full-term neonatal and adult rat lungs demonstrated that lung SOD mRNAs, proteins, and activities increase after exposure to sublethal oxygen tensions compared with values in air-breathing control animals. These associated increases are implicated in adaptation (5, 8, 11, 18, 20, 26, 31, 42, 46). Relatively little information is known about the activities of the SODs in the lungs of premature humans or primates (3, 28). Because SOD activities ultimately determine the rates of superoxide detoxification in vivo, the second question addressed in the present study was what effect hyperoxic exposure has on lung SOD activities in the premature baboon. A study (40) in rabbits indicated that premature animals do not increase lung AOE activity in response to a hyperoxic challenge. Because primates can be supported successfully at much more extreme levels of prematurity and do develop marked lung injury due to hyperoxia, we hypothesized that they would not develop adaptive increases in lung AOE activity reported in term newborns of lesser species (10, 18, 19, 29, 41, 47). To address this hypothesis, we studied lung tissue from baboons of different gestational ages exposed to varying oxygen tensions in a premature primate model of bronchopulmonary dysplasia (BPD) (14).
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MATERIALS AND METHODS |
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Reagents. SOD, horse heart cytochrome c, xanthine, xanthine oxidase, sodium dithionite, diethyldithiocarbamate (DDC), sodium cyanide, and dithiothreitol (DTT) were purchased from Sigma.
Animal care protocols. All animal studies were performed at the Southwest Foundation for Biomedical Research (San Antonio, TX) according to the National Research Council's Guide for the Care and Use of Laboratory Animals. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee. All animals for this project were used in the ongoing multi-investigator National Institutes of Health Collaborative Project on Bronchopulmonary Dysplasia.
All pregnant baboons were treated in identical fashion. Gestational ages were determined by timed matings as previously described (13). Fetal or newborn baboons were delivered at either 140 ± 2 (78% term) or 125 ± 2 days (69% term) gestation by hysterotomy. Gestational control animals were killed at delivery before the onset of breathing. Seventy-one animals were studied (140-day animals, n = 38; 125-day animals, n = 15; 160- and 175-day animals, n = 18). Term delivery occurs in the baboon after ~184 days gestation.
All newborn animals were also treated similarly. Those receiving special treatment protocols were not included. Newborns were delivered and immediately placed on positive-pressure ventilation (PPV). Animals of 140 days gestation were treated at two different levels of oxygenation. They were given either continuous 100% oxygen or as needed [pro re nada (PRN)] oxygen [inspired oxygen fraction (FIO2) ranged from 0.21 to 0.8] necessary to maintain arterial PO2 (PaO2) at 60-70 mmHg. After 6-10 days, animals at 140 days gestation exposed to 100% oxygen develop a lung histopathology characteristic of BPD, whereas the PRN oxygen-exposed animals do not develop BPD at this gestation (14). Animals of 125 days gestation received immediate resuscitation with artificial surfactant, PPV, and PRN oxygen. In the very premature animal (125 days), the histopathology characteristic of BPD develops after receiving lower (PRN) concentrations of oxygen (37). All animals were supported in a state-of-the-art neonatal intensive care unit.
There were significant differences in the treatment of PRN oxygen-exposed animals at the two gestational ages. Animals born at 125 days gestation were treated with artificial surfactant (4 ml/kg body wt; Survanta, Ross Laboratories, Columbus, OH) at delivery, and this treatment was required for their survival at this gestational age. Those born at 140 days gestation did not receive this treatment. Ventilators were adjusted to maintain a PaO2 of 60-70 mmHg and an arterial PCO2 of 45-55 mmHg. Inspired oxygen was weaned as follows in the 125-day newborns. The initial FIO2 after artificial surfactant was generally 0.45-0.5 and usually could be decreased rapidly into the 0.30-0.40 range by age 6 h. Therefore, FIO2 remained at ~0.35-0.45. In the 140-day newborns receiving PRN oxygen and not receiving artificial surfactant, the initial FIO2 was usually 0.6-0.7 for the first 48 h. Then, FIO2 generally could be rapidly weaned such that FIO2 was usually ~0.3-0.4 by age 72 h and thereafter was weaned further. These animals generally did not require any supplemental oxygen by day 14.
Tissue samples. After treatment, the animals were killed by administering intravenous pentobarbital sodium. Peripheral lung tissues for enzyme assays were processed immediately by homogenizing in buffer (50 mM potassium phosphate, pH 7.8, with 1 mM EDTA) with a polytron set at maximal setting for 30 s, centrifuged (12,000 g for 10 min) for separation of pellet and supernatant, frozen in liquid nitrogen, and shipped on dry ice by overnight courier. Samples were held in liquid nitrogen until analysis.
