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Antioxidant enzyme activities and lipid peroxidation levels in exercised and hypertensive rat tissues
Vol. 27, No. 9, pp. 923-931, 1995 0 1995 Elsevier ScienceLtd printed in Great Britain. All rightsrcscrved
hr. J. Biochem.Cell
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Antioxidant Enzyme Activities and Lipid PeroxidMm Levels in Exercised and Hypertensive Rat Tissues HUI HONG, PETER JOHNSON* Department of Chemistry and College of Osteopathic Medicine, Ohio University, Athens, OH 45701, U.S.A. Previous studies have shown that exercise-inducedchanges in muscle antioxidant status oCctlr shortly after exercise.The present studies were de&& to determiue if longer-term exercise-related changesin antioxidant enzymeactivities in both normoteusiveOYKY) and byperteusiverats (SHR) occurred, and if tbese cbauges were related to the levelsof lipid peroxid&on. WKY muI SIR rats were exercised over a l&week period using a progressivetreadmill regimen. After a l-week detraining period, the animals were eutba&ed and measurementsof tissne autioxidant enzyme activities and lipid peroxide levelswere determined iu bDtb exercisedand epgesedentsry groups. Decreasesin antioxidant activities (particularly glutatbioue peroxidase and catalase) in liver, kidney, skeletal and cardiac were associatedwith exercisetrain&g in both WKY and SHR rats (e.g. left ventricular glutathione peroxidase spec& activity in WIN rats was decreasedfrom 234&25 [SD, n=12] to 187+17 ISD, n = 11) m&s/rug proteiu). Elevatious in activities of antioxidant enzymes were generally associated with hype&m&m in these tissues (e.g. k?fi ventricular gIutMii peroxidase specific activity in SHR rats was 275 + 30 [SD, n = 121 units/mg proteiu), but changes in activities were more variable thau those seen in resportseto exercise. Exercise-related chauges in tissue levels of thiobarbituric acid-reactive substances(an indirect measure of tissue lipid peroxide levels)geueraRy did not correlate with exercise-related antioxidant enzymeactivity changes,and hypertension had no effect on &se levelsexcept in liver. Tbe results show that alterations of the activities of tissue antioxidant euzyme activities by exerciseor hypertension have no major effects on tbe levelsof Upfd peroxidation iu these tissues. These readts also suggestthat the mechanismsby which exercise and hypertension affect tissue antioxidant enzyme activities are different. Keywords: SHR rat
Exercise training Muscle Antioxidant enxymes Lipid peroxides
Int. J. Biochem. Cell Biol. (1995) 27,923-931
potential for generation of increased amounts of reactive oxygen species and elevated levels of oxidative damage (Alessio and Goldfarb, 1988; Kihlstrom, 1992). However, a number of studies have shown that there is an adaptive response in exercise-trained subjects in which oxidative capacity (Favier et al., 1989; Laughlin et al., 1990) and tissue resistance to oxidative damage (Kihlstrom, 1992; Witt et al., 1992, Alessio, 1993) are increased. In the latter case, alterations in the activities of the enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase have been implicated (Harris, 1992; Sies, 1993, Higuchi et al., 1985; Powers et al., 1994), although some of these enzymes have other functions including
INTRODUCTION
Aerobic organisms have evolved a number of antioxidant mechanisms which function to relieve oxidative stress either by preventing reactive oxygen species formation or by mitigating the damage that such species can cause (Halliwell, 1992; Sies, 1993). Exercise presents a particularly important type of oxidative stress to animal systems because the increased consumption of molecular oxygen for respiration has the *To whom correspondence should be addressed. Abbreviations: TBARS, thiobarbituric acid-reactive substances. Received 7 February 1995; accepted 25 April 1995. 923
924
Hui
Hong
and Peter
prostanoid metabolism and the maintenance of adequate glutathione levels for normal tissue metabolism (Halliwell and Gutteridge, 1992). Increases in skeletal muscle antioxidant enzyme activities (particularly of glutathione peroxidase) occur immediately (Lawler et al., 1993; Ji and Fu, 1992; Ji et al., 1992) or shortly after (Ji et al., 1988; Favier et al., 1989; Ji et al., 1991; Hammeren et al., 1992; Criswell et al., 1993) exercise, although no changes or decreases may occur in some antioxidant enzyme activities (Salminen and Vihko, 1983; Higuchi et al., 1985; Laughlin et al., 1990; Schauer et al., 1990; Lee et al., 1991). In the myocardium of exercised animals, reports of increases (Lew and Quintanilha, 1991; Ito et al., 1992) decreases (Kihlstrom et al., 1989, Schauer et al., 1990) or unaltered (Kihlstrom et al., 1989; Ji, 1993) activities of antioxidant enzymes have also been made, and it has been suggested that myocardia from exercise-trained rats and mice may be less susceptible to lipid peroxidation damage (Kihlstrom, 1992). Most of the above studies have focused on muscle sampling in the immediate post-exercise period, and may reflect a very short-term response to an exercise episode. The current studies were designed to examine antioxidant status at a post-exercise period much later than was previously used in order to determine if long-term adaptive changes in antioxidant enzyme activities were similar in tissues with different metabolic functions, and if these changes were related to the levels of lipid peroxidation (as reflected by tissue TBARS levels) that exist in trained subjects. Such longterm changes in enzyme expression in response to chronic exercise are known to occur for non-antioxidant enzymes in muscle (Falduto et al., 1992; Lawler et al., 1993; Staron and Johnson, 1993), although in some cases the exercise-induced changes are reversed during the post-exercise detraining period (Ladu et al., 1991; Neufer et al., 1992). In addition to exercise training, hypertension may also result in changes in tissue morphology and metabolism (Tomanek et al., 1984; Pauletto et al., 1988; Ito et al., 1992; Lakatta, 1990; Smith et al., 1990), including changes in tissue lipid peroxidation (Schimke et al., 1987; Papies et al., 1989; Ito et al., 1992; Uotila et al., 1993) and decreases in the levels of the putative (Boldyrev and Severin, 1990) antioxidant histidine dipeptides (Johnson and Hammer, 1992). To date, the only reports concerning the status of antioxidant enzyme activities in the myocar-
Johnson
dium of hypertensive animals have indicated that the activity of catalase is unaltered in comparison to normotensive activity (Tomanek et al., 1984), whereas glutathione peroxidase activity differs from that in normotensive rats and is age-dependent (Ito et al., 1992). Studies on the effects of exercise training on the myocardium of hypertensive rats have reported changes in morphology (Crisman and Tomanek, 1985) and isoenzyme expression (Schaible et al., 1987; Rupp, 1989) although exercise did not affect heart or left ventricular weight in SHR rats (Crisman and Tomanek, 1985). Part of the significance of the studies on exercised hypertensive animals is that exercise has been suggested as a component in the management of certain types of hypertension (Crisman and Tomanek, 1985; Sleight, 1990) and it seemed important to determine if exercise has advantageous or disadvantageous effects in hypertensive animals with respect to antioxidant status. As previous reports have indicated that some antioxidant enzyme activities are changed as a result of hypertension or exercise in muscle tissue (Ito et al., 1992; Ji and Fu, 1992; Lew and Quintanilha, 1991; Powers et al., 1994) the activities of four key antioxidant enzyme activities in muscle were measured in order to determine if these activities were changed as a result of hypertension and/or exercise and if such changes were related to prevailing TBARS levels in the tissue. For the present studies, we selected a part of the myocardium (the outer wall of the left ventricle) that has been shown to undergo major morphological and biochemical changes in exercise-induced hypertrophy and hypertension (Pauletto et al., 1988; Rupp, 1989; Kihlstrom et al., 1989; Smith et al., 1990), and we have also analyzed changes in the superficial vastus lateralis of the quadriceps femoris, a white anaerobic skeletal muscle (Higuchi et al., 1985) and the longissimus dorsi which is a red aerobic skeletal muscle (Dr R. S. Staron, pers. commun.). In order to ascertain if possible changes in antioxidant enzyme activities and TBARS levels were unique to muscle systems under our experimental conditions, we also included two aerobic non-muscle tissues (liver and kidney) in this study. MATERIALS
