The Protection of Selenium on Adriamycin-Induced Mitochondrial Damage in Rat
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Although adriamycin (ADR) exhibits high anti-tumor efficacy in vitro, its clinical use in cancer chemotherapy is limited due to its high renal toxicity. This study investigated the mechanism of ADR nephropathy and the protective effect of selenium on ADR-induced kidney damage by analyzing of the relationship between selenium and mitochondria. Rats were divided into four groups. The first group was injected with saline i.p. for 21 days, the second group received the 4 mg/kg i.p. ADR every alternate day for 8 days, the third group received the 50 μg/kg i.p. Se for 21 days, and the fourth group received the Se. ADR co-administration i.p. blood pressures were assessed, the mitochondrial membrane potential (MMP) was assessed, and the adenosine triphosphate (ATP) levels were determined. The total antioxidant (TAS) and oxidant status (TOS) in cytosol, the mitochondria of kidney cells, and plasma were measured. Mitochondrial TAS decreased and TOS increased in the ADR group compared to the Se group. ADR-treated rats showed significantly lower MMP than did the control and Se groups. MMP was significantly restored in the Se + ADR group through selenium treatment compared to the ADR group (p < 0.01). In the ADR group, a reduction in ATP content was seen compared to the control and Se groups (p < 0.01). ATP level was significantly restored through treatment with selenium in the Se + ADR group compared to the ADR group (p < 0.01). We concluded that selenium is effective in vivo against ADR-induced kidney damage via the restoration of TAS and TOS, which prevented mitochondrial damage.
KeywordsAdriamycin Selenium Mitochondrial membrane potential ATP Total antioxidant status Total oxidant status
Adriamycin (ADR) nephropathy is a classic experimental model of kidney disease that results from ADR’s selective injury to glomerular podocytes, which are the visceral epithelial cells that maintain the kidney filtration barrier [1, 2]. Genetic or acquired defects that reduce as little as 10–20% of the podocyte cell mass are sufficient to initiate glomerulosclerosis and nephropathy [3, 4]. In the ADR nephropathy model, a single dose of ADR produces a loss of podocyte foot process architecture and causes progressive podocyte depletion, resulting in persistent proteinuria, followed by the development of focal segmental glomerulosclerosis and, finally, global sclerosis . This model is frequently used to unmask susceptibility to glomerulosclerosis in genetically manipulated mice or to test the relevance of specific pathways or interventions in the development of nephropathy [1, 2, 5, 6]. However, interpretation of studies using the ADR nephropathy model is limited by our lack of understanding of the underlying mechanism of injury. Therefore, elucidation of the mechanisms of tissue injury in this trait can provide insight into pathways mediating glomerulosclerosis and can establish a biological context for studies using this model. Moreover, because ADR is a commonly used chemotherapeutic drug, a deeper understanding of ADR nephropathy can also offer insight into mechanisms of ADR tissue toxicity.
ADR is an anthracycline antibiotic with pleiotropic cytotoxic effects. It is used for the treatment of solid and hematogenous tumors. Proposed mechanisms of ADR-induced tissue damage include the introduction of double-stranded DNA breaks, lipid peroxidation, inhibition of protease activity, disruption of the cytoskeletal and extracellular matrix, and inhibition of the topoisomerase II-mediated religation of the broken DNA strands [7, 8, 9]. ADR can damage mtDNA directly by intercalating into mtDNA or indirectly by generating reactive oxygen species (ROS) in the kidneys and heart after short-term treatment [10, 11, 12]. Cardiomyopathy, the most common side effect of ADR therapy in humans, is also associated with mtDNA damage, and interventions that improve mitochondrial biogenesis are protective against cardiac dysfunction [11, 12].
ADR undergoes a futile redox cycling on complex I of the mitochondrial respiratory chain to liberate highly reactive oxygen–free radicals. Associated with this is the oxidative modification of a specific calcium-conducting channel leading to the induction of the mitochondrial permeability transition, interference with the mitochondrial calcium homeostasis, and the disruption of calcium-dependent mitochondrial bioenergetics . In view of the implication of oxygen free radicals in the pathogenic process, one of the approaches to minimizing ADR toxicity has been the use of free radical scavengers and other antioxidants.
