Identifying Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore in Isolated Rat Liver Mitochondria
UNIT 25.4
Lisa Marroquin,1 Rachel Swiss,1 and Yvonne Will1 1
Compound Safety Prediction, Worldwide Medicinal Chemistry, Pfizer Inc., Groton, Connecticut
ABSTRACT The mitochondrial permeability transition pore (MPTP) is a protein pore that forms in the inner mitochondrial membrane and allows the membrane to be permeable to all molecules of less than 1500 Da. Ca2+ , numerous reactive chemicals, and oxidative stress induce MPTP opening, whereas cyclosporin A (CsA) or bongkrekic acid block it. In addition, several drugs have been shown to induce MPTP opening, leading to the loss of mitochondrial membrane potential, swelling of the matrix because of water accumulation, rupture of the outer mitochondrial membrane, and release of intermembrane space proteins into the cytosol. This ultimately leads to the rupture of the outer mitochondrial membrane and cell demise. Here, we describe an assay using isolated rat liver mitochondria that can detect Ca2+ -dependent drug-induced opening of the MPTP, providing protocols for screening in both cuvette and 96-well format. Curr. Protoc. Toxicol. 60:25.4.1:-25.4.17. C 2014 by John Wiley & Sons, Inc. Keywords: mitochondria permeability transition pore r mitochondrial toxicity r mitochondrial swelling
INTRODUCTION Due to the complexity of mitochondria, it is not surprising that adverse drug events often develop due to drug-induced mitochondrial injury. Drug-induced mitochondrial toxicity can occur through several mechanisms, including induction of the mitochondrial permeability transition pore (MPTP), and this could be a reason for organ-related toxicity. For example, hepatic or gastric toxicity by non-steroidal anti-inflammatory drugs (NSAIDS), such as diclofenac and nimesulide, has been postulated to be due at least in part to their ability to induce MPTP opening, with nimesulide being withdrawn from the market due to severe hepatotoxicity (Masubuchi et al., 2002; Tay et al., 2005; Berson et al., 2006). In addition, troglitazone, a diabetic drug that was withdrawn from the market due to idiosyncratic liver toxicity, also induces MPTP opening, and this may contribute to its hepatotoxicity (Masubuchi et al., 2006; Okuda et al., 2010). Described by Hunter and Haworth (1979), the MPTP is a protein pore that forms in the inner mitochondrial membrane and allows membrane permeability to all molecules of less than 1500 Da. The molecular identity of the pore is under debate, but proteins such as cyclophilin D, voltage-dependent anion channel, and adenine nucleotide translocator (ANT) are thought to contribute to MPTP formation. The MPTP is promoted by the accumulation of excessive Ca2+ and stimulated by various compounds and conditions such as oxidative stress, elevated phosphate levels, and adenine nucleotide depletion. The induction of the MPTP can lead to dissipation of membrane potential, uncoupling of oxidative phosphorylation, loss of preaccumulated Ca2+ , expansion of the matrix volume, Mitochondrial Toxicity Current Protocols in Toxicology 25.4.1-25.4.17, May 2014 Published online May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471140856.tx2504s60 C 2014 John Wiley & Sons, Inc. Copyright
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release of cytochrome c to the cytosol, and ultimately death of the cell. Cytochrome c release can trigger a cascade of events that lead to either apoptosis or necrosis (Kim et al., 2003). Induction of the MPTP can be blocked by cyclosporin A (CsA), which binds to cyclophilin D (Crompton et al., 1988; Tanveer et al., 1996), or bongkrekic acid, a ligand of ANT (Halestrap and Brenner, 2003). A common way to measure MPTP opening in isolated mitochondria is by following mitochondrial swelling (Hunter and Haworth, 1979). Mitochondrial swelling can be measured by monitoring changes in light scattering, which inversely correlates with mitochondrial volume (Beavis et al., 1985). The changes in light scattering can be recorded by following the decrease in absorbance at 540 nM after the addition of Ca2+ and drug. To determine if the decrease in absorbance is due to opening of the MPTP, CsA is added to block the opening. The following protocol outlines how to determine Ca2+ -dependent drug-induced opening of the MPTP in isolated rat liver mitochondria by following mitochondrial swelling. The protocol is divided into two parts, including measuring mitochondrial swelling in both cuvette and 96-well formats (Basic Protocols 1 and 2), and assay considerations (Support Protocols 1 to 4). NOTE: Protocols employing live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to government regulations regarding the care and use of laboratory animals. BASIC PROTOCOL 1
ASSESSMENT OF MITOCHONDRIAL SWELLING OF COMPOUNDS IN A 96-WELL FORMAT This protocol outlines the procedure for testing a compound’s effect on mitochondrial swelling in a 96-well plate format. The 96-well format is an efficient way to test a higher number of compounds in a screening paradigm. This protocol is designed for testing rat liver mitochondria and is not optimized for testing other tissues or species.
Materials Sprague-Dawley male rats (150 to 250 g; Charles River) or equivalent strain BCA protein assay kit (Pierce) 1 M succinate stock solution (see recipe) 10 mM CaCl2 stock solution (see recipe) 10 mM rotenone stock solution (see recipe) 30 mM oligomycin stock solution (see recipe) 1 mM cyclosporin A stock solution (see recipe) 100 mM phosphate stock solution (see recipe) Compound stocks dissolved in DMSO, for testing in assay (concentration will depend on highest dose tested) Dimethylsulfoxide (DMSO) Mitochondrial swelling buffer (see recipe)
Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
15-ml tubes, polystyrene, disposable (e.g., BD Falcon) 30°C water bath Multichannel pipettors Standard clear-bottom 96-well plate Absorbance plate reader (capable of measuring absorbance at kinetic interval) Computer running Microsoft Excel Program capable of calculating IC50 (e.g., GraphPad Prism)
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Cmpd 1 100 nmoles/mg
Cmpd 2 100 nmoles/mg Cmpd 3 100 nmoles/mg Positive Control 100 nmoles/mg Cmpd 4 100 nmoles/mg Cmpd 5 100 nmoles/mg Cmpd 6 100 nmoles/mg Cmpd 7 100 nmoles/mg
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Figure 25.4.1 Plate map for compound screening for mitochondrial swelling in isolated mitochondria. Concentrations are in nmoles/mg and are final concentrations. Vehicle is DMSO and positive control is phosphate. Compounds are in duplicate for testing with and without cyclosporin A. Wells in gray color are tested without cyclosporin A and wells in blue color are tested with cyclosporin A.
Additional reagents and equipment for isolation of mitochondria from rat liver (Support Protocol 1), mitochondrial protein assay (Support Protocol 2), optimizing mitochondrial protein concentration (Support Protocol 3), and optimizing CaCl2 concentration (Support Protocol 4) Assay procedure 1. Isolate mitochondria from rat liver (see Support Protocol 1). 2. Keep isolated mitochondria on ice at all times. 3. Determine the protein concentration of the isolated mitochondria using the BCA assay (see Support Protocol 2). 4. Thaw succinate stock solution, CaCl2 stock solution, rotenone, oligomycin, and cyclosporin A stock solutions on ice. 5. Warm mitochondrial swelling buffer to 30°C. 6. Prepare phosphate stock solution and adjust to the correct pH (see Reagents and Solutions). Phosphate stock solution must be made fresh for each experiment.
7. Prepare dilutions of compounds in DMSO in a separate 96-well plate according to the plate map (See Fig. 25.4.1 for plate map). Different dilutions need to be prepared depending on the chosen mitochondrial test concentration. The final DMSO content (in assay wells) should not exceed 0.5% v/v. The authors typically test compounds starting at a high dose of 100 nmol/mg final concentration. The dose-response curve is six points, with a 1:2 serial dilution
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8. Add 10 μl of rotenone stock, 3.3 μl of oligomycin stock, and 1 ml of succinate stock to 100 ml of warmed mitochondrial swelling buffer. This buffer will be used for the remainder of the experiment and referred to as swelling buffer mix. Final concentrations for the reagents are as follows: rotenone, 1 μM; oligomycin, 1 μM; and succinate, 10 mM.
9. Add 10 ml of swelling buffer mix to two 15-ml tubes—one tube will be for half of the 96-well plate for compound testing without cyclosporin A (tube A), and one will be for the remaining half of the plate for compound testing with cyclosporin A (tube B). 10. To tube A, add appropriate quantity of mitochondria (for guidelines on optimizing protein concentration, see Support Protocol 3) and 50 μl CaCl2 stock solution (actual amount will vary depending on the isolated mitochondria prep; for tips on optimizing CaCl2 concentration, see Support Protocol 4). Authors use 1 mg/ml of mitochondria and 50 μM of CaCl2 , but both concentrations are dependent on the quality of the mitochondrial isolation and will be user specific. Therefore, optimization of both of these parameters is recommended. Also, the order of the addition of these reagents is important. Reagents must be added in the specific order described.
11. To tube B, add 1 μM of cyclosporin A (10 μl of stock solution), the appropriate quantity of mitochondria (for guidelines on optimizing protein concentration, see Support Protocol 3), and 50 μl CaCl2 stock solution (actual amount will vary depending on the isolated mitochondria prep; for tips on optimizing CaCl2 concentration, see Support Protocol 4). 12. Use a multichannel pipettor to dispense 200 μl from tube A to each well on the left side of the 96-well plate (see Fig. 25.4.1 for plate map). 13. Use multichannel pipettor to dispense 200 μl from tube B to each well on the right side of the 96-well plate (see Fig. 25.4.1 for plate map). 14. Measure plate absorbance at 540 nm on a plate reader for 2 min at 30-sec intervals to obtain a baseline absorbance reading. 15. Remove plate from plate reader and use a multichannel pipettor to dispense 1 μl of test compound from the 96-well compound stock solution plate (see step 7) into each well. Add 6 μl of phosphate stock to appropriate control wells (see Fig. 25.4.1 for plate map). Addition of compounds or controls should be done as quickly as possible because, after addition of compounds, swelling may begin to occur. Phosphate is at a final concentration of 3 mM. The compound concentration range can be defined by the user, and drug concentrations are usually expressed in nmol/mg mitochondrial protein.
16. Place the plate back into the plate reader and measure the absorbance at 540 nm kinetically over 20 min, with 30-sec intervals.
Data analysis 17. Export data from absorbance reader into Excel format. Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
18. Graph the data with absorbance on the y axis and time on the x axis. 19. Calculate the slope of the steepest portion of the line.
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20a. If using a dose-response curve: Graph the slopes of each dose to calculate an IC50 using a program like GraphPad Prism. Use DMSO as a 0% control and the positive control as the 100% control. 20b. If using a single dose: Use the DMSO as a 0% control and the positive control as the 100% control, and calculate the % induction of the compound as compared to the controls.