AND
METHODS
Materials and animals Reagents and auxiliary enzymes for enzyme assays were purchased from Sigma Chemical
Exercise, hypertension and antioxidant enzymes
Co., St Louis WKY (normotensive) and SHR (spontaneously hypertensive) male rats (Rattus norvegicus) were purchased from Harlan Sprague-Dawley, Indianapolis at an age of 11-12 weeks. Exercise training and animal care
In these studies, four groups of rats were established: exercised or sedentary SHR, and exercised or sedentary WKY. Each group contained 12 rats, with the exception of the exercised normotensive group which contained 11 rats. Each animal was housed in a separate cage and was fed a standard rat diet and allowed access to water ad libitum. The exercise program was commenced one week after the animals were obtained from the supplier, and animals in the exercised groups were subjected to a graded exercise training regimen over a lo-week period using a variable-speed moving-belt treadmill with an adjustable incline. The regimen, which was based on previous procedures designed to elicit sub-maximal O2 consumption (Crisman and Tomanek, 1985), consisted of exercise for five out of seven weekdays as follows: week l-15 min duration, 10 m/min (belt speed), 0” (incline); week 2-30 min, 10 m/min, 0”; week 3-30 min, 10 m/min, 5”; week 4-30 min, 20 m/min, 5”; week 5-45 min, 20 m/min, 5”; weeks 6-lO-60min, 20m/min, 5”. Rats in the control (sedentary) group were removed from and placed into their cages five days per week over a lo-week period to account for any handling-induced stress. After a sedentary (detraining) period of one week following termination of the exercise/handling period, systolic blood pressures were measured by the tail-cuff technique (Henley et al., 1992) and body weights were measured. Tissue collection, preparation
storage
and
homogenate
Following measurement of body weights and blood pressures, the animals were euthanized by CO,-induced asphyxiation. The selected tissues (septum-free outer wall of the left ventricle, superficial vastus lateralis muscle of the quadriceps femoris, longissimus dorsi muscle, liver and kidney) were then removed immediately and washed briefly in cold phosphate buffer in order to minimize interference by erythrocyte antioxidant enzymes (Criswell et al., 1993). The tissues were cut into approx. 50-1OOmg portions and stored separately at -70” in plastic vials. Homogenates from these samples were then
925
prepared after the addition of 1.0 ml phosphate buffer per 100 mg of tissue as described previously (Johnson and Hammer, 1993), and protein concentrations of the preparations were measured by the Bio-Rad Bradford protein assay kit. Enzyme assays
Total superoxide dismutase activity was measured by the NADPH oxidation procedure (Paoletti and Mocali, 1990) using 5-40 ~1 aliquots of homogenate sample in a final assay volume of 1.065 ml. The unit of enzyme activity is the amount of enzyme that gave 50% inhibition of the control rate of NADPH oxidation. Catalase activity was measured by spectophotometric analysis of the rate of hydrogen peroxide decomposition (Aebi, 1984) when 1-50~1 aliquots of homogenate sample were used in a final assay volume of 0.5 ml. The arbitrary unit of catalase activity is defined as the observed firstorder rate constant (U set’) for H,Oz degradation. Glutathione peroxidase activity was measured using t-butyl hydroperoxide and glutathione as substrates in the glutathione reductase-linked “continuous monitoring” procedure (Flohe and Gunzler, 1984) with sample aliquots of 4-20 ,ul used in a final assay volume of 0.5 ml. A unit of glutathione peroxidase activity is defined as the amount of enzyme which gives a 90% decrease in glutathione concentration per min at a 1 mM starting glutathione concentration. Glutathione reductase activity was determined by measurement of the rate of NADPH oxidation in the presence of oxidized glutathione (Carlberg and Mannervik, 1985), using 2-50 ~1 sample aliquots in a final assay volume of 0.5 ml. The unit of enzyme activity is defined as the amount of enzyme which oxidizes 1 mmol of NADPH per min. Enzyme specific activities (using the standard definition of an enzyme activity unit from the method for the assay) and TBARS levels were expressed in terms of protein content of tissue extracts. When the data are expressed on a wet weight basis (results not shown), very similar patterns of results were obtained but the data are presented on a protein content basis for comparison with previous studies (Kainulainen and Komulainen, 1989; Kihlstrom, 1992; Criswell et al., 1993; Powers et al., 1993; Powers et al., 1994) and because protein content is more accurately measured and is unaffected by exercise in rat myocardium (Kihlstrom et al., 1989).
926
Hui Hong and Peter Johnson
Thiobarbituric acid-reactive (TBARS) measurement
substances
Estimates of lipid peroxidation levels were performed by the thiobarbituric acid-based procedure for the fluorometric determination of malonaldehyde (Yagi, 1976) using a PerkinElmer LS-5 Fluorescence Spectrophotometer. Homogenate sample volumes of 0.25 ml were used in a final assay volume of 1.25 ml, and assays were performed in triplicate. The assay procedure was calibrated using tetraethoxypropanone as a malonaldehyde source, and levels of tissue sample TBARS were calculated as fmol malonaldehyde per mg of sample protein. Statistical analysis
Mean values 4 SD were calculated for each data set after elimination of outliers by the Dixon test (Sokal and Rohlf, 1981), and significant differences between data sets were detected by one-way ANOVA. Values for P > 0.05 were taken to indicate no significant difference. RESULTS
E&ct of exercise training on body weight and blood pressure
The effects on body weight and blood pressure of the training regimen are shown in Table 1. In agreement with earlier studies using this regimen (Crisman and Tomanek, 1985; Friberg et al., 1988; Tipton et al., 1991), no significant decrease in blood pressure in hypertensive SHR rats was observed as a result of exercise, although a significant decrease in blood pressure in response to exercise was observed in the normotensive WKY rats. In contrast to the previous studies (Crisman and Tomanek, 1985), small but significant decreases (of about 10%) in body weight for both the
exercised WKY and SHR rats were observed in comparison to sedentary controls. Eflects of exercise training on antioxidant enzyme activities and TBARS 1eveIs in SHR and WKY rats
Table 2 shows that exercise training resulted in decreases in glutathione peroxidase and catalase activities in all of the tissues studied in both WKY and SHR rats. In general, the decreases for each enzyme were similar within the same tissue for both SHR and WKY rats, with catalase activities generally showing larger decreases than glutathione peroxidase. The largest decreases were seen in catalase activities in the left ventricular outer wall, where the exercised animals had activities which were down to 46 and 49% of the respective sedentary WKY and SHR activities. For total superoxide dismutase activity, exercise training caused decreases in WKY and SHR liver activities down to approx. 70% of sedentary animal activities, whereas no changes were seen in kidney, and 93 and 25% increases in activity were seen in longissimus dorsi of WKY and SHR rats, respectively. Glutathione reductase activities were decreased by exercise training in the left ventricular outer wall of both WKY and SHR rats (down to 86 and 89% of sedentary animal activities, respectively), whereas only the quadriceps femoris of WKY rats showed a decrease, with the activity in exercised SHR animals remaining unchanged. In the longissimus dorsi, exercise also had no effect on glutathione reductase activity in SHR rats, but resulted in a 26% increase in WKY rats. Liver and kidney glutathione reductase activities were unaffected by exercise in WKY rats, and in SHR rats, kidney glutathione reductase was also unchanged, whereas an activity decrease was observed in liver of SHR rats.
Table 1. The effect of exercise training on body weight and blood nressure in WKY and SHR rats Body weight Blood pressure Rat Statusa (g; mean k SD) (torr; mean If: SD) WKY Sedentary (12) 375.2 f 30.2 140.8 If: 11.1 P <0.002* PiO.003 Exercised (11) 339.5 & 20.2 121.7 + 14.0 SHR Sedentary (12) 409.0 f 14.0 180.4 + 20.8 P 0.05.
