Dietary antioxidants and the biochemical response to oxidant inhalation

TOXICOLOGY AND APPLIED PHARMACOLOGY71, 398-406 (1983)

Dietary Antioxidants

and the Biochemical Response to Oxidant Inhalation

Il. Influence of Dietary Selenium on the Biochemical Effects of Ozone Exposure in Mouse Lung NABIL M. ELSAYED, ALLEN D. HACKER, KLAUS KUEHN,’ MOHAMMAD G. MUSTAFA,~ AND GERHARD N. SCHRAUZER Division of Environmental Department of Medicine, and Department

and Nutritional Sciences, School of Public Health, and Division of Pulmonary Center for the Health Sciences, University of Cahyornia, Los Angeles, Calijornia of Chemistry, University of Cal$orrzia. San Diego, La Jolla, Callyornia 92093

Received

March

30, 1983:

accepted

July

Disease, 90024,

24, 1983

Dietary Antioxidants and the Biochemical Response to Oxidant Inhalation. II. Influence of Dietary Selenium on the Biochemical Effects of Ozone Exposure in Mouse Lung. ELSAYED, N. M., HACKER, A. D., KUEHN, K., MUSTAFA, M. G., AND SCHRAUZER,G. N. (1983). Toxicol. App/. Pharmacol. 71, 398-406. We examined the influence of dietary selenium (Se) on the pulmonary biochemical response to ozone (03) exposure. For I 1 weeks, weanling female strain A/St mice were fed a test diet containing Se either at 0 ppm (-Se) or 1 ppm (+Se). Each diet contained 55 ppm vitamin E (vit E). Mice from each dietary group were exposed to 0.8 & 0.05 ppm (1568 + 98 pg/m3) O3 continuously for 5 days. Afier O3 exposure, they were killed along with a matched number of unexposed controls, and their lungs were analyzed for various biochemical parameters. The Se contents of lung tissue and whole blood were determined, and the levels were seven- to eightfold higher in +Se mice than in -Se mice, reflecting the Se intake of the animals. In unexposed control mice, Se deficiency caused a decline in ghnathione peroxidase (GP) activity relative to +Se group. After OJ exposure, the GP activity in the -Se group was associated with a lack of stimulation of ghnathione reductase (GR) activity and the pentose phosphate cycle (PPC) as assessed by measuring glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities. In contrast, the +Se group after Ox exposure exhibited increases in all four enzyme activities. Other parameters, e.g., lung weight, total lung protein, DNA and nonprotein sulfbydryl contents, and O* consumption, were not affected by dietary Se in the presence or absence of O3 exposure. The data indicate that dietary Se alters the GP activity, which in turn influences the GR and PPC activities in the lung evidently through a reduced demand for NADPH. The level of vit E in the lung was found to be twofold higher in the -Se group than in the +Se group, suggesting a compensatory relationship between Se and vit E in the lung. With 0s exposure, both Se and vit E contents further increased in the lungs of each dietary group. It is plausible that Se and vit E under oxidant stressare “mobilized” to the lung from other body sites.

Ozone is a major oxidant component of photochemical smog in urban air. An exposure to 0s at or near ambient concentrations has

been shown to produce biochemical and morphological alterations in the lung (DeLucia et ul., 1972; Dungworth et al., 1975; Evans et al., 1976; Mustafa et al., 1977; Mustafa and Tierney, 1978). The response of the lung to 0s was reported to extend from the conducting airways to the gas-exchange region, particularly affecting the terminal bronchioles and

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$3.00

Copyright 0 I983 by Academic Press. Inc. All rights of reproductmn m any form reserved.

