This article was downloaded by: [New York University] On: 17 February 2015, At: 14:59 Publisher: Routledge Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Archives of Environmental Health: An International Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/vzeh20
Long-Term NO2 Exposure of Mice in the Presence and Absence of Vitamin E. II. Effect of Glutathione Peroxidase a
K. Lund Ayaz M.S. & A. Saari Csallany D.Sc.
a
a
Department of Food Science and Nutrition , University of Minnesota , St. Paul , Minnesota Published online: 17 Apr 2013.
To cite this article: K. Lund Ayaz M.S. & A. Saari Csallany D.Sc. (1978) Long-Term NO2 Exposure of Mice in the Presence and Absence of Vitamin E. II. Effect of Glutathione Peroxidase, Archives of Environmental Health: An International Journal, 33:6, 292-296, DOI: 10.1080/00039896.1978.10667350 To link to this article: http://dx.doi.org/10.1080/00039896.1978.10667350
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Long-Term N 0 2Exposure of Mice in the Presence and Absence of Vitamin E. II. Effect of Glutathione Peroxidase
Downloaded by [New York University] at 14:59 17 February 2015
K. LUND AY AZ, M.S. A. SAARI CSALLANY, D.Se. Department of Food Science and Nutrition University of Minnesota St. Paul, Minnesota
ABSTRACT Oile humMed aIIld twenty female mice fed diets containing various levels of vitamin E were continuously exposed to 0.5 ppm, 1.0 ppm nitrogen dioxide (N02)' and filtered air for 17 months. Blood, lung, and liver tissues were assayed for glutathione peroxidase (GSH-peroxidase) activity. Exposure to O.S ppm N0 2 did not affect blood and hmg GSH-pemxidase activity; 1.0 ppm N0 2 exposure, however, caused suppression of the enzyme. A combination of vitamin E deficiency and 1.0 ppm N0 2 exposure resulted in the lowest GSHperoxidase activities in the blood ;rna lung. High levels of vitamin E in the diet resulted ill elevated GSH-peroxidase in the blood and lung. Liver GSH-permddase activity was unaffected by either dietary vitamin E or N0 2 exposure. No inverse relationship was found between GSH-peroxidase levels and concentrations of organic solvent soluble lipofuscin pigments present in tissues.
GLUTATHIONE PEROXIDASE (Glutathione: hydrogenperoxide oxidoreductase, Ee 1.11.1.9) is an enzyme presently believed to play an important protective role in animal tissues because of its participation in interrupting the chain reactions involved in lipid peroxidative processes. 1 - 4 Lipid peroxidation is a basic deteriorative reaction in which cellular polyunsaturated fatty acids (PUF A) are oxidized. The reactions are initiated by free radicals and the lipids are oxidized to hydroperoxides. It is postulated that these hydroperoxides are further metabolized to malondialdehyde (MA) and that MA then reacts with amino groups of other biological molecules to form Schiff-Base class compounds known as lipofuscin pigments (LFP), which are believed by some to be metabolic end products of the lipid per oxidative processes. 5 , 6 Some data, however, do suggest the possibility that these pigments are not slowly accumulating end products of lipid peroxidation but in fact may be further metabolized. 7 -9 Vitamin E is believed to provide a defense against lipid peroxidation.because of its role as a free radical scavenger, which could interrupt the chain reactions occurring in peroxidi.zing systems or prevent them from being initiated. 10 Glutathione peroxidase (GSH-peroxidase), a selenoenzyme,11 has been shown to be involved in the conversion of hydroperoxides to hydroxy acids. l -3 Recently McCay and co-workers have shown some evidence to suggest that GSH-peroxidase also inhibits lipid peroxidative processes by preventing the initial free radical attack on the PUF A in membranes. 4 Whatever mechanism is involved, however,
292
GSH-peroxidase has been shown to be important in reducing lipid peroxidative damage in tissues. Some evidence for the adaptability of the GSH-peroxidase system to oxidative stresses has been shown in some animals. Nitrogen dioxide (N0 2 ) and ozone (0 3 ) are two oxidizing pollutants commonly found in polluted air. 12 Some of the damaging effects of these gases are believed to be due to their capability of forming free radicals, which are then able to initiate lipid peroxidative processes that lead to the damage of the lipids and of other oxidizable substances present in living systems. 13 , 14 Some investigators have reported elevated levels of GSH-peroxidase activity in monkeyslS , 16 and rats 17 , 18 exposed to 3 , indicating the possibility of a protective mechanism against the oxidative stress of pollutants. Lung tissue GSH-peroxidase seems to be the most affected by 03 exposure, whereas in other tissues such as blood the enzyme has not been affected. ls Exposure to N0 2 for short periods did not appear to affect lung GSH-peroxidase activity.!7 The experiments discussed in this paper were designed to investigate whether there is an effect of a continuous lowlevel N0 2 exposure on GSH-peroxidase activities in various tissues, and if changes in GSH-peroxidase activity would result in changes in the levels of lipofuscin pigments found in these tissues.
