Implementation of the aldehyde deshydrogenase (ALDH) activity assay in Caenorhabditis elegans

ROMERO VL; FERNANDEZ-HUBEID, LE.; VIRGOLINI MB

Valparaíso

Congreso; Third Latin Worm Meeting; 2023

Implementation of the aldehyde deshydrogenase (ALDH) activity assay in Caenorhabditis elegansCaenorhabditis elegans (C. elegans) is a model organism that has been used to study the effects of neurotoxicants, including rotenone and benomyl, both widely used in agrochemistry. The toxic mechanism behind these pesticides is ALDH inhibition, either directly (benomyl) or through ALDH cofactor (rotenone). Considering the critical role of this enzyme in the detoxification of reactive aldehydes, the aim of this work was to set up an assay to determine ALDH 2 activity in C. elegans and to assess the consequences of rotenone or benomyl exposure. Wild type N2 nematodes in the L4 stage were exposed for one hour to either rotenone or benomyl at a final concentration of 10 uM. Thereafter they were washed 3 times with M9 buffer, collected, and stored at -80 C. On the day of the assay, the samples were defrosted and homogenized in 0.25 M sucrose/0.1 mM EDTA; the volume was equivalent to 10% w/v of the nematode mass. The homogenates were centrifuged at 800 g for 10 min at 4 °C and the supernatant further centrifuged at 10,000 g for 10 min at 4 °C. The precipitate was suspended in sucrose 0.25 M/triton X100 1% v/v solution, frozen at −80 °C for 30 min, defrosted and centrifuged at 10,000 g for 10 min at 4 °C. Subsequently, 300 μl of the supernatant was incubated at room temperature for 20 min with 500 μl of the reaction mixture containing adenine β-nicotinamide dinucleotide (NAD). ALDH activity was measured spectrophotometrically by following the production of NADH at 340 nm. The reaction was started by the addition of 0.1 ml of acetaldehyde and followed over a 10 min period. The results demonstrated that both rotenone (230 fold than control) and benomyl (13 fold than control) inhibited the enzyme activity so the technique was successful to determine ALDH activity. Further analysis are requested to evaluate compounds that can restore the enzyme activity.