ASSESSMENT OF MITOCHONDRIAL SWELLING OF COMPOUNDS IN A CUVETTE FORMAT
BASIC PROTOCOL 2
This protocol outlines the procedure for testing a compound’s effect on mitochondrial swelling in a cuvette format. The cuvette format is a useful way to investigate a small number of compounds at a single dose or to investigate a single compound in a thorough manner. This protocol is designed for testing rat liver mitochondria and not optimized for testing other tissues or species.
Materials Sprague-Dawley male rats (150 to 250 g; Charles River) or equivalent strain BCA protein assay kit (Pierce) 1 M succinate stock solution (see recipe) 10 mM CaCl2 stock solution (see recipe) 10 mM rotenone stock solution (see recipe) 30 mM oligomycin stock solution (see recipe) 1 mM cyclosporin A stock solution (see recipe) 100 mM phosphate stock solution (see recipe) Compound stocks dissolved in DMSO, for testing in assay (concentration will depend on highest dose tested) Mitochondrial swelling buffer (see recipe) Compound stocks dissolved in DMSO, for testing in assay (concentration will depend on highest dose tested) 30°C water bath Standard cuvettes for absorbance Absorbance cuvette reader (spectrophotometer capable of measuring absorbance at kinetic intervals) Computer running Microsoft Excel Program capable of calculating IC50 (e.g., GraphPad Prism) Additional reagents and equipment for isolation of mitochondria from rat liver (Support Protocol 1), mitochondrial protein assay (Support Protocol 2), optimizing mitochondrial protein concentration (Support Protocol 3), and optimizing CaCl2 concentration (Support Protocol 4) Assay procedure 1. Isolate mitochondria from rat liver (see Support Protocol 1). Keep isolated mitochondria on ice at all times.
2. Determine the protein concentration of the isolated mitochondria using the BCA assay (see Support Protocol 2). 3. Thaw succinate stock solution, CaCl2 stock solution, rotenone, oligomycin, and cyclosporin A stock solutions on ice. 4. Warm mitochondrial swelling buffer to 30°C.
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5. Prepare phosphate solution and adjust to the correct pH (see Reagents and Solutions). Phosphate solution must be made fresh for each experiment.
6. Add 10 μl of rotenone stock, 3.3 μl of oligomycin stock, and 1 ml of succinate stock to 100 ml of warmed mitochondrial swelling buffer. This buffer will be used for the remainder of the experiment and referred to as swelling buffer mix. Final concentrations for the reagents are as follows: rotenone, 1 μM; oligomycin, 1 μM; and succinate, 10 mM.
7. Add 2 ml of swelling buffer mix to a standard cuvette. 8. To the cuvette, add appropriate quantity of mitochondria (for guidelines on optimizing protein concentration, see Support Protocol 3) and mix by gently pipetting up and down. Then, add 5 μl CaCl2 stock solution (actual amount will vary depending on the isolated mitochondria prep; for tips on optimizing CaCl2 concentration, see Support Protocol 4) and mix gently by pipetting up and down. The authors use 0.25 mg/ml of mitochondria and 25 μM of CaCl2 , but both concentrations are dependent on the quality of the mitochondrial prep and will be user specific. Therefore, optimization of both of these parameters is recommended. Also, the order of the addition of these reagents is important. Reagents must be added in this specific order. The protein concentrations and calcium concentrations need to be optimized separately from the 96-well format; however, the volumes are ten-fold higher. Be sure to mix by gently pipetting up and down after compound addition, unless the spectrophotometer has a stirrer feature available.
9. Measure cuvette absorbance at 540 nm for 2 min at 30-sec intervals to obtain a baseline absorbance reading. 10. Remove cuvette from absorbance reader and add 10 μl of test compound. Mix gently by pipetting up and down. In the cuvette format, only one concentration of one compound can be tested at a time. Separate cuvettes need to be set up to measure each condition. The authors typically test compounds at a high dose of 100 nmol/mg final concentration.
11. Place the cuvette back into the absorbance reader and measure the absorbance at 540 nm kinetically over 20 min, with 30-sec intervals. 12. Repeat steps 7 to 11 with the same compound or DMSO, but first add 2 μl of cyclosporin A stock prior to adding the mitochondria to the cuvette. Final concentration of cyclosporin A is 1 μM.
13. Repeat steps 7 to 12 with all remaining compounds.
Data analysis 14. Export data from absorbance reader into Excel format. 15. Graph the data with absorbance on the y axis and time on the x axis. 16. Calculate the slope of the steepest portion of the line.
Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
17a. If using a dose-response curve: Graph the slopes of each dose to calculate an IC50 using a program like GraphPad Prism. Use DMSO as a 0% control and the positive control as the 100% control. 17b. If using a single dose: Use DMSO as a 0% control and the positive control as the 100% control and calculate the % induction of the compound as compared to the controls.
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ISOLATION OF RAT LIVER MITOCHONDRIA This protocol outlines the procedure for isolating mitochondria from rat liver (Lapidus and Sokolove, 1993). Other tissues may also be used, but optimization must be done based on the specific tissue being used. The liver has a large amount of mitochondria, providing for a larger prep than most other organs. If using other tissues, for example heart, then two or more rats may be needed to provide sufficient mitochondrial yield.
SUPPORT PROTOCOL 1
Materials Sprague-Dawley male rats (150 to 250 g; Charles River) or equivalent strain Rat food Isolation buffer I (see recipe), ice cold Isolation buffer II (see reagents and solutions) BCA protein assay kit (Pierce) Rat housing (21°C with 12-hr light/dark cycle capabilities) Sterile dissection tools including scissors 100-ml glass tissue homogenizer with Teflon pestle Power drill (hand-held or static) Refrigerated high-speed centrifuge Cheesecloth Additional reagents and equipment for euthanasia of rats (Donovan and Brown, 2006) Collect liver 1. Use Sprague-Dawley rats or equivalent strain. Provide care and maintenance in accordance with the principles described in the Guide for Care and Use of Laboratory Animals (National Institutes of Health, 2011) or equivalent. House rats in pairs in a controlled environment with constant temperature (21°C ±2°C) and a 12-hr light/dark cycle. Provide food and water ad libidum. For best results, the animals should be young and weigh between 150 and 250 g.
2. Euthanize animals with an overdose of carbon dioxide (Donovan and Brown, 2006). Avoid anesthetics, as they can have adverse effects on mitochondrial quality.
3. Excise organs rapidly and place them into ice-cold isolation buffer I (10 ml/g of liver). 4. Mince 5 to 7 g of liver very finely with scissors and wash several times in 20 ml isolation buffer I until the homogenate is blood free.
Homogenize tissue 5. Add 5 volumes isolation buffer I and homogenize the tissue using a smooth glass grinder with Teflon pestle driven by a power drill on low speed (six to eight passes). 6. Adjust the volume of the homogenate to 8 volumes with isolation buffer I and centrifuge 10 min at 700 × g, 4°C, to remove debris.
Isolate mitochondria 7. Filter homogenate/supernatant through two layers of cheesecloth and centrifuge 10 min at 14,000 × g, 4°C, to precipitate the mitochondrial fraction. 8. Discard the resultant supernatant and wash the mitochondrial pellet by resuspending in 20 ml isolation buffer I using a disposable transfer pipet and centrifuging 10 min at 10,000 × g, 4°C.
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9. Repeat washing step 8 using isolation buffer II. 10. Resuspend the mitochondria in 0.75 ml isolation buffer II and store on ice until use. 11. Determine protein concentration using a BCA kit according to the manufacturer’s instructions, in accordance with Support Protocol 3. Mitochondria should be stored on ice at a protein concentration >30 mg/ml, and used within 4 to 6 hr. SUPPORT PROTOCOL 2
MITOCHONDRIAL PROTEIN ASSAY This protocol describes the quantification of mitochondrial protein concentration.
Materials Triton X-100 2 mg/ml albumin stock solution (Pierce, cat. no. 23209) Isolated mitochondria (see Support Protocol 1) BCA protein assay kit (Pierce, cat. no. 23225) containing: Protein reagent A Protein reagent B 2-ml microcentrifuge tubes Standard clear-bottom 96-well plates 37°C heating block Absorbance plate reader Prepare 1% Triton X-100 1. To prepare a 1% (v/v) Triton X-100 solution, mix 1 ml of Triton X-100 stock solution with 99 ml water. Prepare protein standards 2. Using 1% Triton X-100 as diluent, prepare the following standard solutions: 1.5 mg/ml albumin in 1% Triton X-100 1.0 mg/ml albumin in 1% Triton X-100 0.75 mg/ml albumin in 1% Triton X-100 0.5 mg/ml albumin in 1% Triton X-100 0.25 mg/ml albumin in 1% Triton X-100 0.125 mg/ml albumin in 1% Triton X-100 0.025 mg/ml albumin in 1% Triton X-100 1% Triton X-100. Protein standards can be made in batches and stored for several weeks at 4°C. Albumin is provided as a 2 mg/ml stock solution.
Prepare mitochondrial dilutions 3. In 2-ml microcentrifuge tubes, prepare mitochondrial dilutions 1:60, 1:80, and 1:100 using 1% Triton X-100 as diluent:
Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
1:60—590 μl diluent and 10 μl mitochondrial prep 1:80—790 μl diluent and 10 μl mitochondrial prep 1:100—990 μl diluent and 10 μl mitochondrial prep. Construct standard curve and generate calculations 4. Pipet 20 μl of each standard and sample into a 96-well plate.
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5. Mix 9.8 ml protein reagent A with 200 μl protein reagent B. Solution will turn green (developing reagent).
6. Add 200 μl of developing reagent from step 5 to each sample or standard in wells. 7. Incubate the plate 30 min at 37°C and then read absorbance at 520 nm. 8. Calculate mitochondrial protein concentration based on comparison of sample values with standard curve. Dilute samples to the desired value, e.g., >30 mg/ml, and store on ice. Mitochondria should be stored on ice at a protein concentration >30 mg/ml and used within 4 to 6 hr.
OPTIMIZING PROTEIN CONCENTRATION FOR MITOCHONDRIAL SWELLING MEASUREMENT
SUPPORT PROTOCOL 3
This protocol describes how to optimize the mitochondrial protein concentration when setting up the assay.
