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Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

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Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides
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All experiments and the experiment protocols were performed in accordance with relevant guidelines and regulations and approved by the local Ethical Committee of Nanjing Medical University.

1. Mitochondrial ultrastructure by TEM

  1. Collecting HepG2 cells
    1. Seed HepG2 cells in 100 mm dishes. Store at 37 °C and 5% CO2.
    2. Digest cells with 0.25% EDTA in 1.5 mLEP tube.
    3. Centrifuge at 1000 x g for 3 min at room temperature (RT). Discard the supernatant.
    4. Collect 4-6 x 105 HepG2 cells.
  2. Add 1 mL of 5% glutaraldehyde (Solvent: double distilled water) with pipettes and incubate at 4°C for 2 h.
  3. Add and wash in 4 changes of 1 mL of phosphate buffer (containing Na2HPO4, KH2PO4, NaCl and KCl, pH 7.4), 15 min each. Suck out phosphate buffer with pipettes.
  4. Add 200 µm of 1% osmium (Solvent: double distilled water) and incubate at 4 °C for 2 h to black sample.
    Caution: Osmium is highly toxic and volatile substance. It should be operated in the drug cabinet carefully.
  5. Add and wash in 2 changes of 1 mL of phosphate buffer, 5 min each. Suck out phosphate buffer.
  6. Stain in 2% uranyl acetate solution (Solvent: double distilled water and acetic acid) in 1.5 mL EP tube for 2 h.
  7. Dehydrate and submerge through 50% acetone, 70% acetone, 90% acetone (Solvent: double distilled water), 2 changes of absolute acetone, 15 min each.
  8. Penetrate in 2 drops acetone (100%)/embedding agent (1:1) for 1.5 h at RT.Epon812 embedding agent, the recipe is as follows: A: Epon812, 62 mL and DDSA, 100 mL; B: Epon812, 100 mL and MNA 89 mL. A:B=2:8 (v:v, in winter), A:B=1:9 (v:v, in summer). Embedding in embedding molds (Soft plastic plate with checkered grid).
  9. Incubate sequentially at 37 °C for 12 h, 45 °C for 12 h, and then 60 °C for 48 h.
  10. Prepare and observe ultrathin sections (the largest area cannot exceed 0.5 mm × 0.3 mm) by ultrathin sections machine and TEM (2 µm and 500 nm).

2. Mitochondrial fluorescence intensity detection

  1. Seed 2x 104 HepG2 cells in 6-well plates. Add β-HCH for 24 h.
  2. Add anhydrous DMSO to formulate a final concentration of 1 mM mitochondrial green fluorescent probe.
  3. Add 1 mL mitochondrial green fluorescent probe solution (final concentration: 200 nM) to each well of 6-well plates, incubate at 37 °C for 45 min.
  4. Remove the mitochondrial green fluorescent probe solution and add freshly prepared cell culture medium (DMEM) at 37 °C prior to imaging.
  5. Observe mitochondrial green fluorescence by a fluorescence microscope (10x). The maximum excitation wavelength at detection is 490 nm and the maximum emission wavelength is 516 nm.

3. Assay of the cellular ATP levels

  1. Seed HepG2 cells in 6-well plates. Add β-HCH for 24 h.
  2. Add 200 µL of lysis buffers from the luciferase-luciferin ATP assay kit to each well of 6-well plates. Centrifuge at 12,000 x g for 5 min at 4 °C, collect the supernatant with pipettes into new tubes.
  3. Dilute the ATP detection reagent with the ATP dilution at a ratio of 1: 5, as ATP detection buffer.
  4. Prepare standard curve determination: dilute the 0.5 mM of ATP standard solution to 10, 5, 1, 0.5, 0.1, 0.05, 0.01 µM by ATP lysis buffers.
  5. Add 100 µL of ATP detection buffer in a 96-well plate for 5 min at RT, and add 100 µL of supernatant of cell, detect by a luminometer (chemiluminescence detector). Calculate ATP concentration (nmol/L) though the standard curve.
  6. Detect protein concentration by a BCA Protein Assay Kit with multimode reader.
    1. Prepare of standard curve determination: dilute the 5 mg/mL of protein standard solution to 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL by double distilled water.
    2. Add 1 µL of supernatant of cell in a 96-well plate and add 19 µL of double distilled water.
    3. Add 200 µL of BCA detection solutions for each well. Incubate at 37 °C for 30 min. Detect OD (optical density) value by a multimode reader with 562 nm. Calculate the protein concentration (mg/mL) though the standard curve.
  7. Correct the ATP content per mg protein concentration (unit: ATP concentration/protein concentration, nmol/mg).

