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Cardiovascular Research 2000 46(3):523-530; doi:10.1016/S0008-6363(00)00039-0
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Copyright © 2000, European Society of Cardiology

Effects of probucol on changes of antioxidant enzymes in adriamycin-induced cardiomyopathy in rats

Timao Lia,b, Igor Danelisena,b, Adriane Belló-Kleina,b and Pawan K. Singala,b,*

aInstitute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, R2H 2A6, Canada
bDepartment of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, R2H 2A6, Canada

* Corresponding author. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Room R3022, 351 Tache Avenue, Winnipeg, Manitoba, R2H 2A6, Canada. Tel.: +1-204-235-3416; fax: +1-204-233-6723 psingal{at}sbrc.umanitoba.ca

Received 23 September 1999; accepted 3 February 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The clinical usefulness of doxorubicin (adriamycin, ADR) is restricted by the risk of developing congestive heart failure. Probucol has been reported to completely prevent ADR cardiomyopathy without interfering with its antitumor effects. The current study investigated the effects of ADR and probucol on antioxidant enzyme gene expression during adriamycin-induced cardiomyopathy in a rat model. Methods: The mRNA abundance by Northern and immunoreactive protein levels by Western blotting of myocardial antioxidant enzymes, glutathione peroxidase (GSHPx), manganese superoxide dismutase (MnSOD) and catalase (CAT) were examined in relation to the enzyme activities in hemodynamically assessed control and treated animals. Results: At 3 weeks post-treatment duration, ADR caused heart failure which was prevented by probucol. MnSOD mRNA abundance as well as protein levels were depressed by ADR treatment by 45% and 20%, respectively, and this change was prevented by probucol. However, the mRNA and protein levels of GSHPx and CAT were not significantly changed by ADR or probucol. ADR had no effect on SOD activity but this enzyme activity was increased by probucol and probucol plus ADR. GSHPx enzyme activity was decreased and oxidative stress as indicated by TBARS was increased by ADR and these changes were also modulated by probucol. Conclusion: An increase in oxidative stress, GSHPx inactivation and MnSOD downregulation during ADR cardiomyopathy were prevented by probucol treatment.

KEYWORDS Cardiomyopathy; Free radicals; Gene expression; Heart failure; Enzyme (kinetics)


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Adriamycin is one of the most effective and widely used chemotherapeutic agents against leukemia, lymphomas and various solid tumors of the lung, breast, thyroid and ovary [1]. However, this drug has cardiotoxic effects that restrict its full clinical potential. The congestive heart failure induced by adriamycin is proven refractory to commonly used therapeutic procedures [2,3].

Great efforts have been directed in dissecting out the mechanisms responsible for its antitumor and cardiotoxic effects. Ample evidence is now available supporting the view that increased oxidative stress, because of the adriamycin-induced increase in production of free radicals and the deficit of antioxidants, plays an important role in the development of cardiomyopathy and congestive heart failure [4–8]. The non-radical dependent mechanisms, including inhibition of the topoisomerase II, adriamycin-iron complex linking to DNA and intercalation of the drug between DNA base pairs, may explain its antitumor action [6,9–11]. Based on the hypothesis of free radical mediated cardiotoxicity, probucol, a lipid-lowering agent with known antioxidant properties and a relative high distribution in heart tissue due to its binding ability to cardiolipin, completely prevented adriamycin cardiomyopathy without interfering with its antitumor effects [8,12]. However, the molecular mechanisms of adriamycin-induced antioxidant enzyme changes and their prevention with probucol remain to be understood.

