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TOXICOLOGY
Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, Colorado (P.R.C., D.A.D., M.P.); and Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado (B.J.D.)
Received for publication
September 27, 2007
Accepted
December 5, 2007.
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Abstract |
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). Metalloporphyrins are a class of catalytic antioxidants that are capable of detoxifying a wide range of reactive oxygen species (ROS), such as superoxide (
), H2O2, peroxynitrite, and lipid peroxyl radicals (Patel and Day, 1999). Several water-soluble metalloporphyrin compounds, including manganese (III) meso-tetrakis (4-carboxyphenyl or benzoic acid) porphyrin (MnTBAP), AEOL10150, and AEOL10113, have been shown to be efficacious in animal models of central nervous system disorders, including status epilepticus, (Liang et al., 2000), stroke (Mackensen et al., 2001), and amyotrophic lateral sclerosis (Crow et al., 2005). Previous work has also demonstrated that manganic porphyrins can protect mature neuronal cultures from excitotoxic injury by scavenging intracellular (Patel et al., 1996; Li et al., 2001). These compounds contain a manganese center that catalytically dismutes both and H2O2 (Pasternack and Skowronek, 1979; Day et al., 1997) Previous meso-substituted porphyrin rings contained positively charged pyridyl (AEOL10113) or imidazole (AEOL10150) groups to electrostatically facilitate reaction with negatively charged (Batinic-Haberle et al., 1998; Kachadourian et al., 2004). However, the charged nature of the water-soluble pyridine- and imidazole-substituted metalloporphyrins makes them less efficient in crossing lipid membranes. To overcome these issues, a series of novel glyoxylate metalloporphyrins (AEOL112 series) with improved lipid solubility have been developed and chemically characterized (Trova et al., 2003) to improve the potential for in vivo therapeutic use in neurological disorders characterized by increased ROS levels and oxidative stress. This newly developed glyoxylate series of metalloporphyrins (Fig. 1) have been shown to dismute H2O2 in a catalase-like reaction to generate O2 and inhibit lipid peroxidation in cell-free systems (Kachadourian et al., 2003, 2004; Trova et al., 2003; Liang et al., 2007).
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). The goals of this study were to 1) develop an in vitro assay that generated physiologically relevant H2O2 levels and 2) identify lead metalloporphyrin compounds based on rank order of potency for scavenging endogenously generated H2O2.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Isolation of Purified Rat Brain Mitochondria. Animal housing was conducted in compliance with University of Colorado at Denver and Health Sciences Center (Denver, CO) procedures. Mitochondria were isolated from adult male Sprague-Dawley rats using Percoll gradient density centrifugation as described previously (Anderson and Sims, 2000) with minor modifications (Castello et al., 2007). The purity of mitochondrial fractions was assessed using Western blotting techniques. In brief, denatured protein fractions of cytosol, mitochondria, and whole-cell homogenate were separated by electrophoresis on a 10% polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membrane. Membrane blots were incubated with primary antibodies against lactate dehydrogenase (LDH) (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or cytochrome c oxidase subunit IV (COX), (1:1000; Molecular Probes, Eugene, OR). LDH and COX membranes were incubated with horseradish peroxidase-conjugated anti-goat or anti-mouse secondary antibodies, respectively. Membranes were developed using an ECL Western blotting detection reagent (GE Healthcare, Buckinghamshire, UK). Figure 2 shows that COX was undetectable in cytosolic fractions and robustly expressed in mitochondrial fractions. In contrast, LDH was undetectable in mitochondrial samples indicating highly purified isolated fractions (Fig. 2).