398

INFLUENCE

OF

SELENIUM

the adjacent alveoli (Dungworth ef uZ., 1975: Evans et al.- 1976; Mustafa and Tiemey, 1978). The reactions of OJ with lung tissue have been reported to involve its oxidization properties, producing harmful free radicals which result in the oxidation of various functional groups and membrane lipids (Roehm et ul., 1971: Menzel, 1976). Based on this concept, a variety of biological antioxidants, such as vitamin E and Se, have been investigated for their protective role against hyperoxia, OX, NO1, and paraquat with variable degrees of success (Fletcher and Tappel, 1973; Cross et ul., 1977: Omaye et al., 1978; Burk et al., 1980: Evans et ul., 1980; Elsayed and Mustafa, 1982: Combs and Peterson, 1983). In a preliminary study (Elsayed ef ul., 1982a), we examined the influence of three levels of dietary Se, viz. 0.0, 0.15, and 1.O ppm, and reported that dietary Se influenced the pulmonary glutathione peroxidase (GP) activity, which in turn affected the glutathione reductase (GR) and pentose phosphate cycle (PPC) activities when the animals were exposed to Oj. Although the differences in effects at Se levels between 0.0 and 0.15 or 1.O ppm were highly significant, those between 0.15 and 1.0 ppm were negligible. In view of the findings of this study, we designed a second study the purpose of which was to carry out a systematic examination of the influence of dietary Se on O3 effects in the lung. In this study we used two levels of dietary Se. viz. 0.0 and 1.O ppm, and a series of physical and biochemical parameters. These parameters were selected to determine a range of Se effects, including those on GP and the ensuing GR and PPC activities, that could be discernible under oxidant stress. In addition, we examined whether there was an interrelationship between Se and vitamin E in the lung under oxidant stress. METHODS himuls, &f, arzd exposure. Weanhng female strain A/St mice. bred in our laboratory. were fed a test diet (Table I) containing 55 ppm vitamin E and either 0 ppm Se (-Se group) or I ppm Se (+Se group) for 11 weeks. Torula yeast containing organically bound Se. donated

ON

OZONE

399

BIOCHEMISTRY TABLE

I

DIET COMPOSITION’ Ingredient Sucrose Torula yeast’ Lard (tocopherol Mineral mixture’ Vitamin mixtured

Composition

stripped)

(% j

59.8 30.0 5.0 5.0 0.2

’ Teklad Se-deficient diet # 170698 (TekIad, Madison Wis.) ’ Special torula yeast, containing the desired Se concentration and analyzed in our laboratory, was added. ‘ Hubbel-Mandel-Wakeman mineral mix,. Teklad # 170790. ‘Vitamin contents (g/kg): thiamine-HCl. 0.0004: riboflavin, 0.0025; pyridoxine-HCI, 0.002; vitamin B,J, 0, I: choline chloride. I .O; menadione, 0.001: vitamin A palmitate, 14,000 units; vitamin Dz, 3200 units: and vitamin E, 55 units.

by Cell Life Corp. (San Diego, Calif.) was analyzed in our laboratory for Se content before being mixed with the diet to give the desired Se concentration. The mice were housed in a laminar flow isolation unit (BurIeson Air Tech. Corp., Orange, Calif.) with free access to respective -Se or +Se diet and glass-distilled water. Mice from each dietary group were exposed to 0.8 ? 0.05 ppm (I 568 & 98 pg/m3) OX continuously for 5 days m 300-liter stainless-steel inhalation chambers (Young and Bertke, Cincinnati. Ohio) similar to those described by Hinners ef u/. (1968). The air flow in the chambers was kept at 20 volume changes per hour. A corresponding number of mice from each dietary group were placed in an identical chamber receiving filtered room air. Ozone was generated by passing 100% Oz through an ozoniTer (Sander, Peine Am Osterberg. West Germany) at a rate of 0.5 liter/min. The level of 0, was continuously monitored with an ozone monitor (Daisibi, model 1003-PC. Environmental Corp., Glendale, Calif.) and recorded on a strip chart recorder. For the calibration of O3 monitor. a secondary calibration standard was used according :o the method described by the APHA Intersociety Committee ( 1972). The mice in both exposure and control chambers were housed in open mesh stainless-steel cages and allowed free access to the respective -Se or +Se diet and glass-distilled water. Tissuepreparafion. Immediately after Oj exposure, mice from both exposed and unexposed groups were anesthetized with an ip injection of sodium pentobarbital (60 mg/kg body weight) and weighed. A thorocotomy was performed to expose the lung and heart, and a blood sample (0.5-l ml) collected by cardiac puncture. was pooled from two mice. The lungs were removed. dissected

