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

Summary

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

Abstract

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.

Introduction

The effects of organochlorine pesticides (OCPs) on health, e.g. reproductive interference, immunological toxicity, metabolic changes have been previously studied^1,2,3. The methods to detect cellular metabolism and find out mitochondrial dysfunction have enabled scientists to understand the role of mitochondrial function (i.e., mitochondrial STAT3 levels, lactate, pyruvate, lactate-to-pyruvate ratio, coenzyme Q10, mitochondrial proton leak, bioenergetics, biogenesis, and dynamics) in areas such as aging, obesity, diabetes, cardiovascular function, cancer, and....

Protocol

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 distille....

Results

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 (

Discussion

Critical to the success of the detection protocol is the use of a variety of experimental methods that have been covered the study from phenotype to mechanism. In this study, HepG2 cells were cultured in DMEM with penicillin and streptomycin and 10% fetal bovine serum. When cells reached 40-50% confluence, β-HCH (0, 10, 100 ng/mL) were added and incubated for 24 h. We firstly used TEM which showed the ultrastructural changes in hepatocyte caused by the representative OCPs, β-HCH, showing the impairment of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.