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Polarographic Measurement of Net H2O2 Production. H2O2 net production by isolated brain mitochondria was measured using an Apollo 4000 Free Radical Analyzer (WPI, Sarasota, FL) equipped with a 100-µmH2O2 sensor. The measurements were conducted in a thermostatted open chamber at 30°C with a final reaction volume of 2 ml. Each measurement was started with the addition of reaction buffer (100 mM KCl, 75 mM mannitol, 25 mM sucrose, 10 mM Tris-Cl, and 10 mM KH2PO4, pH 7.4) to the chamber. Once the output signal stabilized, the following were consecutively added: respiration substrate (2.5 mM malate + 5 mM pyruvate) and mitochondrial protein (200 µg of protein). The output signal was allowed to stabilize subsequent to each addition, followed by the addition of 250 µMPQ2+ to the chamber, and the trace was recorded. After 2 to 3 min of recording, vehicle [1 µl of dimethyl sulfoxide (DMSO)] or tested compound (dissolved in 1 µl of DMSO) was added. The antioxidant activity of the metalloporphyrins was evaluated by measuring the change in the rate of H2O2 net production after the addition of the tested compound. The activity was expressed by the percentage of decrease in the rate of H2O2 net increase by mitochondria after the addition of the compound.
Fluorometric Detection of H2O2. H2O2 was measured using the horseradish peroxidase (HRP)-linked fluorometric assay (Amplex Ultra Red; Invitrogen, Carlsbad, CA). Brain mitochondria (10 µg) were added to a 96-well plate containing 100 µl of reaction buffer containing 0.1 U/ml HRP, 50 µM Amplex UltraRed and 2.5 mM malate + 5 mM pyruvate, 250 µMPQ2+, and 1 µl of DMSO control or tested compound dissolved in 1 µl of DMSO. Resorufin fluorescence was followed by a Gemini fluorescence microplate reader (Molecular Devices, Sunnyvale, CA). Superoxide dismutase (SOD) and catalase were added as controls at concentrations of 500 and 40 U/ml, respectively. The antioxidant activity of the compounds was expressed by the percentage of inhibition of the rate of H2O2 net increase by mitochondria in the presence of the compound.
SOD Screening Assay. The SOD-like activities were measured using the xanthine/xanthine oxidase system as a source of and ferricytochrome c as its indicating scavenger. was produced at the rate of 1.2 µM/min, and reduction of ferricytochrome c was followed at 550 nm. Assays were conducted in the presence of 0.1 mM EDTA in 0.05 M phosphate buffer, pH 7.8, at 25°C. Some metalloporphyrins that were analyzed interfered with the activity of xanthine oxidase, as checked by following urate production at 295 nm in the absence of cytochrome c, or they reoxidized cytochrome c at concentrations necessary to measure SOD activity.
Lipid Peroxidation Screening Assay. The concentration of thiobarbituric acid (TBA) reactive species (TBARS) in rat brain homogenates was used as an index of lipid peroxidation (Bernheim et al., 1948). Malondialdehyde (MDA) standards were obtained by adding 8.2 µl of 1,1,3,3-tetramethoxypropane in 10 ml of 0.01 M HCl and mixing for 10 min at room temperature. This stock was further diluted in water to give standards that ranged from 0.25 to 25 µM. Samples or standards (200 µl) were acidified with 200 µl of 0.2 M phosphoric acid in 1.5 ml of locking microfuge tubes. The color reaction was initiated by the addition of 25 µl of a 0.11 M thiobarbituric acid solution, and samples were placed in a 90°C heating block for 45 min. TBARS were extracted with 0.5 ml of n-butanol by vortexing samples for 3 min and chilling on ice for 1 min. The samples were then centrifuged at 12,000g for 3 min, and 150 µl of aliquots of the n-butanol phase were placed in each well of a 96-well plate and read at 535 nm in a plate-reader (Spectramax 340PC; Molecular Devices) at 25°C. Sample absorbances were converted to MDA equivalencies (micromolar) by extrapolation from the MDA standard curve. None of the antioxidants at concentrations used in these studies affected the reaction of MDA standards with TBA, and reactions without TBA were used as subtraction blanks.
Statistical Analysis. All experimental data shown were derived from at least two or three independent experiments. Nonlinear regression was performed with GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA) adjusting a sigmoidal dose (concentration)-response equation (variable slope).