Materials Sprague-Dawley male rats (150 to 250 g; Charles River) or equivalent strain BCA protein assay kit (Pierce) 1 M succinate stock solution (see recipe) 10 mM CaCl2 stock solution (see recipe) 10 mM rotenone stock solution (see recipe) 30 mM oligomycin stock solution (see recipe) 1 mM cyclosporin A stock solution (see recipe) 100 mM phosphate stock solution (see recipe) Mitochondrial swelling buffer (see recipe) Test compound stock: 30 mM troglitazone dissolved in DMSO Dimethylsulfoxide (DMSO) 30°C water bath Standard clear bottom 96-well plate 15-ml tubes, polystyrene, disposable (e.g., BD Falcon) Absorbance plate reader (capable of measuring absorbance at kinetic interval) Additional reagents and equipment for isolation of mitochondria from rat liver (Support Protocol 1) and mitochondrial protein assay (Support Protocol 2) Assay procedure 1. Isolate mitochondria from rat liver (see Support Protocol 1). Keep isolated mitochondria on ice at all times.
2. Determine the protein concentration of the isolated mitochondria using the BCA assay (see Support Protocol 2). 3. Thaw succinate stock solution, CaCl2 stock solution, rotenone, oligomycin, and cyclosporin A stock solutions on ice. 4. Warm mitochondrial swelling buffer to 30°C. 5. Prepare phosphate solution and adjust to the correct pH (see Reagents and Solutions). Phosphate solution must be made fresh for each experiment. Mitochondrial Toxicity
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Figure 25.4.2 Recommended plate map for optimization of protein concentration for mitochondrial swelling in isolated mitochondria. Concentrations are final concentrations. Blue wells are 1.5 mg/ml, purple wells are 1.0 mg/ml, pink wells are 0.5 mg/ml, and orange wells are 0.25 mg/ml.
6. Prepare dilutions of test compounds (e.g., troglitazone) in DMSO in a separate 96-well plate according to plate map (See Fig. 25.4.2 for plate map). The final DMSO content (in assay wells) should not exceed 0.5% (v/v).
7. Add 10 μl of rotenone stock, 3.3 μl of oligomycin stock, and 1 ml of succinate stock to 100 ml of warmed mitochondrial swelling buffer. This buffer will be used for the remainder of the experiment and referred to as swelling buffer mix. Final concentrations for the reagents are as follows: rotenone, 1 μM; oligomycin, 1 μM; and succinate, 10 mM.
8. Add 4 ml of swelling buffer mix to four 15-ml tubes. Each tube will be for a portion of the 96-well plate for optimizing protein concentration (see Fig. 25.4.2 for plate map).
9. To tube A, add 6 mg of mitochondria (1.5 mg/ml concentration) and dispense 200 μl into wells A1-A6, B1-B6, and C1-C6 (see Fig. 25.4.2 for plate map). 10. To tube B, add 4 mg of mitochondria (1.0 mg/ml concentration) and dispense 200 μl into wells A7-A12, B7-B12, and C7-C12 (see Fig. 25.4.2 for plate map). 11. To tube C, add 2 mg of mitochondria (0.5 mg/ml concentration) and dispense 200 μl into wells D1-D6, E1-E6, and F1-F6 (see Fig. 25.4.2 for plate map). 12. To tube D, add 1 mg of mitochondria (0.25 mg/ml concentration) and dispense 200 μl into wells D7-D12, E7-E12, and F7-F12 (see Fig. 25.4.2 for plate map). Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
13. Measure plate absorbance on plate reader for 2 min at 30-sec intervals to obtain a baseline absorbance reading. An appropriate protein concentration will have an absorbance in the range of 0.6 to 0.8. The authors use 1 mg/ml and have a starting absorbance at 0.8.
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14. Remove plate from plate reader and use a multichannel pipettor to dispense 1 μl of test compound from 96-well stock solution plate into each well. Add 6 μl of phosphate stock to appropriate control wells (See Fig. 25.4.2 for plate map). Addition of compounds or controls should be done as quickly as possible, because after addition of compounds swelling may begin to occur. Final concentration for phosphate is 3 mM.
15. Place the plate back into the plate reader and measure the absorbance kinetically over 20 min, with 30-sec intervals. An appropriate protein concentration will have at least a 0.2 shift with the phosphate and troglitazone controls. The window between the vehicle (DMSO) and positive control (phosphate or troglitazone) should be at least two-fold larger than the deviation from the replicates of the DMSO wells.
OPTIMIZING CaCl2 CONCENTRATION FOR THE MITOCHONDRIAL SWELLING ASSAY
SUPPORT PROTOCOL 4
This protocol describes how to optimize the CaCl2 conditions when setting up the mitochondrial swelling assay.
Materials Sprague-Dawley male rats (150 to 250 g; Charles River) or equivalent strain BCA protein assay kit (Pierce) 1 M succinate stock solution (see recipe) 10 mM CaCl2 stock solution (see recipe) 10 mM rotenone stock solution (see recipe) 30 mM oligomycin stock solution (see recipe) 1 mM cyclosporin A stock solution (see recipe) Mitochondrial swelling buffer (see recipe) 30°C water bath 15-ml tubes, polystyrene, disposable (BD Falcon) Standard clear bottom 96-well plate Absorbance plate reader (capable of measuring absorbance at kinetic interval) Assay procedure 1. Isolate mitochondria from rat liver (see Support Protocol 1). Keep isolated mitochondria on ice at all times.
2. Determine the protein concentration of the isolated mitochondria using the BCA assay (see Support Protocol 2). 3. Thaw succinate stock solution, CaCl2 stock solution, rotenone, oligomycin, and cyclosporin A stock solutions on ice. 4. Warm mitochondrial swelling buffer to 30°C. 5. Add 10 μl of rotenone stock, 3.3 μl of oligomycin stock, and 1 ml of succinate stock to 100 ml of warmed mitochondrial swelling buffer. This buffer will be used for the remainder of the experiment and referred to as swelling buffer mix.
6. Add 2 ml of swelling buffer mix to ten 15-ml tubes labeled A-J. Each tube will be for a portion of the 96-well plate for optimizing calcium concentration (see Fig. 25.4.3 for plate map).
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Figure 25.4.3 Recommended plate map for optimization of calcium concentration for mitochondrial swelling in isolated mitochondria. Wells in gray color are without cyclosporin A and wells in blue color are with cyclosporin A.
7. To tube A, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and dispense 200 μl into wells A1-A6 (see Fig. 25.4.3 for plate map). 8. To tube B, add 2 μl of cyclosporin A, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and dispense 200 μl into wells A7-A12 (see Fig. 25.4.3 for plate map). 9. To tube C, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 5 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells B1-B6 (see Fig. 25.4.3 for plate map). Final concentration of CaCl2 is 25 μM.
10. To tube D, add 2 μl of cyclosporin A, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 5 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells B7-B12 (see Fig. 25.4.3 for plate map). 11. To tube E, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 10 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells C1-C6 (see Fig. 25.4.3 for plate map). Final concentration of CaCl2 is 50 μM.
12. To tube F, add 2 μl of cyclosporin A, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 10 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells C7-C12 (see Fig. 25.4.3 for plate map). Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
13. To tube G, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 20 μl of 10 mM
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Figure 25.4.4 Calcium concentration curve for optimization of calcium concentration for setup of the mitochondrial swelling assay.
CaCl2 stock. Mix gently and dispense 200 μl into wells D1-D6 (see Fig. 25.4.3 for plate map). Final concentration of CaCl2 is 100 μM.
14. To tube H, add 2 μl of cyclosporin A, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 20 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells D7-D12 (see Fig. 25.4.3 for plate map). 15. To tube I, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 30 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells E1-E6 (see Fig. 25.4.3 for plate map). Final concentration of CaCl2 is 150 μM.
16. To tube J, add 2 μl of cyclosporin A, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 30 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells E7-E12 (see Fig. 25.4.3 for plate map). 17. Measure plate absorbance at 540 nm on plate reader kinetically over 20 min, with 30-sec intervals. Choose the most appropriate concentration of calcium to use in the mitochondrial swelling assay. An appropriate calcium concentration will not greatly affect the absorbance reading as compared to the mitochondria-only wells, but will still allow the positive controls to induce mitochondrial swelling (See Support Protocol 3). Too little calcium may not be enough for the positive controls or compounds to induce mitochondrial swelling. Too much calcium may already induce swelling, and it would be hard in this case to determine if the compound had an effect on mitochondrial swelling. Therefore having varying amounts of calcium should provide a calcium curve which will allow the user to choose an appropriate calcium concentration (see Fig. 25.4.4 for calcium curve example). The authors typically use 50 μM calcium in the 96-well format and 25 μM calcium in the cuvette format, but this is dependent upon mitochondrial prep quality, so the concentration needs to be determined for the specific user. Mitochondrial Toxicity
25.4.13 Current Protocols in Toxicology
Supplement 60
REAGENTS AND SOLUTIONS Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Calcium stock solution, 10 mM Prepare 10 mM CaCl2 in mitochondrial swelling buffer (see recipe) and adjust pH to 7.4 using HCl or KOH (do not use NaOH). Aliquot and store up to 6 months at −20°C. Cyclosporin A stock solution, 1 mM Prepare 1 mM cyclosporin A in DMSO. Aliquot and store up to 2 months at −20°C. Mitochondrial isolation buffer I 210 mM mannitol 70 mM sucrose 5 mM HEPES 1 mM EGTA 0.5% BSA (add on day of use) Adjust pH to 7.4 using HCl or KOH (do not use NaOH) Store without BSA up to 1 week at 4ºC Mitochondrial isolation buffer II 210 mM mannitol 70 mM sucrose 10 mM MgCl2 5 mM K2 HPO4 10 mM MOPS Adjust pH to 7.4 using HCl or KOH (do not use NaOH) Store up to 1 week at 4ºC Mitochondrial swelling buffer 213 mM mannitol 70 mM sucrose 3 mM HEPES Adjust pH to 7.4 using HCl or KOH (do not use NaOH) Store up to 4 weeks at 4ºC Oligomycin stock solution, 30 mM Prepare 30 mM oligomycin in DMSO. Aliquot and store up to 2 months at −20°C. Phosphate stock solution, 100 mM Prepare 100 mM K2 HPO4 in water and adjust pH to 7.4 using HCl or KOH (do not use NaOH). Prepare fresh on the day of the experiment. Rotenone stock solution, 10 mM Prepare 10 mM rotenone (Sigma, cat. no. R8875) in DMSO. Aliquot and store up to 2 months at −20°C.
Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
Succinate stock solution, 1 M Prepare 1 M sodium succinate dibasic hexahydrate (Sigma, cat. no. S2378) in mitochondrial swelling buffer (see recipe) and adjust pH to 7.4 using HCl or KOH (do not use NaOH). Aliquot and store up to 6 months at −20°C.