Because DNA strands of various enzymes can be potentially damaged directly by adriamycin and indirectly by adriamycin-produced free radicals, altered antioxidant enzyme activities could be the result of modulated gene expression at transcriptional and/or translational levels, as well as oxidative inactivation. In the present study, we have tested the following hypotheses: (1) adriamycin and free radicals generated by adriamycin may inhibit endogenous antioxidant gene expression or inactivate antioxidant enzymes; and (2) the protective effect of probucol in adriamycin cardiomyopathy may be mediated by promoting antioxidant gene expression or protecting against antioxidant enzyme inactivation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animal model
Male Sprague–Dawley rats (body weight, 250±10 g) were maintained on normal rat chow. Rats were divided into four groups: CONT (control), ADR (adriamycin treated), PROB (probucol treated), and PROB+ADR (probucol plus adriamycin treated). Adriamycin (doxorubicin hydrochloride, Pharmacia, Canada) was administered intraperitoneally in six equal injections to animals in the ADR and PROB+ADR groups for a cumulative dose of 15 mgkg–1 body weight (2.5 mgkg–1 in each individual injection). Probucol (Sigma, St. Louis, USA) was also administered intraperitoneally to the PROB and PROB+ADR groups in 12 equal injections for a total dose of 120 mgkg–1 body weight (10 mgkg–1 in each injection) over a period of 4 weeks, 2 weeks before adriamycin treatment and 2 weeks alteration with adriamycin injection. The animals in the CONT group were injected with saline in the same regimen with adriamycin treatment. Animals in each group were observed for 3 weeks after the last injection for general appearance, behavior, mortality, and body weights. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and all studies were approved by the Central Animal Care Committee of the University of Manitoba.

2.2 Hemodynamic study
Hemodynamic study was done as described before [7]. Briefly, animals were anesthetized with sodium pentobarbital (50 mgkg–1, i.p.). A miniature pressure transducer (Millar Micro-Tip) was inserted into the left ventricle via the right carotid artery. Left ventricular systolic (LVSP), left ventricular end-diastolic (LVEDP), aortic systolic (ASP), and aortic diastolic (ADP) pressures were recorded using an online computer data acquisition and analysis system.

2.3 Biochemical assays
Ventricles were weighed and cut into two parts along the vertical axis. One part was frozen in liquid nitrogen and kept under –80°C for mRNA and protein immunoblotting assays. The other part was placed in an ice-cold buffer for antioxidant enzyme activities and lipid peroxidation analysis.

2.3.1 Glutathione peroxidase (GSHPx) assay
GSHPx activity was expressed as nanomoles of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidized to nicotinamide adenine dinucleotide phosphate (NADH) per minute per milligram protein, with a molar extinction coefficient for NADPH of 6.22x106 [13]. Cytosolic GSHPx was assayed in a 3-ml cuvette containing 2.4 ml of 75 mM phosphate buffer (pH 7.0). The following solutions were then added: 50 µl of 60 mM reduced glutathione, 100 µl glutathione reductase (30 Uml–1), 50 µl of 120 mM NaN3, 100 µl of 15 mM Na–EDTA, 100 µl of 3.0 mM NADPH, and 100 µl of cytosolic fraction obtained after centrifugation of the heart homogenate at 20 000 g for 25 min. The reaction was initiated by the addition of 100 µl of 7.5 mM H2O2, and the conversion of NADPH to NADP was assayed by measuring the absorbance at 340 nm at 1 min intervals for 5 min.

2.3.2 Superoxide dismutase (SOD) assay
Supernatant (20 000 g for 20 min) was assayed for SOD activity by following the inhibition of pyrogallol autooxidation [14]. Pyrogallol (24 mM) was prepared in 10 mM HCl and stored at 4°C. Catalase 30 µM stock solution was made in an alkaline buffer (pH 9.0). Aliquots of supernatant (150 µg protein) were added to Tris–HCl buffer containing 25 µl pyrogallol and 10 µl catalase stock solutions. The total reaction mixture was brought to 3 ml using the same Tris–HCl buffer. Autooxidation of pyrogallol was monitored by measuring absorbance at 420 nm at 1 min intervals for 5 min.

