This work was supported by the National Natural Science Foundation of China (Grant Nos. 81573174, 81570574); the Outstanding Youth Fund of Jiangsu Province (SBK2014010296); the Research Project of Chinese Ministry of Education (213015A); the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Flagship Major Development of Jiangsu Higher Education Institutions; and the Open Project Program of the State Key Laboratory of Environmental Chemistry and Ecotoxicology (KF2015-01).

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
<ol>
	<li>Collecting HepG2 cells
	<ol>
		<li>Seed HepG2 cells in 100 mm dishes. Store at 37 &#176;C and 5% CO2.</li>
		<li>Digest cells with 0.25% EDTA in 1.5 mLEP tube.</li>
		<li>Centrifuge at 1000 x g for 3 min at room temperature (RT). Discard the supernatant.</li>
		<li>Collect 4-6 x 105 HepG2 cells.</li>
	</ol>
	</li>
	<li>Add 1 mL of 5% glutaraldehyde (Solvent: double distilled water) with pipettes and incubate at 4&#176;C for 2 h.</li>
	<li>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.</li>
	<li>Add 200 &#181;m of 1% osmium (Solvent: double distilled water) and incubate at 4 &#176;C for 2 h to black sample. 
	Caution: Osmium is highly toxic and volatile substance. It should be operated in the drug cabinet carefully.</li>
	<li>Add and wash in 2 changes of 1 mL of phosphate buffer, 5 min each. Suck out phosphate buffer.</li>
	<li>Stain in 2% uranyl acetate solution (Solvent: double distilled water and acetic acid) in 1.5 mL EP tube for 2 h.</li>
	<li>Dehydrate and submerge through 50% acetone, 70% acetone, 90% acetone (Solvent: double distilled water), 2 changes of absolute acetone, 15 min each.</li>
	<li>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).</li>
	<li>Incubate sequentially at 37 &#176;C for 12 h, 45 &#176;C for 12 h, and then 60 &#176;C for 48 h.</li>
	<li>Prepare and observe ultrathin sections (the largest area cannot exceed 0.5 mm &#215; 0.3 mm) by ultrathin sections machine and TEM (2 &#181;m and 500 nm).</li>
</ol>
2. Mitochondrial fluorescence intensity detection
<ol>
	<li>Seed 2x 104 HepG2 cells in 6-well plates. Add &#946;-HCH for 24 h.</li>
	<li>Add anhydrous DMSO to formulate a final concentration of 1 mM mitochondrial green fluorescent probe.</li>
	<li>Add 1 mL mitochondrial green fluorescent probe solution (final concentration: 200 nM) to each well of 6-well plates, incubate at 37 &#176;C for 45 min.</li>
	<li>Remove the mitochondrial green fluorescent probe solution and add freshly prepared cell culture medium (DMEM) at 37 &#176;C prior to imaging.</li>
	<li>Observe mitochondrial green &#64258;uorescence by a &#64258;uorescence microscope (10x). The maximum excitation wavelength at detection is 490 nm and the maximum emission wavelength is 516 nm.</li>
</ol>
3. Assay of the cellular ATP levels
<ol>
	<li>Seed HepG2 cells in 6-well plates. Add &#946;-HCH for 24 h.</li>
	<li>Add 200 &#181;L 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 &#176;C, collect the supernatant with pipettes into new tubes.</li>
	<li>Dilute the ATP detection reagent with the ATP dilution at a ratio of 1: 5, as ATP detection buffer.</li>
	<li>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 &#181;M by ATP lysis buffers.</li>
	<li>Add 100 &#181;L of ATP detection buffer&#160;in a 96-well plate for 5 min at RT, and add 100 &#181;L of supernatant of cell, detect by a luminometer (chemiluminescence detector). Calculate ATP concentration (nmol/L) though the standard curve.</li>
	<li>Detect protein concentration by a BCA Protein Assay Kit with multimode reader.
	<ol>
		<li>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.</li>
		<li>Add 1 &#181;L of supernatant of cell in a 96-well plate and add 19 &#181;L of double distilled water.</li>
		<li>Add 200 &#181;L of BCA detection solutions for each well. Incubate at 37 &#176;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.</li>
	</ol>
	</li>
	<li>Correct the ATP content per mg protein concentration (unit: ATP concentration/protein concentration, nmol/mg).</li>
</ol>
4. Mitochondrial membrane potential (MMP) assessment by JC-1
<ol>
	<li>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.</li>
	<li>Add 50 &#181;L 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.</li>
	<li>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 &#176;C.</li>
	<li>Centrifuge at 600 x g for 3 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Rinse in 2 changes of 1 mL of JC-1 (1X) dyeing buffer, centrifuge at 600 x g for 3 min at 4 &#176;C. And discard the supernatant.</li>
	<li>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.</li>
</ol>
5. Oxygen consumption rates (OCR) measurements
<ol>
	<li>Seed HepG2 cells in cell culture microplates of 96-well at a density of 4500 cells/100 &#956;L per well. Ensure cells covered with each well after 24 h.</li>
	<li>Add the appropriate volume of the prepared reagents into the appropriate injection port, according to previous literature13. Port A: 25 &#181;L 1 &#181;M of oligomycin (ATP Coupler); Port B: 25 &#181;L 0.75 &#181;M of FCCP (Electronic Throttle Control, ETC Accelerator); Port C: 25 &#181;L 0.5 of &#181;M antimycin A/rotenone (mitochondrial Inhibitor A and mitochondrial Inhibitor B).</li>
	<li>Store cartridge in a 37 &#176;C incubator with no CO2 until ready to use.</li>
	<li>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 &#181;L.</li>
	<li>Store the cell plate in a 37 &#176;C incubator with no CO2 for 1 h before the assay.</li>
	<li>Record OCR automatically by software.
	<ol>
		<li>Open the OCR software.</li>
		<li>Choose Cell Mito Stress Test Kit in the Apps drop-down menu.</li>
		<li>Click the Start App button.</li>
		<li>Click the Run Stress Test button. The Cell Stress Test Setup screen appears.</li>
		<li>Do the following steps:
		<ol>
			<li>Enter the number of cells seeded per well in the Cell seeding # box.</li>
			<li>Enter the average OCR in the Average basal OCR box.</li>
			<li>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.</li>
			<li>Click the Next button. The group info screen appears.</li>
			<li>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.</li>
			<li>Click the Next button. The Stress Test Injection Layout screen appears.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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% con&#64258;uence, &#946;-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, &#946;-HCH, showing the impairment of...