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COMMENTARY Background Information Drug-induced mitochondrial toxicity can occur through several mechanisms, including opening of the mitochondrial permeability transition pore (MPTP). Many drugs that have been withdrawn from the market or that carry black box warnings have been shown to have mitochondrial impairment (Dykens and Will, 2007). Therefore, to reduce drug attrition, predictive screens should be positioned early in the drug-discovery process so results can guide chemistry and biology. Here we describe an assay using isolated rat liver mitochondria that can detect Ca2+ -dependent druginduced opening of the MPTP, in both 96-well and cuvette formats. Compounds or excess Ca2+ can enhance opening of the MPTP, which leads to loss of mitochondrial membrane potential, swelling of the matrix because of water accumulation, rupture of the outer mitochondrial membrane, release of intermembrane space proteins into the cytosol, and, ultimately, cell death. The method provided here provides one way to measure induction of the MPTP in isolated rat liver mitochondria by following mitochondrial swelling. This can be measured by monitoring light scattering, which inversely correlates to mitochondrial volume. Therefore, as the mitochondria begin to swell, then a decrease in absorbance will be observed. To determine if the drug-induced swelling was due to opening of the MPTP and not an unregulated pore, cyclosporin A (CsA) is added to block MPTP opening. Another way to measure induction of the MPTP is to track the ability of mitochondria to take up and retain Ca2+ (Ichas et al., 1994). Extramitochondrial Ca2+ levels can be measured with a fluorescence Ca2+ indicator dye such as Calcium green–5N tetrapotassium salt solution (Molecular Probes). With each addition of Ca2+ , the fluorescence increases; however, as the mitochondria take up the added Ca2+ , the fluorescence decreases back to baseline. This will continue until the mitochondria have taken up enough Ca2+ for the induction of MPTP to occur, which then leads to a rapid increases in fluorescence. A combination of the two protocols would allow for more confidence in drug-induced opening of the MPTP. Data analysis here has been demonstrated using slopes of the kinetic reads. It should be noted that there are other ways for the calculations to be completed. These include area under the curve and change in initial and end absorbance. Current Protocols in Toxicology
Critical Parameters and Troubleshooting Phosphate solution not showing a decrease in absorbance or swelling of mitochondria Check to make sure that the phosphate solution has been made fresh on that day. The solution cannot be made ahead of time or frozen to be used at a later time. All buffers must made up with pure Millipore water Assay and isolation buffers should be made with water that is free from Ca2+ . This is critical so that the mitochondria will not swell before assay is run. CsA must be added to assay solution before Ca2+ or drug addition The order of the addition of the mitochondria, Ca2+ , and CsA is critical to the experimental conditions. The order of addition should be: CsA (if using), mitochondria, then Ca2+ . This is because CsA must first be allowed to bind to cyclophilin D and block the MPTP before Ca2+ or drug induces opening of pore. CsA is not blocking induction of MPTP Some compounds will show a decrease in absorbance, but CsA will not block this decrease. This might be due to the fact that some compounds will induce unregulated pores to open that are not activated by Ca2+ and/or are insensitive to CsA (He and Lemasters, 2002). Compounds block MPTP instead of inducing it Some compounds will behave like CsA and block induction of MPTP instead of inducing it. This might also be concentration dependent. For instance, at low concentrations, the compound might induce MPTP, but at higher concentrations they might block it. Compound interferes with absorbance signal It is recommended before testing to check if the compound interferes with the absorbance readings, and correct for these changes if possible during analysis. Mitochondria are no longer functional Isolated mitochondria are very sensitive and have a limited functional time in which they can be used. The mitochondrial preparation should be used immediately after isolation, for up to 4 to 6 hr, during which they must remain on ice at all times. Mitochondria
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25.4.15 Supplement 60
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Figure 25.4.5 Raw absorbance data for vehicle (DMSO) and positive controls (troglitazone and phosphate). For the data analysis, the slope should be calculated for the maximal slope with a minimum time period of at least 5 min. For these compounds, the slope was calculated for the time period from 5 to 12 min.
remain functional for a longer time if they are maintained at a concentration of >30 mg/ml. Optimization of assay conditions is needed when using different tissues or species Assay conditions such as protein and Ca2+ concentrations may differ among mitochondria isolated from different tissues and also mitochondria from different species. Previous studies have shown that rat brain mitochondria require considerably more Ca2+ to induce MPTP opening than liver mitochondria, and that species differences may play a role as well (Panov et al., 2006). In addition, heart mitochondria are not only more resistant to Ca2+ load than liver mitochondria, but neonatal rat heart mitochondria are more resistant to Ca2+ load than adult rat mitochondria (Drahota et al., 2012). Therefore, in these protocols, assay conditions are only optimized for isolated rat liver mitochondria and not for other tissues or species.
Anticipated Results
Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
Raw absorbance data for vehicle (DMSO) and positive controls (troglitazone and phosphate) are shown in Figure 25.4.5. Results from this assay can giving insight into the ability of the compound to induce Ca2+ -dependent MPTP opening in rat liver mitochondria. Therefore, seeing a decrease in absorbance due to mitochondrial swelling after Ca2+ and drug addition suggests that the drug decreases the threshold amount of Ca2+ needed to induce opening of the MPTP or works synergistically with Ca2+ to induce it. Blockage of this induction, or no decrease in absorbance by adding CsA before drug and
Ca2+ addition, ensures that the swelling is caused by the MPTP and not due to another pore opening.
Time Considerations Basic Protocol 1 r Buffer preparation (up to 1 week prior to experiment): 1 to 2 hr r Compound preparation (Day prior to experiment): 1 to 2 hr r Mitochondrial isolation (Day 1): 2 to 3 hr r Compound testing (Day 1): 20 min per plate (total time will depend on how many compounds are being tested) Basic Protocol 2 r Buffer preparation (up to 1 week prior to experiment): 1 to 2 hr r Compound preparation (Day prior to experiment): 1 to 2 hr r Mitochondrial isolation (Day 1): 2 to 3 hr r Compound testing (Day 1): 20 min per cuvette (total time will depend on how many compounds are being tested)
Literature Cited Beavis, A.D., Brannan, R.D., and Garlid, K.D. 1985. Swelling and contraction of the mitochondrial matrix. I. A structural interpretation of the relationship between light scattering and matrix volume. J. Biol. Chem. 260:13424-13433. Berson, A., Cazanave, S., Descatoire, V., Tinel, M., Grodet, A., Wolf, C., Feldmann, G., and Pessayre, D. 2006. The anti-inflammatory drug, nimesulide (4-nitro-2-phenoxymethanesulfoanilide), uncouples mitochondria and induces mitochondrial permeability transition in human hepatoma cells: Protection by albumin. J. Pharmacol. Exp. Ther. 318:444-454.
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Crompton, M., Ellinger, H., and Costi, A. 1988. Inhibition by cyclosporin A of a Ca2+ -dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem. J. 255:357-360. Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1-1.8.4. Drahota, Z., Milerovia, M., Endlicher, R., Rychtromoc, D., Cervinkova, Z., and Ostadal, B. 2012. Developmental changes of the sensitivity of cardiac and liver mitochondrial permeability transition pore to calcium load and oxidative stress. Physiol. Res. 61:S165-S172. Dykens, J.A. and Will, Y. 2007. The significance of mitochondrial testing in drug development. Drug Discov. Today. 12:777785. Halestrap, A.P. and Brenner, C. 2003. The adenine nucleotide translocase: A central component of the mitochondrial permeability transition pore and key player in cell death. Curr. Med. Chem. 10:1507-1525. Haworth, R.A. and Hunter, D.R. 1979. The Ca2+ induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site. Arch. Biochem. Biophys. 195:460-467. He, L. and Lemasters, J.J. 2002. Regulated and unregulated mitochondrial permeability transition pores: A new paradigm of pore structure and function? FEBS Lett. 512:1-7. Hunter, D.R. and Haworth, R.A. 1979. The Ca2+ induced membrane transition in mitochondria. I. The protective mechanisms. Arch. Biochem. Biophys. 195:453-459. Ichas, F., Jouaville, L.S., Sidash, S.S., Mazat, J.P., and Holmuhamedov, E.L. 1994. Mitochondrial calcium spiking: A transduction mechanism based on calcium-induced permeability transition involved in cell calcium signalling. FEBS Lett. 348:211-215. Kim, J.S., He, L., and Lemasters, J.J. 2003. Mitochondrial permeability transition: A common
pathway to necrosis and apoptosis. Biochem. Biophys. Res. Commun. 304:463-470. Lapidus, R.G. and Sokolove, P.M. 1993. Spermine inhibition of the permeability transition of isolated rat liver mitochondria: An investigation of mechanism. Arch. Biochem. Biophys. 306:246253. Masubuchi, Y., Nakayama, S., and Horie, T. 2002. Role of mitochondrial permeability transition in diclofenac-induced hepatocyte injury in rats. Hepatology 35:544-551. Masubuchi, Y., Kano, S., and Horie, T. 2006. Mitochondrial permeability transition as a potential determinant of hepatotoxicity of antidiabetic thiazolidinediones. Toxicology 222:233239. National Institutes of Health. 2011. Guide for the Care and Use of Laboratory Animals, 8th ed. http://grants.nih.gov/grants/olaw/Guide-for-thecare-and-use-of-laboratory-animals.pdf. Okuda, T., Norioka, M., Shitara, Y., and Horie, T. 2010. Multiple mechanisms underlying troglitazone-induced mitochondrial permeability transition. Toxicol. Appl. Pharmacol. 248:242-248. Panov, A., Dikalov, S., Shalbuyeva, N., Hemendinger, R., Greenamyre, J.T., and Rosenfeld, J. 2006. Species- and tissue-specific relationships between mitochondrial permeability transition and generation of ROS in brain and liver mitochondria of rats and mice. Am. J. Physiol. Cell Physiol. 292:C708-718. Tanveer, A., Virji, S., Andreeva, L., Totty, N.F., Hsuan, J.J., Ward, J.M., and Crompton, M. 1996. Involvement of cyclophilin D in the activation of a mitochondrial pore by Ca2+ and oxidant stress. Eur. J. Biochem. 238:166-172. Tay, V.K., Wang, A.S., Leow, K.Y., Ong, M.M., Wong, K.P., and Boelsterli, U.A. 2005. Mitochondrial permeability transition as a source of superoxide anion induced by the nitroaromatic drug nimesulide in vitro. Free Radic. Biol. Med. 39:949-959.