400

ELSAYED

free of major airways, rinsed with cold saline, blotted gently on gauze, and weighed. Portions of lung tissue were frozen along with the blood samples on dry ice, then stored at -7O’C until analyzed for Se and vitamin E contents. The remainder was homogenized in a PotterElvehjem glass-Teflon homogenizer (0. I-O. 15 mm clearance) in an ice cold medium containing 0.15 M sucrose, 0.15 M mannitol, and 1 mM Tris-HCI at pH 7.5. The homogenate (approx. 8% w/v) was filtered through two layers of gauze and adjusted to a final volume of 4 ml. Ahquots of homogenate were removed for various assays, and the remainder was centrifuged at 40,OOOgat 4’C for 30 min to prepare the cytosol (supematant fraction). Biochemicul uhu&ser. In lung homogenate the DNA and protein contents were determined by the methods of Schneider (1945) and Lowry et uf. (195 I), respectively. Total sulfhydryls (TSH) and nonprotein suhhydryls (NPSH) were determined by the method of Sedlak and Lindsay (I 968). The rate of oxygen utilization was determined polarographically at 37’C with an oxigraph (model RI-C, Gilson Medical Electronics, Madison, Wis.) as described previously (Elsayed et ul., 1982b). In lung cytosol, the enzyme activities were determined as follows: glutathione peroxidase (GP) as described by Little et ul. (1970); ghnathione reductase (GR) by the method of Horn (1965); and glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogmconate dehydrogenase (6PGD) by the methods of Lohr and Wailer (1965). Selenium and vitamin E contents were determined fluorometrically in samples of unhomogenized lung tissue and whole blood as described by the Analytical Methods Committee (1979) and Taylor et ul. (I 976) respectively. Dutu presentution. The data in this study are presented as mean ? standard deviation, and expressed per milhhter whole blood or per whole organ (lung). An expression of data per lung was necessaryto avoid the potential influence of plasma fluid, protein accumulation, or cell population changes in the lung; otherwise, these would affect the data if calculated on the basis of conventional lung weight, or per unit of protein or DNA (Witschi, 1975; Mustafa et ul., 1977, Mustafa and Tiemey, 1978; Elsayed et ul., 1982b). Student’s t test at the 5% level of significance was used to examine the differences in mean responsesbetween control and exposed animals in each dietary group (Dixon and Massey, 1969).

RESULTS Tissue Selenium and Vitamin E Levels Selenium contents of whole blood and lung tissue (Figs. 1A and B) in the two dietary groups reflected their dietary Se intake. The levels in the lungs and blood of Se-supplemented mice were consistently seven- to

ET AL.

