(Received for publication, November 20, 1995; and in revised form, March 5, 1996)
From the
Weak antioxidant capacity, particularly low catalase activity in the heart, may be a factor responsible for the high sensitivity of this organ to doxorubicin-induced oxidative damage. To test this hypothesis, a heart-specific promoter was used to drive the expression of murine catalase cDNA in transgenic mice. Fifteen healthy transgenic mouse lines were produced. Cardiac catalase activity was constitutively overexpressed in both atrium and ventricle, ranging from 2- to 630-fold higher than normal. This enzyme activity was not altered in liver, kidneys, lungs, and skeletal muscles. Other antioxidant components, including glutathione, glutathione peroxidase, glutathione reductase, metallothionein, and superoxide dismutase, were not altered in the catalase-overexpressing heart. Mice (7 weeks old) from several transgenic lines and from nontransgenic controls were treated intraperitoneally with doxorubicin at a single dose of 20 mg/kg and sacrificed on the 4th day after treatment. As compared to normal controls, transgenic lines expressing catalase activity 60- or 100-fold higher than normal exhibited a significant resistance to doxorubicin-induced cardiac lipid peroxidation, elevation of serum creatine phosphokinase, and functional changes in the isolated atrium. Interestingly, 200-fold or greater elevation of catalase activity did not provide protection. The results provide direct evidence for the role of catalase in doxorubicin cardiotoxic responses.
Doxorubicin is an anthracycline antibiotic and one of the most important anti-cancer agents. It is effective in treatment of acute leukemias and malignant lymphomas as well as a number of solid tumors. However, the clinical use of doxorubicin is limited by its cardiotoxicity(1, 2, 3) . Studies have suggested that doxorubicin may have at least two mechanisms of action that cause cellular damage. One mechanism involves generation of oxygen free radicals, the damage from which is inhibited by free radical scavengers. This appears to play a major role in the development of cardiomyopathy(4, 5, 6, 7) . The other mechanism is mediated by the intercalation of the drug to DNA and is unaffected by free radical scavengers. This appears to be the major determinant of doxorubicin cytotoxicity to tumor cells(7) .
Catalase is a major enzyme involved in the detoxification of
hydrogen peroxide (HO
). This enzyme catalyzes
the conversion of two molecules of H
O
to
molecular oxygen and two molecules of water. The primary physiological
function of this enzyme is to detoxify the H
O
produced as a result of peroxisomal aerobic dehydrogenase
reactions. The importance of catalase in providing protection against
extraperoxisomal H
O
is not clear. Studies with
isolated hepatocytes, however, have demonstrated that, under conditions
of GSH depletion, catalase functions in the metabolism of
H
O
produced by the cytochrome P-450-linked
monooxygenase system(8) . It is thus possible that, under
conditions of extreme oxidative stress, which in most cases involves
GSH depletion, catalase may become important in providing
cytoprotection.
Catalase activities are highest in liver and
erythrocytes, relatively high in kidney and adipose tissue,
intermediate in lung and pancreas, and very low in heart and
brain(9) . It has been shown that catalase activity in the
heart is about 2% of that in the liver of the human (9) , the
mouse(10) , and the rat(11) . Several studies have been
undertaken to determine the role of catalase in cardioprotection
against oxidative injuries. Supplementation of perfusion medium with
catalase significantly reduced ischemia-reperfusion injury to the
isolated rat heart by detoxifying
HO
(12) . The role of catalase in
metabolism of H
O
in the heart tissue was also
tested directly by adding H
O
into perfusion
medium(13) . Accumulated evidence shows that catalase, although
present at low levels of activity, functions in detoxification of
H
O
in the myocardium.
It is unknown,
however, whether the low catalase activity in the heart is responsible
for the high sensitivity of this organ to oxidative stress. Current
approaches using the isolated heart perfused with catalase have limited
application. Exogenously added catalase may not be able to function
intracellularly, but may react only with HO
directly in the perfusion medium. Thus, the relationship between
the low constitutive catalase activity and the high sensitivity to
oxidative stress in the heart has not been defined. A unique in
vivo model is required to overcome these limitations and to
precisely determine the role of catalase in protection against
oxidative cardiotoxicity.
