Effects of Carboxymethylcellulose and Polysorbate 80 food additive on Nonalcoholic Fatty Liver Disease-associated hepatocarcinogenesis

Abstract

Hepatocellular carcinoma (HCC) features as the 6th/3rd most incident and deadliest, respectively, among all kinds of cancers worldwide, and it has been linked to chronic liver diseases like non-alcoholic fatty liver disease (NAFLD). NAFLD affects ~25% of the global population and contributes to increasing the HCC-related incidence by 26%. Adherence to a western diet (WD) - mostly composed of saturated fatty acids, sugars, and food additives - has been targeted as a driver of NAFLD, also increasing the risk of developing HCC by 80%. Ultra-processed food (UPF) is characterized by its hypercaloric and food additive-enriched profile, of which carboxymethylcellulose (CMC) and polysorbate 80 (P80) show potential effects on the liver-gut-adipose axis and prospecting as potential drivers of HCC. We sought to assess the effects of CMC and/or P80 on both in vivo and in vitro models of HCC-associated NAFLD. Male C57BL/6 mice will receive intraperitoneal injections of diethylnitrosamine [DEN, 25mg/Kg of body weight (b.w.), 1x/week, for 4 weeks] or vehicle, starting on the 14th post-natal day. At the 6th week of life, mice also received a WD (hypercaloric chow: 30% of saturated fat/20% of sucrose/0.2% of cholesterol; and a high-sugar solution: 55/45% of d-frcutose/d-glucose, for drinking) or a basal diet, for further 24 weeks. Simultaneously, mice received intragastric injections of CMC (370.0 or 740.0 mg/Kg of b.w., 5x/week, for 24 weeks) and/or P80 (100.0 or 200.0 mg/Kg of b.w., 5x/week, for 24 weeks) and were euthanized at the end of 24th week of the protocol. Neoplastic and hepatic (histopathological, lipid/collagen content, Ki67, cleaved caspase-3, and ±-smooth muscle actin immunoreactions, metabolomic, and mRNAseq), adipose tissue (histopathological and CD68 immunoreaction), small intestine (zonula occludens-1 and occludin immunoreaction, toluidine blue, and mucin density), feces (microbiome profiling), and serum (glucose tolerance test, alanine aminotransferases, total cholesterol, and triglycerides levels) samples will be further assessed. In a co-culture spheroid model (C3A e LX-2 cells) exposed to a fatty acid-supplemented medium, cell viability, lipid/collagen content, will be assessed. Additionally, based on our findings of mRNA sequencing, a western blotting strategy will be performed in spheroids for further validation of the in vivo findings. Data will be analyzed by one-way ANOVA or Kruskal-Wallis and Tukey pos hoc test and will be considered statistically different when p<0.05. (AU)