Biochemical
and Molecular Roles of Nutrients
Postnatal Selenium Repletion Protects Neonatal Rats from Hyperoxia1'2
Lungs of
Division of Nutritional Sciences and Department of Veterinary Pathobiology, University of Illinois, Urbana, IL 61801 and *Department of Nutrition, Pennsylvania State University, University Park, PA 16802 cantly reduced in pups Se deficient during gestation and early postnatal life compared with pups that are Se adequate during gestation and the first 5 d of life. However, specific histomorphometric indices of lung function in neonatal rat pups that might be affected by Se nutriture have not been investigated. The impact of maternal Se deficiency on low-birthweight (LBW)4 human infants remains to be fully evaluated. Significantly decreased plasma Se concen trations in LBW infants have been documented in recent clinical reports (Amin et al. 1980, Lockitch et al. 1989, VanCaillie-Bertrand et al. 1984), although the exact significance of these findings is not clear. Enterai feeding is often delayed in LBW infants due to the multiple complex problems associated with the short gestational period, including immaturity of organ systems and respiratory distress syndrome. Be cause neither intravenous fluids nor parenteral for mulas contain added Se, LBW infants have dramati cally decreased plasma Se concentrations during first few weeks of life (Huston et al. 1982 and 1987). This, in turn, may accelerate or exacerbate clinical problems associated with the high oxygen environ ments used to assist ventilation in these LBW infants. It has been noted that children receiving long-term total parenteral nutrition have myocardiopathy, pseudoalbinism and macrocytosis that are alleviated by Se therapy (Vinton et al. 1987). Whether a short period of Se repletion in Se-deficient neonates also can protect them from chronic lung disease that may result from
ABSTRACT We reported previously that Se-adequate neonatal rat pups born to Se-adequate dams were resistant to lung damage by hyperoxia. To assess whether early postnatal Se repletion could also protect developing pups reared under hyperoxia, female Sprague-Dawley rats (n - 20) were bred and fed a Sedeficient (0.04 ug/g) diet during pregnancy. On d 1 postpartum, dams were divided into two groups and fed either a Se-deficient diet or a Se-repleted (0.5 ug/g) diet. On d 4 postpartum, litters in each group were randomly assigned to either air or high oxygen (>95% 03) environments. Histologie evaluation of lungs from d8 pups indicated that Se repletion significantly reduced the incidence of lung lesions caused by hyperoxia. Selenium-repleted pups also had significantly greater lung volumes and internal surface areas. The 7-d period of Se repletion resulted in significantly elevated maternal milk Se concentrations compared with a Se-deficient group, which was reflected in the pups by elevated plasma and hepatic Se concentrations and Se-dependent glutathione peroxidase (SeGPx) activities. Pul monary glutathione concentration and SeGPx activity in pups were affected by oxygen exposure only, not by Se nutrition. Therefore, early postnatal Se repletion can protect the developing lung from oxygen-induced injury, a protection that is not entirely due to the effects of Se on pulmonary SeGPx activity and glutathione concen tration. J. Nutr. 122: 1760-1767, 1992. INDEXING KEY WORDS:
•selenium repletion •oxygen toxtcity •lung injury •glutathione •rats
Selenium-deficient newborn rat pups are much less tolerant of high oxygen exposure than Se-adequate pups. Selenium-deficient newborn pups exposed to >95% C>2for 4 d have higher incidences of pulmonary septal attenuation and interstitial inflammation than Se-adequate pups (Kim et al. 1991). Lung development in rat pups also seems to be affected by the Se nutriture of the dam, i.e., mean lung weight is signifi 0022-3166/92
$3.00 ©1992 American Institute
of Nutrition.
'Presented
in part at the 75th Annual Meeting of the Federation
of American Societies for Experimental Biology, April 1991, At lanta, GA [Kirn, H. Y., Picciano, M. F. & Wallig, M. A. (1991) Selenium repletion protects neonatal rat lungs from hyperoxia. FASEB I. 5: A1072 (abs. 4071)]. Supported in part by NICHHD Grant No. 18689. 3To whom correspondence should be addressed. 4Abbreviations used: GSH, glutathione; LBW, low birth weight; SeGPx, selenium-dependent glutathione peroxidase.
Received 26 February 1992. Accepted 29 April 1992. 1760
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HYE YOUNG KIM, MARY F. PICCIANO* AND MATTHEW A. WALLIG3
SELENIUM AND PROTECTION
1761
TABLE 1 Diet composition
Ingredient
Amount
Casein1 Sucrose Cornstarch Com oil Cellulose2 Vitamin mixture3 Se-free AIN-76 mineral mixture4 DL-Methionine Choline bitartrate
g/kg 200 300 331 100 20 10 35 2 2
Vitamin-free test casein (Teklad, Madison, WI). 2Alphacel (ICN Nutritional Biochemical, Cleveland, OH). 3AIN-76 vitamin mixture (AIN 1977). 4AIN-76 mineral mixture prepared without Se (AIN 1977).
MATERIALS AND METHODS Animal care. Nulliparous female Sprague-Dawley rats (HarÃ-anIndustries, Indianapolis, IN), weighing 180 to 200 g, were housed in individual, suspended, stainless steel wire-mesh cages in a room with con trolled temperature (20 to 22°C)and lighting (12-h light:dark cycle). The animals were fed a commercial ration (Purina Rodent Chow, Ralston Purina, St. Louis, MO) for a 2-wk adaptation period. At 200 to 240 g, rats were mated, and d 1 of pregnancy was determined by the presence of vaginal plugs and sperm. On d 1 of pregnancy, all rats were assigned to a semipurified, Se-deficient diet. Rats were given free access to demineralized water (Nanopure, Barnstead, Boston, MA) and experimental diets. The Animal Care and Use Committees of both the Pennsylvania State University and the University of Illinois re viewed and approved the use of experimental animals in these studies. Diets. Diets (Table 1) were formulated to contain all nutrients in quantities adequate for reproduction (NRC 1978) except for Se in the Se-deficient diet. Direct analysis of diets by gas chromatography coupled with electron capture detection was used to confirm dietary Se concentration of 0.04 ug/g (Sedeficient) and 0.5 ug/g (Se-adequate). Experimental design. Twenty female rats were bred and fed the Se-deficient (0.04 ug/g) diet during pregnancy. On d 1 postpartum, dams were divided into two groups and either continued to receive a Sedeficient diet or were fed a new sodium selenitesupplemented (+Se, 0.5 ug/g) diet. Food dishes were positioned so that only dams had access to food, and maternal milk was the only nourishment for pups. The day after parturition (d 2), litters were culled so that there were 10 pups per dam. On d 4, two litters
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high oxygen exposure has not been investigated in animals or humans. The mechanism by which Se deficiency results in increased susceptibility to damage from high oxygen exposure is not known. The protective role of Se against oxygen exposure is, however, not due only to its role as an essential component of the key antioxidant enzyme, glutathione peroxidase (Cross et al. 1977, Forman et al. 1983, Kim et al. 1991). Both marginally Se-deficient and Se-adequate pups have equally enhanced Se-dependent glutathione perox idase (SeGPx, EC 1.11.1.9) activities after oxygen ex posure, but only Se-adequate pups are protected from lung damage. Pulmonary lesions and mortality due to paraquat and diquat poisoning resemble those in duced by high oxygen and are believed to be produced by toxic oxygen intermediates. Selenium deficiency has been found to exacerbate paraquat-induced lung injury in rats (Omaye et al. 1978). In addition, Burk and co-workers (1980) found that the lesions induced by diquat in Se-deficient rats can be reversed by in jection of Se. This protection afforded by Se, however, also could not be related to its role in SeGPx function, and Burk and co-workers (1980) suggested the exis tence of another Se-dependent factor, apart from SeGPx, that protects against cellular peroxidation during oxidant stress. Glutathione (GSH) is another important com ponent of the antioxidant defense system in mammals. Its mechanism of protection against ox idant stress is thought to be related to its ability to quench free radical products (Meister and Anderson 1983). Glutathione content is increased significantly in lung tissue during hyperoxia in animals (Jenkinson et al. 1988). It is reported that intraperitoneal glutathione administration can protect rats from hyperbaric oxygen toxicity, without producing any changes in SeGPx activities (Weber et al. 1990). Di etary selenium has been shown to affect glutathione metabolism in rat liver (Chung and Maines 1981, Hill and Burk 1982, LeBoeuf et al. 1985). However, whether Se has an effect on GSH content in rat lungs under hyperoxia is not known. The present study was designed to determine whether Se repletion during the early postnatal period can, have a positive effect on lung development in neonatal rats and protect the developing rat lung against oxygen-induced injury. We also sought to identify a mechanism by which Se repletion may protect neonatal lung from high oxygen damage. Spec ifically, we assessed histopathological changes using histomorphometry in neonatal lungs after oxygen ex posure in both Se-deficient and Se-repleted rat pups. We also analyzed glutathione content in the neonatal lung to determine whether any alterations in content could be related to damage and/or protection from hyperoxia.
