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

Our research group uses mitochondria as pharmacological target for the development of different products such as chemotheraputes, insecticides, among others. With this video, we want to describe the methodology to evaluate enzymes of the mitochondrial respiration in a more accessible, understandable, and usable way. In our lab, we have established a protocol to assess the different effects on xenobiotics on mitochondria in rats, mosquitoes, and ticks, providing some insights into a specific mitochondrial response.

This approach supports bio-safe pesticide formulation by identifying mitochondrial bioenergetics disruptions critical to minimizing non-target toxicity. Our differential advantage include an insecticide design protocol that starts with in silico analysis of thousand of molecules, followed by in vitro validation of those that meet biosecurity criteria, and concludes with in vivo testing. This method conserves time and resource while adhering to the best practice in pesticide production.

In the medium term, we will select naturally derived molecules that can be combined with genetic modification, targeting insect mitochondria, aiming for enhanced specificity and biosecurity in non-target organisms. To begin, prepare 250 milliliters of mitochondria isolation buffer with or without 0.2 grams of BSA. Aliquot 10 milliliters of the buffer into an ice-cold conical tube.

After euthanizing a rat, dissect its liver completely and place it in the ice-cold isolation buffer to rinse it. After two washes, mince the liver tissue with sharp scissors in a Petri dish containing 10 milliliters of isolation buffer. Place the solution into the Potter-Elvehjem tissue homogenizer tube to homogenize the minced tissue at least 10 times at a low velocity of 390 revolutions per minute.

Then, transfer the homogenized solution into a conical tube and centrifuge at 600 G for 10 minutes at four degrees Celsius. After centrifugation, carefully pour the supernatant into a new conical tube. Centrifuge the supernatant again at 7, 000 G for 10 minutes at four degrees Celsius and discard the resulting supernatant.

Resuspend the isolated mitochondrial pellet in two milliliters of isolation buffer without BSA. Check if the Aedes aegypti eggs are hatched in trays with filtered dechlorinated water one week before the experiment, and monitor the molting process in larvae. Once the larvae reach the L3 stage, wait for a maximum of 12 hours before collecting them.

Using a Pasteur pipette, collect at least 100 Aedes aegypti larvae at instars L3 to L4 in approximately 30 milliliters of chlorine-free water. After filtering, transfer the solution with larvae into Potter-Elvehjem tissue homogenizer tube and homogenize carefully at approximately 390 revolutions per minute at least 10 times. Transfer the homogenized tissue through a glass wool syringe to filter out large exoskeleton residues.

To begin, obtain rat liver mitochondria and homogenized Aedes aegypti L3, L4 larvae. For polarographic assay, calibrate the high resolution respirometer each day prior to starting any assays, following the manufacturer's instructions. Add reaction medium to the chambers, ensuring an excess of at least 0.2 milliliters above the standard experimental chamber volume of two milliliters.

Monitor oxygen consumption to measure NADH and succinate oxidase activities after adding reaction buffer, 0.1 milligrams of protein, and the substrate. Wait until the oxygen concentration signal stabilizes and record it. Add electron transport protein inhibitors to the chamber to confirm that the observed oxygen consumption is due to the mitochondrial respiratory chain.

For NADH dehydrogenase activity, first prepare the reaction system containing all the required components and add the reagents to the spectrophotometer cell. Monitor the NADH dehydrogenase activity with the reduction of potassium ferrocyanide at 420 nanometers. Use the molar extinction coefficient of 1.04 per millimolar per centimeter to calculate concentration.

Finally, divide the reported activity by the protein concentration used to obtain the specific enzyme activity. Similarly, evaluate succinate dehydrogenase and cytochrome c reductase or complex III activity using appropriate reaction mixtures. To measure cytochrome c oxidase or Complex IV activity, prepare the reaction system and mix equal molar amounts of cytochrome c and sodium dithionite, ensuring complete reduction.

Pass the reduced mixture through a cross-linked dextran gel column using the phosphate buffer as the mobile phase. Quantify the fractions collected at 550 nanometers in a spectrophotometer. Finally, calculate the concentration with a molar extinction coefficient of 19.8 per millimolar per centimeter, applying Beer's law.

Divide the reported activity by protein concentration for specific enzyme activity results. Cavacrol at 9.7 parts per million reduced NADH oxidase activity by approximately 9.35%while Cymbopogon flexuosus essential oil, or EO, at 10 parts per million reduced NADH oxidation and oxygen reduction by about 34%indicating inhibition in electron transport between Complex I and Complex IV.Cavacrol reduced succinate oxidase activity by about 10.84%in Aedes aegypti larvae, and Cymbopogon flexuosus EO reduced succinate oxidase activity by up to 50%in isolated rat liver mitochondria, suggesting an inhibition between Complex II and Complex IV.Cavacrol decreased NADH cytochrome c reductase activity and cytochrome c oxidation and oxygen reduction at Complex IV in Aedes aegypti larvae, while Cymbopogon flexuosus EO reduced cytochrome c oxidase activity by around 58%in rat liver mitochondria.