Fetal lung explant cultures, To
examine the intrinsic effects of acute oxygen exposure on SOD
activities in neonatal lungs, an explant model was used. For these
cultures, fetal lung specimens were cut, placed in sterile Waymouth
medium (50 ml), and shipped (4°C) by overnight courier. Distal
fetal lung tissue (gestational ages 125-175 days) was dissected
into 1-mm3 pieces, with removal of
any attached airways. Explant pieces were placed at 4-5/well in
3.5-cm-diameter wells (6-well plates) that had been precoated with 2 ml
of Waymouth A157 medium containing penicillin (50 U/ml) and
streptomycin (50 U/ml) but lacking fetal bovine serum. This coating
material also contained ultrapure agarose (0.5% wt/vol; Fisher) that
had been dissolved by brief boiling (91°C) and was then allowed to
harden on the plates by brief chilling (4°C). After they were
placed on the plates, the explants were then overlaid with 1 ml of
complete Waymouth A157 medium containing penicillin and streptomycin in
addition to 10% fetal bovine serum. The explants were then exposed to
1, 21, or 95% oxygen in plastic modular exposure chambers
(Billups-Rothenburg, La Jolla, CA) for 16 h (37°C). A concentration
of 1% oxygen was chosen to approximate the range of
PO2 likely to be present in the
distal fetal lung, and an exposure duration of 16 h was chosen because
we have not observed significant cell death in hyperoxia after such
brief exposures. After exposure, the explants were homogenized in
buffer (50 mM potassium phosphate, pH 7.8, with 1 mM EDTA), rapidly
frozen in liquid nitrogen, and stored at 70°C for further
biochemical analysis.
DDC treatment of lung homogenates. The samples were incubated with 50 mM DDC in 50 mM potassium phosphate buffer, pH 7.8, with 1 mM EDTA, at 30°C for 1 h. With a SpectraPor microdialyzer (Spectrum, Houston, TX), the samples were dialyzed extensively with three changes of a 400× volume of buffer (50 mM potassium phosphate, pH 7.8, with 1 mM EDTA) overnight (adapted from Ref. 27). DDC is a nonspecific copper chelator that eliminates nearly all (>98%) of the Cu,ZnSOD activity after treatment, thereby allowing the determination of MnSOD activity from the residual activity. For lung samples obtained in vivo and from fetal animals, all MnSOD measurements were made by the DDC method. The data presented for these animals all were obtained with the DDC method.
Cyanide treatment of lung homogenates and explants. Lung MnSOD activity also was determined after the addition of sodium cyanide (2 mM) to the assay wells (35). We found that this concentration of sodium cyanide eliminates 95% of the Cu,ZnSOD activity in the Cu,ZnSOD standards after treatment. This method was used to confirm the results for MnSOD activity obtained with the DDC method in lung homogenates. The lung explants were also treated with sodium cyanide (2 mM) to determine the MnSOD activity. Because the volumes of these samples were extremely limited, this was the only method used to determine MnSOD activity in the explants.
Microtiter SOD assay. SOD activity was determined in a 100-µl assay mixture containing 1 mM acetylated cytochrome c (acetylation of cytochrome c decreases autoxidation of cytochrome c in solution), 1 mM xanthine, and 1 mM EDTA in 50 mM potassium phosphate buffer, pH 7.8, at 25°C. Xanthine oxidase was added to give a rate of reduction of cytochrome c of ~0.0075 absorbance units/min (±10%). The absorbance was measured at 550 nm with a Spectramax 340 microtiter plate reader (Molecular Devices, Sunnyvale, CA) with a 96-well plate (adapted from Ref. 35). SOD activities were calculated from assays in which there was an inhibition of cytochrome c reduction of between 40 and 50%. Although all samples initially were analyzed at the same volume, the volume of sample was then adjusted repeatedly until the level of inhibition fell within this range. Data presented all derive from assays in which these conditions were met. All samples were assayed until these conditions were met. No data were discarded. One unit of SOD activity was defined as the amount of SOD that gives 50% inhibition of the rate of cytochrome c reduction (34). An SOD standard curve was plotted with various dilutions of a Cu,ZnSOD standard at 40 McCord-Fridovich (34) units/ml. One unit in the microtiter plate assay was equivalent to 0.027 McCord-Fridovich unit/ml based on the standard curve. Each sample was run with a minimum of three to four replicates, taking an average of the wells. The total SOD (before DDC or cyanide treatment), MnSOD (after DDC or cyanide treatment), and Cu,ZnSOD activities (the difference between total SOD and MnSOD measurements) were determined.