2.3.3 Catalase assay
The ventricles were homogenized in 50 mM potassium phosphate buffer (pH 7.4) using a weight-to-volume ratio of 1:10. The homogenate was centrifuged at 20 000 g for 30 min. Supernatant of 50 µl was added to a cuvette containing 2.95 ml of 19 mM H2O2 solution prepared in potassium phosphate buffer [15]. The disappearance of H2O2 was monitored at 240 nm wavelength at 1 min intervals for 5 min. Specific activity of the enzyme was expressed in µmolesmg–1 protein.

2.3.4 Thiobarbituric acid reactive substances (TBARS) assay
Measurement of lipid peroxidation by determining TBARS was performed by using a modified thiobarbituric acid (TBA) method as described previously [16]. Hearts were quickly excised and washed in buffered 0.9% KCl (pH 7.4). After atria, extraneous fat, and connective tissue were removed, the homogenization (10% w/v) in the same buffer was done. The homogenate was incubated in a 37°C water bath. An aliquot of 2 ml was withdrawn from the incubation mixture and pipetted into an 8-ml Pyrex tube. One milliliter of 40% (w/v) trichloroacetic acid (TCA) and 1 ml of 0.2% (w/v) TBA were promptly added. To minimize peroxidation during the subsequent assay procedure, 2% (w/v) butylated hydroxytoluene was added to the TBA reagent mixture [17]. Contents were vortexed briefly, boiled for 15 min, and cooled on ice for 5 min. Two milliliters of 70% (w/v) TCA was then added and vortexed again. The tubes were allowed to stand for 20 min at room temperature, followed by centrifugation for 20 min at 3500 rpm (Sorvall GLC-1 centrifuge). The color developed was assayed by measuring absorbance at 523 nm, and expressed in nmolesgm–1 heart tissue by comparing to a known MDA standard.

2.4 Northern blot analysis
Total RNA was isolated by the acid guanidium thiocyanate–phenol–chloroform extraction method [18]. Twenty micrograms of total RNA was denatured, fractionated in 1% (w/v) agarose gel and transferred to a Zeta-Probe GT blotting membrane (Bio-Rad, CA, USA) by capillary blot with 10x SSC as the transfer buffer for 16 h. The membranes were prehybridized at 42°C for 2–4 h in a solution containing 50% (v/v) deionized formamide, 5 M NaCl, 0.12 M Na2HPO4(pH 7.2), 7% (w/v) sodium dodecyl sulfate (SDS). Hybridization was carried out in the same solution at 42°C for 12–18 h with 32P-labelled cDNA probes (specific activity>109 cpmµg–1 DNA). Human GSHPx, MnSOD and CAT cDNA inserts were purchased from the American Type Culture Collection (ATCC, MD, USA). Isolated and purified cDNA fragments were labelled with {alpha}32P-dCTP using a random primer DNA labelling kit (Gibco BRL, MD, USA) using a klenow fragment [19]. To control the RNA loading, the membrane was stripped and re-hybridized using a 18S ribosomal RNA oligonucleotide probe (kindly provided by Dr. I.M.C. Dixon, University of Manitoba, Winnipeg, Canada). The 18S oligonucleotide was end-labelled using polynucleotide kinase (Gibco BRL) with {gamma}-32P-dATP [20]. The membranes were washed for 15 min at room temperature with a solution of 2x SSC–0.1% (w/v) SDS, and followed by a wash at 42°C in 0.1x SSC–0.1% (w/v) SDS. The autoradiograph was established by exposing the filter for 24–48 h to X-ray film (DuPont Reflection) at –70°C with intensifying screens. In addition, the RNA load per lane was also assessed by ethinium bromide staining of the original agarose gel.

The relative levels of mRNA signals of GSHPx, MnSOD and CAT were quantified from autoradiographs by a scanning densitometer (Bio-Rad imaging densitometer GT-670). The strength of the message was presented as the ratio of expression of enzyme vs. 18S rRNA. The MnSOD scanning values represented the total densities of 3.8, 2.7, 2.2, 1.3 and 1.1 kb, corresponding to polyadenylated isoforms [21]. All the quantitative data were presented as percentages of the values in the control group.