Mitochondrial Toxicity
25.4.17 Current Protocols in Toxicology
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UNIT 25.4
Lisa Marroquin,1 Rachel Swiss,1 and Yvonne Will1 1
Compound Safety Prediction, Worldwide Medicinal Chemistry, Pfizer Inc., Groton, Connecticut
ABSTRACT The mitochondrial permeability transition pore (MPTP) is a protein pore that forms in the inner mitochondrial membrane and allows the membrane to be permeable to all molecules of less than 1500 Da. Ca2+ , numerous reactive chemicals, and oxidative stress induce MPTP opening, whereas cyclosporin A (CsA) or bongkrekic acid block it. In addition, several drugs have been shown to induce MPTP opening, leading to the loss of mitochondrial membrane potential, swelling of the matrix because of water accumulation, rupture of the outer mitochondrial membrane, and release of intermembrane space proteins into the cytosol. This ultimately leads to the rupture of the outer mitochondrial membrane and cell demise. Here, we describe an assay using isolated rat liver mitochondria that can detect Ca2+ -dependent drug-induced opening of the MPTP, providing protocols for screening in both cuvette and 96-well format. Curr. Protoc. Toxicol. 60:25.4.1:-25.4.17. C 2014 by John Wiley & Sons, Inc. Keywords: mitochondria permeability transition pore r mitochondrial toxicity r mitochondrial swelling
INTRODUCTION Due to the complexity of mitochondria, it is not surprising that adverse drug events often develop due to drug-induced mitochondrial injury. Drug-induced mitochondrial toxicity can occur through several mechanisms, including induction of the mitochondrial permeability transition pore (MPTP), and this could be a reason for organ-related toxicity. For example, hepatic or gastric toxicity by non-steroidal anti-inflammatory drugs (NSAIDS), such as diclofenac and nimesulide, has been postulated to be due at least in part to their ability to induce MPTP opening, with nimesulide being withdrawn from the market due to severe hepatotoxicity (Masubuchi et al., 2002; Tay et al., 2005; Berson et al., 2006). In addition, troglitazone, a diabetic drug that was withdrawn from the market due to idiosyncratic liver toxicity, also induces MPTP opening, and this may contribute to its hepatotoxicity (Masubuchi et al., 2006; Okuda et al., 2010). Described by Hunter and Haworth (1979), the MPTP is a protein pore that forms in the inner mitochondrial membrane and allows membrane permeability to all molecules of less than 1500 Da. The molecular identity of the pore is under debate, but proteins such as cyclophilin D, voltage-dependent anion channel, and adenine nucleotide translocator (ANT) are thought to contribute to MPTP formation. The MPTP is promoted by the accumulation of excessive Ca2+ and stimulated by various compounds and conditions such as oxidative stress, elevated phosphate levels, and adenine nucleotide depletion. The induction of the MPTP can lead to dissipation of membrane potential, uncoupling of oxidative phosphorylation, loss of preaccumulated Ca2+ , expansion of the matrix volume, Mitochondrial Toxicity Current Protocols in Toxicology 25.4.1-25.4.17, May 2014 Published online May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471140856.tx2504s60 C 2014 John Wiley & Sons, Inc. Copyright
25.4.1 Supplement 60
release of cytochrome c to the cytosol, and ultimately death of the cell. Cytochrome c release can trigger a cascade of events that lead to either apoptosis or necrosis (Kim et al., 2003). Induction of the MPTP can be blocked by cyclosporin A (CsA), which binds to cyclophilin D (Crompton et al., 1988; Tanveer et al., 1996), or bongkrekic acid, a ligand of ANT (Halestrap and Brenner, 2003). A common way to measure MPTP opening in isolated mitochondria is by following mitochondrial swelling (Hunter and Haworth, 1979). Mitochondrial swelling can be measured by monitoring changes in light scattering, which inversely correlates with mitochondrial volume (Beavis et al., 1985). The changes in light scattering can be recorded by following the decrease in absorbance at 540 nM after the addition of Ca2+ and drug. To determine if the decrease in absorbance is due to opening of the MPTP, CsA is added to block the opening. The following protocol outlines how to determine Ca2+ -dependent drug-induced opening of the MPTP in isolated rat liver mitochondria by following mitochondrial swelling. The protocol is divided into two parts, including measuring mitochondrial swelling in both cuvette and 96-well formats (Basic Protocols 1 and 2), and assay considerations (Support Protocols 1 to 4). NOTE: Protocols employing live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to government regulations regarding the care and use of laboratory animals. BASIC PROTOCOL 1
ASSESSMENT OF MITOCHONDRIAL SWELLING OF COMPOUNDS IN A 96-WELL FORMAT This protocol outlines the procedure for testing a compound’s effect on mitochondrial swelling in a 96-well plate format. The 96-well format is an efficient way to test a higher number of compounds in a screening paradigm. This protocol is designed for testing rat liver mitochondria and is not optimized for testing other tissues or species.
Materials Sprague-Dawley male rats (150 to 250 g; Charles River) or equivalent strain BCA protein assay kit (Pierce) 1 M succinate stock solution (see recipe) 10 mM CaCl2 stock solution (see recipe) 10 mM rotenone stock solution (see recipe) 30 mM oligomycin stock solution (see recipe) 1 mM cyclosporin A stock solution (see recipe) 100 mM phosphate stock solution (see recipe) Compound stocks dissolved in DMSO, for testing in assay (concentration will depend on highest dose tested) Dimethylsulfoxide (DMSO) Mitochondrial swelling buffer (see recipe)
Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
15-ml tubes, polystyrene, disposable (e.g., BD Falcon) 30°C water bath Multichannel pipettors Standard clear-bottom 96-well plate Absorbance plate reader (capable of measuring absorbance at kinetic interval) Computer running Microsoft Excel Program capable of calculating IC50 (e.g., GraphPad Prism)
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Figure 25.4.1 Plate map for compound screening for mitochondrial swelling in isolated mitochondria. Concentrations are in nmoles/mg and are final concentrations. Vehicle is DMSO and positive control is phosphate. Compounds are in duplicate for testing with and without cyclosporin A. Wells in gray color are tested without cyclosporin A and wells in blue color are tested with cyclosporin A.
Additional reagents and equipment for isolation of mitochondria from rat liver (Support Protocol 1), mitochondrial protein assay (Support Protocol 2), optimizing mitochondrial protein concentration (Support Protocol 3), and optimizing CaCl2 concentration (Support Protocol 4) Assay procedure 1. Isolate mitochondria from rat liver (see Support Protocol 1). 2. Keep isolated mitochondria on ice at all times. 3. Determine the protein concentration of the isolated mitochondria using the BCA assay (see Support Protocol 2). 4. Thaw succinate stock solution, CaCl2 stock solution, rotenone, oligomycin, and cyclosporin A stock solutions on ice. 5. Warm mitochondrial swelling buffer to 30°C. 6. Prepare phosphate stock solution and adjust to the correct pH (see Reagents and Solutions). Phosphate stock solution must be made fresh for each experiment.
7. Prepare dilutions of compounds in DMSO in a separate 96-well plate according to the plate map (See Fig. 25.4.1 for plate map). Different dilutions need to be prepared depending on the chosen mitochondrial test concentration. The final DMSO content (in assay wells) should not exceed 0.5% v/v. The authors typically test compounds starting at a high dose of 100 nmol/mg final concentration. The dose-response curve is six points, with a 1:2 serial dilution
Mitochondrial Toxicity
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8. Add 10 μl of rotenone stock, 3.3 μl of oligomycin stock, and 1 ml of succinate stock to 100 ml of warmed mitochondrial swelling buffer. This buffer will be used for the remainder of the experiment and referred to as swelling buffer mix. Final concentrations for the reagents are as follows: rotenone, 1 μM; oligomycin, 1 μM; and succinate, 10 mM.
9. Add 10 ml of swelling buffer mix to two 15-ml tubes—one tube will be for half of the 96-well plate for compound testing without cyclosporin A (tube A), and one will be for the remaining half of the plate for compound testing with cyclosporin A (tube B). 10. To tube A, add appropriate quantity of mitochondria (for guidelines on optimizing protein concentration, see Support Protocol 3) and 50 μl CaCl2 stock solution (actual amount will vary depending on the isolated mitochondria prep; for tips on optimizing CaCl2 concentration, see Support Protocol 4). Authors use 1 mg/ml of mitochondria and 50 μM of CaCl2 , but both concentrations are dependent on the quality of the mitochondrial isolation and will be user specific. Therefore, optimization of both of these parameters is recommended. Also, the order of the addition of these reagents is important. Reagents must be added in the specific order described.
11. To tube B, add 1 μM of cyclosporin A (10 μl of stock solution), the appropriate quantity of mitochondria (for guidelines on optimizing protein concentration, see Support Protocol 3), and 50 μl CaCl2 stock solution (actual amount will vary depending on the isolated mitochondria prep; for tips on optimizing CaCl2 concentration, see Support Protocol 4). 12. Use a multichannel pipettor to dispense 200 μl from tube A to each well on the left side of the 96-well plate (see Fig. 25.4.1 for plate map). 13. Use multichannel pipettor to dispense 200 μl from tube B to each well on the right side of the 96-well plate (see Fig. 25.4.1 for plate map). 14. Measure plate absorbance at 540 nm on a plate reader for 2 min at 30-sec intervals to obtain a baseline absorbance reading. 15. Remove plate from plate reader and use a multichannel pipettor to dispense 1 μl of test compound from the 96-well compound stock solution plate (see step 7) into each well. Add 6 μl of phosphate stock to appropriate control wells (see Fig. 25.4.1 for plate map). Addition of compounds or controls should be done as quickly as possible because, after addition of compounds, swelling may begin to occur. Phosphate is at a final concentration of 3 mM. The compound concentration range can be defined by the user, and drug concentrations are usually expressed in nmol/mg mitochondrial protein.
16. Place the plate back into the plate reader and measure the absorbance at 540 nm kinetically over 20 min, with 30-sec intervals.
Data analysis 17. Export data from absorbance reader into Excel format. Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
18. Graph the data with absorbance on the y axis and time on the x axis. 19. Calculate the slope of the steepest portion of the line.
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20a. If using a dose-response curve: Graph the slopes of each dose to calculate an IC50 using a program like GraphPad Prism. Use DMSO as a 0% control and the positive control as the 100% control. 20b. If using a single dose: Use the DMSO as a 0% control and the positive control as the 100% control, and calculate the % induction of the compound as compared to the controls.
ASSESSMENT OF MITOCHONDRIAL SWELLING OF COMPOUNDS IN A CUVETTE FORMAT
BASIC PROTOCOL 2
This protocol outlines the procedure for testing a compound’s effect on mitochondrial swelling in a cuvette format. The cuvette format is a useful way to investigate a small number of compounds at a single dose or to investigate a single compound in a thorough manner. This protocol is designed for testing rat liver mitochondria and not optimized for testing other tissues or species.