Treatment of lung homogenates with DTT. Samples were incubated with DTT (2.5 mM) and EDTA (2 mM) in buffer (50 mM potassium phosphate with 1 mM EDTA) at 25°C for 30 min. Next, the samples were dialyzed extensively against a 3,000× volume of buffer that was changed three times to remove DTT (11). The DTT-treated samples were then assayed both for total SOD and MnSOD activities.
Western blot analyses of Cu,ZnSOD and MnSOD proteins. Whole lung tissue stored in liquid nitrogen was homogenized in buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml of leupeptin, and 20 µg/ml of aprotinin) followed by centrifugation at 14,000 g for 30 min. A Bradford protein assay (Bio-Rad) was performed with Coomassie blue and with bovine serum albumin as the standard to determine the protein concentration of the supernatant. For Cu,ZnSOD protein analysis, 15 µg of sample protein were loaded per lane in a 15% denaturing and reducing polyacrylamide minigel (Bio-Rad). Purified baboon Cu,ZnSOD protein was used as a standard. Protein was transferred from the gel onto a nitrocellulose filter (Amersham) as a solid support with a Transblot apparatus (Bio-Rad). Cu,ZnSOD proteins were visualized with a rabbit anti-baboon Cu,ZnSOD polyclonal antibody (a generous gift from Dr. Ling-Yi Chang, National Jewish Medical and Research Center, Denver CO) and enhanced chemiluminescence detection (Amersham) with an anti-rabbit-horseradish peroxidase conjugate (Pierce, Rockford, IL). When the samples for MnSOD protein were analyzed, 60 µg of sample protein were loaded per lane in a 12% denaturing and reducing polyacrylamide minigel (Bio-Rad). Human recombinant MnSOD protein (Boehringer Ingelheim) was used as a standard. MnSOD proteins were visualized with a rabbit anti-human MnSOD polyclonal antibody (a generous gift from Dr. Ling-Yi Chang) and enhanced chemiluminescence detection with Supersignal Ultradetection Reagent (Pierce) with an anti-rabbit-horseradish peroxidase conjugate (Pierce). Densitometry was performed with Image 1.61 software (National Institutes of Health) on the scanned blots.
Northern blot analysis for SOD mRNAs.
To isolate and preserve RNA, whole lung tissue was homogenized 25 mM
sodium citrate buffer containing 4 M guanidine isothiocyanate, 0.5%
sodium N-laurylsarcosine, and 0.1 M
-mercaptoethanol, rapidly frozen in liquid nitrogen, shipped
overnight, and stored at
70°C until further analysis (9).
RNA was purified by centrifugation through a cesium chloride gradient
(25 mM sodium citrate, pH 7.0, containing 0.1 M EDTA and 5.7 M cesium
chloride) and quantified by measuring its ultraviolet absorption
spectrophotometrically at 260 nm (36). 28S rRNA was used as an internal
standard to ensure uniformity of loading (4). The cDNA probes for human
Cu,ZnSOD and MnSOD were purchased from American Type Culture Collection
(Manassas, VA). Plasmids were amplified in Escherichia
coli and purified with a Qiagen (Chatsworth, CA)
plasmid preparation kit. The Cu,ZnSOD cDNA (560 bp, chromosome 21q22.1,
62 bp upstream from the Taq 1 site)
was isolated from the vectors by treatment with
Pst I and gel purified (38). The MnSOD
cDNA (976 bp, chromosome 6) was isolated from the vectors by treatment
with EcoR I and gel purified (23). The
probes were labeled with
[
-32P]CTP (ICN,
Irvine, CA) by random priming (RadPrime Kit, GIBCO, Grand Island, NY).
The RNA was separated under denaturing conditions by electrophoresis
with a 2.5 M formaldehyde-1% agarose gel, transferred to a Nytran
membrane (Magnagraph, Westboro, MA) by capillary action, and hybridized
with the labeled cDNA probes (36). The labeled membranes were placed in
storage phosphor cassettes (Molecular Dynamics, Sunnyvale, CA) until
adequate development of an image was noted with a Molecular Dynamics
phosphorimager. The images were quantified with ImageQuant software
version 3.35 (Molecular Dynamics).
Protein assay. The protein concentration in the lung homogenate was determined by the Coomassie blue dye binding method (6) with a Spectramax 340 microtiter plate reader (Molecular Devices) with a 96-well plate and bovine serum albumin, fraction V (Calbiochem), as a standard. Data analysis was done with Softmax Pro 1.2 software (Molecular Devices).