2.5 Western blot analysis
The ventricular tissue was immersed immediately in liquid nitrogen and stored at –80°C until the protein was isolated. For protein isolation, the tissue samples were thawed in ice-cold Tris–EDTA buffer (100 mM Tris–HCl, 5 mM EDTA, pH 7.4) and homogenized using a Polytron homogenizer with two 15-s pulses and an intervening 10-s rest period. Aprotinin (10 µgml–1), leupepsin (10 µgml–1), pepstinin A (10 µgml–1) and phenylmethylsulfonyl fluoride (20 µM) were included in the buffer to prevent protein degradation. Protein concentrations were determined and used to normalize the protein loading.

Forty micrograms of protein were subjected to one-dimensional sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) in a discontinuous system using 15% (w/v) separating gel and 5% (w/v) stacking gel [22]. The separated proteins were electrophoretically transferred to nitrocellulose membranes using a modified Towbin buffer containing 20 mM Tris, 150 mM glycine, 20% (v/v) methanol, 0.02% (w/v) SDS (pH8.3) in a cooled Bio-Rad TransBlot unit. After non-specific protein-binding sites were blocked with 1-h incubation with 5% (w/v) non-fat milk in Tris-buffered saline–0.1% (w/v) Tween-20, the membranes were processed for immunodetection using rabbit anti-human GSHPx antibody (kindly provided by Dr. I. Singh, Medical University of South Carolina, Charleston, SC), rabbit anti-MnSOD antibody (kindly provided by Dr. L. W. Oberley, University of Iowa, Iowa City, IA) and sheep anti-CAT polyclonal antibody (The Binding Site, Birmingham, UK) as primary antibody. The bound primary antibodies were detected using a donkey anti-rabbit or anti-sheep horseradish peroxidase-conjugated secondary antibody and an ECL Western blotting detection system (Amersham, IL, USA). The photographs generated were quantitatively analyzed for the GSHPx, MnSOD and CAT protein levels with a Bio-Rad GS-670 image densitometer. The molecular weights of the protein bands were determined by reference to the standard molecular weight markers (Bio-Rad, CA, USA).

2.6 Protein determination and statistical analysis
Proteins were determined using the methods of Lowry et al. [23]. Data were expressed as the means±S.E. For a statistical analysis of the data, group means were compared by one-way analysis of variance and Bonferroni's test was used to identify differences between groups. Statistical significance was acceptable to a level of P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 General observations, mortality and weight data
In the first 2 weeks of probucol treatment, animals in the PROB and PROB+ADR groups did not show any significant change in their behavior or appearance as compared with the CONT group. The most predominant feature of animals in the ADR group was the development of a progressively enlarged abdomen, ascites and dyspnea. At the time of sacrifice, all animals in the ADR group showed a significant amount of fluid in the abdominal cavity. In addition, the livers of adriamycin-treated animals were enlarged and appeared dark in color. There was a significant increase in the wet to dry lung weight ratio (Table 1). Probucol treatment in the PROB+ADR group prevented these changes due to adriamycin administration. Adriamycin caused 40% mortality in 3 weeks of post-treatment period, and there was no death in the PROB+ADR group (Table 1).


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  		Table 1 Effects of probucol on adriamycin-induced changes in body weight, heart weight, lung weight and mortality of ratsa

 
Body weight was significantly lower in the ADR and PROB+ADR groups compared to those in the CONT and PROB groups (Table 1). The difference in body weight between the ADR and PROB+ADR was attributed to the ascites which was seen only in the ADR group but not in the PROB+ADR group. Adriamycin treatment resulted in a significant decrease in the heart weight of both the ADR and PROB+ADR groups (Table 1). Heart weight was significantly improved in the PROB+ADR group but it was still below the control level. Heart weight/body weight ratio was depressed only in the ADR group.

3.2 Hemodynamic studies
Aortic systolic pressure (ASP) and left ventricular peak systolic pressure (LVSP) were significantly lower in the ADR group, while left ventricular end diastolic pressure (LVEDP) was elevated 4-fold by adriamycin administration (Table 2). These altered parameters in the ADR group were normalized by probucol to control values in the PROB+ADR group although there were no significant changes in the PROB group (Table 2).


