Superoxide dismutase, glutathione peroxidase, and glutathione reductase in sheep organs

Comp. Biochem. Physiol. Vol. l15B, No. 4, pp. 451-456, 1996 Copyright © 1996 Elsevier Science Inc.

ISSN 0305-0491/96/$15.00 PII S0305-0491(96)00132-0 ..

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ELSEVIER

Superoxide Dismutase, Glutathione Peroxidase, and Glutathione Reductase in Sheep Organs K. Holovskd,* V. Lendrtovd,* J. R. Pedrajas,~ J. Peinado,~c J. L6pez-Barea,~7 I. Rosival,* and J. Legdth* *DEPARTMENT OF CHEMISTRY, BIOCHEMISTRY AND BIOPHYSICS,UNIVERSITY OF VETERINARY MEDICINE, KOSlCE, SLOVAKIA AND tDEPARTMENT OF BIOCHEMISTRYAND MOLECULARBIOLOGY,FACULTYOF VETERINARY MEDICINE, CORDOBA, SPAIN

ABSTRACT. The enzyme activities of the superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione reductase (GR) and thiobarbituric acid reactive substances (TBARS) content were measured in tissue extracts of the liver, kidney and lung of sheep in a nonpoUuted control area (C), a polluted area pasture (PP) and those from polluted areas but fed in the laboratory with an experimental emission supplement diet (EEF). Compared with the control SOD, activity was significantly increased (1.75 times) only in the liver of the PP group. In the EEF group there was a tendency toward lower activities in all organs. The Cu,Zn-SOD isoenzymes pattern analyzed by isoelectrofocusing was different in the organs of the animals exposed to pollutants when compared with those of the controls. In the liver, two new isoenzymes with pI 5.30 and 5.70 were found in the PP group and an additional isoenzyme with pI 5.10 in the EEF group. The kidney isoenzymes with pl 5.30 and 5.40 were inhibited in the EEF group. In the lung, two new isoenzymes appeared with pl 5.30 and 5.40 in the PP group and two new isoenzymes with pI 6.10 and 6.50 in the EEF group. GSHPx activity was inhibited in the liver and kidney of the sheep exposed to pollutants. GR activity was significantly changed only in the liver. The activity in the PP group was 2.30 and 2.10 times higher than in the C and EEF groups, respectively. TBARS content was increased in the liver and kidney of the EEF group compared with the control. Copyright © 1996 Elsevier Science Inc. COMPmOCHEMPHYSIOL115B;4:451-456, 1996 KEY WORDS. Superoxide dismutase, glutathione peroxidase, glutathione reductase, thiobarbituric acid reactive substances, sheep, liver, kidney, lung, heavy metals pollutants INTRODUCTION Animals can be exposed to toxic concentrations of heavy metals by the consumption of contaminated feed and water. Frequent sources of heavy metals intoxication are compounds of Cd, Pb, Hg, Cu and others (6). It is known that heavy metals can generate the extremely reactive hydroxyl radicals (in a reaction known as the Fenton reaction) that attack cell components (31 ). To prevent oxidative damage, the cells are equipped with a scavenging system (32), consisting of low molecular weight antioxidants such as glutathione (22), ascorbic acid (39) and others; cytosolic enzymes such as glutathione peroxidase (GSHPx) (9), glutathione-S-transferase (16) and superoxide dismutase (SOD) (21) and ancillary enzymes such as glutathione reductase (GR) or glucose-6-phosphate dehydrogenase (7,32). The aim of our work was to compare the enzyme activities of SOD, GSHPx and GR in the liver, kidney and lung of sheep in a polluted area, sheep originating from the same polluted area and receiving subchronic doses of emission Address reprint requests to: K. Holovskfi; Department of Chemistry, Bio-

chemistry and Biophysics, University of Veterinary Medicine, Kosice, Slovakia. Received 30 November 1995; accepted 1 May 1996

and those from a nonpolluted area. Activities were observed in the liver, because it is one of the tissues showing a high rate of free radical generation, with high metabolic capability and a detoxifying capacity; in the kidney, which is preferentially exposed to xenobiotics and the place of highest Hg accumulation, and in the lung, which is primarily exposed to the environment and may be subject to irritation and specific toxicity.