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KIM ET AL.
oxygen was continuous except for a brief period daily, when chambers were opened for rotation of dams between oxygen- and air-housed litters. This rotation was done to prevent oxygen-induced illness in the dams. In a previous study, we showed that no dams had pulmonary lesions by this procedure. On d 8 of lactation, dams and pups were separated for 2 h to allow for accumulation of milk. Dams were anesthe tized with ketamine HCl (10 mg/100 g body wt) containing acepromazine (l mg/100 g body wt) and injected intraperitoneally with oxytocin (0.25 U/100 g body wt) to facilitate milking. Milk was collected with a suction apparatus in which milk flowed through polyethylene capillary tubing attached to a test tube kept in an ice bath. Pups were weighed and killed at d 8 of age by decapitation, heparinized blood was collected, and perfused lungs and liver were ex cised. Analytical procedures. Four pups in each litter were used for histopathology and histomorphometry. Lungs were fixed via intratracheal instillation of 10% buffered formalin at an inflation pressure of 22 cm of H2Û and kept in the same buffer solution. At least 3 d after fixation, total lung volume was measured by water displacement. Three standard cross-sections from each lung (right and left) were examined. These sections were taken from the same area of apical, middle and diaphragmatic portions of each lung. Sec tions were dehydrated in graded alcohols, embedded in paraffin, sectioned at 5 urn and stained with hematoxylin and eosin. Sections were examined at low (lOx) and high (40x) magnification by light mi croscopy to determine the presence of either of two lesion patterns: septal attenuation with enlarged al veolar spaces and interstitial inflammation. Each section was evaluated twice in a double-blind manner and the average was used as a final score. Each lesion received a score ranging from 0 (no lesion) to 3 (severe lesion). For septal attenuation, a score of 1 (mild lesion) was recorded where there were only mild, occasional foci of septal bud stunting and alveolar enlargement, score 2 (moderate lesion) when there was multiple diffusely scattered but still mild septal bud stunting, and score 3 (severe lesion) when there were many, coalescing foci of moderate septal stunting and alveolar enlargement. For inflammatory lesions, a score of 1 (mild lesion) was recorded if there were occasional, small foci of interstitial inflam mation occupying 10% but 50% of the alveolar tissue in a section. For histomorphometry, a standard integrating eye piece was used. The number of bars that intersected lung tissue per field and the number of times the lines were crossed by tissue septa per field were counted. Mean air space size (Lm) was calculated using the following formula (Thurlbeck et al. 1981): Lm = length of line x number of lines x number number of tissue intercepts
of fields
Mean internal surface area (ISA) was calculated the following formula (Weibel 1963): ISA = 4 x volume
using
of lung parenchyma Lm
and mean percent Percent
air space by: air space = Pa/(Pa + Pt),
where Pa is the number of intercept bars hitting air and Pt is the number of intercept bars hitting tissue. The remaining six pups in each litter were used for biochemical assays. Three pups from the same litter were pooled and used as one sample to achieve enough sample for biochemical evaluation. Whole blood was centrifuged (800 x g) and plasma was col lected. Lungs used for biochemical analysis were first carefully perfused through the pulmonary artery with ice-cold isotonic buffer (0.1 mol/L potassium phos phate, 0.15 mol/L KCl, pH 7.4). Lung and liver were surgically removed, rinsed and blotted dry. An aliquot of fresh lung tissue was homogenized in ice-cold iso tonic KCl buffer, and glutathione content was mea sured using the GSH S-transferase linked method of Asaoka and Takahashi (1981). Remaining tissue samples were stored at -70"C until subsequent bio chemical assays were performed. Selenium concentrations of diet, lung, plasma and liver were determined according to the method of McCarthy et al. (1981) using a gas Chromatograph equipped with an electron capture detector (HewlettPackard 5710A, Avondale, PA) and a 0.53-mm i.d. fused silica capillary column (Supelco, Belief onte, PA). The activities of SeGPx were determined in blood and tissue homogenates by the coupled assay of Paglia and Valentine (1967) as modified by Levander et al. (1983). Hydrogen peroxide was used as the sub strate in this assay. Protein content of tissues and blood was determined by a modified Lowry method (Lowry et al. 1951). Statistics. Biochemical data and histomorphometrical data were evaluated using ANO VA (2x2
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from dams fed the same diet were mixed, with half of the pooled litter assigned to an air environment and the other half to an oxygen environment. The two matched dams (cross-fostering dams) were then ro tated between their air- and oxygen-housed litters for the next 4 d. In the latter environment, which was set up to mimic clinical ventilators for LEW infants, exposure to oxygen was conducted with careful moni toring of oxygen concentration (>95%), temperature (22-25°C), and humidity (55-75%). The exposure to
SELENIUM AND PROTECTION
FROM HYPEROXIA
1763
TABLE 2 Severity scores of lung interstitial
inflammation
and septa! attenuation from Se-deficient oxygen environment for 4 d1
and Se-repleted
Air
Oxygen
-Se
+Se
-Se
0.175 ±0.063 0.387 ±0.121 0.562 ±0.140
0.031 ±0.031 0.219 ±0.091 0.250 ±0.102
0.375 ±0.113 0.825 ±0.163 1.200 ±0.248
0.188 ±0.101 0.547 ±0.141 0.734 ±0.228
'Values are means ±SEMfor 16 to 20 pups. Each lung tissue received a score ranging from 0 (no lesion) to 3 (severe lesion) in a double-blind evaluation. Data analyzed by 2 x 2 factorial ANOVA (P < 0.05): "significant effect of dietary selenium; Significant effect of oxygen environment.
factorial), followed by least significant difference tests (Steel and Torrie 1980). Data for incidence of lung injury were assessed by Fisher exact probability test (SAS 1985). The value of P < 0.05 was chosen as the level of statistical significance. RESULTS Maternal food intake, body weights and tissue Se concentrations. Throughout the study, no significant differences in mean food intake and body weight of dams were observed between groups. The mean food intake of all dams was 17.1 ±1.0 g/d. The mean body weight of dams was 324.7 ±9.8 g at the end of pregnancy and 230.7 ±10.8 g at d 8 of lactation. Feeding the Se-supplemented diet to marginally Sedeficient dams resulted in a significant increase in Se concentrations in liver and milk. Mean concentra tions of Se in milk from Se-deficient and Se-repleted dams were 0.63 ±0.05 and 1.03 ±0.03 nmol/L, respec tively; in liver the concentrations were 4.75 ±0.54 and 7.58 ±0.64 nmol/g, respectively. Histopathology and bistomorphontetry of pups. Selenium repletion via the dam significantly reduced the total pulmonary damage (P < 0.05) and tended to decrease the severity of inflammation and septal at tenuation (P < 0.10) (Table 2). Seventy-five percent of oxygen-exposed, Se-deficient pups had lung lesions, whereas only 43.8% of oxygen-exposed, Se-repleted pups had lesions (Fig. 1). Typical microscopic charac teristics of lungs from the experimental rat pups are shown in Figures 2 and 3. The alterations associated with septal attenuation included stunting of septal buds, a decrease in the number of alveolar septa, thinning of remaining septal buds and enlarged al veolar spaces (Fig. 3A). In lungs with interstitial in flammation (Fig. 3B), there were multiple foci of thickening and hypercellularity of alveolar septa, mainly due to infiltration by macrophages and some neutrophils.
