T I S S U E D I F F E R E N C E S I N A N T I O X I D A N T E N Z Y M E G E N E E X P R E S S I O N
I N R E S P O N S E T O E N D O T O X I N
BIDYUT GHOSH, CORAL DAWN HANEVOLD, KAZUSHIGE DOBASHI, JOHN KENNETH ORAK, and INDERJIT SINGH
Department of Pediatrics, Medical University of South Carolina, Charleston, SC 29425, USA
(Received 7 August 1995; Revised 10 November 1995; Re-Revised 9 January 1996; Accepted 24 January 1996)
Abstract--The effect of endotoxin on antioxidant gene expression and antioxidant enzyme activity in homogenates of the heart, liver, and kidney from Sprague-Dawley rats was compared by quantitation of m-RNA and enzyme activities. Alterations in the message level for Cu-Zn superoxide dismutase (SOD), Mn SOD, and catalase varied with the tissue type, length of exposure to endotoxin, and dose of endotoxin. In general, endotoxin treatment reduced Cu-Zn SOD expression in the heart and liver, but had no noticeable effect in the kidney. Mn SOD message levels were increased in the heart and kidney but decreased in the liver. Catalase expression was reduced in the kidney and increased marginally in the heart and liver. With regard to enzyme activity, endotoxin treatment reduced Cu-Zn SOD activity in the heart, liver, and kidney. Mn SOD activity showed little change in the heart, but increased in the liver and, to a lesser extent, in the kidney. Catalase activity showed little change in the heart and kidney but was decreased at 12 h in the liver. The differing responses of tissues to the oxidant stress of endotoxin exposure should be considered when evaluating the effect of endotoxin on antioxidant enzymes.
There is increasing evidence that cellular damage in endotoxin-induced shock is due, at least in part, to the generation o f free oxygen radicals.l'2 Paradoxically, a sublethal dose o f endotoxin has also been shown to provide protection against oxidant stresses such as myocardial ischemia-reperfusion injury and hyper- oxia. 3-5 Investigators have linked this protective effect with an increase in the activity of various antioxidant enzymes including catalase, superoxide dismutase (SOD), glutathione peroxidase (GPX), and glucose-6- phosphate dehydrogenase. 3 5 However, there has been
1 3~5 considerable variation in these findings, ' perhaps be- cause o f differences in the overall responsiveness o f various tissues and animal species to oxidative stress and endotoxin. 5'7-~° We evaluated the effect of endo- toxin treatment on the gene expression and activity of
Address correspondence to: Inderjit Singh, Ph.D., Medical Uni- versity of SC, Pediatrics Department, Director, Division of Devel- opmental Neurogenetics, 171 Ashley Ave., Charleston, SC 29425.
the antioxidant enzymes, Cu-Zn SOD, Mn SOD, and catalase in the rat heart, liver, and kidney.
MATERIALS AND METHODS
Endotoxin treatment
Endotoxin (Salmonella typhimurium phenol extract, Sigma Chemical Co., St. Louis, MO) was dissolved in 5 m M phosphate buffer, pH 7.4 and injected intraperi- toneally into S p r a g u e - D a w l e y rats (male, 2 5 0 - 2 7 5 g) at doses o f 250, 500, and 750 #g/kg. Saline (0.9% NaC1) was given to another set o f rats who served as controls. After 12 or 24 h o f endotoxin or saline treat- ment the rats were sacrificed, and the heart, liver, and kidneys were immediately removed.
RNA isolation
The organs were placed into ultraspecII R N A solu- tion (Biotecx Corporation), and then total R N A was
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isolated according to Biotecx Corporation's protocol. Briefly, the tissue was homogenized in a guanidium isothiocyanate and phenol containing solution by a polytron homogenizer. After centrifugation, the RNA was precipitated from the aqueous phase with isopro- panol. The RNA pellet was washed in 70% ethanol, dried and resuspended in DEPC-treated water.
