Non-alcoholic fatty liver disease (NAFLD), and its most advanced stage, the non-alcoholic steatohepatitis (NASH) affect 25% and 5% of the world's population, respectively. The NAFLD/NASH are complex and multifactorial conditions involving genetic, epigenetic and environmental risk factors, as the consumption of a saturated fat- and carbohydrate-rich diet. NASH development involves molecular alterations and interactions among different organs, including the liver, adipose tissue and the intestinal microbiota. The herbicides glyphosate (N- phosphonomethyl)glycine) and 2,4-D (2,4-dichlorophenoxyacetic) are widely applied in agriculture and their residues are frequently identified in food and environment. These herbicides have already demonstrated hepatic pro-oxidant, lipogenic and/or inflammatory effects in preclinical studies at doses below the toxicological limits adopted by governmental agencies. Thus, it will be assessed whether glyphosate and 2,4D, alone or in combination, promote NASH in vivo. Then, a NASH model will be established in male C57BL/6J mice (n=10/group) by providing a fat- (20% lard) and sucrose-rich (20%) diet, and a high sugar solution (23.1 g/L fructose and 18.9 g/L glucose) for 6 months. Concomitantly, animals will receive glyphosate (0.05, 5 or 50 mg/kg/day), 2,4-D (0.02, 2 or 20 mg/kg/day) or a combination of both (0.05+0.02; 5+2 or 50+20 mg/kg/day) intrasgastrically (5 times/week). The doses were based on the Acceptable Daily Intake (ADI) or on the No Observed Adverse Effect Level (NOAEL) values. The glycemic curve will be calculated, and serum samples will be obtained for biochemical analyses (ALT, triglycerides and cholesterol). Liver samples will be obtained for histological (NASH grade, collagen and immunohistochemistry for Ki-67, CD68 and alpha-SMA) and molecular (transcriptome by RNAseq) analyses. The adipose tissue will be sampled for histological analysis (morphometry, mast cells, immunohistochemistry for CD68). Small intestine samples will be obtained for immunohistochemistry (ZO-1 and occludin), and the cecal fecal pellets for microbiome analysis (16S rRNA sequencing).
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