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U.S. Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, ND 58202-9034
5To whom correspondence should be addressed. E-mail: tjohnson{at}gfhnrc.ars.usda.gov.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • heme oxygenase • mitochondria • hydrogen peroxide • respiratory complexes • copper deficiency
Heme oxygenase (HO)6 catalyzes the initial rate-limiting step in heme catabolism, producing equimolar quantities of iron, carbon monoxide, and biliverdin from the oxidative cleavage of b-type heme. The HO family consists of the following 3 isozymes: an inducible form, HO-1, a constitutively expressed form, HO-2, and a cloned form, HO-3, identified in the brain (1,2). The HO-1 form is induced by its heme substrate (3) and also by a number of stress factors including heavy metals, certain organic compounds, hypoxia, hyperoxia, heat shock, ultraviolet radiation, hydrogen peroxide, and cytokines (4–10). Although the exact functional role of HO-1 induction is not fully understood, the realization that many inducers of HO-1 are either oxidants themselves or are able to generate reactive oxygen species (ROS) led to the hypothesis that HO-1 induction is an adaptive cellular defense mechanism against oxidative stress (11,12). Confirmation of the role of HO-1 in defense against oxidative stress comes from the observation that cultured fibroblasts from HO-1 knockout mice are highly susceptible to hydrogen peroxide– and heme-mediated toxicity (13).
Elevated HO activity was reported in livers of copper-deficient rats (14). Although the increase in HO activity may be caused by alterations in iron and selenium metabolism that occur during copper deficiency, a mechanism for the increase has not been clearly identified. However, copper deficiency, by decreasing the activities of copper-dependent antioxidant enzymes, primarily superoxide dismutase (SOD)-1 and ceruloplasmin, results in an increase in oxidative stress (15) that may be an important determinant of HO activity in copper-deficient rats through the induction of HO-1. Cytochrome c oxidase (CCO), the terminal respiratory complex (complex IV) of the mitochondrial respiratory chain, is a copper-dependent enzyme whose activity is also reduced during copper deficiency (16,17). Although CCO is not an antioxidant enzyme per se, the reduction in CCO activity may also contribute to oxidative stress during copper deficiency by promoting increased mitochondrial ROS production. Although the mitochondrial electron transport chain converts 85–90% of the oxygen utilized by cells to water, 
1–2% of the oxygen is converted to ROS (18,19). Mitochondrial ROS production is largely determined by the redox state of the respiratory complexes and is highest when the complexes are highly reduced (18). Blockage of electron flow near the terminus of the electron transport chain increases the reducing potential of the respiratory complexes upstream from the blockage, causing increased ROS production through single electron transfer to molecular oxygen. This principle was demonstrated by showing that partial inhibition of CCO in mitochondria from the flight muscles of house flies increases the rate of mitochondrial H2O2 production (20). Thus, reduction in CCO activity caused by copper deficiency may result in overproduction of mitochondrially generated H2O2. The overproduction of H2O2 combined with compromised enzymatic antioxidant defenses may produce sufficient oxidative stress during copper deficiency to induce stress proteins such as HO-1. Accordingly, the present study examined the effect of copper deficiency on mitochondrial respiratory complex activities as a potential model for investigating the relationship between mitochondrially generated ROS and the expression of HO-1. In addition, the tissue specificity of HO-1 overexpression during copper deficiency was examined by comparing HO-1 induction in liver and heart.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Immunoblot analysis. Liver and heart samples were weighed and homogenized in 10 volumes of either liver homogenizing buffer (0.25 mol/L sucrose, 10 mmol/L HEPES, 0.1 mmol/L EGTA, pH 7.4) or heart homogenizing buffer (0.225 mol/L mannitol, 0.075 mol/L sucrose, 20 mmol/L HEPES, 1 mmol/L EGTA, pH 7.4). Homogenates were centrifuged at 600 x g for 10 min. After the pellet was discarded, the supernatant fractions were centrifuged at 9000 x g for 10 min. The resulting supernatants, which contained suspended microsomes, were used for immunoblot detection of HO-1.