Reverse transcript ion-polymerase chain reaction
Total RNA (5 #g) was reverse transcribed with oligo dT primer using 1 mM of each dNTP, 40 U of RNase inhibitor (Promega Corporation), 50 U of Moloney mu- rine leukemia virus (M-MLV) reverse transcriptase ob- tained from Stratagene, 1 × reverse transcriptase buffer (Stratagene) in a 50 #1 reaction volume. The reaction was carried out at 37°C for 1 h.
For PCR, 5/~1 of cDNA from the RT reaction mix- ture was taken to amplify in a 100 #1 reaction volume containing 0.2 /.tM of each primer, 200 #M of each dNTP, IX buffer containing 1.5 mM MgC12 (Strata- gene), and 2.5 U of Taq Polymerase (Stratagene). The reaction was denatured at 91°C for 5 rain and at 54°C for 5 min for primer annealing. A total of 35 cycles were run, with each cycle having denaturation at 91°C for 1 min, annealing at 54°C for 1 min, and extension at 72°C for 2 rain. Final extension was carried out at 72°C for 10 min. RT-PCR was carried using the pro- tocol supplied by Stratagene. Gene specific primers were synthesized at the DNA-core facilities at the Med- ical University of South Carolina.
A 10 #1 portion of the PCR product was electropho- resed in a 1.5% agarose gel. The ethidium bromide stained gel was photographed with a DS-34 type Po- laroid camera and the band was scanned with a Biorad (Model GS-670) imaging densitometer.
G l y c e r a l d e h y d e p h o s p h a t e d e h y d r o g e n a s e (GAPDH) was used as the internal standard for all RT- PCR reactions. To quantitate the message level, the ra- tio of the corresponding gene product to the GAPDH gene product was calculated. Experiments were per- formed in duplicate.
Measuremen t o f antioxidant enzyme activit ies
Tissues were homogenized in 10 vol (w/v) of ice cold homogenizing buffer consisting of 0.25 M su- crose, 3 mM imidazole, 1 mM EDTA, 0.1% ethanol, and protease inhibitors (0.2 mM phenylmethyl sulfo- nyfluoride, 0.7 #g/ml pepstatin, 1 #g/ml leupeptin, 1 #g/m antipain, and 2 #g/ml aprotinin) at pH 7.4. The catalase activity was measured according to the method of Baudhuin et al. 11 The total superoxide dismutase ac- tivity was determined by monitoring the rate of reduc-
tion of nitroblue tetrazolium by superoxide radical, util- izing a xanthine-xanthine oxidase system as the source of 0 2 ] 2 Cu-Zn SOD and Mn SOD activities were dif- ferentiated by measuring the enzyme activity in the presence of 2 mM NaCN. One enzyme unit of super- oxide dismutase is defined as the amount that showed 50% inhibition at room temperature, pH 7.8. Protein was estimated using the method of Bradford. ~3
RESULTS AND DISCUSSION
Evaluation of the effect of endotoxin alone (without a subsequent oxidant stress) on antioxidant enzyme ac- tivity has yielded variable results. ~'5'14-16 As an exam- ple, work in our laboratory has demonstrated that en- dotoxin exposure increased total SOD activity, decreased catalase activity, and did not alter GPX ac- tivity in rat liver homogenates. 17 In contrast, Daryani et al. found that endotoxin treatment decreased catalase activity in sheep lung but not in liver homogenates) Several investigators have noted variability in the re- sponsiveness of different tissues t° and species 5'8'9 to en- dotoxin. Therefore, endotoxin-induced alterations in antioxidant enzyme activity may be expected to vary, depending on the sensitivity of the tissue and animal species to endotoxin.