921
Exercise, hypertension and antioxidant enzymes Table 2. Antioxidant
Parameter SOD
CAT
Tissue OWLV Liver Kidney OWLVT
LDt QFt
GPX
Liver Kidney OWLV
GR
& Liver Kidney OWLV
TBARS
g Liver Kidney OWLV
enzyme specific activities* and TBARS levels in tissues of exercised and sedentary rats SHR Rats WKY Rats Sedentary (1) 3.06 +0.59 3.05
zo.71
4.18 k 1.23 12.3 + 1.8 12.9 I5.1 26.1 f 4.11 5.53 * 1.55 2.90 f 0.90 1.11 +0.16 0.287 &0.047 234+25 88.4 + 11.3 55.9 f 14.51 107Ok98 571+69f 20.4 + 1.7 9.86 + 1.14 14.9 * 2.1 99.3 f 9.2 236+14$ 230+23 252f54 204k26 24Ok56 250*43$
Exercised
P1.2
nn n: iiII I/ II I/ II -
(2)
P 1.3
(3)
k& II-7 f+f 0.60# n7 5 r11 -II f f n --
4.07 5.88 2.303 2.111 4.03 8.9k2.55 +0.60$
12.1 14.0 5.31 1.51 3.87 2.00 +0.92$
0.569 +0.033# 0.235 0.099 1872171 66.9* ll.l$ 31.2 f 10.23 103593$ 17.6 37lf50$ 1.2:
I/
14.7 11.0 f 1.731 1.41 103* 111 239*9$ 172&40# 288+41§
11
Exercised
Sedentary 2.96 kO.83 4.82 + 1.07$ 2.86 50.79 11.7 f 2.3 14.9 * 3.7 34.9 k 7.2 7.25 kO.783 3.36 kO.79 1.26*0.18 0.393 +0.048 275+30 126f21.0$ 55.9 + 14.6 647559 573+5q 21.7 &- 1.6 18.0 k 1.71 9.99 + 1.21 143+6 262+20 221*55 263+88$ 190+29 249k53 256+39
-iII n:
PM
/i
: IIII n nn
P2.4
(4)
3.51 + 1.69
fk *f k + f +
5.28 kO.92 3.58 1.95 8.32 2.0 17.0 14.5 4.9 4.4$ 2.4Of0.90 5.91 1.20
0.323 0.661 208 87.5 39.5
f 0.127 0.051 24 12.3 10.3
-
518k83 43lk78 19.3 + 1.1 16.6 2.13
-
10.7 126+ 2.5 11 268*18
+ f
258f41$ 175*44
---7II 11 -: II 1 --II n-
-
-
n
-i
g 166+30# 186&44 Liver 173+32# 250*60$ Kidney 284*51$ 315+39 *Enzyme specific activities and TBARS levels in the outer wall of the left ventricle (OWLV), longissimus dorsi (LD), quadriceps femoris (QF), liver and kidney are expressed as the mean & SD in units per mg of homogenate protein as defined in the assay procedure described in “Materials and Methods”. For clarity, mean values are simplified to three significant figures. The enzyme names are abbreviated as superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR), and catalase (CAT). tValues shown are actual x 10’. Values were obtained from assays on 12 different animals in each group, except for those values marked with $(n = 11) and $(a = 10). P values for statistically significant differences between data sets for animal groups 1 and 2, 1 and 3, 3 and 4, and 2 and 4 are indicated as P,,,, P,,,, P3,4and PzAand the symbols used are-for no significant difference (P > 0.05), 11for P between 0.05 and 0.005, and 7 for P < 0.005.
With respect to TBARS levels (Table 2), the only tissue which showed similar exerciserelated changes in both WKY and SHR rats was longissimus dorsi (decreases down to about 67% of sedentary levels). In WKY animals only, exercise-related decreases in TBARS levels occurred in quadriceps femoris and liver, whereas an increase was found in the left ventricular outer wall. TBARS levels in kidney were unaffected by exercise in WKY rats but showed a 23% increase in SHR animals. Hypertension-related changes in antioxidant enzyme activities and TBARS levels
Table 2 shows that the only hypertensionrelated changes in total superoxide dismutase activity were in the longissimus dorsi (a 58% increase over normotensive activity) and in the quadriceps femoris (a decrease down to 68% of the normotensive activity) of sedentary animals. In the case of catalase activity, increases were seen in the left ventricular outer wall, longis-
simus dorsi and kidney for both sedentary and exercised groups, and in liver of sedentary animals. For both the sedentary and exercised groups, hypertension-related increases in glutathione peroxidase activity were also seen in the left ventricular outer wall and longissimus dorsi, whereas no changes were seen in quadriceps femoris and activity decreases were seen in liver. With glutathione reductase, hypertensionrelated increases in activity were found in longissimus dorsi, liver and kidney of both sedentary and exercised groups, with the largest increase (83% above normotensive activity) being observed in the longissimus dorsi of sedentary animals. The activity of glutathione reductase in the left ventricular outer wall was unaffected by hypertension in sedentary animals, although an increase of 11% was seen in the exercised group. In the case of glutathione reductase in the quadriceps femoris, a decrease in activity was observed in sedentary animals, whereas no change was found in the exercised group.
Hui Hong and Peter Johnson
928
Table 2 also shows that TBARS levels were unaffected by hypertension in sedentary rats, and the only hypertension-related change in TBARS levels in the exercised animals was in liver, where a 59% increase over normotensive levels was observed. DISCUSSION
Weight loss caused by starvation is associated with changes in antioxidant enzyme activities in rat heart, liver and kidney (Wohaieb and Godin, 1987). Although the exercise regimen in the present studies did cause a statistically-significant weight loss, this weight loss was much smaller than that observed in the foregoing starvation studies (about 10% compared to 20%), and with the exception of the decrease in liver catalase, the exercised-related enzyme activity changes were quite different from those observed in these starvation studies. These differences strongly suggest that the enzyme activity and TBARS level changes observed in the present studies are exercise-related and not a result of weight loss per se. Exercise training in both WKY and SHR rats resulted in long-term decreases in the activities of glutathione peroxidase and catalase in all of the tissues examined. These results are in agreement with earlier studies which have reported exercise-related decreases in myocardial and skeletal muscle catalase activities (Kihlstrom et al., 1989; Powers et al., 1994), but not with a previous report of post-exercise activity increases in skeletal and cardiac muscle catalase activities (Lew and Quintanilha, 1991). Our findings of decreases in glutathione peroxidase activities also differ from a number of previous reports which have shown immediate postexercise activity increases (Ji et al., 1990; Ji et al., 1991; Hammeren et al., 1992; Criswell et al., 1993; Lawler et al., 1993; Powers et al., 1994; Schauer et al., 1990; Lew and Quintanilha, 199 1; Kumar et al., 1992) or no activity change (Schauer et al., 1990; Ji, 1993) in this enzyme. In the case of superoxide dismutase activities in skeletal muscles, Criswell et al. (1993) concluded that exercise-related upregulation of superoxide dismutase was limited to highly oxidative skeletal muscle, and our studies confirm these findings for WKY rats. However, in the case of the SHR rats, an exercise-related increase in total superoxide dismutase activity occurred in the anaerobic quadriceps femoris but not the aerobic longissimus dorsi muscle,
which implies that the regulation of skeletal muscle superoxide dismutase activities is different in SHR and WKY rats. No changes were found in left ventricular total superoxide dismutase activity as a result of exercise and/or hypertension, which differs from earlier reports of an exercise-related activity decrease in the Cu,Zndependent enzyme (Kihlstrom et al., 1989) and of an apparent increase in Mn-dependent superoxide dismutase activity (Powers et al., 1993). It is not yet clear if the lack of change in total superoxide dismutase activity in the myocardium after detraining represents compensatory changes in the two forms of superoxide dismutase, or a return to normal levels of one or both of these enzyme forms, and further studies will be necessary to investigate these possibilities. Some of the above-mentioned disparities between previous and current results may be related to the choice of post-exercise times at which tissue samples were obtained. For superoxide dismutase in the left ventricular outer wall, where previous studies have shown immediate post-exercise activity changes (Kihlstrom et al., 1989) the lack of long-term activity changes observed in our studies may indicate that the length of the detraining period (7 days) before measurement permitted a return to control levels, as has previously been reported for other muscle non-antioxidant proteins and enzymes (Ladu et al., 199 1; Neufer et al., 1992). However, for superoxide dismutase and glutathione reductase in longissimus dorsi muscle, the long-term enzyme activity changes were in the same direction as the immediate post-exercise changes (Ji et al., 1990; Criswell et al., 1993; Lawler et al., 1993; Powers et al., 1994), indicating that the activity changes were not reversed during the detraining period and that the training effect was of a prolonged duration for these enzymes. Such differences in post-exercise enzyme activities suggest that the regulation of these activities may be quite complicated, and we are currently investigating the roles that transcriptional and translational controls may play under such circumstances. In previous studies (Godin and Garnett, 1992a) on antioxidant enzymes in erythrocytes and myocardium, patterns of activity changes were examined for the functionally coupled pairs of antioxidant enzymes glutathione peroxidase/glutathione reductase and superoxide dismutase/catalase, and it was concluded that the enzyme levels in these pairs may be independently regulated. In the present studies, parallel
Exercise, hypertension and antioxidant enzymes
929