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  		Table 2 Effects of probucol on adriamycin-induced hemodynamic changes in ratsa

 
3.3 Antioxidant enzyme activities and lipid peroxidation
The activities of myocardial endogenous antioxidant enzymes glutathione peroxidase (GSHPx), superoxide dismutase (SOD), and catalase (CAT) were measured at the end of the 3 weeks post-treatment period and these data are shown in Table 3. Adriamycin treatment resulted in 30% decrease of GSHPx activity compared to the control value (P<0.05). In the PROB+ADR group, GSHPx activity was maintained and it was significantly higher than in the ADR group. SOD activity was increased in the PROB group (41%) and PROB+ADR group (62%). CAT activity did not change significantly in any of the groups. Adriamycin treatment led to 45% increase in lipid peroxidation compared to the control (Table 3). Probucol prevented this increase in the PROB+ADR group.


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  		Table 3 Effect of probucol on adriamycin-induced changes in antioxidant enzyme activities and lipid peroxidationa

 
3.4 mRNA levels of different antioxidant enzymes
In order to examine the molecular mechanism responsible for changes in the activities of these antioxidant enzymes, mRNA levels of GSHPx, MnSOD, and CAT in myocardium were analysed using the Northern blot technique. Autoradiographs as well as relative levels of GSHPx, MnSOD and CAT mRNA in all experimental groups are shown in Fig. 1. GSHPx mRNA level was relatively stable at the end of 3 weeks after adriamycin, probucol, and their combination treatment. In contrast, MnSOD mRNA abundance was reduced by 45% by adriamycin. Probucol treatment demonstrated a protective effect and the MnSOD mRNA expression in the PROB+ADR group was close to the normal level. CAT mRNA expression did not significantly change in any of the groups.


Figure 1
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  		Fig. 1 (A), (B) and (C): Northern blot assay of GSHPx, MnSOD and CAT mRNA abundances in the ventricles. Total RNA was isolated at the end of 3 weeks after the last injection. Twenty micrograms of total RNA was separated electrophoretically, transferred to a membrane and sequentially hybridized with 32P-labelled GSHPx, MnSOD and CAT cDNA probes. 18S rRNA was used as the control for uniformity of RNA loading and transfer. (A) Representative autoradiographs: CONT, control; ADR, adriamycin; PROB, probucol, and PROB+ADR, probucol+adriamycin. (B) Ethidium bromide stained gel shown as control for equivalent loading of RNA. (C) Densitometric analysis of the relative levels of GSHPx, MnSOD and CAT mRNA abundance. Obtained densities were corrected for those of 18S rRNA and represented as a percentage of the control value. Data are mean±S.E. of 5 animals in each group. *, P<0.05. MnSOD shows as five bands because of alternative adenylation of transcripts from the same gene. For densitometric analysis, all bands were counted.

 
3.5 Protein levels of different antioxidant enzymes
Protein levels of antioxidant enzymes in each group were analyzed by Western blot analysis. Representative immunoblots corresponding to GSHPx, MnSOD and CAT, and densitometer scanning analysis are shown in Fig. 2(A) and (B). The protein level of MnSOD was significantly reduced by 20% at the end of 3 weeks in the ADR group. This change was prevented by probucol in the PROB+ADR group. Protein levels for GSHPx and CAT were not affected in any of the groups.


Figure 2
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  		Fig. 2 (A) and (B): Western blot analysis of myocardial proteins using antibodies against GSHPx, MnSOD and CAT, respectively. (A) Representative immunoblots: CONT, control; ADR, adriamycin; PROB, probucol; and PROB+ADR, probucol+adriamycin. (B) Densitometric analysis of GSHPx, MnSOD and CAT immunoreactive proteins. Data are mean±S.E. of 4 animals in each group. *, P<0.05.