The effects of organochlorine pesticides (OCPs) on health, e.g. reproductive interference, immunological toxicity, metabolic changes have been previously studied1,2,3. 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 safety toxicity4,5,6,7. In this paper, we describe the methods on assessing mitochondrial dysfunction caused by OCPs.
We exposed HepG2 cells to &#946;-hexachlorocyclohexane (&#946;-HCH), one representative OCPs, for 24 h at a dose equivalent to internal exposure of human. Firstly, TEM was applied to observe the ultrastructure of hepatocytes, such as nuclei, mitochondria and endoplasmic reticulum8. Compared with ordinary microscopes, TEM enables to explore the 2D and 3D ultra-structure of cells and cell components (cell lines or tissue), the morphology, chemical composition, as well as function of natural or artificial materials that play a pivotal role in modern science and technology. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5&#39;-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP)in HepG2 cells incubated with &#946;-HCH. Mito-tracker green is a mitochondria green fluorescent probe that can be used for live cell mitochondrial-specific fluorescent staining. The mitochondria in hepatocytes were stained by mito-tracker green solution and mitochondrial fluorescence intensity, number and pattern were observed with a confocal microscopy9. Mitochondrial green fluorescent probe can be used to stain live cells. Compared with rhodamine 123 or JC-1, mitochondrial green fluorescent probe does not depend on mitochondrial membrane potential for mitochondrial staining. ATP levels were determined by a luciferase-luciferin kit and normalized by protein concentration. ATP assay kit can be used to detect ATP levels in common solutions, cells or tissues. This kit is based on firefly luciferase catalyzed by fluorescein to generate fluorescence, when ATP is required to provide energy. When the firefly luciferase and fluorescein are excessive, in a certain concentration range, the generation of fluorescence is proportional to the concentration of ATP. In addition, this kit has been specially designed to optimize the chemiluminescence of ATP. ATP, as the most important energy molecules, plays an important role in the various physiological or pathological processes of cells. Changes in ATP levels can reflect defects in cell function, especially mitochondrial energy production. Usually under apoptosis, necrosis or in some toxic state, cellular ATP levels reduced 10. MMPs assay kit with JC-1 is a kit that uses JC-1 as a fluorescent probe to detect cells, tissues or purified MMPs quickly and sensitively. It can be used for early detection of apoptosis. The cationic dye JC-1 is a fluorescent probe used to detect the MMP which can be analyzed under flow cytometry indicated by changes of green and red fluorescence ratio. When the MMP is high, JC-1 is aggregated in the matrix of the mitochondria to form a polymer (J-aggregates), which produces red fluorescence. When the MMP is low, JC-1 cannot accumulate, and forms monomer which produces green fluorescence11. So the ratio of red and green fluorescence can reflect the level of MMP. The OCR of cells is a crucial indicator of normal cellular function12. It is regarded as a parameter to research mitochondrial function. Cell mito stress test kit provides a stable method for analyzing key parameters of mitochondrial function. The kit provides quality control and predictive reagents as well as a standard method for performing cell mitochondrial stress test. It can be used to detect all cell types, including primary cells, cell lines, suspended cells, and also for islets, nematodes, yeasts and isolated mitochondria.
OCR measurement can provide valuable insight into the physiological status or alterations of cells. It was determined with an extracellular flux analyzer to detect breathing baseline, proton leak, maximal respiratory, ATP turnover and reserve capacity. In brief, after baseline measurements of OCR, OCR was detected after sequentially adding to oligomycin (ATP Coupler), FCCP (mitochondrial oxidative phosphorylation uncoupler) and antimycin A/rotenone (an inhibitor of oxygen consumption)per well.
In an effort to facilitate the development of more specific protocol for detecting mitochondrial function in hepatocytes in vitro, we present here experiments by TEM, confocal microscopy, luminometer, flow cytometry and extracellular flux analyzer with future application in studying mitochondria damage related adverse outcomes.