MATERIALS AND METHODS Animals and Diets Fifteen Slovak Merino sheep divided into three groups were included in the experiment (3 years old, 44-65 kg b.w.). The control group consisted of five sheep from a nonpolluted area (C group). Another 10 sheep came from an industrial area polluted by heavy raetals with a predominance of Hg and Cu. Five sheep taken from the polluted pasture were killed immediately (PP group). Another five sheep also taken from the polluted pasture were subjected to experimental emission feeding (EEF group) for 28 days before killing. They were given a balanced diet (CSN 46 70 70) of hay, molasses food and industrial emission in capsules of following composition: Hg (675 mg), Pb (438 mg), Cd (74

K. Holovsk~ et al.

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mg), Cu (2218 mg), Zn (1478 mg) and Cr (125 mg) per kg of body weight per day. Doses were - 2 0 times higher than was the daily intake of emissions under natural conditions. Water was supplied ad libitum. Immediately after slaughtering, liver samples taken from the region of the vena portae, lung samples from the upper lobes and the kidney were all homogenized.

Preparation of Tissue Extracts The liver, kidney and lung were washed two times with cooled physiological solution, cut into pieces and homogenized in Ultra-Turrax homogenizer to make a 25% (w/v) homogenate in 5 mM Tris-HC1 buffer, pH 7.8, containing 0.15 M KCI, 1 mM EDTA and 2 mM GSH. Homogenates were centrifuged for 15 min at 9000 g. Supematants were filtered through glass wool and centrifuged again for 60 rain at 105,000 g using a Beckman L8-60 ultracentrifuge. All procedures were performed at 4°C. The supematants were taken for all the biochemical assays.

Protein concentration was determined by the method of Bradford (4). Mean values for each enzyme activity were obtained from at least five separate determinations.

Statistics The results are means -+ SEM. Statistical analysis was done by Student's t-test with a significance level of P < 0.05.

Isoelectric Focusing Isoelectrofocusing was carried out in a PhastSystem equipment (Pharmacia) in gels with pH gradient 5-8. pI values of the SOD isoenzymes were determined in gels with pH gradient using isoelectric focusing calibration kits (Pharmacia). Staining for SOD activity was done immediately after electrophoresis using nitroblue tetrazolium, riboflavin and TEMED. Incubation time with nitroblue tetrazolium and riboflavin was only 5 rain (1).

Enzyme Assays

Assay of Metals

SOD ( O s -O~ oxido-reductase, EC 1.15.1.1) was measured at 550 nm (25°C) according to Floh4 and Otting (11), through the inhibition in the reduction rate of cytochrome c. Aliquots of the supernatants were added to a cuvette, containing a 50 mM sodium phosphate buffer (pH 7.8), 0.1 mM EDTA, 20/aM cytochrome c, 50 2/aM xanthine and xanthine oxidase. One unit of SOD was defined as the amount of enzyme that inhibits the rate of cytochrome c reduction, under the condition specified, by 50%. Total GSHPx (glutathione: H202 oxidoreductase, EC 1.11.1.9) was measured according to Flohd and Gunzler (10). Freshly prepared GR solution was added to a medium containing 0.1 M sodium phosphate buffer (pH 7.0), 10.0 mM GSH, 1.5 mM NADPH and after incubation 10 min at 37°C, 40.0 mM cumene hydroperoxide was added. The decrease in absorption at 340 nm was monitored. GR (NAD(P)H:oxidized glutathione oxidoreductase, EC 1.6.4.2) was determined at 30°C following the decrease in NADPH absorbance at 340 nm due to 2 GSSG reduction in 0.1 M potassium phosphate buffer (pH 7.5), containing 1 mM EDTA, 0.125 mM NADPH and 2.5 mM GSSG in 1 ml final volume (27). Enzyme activities were expressed in U/mg of prot. or mU/ mg of prot., respectively. Lipid peroxide formation was measured as malondialdehyde and other aldehydes, by reaction with thiobarbituric acid yielding colored products that absorb at 535 nm, named thiobarbituric acid reactive substances (TBARS) (15), with minor differences: incubations were made in 3 ml total volume and 2.8% (w/v) trichloroacetic acid was used instead of HCI. Content of TBARS was expressed in absorbance/ g wet tissue.