°
Materials and Methods One hundred and twenty mice of the C57BL/6J (Jackson Laboratories) strain were fed one of the following four
Archives of Environmental Health
}II
whole
2
3
4
10
15
20
blood/assay
5
6
7
8
9
25
30
35
40
45
60 ~
I
50
&\
&
&
'">C
1/1 1/1
I
C
...... 40
1/1
c:
Downloaded by [New York University] at 14:59 17 February 2015
:::I Q)
E
30
1 &
>-
N
c: W >C
a..
20
I
I
en
(!)
10
5
mg
wet
tissue / assay
Fig. 1. Glutathione peroxidase activity versus enzyme concentration in assay mixture. GSH·peroxidase assay as described in Methods sec· tion. (e - e) Whole blood (0 - 0) lung, (. - .) liver. *Assay conditions as described in Methods section with total volume of 5.0 ml.
diets: (1) a basal diet deficient in vitamin E, yet adequate in all other respects; (2) a basal diet supplemented with 30 ppm vitamin E; (3) a basal diet supplemented with 300 ppm vitamin E; or (4) a basal diet supplemented with 30 ppm N, N-diphenyl-p-phenylenediamine (DPPD), a synthetic antioxidant. The basal diet was prepared as described previously by Csallany et al. 9 and contained 8% vacuumdistilled corn oil. Elemental analysis showed the average selenium content of the diet to be 0.05 ppm. (Analysis of the diets for selenium content was performed by Dr. L. M. Scott of Cornell University, Ithaca, New York.) The mice were exposed continuously for 17 months to either 0.5 ppm N0 2 , 1.0 ppm N0 2 , or fIltered room air, as previously described. 19 At 18 months of age, the mice were killed by decapitation and blood samples were collected from the severed jugular vein. Portions of lung and liver were washed by immersion in fresh, cold, physiologic saline solution, were blotted dry, and were then frozen immediately at -70°C: Hematocrits of each blood sample were determined an4 the percentage of red blood cells (RBC) recorded. Fresh whole-blood samples were hemolyzed with cold dis-
November/December 1978
tilled water and assayed for GSH-peroxidase activity immediately, using the method described by Hafeman et al. 20 This method was modified by using cumene hydroperoxide as substrate instead of hydrogen peroxide. Tissue homogenates were prepared using a ground glass, motor~driven pestle to homogenize the frozen tissues in 0.15 M KCI immediately before assaying. The optimum concentrations of blood, lung, and liver enzyme for the assay were determined for each tissue (Fig. 1). Amounts of tissue used for the assays were determined from the linear portion of the curve for each tissue and were as follows: lung, 6 to 12 mg wet tissue per assay; liver, 0.5 to 1.5 mg wet tissue per assay; and blood, 3 to 6 pi whole blood per assay. The incubation mixture for measing GSH-peroxidase activity consisted of: 1.0 m1 of 2 mM reduced glutathione (prepared fresh daily); 1.0 m1 O.4M sodium phosphate buffer, pH 7.0, containing 4 X 1O- 4 M ethylenediaminetetraacetic acid (EDTA); 0.5 ml 0.01 N sodium azide; and 0 to 1.5 m1 blood hemolysate or tissue homogenate (volume of enzyme made up to 1.5 m1 with H 2 0). At t = 0 min, to start the assay, 1.0 ml of 7.3 mM cumene hydroperoxide
293
Downloaded by [New York University] at 14:59 17 February 2015
(0.1 ml of cumene hydroperoxide made up to 100 ml with water and made fresh daily) was added to a 37°C assay mixture that had been pre-incubated for 5 min. Total volume of assay mixture was 5.0 ml. Aliquots of the assay mixture were removed at 3-min intervals and added to 4.0 ml of a solution of metaphosphoric acid, NaCI, and EDTA (I.67 g metaphosphoric acid, 30 g NaCI, and 0.2 g EDTA, made up to 100 rn1 with H2 0) to stop the reaction by precipitation of the enzyme. After fIltration, a 2.0-ml aliquot of the fIltrate was added to 2.0 ml of 0.4 M Na 2HP0 4 and 1.0 ml of Ellman's reagent [0.04% 5, 5' -dithio-bis-(2-nitrobenzoic acid) in 1% sodium citrate]. The absorbance was measured at 412 nm, on a Bausch and Lomb Spectronic 20 spectrophotometer, and the concentration of GSH was determined, using a molar extraction coefficient of 13,600 liter· cm- 1 • rriole- 1 for GSH. One GSH-peroxidase enzyme unit was expressed as a decrease in the 10g([GSH] X 104 ) of 0.001 per min after subtraction of the non -enzymatic rate of GSH destruction. The non-enzymatic rate of GSH destruction was determined by using an incubation mixture containing distilled H2 instead of tissue homogenate or blood hemolysate. Reproducibility of the assay was 97% ± standard deviation of 3.0%; it was calculated for 27 assays, each done in duplicate.