<table><tbody><tr><td>Transmission electron microscope&amp;nbsp;</td><td>FEI</td><td>Tecnai G2 Spirit Bio TWIN</td><td>High-contrast, high-resolution imaging, Low-dose observation and imaging, Low-temperature observation, Outstanding analytical performance, Automation for convenience and performance</td></tr><tr><td>Mito-Tracker Green</td><td>Beyotime</td><td>C1048</td><td>Mito-Tracker Green is a mitochondrial green fluorescent probe that can be used for live cell mitochondrial-specific fluorescent staining.</td></tr><tr><td>Laser scanning confocal microscope</td><td>Zeiss</td><td>700B</td><td>The design is compact, stable, light path is the shortest, high light precision, creative technology and sophisticated scanning technology together to&amp;nbsp; produce a perfect 3-dimensional specimen image.</td></tr><tr><td>Enhanced ATP Assay Kit</td><td>Beyotime</td><td>S0027</td><td>Enhanced ATP Assay Kit can be used to detect ATP (adenosine 5&#039;-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.</td></tr><tr><td>Luminometer</td><td>Berthold</td><td>Centro LB 960</td><td>Luminometer is chemiluminescence detector, the test sample itself can be light, do not need to stimulate. Luminometer is the instrument that detects chemiluminescence.</td></tr><tr><td>BCA Protein Assay Kit</td><td>Beyotime</td><td>P0012</td><td>BCA Protein Assay Kit is one of the most commonly used methods for detecting protein concentrations.</td></tr><tr><td>Multimode reader</td><td>TECAN</td><td>InfiniteM200</td><td>Multimode reader be used to detect protein consentration.</td></tr></tbody></table>

experimental protocol for detecting mitochondrial function in hepatocytes exposed to organochlorine pesticides

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 &#946;-hexachlorocyclohexane (&#946;-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 &#946;-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.