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Results |
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). The consistent and stable increase in H2O2 allows for the analysis of antioxidant activity of added compounds over a short period of time. To validate this method as a tool for identifying antioxidant molecules, we analyzed the effect of externally added endogenous antioxidant enzymes, catalase, and SOD. SOD had no effect on net H2O2 production at a saturating concentration of 500 U/ml, whereas catalase produced a concentration-dependent inhibition of PQ2+-induced mitochondrial H2O2 (Figs. 3, A and B, and 4).
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Inhibition of PQ2+-Induced Mitochondrial H2O2 by AEOL112 Compounds. Using the polarographic method described above, we were able to measure the H2O2-scavenging activities of metalloporphyrin compounds. Figure 4 shows an example of the results using AEOL11207. After the addition of PQ2+, H2O2 levels increased at a steady rate for several minutes at which point compounds were added to determine inhibitory effects. Catalase at a concentration of 12 nM was chosen as a positive control and was considered to represent 100% inhibition of the H2O2 net increase. This concentration of catalase caused no significant change in levels of H2O2, indicating a balance between the production and removal of H2O2 in the system. Increasing concentrations of catalase (125 nM) led to a decrease in H2O2 signal corresponding with the enzymatic removal of endogenously generated H2O2 in the system. Addition of SOD at saturating concentrations had no effect on the rate of H2O2 net production. A full range of inhibition curves was determined for different concentrations of AEOL112 compounds (Fig. 5). Figure 5 represents the percentage of inhibition of steady state of PQ2+-induced mitochondrial H2O2 as a function of the logarithm concentration of each compound. Using nonlinear regression, the best fit for each concentration-response curve was obtained to determine IC50 values for each compound. Compounds were divided into two groups based on their observed H2O2-scavenging activities: 1) those compounds exhibiting a strong concentration-response relationship (Fig. 5; Table 1) and 2) those compounds exhibiting a less optimal concentration-response relationship (Table 1). The former group included compounds showing canonical concentration-response relationship with IC50 values obtained after nonlinear regression of the activity data. The latter contains compounds that do not demonstrate any inhibition of H2O2 net increase and/or have an IC50 value higher than 3 µM. Table 1 shows the IC50 values for the compounds that exhibited a strong concentration-response relationship. A notable exception is AEOL10150, which interfered with the polarographic detection. To ascertain whether the observed inhibition of H2O2 net production by metalloporphyrins was not due to a change in the sensitivity of the electrode for the H2O2 after addition of the compound, the effect of exogenously added H2O2 was determined. No change in signal was observed after the addition of the AEOL112 series compounds listed in Table 1 to the reaction buffer + mitochondria + malate + pyruvate + 2 µMH2O2. The most potent metalloporphyrins identified with IC50 < 1 µM are as follows: AEOL11209 (IC50 = 17 nM) > AEOL11216 (IC50 = 93 nM) 
AEOL11207 (IC50 = 104 nM) > AEOL11215 (IC50 = 206 nM) > AEOL11223 (IC50 = 408) > AEOL11210 (IC50 = 725 nM) > AEOL11202 (IC50 = 1642 nM).
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Comparison of the Inhibition of PQ2+-Induced Mitochondrial H2O2 by AEOL10150 and AEOL112 Series. To validate the polarographic method and overcome its interference with AEOL10150, a HRP-linked fluorometric method (Amplex Red assay) was used. The values obtained were used to construct the concentration-response curve shown in Fig. 6. To compare the IC50 values obtained using the fluorometric assay with the polarographic method, AEOL11207 was used as a positive control. AEOL11207 showed a concentration-response relationship comparable to that obtained using the polarographic method. The IC50 values of AEOL11207 using the fluorometric and the polarographic assays were 30 and 104 nM, respectively. AEOL10150 showed a concentration-response relationship with an IC50 value of 3 µM, using the fluorometric assay.
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To determine whether inhibition of PQ2+-induced H2O2 by metalloporphyrins showing an optimal concentration-response relationship was due to the manganese moiety, the effects of metal-substituted analogs of AEOL11215 (AEOL11249, Zn2+ analog; AEOL11250, Fe2+ analog; AEOL11251, Co2+ analog) were evaluated. Figure 7 shows that none of these analogs was able to inhibit the net production of H2O2 in a broad range of concentrations. These studies suggest that manganese is the optimal metal to support H2O2-scavenging activity.