Clinical course. Clinical data including birth weight, gender, airway pressures, blood gas analysis, and vital signs were collected sequentially and analyzed on all 125- and 140-day-gestation baboons studied. All animals were ventilated with conventional PPV, with inspiratory times ranging from 0.4 to 0.6 s and respiratory rates from 20 to 40 breaths/min. Ten animals required high-frequency oscillatory ventilation at some point during their course because of worsening acidosis or pulmonary complications (1 pulmonary hemorrhage, 1 bilateral pneumothoraces). The newborns on PPV received peak pressures ranging from 18 to 35 cmH2O and peak end-expiratory pressures from 2 to 4 cmH2O. The mean airway pressure during the course of ventilation ranged from 5 to 15 cmH2O (overall mean 7 cmH2O). Two animals were bacteremic with Candida albicans during the course of the study. The inclusion of results from these animals in our analysis did not bias our results.
Statistics. Throughout the studies,
data (enzyme activities, mRNAs, and protein measurements) from newborns
exposed to oxygen for 6 and 10 days were compared. Within each level of
oxygen treatment (PRN or 100% oxygen) and each gestational age, there
were no differences between 6- and 10-day-exposed animals. Therefore,
data from animals exposed for 6 and 10 days were pooled throughout.
Comparisons of response variables between levels of oxygenation were
made within gestational age groups by one-way analysis of variance or
independent sample t-tests depending
on the number of levels of oxygenation. Multiple comparisons between
individual levels were made with Fisher's protected least significant
difference procedure. Data within groups were summarized with means ± SE. The mRNA data at 140 days gestation were log transformed to
meet the analytic assumption of normality, and these data are reported as geometric means and 95% confidence intervals. All tests were two
tailed, and an level of 0.05 was used as the standard for assessing
significance. All statistical analyses were performed with JMP Version
3.1 software (SAS Institute) running on a PowerPC computer (Macintosh).
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RESULTS |
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Effect of gestation on SOD activities in the developing baboon lung. Lung Cu,ZnSOD specific activity decreased in the late-gestation fetal lung (175 days) compared with earlier times in gestation (Fig. 1A). In contrast, lung MnSOD activity was lowest during early gestation (125 and 140 days) and tended to increase throughout the remainder of the third trimester (Fig. 1B).
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Effect of oxygen exposure on lung SOD activities in the 140-day-gestation newborn baboon. In the 140-day-gestation newborn at age 6-10 days, lung total SOD and Cu,ZnSOD specific activities (Fig. 2, A and B, respectively) were lower in the 100% and PRN oxygen exposure groups compared with 140-day fetal control animals (P < 0.0001). However, there was no significant difference in lung MnSOD activity among any of the groups studied (P = 0.2; Fig. 2C).
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Effect of oxygen exposure on lung SOD activities in the 125-day-gestation newborn baboon. Lungs of the 125-day-gestation (6-10 day old) PRN oxygen-treated baboon had lower total SOD and Cu,ZnSOD specific activities (Fig. 3, A and B, respectively) compared with 125-day fetal control animals (P < 0.05). No difference in lung MnSOD activity between the PRN oxygen exposure group and 125-day fetal control animals was found (P = 0.5; Fig. 3C).
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Effect of DTT on lung SOD activities in the 140-day gestation newborn baboon. No significant difference was found in Cu,ZnSOD or MnSOD specific activity in the lung homogenates of any of the groups of 140-day-gestation baboons when compared before and after DTT treatment. Neither was there a significant difference observed in Cu,ZnSOD or MnSOD specific activity measured before and after treatment of the lung homogenates with DTT when the results from all lungs were analyzed together.
Effect of gestation and oxygen exposure on Cu,ZnSOD and MnSOD protein expression. No difference occurred in lung expression of Cu,ZnSOD protein as detected by Western blot analysis during the final third of gestation (125-175 days; P = 0.7; Fig. 4A). In addition, there was no significant change in expression of Cu,ZnSOD protein in the lung after ventilation with PRN or 100% O2 for 6-10 days in 140-day-gestation newborns (P = 0.2; Fig. 4B). Nonetheless, there was considerable variation in the level of Cu,ZnSOD protein expressed in these animals after 100% oxygen exposure, and some individuals had low values. By contrast, lung Cu,ZnSOD protein decreased by almost one-half in newborns of 125 days gestation exposed to only PRN oxygen (P < 0.0001; Fig. 4C). Densitometric quantitation of the results of the Western blot analyses is provided (Fig. 4, D-F).