Analysis of histomorphometric data showed that Se repletion significantly enhanced lung volume (P < 0.001) and internal surface area (P < 0.05), whereas oxygen exposure significantly decreased the values for these measures regardless of Se status. Mean air space size and percent air space of the pups were not af fected by either Se repletion or oxygen exposure (Table 3). Pup body and organ weights, Se concentrations, SeGPx activities and GSH content in organs and plasma. Pup body weights were similar among groups throughout the study. Mean body weights for all pups at d 2 and at the beginning (d 4) and end (d 8) of oxygen exposure were 5.94 ±0.17, 8.20 ±0.17 and 12.20 ±0.33, respectively. Organ weights were similar on d 8 among treatment groups. The mean lung and liver weights of pups were 0.23 ±0.01 and 0.40 ±0.02 g, respectively.
Air
Oxygen
Environment FIGURE 1 Percent incidence of lung injury from selenium-deficient (Se-) or selenium-repleted (Se+) pups reared in air or oxygen environment for 4 d (n - 16 to 20 pups/group). Unlike superscripts represent significant differ ences as determined by Fisher's exact probability test (P < 0.05).
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Interstitial inflammation Septal attenuation Total histological score
pups reared in air or
1764
KIM ET AL. •*
/"
**•"
ï 4«*•% s> » ^ /> iv
Data summarizing pulmonary, liver and plasma Se concentrations and SeGPx activities in pups are pre sented in Table 4. Lung Se concentration and SeGPx activity of air-exposed pups were similar, regardless of the Se nutrition of parent dams. However, when pups were reared in oxygen, activity of lung SeGPx was significantly elevated in both Se-deficient and Serepleted groups, but the concentration of lung Se did not change. In contrast, Se concentrations and SeGPx activities in liver and plasma were significantly elevated in Se-repleted pups regardless of whether they were reared in air or oxygen. The mean GSH concentration of pup lungs was significantly increased at the end of 4 d of oxygen exposure, and unchanged by Se repletion (Fig. 4).
DISCUSSION Results from the present study provide evidence that Se repletion of Se-deficient rats during the early postnatal period enhances lung development and pro tects the neonatal lung against oxygen-induced injury. It is evident that Se has a primary effect on pul monary development by increasing lung volume and internal surface area of Se-repleted pups compared with Se-deficient pups. Furthermore, this short period of dietary Se-repletion (7 d) also partially protects the neonatal pups from lung damage under conditions of high Û2 exposure. At the time of parturition, the lung is structurally immature in both humans and rats. Extensive post natal alveolarization occurs during the neonatal period, and this accounts for 80 to 90% of alveoli eventually formed (Burri 1984). During this alveolari zation stage, lung volume and internal surface area increase, and mean air space size of alveoli and
FIGURE 3 Lungs from Se-deficient rat pups exposed to >95% Û2for 4 d. Hematoxylin and eosin, bar = 60 urn. A. Lung with septal attenuation. In this field, alveolar sacs are much enlarged. There are fewer and thinner septal buds (arrows). B. Lung with interstitial inflammation. There is prominent thickening and hypercellularity of alveolar septa (large arrows), mainly due to infiltration by inflammatory cells. There are also increased alveolar macrophages (small arrows) and other inflammatory cells within alveolar spaces.
percent air space in the lung decrease. In this ex periment, enhanced lung development in Se-repleted pups was indicated by their increased lung volume and internal surface area, regardless of environmental status. Selenium-repleted pups also had increased re sistance to damage by hyperoxic conditions, a re sponse possibly related to increased maturity of the lung directly associated with Se repletion. Thus ade quate Se nutriture is apparently important not only for protection against lung damage under conditions of oxidant stress, but also for maximal development of the lung under normal environmental conditions. The concentration of Se in milk collected from cross-fostering dams was significantly greater when maternal diet was supplemented with Se. The con centrations of Se and SeGPx activities in pup plasma
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FIGURE 2 Air control lung from a Se-deficient neonatal rat pup exposed to room air for 4 d. The lung has many small alveoli and numerous septal buds (arrows). Hematoxylin and eosin, bar - 60 urn.
SELENIUM AND PROTECTION
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TABLE 3 Percent air space, mean air space size (Lm), internal surface area (ISA) and volume of lungs from Se-deficient reared in air or oxygen environment for 4 d1
Air
2.1069.45± 1.0967.99± 1.10464.52± 1.97514.58± 13.500.793 ± 20.980.882 ± 0.0247.175 ± 0.0206.647 ± 0.1673893.7± 0.1984183.6± ±113.273.39 ±170.676.78
-Se
+Se
1.5269.12± 0.9272.00 ± 1.48435.97± 1.70469.43± 18.470.728 ± 21.500.833 ± 0.0326.113 ± 0.0236.570 ± 0.2663663.6± 0.1823702.1± ±155.274.66 ±169.6 0.05): "significant
effect of dietary selenium;
effect of oxygen environment.
and liver directly reflected the milk Se concentrations of the dams. In contrast, the level of dietary Se had no effect on the concentration of Se and SeGPx activity in pup lung tissue. It has been suggested that de veloping lung may preferentially utilize Se over other tissues and that such utilization is accelerated under conditions of hyperoxia (Kim et al. 1991). Stable Se concentrations within neonatal lung tissue under con ditions of oxidant stress, and a concurrent but slight decrease in hepatic Se, support this hypothesis. Both Se-deficient and Se-repleted pups in this study had increased pulmonary SeGPx activity under hyperoxia, which is consistent with our previous findings (Kim et al. 1991). However, despite the increase in SeGPx activity to equivalent levels, Se-repleted pups had fewer lesions and greater lung development compared with Se-deficient pups. The precise mechanism of Se-mediated protection of neonatal rat lungs during high oxygen exposure is unknown. It is possible, however, that faster alveolar-
ization of lung tissues in Se-repleted pups provides a greater "cushion" against the effects of hyperoxia. In this experiment, we assessed the GSH concentration in pup lungs to determine whether Se status affects levels of this essential antioxidant under hyperoxia. Enhancement of GSH by Se repletion would suggest that Se exerts a protective effect on the lung against the stress of hyperoxia by enhancing the availability of this antioxidant. However, basal GSH concen tration of the lung was not changed by Se nutriture and was increased to the same degree in both Sedeficient and Se-repleted pups under oxygen exposure, implying a generalized response to oxidant stress in dependent of Se status. The mechanism by which Se enhances lung de velopment in pups also is not known. The role of Se in lung development may be due to an effect on thyroid hormone. It has been noted that Se-deficient rats have lower activities of type I iodothyronine 5'-deiodinase, the enzyme that converts thyroxine
TABLE 4 Mean lung, liver, and plasma Se concentrations and selenium-dependent glutathione peroxidase (SeGPx) activities and Se-repleted pups reared in air or oxygen environment for 4 d1
Air -Se
of Se-deficient
Oxygen
+Se
-Se
+Se
Se concentration, \imol/gLungLiver3Plasma3SeGPx
0.08(imo] NADPH oxidized/(min-g activity,LungbLiver3Plasma"1.42
0.142.37 ± 0.160.90 ± ± protein)36.3 2.530.0± 1.710.7± ±1.71.74
0.142.79 ± ±0.171.32 0.1237.9 ±
0.122.08 ± 0.100.86 ± 0.0648.4±
0.162.61 ± 0.201.43 ± 0.1151.5 ±
2.842.5± 5.127.9± 5.339.6± 2.818.2± 3.09.7± 2.013.8± ±2.91.48 ±1.51.42 ±2.1 'Values are means ±SEMfor 9-10 samples. Data analyzed by 2 x 2 factorial ANOVA (P < 0.05): "significant effect of dietary selenium; bsignificant effect of oxygen environment.