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Antioxidant Properties of AEOL112 Compounds. Table 2 shows the antioxidant activity of hydrophobic metalloporphyrins evaluated as SOD-like activity and inhibition of lipid peroxidation (TBARS). Overall, the data indicate that AEOL112 compounds have very low SOD-like activity and very high lipid peroxidation inhibition activity. These properties, together with the high capacity to dismute H2O2, suggest that these compounds have an alternative antioxidant mechanism compared with that observed in known SOD mimetics.
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Discussion |
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A polarographic method was established for the sensitive, accurate, and reproducible detection of H2O2 scavenging by AEOL compounds in a physiologically relevant in vitro model involving rat brain mitochondrial H2O2. The method is rapid and sensitive, with low level of interferences, and has the potential for high-throughput analysis. The assay uses respiring mitochondria and PQ2+ to generate endogenous H2O2. The system is specific for H2O2 as shown by the high sensitivity to the addition of catalase but not SOD (Fig. 4). The ability of catalase to inhibit the H2O2 signal is due to the ability of intramitochondrially generated H2O2 to cross mitochondrial membranes, which then can be readily dismuted by catalase. The lack of effect with SOD is probably due to the inability of this large protein to penetrate mitochondrial membranes to dismute intramitochondrial , which is short-lived and not very permeable to biological membranes. Once the steady-state net increase of H2O2 was induced by PQ2+ in mitochondria, the addition of either several metalloporphyrins or catalase (but not SOD) changed the steady-state net production of H2O2. The velocity of H2O2 net production in the lower steady state was used as an indicator of compound potency. This approach has the advantage of using the initial steady state as a control, eliminating the random differences between different measurements and preparations in which the initial steady state may vary.
It is worth mentioning that in vitro H2O2 concentrations previously used in the measurement of catalase activity of AEOL compounds was 1 mM (Day et al., 1997). This concentration is orders of magnitude higher than H2O2 steady-state concentrations in physiological systems that are in the nanomolar range (Chance et al., 1979). Because catalase activity is assumed to follow pseudo-first-order kinetics, the use of such high amounts of H2O2 can result in an overestimation of the pseudo-first-order rate constant and, therefore, of catalase activity. Moreover, it has been reported that high amounts of H2O2 can inactivate the metalloporphyrins (Day et al., 1997). This new assay presents several advantages over the established ones. First, it uses concentrations of H2O2 (
1–100 nM) that may be achieved physiologically. Steady-state concentrations of H2O2 are estimated to 10–9 to 10–7 M (Chance et al., 1979; Gardner et al., 2006). Second, it uses H2O2 produced by brain mitochondria, which are an important cellular source of ROS contributing to neurodegeneration and aging. Third, it is based in an in vitro system using the redox-cycling agent PQ2+, an environmental toxin implicated in the etiology of Parkinson's disease (Di Monte, 2003).
The results presented in Table 1 reveal the following order of potencies of the metalloporphyrins tested in this study that showed an IC50 < 1 µM: AEOL11209 (IC50 = 17 nM) > AEOL11216 (IC50 = 93 nM) > AEOL11207 (IC50 = 104 nM) > AEOL11215 (IC50 = 206 nM) > AEOL11223 (IC50 = 408) > AEOL11210 (IC50 = 725 nM) > AEOL11202 (IC50 = 1642 nM). The potencies of the compounds obtained in our in vitro assay have been validated in the in vivo setting by the demonstration that orally administered AEOL11207 achieving brain concentrations of 200 nM inhibited oxidative stress indices and neuronal damage in a mouse model of mitochondrial oxidative stress (Liang et al., 2007).
The ability of manganese-substituted, but not zinc-, cobalt-, and iron-substituted, metalloporphyrins to inhibit the net production of H2O2 illustrates the importance of manganese as the optimal metal in the H2O2-scavenging effects of the compounds. Although the glyoxylate metalloporphyrins are lipid-soluble, the control studies described under Results suggest that most AEOL compounds do not inhibit the net production of H2O2 by interfering with the redox-cycling mechanism of PQ2+ in the mitochondria. Their ability to remove H2O2 is probably not caused by scavenging of intramitochondrial . This observation is based on their low SOD activity in cell-free assays (Trova et al., 2003), which renders the compounds less suitable as SOD mimetics.