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Fetal lung expression of MnSOD protein was not detected at 140 days gestation as measured by Western blot (Fig. 5A). However, MnSOD protein became detectable after exposure to PRN or 100% oxygen, with the greatest and most consistent expression of the protein in the 100% oxygen-exposed group (Fig. 5A). Densitometric quantitation of the results of the Western blot analysis for MnSOD protein at 140 days gestation is provided (Fig. 5B). As seen at 140 days, lung MnSOD protein was not detectable in 125-day fetal lungs. However, it remained undetectable in the lungs of newborns at this gestation even after PRN oxygen exposure for 6-10 days (data not shown).
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Effect of gestation and oxygen exposure on lung Cu,ZnSOD and MnSOD mRNA expression. Throughout these studies, a single Cu,ZnSOD transcript was expressed in baboon distal lung. For MnSOD, there were two primary transcripts (4 and 1 kb) expressed in parallel in all studies. Densitometric measures of the intensity of the 4-kb band are reported, although densitometry from the 1-kb band was proportionately equivalent.
No difference in lung Cu,ZnSOD or MnSOD mRNA expression was observed between 140- and 160-day-gestation animals (P = 0.8; Table 1).
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At 140 days gestation, lung MnSOD mRNA expression increased after 6-10 days of PRN or 100% oxygen exposure compared with 140-day fetal control animals (P < 0.05; Table 1). Lung MnSOD mRNA also increased after 6-10 days of 100% oxygen exposure relative to 160-day fetal control animals (P < 0.05; Table 1). Lung Cu,ZnSOD mRNA was not significantly changed after either PRN or 100% oxygen exposure in 140-day-gestation newborns relative to either fetal control group (P = 0.3; Table 1). All mRNA values were normalized to 28S rRNA as a standard.
At 125 days gestation, lung Cu,ZnSOD mRNA expression increased after 6-10 days of PRN oxygen exposure relative to 125-day fetal control animals (P = 0.0007; Table 2). Lung MnSOD mRNA expression also increased after PRN oxygen exposure relative to fetal control animals (P < 0.05; Table 2). Again, mRNA values were normalized to 28S rRNA.
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Effect of oxygen exposure on lung SOD activities in fetal lung explant cultures. To examine the intrinsic effects of acute oxygen exposure on premature fetal lung tissue, a fetal lung explant exposure system was used. Total SOD and MnSOD activities decreased in 140-day fetal explants after 95% oxygen exposure for 16 h relative to 1% oxygen exposure (P < 0.02; Fig. 6), with a comparable trend in Cu,ZnSOD activity (P = 0.06). In lung explants from very premature animals (125 days), total SOD and Cu,ZnSOD activities decreased after 21 or 95% oxygen exposure relative to 1% oxygen exposure (P = 0.01; Fig. 7). MnSOD activity was low in 125-day fetal explants and was not different in 1, 21, or 95% oxygen. No difference was observed between activities measured after 21 and 95% oxygen exposures at either 140 or 125 days gestation in fetal explant tissue (P > 0.05). Taken together, these data indicate that oxygen tension by itself exerts effects on SOD activities in fetal lung tissue in vitro.
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Clinical data. In the 140-day-gestation 100% oxygen group. The PaO2 ranged from 98 to 420 mmHg at age 6- 10 days. At this age, the mean FIO2 in the 140-day-gestation PRN oxygen group was 0.33, with a PaO2 ranging from 58 to 84 mmHg. By contrast, the mean FIO2 in the 125-day-gestation PRN oxygen group was 0.42, with a PaO2 ranging from 41 to 86 mmHg. A trend was observed for a decrease in lung total SOD specific activity, with an increasing alveolar-arterial oxygen gradient in all 140-day-gestation animals studied (P < 0.05; r = 0.54). No gender effect on Cu,ZnSOD or MnSOD specific activity was observed between oxygen exposure groups at either 125 or 140 days gestation (data not shown).
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DISCUSSION |
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Fetal expression of lung SOD activities. We initially addressed the developmental profile of lung SOD activities in the late-gestation primate. First, the developmental profile of lung total SOD and Cu,ZnSOD specific activities was evaluated. In contrast to some previous studies in rodents and ruminants that described an increase in total SOD (19, 41) and Cu,ZnSOD (44) activities during late gestation, we found lung total SOD and Cu,ZnSOD specific activities decreased in the late-gestation fetal primate lung (175 days, term 184 days) compared with earlier time points in gestation (125 or 140 days). This is especially interesting given that the level of immunodetectable Cu,ZnSOD protein did not change throughout the final third of gestation, and it suggests that additional factors may modify the activity of the Cu,ZnSOD protein after synthesis in vivo. Our findings are compatible with those of others (8, 22, 44), who also found a declining pattern of lung Cu,ZnSOD activity in the late-gestation fetal rat and lamb.
