Implementation of the aldehyde dehydrogenase (ALDH) activity assay in Caenorhabditis el-egans.

VERONICA ROMERO; LUCIA FERNANDEZ HUBEID; MIRIAM VIRGOLINI

Congreso; Third Latin American C. elegans Meeting; 2023

Implementation of the aldehyde dehydrogenase (ALDH) activity assay in Caenorhabditis elegansVeronica L. Romero, Lucia E. Fernandez-Hubeid and Miriam B. VirgoliniIFEC-CONICET, Departamento de Farmacología Otto Orsingher, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina. E mail: veronica.romero@unc.edu.arCaenorhabditis 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 the agroindustry. The toxic mechanism behind these pesticides is ALDH inhibition, either directly (benomyl) or through the 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 activity in C. elegans to assess the consequences of rotenone or benomyl exposure. Wild-type nematodes in the L4 stage were exposed for one hour to either rotenone or benomyl at a final concentration of 10 uM. They were washed thereafter 3 times with M9 buffer, collected, and stored at -80 oC. On the day of the assay, the samples were defrosted and homogenized in 0.25 M sucrose/0.1 mM EDTA in a volume equivalent to 10% w/v of the nematode mass. 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. As expected, the results show that both rotenone and benomyl inhibited ALDH activity with respect to control animals (230 fold and 13 fold, respectively) demonstrating that this technique was sensitive enough to detect changes in ALDH enzymatic activity. Analysis in progress is evaluating compounds that can restore or enhance the enzyme activity.Financing agencies: PICT 2017-0874 and PICT 2019-02444 from ANPCyT (MBV).