eightfold higher than those in the Se-deficient mice. The blood and lung levels increased in both dietary groups with 0s exposure, but the magnitude of increases was higher in -Se than in +Se animals, viz. 20 versus 9% for whole blood, and 45 versus 27% for lung tissue. Vitamin E levels in lung tissue of the two dietary groups (Fig. 1C) showed an inverse relationship with Se contents; the vitamin E level was consistently higher (70%) in -Se group than in +Se group. After O3 exposure, vitamin E levels showed an increase similar to that observed for Se levels in both dietary groups. Body Weight and Lung Weight The presence or absence of Se in the diet did not influence the body weight or wet lung weight of mice (Table 2). However, 0s exposure resulted in a slight decrease in body weights (9 to 1 l%, NS). The lung weights showed significant increases in both dietary groups after OX exposure, except that the increase was less in the Se-deficient group (38 and 15%, respectively, for +Se and -Se groups). Total Lung Protein and DNA Contents There was no difference in mean protein contents of the lungs in the two dietary groups. After O3 exposure, the protein content showed a significant increase for -Se animals (35%, JI < 0.05) but not for +Se animals (14%, NS) (Table 2). Mean DNA contents of mouse lungs were virtually unaffected by dietary Se levels or by OJ exposure (Table 2). Oxygen Utilization The rate of oxygen utilization was measured in lung homogenate by using succinate as a substrate (Fig. 2A). The rate was essentially the same for the two dietary groups. After OJ exposure, the rate increased in both dietary groups (56 and 47%, respectively, for -Se and +Se mice).

INFLUENCE

OF

SELENIUM

ON

OZONE

40 I

BIOCHEMISTRY

.20

C .16

F-4 E .6 \ L? .I3

J

T.12 T !? .06

.4 .04 .2

FIG. 1. Selenium ppm (1568 &m3) vitamin E content

and vitamin E contents of mouse blood and O3 for 5 days. (A) Se content of whole blood; of lung tissue. Asterisk (8) refers to statistically

SuUhydryl Metabolism Mean TSH and NPSH contents measured in lung tissue homogenate were not different for the two dietary groups (Figs. 2B and C). After O3 exposure both parameters increased significantly in the two dietary groups. The GP activity in lung cytosol of the two dietary groups reflected their Se intake and was threefold higher in +Se than in -Se mice (Fig. 3A). After Oj exposure, there were small but statistically nonsignificant increases in both dietary groups. The activity of GR (Fig. 3B) was not affected by dietary Se. After OX exposure, a decrease (25% p < 0.05) was observed for -Se mice but an increase ( 18% NS) for +Se mice.

Activities ofthe Pentose Phosphate Cycle (PPC) Enzymes The PPC activity was assessed by measuring the activities of G6PD (Fig. 3C) and 6PGD

lungs after continuous exposure to 0.8 (B) Se content of lung tissue: and (C) significant (p < 0.05).

(Fig. 3D) in lung cytosol. In general, the activities of the two enzymes in control mice were not affected by the dietary Se levels. After OJ exposure, these activities in -Se mice remained essentially unchanged, but in -tSe mice significant increases were observed (7 I and 59%. p < 0.05, for G6PD and 4PGD, respectively). DISCUSSION The results of this study demonstrate that Se levels in the diet can influence the tissue and blood levels of Se as well as the pulmonary biochemical response to O3 exposure. The study also demonstrates that dietary Se intake has no effect on the growth of mice as indicated by their body weights. A small decrease in body weight of both dietary groups observed with Oj exposure can be attributed to a reduction in food intake during the oxidant stress. However, the lung enzymatic changes seen after Oj exposure might not be related to the decrease in body weight or food intake.

402

ELSAYED TABLE

2

BODY WHGHT, WET LUNG WEIGHT, AND TOTAL LUNG PROTEIN AND DNA CONTENTS IN SE-SUPPLEMENTED AND SE-DEFICIENT MICE AVER 0.8 ppm (I 568 @/m3) O3 EXFQSURECONTINUOUSLY FOR 5 DAYS

Snpplemented

Deficient

20.4 k 2.1 18.5 L I.7

19.3 ? 1.4 17.2 ic I.9

-9

-11

Parameter Body weight Cd

Control Exposed Percentage change’ Control Exposed Percentage change’ Control Exposed Percentage changeb Cow01 Exposed Percentage changeb

0.13 k 0.02 0.18 + 0.01

0.13 * 0.01 0.15 * 0.01

+38’

+15c

13.3 * 3.1 15.2 2 2.4

13.1 * 2.7 17.7 * 3.8

+14

+35c

1.48 k 0.69 1.43 k 0.45 -3

I .33 k 0.40 I.34 * 0.15 +I

a Data representmean k SQ n = 8. ‘Change in exposedrelative to control: (-) sign denotesa decrease, (+) sign denotes an increase.