Second, a trend for MnSOD specific activity to increase from 125 through 175 days gestation in the premature baboon model was observed. Our findings are in agreement with several animal models describing the effect of fetal maturation on the development of different antioxidant enzyme activites (41, 44). Tanswell and Freeman (41) found that lung MnSOD activity increased during late gestation (from 4 days before delivery until term) in fetal rats. In addition, Chen and Frank (8) found a nearly twofold increase in MnSOD activity in rat lungs at birth (between fetal day 22 to postnatal day 1). Based on the gestational patterns seen in lung CuZn and MnSOD activities in our model, we speculate that the trend for increasing MnSOD enzyme activity during late gestation emphasizes the relatively greater importance of mitochondrial respiration in the newborn animal than in the late-gestation fetus (1, 43).
Cu,ZnSOD expression in lungs of premature newborns. Our second question addressed the effects of hyperoxia on expression of SOD activities in the premature primate model of BPD. In marked contrast to the term newborn rat, in sick premature newborn baboons, total SOD and Cu,ZnSOD activities decreased in lung homogenates of 140-day-gestation baboons exposed to PRN or 100% oxygen compared with fetal control animals (P < 0.0001; Fig. 2, A and B, respectively). This greatly exceeded the minor, nonsignificant decline in Cu,ZnSOD specific activity noted between gestational ages 140 and 160 days. We noted a large variance in the data in the 100% oxygen exposure group, possibly related to the genetic diversity within the baboon population or to the heterogeneity of disease severity within this group. In the very premature animal (125 days), total SOD and Cu,ZnSOD activities decreased after only PRN oxygen exposure compared with fetal control animals. Thus lung total SOD and Cu,ZnSOD activities decreased in premature primates ventilated with supplemental oxygen at either of the two gestational ages.
To better define the level at which Cu,ZnSOD activity was downregulated in premature primates exposed to hyperoxia, we also measured the expression of Cu,ZnSOD mRNA and protein. The consistent expression of Cu,ZnSOD mRNA in fetal (140 and 160 days) PRN and 100% oxygen-exposed newborns indicates that the decreased lung Cu,ZnSOD activity expressed in these premature newborns occurs at a level other than that of mRNA expression. In 100% oxygen, Cu,ZnSOD protein expression was inconsistent, suggesting that variable mRNA translation or protein degradation may have contributed to diminished activity in some animals. However, in the 140-day PRN oxygen-exposed animals, lung Cu,ZnSOD activitiy was also decreased despite the fact that protein expression was not decreased relative to fetal control animals. This indicates that posttranslational factors must play a role in decreasing lung Cu,ZnSOD activity in this group.
In the case of the 125-day premature primate, Cu,ZnSOD mRNA increased in response to oxygen. Such increases in mRNA could be due to increased transcriptional rates and/or enhanced mRNA stability. In contrast to the 140-day model, Cu,ZnSOD protein expression was consistently decreased by exposure even to PRN oxygen in 125-day newborns. This occurred despite constant Cu,ZnSOD protein expression throughout this period in fetal life. These data indicate that impaired translation of the mRNA and/or increased degradation of the protein may contribute to the decline in Cu,ZnSOD activity in this more premature model.
Our findings are in relative agreement with those of Sosenko et al. (40), who found that premature rabbits exposed briefly to hyperoxia (48 h) had increased lung Cu,ZnSOD mRNA without a change in activity. In a recent human study (2), lung Cu,ZnSOD mRNA increased during late gestation, whereas Cu,ZnSOD specific activity did not increase during late gestation or the postnatal period. In addition, support for the decrease in lung Cu,ZnSOD activity found in our study after hyperoxic exposure was found in a study by Dobashi et al. (17) in the premature human. In this study, immunohistochemistry was performed in lungs of newborns who died from respiratory distress syndrome (RDS) or BPD, and these lungs were compared with the fetal lung. Although a relatively small number of cases were studied, positive staining for Cu,ZnSOD was markedly decreased in terminal and respiratory bronchioles of infants with RDS or BPD compared with gestational age-matched control fetuses. This decreased staining for Cu,ZnSOD observed in the lungs of human infants with RDS and subsequent BPD is consistent with the decreased lung Cu,ZnSOD activity we observed in premature baboons developing BPD.