GSHPx catalyses the reduction of hydrogen peroxide to water, with the simultaneous conversion of reduced glutathione to oxidized glutathione. The origin of the formed H2O2 is primarily the lipid hydroperoxides which are released from membrane phospholipids. GSHPx de-toxifies this H2O2 by reducing it to water, while at the same time oxidizing glutathione. Any deficiency in this de-toxification cycle (e.g., of GSHPx or of glutathione itself) puts the cell at risk from the potentially mutagenic effects of lipid hydroperoxides. GSHPx thus protects against lower physiological levels of H2O2 than does the enzyme catalase, which reduces the higher concentrations of H2O2 formed during respiration. GSHPx contains one residue per mole of selenocysteine, an analog of cysteine in which selenium is substituted for sulfur. Deficiency of selenium therefore greatly decreases the activity of this enzyme .
Selenium (Se) is an essential trace element, which means that it must be supplied through daily diet . The protective mechanism of selenium includes a significant reduction in the intracellular ROS, the reversal of DNA fragmentation, and the suppression of caspase and apoptosis signal-regulating kinase 1 activity .
We showed that ADR administration decreased the mitochondrial membrane potential (MMP), ATP level, and thioredoxin reductase activity (TrxR) in rat myocyte mitochondria. Cytosolic and mitochondrial total antioxidant (TAS) decreased, and plasma oxidant status (TOS) increased in the group receiving ADR compared to the control. The co-administration of selenium with ADR improved MMP and ATP levels, and it prevented oxidative stress by increasing antioxidants (especially TrxR) and decreasing oxidants .
For first time, this study investigated the mechanism of ADR nephropathy and the protective effect of selenium on ADR-induced kidney damage by analyzing the relationship between selenium and the mitochondria.
Materials and Methods
Chemicals and Reagents
Reagents used in the experiments were as follows: sodium selenite, Tris HCl, EGTA, sucrose from Sigma Chemical (St. Louis, MO, USA); adriamycin HCl Pharmacia (Milan, Italy); JC-1 Mitochondrial Membrane Potential Assay (MMP) Kit from Cayman Chemical Company, USA; TAS, TOS kits from Rel Assay Diagnostics, TURKEY; and the adenosine triphosphate (ATP) kit from Cambrex Bio Science, Rockland, MA, USA.
Rats were observed for their general appearance, behavior, and mortality for 3 weeks after the first injection. After measurement of arterial blood pressures, blood samples were collected, and plasma was separated by centrifugation and used for biochemical assays; then, the kidneys were also removed from the rats, weighed and kept at −80°C until use.
Measurement of Hemodynamic Parameters
Animals were anesthetized with ketamin (39.35 mg/kg, i.m.) plus xylasine (4.96 mg/kg, i.m.). Systolic, diastolic and mean blood pressures and heart rate were directly measured through the femoral artery. A polyethylene catheter (PE-50, Intramedic, Clay Adams, MD, USA) was implanted in the femoral artery. The measurement of blood pressure was begun 20–30 min after connecting the catheter to the transducer. The transducer output was amplified, and the arterial pressure signal passed to an analog-digital converter installed (MP30, Biopac System, Inc., CA, USA).
Preparation of Mitochondria
Mitochondria from fresh rat kidneys were isolated as described [21, 22]. The kidneys were homogenized in an ice-cold buffer A (250 mM sucrose, 2 mM EGTA, 5 mM Tris HCl) with a homogenizer and centrifuged at 2,000×g for 8 min at 4°C. The supernatant (cytosol) was further centrifuged at 12,000×g for 10 min at 4°C in a new tube. The pellet of mitochondria was suspended in an ice-cold buffer B (140 mM potassium, 20 mM Tris HCl). The mitochondria were kept at −80°C until use.