°
Results Hematocrit values for the blood from the mice deficient in vitamin E generally were lower than the groups supplemented with vitamin E and DPPD under the same exposure
Table i.-Hematocrit Values for Blood from 18-Month-Old Mice* lHematocrit Valuest after Continuous Gas Exposure from Weaning [or 17 Months Filtered 0.5 ppm 1.0 ppm Diet air N02 N02 Deficient in vitamin E
40 ±2taA
39 ± 3a
46 ± I bA
Supplement of 30 ppm vitamin E
48 ± IB
45 ±2
49 ±1
Supplement of 300 ppm vitamin E
47 ±2B
45 ± 1
48 ±2AB
Supplement of 30 ppm DPPD§
45 ± IB
45 ±1
47 ±IA
Comment
* t
Six to ten animals in each group.
t
Means which share common small letter superscripts in the same line and common capital letter superscripts in the same column are not significantly different (P .05 using Student's t Test). Means with no superscript(s) indicate no significant difference.
%RBC, mean for group ± SEM.
Archives of Environmental Health: An International Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/vzeh20
Long-Term NO2 Exposure of Mice in the Presence and Absence of Vitamin E. II. Effect of Glutathione Peroxidase a
K. Lund Ayaz M.S. & A. Saari Csallany D.Sc.
a
a
Department of Food Science and Nutrition , University of Minnesota , St. Paul , Minnesota Published online: 17 Apr 2013.
To cite this article: K. Lund Ayaz M.S. & A. Saari Csallany D.Sc. (1978) Long-Term NO2 Exposure of Mice in the Presence and Absence of Vitamin E. II. Effect of Glutathione Peroxidase, Archives of Environmental Health: An International Journal, 33:6, 292-296, DOI: 10.1080/00039896.1978.10667350 To link to this article: http://dx.doi.org/10.1080/00039896.1978.10667350
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Long-Term N 0 2Exposure of Mice in the Presence and Absence of Vitamin E. II. Effect of Glutathione Peroxidase
Downloaded by [New York University] at 14:59 17 February 2015
K. LUND AY AZ, M.S. A. SAARI CSALLANY, D.Se. Department of Food Science and Nutrition University of Minnesota St. Paul, Minnesota
ABSTRACT Oile humMed aIIld twenty female mice fed diets containing various levels of vitamin E were continuously exposed to 0.5 ppm, 1.0 ppm nitrogen dioxide (N02)' and filtered air for 17 months. Blood, lung, and liver tissues were assayed for glutathione peroxidase (GSH-peroxidase) activity. Exposure to O.S ppm N0 2 did not affect blood and hmg GSH-pemxidase activity; 1.0 ppm N0 2 exposure, however, caused suppression of the enzyme. A combination of vitamin E deficiency and 1.0 ppm N0 2 exposure resulted in the lowest GSHperoxidase activities in the blood ;rna lung. High levels of vitamin E in the diet resulted ill elevated GSH-peroxidase in the blood and lung. Liver GSH-permddase activity was unaffected by either dietary vitamin E or N0 2 exposure. No inverse relationship was found between GSH-peroxidase levels and concentrations of organic solvent soluble lipofuscin pigments present in tissues.