MnSOD expression in lungs of premature newborns. At the mRNA level, we found that the lung MnSOD message increased after 100% and PRN oxygen exposures compared with fetal control animals at 140 days gestation, a pattern similar to what was observed for MnSOD protein. Together, these findings indicate that the reason we observed no difference in lung MnSOD activity was due to posttranslational modification of the protein. Because both MnSOD activity and protein were normalized to lung protein, it was apparent that activity did not increase concomitantly with MnSOD protein at this gestation. In contrast to the 140-day model, the amount of MnSOD protein in the 125-day newborn lung remained undetectable during PRN oxygen exposure despite measurable and increasing MnSOD mRNA at this time. This suggests that effects occurring after expression of mRNA also could contribute to the failure to increase lung MnSOD in response to oxygen at this gestation. It is quite possible that impaired translation of the MnSOD protein contributed to the decrease in activity expressed, especially in the 125-day model. Such a mechanism has been identified in an adult animal model (12).
In human newborns at 25 wk gestation, a developmental stage comparable to the 125-day baboon, the intensity of MnSOD staining decreased in the lungs of infants dying acutely from RDS. However, the intensity of MnSOD staining increased in the lungs of infants exposed to oxygen who survived and subsequently developed BPD and was associated with an increased number of type II alveolar cells (17). In another human study (2), lung MnSOD mRNA and immunoreactive protein increased in late gestation, whereas MnSOD specific activity did not increase, indicating that regulation can be complex in both pre- and postnatal periods. The findings from these studies in very premature human fetuses and newborns tend to parallel our findings in both the in vitro and in vivo models.
In the premature baboon (14), other investigators (13) found no difference in lung MnSOD mRNA concentrations between 140-day 100% oxygen-exposed animals who developed BPD and control animals. However, the MnSOD protein content was significantly increased in the lungs of those BPD animals compared with PRN oxygen-exposed and fetal control animals (13). In that investigation, the total study was 16 days, with 100% oxygen for the first 11 days. Thereafter, there was a period of recovery during which oxygen therapy was tapered to a minimum level (PRN oxygen exposure) on days 11-16. Those conditions were significantly different from the ones employed in this study. Here, we sought to determine whether a change in biologically active MnSOD protein occurred during a relatively briefer exposure period (6-10 days). There was no period of recovery from 100% oxygen before death in our study. After 140 days gestation, we found no difference in lung MnSOD specific activity among the 100% oxygen-exposed group, the PRN oxygen-exposed group, and the fetal control group. We also found no difference in lung MnSOD specific activity between the PRN oxygen-exposed group and the fetal control group at 125 days gestation. This occurred despite the fact that fetal lung MnSOD specific activity showed an increasing trend from 125 days through term gestation. Our findings show relative agreement with those previously obtained in premature rabbits (40). In that investigation, brief exposure to hyperoxia (48 h) tended to increase MnSOD mRNA, whereas a greater, although nonsignificant, decline in activity was observed. Overall, those findings and ours raise the possibility of MnSOD inactivation in vivo.
Potential mechanisms for failure to increase MnSOD activity. Prior investigations have shown a decrease in lung MnSOD protein and/or activity after exposure of adult rats to >95% oxygen (11, 31, 42). Because DTT could selectively increase lung MnSOD activity in the lung homogenates of such oxygen-exposed rats (11), we sought to determine whether this reducing agent could increase the MnSOD activity of lung homogenates in 140-day newborn baboons after hyperoxic exposure. However, no such increase in activity was observed in our study. These results could indicate that oxidized MnSOD was further degraded by proteolysis, as may occur with other oxidized proteins. Nonetheless, increased immunodetectable MnSOD protein was present in the lungs of hyperoxia-exposed newborns at this gestation, suggesting that if MnSOD proteolysis occurred, it was incomplete. Alternatively, another biochemical process besides reversible oxidation and/or proteolysis could be responsible. One mechanism that could cause such a loss of activity is nitration of the protein by peroxynitrite. Such nitration and inactivation of MnSOD recently was demonstrated in rejected transplanted human kidney tissues (33). Because increased nitrotyrosine residues have already been detected in hyperoxic lung injury (21), we speculate that peroxynitrite production and action on MnSOD could contribute to the lack of increase in its activity. A very recent study (12) indicates that both hyperoxia-exposed adult rat and adult baboon lungs have decreased MnSOD activity relative to air-exposed lungs. This mechanistic study in the rat indicated that this decline was due to both translational and posttranslational effects.