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+Se
'Values are means ±SEMfor 16 to 20 pups. Data analyzed by 2 x 2 factorial ANOVA (P significant
pups
Oxygen
-Se Air %Lm,space, limISA,3*5 cm2Volume,3b mlVolume,ab wtSpecific mL/100 g body ISA,b cm2/ 100 g body wt73.17
and Se-repleted
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KIM ET AL.
Oxygen
Environment
FIGURE 4 Glutathione concentration of lung from selenium-deficient (Se-) or selenium-repleted (Se+) pups reared in air or oxygen environment for 4 d. Values are means ±SEM,n - 9 to 10 pups/group. Unlike superscripts represent significant differences (P < 0.05) as determined by factorial ANOVA, followed by least significant difference tests.
into the more metabolically active form of the hormone, 3,3',5-triiodothyronine (Arthur et al. 1990). It has recently been found that this enzyme is a selenocysteine-containing enzyme (Berry et al. 1991). Thyroid hormone is thought to promote pulmonary alveolarization during the postnatal period (Massaro et al. 1985) and also to enhance the rate of synthesis and release of surfactant phospholipids (Ballard et al. 1984). Specific receptors for thyroid hormones have been described in the fetal lung (Gonzales and Ballard 1982), but the precise mechanism whereby thyroid hormone acts on the lung has not been elucidated. The effect of Se repletion on thyroid hormone status was not investigated in this experiment, and further investigation is needed to determine whether Se repletion can affect thyroxine status and lung de velopment. Another possible explanation of Se-mediated pro tection from hyperoxia is that the neonatal lung con tains a specific Se-binding protein, different from SeGPx, that protects neonatal lung from oxidative damage. Recent investigations (Behne et al. 1988, Evenson and Sunde 1988) have reported that most tissues, including lung, contain several selenoproteins in addition to SeGPx. However, the functions of most selenoproteins other than SeGPx in mammalian tissues are not well established, and how any of these selenoproteins may be involved in protection against hyperoxia has not been investigated. In summary, results from the present study show that a short period of Se repletion in the early post
ACKNOWLEDGMENTS The authors thank Wanda Haschek for scientific advice, Janice Rutherford, Sasha Cavanagh and Hen rietta Tabe for technical assistance, Duane Steiner for animal care, and the word processing center for secre tarial assistance. LITERATURE CITED American Institute of Nutrition (1977} Report of Institution of Nutrition ad hoc committee on nutritional studies. I. Nutr. 107: 1340-1348. Amin, S., Chen, S. V., Collipp, P. f., Castro-Magana, V. T. & Klein, S. W. (1980) Selenium in premature Metab. 24: 331-340. Arthur, J. R., Nicol, F. & Beckett, G. J. (1990) Hepatic 5'-deiodinase: the role of selenium. Biochim. J.
the American standards for M., Maddaiah, infants. Nutr. iodothyronine 272: 537-540.
Asaoka, K. & Takahashi, K. (1981) An enzymatic assay of reduced glutathione S-aryltransferase with o-dinitrobenzene as a sub strate. J. Biochem. (Tokyo) 90: 1237-1242. Ballard, P. L., Hovey, M. L. & Gonzales, L. K. (1984) Thyroid hormone stimulation of phosphatidylcholine synthesis in cul tured fetal rabbit lung. J. Clin. Invest. 74: 898-905. Behne, D., Hilmert, H., Scheid, S., Gessner, H. & Elger, W. (1988) Evidence for specific selenium target tissues and new biolog ically important selenoproteins. Biochem. Biophys. Acta 966: 12-21. Berry, M. J., Banu, L. & Larsen, P. R. (1991) Type I iodothyronine deiodinase is a selenocysteine-containing enzyme. Nature (Lond.) 349: 438^40. Burk, R. F., Lawrence, R. A. & Lane, J. M. (1980) Liver necrosis and lipid peroxidation in the rat as the result of paraquat and diquat administration. J. Clin. Invest. 65: 1024-1031. Burri, P. H. (1984) Fetal and postnatal development of the lung. Annu. Rev. Physiol. 46: 617-628. Chung, A. S. &. Maines, M. A. (1981) Effect of selenium on
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Air
natal period can significantly enhance lung de velopment in rat pups and also protect the developing lung from damage by hyperoxia. Although the effect of Se on enhancing pulmonary GSH content does not provide an explanation for the observed protective effects of Se repletion during hyperoxia, the possible role of Se on neonatal lung development and pro tection via Se-containing type I iodothyronine S'-deiodinase has been suggested. An extension of these results to the clinical practice of medicine is that LBW infants, when treated with high oxygen concen trations for respiratory distress syndrome, may benefit from Se repletion, especially because these infants generally have significantly reduced stores of Se compared with term infants and because deterio ration of Se status of such infants during intensive care is documented (Amin et al. 1980, Huston et al. 1987, Lockitch et al. 1989, VanCaillie-Bertrand et al. 1984). Maintenance of adequate Se status in LBW infants has the potential to reduce the risks for com plications associated with positive ventilation in cluding bronchopulmonary dysplasia.