Measurement of Mitochondrial Membrane Potential
The fluorescent mitochondrial-specific cationic dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolyl carbocyanine iodide (JC-1) was used to assess the status of MMP JC-1 undergoes potential-dependent accumulation in the mitochondria. Mitochondria with a normal MMP aggregated JC-1 (red fluorescence) and in depolarized mitochondria, JC-1 formed monomers (green fluorescence). Mitochondria were added to each well, preincubated with 10 μM JC-1 for 20 min at 37°C in the dark; then, the mitochondria were analyzed by a fluorescent plate reader (Biotek, Synergy HT). In healthy cells, JC-1 forms J-aggregates which displayed strong fluorescent intensity with excitation and emission at 560 and 595 nm, respectively. In apoptotic or unhealthy cells, JC-1 existed as monomers which showed strong fluorescence intensity with excitation and emission at 485 and 535 nm, respectively. The ratio of fluorescent intensity of J-aggregates to fluorescent intensity of monomers was used as an indicator of cell health.
Determination of ATP Content
ATP can be used to assess the functional integrity of living cells since all cells require ATP to remain alive and carry out their specialized functions. The kit allows measurements of the bioluminescence of ATP that is present in all metabolically active cells. The bioluminescence method uses an enzyme, luciferase, to cause ATP to luminesce.
Briefly, a 10-μl aliquot of a mitochondria sample was mixed with 100 μl Cell Lysis Reagent and incubated at room temperature for 10 min to extract ATP from cells. Following the addition of 100 μl ATP Monitoring Reagent, luminescence was measured using a luminometer (Biotek, Synergy HT). A standard curve was generated from known concentrations of ATP and used to calculate the concentration of ATP in each sample. Luminescence increased linearly with the negative log of the ATP concentration in the samples over the range of measured concentrations. Mitochondrial ATP content from each probe was assessed in duplicate.
Measurement of Total Antioxidant Status
Total antioxidant status (TAS) of the cytosol and of the kidney cell mitochondria were measured using Rel Assay Kit. The TAS method is based on the bleaching of the characteristic color of a more stable 2,2′-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) radical cation by antioxidants. The results were expressed in millimoles Trolox equivalent per liter.
Measurement of Total Oxidant Status
The total oxidant status (TOS) of the cytosol and of the kidney cell mitochondria were measured using Rel Assay Kit. The TOS method is based on the oxidation of ferrous ion to the ferric ion in the presence of various oxidizing species in an acidic medium and the measurement of the ferric ion by xylenol orange. The results were expressed in micromoles H2O2 per liter.
Measurement of Oxidative Stress Index
The TOS to TAS ratio in the plasma was regarded as the oxidative stress index (OSI). To perform the calculation, the resulting unit of TAS, micromoles Trolox equivalent per liter, was changed to micromoles Trolox equivalent per liter, and the OSI value was calculated as follows: OSI = [(TOS, micromoles per L)/(TAS, micromoles Trolox equivalent per liter)/100].
Statistical Data Analysis
Statistical analyses were conducted using Excel and SPSS version 11.0. All results were expressed as mean ± SEM. Comparisons among different groups were made using multiple analyses of variance followed by a post-hoc protected Tukey test. In all cases, p < 0.05 was considered to be significant.
Effects of Adriamycin and Selenium on Hemodynamic Parameters
Systolic blood pressures in the ADR group significantly lower than in the CONT and Se groups
Se + ADR
84 ± 11
78 ± 7
66 ± 10
80 ± 13
106 ± 14
111 ± 9
75 ± 13 *,**
92 ± 10
69 ± 8
60 ± 9
57 ± 8
69 ± 16
318 ± 39
251 ± 37
304 ± 42
239 ± 47
Effects of Selenium on Total Antioxidant, Oxidant Status, and the Oxidative Stress Index
In normal rats, the OSI, which stands for the ratio of TOS and TAS in the mitochondria and the cytosol, was in mitochondria and cytosol was 61 ± 51, 13.7 ± 0.4, respectively. The administration of ADR caused the OSI to increase to 264 ± 38, a value significantly higher (p < 0.05) than that seen in the control and selenium groups not treated with ADR vs. control and Se groups. The OSI was maintained by selenium in the Se + ADR group (p < 0.05 vs. control, ADR, and Se groups, Fig. 2c)
Effects of Selenium on Mitochondrial Membrane Potential
Effects of Selenium on Mitochondrial ATP Content
No significant change in ATP content was observed when a comparison was made between the CONT and selenium groups. In the ADR group, a reduction in ATP content was seen compared to the CONT and Se groups (p < 0.01). This decrease in ATP level was significantly restored by administered with selenium in the Se + ADR group compared to the ADR group (p < 0.01; Fig. 3b).