 
3.6 Correlation between GSHPx activity and lipid peroxidation
The relationship between glutathione peroxidase and oxidative stress as indicated by lipid peroxidation in the CONT and ADR groups was also analyzed and these data are plotted in Fig. 3. There was an inverse correlation between these two measures.


Figure 3
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  		Fig. 3 The correlation between glutathione peroxidase (GSHPx) activity and lipid peroxidation as indicated by TBARS following adriamycin treatment.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In order to eliminate adriamycin cardiotoxicity without interfering with its antitumor property, every possible approach, including modification of drug delivery and chemical structure as well as combination therapy with antioxidants, has been tried, but the problem still remains [3]. In this study, clinical observations and hemodynamic as well as mortality data demonstrate the presence of adriamycin-induced cardiomyopathy and congestive heart failure. Lung congestion in the adriamycin group was indicated by an increase in the wet to dry lung weight ratio as well as dyspnea. Poor performance of the left ventricle may have caused this change subsequent to an increase in LVEDP. The latter change in the present study was confirmed by the hemodynamic data. Treatment with probucol, an antioxidant, prevented these adriamycin-induced changes, thus confirming our previous findings on modulation of adriamycin-induced cardiomyopathy by probucol [8]. It is interesting to note that in adriamycin cardiomyopathic hearts, the decrease in GSHPx enzyme activity was not accompanied by changes in its mRNA and protein levels. Furthermore, SOD enzyme activity in the ADR group did not change while MnSOD mRNA and protein levels were depressed. These data suggest, for the first time, that the alterations in antioxidant enzyme activity may be independent of changes in the gene expression by adriamycin treatment.

The decreased enzyme activity of GSHPx without changes in mRNA and protein levels suggest that GSHPx might be inactivated through oxidation either by free radicals [24–27] or lipid peroxides [27]. With the overproduction of oxygen free radicals in skeletal muscle during sepsis in rats, GSHPx activity was reduced to 55% [25]. In addition, it has also been demonstrated that incubation of GSHPx in vitro with a source of superoxide anion significantly diminishes the enzyme activity [26]. Thus, adriamycin free radical metabolites and/or activated oxygen radicals could be directly responsible for the decrease in the enzyme activity. Alternatively, malondialdehyde, a by-product of lipid peroxidation may also diminish enzyme activity by oxidizing the active site or by forming protein cross-links [28]. In our study, lipid peroxidation indicated by TBARS was significantly increased suggesting an increase in oxidative stress. GSHPx has shown to be relatively more sensitive to oxidative stress compared to other antioxidant enzymes [29]. In this regard, the activities of CAT and glutathione reductase were not affected in cultured Chinese hamster V79 cells incubated with an organic hydroperoxide (tertiary-butyl hydroperoxide), while the activity of GSHPx was significantly inhibited [24]. In addition, the decreased selenium concentration may also contribute to GSHPx activity changes because this enzyme requires selenium for activity [30]. The selenocysteine molecule at the active site of GSHPx can be oxidized to a diselenide that is resistant to reduction [31]. However, these correlative changes in the GSHPx activity need further examination to check the cause and effect relationship.

In the heart where GSHPx is a major enzymatic mechanism for the disposal of peroxides, a prolonged depression in the level of this enzyme might lead to intracellular peroxide accumulation. Since adriamycin increases cardiac superoxide anion generation, the potential exists for hydrogen peroxide formation at a time when peroxide removal is impaired. The hydrogen peroxide concentration could be reached at a level exceeding the detoxification ability of the myocardial cell, resulting in an increase in TBARS. The data indeed showed an inverse relationship between GSHPx activity and lipid peroxidation. Furthermore, there was a correlation between the decrease in GSHPx and depressed contractile function. Probucol, a strong antioxidant, offered complete protection of this enzyme activity. Thus, we suggest that the protective effect of probucol in adriamycin cardiomyopathy may partially be mediated by a modulation of the oxidative inactivation of GSHPx.