changes for theseenzymesin both WKY and enzyme activities may be involved in minimizing the extent of tissue lipid peroxidation associated SHR rats were seen for glutathione peroxidase/ glutathione reductase in the outer wall of the left with exercise. In addition to the factors discussed in terms of the TBARS assay, such ventricle, and for superoxide dismutaselcatalase in liver, where decreases in activity were ob- factors may include decreases in O2 uptake in served. Parallel decreases in activity for gluta- trained hypertensive rats (F&erg et al., 1988) thione peroxidase/glutathione reductase were changes associated with increases in glucose uptake in hypertensive rats (Leipala et al., also seen in the quadriceps femoris of WKY (but not SHR) rats and in liver of SHR (but not 1989) changes in the levels of non-enzymatic WKY) rats. These reults indicate that there may tissue antioxidants (Janero and Burghardt, be an exercise-related co-ordinated regulation of 1988; Lew and Quintanilha, 1991), and changes enzyme levels in some tissues such as the left in the activities of other antioxidant enzymes ventricular outer wall, whereas such changes which were not part of this study. In contrast to the exercise-related decreases in may not occur in other tissues or may be changed as a result of hypertension. glutathione peroxidase and catalase, elevated glutathione peroxidase and catalase activities In liver, skeletal muscle and erythrocytes, there did not appear to be a causal relationship were associated with hypertension in the left between tissue activities of antioxidant enzymes ventricular outer wall and longissimus dorsi, and the prevailing levels of lipid peroxidation whereas hypertension had no effect on gluta(Godin and Garnet& 1992b; Lawler et al., 1993; thione reductase activity in the anaerobic Ji et al., 1990), whereas in rat myocardium, muscle (quadriceps femoris). In the case of liver antioxidant enzyme levels were shown to be and kidney, both of which are aerobic tissues, inversely correlated with susceptibility to lipid activity increases associated with hypertension peroxidation as a result of experimental hyper- were only seen for catalase in kidney, whereas trophy and spontaneous hypertension (Ito et al., for liver glutathione peroxidase, decreases in 1992; Kirshenbaum and Singal, 1993). In some activity were observed. These results indicate of these previous studies and in the present that the effect of hypertension on antioxidant studies, lipid peroxidation levels were measured enzyme activities is different than the effect of by the widely-used indirect TBARS procedure exercise, and suggest that different mechanisms which is designed to measure malonaldehyde may exist in various tissues for regulation of levels. Although the procedure measures malonenzyme activities in response to exercise and aldehyde levels accurately, the assay is subject to hypertension. interference by a number of substances present No changes in TBARS levels in the left in biological samples which can cause an overes- ventricular outer wall, skeletal muscles or kidtimation of tissue lipid peroxide levels (Slater, ney were associated with hypertension, although 1984). A further complication in the use of a large increase in the TBARS level was found TBARS data to indirectly measure free radical in liver of exercised SHR rats (59% above the generation in tissues is that endogenous tissue exercised WKY rat level). In exercise-trained antioxidants such as ascorbate and u-tocoSHR rats, some tissue enzyme activities appherol can effectively scavenge oxygen-derived proached sedentary normotensive values withfree radicals, thereby preventing propagation of out increases in TBARS levels, except in the lipid peroxidation (Krinsky, 1992) and causing case of kidney where an increase in TBARS an underestimation of free radical generation in level occurred. These results indicate that the the tissue. Bearing in mind these uncertainties processes for maintaining TBARS levels in hyinherent in the use of the TBARS assay to pertensive animals in exercise training may be measure tissue lipid peroxide levels, our exercise different from those in normotensive animals, training results showed that an inverse relationand also support the conclusion previously disship between tissue TBARS levels and enzyme cussed that alterations in tissue susceptibilities activities did exist in some tissues (longissimus to lipid peroxidation (measured as TBARS) dorsi, left ventricular outer wall and kidney), may not be inversely correlated with the activibut that this relationship could be altered in ties of the antioxidant enzymes studied. SHR rats. Because there was not a consistent In conclusion, the present study has shown picture of an inverse correlation between that long-term decreases in some tissue antiTBARS levels and antioxidant enzyme activi- oxidant enzyme activities were associated ties, it is possible that factors other than these with chronic exercise in both normotensive and
930
Hui Hong and Peter Johnson
hypertensive rats, whereas increases in some tissue antioxidant enzyme activities were associated with hypertension. The patterns of enzyme activity changes associated with exercise training or hypertension were generally not consistent for a particular enzyme from tissue to tissue, and changes in tissue TBARS levels did not appear to be related to the observed enzyme activity changes in most cases. Previous studies have shown that in tissues such as myocardium, antioxidant enzyme activities may be rate-limiting for oxidation processes (Wohaieb and Godin, 1987), and in such cases, changes in enzyme activities could alter in vivo rates of oxidative processes if such enzymes were substrate-saturated (Newsholme and Leech, 1983). More detailed information about the rate-limiting features of enzyme concentration and substrate concentrations will therefore be needed to establish if the changes in enzyme activity measured by in vitro assays actually reflect changes in the rates of oxidative process in vivo. Acknowledgements-These studies were supported by a grant from the American Heart Association, Ohio Alhhate, Inc., Columbus, Ohio. We thank MS M. Halterman and MS M. A. Notestine for excellent technical assistance. REFERENCES
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Crisman R. P. and Tomanek R. J. (1985) Exercise training modifies myocardial mitochondria and myofibril growth in spontaneously hypertensive rats. Am. J. Physiol. 248, H8-H14. Criswell D., Powers S., Dodd S., Lawler J., Edwards W., Renshler K. and Grinton S. (1993) High intensity training-induced changes in skeletal muscle antioxidant enzyme activity. Med. Sci. Sports Exer. 25, 1135-1140. Fad&o M. T., Young A. P. and Hickson R. C. (1992) Exercise inhibits glucocorticoid-induced glutamine synthetase expression in red skeletal muscles. Am. J. Physiol. 262, c214C220. Favier R. J., Ghaemmaghami F., Sempore B., Desplanches D., Mayet M. H., Frutoso J., Gharib C. and Flandrois R. (1989) Skeletal muscle adaptation to physical training and beta-adrenergic blockade in spontaneously hypartensive rats. Eur. J. appl. Physiol. 58, 652-660.
Flohe L. and Gunzler W. F. (1984) Assays of glutathione peroxidase. Merh. Enzym. 105, 114121. Friberg P., Hoffman P., Norlander M. and Thoren P. (1988) Effects of voluntary physical exercise on cardiac function and energetics in spontaneously hypertensive rats. Acra physiol.
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Godin D. V. and Garnett M. E. (1992a) Species-related variations in tissue antioxidant status--I. Differences in antioxidant enzyme profiles. Comp. Biochem. Physiol. 103B,
737-742.
Godin D. V. and Garnett M. E. (1992b) Species-related variations in tissue antioxidant status--II. Differences in susceptibility to oxidative challenge. Comp. Biochem. Physiol.
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743-748.
Halliwell B. (1992) Reactive oxygen species and the central nervous system. J. Neurochem. 59, 16091623. Halliwell B. and Gutteridge J. M. C. (1992) Free Radicals in Biology and Medicine, 2nd edn, pp. 399-107. Oxford University Press, Oxford. Hammeren J., Powers S., Lawler J., Criswell D., Martin D., Lowenthal D. and Pollock M. (1992) Exercise traininginduced alterations in skeletal muscle oxidative and antioxidant enzyme activity in senescent rats. Int. J. Sports Med. 13, 412416. Harris E. D. (1992) Regulation of antioxidant enzymes. FASEB
J. 6, 2675-2683.
Henley W. N., Bellush L. L. and Notestine M. A. (1992) Reemergence of spontaneous hypertension in hypoxiaprotected rats returned to normoxia as adults. Brain Res. 579, 211-218. Higuchi M., Cartier L. J., Chen M. and Holloszy J. 0. (1985) Superoxide dismutase and catalase in skeletal muscle: adaptive response to exercise. J. Gerontol. 40, 281-286. Ito H., Torii M. and Suzuki T. (1992) A comparative study on defense systems for lipid peroxidation by free radicals in spontaneously hypertensive and normotensive rat myocardium. Comp. Biochem. Physiol. MB, 3740. Janero D. R. and Burghardt B. (1988) Myocardial vitamin E (alpha-tocopherol) contents of spontaneously hypertensive and Wistar-Kyoto normotensive rats. Int. J. Vit. Nutr.
Res. 58, 292-294.
Ji L. L. (1993) Antioxidant enzyme response to exercise and aging. Med. Sci. Sports Exer. 25, 225-231. Ji L. L. and Fu R. (1992) Responses of ghttathione system and antioxidant enzymes to exhaustive exercise and hydroperoxide. J. appl. Physiol. 72, 549-554. Ji L. L., Fu R. and Mitchell E. W. (1992) Glutathione and antioxidant enzymes in skeletal muscle: effects of fiber type and exercise intensity. J. appl. Physiol. 73, 18541859. Ji L. L., Wu E. and Thomas D. P. (1991) Effect of exercise. training on antioxidant and metabolic functions in senescent rat skeletal muscle. Gerontology 37, 3 17-325. Ji L. L., Dillon D. and Wu E. (1990) Alteration of antioxidant enzymes with aging in rat skeletal muscle and liver. Am. J. Physiol. 258, R918-R923. Ji L. L., Stratman F. W. and Lardy H. A. (1988) Enzymatic down regulation with exercise in rat skeletal muscle. Archs biochem. Biophys. 263, 137-149. Johnson P. and Hammer J. L. (1992) Histidine dipeptide levels in ageing and hypertensive rat skeletal and cardiac muscles. Comp. Biochem. Physiol. 103B, 981-984. Johnson P. and Hammer J. L. (1993) Cardiac and skeletal muscle enzyme levels in hypertensive and ageing rats. Comp.