In the present study, we have developed a
transgenic mouse model in which catalase is overexpressed only in the
heart. To determine whether catalase elevation provides protection
against doxorubicin cardiotoxicity, lipid peroxidation in the heart and
serum creatine phosphokinase (CPK) ()activity were measured
in the doxorubicin-treated transgenic mice and nontransgenic controls.
In addition, the atria isolated from the transgenic mice and
nontransgenic controls were used to determine the effect of catalase on
doxorubicin-induced functional changes. The results demonstrate that
overexpression of catalase in the heart provides protection against
doxorubicin cardiotoxicity.
Figure 1: Structure of the MyCat transgene. The black boxes indicate the promoter and first three noncoding exons of the alpha cardiac MHC gene(15) . Lines between the boxes designate two MHC introns. The open box designates the rat catalase cDNA(14) , and the slashed box indicates the portion of the transgene derived from the rat insulin II gene(16) . Arrow 1 shows the position of the transcription initiation site of the alpha cardiac MHC gene. Arrows 2 and 3 show the positions of the native start and stop codons of rat catalase. Arrow 4 indicates the position of the polyadenylation sequence from the rat insulin II gene. Restriction sites used to construct the transgene, to purify the transgene, or to obtain probes are indicated. Restriction sites shown in parentheses were destroyed during construction of MyCat. The bars designated as A and B indicate the portions of the transgene which were used to prepare probes of the Northern and Southern blots, respectively.
Standard procedures (17) were employed for development of transgenic mice. The 8-kb MyCat transgene (the NotI fragment, Fig. 1) was purified (18) on a matrix of diatemaceous earth (Prepagene, Bio-Rad) and filtered through a 0.22-µm filter. Approximately 100 copies of the purified transgene insert were microinjected into one pronucleus of each one-cell mouse embryo of the inbred strain FVB(19) . Twenty microinjected embryos were implanted into each Avertin-anesthetized pseudopregnant female and allowed to come to term. To identify transgenic founder mice, genomic DNA was isolated from 1-cm tail clips from 3-4-wk-old mice. DNA was subjected to Southern and dot blot analyses which were probed with a 550-bp SmaI/NotI fragment derived from the rat insulin II portion (16) of the MyCat transgene. This probe hybridized to an 8000-bp EcoRI fragment of the transgene, consistent with the presence of a unique EcoRI site in the MyCat transgene (600 bp upstream of the MHC transcription initiation site)(15) . Founder mice were bred with mice of the same strain and transgenic offspring were routinely identified by a polymerase chain reaction using a primer pair derived from the MHC promoter and rat catalase cDNA. Transgenic positive mice (heterozygotes) and negative littermates were then used for experiments.
Cardiac catalase activity was analyzed in six animals (three males and three females) of each transgenic line and nontransgenic controls. As shown in Fig. 2, catalase activity was markedly elevated in the transgenic hearts. This elevation ranges from 2-fold in line 786 to 630-fold in line 784. There was no significant difference (p > 0.10) in the catalase activity between males and females within the same transgenic line (data not shown). The level of mRNA for catalase was also increased in the transgenic heart as shown in Fig. 3. This overexpression was rather stable as evidenced by consistent results obtained in several assays performed over one year. The results clearly demonstrate that the increased catalase activity results from expression of the MyCat transgene. We tested whether the catalase activity is elevated to the same extent in both atria and ventricles. As shown in Fig. 4, the elevated catalase activity was the same in atria and ventricles from five representative transgenic mouse lines. We also determined whether the elevated catalase expression was specific to the heart. Catalase activities in liver, kidneys, lungs, and skeletal muscles of the transgenic mice were measured. As shown in Fig. 5, catalase activities in all of these tissues were the same as that of controls.
Figure 2: Catalase activities in the heart from each of the 15 different transgenic mouse lines in comparison with that of controls (Cont). Each value is the average of six determinations (mean ± S.D.).
Figure 3: Representative Northern blot analysis of catalase mRNA in the hearts of the 15 different transgenic mouse lines and the controls. The amount of mRNA detected in each line correlates with the level of catalase activity in the heart. Three bands hybridized with the catalase probe in transgenic samples. Only the smallest band corresponds to the expected, full processed transgenic mRNA. The arrow indicates the position of the single control band.