Materials Sprague-Dawley male rats (150 to 250 g; Charles River) or equivalent strain BCA protein assay kit (Pierce) 1 M succinate stock solution (see recipe) 10 mM CaCl2 stock solution (see recipe) 10 mM rotenone stock solution (see recipe) 30 mM oligomycin stock solution (see recipe) 1 mM cyclosporin A stock solution (see recipe) 100 mM phosphate stock solution (see recipe) Compound stocks dissolved in DMSO, for testing in assay (concentration will depend on highest dose tested) Mitochondrial swelling buffer (see recipe) Compound stocks dissolved in DMSO, for testing in assay (concentration will depend on highest dose tested) 30°C water bath Standard cuvettes for absorbance Absorbance cuvette reader (spectrophotometer capable of measuring absorbance at kinetic intervals) Computer running Microsoft Excel Program capable of calculating IC50 (e.g., GraphPad Prism) Additional reagents and equipment for isolation of mitochondria from rat liver (Support Protocol 1), mitochondrial protein assay (Support Protocol 2), optimizing mitochondrial protein concentration (Support Protocol 3), and optimizing CaCl2 concentration (Support Protocol 4) Assay procedure 1. Isolate mitochondria from rat liver (see Support Protocol 1). Keep isolated mitochondria on ice at all times.
2. Determine the protein concentration of the isolated mitochondria using the BCA assay (see Support Protocol 2). 3. Thaw succinate stock solution, CaCl2 stock solution, rotenone, oligomycin, and cyclosporin A stock solutions on ice. 4. Warm mitochondrial swelling buffer to 30°C.
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5. Prepare phosphate solution and adjust to the correct pH (see Reagents and Solutions). Phosphate solution must be made fresh for each experiment.
6. Add 10 μl of rotenone stock, 3.3 μl of oligomycin stock, and 1 ml of succinate stock to 100 ml of warmed mitochondrial swelling buffer. This buffer will be used for the remainder of the experiment and referred to as swelling buffer mix. Final concentrations for the reagents are as follows: rotenone, 1 μM; oligomycin, 1 μM; and succinate, 10 mM.
7. Add 2 ml of swelling buffer mix to a standard cuvette. 8. To the cuvette, add appropriate quantity of mitochondria (for guidelines on optimizing protein concentration, see Support Protocol 3) and mix by gently pipetting up and down. Then, add 5 μl CaCl2 stock solution (actual amount will vary depending on the isolated mitochondria prep; for tips on optimizing CaCl2 concentration, see Support Protocol 4) and mix gently by pipetting up and down. The authors use 0.25 mg/ml of mitochondria and 25 μM of CaCl2 , but both concentrations are dependent on the quality of the mitochondrial prep and will be user specific. Therefore, optimization of both of these parameters is recommended. Also, the order of the addition of these reagents is important. Reagents must be added in this specific order. The protein concentrations and calcium concentrations need to be optimized separately from the 96-well format; however, the volumes are ten-fold higher. Be sure to mix by gently pipetting up and down after compound addition, unless the spectrophotometer has a stirrer feature available.
9. Measure cuvette absorbance at 540 nm for 2 min at 30-sec intervals to obtain a baseline absorbance reading. 10. Remove cuvette from absorbance reader and add 10 μl of test compound. Mix gently by pipetting up and down. In the cuvette format, only one concentration of one compound can be tested at a time. Separate cuvettes need to be set up to measure each condition. The authors typically test compounds at a high dose of 100 nmol/mg final concentration.
11. Place the cuvette back into the absorbance reader and measure the absorbance at 540 nm kinetically over 20 min, with 30-sec intervals. 12. Repeat steps 7 to 11 with the same compound or DMSO, but first add 2 μl of cyclosporin A stock prior to adding the mitochondria to the cuvette. Final concentration of cyclosporin A is 1 μM.
13. Repeat steps 7 to 12 with all remaining compounds.
Data analysis 14. Export data from absorbance reader into Excel format. 15. Graph the data with absorbance on the y axis and time on the x axis. 16. Calculate the slope of the steepest portion of the line.
Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
17a. If using a dose-response curve: Graph the slopes of each dose to calculate an IC50 using a program like GraphPad Prism. Use DMSO as a 0% control and the positive control as the 100% control. 17b. If using a single dose: Use DMSO as a 0% control and the positive control as the 100% control and calculate the % induction of the compound as compared to the controls.
25.4.6 Supplement 60
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ISOLATION OF RAT LIVER MITOCHONDRIA This protocol outlines the procedure for isolating mitochondria from rat liver (Lapidus and Sokolove, 1993). Other tissues may also be used, but optimization must be done based on the specific tissue being used. The liver has a large amount of mitochondria, providing for a larger prep than most other organs. If using other tissues, for example heart, then two or more rats may be needed to provide sufficient mitochondrial yield.
SUPPORT PROTOCOL 1
Materials Sprague-Dawley male rats (150 to 250 g; Charles River) or equivalent strain Rat food Isolation buffer I (see recipe), ice cold Isolation buffer II (see reagents and solutions) BCA protein assay kit (Pierce) Rat housing (21°C with 12-hr light/dark cycle capabilities) Sterile dissection tools including scissors 100-ml glass tissue homogenizer with Teflon pestle Power drill (hand-held or static) Refrigerated high-speed centrifuge Cheesecloth Additional reagents and equipment for euthanasia of rats (Donovan and Brown, 2006) Collect liver 1. Use Sprague-Dawley rats or equivalent strain. Provide care and maintenance in accordance with the principles described in the Guide for Care and Use of Laboratory Animals (National Institutes of Health, 2011) or equivalent. House rats in pairs in a controlled environment with constant temperature (21°C ±2°C) and a 12-hr light/dark cycle. Provide food and water ad libidum. For best results, the animals should be young and weigh between 150 and 250 g.
2. Euthanize animals with an overdose of carbon dioxide (Donovan and Brown, 2006). Avoid anesthetics, as they can have adverse effects on mitochondrial quality.
3. Excise organs rapidly and place them into ice-cold isolation buffer I (10 ml/g of liver). 4. Mince 5 to 7 g of liver very finely with scissors and wash several times in 20 ml isolation buffer I until the homogenate is blood free.
Homogenize tissue 5. Add 5 volumes isolation buffer I and homogenize the tissue using a smooth glass grinder with Teflon pestle driven by a power drill on low speed (six to eight passes). 6. Adjust the volume of the homogenate to 8 volumes with isolation buffer I and centrifuge 10 min at 700 × g, 4°C, to remove debris.
Isolate mitochondria 7. Filter homogenate/supernatant through two layers of cheesecloth and centrifuge 10 min at 14,000 × g, 4°C, to precipitate the mitochondrial fraction. 8. Discard the resultant supernatant and wash the mitochondrial pellet by resuspending in 20 ml isolation buffer I using a disposable transfer pipet and centrifuging 10 min at 10,000 × g, 4°C.
Mitochondrial Toxicity
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9. Repeat washing step 8 using isolation buffer II. 10. Resuspend the mitochondria in 0.75 ml isolation buffer II and store on ice until use. 11. Determine protein concentration using a BCA kit according to the manufacturer’s instructions, in accordance with Support Protocol 3. Mitochondria should be stored on ice at a protein concentration >30 mg/ml, and used within 4 to 6 hr. SUPPORT PROTOCOL 2
MITOCHONDRIAL PROTEIN ASSAY This protocol describes the quantification of mitochondrial protein concentration.
Materials Triton X-100 2 mg/ml albumin stock solution (Pierce, cat. no. 23209) Isolated mitochondria (see Support Protocol 1) BCA protein assay kit (Pierce, cat. no. 23225) containing: Protein reagent A Protein reagent B 2-ml microcentrifuge tubes Standard clear-bottom 96-well plates 37°C heating block Absorbance plate reader Prepare 1% Triton X-100 1. To prepare a 1% (v/v) Triton X-100 solution, mix 1 ml of Triton X-100 stock solution with 99 ml water. Prepare protein standards 2. Using 1% Triton X-100 as diluent, prepare the following standard solutions: 1.5 mg/ml albumin in 1% Triton X-100 1.0 mg/ml albumin in 1% Triton X-100 0.75 mg/ml albumin in 1% Triton X-100 0.5 mg/ml albumin in 1% Triton X-100 0.25 mg/ml albumin in 1% Triton X-100 0.125 mg/ml albumin in 1% Triton X-100 0.025 mg/ml albumin in 1% Triton X-100 1% Triton X-100. Protein standards can be made in batches and stored for several weeks at 4°C. Albumin is provided as a 2 mg/ml stock solution.
Prepare mitochondrial dilutions 3. In 2-ml microcentrifuge tubes, prepare mitochondrial dilutions 1:60, 1:80, and 1:100 using 1% Triton X-100 as diluent:
Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
1:60—590 μl diluent and 10 μl mitochondrial prep 1:80—790 μl diluent and 10 μl mitochondrial prep 1:100—990 μl diluent and 10 μl mitochondrial prep. Construct standard curve and generate calculations 4. Pipet 20 μl of each standard and sample into a 96-well plate.
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5. Mix 9.8 ml protein reagent A with 200 μl protein reagent B. Solution will turn green (developing reagent).
6. Add 200 μl of developing reagent from step 5 to each sample or standard in wells. 7. Incubate the plate 30 min at 37°C and then read absorbance at 520 nm. 8. Calculate mitochondrial protein concentration based on comparison of sample values with standard curve. Dilute samples to the desired value, e.g., >30 mg/ml, and store on ice. Mitochondria should be stored on ice at a protein concentration >30 mg/ml and used within 4 to 6 hr.
OPTIMIZING PROTEIN CONCENTRATION FOR MITOCHONDRIAL SWELLING MEASUREMENT
SUPPORT PROTOCOL 3
This protocol describes how to optimize the mitochondrial protein concentration when setting up the assay.
Materials Sprague-Dawley male rats (150 to 250 g; Charles River) or equivalent strain BCA protein assay kit (Pierce) 1 M succinate stock solution (see recipe) 10 mM CaCl2 stock solution (see recipe) 10 mM rotenone stock solution (see recipe) 30 mM oligomycin stock solution (see recipe) 1 mM cyclosporin A stock solution (see recipe) 100 mM phosphate stock solution (see recipe) Mitochondrial swelling buffer (see recipe) Test compound stock: 30 mM troglitazone dissolved in DMSO Dimethylsulfoxide (DMSO) 30°C water bath Standard clear bottom 96-well plate 15-ml tubes, polystyrene, disposable (e.g., BD Falcon) Absorbance plate reader (capable of measuring absorbance at kinetic interval) Additional reagents and equipment for isolation of mitochondria from rat liver (Support Protocol 1) and mitochondrial protein assay (Support Protocol 2) Assay procedure 1. Isolate mitochondria from rat liver (see Support Protocol 1). Keep isolated mitochondria on ice at all times.