The mitochondria cristae of HepG2 cells exposed to &#946;-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 &#946;-HCH (<stro...

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

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.

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

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Research

JoVE Journal

Environment

7.9K Views.  Nanjing Medical University. Het is een messer die oproep antwoord op een belangrijke vraag die je hebt in ook een functie van het veld, zoals ATP-niveau en het zuurstofverbruik tarieven. De nieuwe of de ene pagina oud, deze techniek is dat het gemakkelijk uit te voeren en het is veel voor een speciale evaluatie van cellulaire mitochondriale functie. Om dit experiment te starten, zaad 20,000 HepG2 cellen in een glazen bodem Petri schotel.Voeg bèta-hexachlooohohexaan of B-HCH toe in couveuse bij 37 graden Celsius ...

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

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

Biochemistry

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule synthesis. Genetic and functional adaptations of mitochondria to the tumor environment drive proliferative and metastatic potential. The advent of DNA and RNA sequencing removed critical barriers to the evaluation of genetic mediators of tumorigenesis. However, to date, methodological approaches to evaluate tumor mitochondrial function remain elusive and require technical proficiency limiting the feasibility, ultimately diminishing diagnostic and prognostic value in both experimental and clinical settings. Here, we&#160;outline a simple and rapid method to quantify rates of oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacity in freshly excised solid tumor homogenates using high-resolution respirometry. The protocol can be reproducibly applied across species and tumor types as well as adapted to evaluate a diversity of mitochondrial ET pathways. Using this protocol, we demonstrate that mice bearing a luminal B mammary cancer exhibit defective nicotinamide adenine dinucleotide-linked respiration and reliance on succinate to generate adenosine triphosphate via OXPHOS.

We developed a practical protocol and analytical approach to evaluate mitochondrial oxidative phosphorylation and electron transfer capacity in fresh tumor homogenates. This protocol can be easily adapted to survey various mitochondrial functions that contribute to cancer initiation, progression, and treatment response.

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule ...

Cancer Research

Estimating the Integral Metabolite Cytotoxicity of Triazole Pesticides in Plants

Assessing Cytotoxicity of Metabolites of Typical Triazole Pesticides in Plants

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, causing potential threats to the ecosystem and human health throughout the food chain. The biotransformation of pollutants in plants generates a number of metabolites that may be more toxic than their parent compounds, implying that the metabolites should be taken into account during the toxicity assessment. However, the metabolites of pollutants in plants are extremely complex, making it difficult to comprehensively obtain the toxicological information of all metabolites. This study proposed a strategy to assess the integral cytotoxicity of pollutant metabolites in plants by treating them as a whole during toxicological tests. Triazole pesticides, a class of broad-spectrum fungicides, have been widely applied in agricultural production. Their residue pollution in farmland has drawn increasing attention. Hence, four triazole pesticides, including flusilazole, diniconazole, tebuconazole, and propiconazole, were selected as the tested pollutants. The metabolites were generated by the treatment of carrot callus with tested triazole pesticides. After treatment of 72 h, the metabolites of pesticides in carrot callus were extracted, followed by toxicological tests using the Caco-2 cell line. The results showed that the metabolites of tested pesticides in carrot callus did not significantly inhibit the viability of Caco-2 cells (P>0.05), demonstrating no cytotoxicity of pesticide metabolites. This proposed method opens a new avenue to assess the cytotoxicity of pollutant metabolites in plants, which is expected to provide valuable data for precise toxicity assessment.

Author Spotlight: A New and Efficient Method for Comprehensive Metabolite Cytotoxicity Assessment of Triazole Pesticides in Plants

The protocol describes a new method to assess the integral cytotoxicity of metabolites of triazole pesticides in plants.