Figure 4: Comparison of elevated catalase activities in the atria and ventricles in 5 representative transgenic mouse lines. Each value is the average of three determinations (mean ± S.D.). There is no significant difference (p > 0.10) in the catalase activity between the atria and ventricles in any of these transgenic lines.
Figure 5: Comparison of catalase activities in the liver, kidney, lung, and skeletal muscles from the left rear leg (M1) and the abdominal external oblique muscles (M2) of the five transgenic mouse lines as shown in Fig. 4(lines 738, 742, 782, 776, 777). The values represent the pooled data from the five lines with three determinations of each line (mean ± S.D., n = 15) for MyCat and from six control animals (Cont).
We then determined whether other antioxidant enzyme activities in the transgenic heart were altered due to changes in catalase activity. The activities of SOD, GSH peroxidase, and GSH reductase were measured in the catalase-enriched transgenic hearts. As shown in Table 1, none of these enzyme activities was changed. We also measured the concentrations of two important nonenzymatic antioxidant components, GSH and MT. There is no alteration in either of the two components in the catalase-enriched transgenic hearts (Table 1).
Four transgenic lines were then selected to determine the effect of elevated catalase activity on doxorubicin-induced oxidative damage to the heart. These four transgenic lines have cardiac catalase activity about 15-, 60-, 100-, and 200-fold higher than normal. Transgenic mice and nontransgenic controls were treated with a single dose of doxorubicin at 20 mg/kg. Four days after the treatment, the lipid peroxide products, MDA, and 4-hydroxyalkenals were determined. As shown in Fig. 6, the level of the lipid peroxidation products was markedly elevated by doxorubicin in nontransgenic mice. Overexpression of catalase suppressed this effect of doxorubicin in a catalase activity-dependent fashion. The highest protection was observed in the hearts expressing this enzyme activity about 60-100-fold higher than normal. However, the transgenic line overexpressing catalase activity about 200-fold higher than normal demonstrated no protection from doxorubicin.
Figure 6: Doxorubicin-induced lipid peroxidation in the hearts of normal and transgenic mice. The levels of MDA and 4-hydroxyalkenals were measured to estimate the extent of lipid peroxidation in the tissue. The concentration of the lipid peroxides in nontransgenic saline-injected mice (Cont, n = 15) was 56.8 nmol/g of wet weight. Controls for each transgenic mouse line (saline injection only) showed no significant difference in the cardiac lipid peroxide level from the nontransgenic controls (data not shown). Doxorubicin significantly (p < 0.05) increased the lipid peroxide level in non-transgenic normal mouse hearts (DOX, n = 15). Fifteen-fold elevation of catalase activity in the heart slightly decreased the level of doxorubicin-induced lipid peroxides (line 738, n = 8). Sixty (line 742, n = 10) or 100-fold (line 776, n = 10) elevation significantly inhibited doxorubicin-induced lipid peroxidation (p < 0.05). The level of lipid peroxides in these hearts was not significantly different from that in the normal saline-treated hearts. Two hundred-fold elevation did not significantly decrease the level of lipid peroxides (line 777, n = 10).
Doxorubicin toxicity was also revealed by elevated serum CPK in nontransgenic mice (Fig. 7). Catalase overexpression provided essentially the same protection against CPK release as it did for lipid peroxidation. Again, the protection was roughly activity dependent except for the line with 200-fold overexpression, which failed to demonstrate any protection.
Figure 7: Doxorubicin-induced CPK release from the heart. Serum CPK was measured using a CK-20 kit from Sigma. Sera from the same animals treated for determination of lipid peroxidation were collected for the CPK measurement. As shown, doxorubicin significantly increased serum CPK level in the nontransgenic control mice (DOX). Elevation of catalase in the heart to about 60- (line 742) or 100-fold (line 776) higher than normal completely inhibited the increase in the serum CPK level (p < 0.05). However, elevation of cardiac catalase activity to about 200-fold (line 777) higher than normal did not protect against this toxic effect of doxorubicin.