Although cytosolic CuZnSOD and MnSOD have the same function in dismutating superoxides, these two enzymes differ in their intracellular distribution, composition and sequences of nucleotides in their mRNAs and amino acids in protein. Therefore, different cDNA probes and antibodies are required for the Northern and Western blot analysis. It is important to note that MnSOD mRNA levels were reduced in the hearts of adriamycin-treated animals while there was no change in the SOD enzyme activity. In the PROB and PROB+ADR groups, the SOD activity was even increased. Reasons for this disparity between changes in the mRNA level and enzyme activity are not immediately apparent. One possibility is that the mRNA monitored in our study was strictly for the mitochondrial SOD while the enzyme activity monitored was inclusive of CuZnSOD (cytosolic) and MnSOD (mitochondrial). Furthermore, in the enzyme activity measuring procedure used by us, centrifugation at 20 000 g must eliminate a greater proportion of mitochondria, thus the SOD activity monitored may in fact be principally cytosolic. Further studies of the CuZnSOD mRNA as well as of the enzyme characteristics will resolve this issue. The decrease of MnSOD mRNA was prevented by probucol treatment, it will be very interesting to further study the mechanism for the changes of the levels of MnSOD in mRNA.

In summary, the study shows that the molecular mechanism for a reduction in glutathione peroxidase activity may not involve downregulation of the enzyme gene transcription or translation. Rather, oxidative stress mediated changes at the enzyme protein level may have an important role to play. Further studies are required to describe the mechanism of downregulation of MnSOD gene expression as well as its prevention by probucol.

Time for primary review 26 days.