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Exercise, hypertension and antioxidant enzymes Kainulainen H. and Komulainen J. (1989) Effects of training on regional substrate oxidation in the hearts of ageing rata. Gerontology 35, 289-296. Kihlstrom M. T. (1992) Lipid peroxidation capacities in the myocardium of endurance-trained rats and mice in vitro. Acta physiol. &and. 146, 177-183. Kihlstrom M., Ojala J. and Salminen A. (1989) Decreased level of cardiac antioxidants in endurance-trained rats. Acta
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Kirshenbaum L. A. and Singal P. K. (1993) Increase in endogenous antioxidant enzymes protects hearts against reperfusion injury. Am. J. Physiol. 265, H484H493. Krinsky N. I. (1992) Mechanism of action of biological antioxidants. Proc. Sot. Exp. Biol. Med. 200, 248-254. Kumar C. T., Reddy V. K., Prasad M., Thyagaraju K. and Reddanna P. (1992) Dietary supplementation of vitamin E protects heart tissue from exercise-induced oxidant stress. Molec. Cell. Biochem. 111, 109-115. Ladu M. J., Kapsas H. and Palmer W. K. (1991) Regulation of lipoprotein lipase in muscle and adipose tissue during exercise. J. appl. Physiol. 71, 404-409. Lakatta E. G. (1990) Similar myocardial effects of aging and hypertension. Eur. Heart J. 11, G29-G38. Laughlin M. H., Simpson T., Sexton W. L., Brown 0. R., Smith J. K. and Korthius R. J. (1990) Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training. J. appl. Physiol. 68, 2337-2343. Lawler J. M., Powers S. K., Visser T., Van Dijk H., Kordus M. J. and Ji L. L. (1993) Acute exercise and skeletal muscle antioxidant and metabolic enzymes: effects of fiber type and age. Am. J. Physiol. 265, RI344Rl350. Lee E., Miki Y. and Kariya K. (1991) Subunit selective alteration of hepatic glutathione transferases in spontaneously hypertensive rats. Comp. Biochem. Physiol. 99, 93-97. Leipala J. A., Virtanen P., Ruskoaho H. J., Hassinen I. E. and Takala T. E. (1989) Transmural distribution of left ventricular glucose uptake in spontaneously hypertensive rats during rest and exercise. Acta physiol. &and. 135, 435-442.
Lew H. and Quintanilha A. (1991) Effects of endurance training and exercise on tissue antioxidative capacity and acetaminophen detoxification. Eur. J. drug Metab. Pharmacokin.
16, 5948.
Neufer P. D., Shinebarger M. H. and Dohm G. L. (1992) Effect of training and detraining on skeletal muscle glucose transporter (GLUT4) content in rats. Can. J. Physiol. Pharmac. 70, 12861290. Newsholme E. A. and Leech A. R. (1983) Biochemistryfor the Medical Sciences, pp. 4142. John Wiley, Chichester. Paoletti F. and Mocali A. (1990) Determination of superoxide dismutase activity by purely chemical system based on NAD(P)H oxidation. Merh. Enzym. 186, 209-220. Papies B., Schimke I. and Moritz V. (1989) Enhanced formation of lipid peroxides might contribute to the high sensitivity of spontaneously hypertensive rats towards isoproterenol-induced myocardial damage. Biomed. biochim. Acta 48, 681687. Pauletto P., Piccolo D., Scannapieco G., Vescovo G., Dalla Libera L. and Dal Palu C. (1988) Left ventricular hypertrophy in hypertension. Changes in isomyosins and creatine-kinase isoenzymes. Am. J. Med. 84, 122-124.
931
Powers S. K., Criswell D., Lawler J., Ji L. L., Martin D., Herb R. A. and Dudley G. (1994) Influence of exercise and fiber type on antioxidant enzyme activity in rat skeletal muscle. Am. J. Physiol. 266, R375-R380. Powers S. K., Criswell D., Lawler J., Martin D., Lieu F.-K., Ji L. L. and Herb R. A. (1993) Rigorous exercise training increases superoxide dismutase in ventricular myocardium. Am. J. Physiol. 265, H2094-H2098. Rupp H. (1989) Differential effect of physical exercise routines on ventricular myosin and peripheral catecholamine stores in nonnotensive and spontaneously hypertensive rats. Circ. Res. 65, 370-377. Salminen A. and Vihko V. (1983) Lipid peroxidation in exercise myopathy. Exp. Molec. Path. 38, 380-388. Schaible T., Malhotra A., Ciambrone G., Buttrick P. and Scheuer J. (1987) Combined effects of hypertension and chronic running program on rat heart. J. appl. Physiol. 63, 322-327.
Schauer J. E., Schelin A., Hanson P. and Stratman F. W. (1990) Dehydroepiandrosterone and a beta-agonist, energy transducers, alter antioxidant enzyme systems: influence of chronic training and acute exercise in rats. Archs biochem. Biophys. 283, 503-5 11. Schimke I., Papies B. and Moritz V. (1987) Importance of the antioxidative potential for free radical induced heart damage. Biomed. biochim. Acta 46, S576-S579. Sies H. (1993) Strategies of antioxidant defense. Eur. J. Biochem. 215, 213-219. Slater T. F. (1984) Overview of methods used for determining lipid peroxidation. Meth. Enzym. 105, 283-293. Sleight P. (1990) Hypertension. In Oxford Textbook of Medicine (Edited by Weatherall D. J., Ledingham J. G. G. and Warrell D. A.), 2nd edn, pp. 13.36sl3.382. Oxford University Press, Oxford. Smith S. H., Kramer M. F., Reis I., Bishop S. P. and Ingwall J. S. (1990) Regional changes in creatine kinase and myocyte size in hypertensive and nonhypertensive cardiac hypertrophy. Circ. Res. 67, 13341344. Sokal R. R. and Rohlf F. J. (1981) Biometry, 2nd edn, pp. 412414. W. H. Freeman, New York. Staron R. S. and Johnson P. (1993) Myosin polymorphism and differential expression in adult human skeletal muscle. Camp. Biochem. Physiol. MB, 463475. Tipton C. M., Sebastian L. A., Overton J. M., Woodman C. R. and Williams S. B. (1991) Chronic exercise and its hemodynamic influences on resting blood pressure of hypertensive rats. J. appl. Physiol. 71, 2206-2210. Tomanek R. J., Trout J. J. and Lauva I. K. (1984) Cytochemistry of myocardial structures related to degenerative processes in spontaneously hypertensive and normotensive rats. J. Molec. Cell. Cardiol. 16, 227-237. Uotila J., Tuimala R., Pykko K. and Ahotupa M. (1993) Pregnancy-induced hypertension is associated with changes in maternal and umbilical blood antioxidants. Gynecol. Obst. Invest. 6, 153-157. Witt E. H., Reznick A. Z., Viguie C. A., Starke-Reed P. and Packer L. (1992) Exercise, oxidative damage and effects of antioxidant metabolism. J. Nutr. 122, 766-773. Wohaieb S. A. and Godin D. V. (1987) Starvation-related alterations in free radical tissue defense mechanisms in rats. Diabetes 36, 169-173. Yagi K. (1976) A simple fluorometric assay for lipoperoxide in blood plasma. Biochem. Med. 15, 212-216.