ADR is a commonly used chemotherapeutic drug, so a deeper understanding of ADR nephropathy and the factors that reduce it can prevent ADR tissue toxicity. ADR accumulates in higher quantities in the kidney than it does in any other organ . The accumulation of the drug in the kidney and its concentration in the kidney nuclei and mitochondria are much higher than its concentration levels observed in the heart. The mitochondria have the ability to accumulate ADR in vivo. Intravenous injections of adriamycin resulted in decreased activities of the glycolytic enzymes, hexokinase, phosphoglucoisomerase, aldolase, and lactate dehydrogenase in rat kidney tissue . The transmembrane enzymes, namely the Na+, K+-ATPase, Ca+2-ATPase, Mg2+ and -ATPase, showed a decrease in their activities . The results of this study suggested that the accumulation of the drug in the kidney mitochondria affects energy metabolism. The exact mechanisms of the ADR inhibition of mitochondrial electron transport and oxidative phosphorylation are not clearly understood. An emerging hypothesis expands this concept to implicate free radical-mediated interference with mitochondrial bioenergetics and calcium regulation as being definitive in the pathogenic process . When mitochondrial permeability transition (MPT) is promoted by the oxidation of protein thiol groups and through increases in cytosolic Ca+2 concentration, cytochrome c is released. This cytochrome is associated with MPT and results in a loss of H+2 gradient and mitochondrial membrane potential (MMP) across the mitochondrial inner membrane, an increase in the matrix volume, and disruption of the outer membrane . Additional indications of oxidative injury to mitochondria include membrane lipid peroxidation, inhibition of respiration, and oxidative phosphorylation, decreased mitochondrial ATPase activity, and a net decrease in the redox state of respiratory carriers .
In the present study, we showed important impairments, including in the antioxidant, oxidant status, and MMP and ATP levels in kidney mitochondria when ADR was administered at 16 mg/kg. In our current study, the onset and extent of an oxidative condition following ADR administration was clearly indicated by the increase in mitochondria TOS, which has been reported by different investigators as a valid indicator for oxidative damage measurements. ADR administration significantly decreased mitochondrial TAS, which can be considered the aggregation of the interactions among various antioxidants. This reduction in TAS reflected a consumption of antioxidant molecules because of the ADR-induced increase in mitochondrial TOS. The plasma OSI was higher in the ADR group than it was other groups. Additional indications of oxidative injury of ADR in the kidney were increased apoptosis (i.e., MMP) and decreased energy production (i.e., ATP) in the kidney mitochondria.
Selenium co-administered with ADR aids recovery within the parameters of antioxidant, oxidant, MMP, and ATP levels in kidney tissue. We suggest that selenium is probably a protective agent against excessive raised radical products due to its increase of antioxidant enzyme activities, such as TrxR and GPx activities. Our previous study showed that TrxR activity decreased in the ADR group compared to the Se-ADR co-administered group . TrxR displays a broad specificity and plays an important antioxidant role, not only by supplying reducing equivalents to the thioredoxin/thioredoxin peroxidase systems, but also by indirectly reducing H2O2 and lipid peroxidase.
A study of porcine kidney epithelial cells (LLC-PK1 cell) emphasized that selenium had an ability to inhibit cadmium-induced apoptosis and provided mechanistic evidence that cytoprotective effects were mediated through blocking the ROS generation, restoring the mitochondrial potential collapse, preventing cytochrome c release, subsequent inhibition of caspase activation, and the changed expression of Bcl-2, Bax . In another study, the results indicate that selenite induces the MPT as a result of direct modifications of protein thiol groups, resulting in the release of cytochrome c and the loss of mitochondrial membrane potential in rat liver .
Although it is difficult to extrapolate rat data to humans, it is possible that a selenium supplement for patients on ADR chemotherapy for the management of cancers may provide a survival benefit and protect their vital organs, including the heart, kidney, and liver against ADR-induced toxicity and organ dysfunction.