   Acknowledgements
 
The study was supported by a grant from the Heart and Stroke Foundation of Manitoba. PKS is a career investigator supported by the Medical Research Council. TL is supported by a Traineeship from the Heart and Stroke Foundation of Canada. ABK is supported by a scholarship from the Brazilian Federal Government (CAPES).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Young R.C., Ozols R.F., Myers C.E. The anthracycline antineoplastic drugs. N. Engl. J. Med. (1981) 305:139–153.[ISI][Medline]
  2. Lefrak E.A., Pitha J., Rosenheim S., Gottlieb J.A. A clinicopathologic analysis of adriamycin cardiotoxicity. Cancer (1973) 32:302–314.[CrossRef][Medline]
  3. Singal P.K., Iliskovic N. Doxorubicin-induced cardiomyopathy. N. Engl. J. Med. (1998) 339:900–905.[Free Full Text]
  4. Myers C.E., McGuire W.P., Liss R.H., et al. Adriamycin: The role of lipid peroxidation in cardiac toxicity and tumor response. Science (1977) 197:165–167.[Abstract/Free Full Text]
  5. Singal P.K., Deally C.M., Weinberg L.E. Subcellular effects of adriamycin in the heart: A concise review. J. Mol. Cell Cardiol. (1987) 19:817–828.[ISI][Medline]
  6. Singal P.K., Iliskovic N., Li T., Kumar D. Adriamycin cardiomyopathy: Pathophysiology and prevention. FASEB J. (1997) 11:931–936.[Abstract]
  7. Siveski-Iliskovic N., Kaul N., Singal P.K. Probucol promotes endogenous antioxidants and provides protection against adriamycin-induced cardiomyopathy in rats. Circulation (1994) 89:2829–2835.[Abstract/Free Full Text]
  8. Siveski-Iliskovic N., Hill M., Chow D.A., Singal P.K. Probucol protects against adriamycin cardiomyopathy without interfering with its antitumor effect. Circulation (1995) 91:10–15.[Abstract/Free Full Text]
  9. Bachur N.R. Adriamycin (NSC-123127) pharmacology. Cancer Chemother Rep, Part 3 (1975) 6:153–158.
  10. Muindi J.R., Sinha B.K., Gianni L., Myers C.E. Hydroxyl radical production and DNA damage induced by anthracycline– iron complex. FEBS Lett. (1984) 172:226–230.[CrossRef][ISI][Medline]
  11. Potmesil M. Anthracycline and anthracenedione-based anticancer agents. Lown J.W., ed. (1988) Amsterdam: Elsevier. 447–474.
  12. Singal P.K., Siveski-Iliskovic N., Hill M., Thomas T.P., Li T. Combination therapy with probucol prevents adriamycin-induced cardiomyopathy. J. Mol. Cell Cardiol. (1995) 27:1055–1063.[CrossRef][ISI][Medline]
  13. Paglia D.E., Valentine W.N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. (1967) 70:158–169.[ISI][Medline]
  14. Marklund S.L. Handbook of methods for oxygen radical research. Greenwald R.A., ed. (1985) Boca Raton, FL: CRC Press. 243–247.
  15. Clairborne A. Handbook of methods for oxygen radical research. Greenwald R.A., ed. (1985) Boca Raton, FL: CRC Press. 283–284.
  16. Singal P.K., Pierce G.N. Adriamycin stimulates low-affinity Ca2+ binding and lipid peroxidation but depresses myocardial function. Am. J. Physiol. (1986) 250:H419–H425.[ISI][Medline]
  17. Aust S.D. Handbook of methods for oxygen radical research. Greenwald R.A., ed. (1985) Boca Raton, FL: CRC Press. 203–207.
  18. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem. (1987) 162:156–159.[ISI][Medline]
  19. Feinberg A.P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. (1984) 137:266–267.[CrossRef][ISI][Medline]
  20. Maxam A.M., Gilbert W. A new method for sequencing DNA. Biotechnology (1977) 24:99–103.
  21. Hurt J., Hsu J.L., Dougall W.C., et al. Multiple mRNA species generated by alternate polyadenylation from the rat manganese superoxide dismutase gene. Nucleic. Acids. Res. (1992) 20:2985–2990.[Abstract/Free Full Text]
  22. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (1970) 227:680–685.[CrossRef][Medline]
  23. Lowry O.H., Rosenbrough N.T., Farr A.L., Randall A.T. Protein measurements with the Folin phenol reagent. J. Biol. Chem. (1951) 193:265–275.[Free Full Text]
  24. Ochi T. Effects of an organic hydroperoxide on the activity of antioxidant enzymes in cultured mammalian cells. Toxicology (1990) 61:229–239.[CrossRef][ISI][Medline]
  25. Peralta J.G., Llesuy S., Evelson P., et al. Oxidative stress in skeletal muscle during sepsis in rats. Circ. Shock (1993) 39:153–159.[ISI][Medline]
  26. Rister M., Baehner R.L. The alteration of superoxide dismutase, catalase, glutathione peroxidase, and NAD(P)H cytochrome c reductase in guinea pig polymorphonuclear leukocytes and alveolar macrophages during hyperoxia. J. Clin. Invest. (1976) 58:1174–1184.[ISI][Medline]
  27. Wills E.D. Effects of unsaturated fatty acids and their peroxides on enzymes. Biochem. Pharmacol. (1961) 7:7–16.[CrossRef][ISI][Medline]
  28. Choi K.S., Tappel A.L. Inactivation of ribonuclease and other enzymes by peroxidizing lipids and by malonaldehyde. Biochemistry (1969) 8:2827–2832.[CrossRef][ISI][Medline]
  29. Lores A.S., Llesuy S., Cutrin J.C., Boveris A. Oxidative stress by acute acetaminophen administration in mouse liver. Free Radic. Biol. Med. (1995) 19:303–310.[CrossRef][ISI][Medline]
  30. Doroshow J.H., Locker G.Y., Myers C.E. Enzymatic defenses of the mouse heart against reactive oxygen metabolites: Alterations produced by doxorubicin. J. Clin. Invest. (1980) 65:128–135.[ISI][Medline]
  31. Tappel A.L., Forstrom J.W., Zakowski J.J., Lyone D.E., Hawkes W.C. The catalytic site of rat liver glutathione peroxidase as selenocysteine in rat liver. Fed. Proc. (1978) 37:706–706. (Abstract).

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