SELENIUM AND PROTECTION
ronine receptors in rabbit lung: characterization and develop mental changes. Endocrinology 111: 542-552. Hill, K. E. & Burk, R. F. |1982) Effect of selenium deficiency and vitamin E deficiency on glutathione metabolism in isolated rat hepatocytes. J. Biol. Chem. 257: 10668-10672. Huston, R. K., Shearer, T. R., lelen, B. J., Whall, P. D. & Reynolds, I. W. (1987) Relationship of antioxidant enzymes to trace metals in premature infants. J. Parenter. Enterai Nutr. 11: 163-168. Huston, R. K., Benda, G. I., Carlson, C. V., Shearer, T. R., Reynolds, J. W. & Neerhout, R. C. (1982) Selenium and vitamin E suffi ciency in premature infants requiring total parenteral nutrition. J. Parenter. Enterai Nutr. 6: 507-510. Jenkinson, S. G., Black, R. D. & Lawrence, R. A. (1988) Glutathione concentrations in rat lung bronchoalveolar lavage fluid: effects of hyperoxia. J. Lab. Clin. Med. 112: 345-351. Kim, H. Y., Picciano, M. F., Wallig, M. A. & Milner, J. A. (1991) The role of selenium in the development of neonatal rat lung. Pediatr. Res. 29: 44CM45. LeBoeuf, R. A., Zentner, K. L. & Hoekstra, W. G. (1985) Effect of dietary selenium concentration and duration of selenium feeding on hepatic glutathione concentrations in rats. Proc. Soc. Exp. Biol. Med. 180: 348^52. Levander, O. A., DeLoach, D. P., Morris, V. C. & Moser, P. B. (1983) Platelet glutathione peroxidase activity as an index of selenium status in rats. J. Nutr. 113: 55-63. Lockitch, G., Jacobson, B., Quigley, G., Dison, P. & Pendray, M. (1989) Selenium deficiency in low birth weight neonates: an
1767
unrecognized problem. J. Pediatr. 114: 865-870. Lowry, O. H., Rosebrough, N. J., Farr, A. L. &. Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Massaro, D., Teich, N., Maxwell, S., Massaro, G. D. & Whitney, P. (1985) Postnatal development of alveoli. J. Clin. Invest. 76: 1297-1305. McCarthy, T. P., Brodie, B., Milner, J. A. &. Bevili, R. F. (1981) Improved method for selenium determination in biological samples by gas chromatography. J. Chromatogr. 225: 9-16. Meister, A. & Anderson, M. E. (1983) Glutathione. Annu. Rev. Biochem. 52: 711-760. National Research Council (1978) Nutrient Requirements of Labo ratory Animals, no. 10, 3rd rev. ed. National Academy of Sciences, Washington, DC. Omaye, S. T., Reddy, K. A. & Cross, C. E. (1978) Enhanced lung toxicity of paraquat in selenium-deficient rats. Toxicol. Appi. Pharmacol. 43: 237-247. Paglia, D. E. & Valentine, W. N. (1967) Studies on the quantitative and qualitative characterization of erythrocyte glutathione per oxidase. J. Lab. Clin. Med. 70: 158-169. SAS Institute Inc. (1985) SAS User's Guide: Statistics, 5th éd. SAS Institute, Cary, NC. Steel, R.G.D. & Torrie, ]. H. (1980) Principles and Procedures of Statistics: A Biometrical Approach, 2nd ed. McGraw-Hill, New York, NY. Thurlbeck, W. M., Galaugher, W. & Mathers, f. (1981) Adaptive response to pneumonectomy in puppies. Thorax 36: 414-427. VanCaillie-Bertrand, M., Degenhart, H. J. & Fernandes, J. (1984) Selenium status of infants on nutritional support. Acta Paediatr. Scand. 73: 816-819. Vinton, N. E., Dahlstrom, K. A., Strobel, C. T. & Ament, M. E. (1987) Macrocytosis and pseudoalbinism: manifestations of selenium deficiency. J. Pediatr. Ill: 711-717. Weber, C. A., Duncan, C. A., Lyons, M. J. & Jenkinson, S. G. (1990) Depletion of tissue glutathione with diethyl maléateenhances hyperbaric oxygen toxicity. Am. J. Physiol. 258: L308-L312. Weibel, E. R. (1963) Morphometry of the Human Lung, pp. 51-73. Academic Press, New York, NY.
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glutathione metabolism. Induction of y-glutamylcysteine synthetase and glutathione reducÃ-ase in the rat liver. Biochem. Pharmacol. 30: 3217^223. Cross, E. E., Hasegawa, G., Reddy, K. A. & Omaye, S. T. (1977) Enhanced lung toxicity of oxygen in selenium-deficient rats. Res. Commun. Chem. Pathol. Pharmacol. 16: 695-706. Evenson, f. K. &. Sunde, R. A. (1988) Selenium incorporation into selenoproteins in the Se-adequate and Se-deficient rat. Proc. Soc. Exp. Biol. Med. 187: 169-180. Forman, H. J., Rotman, E. I. & Fisher, A. B. (1983) Roles of selenium and sulfur-containing amino acids in protection against oxygen toxicity. Lab. Invest. 49: 148-153. Gonzales, L. W. & Ballard, P. L. (1982) Nuclear 3,5,3'-triiodothy-
FROM HYPEROXIA
and Molecular Roles of Nutrients
Postnatal Selenium Repletion Protects Neonatal Rats from Hyperoxia1'2
Lungs of
Division of Nutritional Sciences and Department of Veterinary Pathobiology, University of Illinois, Urbana, IL 61801 and *Department of Nutrition, Pennsylvania State University, University Park, PA 16802 cantly reduced in pups Se deficient during gestation and early postnatal life compared with pups that are Se adequate during gestation and the first 5 d of life. However, specific histomorphometric indices of lung function in neonatal rat pups that might be affected by Se nutriture have not been investigated. The impact of maternal Se deficiency on low-birthweight (LBW)4 human infants remains to be fully evaluated. Significantly decreased plasma Se concen trations in LBW infants have been documented in recent clinical reports (Amin et al. 1980, Lockitch et al. 1989, VanCaillie-Bertrand et al. 1984), although the exact significance of these findings is not clear. Enterai feeding is often delayed in LBW infants due to the multiple complex problems associated with the short gestational period, including immaturity of organ systems and respiratory distress syndrome. Be cause neither intravenous fluids nor parenteral for mulas contain added Se, LBW infants have dramati cally decreased plasma Se concentrations during first few weeks of life (Huston et al. 1982 and 1987). This, in turn, may accelerate or exacerbate clinical problems associated with the high oxygen environ ments used to assist ventilation in these LBW infants. It has been noted that children receiving long-term total parenteral nutrition have myocardiopathy, pseudoalbinism and macrocytosis that are alleviated by Se therapy (Vinton et al. 1987). Whether a short period of Se repletion in Se-deficient neonates also can protect them from chronic lung disease that may result from
ABSTRACT We reported previously that Se-adequate neonatal rat pups born to Se-adequate dams were resistant to lung damage by hyperoxia. To assess whether early postnatal Se repletion could also protect developing pups reared under hyperoxia, female Sprague-Dawley rats (n - 20) were bred and fed a Sedeficient (0.04 ug/g) diet during pregnancy. On d 1 postpartum, dams were divided into two groups and fed either a Se-deficient diet or a Se-repleted (0.5 ug/g) diet. On d 4 postpartum, litters in each group were randomly assigned to either air or high oxygen (>95% 03) environments. Histologie evaluation of lungs from d8 pups indicated that Se repletion significantly reduced the incidence of lung lesions caused by hyperoxia. Selenium-repleted pups also had significantly greater lung volumes and internal surface areas. The 7-d period of Se repletion resulted in significantly elevated maternal milk Se concentrations compared with a Se-deficient group, which was reflected in the pups by elevated plasma and hepatic Se concentrations and Se-dependent glutathione peroxidase (SeGPx) activities. Pul monary glutathione concentration and SeGPx activity in pups were affected by oxygen exposure only, not by Se nutrition. Therefore, early postnatal Se repletion can protect the developing lung from oxygen-induced injury, a protection that is not entirely due to the effects of Se on pulmonary SeGPx activity and glutathione concen tration. J. Nutr. 122: 1760-1767, 1992. INDEXING KEY WORDS:
•selenium repletion •oxygen toxtcity •lung injury •glutathione •rats
Selenium-deficient newborn rat pups are much less tolerant of high oxygen exposure than Se-adequate pups. Selenium-deficient newborn pups exposed to >95% C>2for 4 d have higher incidences of pulmonary septal attenuation and interstitial inflammation than Se-adequate pups (Kim et al. 1991). Lung development in rat pups also seems to be affected by the Se nutriture of the dam, i.e., mean lung weight is signifi 0022-3166/92
$3.00 ©1992 American Institute
of Nutrition.
'Presented
in part at the 75th Annual Meeting of the Federation
of American Societies for Experimental Biology, April 1991, At lanta, GA [Kirn, H. Y., Picciano, M. F. & Wallig, M. A. (1991) Selenium repletion protects neonatal rat lungs from hyperoxia. FASEB I. 5: A1072 (abs. 4071)]. Supported in part by NICHHD Grant No. 18689. 3To whom correspondence should be addressed. 4Abbreviations used: GSH, glutathione; LBW, low birth weight; SeGPx, selenium-dependent glutathione peroxidase.