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, ...

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests phase of drug developing process because they are genetically human and recapitulate the interplay among tissues. In this study, the human intestinal barrier (emulated by a co-culture of Caco-2 and HT-29 cells) and the liver equivalent (emulated by spheroids made of differentiated HepaRG cells and human hepatic stellate cells) were integrated into a two-organ chip (2-OC) microfluidic device to assess some acetaminophen (APAP) pharmacokinetic (PK) and toxicological properties. The MPS had three assemblies: Intestine only 2-OC, Liver only 2-OC, and Intestine/Liver 2-OC with the same media perfusing both organoids. For PK assessments, we dosed the APAP in the media at preset timepoints after administering it either over the intestinal barrier (emulating the oral route) or in the media (emulating the intravenous route), at 12 &#181;M and 2 &#181;M respectively. The media samples were analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Organoids were analyzed for gene expression, for TEER values, for protein expression and activity, and then collected, fixed, and submitted to a set of morphological evaluations. The MTT technique performed well in assessing the organoid viability, but the high content analyses (HCA) were able to detect very early toxic events in response to APAP treatment. We verified that the media flow does not significantly affect the APAP absorption whereas it significantly improves the liver equivalent functionality. The APAP human intestinal absorption and hepatic metabolism could be emulated in the MPS. The association between MPS data and in silico modeling has great potential to improve the predictability of the in vitro methods and provide better accuracy than animal models in pharmacokinetic and toxicological studies.

We exposed a microphysiological system (MPS) with intestine and liver organoids to acetaminophen (APAP). This article describes the methods for organoid production and APAP pharmacokinetic and toxicological property assessments in the MPS. It also describes the tissue functionality analyses necessary to validate the results.

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests ...

Chemistry

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Mitochondrial Functional Assessment in Brush Lizard Liver Mitochondria

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

Assessment of oxygen consumption provides integral information about mitochondrial function. Using a phosphorescent probe with a fluorescent plate reader, accurate and reproducible data can be obtained easily without specialized equipment. This assay enables any lab to measure the oxygen consumption of isolated mitochondria and calculate respiratory control ratios.

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of ...

Changing Mitochondrial Function for Therapeutic and Bioinsecticide Applications

Unveiling Xenobiotic Transport and Effects in Isolated Mitochondria: Insights from Respirometric and Enzymatic Assays

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). However, their significance extends far beyond energy production. Dysregulation of mitochondrial bioenergetics can trigger intracellular cascades that impact health and cellular function. Given their physiological importance, there is growing interest in exploring the pharmacological potential of mitochondrial function for developing new therapies and bioproducts. Modulating mitochondrial bioenergetics could provide innovative approaches to address challenges such as neurodegenerative disorders, metabolic diseases, and cancer, as well as to develop bioproducts for controlling pests and vector-borne diseases.
This work presents a method for assessing the effects of xenobiotics on the electron transport chain (ETC) using various substrates, including glutamate and NADH for complex I, succinate for complex II, and cytochrome c (both oxidized and reduced) for complexes III and IV. This methodology allows the activation or inhibition of electron transport through mitochondrial complexes to be evaluated using a respirometer and spectrophotometer. The mitochondria can be sourced from isolated mitochondria, fragmented cells, or homogenized tissue from various species.
In the laboratory, mitochondrial function has been analyzed in Aedes aegypti and Wistar rats, but this method is also applicable to other species, such as Rhipicephalus microplus and Rhodnius prolixus. This approach provides the basis for theorizing about the existence of uncoupler proteins and species-specific oxidizable substrate preferences influenced by their unique energetic demands. Recent findings offer valuable insights into innovative bioinsecticide design strategies that target mitochondrial function, holding significant potential for effectively controlling vector-borne diseases and pest infestations.

This study introduces a methodology for studying xenobiotic effects on mitochondrial function via respirometric and enzymatic assays. It examines electron transport chain modulation in various species, offering insights for targeted biopesticide development against insect vector-borne diseases.

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Biology

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

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

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