Metals (Hg, Pb, Cd, Cu, Zn, Cr) were determined by the atomic absorption spectroscopy method using the Varian spectrophotometer (18).

Chemicals All reagents used were of the highest analytical grade available and were obtained from Sigma, Merck and Boehringer. RESULTS A N D D I S C U S S I O N

A few studies have examined the effects of heavy metals on the antioxidant enzyme activities in various organisms (14,29,30,36). However, the influence of chronic or subchronic heavy metals exposure to antioxidant enzyme activities in sheep was not described. The accumulation of heavy metals in the organs of sheep in the metal-contamined area (PP) was observed (Tables 1 and 2). In the liver, compared with the control, the concentration of Hg was 15 times higher and Cu 500 times higher. In the kidney, the concentration of Hg was 27 times higher and Cu 10 times higher. Since Hg and Cu catalyze the generation of oxygen active species, animals living in metalpolluted areas would be subjected to oxidative stress. Numerous studies indicate that in both prokaryotes and eukaryotes, oxidative stress induces or enhances the activity of SOD (3,29,30). SOD as a scavenger of superoxide radicals in biological tissues is an important factor in the protection against free radical damage (12). Figure 1 shows the specific activity of SOD found in the liver, kidney and lung. The activity of SOD compared with other observed antioxidant enzymes was relatively high.

Superoxide Dismutase, Glutathione Peroxidase, Glutathione

453

TABLE 1. Metal Contents in Liver Body Weight (mg/kg)

C group (n = 5) PP group (n = 5) EEF group (n = 5)

Hg

Pb

Cd

Cu

Zn

Cr

0.016 _+ 0.005 0.016 + 0.009 0.004 -+ 0.001 0.366 -+ 0.145 18.040 -+ 4.158 0.010 + 0.000 0.246 _ 0.106" 0.020 _+ 0.073* 0.006 -+ 0.006* 188.78 -+ 33.70* 24.90 + 4.36* 0.262 + 0.067* 0.788 _+ 0.096"t 0.166 -+ 0.064"t 0.008 _+ 0.002*t 368.07 -+ 45.80"t 60.80 _+ 13.59"t 0.236 ± 0.059"t

*Significantly different from respectivecontrol (P < 0.05). "~Significantlydifferent from respective PP group (P < 0.05).

The highest activity was in the kidney and the lowest in the liver and lung. A significant increase of SOD activity was observed only in the liver. The activity in the PP group was 1.75 times higher than in the C group. The activities in the EEF group were lower in all organs, but not significantly. Eukaryotes have two major kinds of SOD, a dimeric form containing two atoms of Cu and two atoms of Zn (Cu,ZnSOD) and a tetrameric form that contains one Mn atom per subunit (Mn-SOD). Cu,Zn-SOD is found predominantly in the cytosolic fraction and shows very sensitive behavior to cyanide (8,17). Mn-SOD is associated with the mitochondria and is not sensitive to cyanide (33). Cu,Zn-SOD is not a single entity. This enzymatic activity shows several isoenzymatic forms in tissues from different organisms (20,25). In our experiments we noticed a difference between Cu,Zn-SOD isoenzyme patterns in the examined organs of sheep not exposed to pollutants analyzed by isoelectrofocusing. In the control group (C), two isoenzymes were found in the liver: one Mn-SOD with pI 6.20 and one Cu,ZnSOD with pI 5.45 (Fig. 2). Two isoenzymes were found in the lung: one Mn-SOD with pI 6.20 and one Cu,Zn-SOD with pl 5.45 (Fig. 3A). Four isoenzymes were detected in the kidney: one Mn-SOD with pI 6.20 and three Cu,ZnSOD with pI 5.30, 5.40 and 5.45 (Fig. 3B). Further, our results showed that the Cu,Zn-SOD isoenzyme pattern of the organs examined differed between the animals exposed to pollutants and those from nonpolluted areas. In the liver, from the PP group, two new isoenzymes with pI 5.30 and 5.70 were detected. All the isoenzymes (pI 5.30, 5.45, 5.70) were more intense in the EEF group. In addition, a new isoenzyme with pI 5.10 was found in the EEF group. In the kidney, there was no change in the isoenzymes of the PP group compared with the control. The isoenzymes with pI 5.30 and 5.40 were inhibited in the EEF group. In the lung in the PP and EEF groups, the band of