Because the loss of protection with 200-fold overexpression of catalase activity was unexpected, we performed preliminary studies with 500-fold catalase overexpressing mice (line 756). The doxorubicin increased MDA levels to 93.1 ± 10.9 nmol/g (n = 4) in the transgenic heart and 80.6 ± 2.2 nmol/g (n = 4) in the nontransgenic heart. The level of serum CPK was 1.70 ± 0.15 units/ml (n = 4) in the doxorubicin-treated transgenic mice and 1.54 ± 0.20 units/ml (n = 4) in the doxorubicin-treated nontransgenic mice. These preliminary results suggested that 500-fold higher levels of catalase may have enhanced doxorubicin-induced lipid peroxidation in the heart and CPK release from the heart.
Isolated atria from transgenic mice overexpressing cardiac catalase about 60-fold were used to determine the effect of doxorubicin on atrial function. Spontaneous contractions were recorded. When doxorubicin was added to the incubation buffer, inhibition of both contraction frequency and contractile force occurred. As shown in Fig. 8, the catalase overexpressing atrium displayed a marked resistance to the toxic effect of doxorubicin. In addition, the function of the transgenic atrium completely recovered after removal of doxorubicin, but the normal atrium did not. To precisely determine the effect of doxorubicin on contractile force (inotropy) and heart rate (chronotropy), left and right atria were separated. The left atrium was paced by a stimulator at a fixed frequency of 4 Hz and a voltage of 1.5 times the threshold value. As shown in Fig. 9, 60-fold elevation of catalase activity markedly suppressed doxorubicin-reduced changes in inotropy (left atrium) and chronotropy (right atrium).
Figure 8: Functional changes induced by doxorubicin in the isolated atria from normal and transgenic mice (line 742). The transgenic atrium contains catalase activity about 60-fold higher than the normal control. The atria were incubated with 100 µM doxorubicin (final concentration) in the perfusion buffer. Changes in spontaneous contraction of the atria were recorded at the times before (0 min) and post doxorubicin treatment (10, 20, and 30 min) as shown. This experiment was repeated five times with atria isolated from different animals. The same result was obtained with the repeated experiments.
Figure 9: Effect of catalase elevation on doxorubicin-reduced inotropy (A, left atrium) and chronotropy (B, right atrium). The transgenic atria (line 742) contain catalase activity about 60-fold higher than the nontransgenic control and doxorubicin was added into the incubation buffer to a final concentration of 100 µM. The left atria were paced by a fixed frequency of 4 Hz and a voltage of 1.5 times the threshold value. This measurement was repeated three times with atria isolated from different animals and consistent results were obtained.
Cardiotoxicity has long been recognized as a complicating factor of cancer chemotherapy with doxorubicin. There are several hypotheses to explain doxorubicin cardiotoxicity. Among these the free radical hypothesis is the most thoroughly investigated. Doxorubicin undergoes one-electron reduction through a metabolic activation caused by NADPH-cytochrome-P-450 reductase, or other flavin-containing enzymes (30) . This reduction generates a doxorubicin semiquinone free radical. In the presence of molecular oxygen, the semiquinone rapidly reduces the oxygen to superoxide with regeneration of intact doxorubicin. Superoxide is rapidly converted to hydrogen peroxide spontaneously or by superoxide dismutase. The doxorubicin semiquinone can then react with the hydrogen peroxide to yield a hydroxyl radical(31) . These highly toxic reactive oxygen species react with cellular molecules including nucleic acids, protein, and lipids, thereby causing cell damage. Most supportive evidence for this free radical hypothesis has been obtained from in vitro studies including reports that: 1) doxorubicin increases lipid peroxidation and free radical production in the heart tissue(32) ; 2) free radical scavengers such as N-acetylcysteine(33) , vitamin E(34) , superoxide dismutase (34) , and catalase (7) decrease the severity of doxorubicin-induced oxidative damage; 3) suppression of antioxidant activities enhances doxorubicin toxicity in cultured cardiac cells(35) . However, contradictory results have been reported. Some in vivo studies have shown that free radical scavengers failed to prevent cumulative doxorubicin cardiotoxicity(36, 37) .