Comparison of the data obtained in this study (Table 1) with previously published values for the AEOL112 series (Gauuan et al., 2002; Trova et al., 2003) demonstrates that compounds exhibiting a strong concentration-response relationship (Fig. 5; Table 1) have an average catalase activity 145% higher than the average activity of the compounds exhibiting a less optimal concentration-response relationship (Table 1). On the other hand, the compounds demonstrating a strong concentration-response relationship (Fig. 5; Table 1) display average TBARS levels that are eight times lower than the average of the compounds exhibiting a less optimal concentration-response relationship, indicating a greater ability to remove lipid peroxides (Table 2). Together, these results conclude that the grouping of compounds according to their antioxidant properties was reflected not only by the current method but also by other previous screening methods.
One interesting finding that has emerged from several in vitro models of neuronal injury is a discrepancy between antioxidant potency and neuroprotective efficacy of metalloporphyrins. For example, although the water-soluble metalloporphyrin MnTE-2-PyP (AEOL10113) has at least 20 times more SOD activity in cell-free assays compared with MnTBAP, it was only two to three times more potent in its efficacy as a neuroprotective agent in Sod2–/– cultures (Patel, 2003). This paradoxical difference between antioxidant activities and neuroprotective efficacy has also been observed in other in vitro models involving glutamate excitotoxicity and oxygen-glucose deprivation injury (Li et al., 2001). These observations suggest that high SOD activity in a cell-free assay per se may not be sufficient to predict neuroprotection in vivo (cells or animals) and provides the rationale for the development of metalloporphyrins with broad antioxidant properties other than antioxidant activities derived from cell-free assays. The glyoxylate (AEOL112) series of metalloporphyrins have modest SOD activity but show high potencies as inhibitors of lipid peroxidation and cell and tissue injury (Choudhary et al., 2001; Kachadourian et al., 2003, 2004; Trova et al., 2003; Liang et al., 2007).
The ability of metalloporphyrins to scavenge mitochondrially generated H2O2 is highly significant based on previous demonstrations that mitochondrial overexpression of catalase provides protection against menadione toxicity, a chemical agent that preferentially generates intramitochondrially (Gurgul et al., 2004). Moreover, median and maximal life spans were increased the most in animals overexpressing mitochondrial catalase due to a reduction in mitochondrial generated oxidative stress (Schriner et al., 2005). Although the overexpression of mitochondrial catalase is difficult to achieve, the particular scavenging of mitochondrial H2O2 by AEOL compounds opens a new paradigm in the therapeutic treatment of neuronal diseases in which mitochondrial oxidative stress is a major contributor.
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Acknowledgements |
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Footnotes |
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B.J.D. is a consultant for and holds equity in Aeolus Pharmaceuticals, which is developing catalytic antioxidants as therapeutic agents.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ROS, reactive oxygen species; MnTBAP, manganese (III) meso-tetrakis (4-carboxyphenyl or benzoic acid) porphyrin; AEOL10150, manganese (III) meso-tetrakis (N,N'-diethylimidazolium-2-yl) porphyrin; AEOL10113, manganese (III) meso-tetrakis (N-ethyl pyridinium-2-yl) porphyrin; PQ2+, paraquat; LDH, lactate dehydrogenase; COX, cytochrome c oxidase; DMSO, dimethyl sulfoxide; HRP, horseradish peroxidase; SOD, superoxide dismutase; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive species; MDA, malondialdehyde; MnTE-2-PyP, manganese tetrakis-(N-ethyl-2-pyridyl) porphyrin.
1 Current affiliation: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado. 
Address correspondence to: Dr. Manisha Patel, Department of Pharmaceutical Sciences, 4200 East Ninth Ave., School of Pharmacy, C238, Denver, CO 80262. E-mail: manisha.patel{at}uchsc.edu
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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