In this study we evaluated the effect of endotoxin on antioxidant enzyme gene expression and enzyme activities in three different tissues within one animal species. At 12 and 24 h after treatment with endotoxin the rats were sacrificed and gene expression of three antioxidant enzymes was quantitated by reverse tran- scriptase PCR. As shown in Fig. 1, the major effect of endotoxin on Cu-Zn mRNA levels occurred in the heart. Endotoxin treatment decreased Cu-Zn SOD ex- pression in heart homogenates at both time points, with greater inhibition observed at higher doses. In liver, little effect was noted after 12 h, but expression was inhibited after 24 h, though to a lesser extent than in the heart. No effect was noted in the kidney homoge- nates at either time point. Effects of endotoxin on Mn SOD mRNA varied between tissue types (Fig. 2). In the heart, gene expression was increased at higher doses after 24 h with no effect seen at 12 h. In liver, Mn SOD mRNA levels were decreased 24 h after en- dotoxin treatment. In the kidney, Mn SOD expression was increased 12 h after treatment with the maximal dose of endotoxin, but negligible changes were seen at 24 h. As shown in Fig. 3, catalase mRNA levels were increased in the heart marginally at the maximal dose of endotoxin after 24 h. Similarly, in the liver, mRNA levels were slightly increased after 24 h at all doses. In contrast, catalase gene expression in the kidney was inhibited at all doses at both time points.
Antioxidant gene expression 535
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mRNA expression of Cu-Zn SOD in rat heart, liver & kidney
Fig. 1. Quantitation of Cu-Zn SOD gene expression after exposure to saline (controls) or endotoxin at doses of 250, 500, and 750 #g/kg for 12 and 24 h in the heart, liver, and kidney. The ratio of the Cu-Zn SOD gene product to the internal standard, glyceraldehyde phosphate dehydrogenase (GAPDH) gene product was used to quantitate the message level. The values given in the parentheses are the result of duplicate experiments from two different sets of animals.
536 B. GHOSH et al.
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mRNA expression of Mn-SOD in rat heart, liver, & kidney Fig. 2. Quantitation of Mn SOD gene expression after exposure to saline (cont) or endotoxin at doses of 250, 500, and 750 #g/ kg for 12 and 24 h in the heart, liver and kidney, The ratio of the Mn SOD gene product to the internal standard, glyceraldehyde phosphate dehydrogenase (GAPDH) gene product was used to quantitate the message level. The values given in the parentheses are the result of duplicate experiments from two different sets of animals.
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Antioxidant gene expression 537
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mRNA expression of Catalase in rat heart, liver, & kidney
Fig. 3. Quantitation of catalase gene expression after exposure to saline (cont) or endotoxin at doses of 250, 500, and 750 #g/ kg for 12 and 24 h in the heart, liver, and kidney. The ratio of the catalase gene product to the internal standard, glyceraldehyde phosphate dehydrogenase (GAPDH) gene product was used to quantitate the message level. Tile values given in the parentheses are the result of duplicate experiments from two different sets of animals.
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Fig. 4. Enzyme specific activities of Cu-Zn SOD (U/mg protein), MnSOD (U/my protein), and catalase (U/rain/my protein) in the heart, kidney, and liver 12 and 24 h after intraperitoneal injection of saline (control) or endotoxin at doses of 250, 500, or 750 g/kg of body weight. Values represent the mean of experiments performed in duplicate.
Antioxidant enzyme activities after treatment with endotoxin are plotted in Fig. 4. As shown, the effect of endotoxin varied with the tissue type, dose and length of exposure. Overall, Cu-Zn SOD activity was decreased in the heart and liver 12 and 24 h after en- dotoxin treatment. In the kidney, 12 h after treatment Cu-Zn SOD activity was reduced at all doses, with a slightly greater effect at the maximal dose. In contrast, at the 24-h time point a decrease in activity was ob- served only with the maximum dose of 750 #g. With regard to Mn SOD, enzyme activity in the heart
showed a decrease at a dose of 500 #g at 12 h, but little change was observed at other doses and time points. In the liver, Mn SOD activity was increased at all doses at 12 h and at 250 and 500 #g doses at 24 h. In the kidney, Mn SOD activity was increased at all doses 12 h after endotoxin treatment, while negligible changes were observed in the same tissue at 24 h. With regard to catalase, enzyme activity was decreased at 12 h in liver, with the greatest effect at the 500 #g dose; little change was observed at the 24-h time points. In the kidney, there was little change in cata-
Antioxidant gene expression 539
lase activity except for a minor decrease in activity at 750 #g after 12 h. In heart there was no change at all time points and doses.