hr. J. Biochem.Cell
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Antioxidant Enzyme Activities and Lipid PeroxidMm Levels in Exercised and Hypertensive Rat Tissues HUI HONG, PETER JOHNSON* Department of Chemistry and College of Osteopathic Medicine, Ohio University, Athens, OH 45701, U.S.A. Previous studies have shown that exercise-inducedchanges in muscle antioxidant status oCctlr shortly after exercise.The present studies were de&& to determiue if longer-term exercise-related changesin antioxidant enzymeactivities in both normoteusiveOYKY) and byperteusiverats (SHR) occurred, and if tbese cbauges were related to the levelsof lipid peroxid&on. WKY muI SIR rats were exercised over a l&week period using a progressivetreadmill regimen. After a l-week detraining period, the animals were eutba&ed and measurementsof tissne autioxidant enzyme activities and lipid peroxide levelswere determined iu bDtb exercisedand epgesedentsry groups. Decreasesin antioxidant activities (particularly glutatbioue peroxidase and catalase) in liver, kidney, skeletal and cardiac were associatedwith exercisetrain&g in both WKY and SHR rats (e.g. left ventricular glutathione peroxidase spec& activity in WIN rats was decreasedfrom 234&25 [SD, n=12] to 187+17 ISD, n = 11) m&s/rug proteiu). Elevatious in activities of antioxidant enzymes were generally associated with hype&m&m in these tissues (e.g. k?fi ventricular gIutMii peroxidase specific activity in SHR rats was 275 + 30 [SD, n = 121 units/mg proteiu), but changes in activities were more variable thau those seen in resportseto exercise. Exercise-related chauges in tissue levels of thiobarbituric acid-reactive substances(an indirect measure of tissue lipid peroxide levels)geueraRy did not correlate with exercise-related antioxidant enzymeactivity changes,and hypertension had no effect on &se levelsexcept in liver. Tbe results show that alterations of the activities of tissue antioxidant euzyme activities by exerciseor hypertension have no major effects on tbe levelsof Upfd peroxidation iu these tissues. These readts also suggestthat the mechanismsby which exercise and hypertension affect tissue antioxidant enzyme activities are different. Keywords: SHR rat
Exercise training Muscle Antioxidant enxymes Lipid peroxides
Int. J. Biochem. Cell Biol. (1995) 27,923-931
potential for generation of increased amounts of reactive oxygen species and elevated levels of oxidative damage (Alessio and Goldfarb, 1988; Kihlstrom, 1992). However, a number of studies have shown that there is an adaptive response in exercise-trained subjects in which oxidative capacity (Favier et al., 1989; Laughlin et al., 1990) and tissue resistance to oxidative damage (Kihlstrom, 1992; Witt et al., 1992, Alessio, 1993) are increased. In the latter case, alterations in the activities of the enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase have been implicated (Harris, 1992; Sies, 1993, Higuchi et al., 1985; Powers et al., 1994), although some of these enzymes have other functions including
INTRODUCTION
Aerobic organisms have evolved a number of antioxidant mechanisms which function to relieve oxidative stress either by preventing reactive oxygen species formation or by mitigating the damage that such species can cause (Halliwell, 1992; Sies, 1993). Exercise presents a particularly important type of oxidative stress to animal systems because the increased consumption of molecular oxygen for respiration has the *To whom correspondence should be addressed. Abbreviations: TBARS, thiobarbituric acid-reactive substances. Received 7 February 1995; accepted 25 April 1995. 923
924
Hui
Hong
and Peter
prostanoid metabolism and the maintenance of adequate glutathione levels for normal tissue metabolism (Halliwell and Gutteridge, 1992). Increases in skeletal muscle antioxidant enzyme activities (particularly of glutathione peroxidase) occur immediately (Lawler et al., 1993; Ji and Fu, 1992; Ji et al., 1992) or shortly after (Ji et al., 1988; Favier et al., 1989; Ji et al., 1991; Hammeren et al., 1992; Criswell et al., 1993) exercise, although no changes or decreases may occur in some antioxidant enzyme activities (Salminen and Vihko, 1983; Higuchi et al., 1985; Laughlin et al., 1990; Schauer et al., 1990; Lee et al., 1991). In the myocardium of exercised animals, reports of increases (Lew and Quintanilha, 1991; Ito et al., 1992) decreases (Kihlstrom et al., 1989, Schauer et al., 1990) or unaltered (Kihlstrom et al., 1989; Ji, 1993) activities of antioxidant enzymes have also been made, and it has been suggested that myocardia from exercise-trained rats and mice may be less susceptible to lipid peroxidation damage (Kihlstrom, 1992). Most of the above studies have focused on muscle sampling in the immediate post-exercise period, and may reflect a very short-term response to an exercise episode. The current studies were designed to examine antioxidant status at a post-exercise period much later than was previously used in order to determine if long-term adaptive changes in antioxidant enzyme activities were similar in tissues with different metabolic functions, and if these changes were related to the levels of lipid peroxidation (as reflected by tissue TBARS levels) that exist in trained subjects. Such longterm changes in enzyme expression in response to chronic exercise are known to occur for non-antioxidant enzymes in muscle (Falduto et al., 1992; Lawler et al., 1993; Staron and Johnson, 1993), although in some cases the exercise-induced changes are reversed during the post-exercise detraining period (Ladu et al., 1991; Neufer et al., 1992). In addition to exercise training, hypertension may also result in changes in tissue morphology and metabolism (Tomanek et al., 1984; Pauletto et al., 1988; Ito et al., 1992; Lakatta, 1990; Smith et al., 1990), including changes in tissue lipid peroxidation (Schimke et al., 1987; Papies et al., 1989; Ito et al., 1992; Uotila et al., 1993) and decreases in the levels of the putative (Boldyrev and Severin, 1990) antioxidant histidine dipeptides (Johnson and Hammer, 1992). To date, the only reports concerning the status of antioxidant enzyme activities in the myocar-
Johnson
dium of hypertensive animals have indicated that the activity of catalase is unaltered in comparison to normotensive activity (Tomanek et al., 1984), whereas glutathione peroxidase activity differs from that in normotensive rats and is age-dependent (Ito et al., 1992). Studies on the effects of exercise training on the myocardium of hypertensive rats have reported changes in morphology (Crisman and Tomanek, 1985) and isoenzyme expression (Schaible et al., 1987; Rupp, 1989) although exercise did not affect heart or left ventricular weight in SHR rats (Crisman and Tomanek, 1985). Part of the significance of the studies on exercised hypertensive animals is that exercise has been suggested as a component in the management of certain types of hypertension (Crisman and Tomanek, 1985; Sleight, 1990) and it seemed important to determine if exercise has advantageous or disadvantageous effects in hypertensive animals with respect to antioxidant status. As previous reports have indicated that some antioxidant enzyme activities are changed as a result of hypertension or exercise in muscle tissue (Ito et al., 1992; Ji and Fu, 1992; Lew and Quintanilha, 1991; Powers et al., 1994) the activities of four key antioxidant enzyme activities in muscle were measured in order to determine if these activities were changed as a result of hypertension and/or exercise and if such changes were related to prevailing TBARS levels in the tissue. For the present studies, we selected a part of the myocardium (the outer wall of the left ventricle) that has been shown to undergo major morphological and biochemical changes in exercise-induced hypertrophy and hypertension (Pauletto et al., 1988; Rupp, 1989; Kihlstrom et al., 1989; Smith et al., 1990), and we have also analyzed changes in the superficial vastus lateralis of the quadriceps femoris, a white anaerobic skeletal muscle (Higuchi et al., 1985) and the longissimus dorsi which is a red aerobic skeletal muscle (Dr R. S. Staron, pers. commun.). In order to ascertain if possible changes in antioxidant enzyme activities and TBARS levels were unique to muscle systems under our experimental conditions, we also included two aerobic non-muscle tissues (liver and kidney) in this study. MATERIALS
AND
METHODS
Materials and animals Reagents and auxiliary enzymes for enzyme assays were purchased from Sigma Chemical
Exercise, hypertension and antioxidant enzymes
Co., St Louis WKY (normotensive) and SHR (spontaneously hypertensive) male rats (Rattus norvegicus) were purchased from Harlan Sprague-Dawley, Indianapolis at an age of 11-12 weeks. Exercise training and animal care
In these studies, four groups of rats were established: exercised or sedentary SHR, and exercised or sedentary WKY. Each group contained 12 rats, with the exception of the exercised normotensive group which contained 11 rats. Each animal was housed in a separate cage and was fed a standard rat diet and allowed access to water ad libitum. The exercise program was commenced one week after the animals were obtained from the supplier, and animals in the exercised groups were subjected to a graded exercise training regimen over a lo-week period using a variable-speed moving-belt treadmill with an adjustable incline. The regimen, which was based on previous procedures designed to elicit sub-maximal O2 consumption (Crisman and Tomanek, 1985), consisted of exercise for five out of seven weekdays as follows: week l-15 min duration, 10 m/min (belt speed), 0” (incline); week 2-30 min, 10 m/min, 0”; week 3-30 min, 10 m/min, 5”; week 4-30 min, 20 m/min, 5”; week 5-45 min, 20 m/min, 5”; weeks 6-lO-60min, 20m/min, 5”. Rats in the control (sedentary) group were removed from and placed into their cages five days per week over a lo-week period to account for any handling-induced stress. After a sedentary (detraining) period of one week following termination of the exercise/handling period, systolic blood pressures were measured by the tail-cuff technique (Henley et al., 1992) and body weights were measured. Tissue collection, preparation
storage
and
homogenate
Following measurement of body weights and blood pressures, the animals were euthanized by CO,-induced asphyxiation. The selected tissues (septum-free outer wall of the left ventricle, superficial vastus lateralis muscle of the quadriceps femoris, longissimus dorsi muscle, liver and kidney) were then removed immediately and washed briefly in cold phosphate buffer in order to minimize interference by erythrocyte antioxidant enzymes (Criswell et al., 1993). The tissues were cut into approx. 50-1OOmg portions and stored separately at -70” in plastic vials. Homogenates from these samples were then
925
prepared after the addition of 1.0 ml phosphate buffer per 100 mg of tissue as described previously (Johnson and Hammer, 1993), and protein concentrations of the preparations were measured by the Bio-Rad Bradford protein assay kit. Enzyme assays
Total superoxide dismutase activity was measured by the NADPH oxidation procedure (Paoletti and Mocali, 1990) using 5-40 ~1 aliquots of homogenate sample in a final assay volume of 1.065 ml. The unit of enzyme activity is the amount of enzyme that gave 50% inhibition of the control rate of NADPH oxidation. Catalase activity was measured by spectophotometric analysis of the rate of hydrogen peroxide decomposition (Aebi, 1984) when 1-50~1 aliquots of homogenate sample were used in a final assay volume of 0.5 ml. The arbitrary unit of catalase activity is defined as the observed firstorder rate constant (U set’) for H,Oz degradation. Glutathione peroxidase activity was measured using t-butyl hydroperoxide and glutathione as substrates in the glutathione reductase-linked “continuous monitoring” procedure (Flohe and Gunzler, 1984) with sample aliquots of 4-20 ,ul used in a final assay volume of 0.5 ml. A unit of glutathione peroxidase activity is defined as the amount of enzyme which gives a 90% decrease in glutathione concentration per min at a 1 mM starting glutathione concentration. Glutathione reductase activity was determined by measurement of the rate of NADPH oxidation in the presence of oxidized glutathione (Carlberg and Mannervik, 1985), using 2-50 ~1 sample aliquots in a final assay volume of 0.5 ml. The unit of enzyme activity is defined as the amount of enzyme which oxidizes 1 mmol of NADPH per min. Enzyme specific activities (using the standard definition of an enzyme activity unit from the method for the assay) and TBARS levels were expressed in terms of protein content of tissue extracts. When the data are expressed on a wet weight basis (results not shown), very similar patterns of results were obtained but the data are presented on a protein content basis for comparison with previous studies (Kainulainen and Komulainen, 1989; Kihlstrom, 1992; Criswell et al., 1993; Powers et al., 1993; Powers et al., 1994) and because protein content is more accurately measured and is unaffected by exercise in rat myocardium (Kihlstrom et al., 1989).