Proteins (15 µg of protein per lane) were separated by SDS-PAGE on 10% polyacrylamide gels and electroblotted to polyvinylidene fluoride membrane (Immobilon-P, Millipore). The blots were dried and incubated, without prior blocking, according to the manufacturer’s recommendation (Millipore Technical Note RP562), with anti-HO-1 (StressGen Biotechnologies) for 1 h at room temperature. After incubation with primary antibody, the blots were rinsed and incubated with horseradish peroxidase-coupled anti-IgG. Visualization of HO-1 was accomplished by chemiluminescence and exposure of the blots to luminescence detection film (ECL Western Blotting detection reagents and Hyperfilm-ECL, Amersham). The ODs of immunoreactive protein in the visualized bands were determined by imaging densitometry (GS-700 Imaging Densitometer, Bio-Rad Laboratories). One lane of each electrophoresis gel, regardless of whether it contained liver or heart samples, was loaded with a standard microsomal sample (15 µg protein) prepared from adult rat liver. This corrects for between-blot variations by allowing the content of HO-1 in liver and heart samples to be represented as a relative OD calculated by dividing the density of the immunoreactive protein in the experimental sample by the OD of the immunoreactive protein in the standard liver microsomal sample. In addition, each electrophoresis gel contained samples from rats fed CuD and CuA to balance between-blot variations for the 2 diet treatment groups.
RNA isolation and PCR amplification. Standard methods were used to isolate total RNA by phenol-guanidine thiocyanate-chloroform extraction and to determine its quality and quantity (24). Real-time RT-PCR was performed using the Smart Cycler II System (Cepheid) with Smart Cycler software (version 2.0c.). Genomic DNA was removed from RNA samples before performing RT-PCR using DNase I amplification grade (Invitrogen Life Technologies) and following the manufacturer’s instructions. Heme oxygenase-1 mRNA was identified using fluorogenic primers targeted towards the rat HO-1 gene (Acc. no. NM 012580). The primer sequences, designed using LUX Designer software (Invitrogen Life Technologies), were as follows: HO-1 forward (FAM labeled), CACGATCCAAGTTCAAACAGCTCTATCGG; and HO-1 reverse, TGAGCAGGAAGGCGGTCTTAG. PCR also was performed in identical tubes with a FAM-labeled primer (mouse/rat PGK1-Certified LUX Primer Set, Invitrogen Life Technologies) targeted toward the phosphoglycerate kinase-1 gene (PGK-1), which was used as the internal control. One-step, real-time RT-PCR was performed using the Quanti Test Probe RT-PCR kit (Qiagen). Reaction mixtures were prepared according to instructions and contained LUX FAM-labeled primer (400 nmol/L), unlabeled primer (400 nmol/L), and 50 ng liver RNA or 100 ng heart RNA in a total volume of 25 µL. Conditions for RT-PCR were as follows: RT (30 min, 50°C; 15 min, 95°C) and 45 cycles of 3-step PCR (15 s, 94°C; 30 s, 50°C; 30 s, 72°C). Relative standard curves for the amplification of the HO-1 and PGK-1 genes were constructed by determining the threshold cycle (Ct) for several dilutions of total liver or heart RNA. The amount of HO-1 RNA relative to PGK-1 RNA in livers and hearts from rats fed copper-deficient and copper-adequate diets was quantified by measuring Ct, determining the corresponding amount of RNA from the appropriate standard curve, and calculating the ratio of HO-1 RNA to PGK-1 RNA (25).
Hepatic mitochondrial enzyme activities and hydrogen peroxide production.
Livers were homogenized as described above. The homogenate was centrifuged at 600 x g for 10 min. After the pellet was discarded, the supernatant was centrifuged at 7700 x g for 10 min. The resulting mitochondrial pellet was washed once and resuspended in liver homogenizing buffer (1 mL/g liver). NADH:cytochrome c reductase (NADHCR), succinate:cytochrome c reductase (SucCR), and CCO activities were assayed at 30°C in isolated mitochondria by a sequential, continuous assay that allows measurement of the 3 enzyme activities in a single sample of mitochondria (26). Briefly, CCO activity was measured by monitoring the change in absorbance at 550 nm (
A550) resulting from the oxidation of reduced cytochrome c. The CCO activity was monitored until A550 was 0.6 U, at which point CCO activity was terminated by adding KCN (0.17 mmol/L final concentration). Succinate was then added (12.5 mmol/L final concentration) and SucCR activity was measured by monitoring A550 resulting from the reduction of the cytochrome c previously oxidized by CCO. SucCR activity was terminated by adding malonate (17 mmol/L final concentration), and NADHCR activity was measured by adding NADH (2.8 nmol/L final concentration) and monitoring A550 resulting from the further reduction of cytochrome c. The rate of cytochrome c oxidation or reduction (nmol/min) was calculated using a molar extinction coefficient of 19,600 for cytochrome c (16). The rate of hydrogen peroxide production was measured fluorometrically (27) using a p-hydroxyphenylacetate-horseradish peroxidase coupled assay as previously described. The rate of hydrogen peroxide release from mitochondria (75–150 µg protein) was determined after the addition of glutamate (final concentration 10 mmol/L).