5.45 was more intense, and two new isoenzymes with pl 5.40 and 5.30 were found compared with the control. In addition to these changes, two new isoenzymes with pl 6.10 and 6.50 were observed in the EEF group. Those of pl 6.10 and 6.50 were of slight intensity. Mn-SOD with pl 6.20 was the same in all groups. As our results show (Fig. 1), total SOD activity was higher in all organs of sheep exposed to pollutants. This increase in activity did not affect the isoenzyme pattern in the same way. In the organs examined, the different induction or inhibition of new isoenzymes was observed; this suggests that they might respond to the metal contaminated environments in different ways (Table 3). It is possible that the new Cu,Zn-SOD isoenzymes are generated as a result of the oxidative stress conditions provoked by the metals. In fact, new more-acidic Cu,Zn-SOD isoforms were generated by the incubation of purified fish Cu,Zn-SOD or cellfree crude extract with moderate H202 concentrations, making the SOD forms disappear at higher oxidant concentrations. The new SOD forms had similar pI to those present in fish that live in polluted littoral areas (mainly by copper pollution) (25,26). GSHPx plays a major role in protecting cells from oxidative damage, especially lipid peroxidation of biological membranes (9). Based on their substrate specificity, two types of GSHPx are generally recognized: selenium-dependent (24) and selenium-independent enzymes (19). In our experiments, GSHPx activity was determined by cumene hydroperoxide, which is active with both seleniumand non-selenium-dependent enzymes, and thus the "total" GSHPx activity was measured (Fig. 4). The enzymatic activities of the control group (C) were rather low; the highest specific activity was observed in the lung and the lowest in the kidney and liver. As with other authors, we also observed the inhibition of GSHPx activity in the presence of the heavy metals (14,34,35,36). In the liver and kidney of

TABLE 2. Metal Contents in Kidney Body Weight (mg/kg)

C group (n = 5) PP group (n = 5) EEF group (n = 5)

Hg

Pb

Cd

Cu

Zn

0.046 _+ 0.038 0.022 +- 0.013 0.002 -+ 0.0005 4.36 -+ 1.88 14.82 -+ 2.16 1.24 _+ 0.351" 0.148 -+ 0.046* 0.006 _+ 0.006* 42.50 -+ 10.56" 18.80 +_ 0.972* 3.35 _+ 0.667"t 0.566 +- 0.154"t 0.017 -+ 0.005*t 188.78 -+ 33.71"t 19.44 + 1.51"t

*Significantly different from respectivecontrol (P < 0.05). tSignificantly different from respective PP group (P < 0.05).

Cr

0.012 +_ 0.005 0.184 _+ 0.083* 0.252 _+ 0.072"t

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FIG. 1. Superoxide dismutase activity in the liver, kidney and lung of tissue extracts. The assays were performed as described in Materials and Methods. C, nonpolluted a r e a - control group; PP, polluted pasture group; EEF, experimental emission feeding group. Asterisks situated between groups represent significant differences between these two groups. * P < 0.05.

sheep exposed to pollutants (groups PP and EEF), total inhibition of GSHPx activity was noticed. In the lung, the activity was 4.60 times lower in the PP group than in the C group. So although GSHPx was inhibited, the organs should be defended against lipid peroxidation by other alternative routes, such as microsomal glutathione transferase, which also has peroxidase activity (23). It is known that G R reduces the GSSG produced by GSHPx during detoxification of H202 and organic hydroperoxides (38). Thus, a similarity in organ distribution could be expected between GR and GSHPx. We did not observe this similarity in our experiments. The liver activity, in the PP group, was 2.30 times higher than the control and 2.10 times higher than in the EEF group. Differences