2. Determine the protein concentration of the isolated mitochondria using the BCA assay (see Support Protocol 2). 3. Thaw succinate stock solution, CaCl2 stock solution, rotenone, oligomycin, and cyclosporin A stock solutions on ice. 4. Warm mitochondrial swelling buffer to 30°C. 5. Prepare phosphate solution and adjust to the correct pH (see Reagents and Solutions). Phosphate solution must be made fresh for each experiment. Mitochondrial Toxicity
25.4.9 Current Protocols in Toxicology
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Figure 25.4.2 Recommended plate map for optimization of protein concentration for mitochondrial swelling in isolated mitochondria. Concentrations are final concentrations. Blue wells are 1.5 mg/ml, purple wells are 1.0 mg/ml, pink wells are 0.5 mg/ml, and orange wells are 0.25 mg/ml.
6. Prepare dilutions of test compounds (e.g., troglitazone) in DMSO in a separate 96-well plate according to plate map (See Fig. 25.4.2 for plate map). The final DMSO content (in assay wells) should not exceed 0.5% (v/v).
7. Add 10 μl of rotenone stock, 3.3 μl of oligomycin stock, and 1 ml of succinate stock to 100 ml of warmed mitochondrial swelling buffer. This buffer will be used for the remainder of the experiment and referred to as swelling buffer mix. Final concentrations for the reagents are as follows: rotenone, 1 μM; oligomycin, 1 μM; and succinate, 10 mM.
8. Add 4 ml of swelling buffer mix to four 15-ml tubes. Each tube will be for a portion of the 96-well plate for optimizing protein concentration (see Fig. 25.4.2 for plate map).
9. To tube A, add 6 mg of mitochondria (1.5 mg/ml concentration) and dispense 200 μl into wells A1-A6, B1-B6, and C1-C6 (see Fig. 25.4.2 for plate map). 10. To tube B, add 4 mg of mitochondria (1.0 mg/ml concentration) and dispense 200 μl into wells A7-A12, B7-B12, and C7-C12 (see Fig. 25.4.2 for plate map). 11. To tube C, add 2 mg of mitochondria (0.5 mg/ml concentration) and dispense 200 μl into wells D1-D6, E1-E6, and F1-F6 (see Fig. 25.4.2 for plate map). 12. To tube D, add 1 mg of mitochondria (0.25 mg/ml concentration) and dispense 200 μl into wells D7-D12, E7-E12, and F7-F12 (see Fig. 25.4.2 for plate map). Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
13. Measure plate absorbance on plate reader for 2 min at 30-sec intervals to obtain a baseline absorbance reading. An appropriate protein concentration will have an absorbance in the range of 0.6 to 0.8. The authors use 1 mg/ml and have a starting absorbance at 0.8.
25.4.10 Supplement 60
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14. Remove plate from plate reader and use a multichannel pipettor to dispense 1 μl of test compound from 96-well stock solution plate into each well. Add 6 μl of phosphate stock to appropriate control wells (See Fig. 25.4.2 for plate map). Addition of compounds or controls should be done as quickly as possible, because after addition of compounds swelling may begin to occur. Final concentration for phosphate is 3 mM.
15. Place the plate back into the plate reader and measure the absorbance kinetically over 20 min, with 30-sec intervals. An appropriate protein concentration will have at least a 0.2 shift with the phosphate and troglitazone controls. The window between the vehicle (DMSO) and positive control (phosphate or troglitazone) should be at least two-fold larger than the deviation from the replicates of the DMSO wells.
OPTIMIZING CaCl2 CONCENTRATION FOR THE MITOCHONDRIAL SWELLING ASSAY
SUPPORT PROTOCOL 4
This protocol describes how to optimize the CaCl2 conditions when setting up the mitochondrial swelling assay.
Materials Sprague-Dawley male rats (150 to 250 g; Charles River) or equivalent strain BCA protein assay kit (Pierce) 1 M succinate stock solution (see recipe) 10 mM CaCl2 stock solution (see recipe) 10 mM rotenone stock solution (see recipe) 30 mM oligomycin stock solution (see recipe) 1 mM cyclosporin A stock solution (see recipe) Mitochondrial swelling buffer (see recipe) 30°C water bath 15-ml tubes, polystyrene, disposable (BD Falcon) Standard clear bottom 96-well plate Absorbance plate reader (capable of measuring absorbance at kinetic interval) Assay procedure 1. Isolate mitochondria from rat liver (see Support Protocol 1). Keep isolated mitochondria on ice at all times.
2. Determine the protein concentration of the isolated mitochondria using the BCA assay (see Support Protocol 2). 3. Thaw succinate stock solution, CaCl2 stock solution, rotenone, oligomycin, and cyclosporin A stock solutions on ice. 4. Warm mitochondrial swelling buffer to 30°C. 5. Add 10 μl of rotenone stock, 3.3 μl of oligomycin stock, and 1 ml of succinate stock to 100 ml of warmed mitochondrial swelling buffer. This buffer will be used for the remainder of the experiment and referred to as swelling buffer mix.
6. Add 2 ml of swelling buffer mix to ten 15-ml tubes labeled A-J. Each tube will be for a portion of the 96-well plate for optimizing calcium concentration (see Fig. 25.4.3 for plate map).
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Figure 25.4.3 Recommended plate map for optimization of calcium concentration for mitochondrial swelling in isolated mitochondria. Wells in gray color are without cyclosporin A and wells in blue color are with cyclosporin A.
7. To tube A, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and dispense 200 μl into wells A1-A6 (see Fig. 25.4.3 for plate map). 8. To tube B, add 2 μl of cyclosporin A, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and dispense 200 μl into wells A7-A12 (see Fig. 25.4.3 for plate map). 9. To tube C, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 5 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells B1-B6 (see Fig. 25.4.3 for plate map). Final concentration of CaCl2 is 25 μM.
10. To tube D, add 2 μl of cyclosporin A, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 5 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells B7-B12 (see Fig. 25.4.3 for plate map). 11. To tube E, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 10 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells C1-C6 (see Fig. 25.4.3 for plate map). Final concentration of CaCl2 is 50 μM.
12. To tube F, add 2 μl of cyclosporin A, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 10 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells C7-C12 (see Fig. 25.4.3 for plate map). Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
13. To tube G, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 20 μl of 10 mM
25.4.12 Supplement 60
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.8 0.8 Mitos Only
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Figure 25.4.4 Calcium concentration curve for optimization of calcium concentration for setup of the mitochondrial swelling assay.
CaCl2 stock. Mix gently and dispense 200 μl into wells D1-D6 (see Fig. 25.4.3 for plate map). Final concentration of CaCl2 is 100 μM.
14. To tube H, add 2 μl of cyclosporin A, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 20 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells D7-D12 (see Fig. 25.4.3 for plate map). 15. To tube I, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 30 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells E1-E6 (see Fig. 25.4.3 for plate map). Final concentration of CaCl2 is 150 μM.
16. To tube J, add 2 μl of cyclosporin A, add appropriate amount of mitochondria (see Support Protocol 3 for guidelines on optimizing protein concentration), and then add 30 μl of 10 mM CaCl2 stock. Mix gently and dispense 200 μl into wells E7-E12 (see Fig. 25.4.3 for plate map). 17. Measure plate absorbance at 540 nm on plate reader kinetically over 20 min, with 30-sec intervals. Choose the most appropriate concentration of calcium to use in the mitochondrial swelling assay. An appropriate calcium concentration will not greatly affect the absorbance reading as compared to the mitochondria-only wells, but will still allow the positive controls to induce mitochondrial swelling (See Support Protocol 3). Too little calcium may not be enough for the positive controls or compounds to induce mitochondrial swelling. Too much calcium may already induce swelling, and it would be hard in this case to determine if the compound had an effect on mitochondrial swelling. Therefore having varying amounts of calcium should provide a calcium curve which will allow the user to choose an appropriate calcium concentration (see Fig. 25.4.4 for calcium curve example). The authors typically use 50 μM calcium in the 96-well format and 25 μM calcium in the cuvette format, but this is dependent upon mitochondrial prep quality, so the concentration needs to be determined for the specific user. Mitochondrial Toxicity
25.4.13 Current Protocols in Toxicology
Supplement 60
REAGENTS AND SOLUTIONS Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Calcium stock solution, 10 mM Prepare 10 mM CaCl2 in mitochondrial swelling buffer (see recipe) and adjust pH to 7.4 using HCl or KOH (do not use NaOH). Aliquot and store up to 6 months at −20°C. Cyclosporin A stock solution, 1 mM Prepare 1 mM cyclosporin A in DMSO. Aliquot and store up to 2 months at −20°C. Mitochondrial isolation buffer I 210 mM mannitol 70 mM sucrose 5 mM HEPES 1 mM EGTA 0.5% BSA (add on day of use) Adjust pH to 7.4 using HCl or KOH (do not use NaOH) Store without BSA up to 1 week at 4ºC Mitochondrial isolation buffer II 210 mM mannitol 70 mM sucrose 10 mM MgCl2 5 mM K2 HPO4 10 mM MOPS Adjust pH to 7.4 using HCl or KOH (do not use NaOH) Store up to 1 week at 4ºC Mitochondrial swelling buffer 213 mM mannitol 70 mM sucrose 3 mM HEPES Adjust pH to 7.4 using HCl or KOH (do not use NaOH) Store up to 4 weeks at 4ºC Oligomycin stock solution, 30 mM Prepare 30 mM oligomycin in DMSO. Aliquot and store up to 2 months at −20°C. Phosphate stock solution, 100 mM Prepare 100 mM K2 HPO4 in water and adjust pH to 7.4 using HCl or KOH (do not use NaOH). Prepare fresh on the day of the experiment. Rotenone stock solution, 10 mM Prepare 10 mM rotenone (Sigma, cat. no. R8875) in DMSO. Aliquot and store up to 2 months at −20°C.
Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
Succinate stock solution, 1 M Prepare 1 M sodium succinate dibasic hexahydrate (Sigma, cat. no. S2378) in mitochondrial swelling buffer (see recipe) and adjust pH to 7.4 using HCl or KOH (do not use NaOH). Aliquot and store up to 6 months at −20°C.