Received 26 February 1992. Accepted 29 April 1992. 1760
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HYE YOUNG KIM, MARY F. PICCIANO* AND MATTHEW A. WALLIG3
SELENIUM AND PROTECTION
1761
TABLE 1 Diet composition
Ingredient
Amount
Casein1 Sucrose Cornstarch Com oil Cellulose2 Vitamin mixture3 Se-free AIN-76 mineral mixture4 DL-Methionine Choline bitartrate
g/kg 200 300 331 100 20 10 35 2 2
Vitamin-free test casein (Teklad, Madison, WI). 2Alphacel (ICN Nutritional Biochemical, Cleveland, OH). 3AIN-76 vitamin mixture (AIN 1977). 4AIN-76 mineral mixture prepared without Se (AIN 1977).
MATERIALS AND METHODS Animal care. Nulliparous female Sprague-Dawley rats (HarÃ-anIndustries, Indianapolis, IN), weighing 180 to 200 g, were housed in individual, suspended, stainless steel wire-mesh cages in a room with con trolled temperature (20 to 22°C)and lighting (12-h light:dark cycle). The animals were fed a commercial ration (Purina Rodent Chow, Ralston Purina, St. Louis, MO) for a 2-wk adaptation period. At 200 to 240 g, rats were mated, and d 1 of pregnancy was determined by the presence of vaginal plugs and sperm. On d 1 of pregnancy, all rats were assigned to a semipurified, Se-deficient diet. Rats were given free access to demineralized water (Nanopure, Barnstead, Boston, MA) and experimental diets. The Animal Care and Use Committees of both the Pennsylvania State University and the University of Illinois re viewed and approved the use of experimental animals in these studies. Diets. Diets (Table 1) were formulated to contain all nutrients in quantities adequate for reproduction (NRC 1978) except for Se in the Se-deficient diet. Direct analysis of diets by gas chromatography coupled with electron capture detection was used to confirm dietary Se concentration of 0.04 ug/g (Sedeficient) and 0.5 ug/g (Se-adequate). Experimental design. Twenty female rats were bred and fed the Se-deficient (0.04 ug/g) diet during pregnancy. On d 1 postpartum, dams were divided into two groups and either continued to receive a Sedeficient diet or were fed a new sodium selenitesupplemented (+Se, 0.5 ug/g) diet. Food dishes were positioned so that only dams had access to food, and maternal milk was the only nourishment for pups. The day after parturition (d 2), litters were culled so that there were 10 pups per dam. On d 4, two litters
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high oxygen exposure has not been investigated in animals or humans. The mechanism by which Se deficiency results in increased susceptibility to damage from high oxygen exposure is not known. The protective role of Se against oxygen exposure is, however, not due only to its role as an essential component of the key antioxidant enzyme, glutathione peroxidase (Cross et al. 1977, Forman et al. 1983, Kim et al. 1991). Both marginally Se-deficient and Se-adequate pups have equally enhanced Se-dependent glutathione perox idase (SeGPx, EC 1.11.1.9) activities after oxygen ex posure, but only Se-adequate pups are protected from lung damage. Pulmonary lesions and mortality due to paraquat and diquat poisoning resemble those in duced by high oxygen and are believed to be produced by toxic oxygen intermediates. Selenium deficiency has been found to exacerbate paraquat-induced lung injury in rats (Omaye et al. 1978). In addition, Burk and co-workers (1980) found that the lesions induced by diquat in Se-deficient rats can be reversed by in jection of Se. This protection afforded by Se, however, also could not be related to its role in SeGPx function, and Burk and co-workers (1980) suggested the exis tence of another Se-dependent factor, apart from SeGPx, that protects against cellular peroxidation during oxidant stress. Glutathione (GSH) is another important com ponent of the antioxidant defense system in mammals. Its mechanism of protection against ox idant stress is thought to be related to its ability to quench free radical products (Meister and Anderson 1983). Glutathione content is increased significantly in lung tissue during hyperoxia in animals (Jenkinson et al. 1988). It is reported that intraperitoneal glutathione administration can protect rats from hyperbaric oxygen toxicity, without producing any changes in SeGPx activities (Weber et al. 1990). Di etary selenium has been shown to affect glutathione metabolism in rat liver (Chung and Maines 1981, Hill and Burk 1982, LeBoeuf et al. 1985). However, whether Se has an effect on GSH content in rat lungs under hyperoxia is not known. The present study was designed to determine whether Se repletion during the early postnatal period can, have a positive effect on lung development in neonatal rats and protect the developing rat lung against oxygen-induced injury. We also sought to identify a mechanism by which Se repletion may protect neonatal lung from high oxygen damage. Spec ifically, we assessed histopathological changes using histomorphometry in neonatal lungs after oxygen ex posure in both Se-deficient and Se-repleted rat pups. We also analyzed glutathione content in the neonatal lung to determine whether any alterations in content could be related to damage and/or protection from hyperoxia.
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KIM ET AL.
oxygen was continuous except for a brief period daily, when chambers were opened for rotation of dams between oxygen- and air-housed litters. This rotation was done to prevent oxygen-induced illness in the dams. In a previous study, we showed that no dams had pulmonary lesions by this procedure. On d 8 of lactation, dams and pups were separated for 2 h to allow for accumulation of milk. Dams were anesthe tized with ketamine HCl (10 mg/100 g body wt) containing acepromazine (l mg/100 g body wt) and injected intraperitoneally with oxytocin (0.25 U/100 g body wt) to facilitate milking. Milk was collected with a suction apparatus in which milk flowed through polyethylene capillary tubing attached to a test tube kept in an ice bath. Pups were weighed and killed at d 8 of age by decapitation, heparinized blood was collected, and perfused lungs and liver were ex cised. Analytical procedures. Four pups in each litter were used for histopathology and histomorphometry. Lungs were fixed via intratracheal instillation of 10% buffered formalin at an inflation pressure of 22 cm of H2Û and kept in the same buffer solution. At least 3 d after fixation, total lung volume was measured by water displacement. Three standard cross-sections from each lung (right and left) were examined. These sections were taken from the same area of apical, middle and diaphragmatic portions of each lung. Sec tions were dehydrated in graded alcohols, embedded in paraffin, sectioned at 5 urn and stained with hematoxylin and eosin. Sections were examined at low (lOx) and high (40x) magnification by light mi croscopy to determine the presence of either of two lesion patterns: septal attenuation with enlarged al veolar spaces and interstitial inflammation. Each section was evaluated twice in a double-blind manner and the average was used as a final score. Each lesion received a score ranging from 0 (no lesion) to 3 (severe lesion). For septal attenuation, a score of 1 (mild lesion) was recorded where there were only mild, occasional foci of septal bud stunting and alveolar enlargement, score 2 (moderate lesion) when there was multiple diffusely scattered but still mild septal bud stunting, and score 3 (severe lesion) when there were many, coalescing foci of moderate septal stunting and alveolar enlargement. For inflammatory lesions, a score of 1 (mild lesion) was recorded if there were occasional, small foci of interstitial inflam mation occupying 10% but 50% of the alveolar tissue in a section. For histomorphometry, a standard integrating eye piece was used. The number of bars that intersected lung tissue per field and the number of times the lines were crossed by tissue septa per field were counted. Mean air space size (Lm) was calculated using the following formula (Thurlbeck et al. 1981): Lm = length of line x number of lines x number number of tissue intercepts
of fields
Mean internal surface area (ISA) was calculated the following formula (Weibel 1963): ISA = 4 x volume
using
of lung parenchyma Lm
and mean percent Percent
air space by: air space = Pa/(Pa + Pt),
where Pa is the number of intercept bars hitting air and Pt is the number of intercept bars hitting tissue. The remaining six pups in each litter were used for biochemical assays. Three pups from the same litter were pooled and used as one sample to achieve enough sample for biochemical evaluation. Whole blood was centrifuged (800 x g) and plasma was col lected. Lungs used for biochemical analysis were first carefully perfused through the pulmonary artery with ice-cold isotonic buffer (0.1 mol/L potassium phos phate, 0.15 mol/L KCl, pH 7.4). Lung and liver were surgically removed, rinsed and blotted dry. An aliquot of fresh lung tissue was homogenized in ice-cold iso tonic KCl buffer, and glutathione content was mea sured using the GSH S-transferase linked method of Asaoka and Takahashi (1981). Remaining tissue samples were stored at -70"C until subsequent bio chemical assays were performed. Selenium concentrations of diet, lung, plasma and liver were determined according to the method of McCarthy et al. (1981) using a gas Chromatograph equipped with an electron capture detector (HewlettPackard 5710A, Avondale, PA) and a 0.53-mm i.d. fused silica capillary column (Supelco, Belief onte, PA). The activities of SeGPx were determined in blood and tissue homogenates by the coupled assay of Paglia and Valentine (1967) as modified by Levander et al. (1983). Hydrogen peroxide was used as the sub strate in this assay. Protein content of tissues and blood was determined by a modified Lowry method (Lowry et al. 1951). Statistics. Biochemical data and histomorphometrical data were evaluated using ANO VA (2x2
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from dams fed the same diet were mixed, with half of the pooled litter assigned to an air environment and the other half to an oxygen environment. The two matched dams (cross-fostering dams) were then ro tated between their air- and oxygen-housed litters for the next 4 d. In the latter environment, which was set up to mimic clinical ventilators for LEW infants, exposure to oxygen was conducted with careful moni toring of oxygen concentration (>95%), temperature (22-25°C), and humidity (55-75%). The exposure to
SELENIUM AND PROTECTION
FROM HYPEROXIA
1763
TABLE 2 Severity scores of lung interstitial
inflammation
and septa! attenuation from Se-deficient oxygen environment for 4 d1
and Se-repleted
Air
Oxygen
-Se
+Se
-Se
0.175 ±0.063 0.387 ±0.121 0.562 ±0.140
0.031 ±0.031 0.219 ±0.091 0.250 ±0.102
0.375 ±0.113 0.825 ±0.163 1.200 ±0.248
0.188 ±0.101 0.547 ±0.141 0.734 ±0.228
'Values are means ±SEMfor 16 to 20 pups. Each lung tissue received a score ranging from 0 (no lesion) to 3 (severe lesion) in a double-blind evaluation. Data analyzed by 2 x 2 factorial ANOVA (P < 0.05): "significant effect of dietary selenium; Significant effect of oxygen environment.