’ StaMically significant @ < 0.05).

Kimball et ul. (1976) evaluated the effects of starvation on body weight and enzyme activities. They noted a decrease in the lung enzyme activities concomitant with the loss in body weight and concluded that a “starvation effect” could not account for the increase in enzyme activities observed after oxidant exposure. The general growth of the lung, as reflected by the lung weight, and total protein and DNA contents (Enesco and Leblond, 1962; Winick and Noble, 1965) were not affected by dietary Se for animals receiving room air only. The changes observed in lung weight and protein contents after Oj exposure might have resulted from an injury to the lung manifested by edema formation, influx of inflammatory cells, and/or proliferation of the alveolar type II cells replacing the injured type I cells (Kimball ef al., 1976: Schwartz et al., 1976; Evans et al., 1976, 1980).

ET

AL.

The lack of significant changes in lung tissue OZ consumption, and TSH and NPSH contents of unexposed control mice possibly indicate that Se levels did not affect some of these normal cellular functions. However, a significant difference was observed in lung GP activity, which was 70% lower in Se-deficient mice compared to Se-supplemented animals. The activities of the other lung enzymes, viz. GR, G6PD, and 6PGD, did not show changes with the dietary Se levels. After Oj exposure, the activities of GP and all other enzymes showed an increase in the lungs of Se-supplemented mice, although only G6PD and 6PGD activities were statistically significant. These activities remained essentially unchanged in Se-deficient mice. Thus, it appears that with the lack of increase in GP activity, the GR and PPC enzyme activities also remained unstimulated in Se-deficient mice when exposed to Oj. Jacob and Jandel ( 1966) in an earlier study showed that a decrease in the ratio of reduced to oxidized glutathione (GSH/GSSG) in the cell by means of a GSH blocker N-ethylmaleimide prevented the stimulation of PPC activity, and they proposed that sulfyhydryls regulated PPC metabolism. There are several possible mechanisms which lead to alterations of GSH/GSSG ratio in animal tissue, e.g., (a) nonenzymatic oxidation catalyzed by metal ions, such as Fe2’, Cu2+, Mn2+, or ascorbic acid; (b) the action of GSH-disulfide transhydrogenase: and (c) the action of GP. Under normal physiological conditions the activity of GP is probably the main reaction for utilization of GSH forming GSSG (Eggleston and Krebs, 1974), and is the only mechanism known to be influenced by dietary Se (Rotruck et ul., 1973; Chow and Tappel, 1974). The concentration of GSH in the cell far exceeds that of GSSG, and the reduction of GSSG via GR reaction utilizes NADPH, which is largely furnished by the PPC, i.e., G6PD and 6PGD activities. Cahill et ul. ( 1958) have established that the rate of PPC is controlled by G6PD activity: G6P + NADP+ =

6PG + NADPH.

INFLUENCE

SELENIUM

ON

A

I

OZONE

BIOCHEMISTRY

5

500

CONTROL i

OF

1.M

El

C

*

EXPOSE0

400

Fzz3

*

y P-4 . .: 300

.e

4

T u-l .-

* i? 3

.6

*

7 4

< c? ;

E 3

200

.4

E L

100

0

FIG. 2. Oxygen homogenate after (B) total sulthydryl

.2

1

0ppm

0.0

1.0ppn

1.0 ppm

0 PP

consumption, and total sulfbydryl and nonprotein sulfiydryl contents in mouse lung continuous exposure to 0.8 ppm (1568 pg/m3) Oj for 5 days. (A) Succinate oxidation: (TSH): and (C) nonprotein sulfiydryl (NPSH). Asterisk (*) refers to statistically significant