Protein assay. The protein concentrations of liver and heart preparations used for enzyme assays, electrophoresis, and measurement of hydrogen peroxide release were determined with bichinchoninic acid (BCA Protein Assay Reagent Kit, Pierce). Volumes of samples applied to electrophoresis gels were adjusted on the basis of sample protein concentrations to ensure that 15 µg of protein was applied to each lane.
Statistics.
Values shown in text and figures are means ± SEM. The data were analyzed for significant differences by a one-tailed t test for 2 samples with unequal variance, 
= 0.05. Associations between HO-1 content and mitochondrial enzyme activities were modeled by stepwise multiple regression analysis (SAS/STAT 8.02, SAS Institute).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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One objective of this investigation was to determine whether copper deficiency affects mitochondrial respiratory complex activities other than CCO. In the present study, CCO activity in mitochondria isolated from livers of copper-deficient rats was 70% lower (P < 0.05) than the activity in mitochondria from copper-adequate controls (Fig. 1A). However, CCO was not the only respiratory complex in hepatic mitochondria affected by copper deficiency. Copper deficiency caused a 42% reduction (P < 0.05) in SucCR activity (Fig. 1B), which represents the combined activities of respiratory complexes II and III, and a 31% (P < 0.05) reduction in NADHCR activity (Fig. 1C), which represents the combined activities of respiratory complexes I and III. These findings indicate that an overall decline in mitochondrial electron transport activity occurs during copper deficiency.
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Cytochrome c oxidase activity was not a significant (P > 0.05) predictor of hepatic HO-1 content.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In the present study, copper deficiency caused significant reductions in the activities of CCO, NADHCR, and SucCR (Fig. 1). NADHCR activity represents the combined electron transport activities of respiratory complexes I + III, and SucCR activity represents the combined electron transport activity of respiratory complexes II + III. Thus, the reductions in CCO, NADHCR, and SucCR indicate that copper deficiency causes general impairment of the mitochondrial electron transport chain by inhibiting copper-dependent and copper-independent respiratory complexes. Complexes I and III are major sites for mitochondrial ROS generation, and the activities of these complexes are determinants of ROS production (33,34). It was shown that genetic or drug-induced inhibition of complex I in cell models increases mitochondrial ROS production (35). Even a partial 25% inhibition of complex I, which was accompanied by only 5% inhibition of respiration, led to a 48% increase in mitochondrial ROS production. Also, fibroblasts from patients with inherited complex I deficiencies exhibit increased mitochondrial ROS production (36). In the present study, copper deficiency led to a 30% inhibition of NADHCR activity and a 40% inhibition of SucCR activity. The degree of NADHCR and SucCR inhibition we observed is similar to that observed by Davies and co-workers (26) in hepatic mitochondria from rats fed CuD for 8 wk. Although the mechanisms through which copper deficiency exerts its inhibitory effect on respiratory complexes upstream from copper-dependent CCO are not clear, the magnitude of the inhibition of respiratory complex activities caused by copper deficiency is within the range that could increase mitochondrial ROS production.
Superoxide is the primary ROS produced by mitochondria, and the rate of its production depends on the metabolic state of the mitochondria; the highest rate occurs in state 4 when ADP is unavailable and the reduction state of the respiratory chain components is high (37). The superoxide produced by the electron transport chain is readily converted to diffusible and more stable H2O2 by Mn-dependent mitochondrial SOD2 located in the mitochondrial matrix. Thus, it is conceivable that conditions that increase superoxide production by respiratory chain components also lead to increased H2O2 release from mitochondria. In the present study, H2O2 production by hepatic mitochondria was measured in the absence of ADP using glutamate, which is an NADH-linked, complex I substrate. Under these conditions, the mitochondria produced measurable amounts of H2O2, indicating that the electron transport chain was functional in the isolated mitochondria and that the mitochondria were likely in state 4. However, the rate of H2O2 production by hepatic mitochondria was increased by copper deficiency (Fig. 2). To our knowledge, this finding is the first to show that copper deficiency stimulates mitochondrial H2O2 release and suggests that the suppression of respiratory complex activities by copper deficiency leads to increased superoxide production at 1 or more sites in the electron transport chain.