factorial), followed by least significant difference tests (Steel and Torrie 1980). Data for incidence of lung injury were assessed by Fisher exact probability test (SAS 1985). The value of P < 0.05 was chosen as the level of statistical significance. RESULTS Maternal food intake, body weights and tissue Se concentrations. Throughout the study, no significant differences in mean food intake and body weight of dams were observed between groups. The mean food intake of all dams was 17.1 ±1.0 g/d. The mean body weight of dams was 324.7 ±9.8 g at the end of pregnancy and 230.7 ±10.8 g at d 8 of lactation. Feeding the Se-supplemented diet to marginally Sedeficient dams resulted in a significant increase in Se concentrations in liver and milk. Mean concentra tions of Se in milk from Se-deficient and Se-repleted dams were 0.63 ±0.05 and 1.03 ±0.03 nmol/L, respec tively; in liver the concentrations were 4.75 ±0.54 and 7.58 ±0.64 nmol/g, respectively. Histopathology and bistomorphontetry of pups. Selenium repletion via the dam significantly reduced the total pulmonary damage (P < 0.05) and tended to decrease the severity of inflammation and septal at tenuation (P < 0.10) (Table 2). Seventy-five percent of oxygen-exposed, Se-deficient pups had lung lesions, whereas only 43.8% of oxygen-exposed, Se-repleted pups had lesions (Fig. 1). Typical microscopic charac teristics of lungs from the experimental rat pups are shown in Figures 2 and 3. The alterations associated with septal attenuation included stunting of septal buds, a decrease in the number of alveolar septa, thinning of remaining septal buds and enlarged al veolar spaces (Fig. 3A). In lungs with interstitial in flammation (Fig. 3B), there were multiple foci of thickening and hypercellularity of alveolar septa, mainly due to infiltration by macrophages and some neutrophils.
Analysis of histomorphometric data showed that Se repletion significantly enhanced lung volume (P < 0.001) and internal surface area (P < 0.05), whereas oxygen exposure significantly decreased the values for these measures regardless of Se status. Mean air space size and percent air space of the pups were not af fected by either Se repletion or oxygen exposure (Table 3). Pup body and organ weights, Se concentrations, SeGPx activities and GSH content in organs and plasma. Pup body weights were similar among groups throughout the study. Mean body weights for all pups at d 2 and at the beginning (d 4) and end (d 8) of oxygen exposure were 5.94 ±0.17, 8.20 ±0.17 and 12.20 ±0.33, respectively. Organ weights were similar on d 8 among treatment groups. The mean lung and liver weights of pups were 0.23 ±0.01 and 0.40 ±0.02 g, respectively.
Air
Oxygen
Environment FIGURE 1 Percent incidence of lung injury from selenium-deficient (Se-) or selenium-repleted (Se+) pups reared in air or oxygen environment for 4 d (n - 16 to 20 pups/group). Unlike superscripts represent significant differ ences as determined by Fisher's exact probability test (P < 0.05).
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Interstitial inflammation Septal attenuation Total histological score
pups reared in air or
1764
KIM ET AL. •*
/"
**•"
ï 4«*•% s> » ^ /> iv
Data summarizing pulmonary, liver and plasma Se concentrations and SeGPx activities in pups are pre sented in Table 4. Lung Se concentration and SeGPx activity of air-exposed pups were similar, regardless of the Se nutrition of parent dams. However, when pups were reared in oxygen, activity of lung SeGPx was significantly elevated in both Se-deficient and Serepleted groups, but the concentration of lung Se did not change. In contrast, Se concentrations and SeGPx activities in liver and plasma were significantly elevated in Se-repleted pups regardless of whether they were reared in air or oxygen. The mean GSH concentration of pup lungs was significantly increased at the end of 4 d of oxygen exposure, and unchanged by Se repletion (Fig. 4).
DISCUSSION Results from the present study provide evidence that Se repletion of Se-deficient rats during the early postnatal period enhances lung development and pro tects the neonatal lung against oxygen-induced injury. It is evident that Se has a primary effect on pul monary development by increasing lung volume and internal surface area of Se-repleted pups compared with Se-deficient pups. Furthermore, this short period of dietary Se-repletion (7 d) also partially protects the neonatal pups from lung damage under conditions of high Û2 exposure. At the time of parturition, the lung is structurally immature in both humans and rats. Extensive post natal alveolarization occurs during the neonatal period, and this accounts for 80 to 90% of alveoli eventually formed (Burri 1984). During this alveolari zation stage, lung volume and internal surface area increase, and mean air space size of alveoli and
FIGURE 3 Lungs from Se-deficient rat pups exposed to >95% Û2for 4 d. Hematoxylin and eosin, bar = 60 urn. A. Lung with septal attenuation. In this field, alveolar sacs are much enlarged. There are fewer and thinner septal buds (arrows). B. Lung with interstitial inflammation. There is prominent thickening and hypercellularity of alveolar septa (large arrows), mainly due to infiltration by inflammatory cells. There are also increased alveolar macrophages (small arrows) and other inflammatory cells within alveolar spaces.
percent air space in the lung decrease. In this ex periment, enhanced lung development in Se-repleted pups was indicated by their increased lung volume and internal surface area, regardless of environmental status. Selenium-repleted pups also had increased re sistance to damage by hyperoxic conditions, a re sponse possibly related to increased maturity of the lung directly associated with Se repletion. Thus ade quate Se nutriture is apparently important not only for protection against lung damage under conditions of oxidant stress, but also for maximal development of the lung under normal environmental conditions. The concentration of Se in milk collected from cross-fostering dams was significantly greater when maternal diet was supplemented with Se. The con centrations of Se and SeGPx activities in pup plasma
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FIGURE 2 Air control lung from a Se-deficient neonatal rat pup exposed to room air for 4 d. The lung has many small alveoli and numerous septal buds (arrows). Hematoxylin and eosin, bar - 60 urn.