926
Hui Hong and Peter Johnson
Thiobarbituric acid-reactive (TBARS) measurement
substances
Estimates of lipid peroxidation levels were performed by the thiobarbituric acid-based procedure for the fluorometric determination of malonaldehyde (Yagi, 1976) using a PerkinElmer LS-5 Fluorescence Spectrophotometer. Homogenate sample volumes of 0.25 ml were used in a final assay volume of 1.25 ml, and assays were performed in triplicate. The assay procedure was calibrated using tetraethoxypropanone as a malonaldehyde source, and levels of tissue sample TBARS were calculated as fmol malonaldehyde per mg of sample protein. Statistical analysis
Mean values 4 SD were calculated for each data set after elimination of outliers by the Dixon test (Sokal and Rohlf, 1981), and significant differences between data sets were detected by one-way ANOVA. Values for P > 0.05 were taken to indicate no significant difference. RESULTS
E&ct of exercise training on body weight and blood pressure
The effects on body weight and blood pressure of the training regimen are shown in Table 1. In agreement with earlier studies using this regimen (Crisman and Tomanek, 1985; Friberg et al., 1988; Tipton et al., 1991), no significant decrease in blood pressure in hypertensive SHR rats was observed as a result of exercise, although a significant decrease in blood pressure in response to exercise was observed in the normotensive WKY rats. In contrast to the previous studies (Crisman and Tomanek, 1985), small but significant decreases (of about 10%) in body weight for both the
exercised WKY and SHR rats were observed in comparison to sedentary controls. Eflects of exercise training on antioxidant enzyme activities and TBARS 1eveIs in SHR and WKY rats
Table 2 shows that exercise training resulted in decreases in glutathione peroxidase and catalase activities in all of the tissues studied in both WKY and SHR rats. In general, the decreases for each enzyme were similar within the same tissue for both SHR and WKY rats, with catalase activities generally showing larger decreases than glutathione peroxidase. The largest decreases were seen in catalase activities in the left ventricular outer wall, where the exercised animals had activities which were down to 46 and 49% of the respective sedentary WKY and SHR activities. For total superoxide dismutase activity, exercise training caused decreases in WKY and SHR liver activities down to approx. 70% of sedentary animal activities, whereas no changes were seen in kidney, and 93 and 25% increases in activity were seen in longissimus dorsi of WKY and SHR rats, respectively. Glutathione reductase activities were decreased by exercise training in the left ventricular outer wall of both WKY and SHR rats (down to 86 and 89% of sedentary animal activities, respectively), whereas only the quadriceps femoris of WKY rats showed a decrease, with the activity in exercised SHR animals remaining unchanged. In the longissimus dorsi, exercise also had no effect on glutathione reductase activity in SHR rats, but resulted in a 26% increase in WKY rats. Liver and kidney glutathione reductase activities were unaffected by exercise in WKY rats, and in SHR rats, kidney glutathione reductase was also unchanged, whereas an activity decrease was observed in liver of SHR rats.
Table 1. The effect of exercise training on body weight and blood nressure in WKY and SHR rats Body weight Blood pressure Rat Statusa (g; mean k SD) (torr; mean If: SD) WKY Sedentary (12) 375.2 f 30.2 140.8 If: 11.1 P <0.002* PiO.003 Exercised (11) 339.5 & 20.2 121.7 + 14.0 SHR Sedentary (12) 409.0 f 14.0 180.4 + 20.8 P
921
Exercise, hypertension and antioxidant enzymes Table 2. Antioxidant
Parameter SOD
CAT
Tissue OWLV Liver Kidney OWLVT
LDt QFt
GPX
Liver Kidney OWLV
GR
& Liver Kidney OWLV
TBARS
g Liver Kidney OWLV
enzyme specific activities* and TBARS levels in tissues of exercised and sedentary rats SHR Rats WKY Rats Sedentary (1) 3.06 +0.59 3.05
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0.323 0.661 208 87.5 39.5
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g 166+30# 186&44 Liver 173+32# 250*60$ Kidney 284*51$ 315+39 *Enzyme specific activities and TBARS levels in the outer wall of the left ventricle (OWLV), longissimus dorsi (LD), quadriceps femoris (QF), liver and kidney are expressed as the mean & SD in units per mg of homogenate protein as defined in the assay procedure described in “Materials and Methods”. For clarity, mean values are simplified to three significant figures. The enzyme names are abbreviated as superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR), and catalase (CAT). tValues shown are actual x 10’. Values were obtained from assays on 12 different animals in each group, except for those values marked with $(n = 11) and $(a = 10). P values for statistically significant differences between data sets for animal groups 1 and 2, 1 and 3, 3 and 4, and 2 and 4 are indicated as P,,,, P,,,, P3,4and PzAand the symbols used are-for no significant difference (P > 0.05), 11for P between 0.05 and 0.005, and 7 for P < 0.005.
With respect to TBARS levels (Table 2), the only tissue which showed similar exerciserelated changes in both WKY and SHR rats was longissimus dorsi (decreases down to about 67% of sedentary levels). In WKY animals only, exercise-related decreases in TBARS levels occurred in quadriceps femoris and liver, whereas an increase was found in the left ventricular outer wall. TBARS levels in kidney were unaffected by exercise in WKY rats but showed a 23% increase in SHR animals. Hypertension-related changes in antioxidant enzyme activities and TBARS levels
Table 2 shows that the only hypertensionrelated changes in total superoxide dismutase activity were in the longissimus dorsi (a 58% increase over normotensive activity) and in the quadriceps femoris (a decrease down to 68% of the normotensive activity) of sedentary animals. In the case of catalase activity, increases were seen in the left ventricular outer wall, longis-
simus dorsi and kidney for both sedentary and exercised groups, and in liver of sedentary animals. For both the sedentary and exercised groups, hypertension-related increases in glutathione peroxidase activity were also seen in the left ventricular outer wall and longissimus dorsi, whereas no changes were seen in quadriceps femoris and activity decreases were seen in liver. With glutathione reductase, hypertensionrelated increases in activity were found in longissimus dorsi, liver and kidney of both sedentary and exercised groups, with the largest increase (83% above normotensive activity) being observed in the longissimus dorsi of sedentary animals. The activity of glutathione reductase in the left ventricular outer wall was unaffected by hypertension in sedentary animals, although an increase of 11% was seen in the exercised group. In the case of glutathione reductase in the quadriceps femoris, a decrease in activity was observed in sedentary animals, whereas no change was found in the exercised group.