GLUTATHIONE PEROXIDASE (Glutathione: hydrogenperoxide oxidoreductase, Ee 1.11.1.9) is an enzyme presently believed to play an important protective role in animal tissues because of its participation in interrupting the chain reactions involved in lipid peroxidative processes. 1 - 4 Lipid peroxidation is a basic deteriorative reaction in which cellular polyunsaturated fatty acids (PUF A) are oxidized. The reactions are initiated by free radicals and the lipids are oxidized to hydroperoxides. It is postulated that these hydroperoxides are further metabolized to malondialdehyde (MA) and that MA then reacts with amino groups of other biological molecules to form Schiff-Base class compounds known as lipofuscin pigments (LFP), which are believed by some to be metabolic end products of the lipid per oxidative processes. 5 , 6 Some data, however, do suggest the possibility that these pigments are not slowly accumulating end products of lipid peroxidation but in fact may be further metabolized. 7 -9 Vitamin E is believed to provide a defense against lipid peroxidation.because of its role as a free radical scavenger, which could interrupt the chain reactions occurring in peroxidi.zing systems or prevent them from being initiated. 10 Glutathione peroxidase (GSH-peroxidase), a selenoenzyme,11 has been shown to be involved in the conversion of hydroperoxides to hydroxy acids. l -3 Recently McCay and co-workers have shown some evidence to suggest that GSH-peroxidase also inhibits lipid peroxidative processes by preventing the initial free radical attack on the PUF A in membranes. 4 Whatever mechanism is involved, however,
292
GSH-peroxidase has been shown to be important in reducing lipid peroxidative damage in tissues. Some evidence for the adaptability of the GSH-peroxidase system to oxidative stresses has been shown in some animals. Nitrogen dioxide (N0 2 ) and ozone (0 3 ) are two oxidizing pollutants commonly found in polluted air. 12 Some of the damaging effects of these gases are believed to be due to their capability of forming free radicals, which are then able to initiate lipid peroxidative processes that lead to the damage of the lipids and of other oxidizable substances present in living systems. 13 , 14 Some investigators have reported elevated levels of GSH-peroxidase activity in monkeyslS , 16 and rats 17 , 18 exposed to 3 , indicating the possibility of a protective mechanism against the oxidative stress of pollutants. Lung tissue GSH-peroxidase seems to be the most affected by 03 exposure, whereas in other tissues such as blood the enzyme has not been affected. ls Exposure to N0 2 for short periods did not appear to affect lung GSH-peroxidase activity.!7 The experiments discussed in this paper were designed to investigate whether there is an effect of a continuous lowlevel N0 2 exposure on GSH-peroxidase activities in various tissues, and if changes in GSH-peroxidase activity would result in changes in the levels of lipofuscin pigments found in these tissues.
°
Materials and Methods One hundred and twenty mice of the C57BL/6J (Jackson Laboratories) strain were fed one of the following four
Archives of Environmental Health
}II
whole
2
3
4
10
15
20
blood/assay
5
6
7
8
9
25
30
35
40
45
60 ~
I
50
&\
&
&
'">C
1/1 1/1
I
C
...... 40
1/1
c:
Downloaded by [New York University] at 14:59 17 February 2015
:::I Q)
E
30
1 &
>-
N
c: W >C
a..
20
I
I
en
(!)
10
5
mg
wet
tissue / assay
Fig. 1. Glutathione peroxidase activity versus enzyme concentration in assay mixture. GSH·peroxidase assay as described in Methods sec· tion. (e - e) Whole blood (0 - 0) lung, (. - .) liver. *Assay conditions as described in Methods section with total volume of 5.0 ml.