Effects of oxygen in fetal lung explant cultures. To isolate the effect of oxygen from potential postnatal factors that could have contributed to our in vivo findings, such as mechanical ventilation, nutritional deficits, microbial colonization, infection, and inflammation (15), we examined the acute (16-h) effects of oxygen in a distal fetal lung explant culture system. In general, our findings parallel those in lungs obtained in vivo. However, there were some important differences that could have been related to in vitro versus in vivo systems and/or to the shorter exposure duration in the former. For example, at 140 days gestation, 95%, but not 21%, oxygen caused a decline in total SOD and MnSOD activities in the explants relative to 1% oxygen, with a comparable trend in Cu,ZnSOD activity (P = 0.06). However, in vivo studies indicated a decline in Cu,ZnSOD activity without significant decreases in MnSOD activity at this gestation. Explants from 125-day animals exposed to either 21 or 95% oxygen had less total SOD and Cu,ZnSOD activities than did those exposed to 1% oxygen, indicating a greater sensitivity of Cu,ZnSOD expression to the effects of oxygen at this early gestation. In the 125-day fetal lung explants, MnSOD activity were uniformly low in 1, 21, or 95% oxygen. One weakness of our studies is the lack of available lungs from animals exposed only briefly to hyperoxia. Hence, we do not know the early time course of potential changes in lung SOD activity in vivo. Although caution is required in extrapolating in vitro findings to the in vivo situation, these findings, taken together, suggest that oxygen could cause or significantly contribute to the postnatal failure to increase lung SOD activity observed in premature primates.
Conclusion and significance. Although these studies do indicate the level(s) at which expression of lung SODs decrease or fail to increase, the mechanisms responsible are not fully defined. In the lungs of the most premature (125-day) animals, all of whom develop BPD, factors other than mRNA expression must account for the majority of the decline in Cu,ZnSOD activity. In less-premature animals (140 day), expression of Cu,ZnSOD protein was impaired inconsistently and then only in 100% oxygen-exposed animals that also develop BPD. Because activity decreased while protein was not significantly less, posttranslational factors are implicated in the oxygen-related decline in Cu,ZnSOD activity at this gestation.
In contrast to Cu,ZnSOD, in the less-premature animals, MnSOD mRNA and protein increased substantially with oxygen exposure, although activity did not change. This indicates that a posttranslational modification of the protein may occur. Because the activity could not be increased with DTT, reversible oxidation does not appear to account for the activity lost. In the most premature newborns in our study, MnSOD protein still could not be detected despite considerably increased mRNA expression in oxygen. Therefore, impaired translation also could contribute to the failure to increase activity at this gestation.
The significance of decreased SOD activities in hyperoxia might seem uncertain based on recent studies in transgenic mice. For example, Ho et al. (26) did not find increased sensitivity to hyperoxic lung injury in mice lacking Cu,ZnSOD. By contrast, mice lacking MnSOD have a limited survival even in air, although their demise appears to be related to failure of the cardiovascular, hematologic, and central nervous systems (30, 32). On the other hand, transgenic mice with increased expression of MnSOD have increased survival in hyperoxia in a manner that appears to be related to the specific activity of the MnSOD protein expressed by the transgene (25, 46). Hence the ability to increase MnSOD activity in response to hyperoxia may potentiate adaptation. Because premature newborns have deficits in multiple antioxidants (18, 19, 39-41, 44, 45, 47), the significance of the diminished expression of lung SOD activities in their lungs may be greater in this context. Thus the premature primate cannot increase SOD enzyme activities in response to oxygen in the same manner as observed in lesser mammals after term or near-term gestation and hence may be more susceptible to acute lung injury. Because limitations in expression of these activities appear to exist in events subsequent to mRNA expression, the efficacy of gene therapy approaches to augment these activities may be limited.
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ACKNOWLEDGEMENTS |
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We acknowledge the superb, dedicated assistance of the numerous physicians, nurses, scientists, technicians, and support personnel at the Bronchopulmonary Dysplasia (BPD) Resource Center located at the Southwest Foundation for Biomedical Research and the University of Texas Health Sciences Center (San Antonio, TX). We acknowledge the helpful suggestions of Drs. John Shannon and Carole Mendelson regarding lung explant culture, the advice and generous provision of antibodies by Dr. Ling-Yi Chang, the helpful comments of Dr. Corrie Allen, and the assistance of Emily Cassidy in preparing the manuscript.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-53636 (to the BPD Resource Center), HL-56263, HL-52732, HL-57144, and HL-07670.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: C. W. White, National Jewish Medical and Research Center, J101, 1400 Jackson St., Denver, CO 80206.
Received 14 May 1998; accepted in final form 29 September 1998.
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