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Table 2 also shows that TBARS levels were unaffected by hypertension in sedentary rats, and the only hypertension-related change in TBARS levels in the exercised animals was in liver, where a 59% increase over normotensive levels was observed. DISCUSSION
Weight loss caused by starvation is associated with changes in antioxidant enzyme activities in rat heart, liver and kidney (Wohaieb and Godin, 1987). Although the exercise regimen in the present studies did cause a statistically-significant weight loss, this weight loss was much smaller than that observed in the foregoing starvation studies (about 10% compared to 20%), and with the exception of the decrease in liver catalase, the exercised-related enzyme activity changes were quite different from those observed in these starvation studies. These differences strongly suggest that the enzyme activity and TBARS level changes observed in the present studies are exercise-related and not a result of weight loss per se. Exercise training in both WKY and SHR rats resulted in long-term decreases in the activities of glutathione peroxidase and catalase in all of the tissues examined. These results are in agreement with earlier studies which have reported exercise-related decreases in myocardial and skeletal muscle catalase activities (Kihlstrom et al., 1989; Powers et al., 1994), but not with a previous report of post-exercise activity increases in skeletal and cardiac muscle catalase activities (Lew and Quintanilha, 1991). Our findings of decreases in glutathione peroxidase activities also differ from a number of previous reports which have shown immediate postexercise activity increases (Ji et al., 1990; Ji et al., 1991; Hammeren et al., 1992; Criswell et al., 1993; Lawler et al., 1993; Powers et al., 1994; Schauer et al., 1990; Lew and Quintanilha, 199 1; Kumar et al., 1992) or no activity change (Schauer et al., 1990; Ji, 1993) in this enzyme. In the case of superoxide dismutase activities in skeletal muscles, Criswell et al. (1993) concluded that exercise-related upregulation of superoxide dismutase was limited to highly oxidative skeletal muscle, and our studies confirm these findings for WKY rats. However, in the case of the SHR rats, an exercise-related increase in total superoxide dismutase activity occurred in the anaerobic quadriceps femoris but not the aerobic longissimus dorsi muscle,
which implies that the regulation of skeletal muscle superoxide dismutase activities is different in SHR and WKY rats. No changes were found in left ventricular total superoxide dismutase activity as a result of exercise and/or hypertension, which differs from earlier reports of an exercise-related activity decrease in the Cu,Zndependent enzyme (Kihlstrom et al., 1989) and of an apparent increase in Mn-dependent superoxide dismutase activity (Powers et al., 1993). It is not yet clear if the lack of change in total superoxide dismutase activity in the myocardium after detraining represents compensatory changes in the two forms of superoxide dismutase, or a return to normal levels of one or both of these enzyme forms, and further studies will be necessary to investigate these possibilities. Some of the above-mentioned disparities between previous and current results may be related to the choice of post-exercise times at which tissue samples were obtained. For superoxide dismutase in the left ventricular outer wall, where previous studies have shown immediate post-exercise activity changes (Kihlstrom et al., 1989) the lack of long-term activity changes observed in our studies may indicate that the length of the detraining period (7 days) before measurement permitted a return to control levels, as has previously been reported for other muscle non-antioxidant proteins and enzymes (Ladu et al., 199 1; Neufer et al., 1992). However, for superoxide dismutase and glutathione reductase in longissimus dorsi muscle, the long-term enzyme activity changes were in the same direction as the immediate post-exercise changes (Ji et al., 1990; Criswell et al., 1993; Lawler et al., 1993; Powers et al., 1994), indicating that the activity changes were not reversed during the detraining period and that the training effect was of a prolonged duration for these enzymes. Such differences in post-exercise enzyme activities suggest that the regulation of these activities may be quite complicated, and we are currently investigating the roles that transcriptional and translational controls may play under such circumstances. In previous studies (Godin and Garnett, 1992a) on antioxidant enzymes in erythrocytes and myocardium, patterns of activity changes were examined for the functionally coupled pairs of antioxidant enzymes glutathione peroxidase/glutathione reductase and superoxide dismutase/catalase, and it was concluded that the enzyme levels in these pairs may be independently regulated. In the present studies, parallel
Exercise, hypertension and antioxidant enzymes
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changes for theseenzymesin both WKY and enzyme activities may be involved in minimizing the extent of tissue lipid peroxidation associated SHR rats were seen for glutathione peroxidase/ glutathione reductase in the outer wall of the left with exercise. In addition to the factors discussed in terms of the TBARS assay, such ventricle, and for superoxide dismutaselcatalase in liver, where decreases in activity were ob- factors may include decreases in O2 uptake in served. Parallel decreases in activity for gluta- trained hypertensive rats (F&erg et al., 1988) thione peroxidase/glutathione reductase were changes associated with increases in glucose uptake in hypertensive rats (Leipala et al., also seen in the quadriceps femoris of WKY (but not SHR) rats and in liver of SHR (but not 1989) changes in the levels of non-enzymatic WKY) rats. These reults indicate that there may tissue antioxidants (Janero and Burghardt, be an exercise-related co-ordinated regulation of 1988; Lew and Quintanilha, 1991), and changes enzyme levels in some tissues such as the left in the activities of other antioxidant enzymes ventricular outer wall, whereas such changes which were not part of this study. In contrast to the exercise-related decreases in may not occur in other tissues or may be changed as a result of hypertension. glutathione peroxidase and catalase, elevated glutathione peroxidase and catalase activities In liver, skeletal muscle and erythrocytes, there did not appear to be a causal relationship were associated with hypertension in the left between tissue activities of antioxidant enzymes ventricular outer wall and longissimus dorsi, and the prevailing levels of lipid peroxidation whereas hypertension had no effect on gluta(Godin and Garnet& 1992b; Lawler et al., 1993; thione reductase activity in the anaerobic Ji et al., 1990), whereas in rat myocardium, muscle (quadriceps femoris). In the case of liver antioxidant enzyme levels were shown to be and kidney, both of which are aerobic tissues, inversely correlated with susceptibility to lipid activity increases associated with hypertension peroxidation as a result of experimental hyper- were only seen for catalase in kidney, whereas trophy and spontaneous hypertension (Ito et al., for liver glutathione peroxidase, decreases in 1992; Kirshenbaum and Singal, 1993). In some activity were observed. These results indicate of these previous studies and in the present that the effect of hypertension on antioxidant studies, lipid peroxidation levels were measured enzyme activities is different than the effect of by the widely-used indirect TBARS procedure exercise, and suggest that different mechanisms which is designed to measure malonaldehyde may exist in various tissues for regulation of levels. Although the procedure measures malonenzyme activities in response to exercise and aldehyde levels accurately, the assay is subject to hypertension. interference by a number of substances present No changes in TBARS levels in the left in biological samples which can cause an overes- ventricular outer wall, skeletal muscles or kidtimation of tissue lipid peroxide levels (Slater, ney were associated with hypertension, although 1984). A further complication in the use of a large increase in the TBARS level was found TBARS data to indirectly measure free radical in liver of exercised SHR rats (59% above the generation in tissues is that endogenous tissue exercised WKY rat level). In exercise-trained antioxidants such as ascorbate and u-tocoSHR rats, some tissue enzyme activities appherol can effectively scavenge oxygen-derived proached sedentary normotensive values withfree radicals, thereby preventing propagation of out increases in TBARS levels, except in the lipid peroxidation (Krinsky, 1992) and causing case of kidney where an increase in TBARS an underestimation of free radical generation in level occurred. These results indicate that the the tissue. Bearing in mind these uncertainties processes for maintaining TBARS levels in hyinherent in the use of the TBARS assay to pertensive animals in exercise training may be measure tissue lipid peroxide levels, our exercise different from those in normotensive animals, training results showed that an inverse relationand also support the conclusion previously disship between tissue TBARS levels and enzyme cussed that alterations in tissue susceptibilities activities did exist in some tissues (longissimus to lipid peroxidation (measured as TBARS) dorsi, left ventricular outer wall and kidney), may not be inversely correlated with the activibut that this relationship could be altered in ties of the antioxidant enzymes studied. SHR rats. Because there was not a consistent In conclusion, the present study has shown picture of an inverse correlation between that long-term decreases in some tissue antiTBARS levels and antioxidant enzyme activi- oxidant enzyme activities were associated ties, it is possible that factors other than these with chronic exercise in both normotensive and
930
Hui Hong and Peter Johnson
hypertensive rats, whereas increases in some tissue antioxidant enzyme activities were associated with hypertension. The patterns of enzyme activity changes associated with exercise training or hypertension were generally not consistent for a particular enzyme from tissue to tissue, and changes in tissue TBARS levels did not appear to be related to the observed enzyme activity changes in most cases. Previous studies have shown that in tissues such as myocardium, antioxidant enzyme activities may be rate-limiting for oxidation processes (Wohaieb and Godin, 1987), and in such cases, changes in enzyme activities could alter in vivo rates of oxidative processes if such enzymes were substrate-saturated (Newsholme and Leech, 1983). More detailed information about the rate-limiting features of enzyme concentration and substrate concentrations will therefore be needed to establish if the changes in enzyme activity measured by in vitro assays actually reflect changes in the rates of oxidative process in vivo. Acknowledgements-These studies were supported by a grant from the American Heart Association, Ohio Alhhate, Inc., Columbus, Ohio. We thank MS M. Halterman and MS M. A. Notestine for excellent technical assistance. REFERENCES
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