4. Mitochondrial membrane potential (MMP) assessment by JC-1

  1. Collect HepG2 cells seeded in 6-well plates. Digest cells with 0.25% EDTA in 1.5 mL EP tube, centrifuge at 1000 x g for 3 min, discard the supernatant and collect cells.
  2. Add 50 µL of JC-1 (200X) to 8 mL of distilled water to vortex and mix. Add 2 mL of JC-1 (5X) staining dyeing buffer as JC-1 detection solution.
  3. Incubate cells with a mixture of 0.5 mL of cell culture medium and 0.5 mL of JC-1 detection solution for 20 min at 37 °C.
  4. Centrifuge at 600 x g for 3 min at 4 °C. Discard the supernatant.
  5. Rinse in 2 changes of 1 mL of JC-1 (1X) dyeing buffer, centrifuge at 600 x g for 3 min at 4 °C. And discard the supernatant.
  6. Suspend cells in 0.5 mL of JC-1 (1X) dyeing buffer, analyze via flow cytometry to detect green and red fluorescence. When the JC-1 polymer is detected, set the excitation light to 490 nm, and the emission light to 530 nm. When the JC-1 polymer is detected, set the excitation light to 525 nm, and the emission light to 590 nm.

5. Oxygen consumption rates (OCR) measurements

  1. Seed HepG2 cells in cell culture microplates of 96-well at a density of 4500 cells/100 μL per well. Ensure cells covered with each well after 24 h.
  2. Add the appropriate volume of the prepared reagents into the appropriate injection port, according to previous literature13. Port A: 25 µL 1 µM of oligomycin (ATP Coupler); Port B: 25 µL 0.75 µM of FCCP (Electronic Throttle Control, ETC Accelerator); Port C: 25 µL 0.5 of µM antimycin A/rotenone (mitochondrial Inhibitor A and mitochondrial Inhibitor B).
  3. Store cartridge in a 37 °C incubator with no CO2 until ready to use.
  4. Make a medium change of the cell plate through removing the running medium from each well and adding to the new DMEM. Final volume for each well is 180 µL.
  5. Store the cell plate in a 37 °C incubator with no CO2 for 1 h before the assay.
  6. Record OCR automatically by software.
    1. Open the OCR software.
    2. Choose Cell Mito Stress Test Kit in the Apps drop-down menu.
    3. Click the Start App button.
    4. Click the Run Stress Test button. The Cell Stress Test Setup screen appears.
    5. Do the following steps:
      1. Enter the number of cells seeded per well in the Cell seeding # box.
      2. Enter the average OCR in the Average basal OCR box.
      3. Enter the final working concentration for each reagent and inject the reagents.
        NOTE: The average basal OCR value for the cell should have been detected before running the optimization assay when optimizing for cell seeding concentration.
      4. Click the Next button. The group info screen appears.
      5. Assign a group to the unassigned wells through choosing a color and giving a name, and then clicking on the appropriate wells.
        NOTE: The different groups are defined as different treatments prior to running of the Cell Mito Stress Test. All wells of microplates will get the same reagent injections when the stress test is run.
      6. Click the Next button. The Stress Test Injection Layout screen appears.
      7. Click Start. The Stress Test is now run on the Analyzer. When the run is over, follow the point s in the software, remove and discard the cartridge and cell plate.

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

Learning Objectives

The mitochondria cristae of HepG2 cells exposed to β-HCH were markedly damaged. Scattered mitochondria were mildly to markedly expanded, irregularly shaped, and mitochondrial ridge disappeared with relatively abnormal mitochondrial architecture (Figure 1).

Average mitochondrial green fluorescence intensity, which represents the mitochondria, decreased in HepG2 cells exposed to β-HCH (Figure 2), as well as in ATP levels (Figure 3). The fluorescence intensity and ATP level decreased gradually with increasing exposure concentrations. Potentially, the reduction in mitochondria number, or damaged mitochondria, caused the observed mitochondrial fluorescence intensity, and consequently the reduced production of ATP.

The results of flow cytometry demonstrated that the ratios of red/green JC-1 fluorescence in β-HCH group were significantly lower than in the control (Figure 4). OCR of HepG2 cells was reduced in a dose-dependent manner after β-HCH exposure. Compared with control group, basal respiration rates, proton leak, maximal respiratory capacity, and ATP turnover were significantly decreased in HepG2 cells exposed to β-HCH (Figure 5). These results indicated that mitochondrial function was impaired after β-HCH exposure.