When the effect of endotoxin on gene expression and enzyme activity were compared, it was noted that the changes were not always similar under the experi- mental conditions tested (Figs. 1--4). For example, Mn SOD mRNA levels were stable or minimally increased in the heart at 12 h, while enzyme activity was de- creased at the 250 and 500 #g doses at 12 h. Similarly, in the liver Mn SOD mRNA values showed very little effect at all doses at 12 h, while Mn SOD activity was increased at the same points. Other investigators have also documented that alterations in gene expression af- ter an oxidant stress are not always reflected in a change in enzyme activity. ~4'18 Iqbal et al. found that endotoxin as a sole stimulus increased lung Cu-Zn SOD mRNA without a corresponding change in enzyme activity. ~4 Unpublished observations by these same investigators indicate that exposure to hyperoxia alone stimulates translation of Cu-Zn SOD synthesis without increasing mRNA levels. These discrepancies in the effect of an oxidant stress on antioxidant gene expression and en- zyme activity may be due to inhibition of enzyme ac- tivity/synthesis or enhanced degradation of the en- zymes or mRNA. 18'w Clerch et al. suggest that variation in the redox status of the cell may alter antioxidant enzyme mRNA binding proteins, thus affecting the sta- bility of mRNA and indirectly enzyme synthesis and activity. ~9
The data presented here suggests that there is consid- erable variability in the capacity of different tissues within the same animal species to modify antioxidant enzyme gene expression in response to an oxidative stress. In con- trast to our findings, previous reports in the literature based on work in cell culture have suggested that endo- toxin selectively induces Mn superoxide dismutase (Mn SOD) mRNA levels with minimal or no effect on the gene expression of Cu-Zn S O D 7'2°'21 or catalase. 21 Aug- mentation of Mn SOD gene expression has been reported in a variety of cell types. 7'2°'21 However, the reported en- hancement of MnSOD mRNA was not universal for all cell types; 7 Visner et al. found that endotoxin dramatically increased Mn SOD mRNA in rat pulmonary epithelial cells but had only a minimal effect on rat pulmonary fi- broblasts. 7 In contrast to these studies in cell culture, work performed in homogenized rat lung by Iqbal et al. dem- onstrated that endotoxin increased Cu-Zn SOD mRNA levels; 14 no other antioxidant enzymes were evaluated in this study. The conflicting results in cell culture and ho- mogenized organs may be related in part to differences in the dose of endotoxin that is delivered to the organ or cell as well as the variation in responsiveness of different cell types to endotoxin.
Earlier work in our laboratory suggests that variation in the antioxidant enzyme response to endotoxin also occurs on a subcellular level.~7 Recently, the presence of Cu-Zn SOD z2 along with glutathione peroxidase (GPX) 23 and Mn SOD (Singh, personal communica- tion) in peroxisomes has been documented. We found that endotoxin treatment enhanced the activity and pro- tein level of peroxisomal Cu-Zn SOD and GPX but had little effect on the mitochondrial and cytosolic fractions of these enzymes, j7 Because Cu-Zn SOD, Mn SOD, and GPX are located in more than one subcellular or- ganelle, these proteins will differ with regard to the topogenic signal that directs their transport to the ap- propriate subcellular site. Although the existence of different proteins suggests that there is more than one mRNA species, it is not known whether there is more than one gene for the antioxidant enzymes. The pos- sibility that one gene is transcribed into different mRNA species through alternate splicing warrants fur- ther evaluation.
In summary, evaluation of the effect of endotoxin on antioxidant enzymes must take into account the vari- able responsiveness of tissues and animal species to this oxidative stimuli. Such differences may contribute to the conflicting findings reported in the literature.
Acknowledgements - - This work was funded in part by grants from the Children's Hospital Fund, Medical University of South Carolina, D.C.I. and from National Institutes of Health N.S. 22576.
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