Although mitochondria isolated from livers of copper-deficient rats exhibited elevated rates of H2O2 release, the question is whether the increase in H2O2 release had any intracellular consequences. The ability of H2O2 to stimulate gene expression is well documented (38), and HO was one of the first enzymes shown to be upregulated by H2O2 (39,40). Our study is the first to show that copper deficiency causes the induction of HO-1 in liver and heart. This finding is consistent with a previous report of increased hepatic HO activity in copper-deficient rats (14) and suggests that HO-1 induction is the most likely explanation for the increased HO activity caused by copper deficiency. Induction of HO-1 is controlled at the transcriptional level by regulatory elements localized in the promoter 5'-flanking region of the HO-1 gene (11). In the extensively studied mouse HO-1 gene, 2 enhancer regions, SX2 and AB1, contain multiple copies of a cis-acting element termed the stress response element (StRE) that mediate transcriptional activation in response to a number of agents, including H2O2. The consensus StRE resembles the consensus binding site for the activator protein (AP)-1 superfamily of transcription factors, which have been implicated in the activation of HO-1 gene transcription by ROS (41). In particular, it was shown that Nrf2, a basic leucine zipper protein, is an important transcription factor for HO-1 gene induction (42). Although the exact mechanisms through which copper deficiency causes the elevation of hepatic HO-1 protein and mRNA are not clear, our findings suggest that the increase in H2O2 release from the mitochondria of copper-deficient rats measured in vitro reflects an intracellular condition in which increased mitochondrial H2O2 production may have initiated the activation of AP-1–dependent HO-1 gene transcription. Although sources of ROS other than the mitochondria may also be involved in stimulating the expression of HO-1, results of the regression analysis showing that variations in the activities of NADHCR and SucCR accounted for 50% of the variation in hepatic HO-1 protein suggest that changes in mitochondrial electron transport chain activity contribute at least partially to the upregulation of HO-1.
The heart is particularly susceptible to oxidative stress during copper deficiency because of its relatively low activities of SOD, catalase, and glutathione peroxidase (43). Thus, our finding that HO-1 protein and mRNA are elevated in hearts of copper-deficient rats (Fig. 4) indicates that the upregulation of HO-1 is not limited to hepatic tissue during copper deficiency, but may be a general response to a shift in redox status associated with copper deficiency in tissues that are susceptible to oxidative stress.
In conclusion, our data indicate that copper deficiency stimulates the gene expression of HO-1 and increases HO-1 protein content in liver and heart. Our findings also suggest that copper deficiency leads to an increase in mitochondrial ROS production through a mechanism that may involve inhibition of respiratory complex activities. Collectively, the results of the present study suggest that the induction of HO-1 during copper deficiency occurs through a series of events in which the respiratory chain is inhibited, causing an increase in mitochondrial generation of H2O2. Release of H2O2 into the cytosol then serves as a signal that stimulates an increase in HO-1 gene expression. Rogers and co-workers (44) suggested recently that ROS may serve as signal transducers in mitochondria-to-nucleus signaling pathways. Although further research is required to establish the exact mechanisms for the induction of stress proteins such as HO-1 during copper deficiency, our results indicate that copper deficiency provides another model in addition to cultured cell models in which genetic, drug, or environmentally induced mitochondrial respiratory chain deficiencies are used to investigate mitochondria-to-nucleus signaling.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by U.S. Department of Agriculture CRIS Project No. 5450–51000-023.
3 The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination.
4 Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
6 Abbreviations used: AP, activator protein; CCO, cytochrome c oxidase; Ct, threshold cycle; CuA, Cu-adequate diet; CuD, Cu-deficient diet; Hgb, hemoglobin; HO, heme oxygenase; NADHCR, NADH:cytochrome c reductase; PKG, phosphoglycerate kinase-1; ROS, reactive oxygen species; SOD; superoxide dismutase; StRE, stress response element; SucCR, succinate:cytochrome c reductase.
Manuscript received 12 January 2004. Initial review completed 30 January 2004. Revision accepted 27 February 2004.
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LITERATURE CITED |
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