All the instruments used in these experiments were show in Supplemental Figure 1.

Figure 1
Figure 1: Representative TEM micrographs of HepG2 cells exposed to β-HCH (A: 6000×, B: 25000×). Typical damages showed mildly enlarged mitochondria (M), electron-lucent matrices, damaged cristae and loose organelle gaps. M: mitochondria. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Detection of mitochondrial fluorescence in HepG2 cells treated with β-HCH13. The amount, location and fluorescence intensity of mitochondria (A) were showed in fluorescence microscopic images and quantitative levels of mitochondrial fluorescence intensity were based on mitochondrial green fluorescent per cell (B). *: P < 0.05, ***: P < 0.001 compared with the control.Each data point was the mean ± SEM from three separate experiments. Please click here to view a larger version of this figure.

Figure 3
Figure 3: ATP levels in HepG2 cells13. ***: P < 0.001 compared with the control, ##: P < 0.01 compared with 10 ng/mL β-HCH.Data were presented as mean ± SEM of three separate experiments. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Effects on MMP by JC-1 staining and flow cytometry13. (A) Flow cytometry plots. The Y-axis showed the ratio of red to green fluorescence. PE: Red fluorescence, FITC: Green fluorescence. (B).**: P < 0.01 compared with the control.Each data point was the mean ± SEM from three separate experiments. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Detection of cellular oxygen consumption rate (OCR). (A) The Effect of β-HCH on cellular OCR was measured by the extracellular flux analyzer. The four periods represent cellular basal respiration rate, ATP-synthase-inhibited rate, maximal uncoupled rate, and rotenone-or antimycin-A-inhibited rate. (B) Quantitative histograms of OCR results for baseline, proton leak, maximal respiratory capacity, ATP turnover andreserve capacity. *: P < 0.05, **: P < 0.01, ***: P < 0.001 compared with the control. Each data point was the mean ± SEM from three separate experiments. Please click here to view a larger version of this figure.

Supplemental Figure 1
Supplemental Figure 1: All the instruments used in protocols. (A) Transmission electron microscopy. (B) Laser scanning confocal microscope. (C) Luminometer. (D) Multimode reader. (E) Flow cytometry. (F) Extracellular flux analyser. Please click here to view a larger version of this figure.

List of Materials

Print Materials List
Transmission electron microscope  FEI Tecnai G2 Spirit Bio TWIN High-contrast, high-resolution imaging, Low-dose observation and imaging, Low-temperature observation, Outstanding analytical performance, Automation for convenience and performance
Mito-Tracker Green Beyotime C1048 Mito-Tracker Green is a mitochondrial green fluorescent probe that can be used for live cell mitochondrial-specific fluorescent staining.
Laser scanning confocal microscope Zeiss 700B The design is compact, stable, light path is the shortest, high light precision, creative technology and sophisticated scanning technology together to  produce a perfect 3-dimensional specimen image.
Enhanced ATP Assay Kit Beyotime S0027 Enhanced ATP Assay Kit can be used to detect ATP (adenosine 5'-triphosphate) levels in common solutions, cells or tissues. Cells and tissue samples can be split to complete the sample preparation, detection sensitivity up to 0.1nmol / L, chemiluminescence can be sustained for 30 minutes.
Luminometer Berthold Centro LB 960 Luminometer is chemiluminescence detector, the test sample itself can be light, do not need to stimulate. Luminometer is the instrument that detects chemiluminescence.
BCA Protein Assay Kit Beyotime P0012 BCA Protein Assay Kit is one of the most commonly used methods for detecting protein concentrations.
Multimode reader TECAN InfiniteM200 Multimode reader be used to detect protein consentration.

Preparação do Laboratório

Print Setup Guide

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to β-hexachlorocyclohexane (β-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5′-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with β-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to β-hexachlorocyclohexane (β-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5′-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with β-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Procedimento

Print Procedure Steps

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to β-hexachlorocyclohexane (β-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5′-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with β-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Tags

Keywords: Mitochondrial FunctionHepatocytesOrganochlorine PesticidesATP LevelOxygen ConsumptionHepG2 CellsBeta-hexachlorcyclohexaneMitochondrial Green Fluorescence ProbeConfocal MicroscopyCellular ATPLuminometerMitochondrial Membrane PotentialJC1 Dye