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

Summary

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.

Abstract

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.

Introduction

Mitochondria have always been called the power plant of cells due to their chemical energy generation during the oxidative phosphorylation process^1,2. Beyond their main core metabolic functions, mitochondria are also implicated in many other cellular processes, ranging from intracellular Ca²⁺ regulation levels, inflammatory processes, and reactive oxygen species production to cell death induction processes³. Mitochondrial bioenergetics and their dysregulation have been associated with intracellular processes that affect cellular function and overall health. This has sparked a....

Protocol

All methods described in this protocol have been approved by the Ethics Committee "Comité de ética en investigación científica CEINCI": Scientific Research Ethics Committee of the Industrial University of Santander. Furthermore, these assays complied with the Arrive guidelines and followed the 1989 Colombian Law 84 (Chapter IV, Art. 23-26) and Resolution 8430 (1993, Title IV, Art. 83-93) that regulates animal research in Colombia.

NOTE: It is strongly recommended to read the oxidative phosphorylation chapter of any biochemistry book before starting this protocol.

1. Reagents and ....

Results

Before analyzing the results obtained with this protocol, it is essential to remember a little about the biochemistry of the electron transport chain in the mitochondria. First, NADH supplies a pair of electrons to the oxidoreduction centers located in complex I. There, it begins the transport of electrons that pass from complex I to ubiquinone, subsequently to complex III, which donates them to cytochrome c, and finally to complex IV, where oxygen is the final electron acceptor, reducing it to water. On the other hand, .......

Discussion

A spectrophotometric and polarographic method with high-resolution O₂ was employed to investigate the effects of xenobiotics on mitochondrial function. The study specifically assessed the impact of Cymbopogon flexuosus essential oil and carvacrol on NADH and succinate oxidation, as well as electron transport through the mitochondrial electron transport chain, using two different biological models: isolated mitochondria from rat liver and homogenized Aedes aegypti larvae. Additionally, potenti.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2,6-dichlorophenolindophenol	Merck	119814	Prepare on the same day of use.
Bradford reagent	Merck	B6916
BSA	Merck	A7030
Carvacrol	Merck	W224511
Cytochrome c	Merck	C2506	Photosensitive conver cover it with aluminum foil. In solution it should be stored in the freezer.
D-mannitol	Merck	M4125
EGTA	Merck	>E3889
Ethylenediaminetetraacetic acid tetrasodium salt dihydrate	Merck	E6511
HEPES	Merck	H3375
KCN	Merck	60178	In solution works weel for 2 weeks.
KH2PO4	Merck	P5655
Multiskan FC Microplate Photometer	Thermo Scientific	51119100
Na2HPO4	Merck	567550
Oroboros O2k	Oroboros Instruments	10203-03	https://www.oroboros.at/index.php/product/o2k-startup/
Phenazine methosulphate	Merck	P9625	Prepare on the same day of use.
Potassium ferricyanide	Merck	244023	Prepare on the same day of use.
Potter Elvehjem tissue homogenizer tube
Rotenone	Merck	557368	Soluble in ethanol. Photosensitive conver cover it with aluminum foil. In solution it should be stored in the freezer.
Sodium succinate	Merck	S9637	In solution it should be stored in the freezer.
Sucrose	Merck	S0389
β-Nicotinamide adenine dinucleotide, reduced Disodium salt hydrate	Merck	N8129	Photosensitive conver cover it with aluminum foil. In solution it should be stored in the freezer.