25.4.14 Supplement 60
Current Protocols in Toxicology
COMMENTARY Background Information Drug-induced mitochondrial toxicity can occur through several mechanisms, including opening of the mitochondrial permeability transition pore (MPTP). Many drugs that have been withdrawn from the market or that carry black box warnings have been shown to have mitochondrial impairment (Dykens and Will, 2007). Therefore, to reduce drug attrition, predictive screens should be positioned early in the drug-discovery process so results can guide chemistry and biology. Here we describe an assay using isolated rat liver mitochondria that can detect Ca2+ -dependent druginduced opening of the MPTP, in both 96-well and cuvette formats. Compounds or excess Ca2+ can enhance opening of the MPTP, which leads to loss of mitochondrial membrane potential, swelling of the matrix because of water accumulation, rupture of the outer mitochondrial membrane, release of intermembrane space proteins into the cytosol, and, ultimately, cell death. The method provided here provides one way to measure induction of the MPTP in isolated rat liver mitochondria by following mitochondrial swelling. This can be measured by monitoring light scattering, which inversely correlates to mitochondrial volume. Therefore, as the mitochondria begin to swell, then a decrease in absorbance will be observed. To determine if the drug-induced swelling was due to opening of the MPTP and not an unregulated pore, cyclosporin A (CsA) is added to block MPTP opening. Another way to measure induction of the MPTP is to track the ability of mitochondria to take up and retain Ca2+ (Ichas et al., 1994). Extramitochondrial Ca2+ levels can be measured with a fluorescence Ca2+ indicator dye such as Calcium green–5N tetrapotassium salt solution (Molecular Probes). With each addition of Ca2+ , the fluorescence increases; however, as the mitochondria take up the added Ca2+ , the fluorescence decreases back to baseline. This will continue until the mitochondria have taken up enough Ca2+ for the induction of MPTP to occur, which then leads to a rapid increases in fluorescence. A combination of the two protocols would allow for more confidence in drug-induced opening of the MPTP. Data analysis here has been demonstrated using slopes of the kinetic reads. It should be noted that there are other ways for the calculations to be completed. These include area under the curve and change in initial and end absorbance. Current Protocols in Toxicology
Critical Parameters and Troubleshooting Phosphate solution not showing a decrease in absorbance or swelling of mitochondria Check to make sure that the phosphate solution has been made fresh on that day. The solution cannot be made ahead of time or frozen to be used at a later time. All buffers must made up with pure Millipore water Assay and isolation buffers should be made with water that is free from Ca2+ . This is critical so that the mitochondria will not swell before assay is run. CsA must be added to assay solution before Ca2+ or drug addition The order of the addition of the mitochondria, Ca2+ , and CsA is critical to the experimental conditions. The order of addition should be: CsA (if using), mitochondria, then Ca2+ . This is because CsA must first be allowed to bind to cyclophilin D and block the MPTP before Ca2+ or drug induces opening of pore. CsA is not blocking induction of MPTP Some compounds will show a decrease in absorbance, but CsA will not block this decrease. This might be due to the fact that some compounds will induce unregulated pores to open that are not activated by Ca2+ and/or are insensitive to CsA (He and Lemasters, 2002). Compounds block MPTP instead of inducing it Some compounds will behave like CsA and block induction of MPTP instead of inducing it. This might also be concentration dependent. For instance, at low concentrations, the compound might induce MPTP, but at higher concentrations they might block it. Compound interferes with absorbance signal It is recommended before testing to check if the compound interferes with the absorbance readings, and correct for these changes if possible during analysis. Mitochondria are no longer functional Isolated mitochondria are very sensitive and have a limited functional time in which they can be used. The mitochondrial preparation should be used immediately after isolation, for up to 4 to 6 hr, during which they must remain on ice at all times. Mitochondria
Mitochondrial Toxicity
25.4.15 Supplement 60
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Figure 25.4.5 Raw absorbance data for vehicle (DMSO) and positive controls (troglitazone and phosphate). For the data analysis, the slope should be calculated for the maximal slope with a minimum time period of at least 5 min. For these compounds, the slope was calculated for the time period from 5 to 12 min.
remain functional for a longer time if they are maintained at a concentration of >30 mg/ml. Optimization of assay conditions is needed when using different tissues or species Assay conditions such as protein and Ca2+ concentrations may differ among mitochondria isolated from different tissues and also mitochondria from different species. Previous studies have shown that rat brain mitochondria require considerably more Ca2+ to induce MPTP opening than liver mitochondria, and that species differences may play a role as well (Panov et al., 2006). In addition, heart mitochondria are not only more resistant to Ca2+ load than liver mitochondria, but neonatal rat heart mitochondria are more resistant to Ca2+ load than adult rat mitochondria (Drahota et al., 2012). Therefore, in these protocols, assay conditions are only optimized for isolated rat liver mitochondria and not for other tissues or species.
Anticipated Results
Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore
Raw absorbance data for vehicle (DMSO) and positive controls (troglitazone and phosphate) are shown in Figure 25.4.5. Results from this assay can giving insight into the ability of the compound to induce Ca2+ -dependent MPTP opening in rat liver mitochondria. Therefore, seeing a decrease in absorbance due to mitochondrial swelling after Ca2+ and drug addition suggests that the drug decreases the threshold amount of Ca2+ needed to induce opening of the MPTP or works synergistically with Ca2+ to induce it. Blockage of this induction, or no decrease in absorbance by adding CsA before drug and
Ca2+ addition, ensures that the swelling is caused by the MPTP and not due to another pore opening.
Time Considerations Basic Protocol 1 r Buffer preparation (up to 1 week prior to experiment): 1 to 2 hr r Compound preparation (Day prior to experiment): 1 to 2 hr r Mitochondrial isolation (Day 1): 2 to 3 hr r Compound testing (Day 1): 20 min per plate (total time will depend on how many compounds are being tested) Basic Protocol 2 r Buffer preparation (up to 1 week prior to experiment): 1 to 2 hr r Compound preparation (Day prior to experiment): 1 to 2 hr r Mitochondrial isolation (Day 1): 2 to 3 hr r Compound testing (Day 1): 20 min per cuvette (total time will depend on how many compounds are being tested)
Literature Cited Beavis, A.D., Brannan, R.D., and Garlid, K.D. 1985. Swelling and contraction of the mitochondrial matrix. I. A structural interpretation of the relationship between light scattering and matrix volume. J. Biol. Chem. 260:13424-13433. Berson, A., Cazanave, S., Descatoire, V., Tinel, M., Grodet, A., Wolf, C., Feldmann, G., and Pessayre, D. 2006. The anti-inflammatory drug, nimesulide (4-nitro-2-phenoxymethanesulfoanilide), uncouples mitochondria and induces mitochondrial permeability transition in human hepatoma cells: Protection by albumin. J. Pharmacol. Exp. Ther. 318:444-454.
25.4.16 Supplement 60
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Crompton, M., Ellinger, H., and Costi, A. 1988. Inhibition by cyclosporin A of a Ca2+ -dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem. J. 255:357-360. Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1-1.8.4. Drahota, Z., Milerovia, M., Endlicher, R., Rychtromoc, D., Cervinkova, Z., and Ostadal, B. 2012. Developmental changes of the sensitivity of cardiac and liver mitochondrial permeability transition pore to calcium load and oxidative stress. Physiol. Res. 61:S165-S172. Dykens, J.A. and Will, Y. 2007. The significance of mitochondrial testing in drug development. Drug Discov. Today. 12:777785. Halestrap, A.P. and Brenner, C. 2003. The adenine nucleotide translocase: A central component of the mitochondrial permeability transition pore and key player in cell death. Curr. Med. Chem. 10:1507-1525. Haworth, R.A. and Hunter, D.R. 1979. The Ca2+ induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site. Arch. Biochem. Biophys. 195:460-467. He, L. and Lemasters, J.J. 2002. Regulated and unregulated mitochondrial permeability transition pores: A new paradigm of pore structure and function? FEBS Lett. 512:1-7. Hunter, D.R. and Haworth, R.A. 1979. The Ca2+ induced membrane transition in mitochondria. I. The protective mechanisms. Arch. Biochem. Biophys. 195:453-459. Ichas, F., Jouaville, L.S., Sidash, S.S., Mazat, J.P., and Holmuhamedov, E.L. 1994. Mitochondrial calcium spiking: A transduction mechanism based on calcium-induced permeability transition involved in cell calcium signalling. FEBS Lett. 348:211-215. Kim, J.S., He, L., and Lemasters, J.J. 2003. Mitochondrial permeability transition: A common
pathway to necrosis and apoptosis. Biochem. Biophys. Res. Commun. 304:463-470. Lapidus, R.G. and Sokolove, P.M. 1993. Spermine inhibition of the permeability transition of isolated rat liver mitochondria: An investigation of mechanism. Arch. Biochem. Biophys. 306:246253. Masubuchi, Y., Nakayama, S., and Horie, T. 2002. Role of mitochondrial permeability transition in diclofenac-induced hepatocyte injury in rats. Hepatology 35:544-551. Masubuchi, Y., Kano, S., and Horie, T. 2006. Mitochondrial permeability transition as a potential determinant of hepatotoxicity of antidiabetic thiazolidinediones. Toxicology 222:233239. National Institutes of Health. 2011. Guide for the Care and Use of Laboratory Animals, 8th ed. http://grants.nih.gov/grants/olaw/Guide-for-thecare-and-use-of-laboratory-animals.pdf. Okuda, T., Norioka, M., Shitara, Y., and Horie, T. 2010. Multiple mechanisms underlying troglitazone-induced mitochondrial permeability transition. Toxicol. Appl. Pharmacol. 248:242-248. Panov, A., Dikalov, S., Shalbuyeva, N., Hemendinger, R., Greenamyre, J.T., and Rosenfeld, J. 2006. Species- and tissue-specific relationships between mitochondrial permeability transition and generation of ROS in brain and liver mitochondria of rats and mice. Am. J. Physiol. Cell Physiol. 292:C708-718. Tanveer, A., Virji, S., Andreeva, L., Totty, N.F., Hsuan, J.J., Ward, J.M., and Crompton, M. 1996. Involvement of cyclophilin D in the activation of a mitochondrial pore by Ca2+ and oxidant stress. Eur. J. Biochem. 238:166-172. Tay, V.K., Wang, A.S., Leow, K.Y., Ong, M.M., Wong, K.P., and Boelsterli, U.A. 2005. Mitochondrial permeability transition as a source of superoxide anion induced by the nitroaromatic drug nimesulide in vitro. Free Radic. Biol. Med. 39:949-959.
Mitochondrial Toxicity
25.4.17 Current Protocols in Toxicology
Supplement 60