SELENIUM AND PROTECTION
FROM HYPEROXIA
1765
TABLE 3 Percent air space, mean air space size (Lm), internal surface area (ISA) and volume of lungs from Se-deficient reared in air or oxygen environment for 4 d1
Air
2.1069.45± 1.0967.99± 1.10464.52± 1.97514.58± 13.500.793 ± 20.980.882 ± 0.0247.175 ± 0.0206.647 ± 0.1673893.7± 0.1984183.6± ±113.273.39 ±170.676.78
-Se
+Se
1.5269.12± 0.9272.00 ± 1.48435.97± 1.70469.43± 18.470.728 ± 21.500.833 ± 0.0326.113 ± 0.0236.570 ± 0.2663663.6± 0.1823702.1± ±155.274.66 ±169.6 0.05): "significant
effect of dietary selenium;
effect of oxygen environment.
and liver directly reflected the milk Se concentrations of the dams. In contrast, the level of dietary Se had no effect on the concentration of Se and SeGPx activity in pup lung tissue. It has been suggested that de veloping lung may preferentially utilize Se over other tissues and that such utilization is accelerated under conditions of hyperoxia (Kim et al. 1991). Stable Se concentrations within neonatal lung tissue under con ditions of oxidant stress, and a concurrent but slight decrease in hepatic Se, support this hypothesis. Both Se-deficient and Se-repleted pups in this study had increased pulmonary SeGPx activity under hyperoxia, which is consistent with our previous findings (Kim et al. 1991). However, despite the increase in SeGPx activity to equivalent levels, Se-repleted pups had fewer lesions and greater lung development compared with Se-deficient pups. The precise mechanism of Se-mediated protection of neonatal rat lungs during high oxygen exposure is unknown. It is possible, however, that faster alveolar-
ization of lung tissues in Se-repleted pups provides a greater "cushion" against the effects of hyperoxia. In this experiment, we assessed the GSH concentration in pup lungs to determine whether Se status affects levels of this essential antioxidant under hyperoxia. Enhancement of GSH by Se repletion would suggest that Se exerts a protective effect on the lung against the stress of hyperoxia by enhancing the availability of this antioxidant. However, basal GSH concen tration of the lung was not changed by Se nutriture and was increased to the same degree in both Sedeficient and Se-repleted pups under oxygen exposure, implying a generalized response to oxidant stress in dependent of Se status. The mechanism by which Se enhances lung de velopment in pups also is not known. The role of Se in lung development may be due to an effect on thyroid hormone. It has been noted that Se-deficient rats have lower activities of type I iodothyronine 5'-deiodinase, the enzyme that converts thyroxine
TABLE 4 Mean lung, liver, and plasma Se concentrations and selenium-dependent glutathione peroxidase (SeGPx) activities and Se-repleted pups reared in air or oxygen environment for 4 d1
Air -Se
of Se-deficient
Oxygen
+Se
-Se
+Se
Se concentration, \imol/gLungLiver3Plasma3SeGPx
0.08(imo] NADPH oxidized/(min-g activity,LungbLiver3Plasma"1.42
0.142.37 ± 0.160.90 ± ± protein)36.3 2.530.0± 1.710.7± ±1.71.74
0.142.79 ± ±0.171.32 0.1237.9 ±
0.122.08 ± 0.100.86 ± 0.0648.4±
0.162.61 ± 0.201.43 ± 0.1151.5 ±
2.842.5± 5.127.9± 5.339.6± 2.818.2± 3.09.7± 2.013.8± ±2.91.48 ±1.51.42 ±2.1 'Values are means ±SEMfor 9-10 samples. Data analyzed by 2 x 2 factorial ANOVA (P < 0.05): "significant effect of dietary selenium; bsignificant effect of oxygen environment.
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+Se
'Values are means ±SEMfor 16 to 20 pups. Data analyzed by 2 x 2 factorial ANOVA (P significant
pups
Oxygen
-Se Air %Lm,space, limISA,3*5 cm2Volume,3b mlVolume,ab wtSpecific mL/100 g body ISA,b cm2/ 100 g body wt73.17
and Se-repleted
1766
KIM ET AL.
Oxygen
Environment
FIGURE 4 Glutathione concentration of lung from selenium-deficient (Se-) or selenium-repleted (Se+) pups reared in air or oxygen environment for 4 d. Values are means ±SEM,n - 9 to 10 pups/group. Unlike superscripts represent significant differences (P < 0.05) as determined by factorial ANOVA, followed by least significant difference tests.
into the more metabolically active form of the hormone, 3,3',5-triiodothyronine (Arthur et al. 1990). It has recently been found that this enzyme is a selenocysteine-containing enzyme (Berry et al. 1991). Thyroid hormone is thought to promote pulmonary alveolarization during the postnatal period (Massaro et al. 1985) and also to enhance the rate of synthesis and release of surfactant phospholipids (Ballard et al. 1984). Specific receptors for thyroid hormones have been described in the fetal lung (Gonzales and Ballard 1982), but the precise mechanism whereby thyroid hormone acts on the lung has not been elucidated. The effect of Se repletion on thyroid hormone status was not investigated in this experiment, and further investigation is needed to determine whether Se repletion can affect thyroxine status and lung de velopment. Another possible explanation of Se-mediated pro tection from hyperoxia is that the neonatal lung con tains a specific Se-binding protein, different from SeGPx, that protects neonatal lung from oxidative damage. Recent investigations (Behne et al. 1988, Evenson and Sunde 1988) have reported that most tissues, including lung, contain several selenoproteins in addition to SeGPx. However, the functions of most selenoproteins other than SeGPx in mammalian tissues are not well established, and how any of these selenoproteins may be involved in protection against hyperoxia has not been investigated. In summary, results from the present study show that a short period of Se repletion in the early post
ACKNOWLEDGMENTS The authors thank Wanda Haschek for scientific advice, Janice Rutherford, Sasha Cavanagh and Hen rietta Tabe for technical assistance, Duane Steiner for animal care, and the word processing center for secre tarial assistance. LITERATURE CITED American Institute of Nutrition (1977} Report of Institution of Nutrition ad hoc committee on nutritional studies. I. Nutr. 107: 1340-1348. Amin, S., Chen, S. V., Collipp, P. f., Castro-Magana, V. T. & Klein, S. W. (1980) Selenium in premature Metab. 24: 331-340. Arthur, J. R., Nicol, F. & Beckett, G. J. (1990) Hepatic 5'-deiodinase: the role of selenium. Biochim. J.
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Air
natal period can significantly enhance lung de velopment in rat pups and also protect the developing lung from damage by hyperoxia. Although the effect of Se on enhancing pulmonary GSH content does not provide an explanation for the observed protective effects of Se repletion during hyperoxia, the possible role of Se on neonatal lung development and pro tection via Se-containing type I iodothyronine S'-deiodinase has been suggested. An extension of these results to the clinical practice of medicine is that LBW infants, when treated with high oxygen concen trations for respiratory distress syndrome, may benefit from Se repletion, especially because these infants generally have significantly reduced stores of Se compared with term infants and because deterio ration of Se status of such infants during intensive care is documented (Amin et al. 1980, Huston et al. 1987, Lockitch et al. 1989, VanCaillie-Bertrand et al. 1984). Maintenance of adequate Se status in LBW infants has the potential to reduce the risks for com plications associated with positive ventilation in cluding bronchopulmonary dysplasia.
SELENIUM AND PROTECTION
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