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FIG. 3. (A) SOD isoenzyme patterns in tissue extracts of the lung. Zymograms obtained after separation by IEF in an 8.05.0 pH gradient, pI values were determined by using isoelec. tric focusing protein markers (from Pharmacia). C, nonpolluted area--control group; PP, polluted pasture group; EEF, experimental emission feeding group. (B) SOD isoenzyme patterns in tissue extracts of the kidney. Zymograms ob. tained after separation by IEF in an 8.0-5.0 pH gradient, pI values were determined by using isoelectric focusing protein markers (from Pharmacia). C, nonpolluted area--control group; PP, polluted pasture group; EEF, experimental emission feeding group.

in GR activities between the groups in the kidney and lung were not significant (Fig. 5). It is known that the stimulatory effects of metals on lipid peroxidation can be manifested by damaging the protective cytosolic system (9,14,16,21). One of the markers of such damage can also be the TBARS content in tissues (28). Therefore, TBARS was detected in the organs of sheep in all groups (Fig. 6). Compared with the control, TBARS content in the liver of the PP group was not increased, but we obtained an 1.80 times increase in the EEF group. Changes in the kidney TBARS content were similar to those in the liver. In the EEF group, the content was 1.70

TABLE 3. SOD Isoenzyme Pattern in Tissue Extracts of Liver, Kidney and Lung pI

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6.20 5.45 5.40 5.30 6.20 5.45 5.40 5.30 6.20 5.45 -----

6.20 5.45 --6.20 5.45 5.40 5.30 6.20 5.45 5.40 5.30 6.10 6.50

PP

EEF PP C

FIG. 2. SOD isoenzyme patterns in tissue extracts of the liver. Zymograms obtained after separation by IEF in an 8.05.0 pH gradient, pI values were determined by using isoelectric focusing protein markers (from Pharmacia). C, nonpolluted area--control group; PP, polluted pasture group; EEF, experimental emission feeding group.

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times higher than in the C group. Lung TBARS content did not show significant changes in any group. Several methods are used for lipid peroxidation identification (5,13,15,37). The method with thiobarbituric acid is not very specific because various substances not related to the peroxidation process can also react with this substrate (2). But from the important increase in TBARS content in the liver and kidney in the EEF group during the same test procedure conditions, we can assume increased lipid peroxidation in this group. From the abovementioned results, it appears that the response to toxic concentrations of heavy metals is specific according to the organ. In the liver of animals of the PP group, where SOD and GR activities were significantly higher, these enzymes can probably play an important role

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FIG. 4. Glutathione peroxidase activity in the fiver, kidney and lung of tissue extracts. The assays were performed as described in Materials and Methods. C, nonpolluted a r e a - control group; PP, polluted pasture group; EEF, experimental emission feeding group. Asterisks situated between groups represent significant differences between these two groups. ND, not detected. * P < 0.05.

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in the elimination of reactive oxygen species, and that is why TBARS content was not increased. These enzymes are already inhibited in the EEF group and membrane damage is likely to be manifested by the TBARS content increase. The kidney is the organ where the accumulation of heavy metals, especially Hg, is higher. This was also verified by our experiment. SOD and GR activities observed were higher than in the liver, but the difference between the groups in their activities was not significant. The TBARS content was increased only in the EEF group. It is known that the elimination of the reactive oxygen species is also performed by other systems (3 2) besides the antioxidant enzymes. It is possible that the TBARS content did not increase due to the function of the other systems in the PP group. Lung TBARS content did not display significant difference in the groups involved in our experiment. Thus, it seems that the antioxidant capacity of the lung is enough to protect its cells against the oxidative stress. According to the enzyme activities determined and TBARS content in particular organs, it can be supposed that an organ's response to oxidative stress may depend on specific organ damage and its protective abilities.

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FIG. 5. Glutathione reductase activity in the fiver, kidney and lung of tissue extracts. The assays were performed as described in Materials and Methods. C, nonpolluted a r e a - control group; PP, polluted pasture group; EEF, experimental emission feeding group. Asterisks situated between groups represent significant differences between these two groups. * P < 0.05.

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