Why have some in vivo studies failed to find antioxidant protection against doxorubicin cardiotoxicity? Under in vivo conditions, three major problems complicate data interpretation: 1) it is impossible to maintain constant plasma antioxidant concentrations and to accurately predict the target tissue concentrations; 2) metabolic activation and inactivation by multiple metabolic organs such as liver and kidney would greatly affect the efficacy of the antioxidants; and 3) high molecular weight antioxidants such as superoxide dismutase and catalase are unlikely transported into intracellular compartments. To overcome the shortcomings of these earlier in vivo studies, we produced a unique transgenic model in the present study. As described under ``Results,'' in this model intracellular catalase activities are maintained at permanently elevated levels specifically in the heart of each transgenic line. Other antioxidant systems were found to be unaffected in the catalase overexpressing heart. Electron microscopic studies (data not shown) demonstrated increased numbers of peroxisomes in transgenic hearts, suggesting a significant amount of elevated catalase is contained in peroxisomes.
The results obtained from this
study clearly demonstrate that elevation of catalase activity to an
optimum level provides protection against doxorubicin-induced cardiac
injury. This suggests that low catalase activity in the heart is a
major factor responsible for the high sensitivity of the heart to
doxorubicin-induced damage. Because catalase is a major enzyme that
metabolizes HO
in the cell and this enzyme has
no activity to react with doxorubicin and its metabolites, the results
thus provide direct evidence to support the oxidative injury hypothesis
for doxorubicin. Lipid peroxidation is a major cardiotoxic indicator of
doxorubicin, particularly under the conditions of acute exposure. It
has been found that doxorubicin more selectively induces this oxidative
damage to the heart relative to other organs such as the liver in mice (38) . We have determined the lipid peroxide levels in the
heart induced by doxorubicin as a function of time (days). We found
that at the selected dose (20 mg/kg) lipid peroxide levels in the heart
reach a peak value on the 4h day after the drug treatment (data not
shown). This peak value coincides with high serum CPK activity at the
same time, which is in agreement with previous studies(39) .
Because the elevation in serum CPK results from the myocardial cell
membrane damage due to lipid peroxidation, the increased serum CPK
activity has been used as another indicator of doxorubicin-induced
heart damage(39, 40) . The results obtained from this
study show that elevated catalase activity protects from
doxorubicin-induced lipid peroxidation, which correlates with the
protection against CPK release. In both cases, 60- or 100-fold
elevation of catalase activity provides maximum protection.
Functionality of the isolated mouse atrium has been increasingly used as a fast screening method for determination of cardiotoxicity induced by doxorubicin(41) . Usually, the mouse atrium is incubated with the tested compound and changes in inotropy (contractile force) and chronotropy (heart rate) are recorded as a measurement of cardiotoxicity. It has been shown that doxorubicin depresses the functionality of isolated mouse atria(42, 43, 44) . Doxorubicin-induced functional changes in the isolated atria were observed in the present study. Both contractile force and heart rate were suppressed. Sixty-fold elevation of catalase activity markedly depressed this effect of doxorubicin on cardiac function. This result demonstrates that doxorubicin-induced cardiac functional alterations most likely result from reactive oxygen species and that the severity of the functional alterations is associated with catalase levels.
In the present study we found that 200-fold elevation of catalase activity in the heart did not provide protection against doxorubicin toxicity and 500-fold elevation may even enhance the doxorubicin cardiotoxicity. The same observation has also been reported in vitro. For instance, transfection of L cells with a human catalase cDNA elevated catalase activity by 100-fold. These catalase-enriched cells, however, were more sensitive rather than resistant to the cytotoxicity of paraquat, bleomycin, and doxorubicin than the untransfected cells from which they were derived(45) . At least two mechanisms have been proposed for the increased sensitivity of the high catalase containing cells: 1) imbalance between SOD and catalase (46, 47, 48) and 2) chelation of heme iron by catalase and release of iron after degradation of catalase. It has been shown that iron-doxorubicin complex is toxic(49) . The relevant mechanism for the effect observed in our mice is uncertain at this time.
The transgenic model produced in this study will provide a valuable experimental approach to understanding mechanisms of other cardiac disease conditions. There is, for example, disagreement on whether ischemia-reperfusion-induced heart damage is mediated by oxidative stress and whether low catalase activity in the heart generally makes the heart more sensitive to a variety of oxidative injuries. This experimental model should provide a means to test these related hypotheses.