Kinase experiment:

To further evaluate apoptosis, cell extracts are collected after 24 h of exposure to Myclobutanil, centrifuged, and analyzed with a multiplex biometric ELISA-based immunoassay containing dyed microspheres conjugated to a monoclonal antibody specific for the target protein. Apoptosis biomarkers are BCL-xL/Bak dimer and Mcl-1/Bak dimer, quantified using RBM Apoptosis Panel 3. Each experiment is performed in triplicate and apoptosis biomarker levels determined using the Bio-Plex Array Reader. The analytic concentrations are calculated using a standard curve, according to the manufacturer’s instructions[1].

Cell experiment:

The hepatoma cell line HepG2 is used in this study. The cells are grown on tissue culture plates in an incubator with a humidified atmosphere (95% air/5% CO2 v/v) at 37℃. Steatosis is induced by incubating the hepatocytes with 6 mM of a 1:1 v/v mixture of oleic (18:1) and linoleic (18:2) fatty acids (Fas) for 24 h. After a wash with PBS, cells are exposed for an additional 24 h to Myclobutanil at 0.1, 1, 10, 100 or 500 ppm. Cytotoxicity is assessed in HepG2 cells (1.0×105 cells/well in 24-well plates) by measuring the reduction of the tetrazolium dye 3-(4, 5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT)[1].

Myclobutanil is a conazole class fungicide widely used as an agrichemical.

Myclobutanil reduces cell viability to<50% at 100 ppm and to<10% at 500 ppm. Myclobutanil promotes a slight, but significant, increase in fatty acid (FA)-induced steatotosis at doses from 1 to 100 ppm. Anti-apoptotic biomarkers are significantly reduced by Myclobutanil[1].

[1]. Stellavato A, et al. Myclobutanil worsens nonalcoholic fatty liver disease: An in vitro study of toxicity and apoptosis on HepG2 cells. Toxicol Lett. 2016 Nov 16;262:100-104.