I3

C

cl

FIG. 3. Sulfhydryi metabolizing and pentow phosphate cycle enzyme activities in mouse lung cytosol after continuous exposure to 0.8 ppm (I 568 &m3) Oj for 5 days. (A) Glutathione peroxidase (GP) activity; (B) glutathione reductase (CR) activity; (C) glucose-6-phosphate dehydrogenase (G6PD) activity; and (D) 6-phosphogluconate dehydrogenase (6PGD) activity. Units are micromoles NADPH oxidized/minute lung for GP and CR, and micromoles NADP’ reduced/minute lung for G6PD and 6PGD. Asterisk (*) refers to statistically significant (p < 0.05).

404

ELSAYED

Since the substrate concentration far exceeds the Km for G6PD, Gumaa et ul. (197 1) have concluded that this control is achieved by a limitation of NADP+ rather than the substrate. In fact, the high ratio of free NADPH/free NADP’ can inhibit the PPC activity (Eggleston and Krebs, 1974). This finding implies that the PPC can be activated by “deinhibition” when NADPH is utilized at an adequate rate by the GP and GR reactions: 2GSH z GSSG GSSG + 2NADPH

2 2GSH + 2NADP+.

Based on our preliminary observations (Elsayed et ul., 1982a), we suggested that the relationship between GSH metabolism and PPC activity was dependent upon GP activity, and that it could be influenced by dietary Se under conditions of oxidant stress. The results of this study confirm our preliminary findings and further suggest that a dietary Se deficiency can influence the pulmonary biochemical response to OX in two ways: (a) directly by a diminution of GP activity, which is thought to be involved in cellular detoxification of lipid peroxides formed as a result of O3 exposure (Chow and Tappel, 1973); and (b) indirectly by preventing a stimulation of the PPC, which is involved in cellular repair mechanism and biosynthetic processes occurring in response to oxidant insult (Tierney et ul., 1973; Mustafa and Tiemey, 1978). In this regard, Se effect or influence seems to be confined to the GP and the ensuing GR and PPC activities, since other biochemical parameters examined are not affected by dietary Se. However, Reddy et al. ( 1982) have recently reported the influence of dietary Se and/or vitamin E on certain enzyme activities associated with xenobiotic metabolism in various organs, but the biochemical mechanisms involved are not clear. The increases in Se levels of lung tissue and whole blood, and vitamin E level of lung tissue observed after Oj exposure might be due to a mobilization under O3 stress. In previous studies (Elsayed and Mustafa, 1982; Sevanian et al., 1982a,b), vitamin E was found to increase in lung tissue of rats following NO2

ET

AL.

exposure. This increase was proposed to represent a mobilization of antioxidants. Such a mobilization of vitamin E and Se to the lung may be physiologically important since it is the target organ for oxidant insult. The increase in blood levels of Se and vitamin E occurs possibly because blood is the vehicle for transport to the lung. Several reports have indicated that the nutritional status of Se and vitamin E and their metabolism are interrelated through a sparing or compensatory mechanism (Scott, 1966, 1970; Combs et al., 1975; Gross, 1979). However, this mechanism was shown only indirectly as a functional compensation, such as through their interchangeable effects on nutritional requirements, body retention, and/or prevention of some deficiency symptoms of each other. In this study, direct measurements of Se and vitamin E levels confirmed the existence of such a compensatory mechanism in terms of actual contents in lung tissue. Disclaimer Although the research described in this article has been funded in part by the U.S. Environmental Protection Agency through a grant CR88066 to M. G. Mustafa, it has not been subjected to the Agency’s required peer and policy review, and therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred. ACKNOWLEDGMENT The authors wish to thank Dr. D. F. Tiemey for his valuable suggestions, Dr. Philip Harber for the use of his computer graphics, and Ms. Christine Quinn for her excellent technical assistance. This study was supported in part by grants from U.S. EPA (CR8806652) NSF (CHE 80- 1OOO), and Deutsche Krebshilfe.

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