diets: (1) a basal diet deficient in vitamin E, yet adequate in all other respects; (2) a basal diet supplemented with 30 ppm vitamin E; (3) a basal diet supplemented with 300 ppm vitamin E; or (4) a basal diet supplemented with 30 ppm N, N-diphenyl-p-phenylenediamine (DPPD), a synthetic antioxidant. The basal diet was prepared as described previously by Csallany et al. 9 and contained 8% vacuumdistilled corn oil. Elemental analysis showed the average selenium content of the diet to be 0.05 ppm. (Analysis of the diets for selenium content was performed by Dr. L. M. Scott of Cornell University, Ithaca, New York.) The mice were exposed continuously for 17 months to either 0.5 ppm N0 2 , 1.0 ppm N0 2 , or fIltered room air, as previously described. 19 At 18 months of age, the mice were killed by decapitation and blood samples were collected from the severed jugular vein. Portions of lung and liver were washed by immersion in fresh, cold, physiologic saline solution, were blotted dry, and were then frozen immediately at -70°C: Hematocrits of each blood sample were determined an4 the percentage of red blood cells (RBC) recorded. Fresh whole-blood samples were hemolyzed with cold dis-
November/December 1978
tilled water and assayed for GSH-peroxidase activity immediately, using the method described by Hafeman et al. 20 This method was modified by using cumene hydroperoxide as substrate instead of hydrogen peroxide. Tissue homogenates were prepared using a ground glass, motor~driven pestle to homogenize the frozen tissues in 0.15 M KCI immediately before assaying. The optimum concentrations of blood, lung, and liver enzyme for the assay were determined for each tissue (Fig. 1). Amounts of tissue used for the assays were determined from the linear portion of the curve for each tissue and were as follows: lung, 6 to 12 mg wet tissue per assay; liver, 0.5 to 1.5 mg wet tissue per assay; and blood, 3 to 6 pi whole blood per assay. The incubation mixture for measing GSH-peroxidase activity consisted of: 1.0 m1 of 2 mM reduced glutathione (prepared fresh daily); 1.0 m1 O.4M sodium phosphate buffer, pH 7.0, containing 4 X 1O- 4 M ethylenediaminetetraacetic acid (EDTA); 0.5 ml 0.01 N sodium azide; and 0 to 1.5 m1 blood hemolysate or tissue homogenate (volume of enzyme made up to 1.5 m1 with H 2 0). At t = 0 min, to start the assay, 1.0 ml of 7.3 mM cumene hydroperoxide
293
Downloaded by [New York University] at 14:59 17 February 2015
(0.1 ml of cumene hydroperoxide made up to 100 ml with water and made fresh daily) was added to a 37°C assay mixture that had been pre-incubated for 5 min. Total volume of assay mixture was 5.0 ml. Aliquots of the assay mixture were removed at 3-min intervals and added to 4.0 ml of a solution of metaphosphoric acid, NaCI, and EDTA (I.67 g metaphosphoric acid, 30 g NaCI, and 0.2 g EDTA, made up to 100 rn1 with H2 0) to stop the reaction by precipitation of the enzyme. After fIltration, a 2.0-ml aliquot of the fIltrate was added to 2.0 ml of 0.4 M Na 2HP0 4 and 1.0 ml of Ellman's reagent [0.04% 5, 5' -dithio-bis-(2-nitrobenzoic acid) in 1% sodium citrate]. The absorbance was measured at 412 nm, on a Bausch and Lomb Spectronic 20 spectrophotometer, and the concentration of GSH was determined, using a molar extraction coefficient of 13,600 liter· cm- 1 • rriole- 1 for GSH. One GSH-peroxidase enzyme unit was expressed as a decrease in the 10g([GSH] X 104 ) of 0.001 per min after subtraction of the non -enzymatic rate of GSH destruction. The non-enzymatic rate of GSH destruction was determined by using an incubation mixture containing distilled H2 instead of tissue homogenate or blood hemolysate. Reproducibility of the assay was 97% ± standard deviation of 3.0%; it was calculated for 27 assays, each done in duplicate.
°
Results Hematocrit values for the blood from the mice deficient in vitamin E generally were lower than the groups supplemented with vitamin E and DPPD under the same exposure
Table i.-Hematocrit Values for Blood from 18-Month-Old Mice* lHematocrit Valuest after Continuous Gas Exposure from Weaning [or 17 Months Filtered 0.5 ppm 1.0 ppm Diet air N02 N02 Deficient in vitamin E
40 ±2taA
39 ± 3a
46 ± I bA
Supplement of 30 ppm vitamin E
48 ± IB
45 ±2
49 ±1
Supplement of 300 ppm vitamin E
47 ±2B
45 ± 1
48 ±2AB
Supplement of 30 ppm DPPD§
45 ± IB
45 ±1
47 ±IA
Comment
* t
Six to ten animals in each group.
t
Means which share common small letter superscripts in the same line and common capital letter superscripts in the same column are not significantly different (P .05 using Student's t Test). Means with no superscript(s) indicate no significant difference.
%RBC, mean for group ± SEM.
