Fibrosis-associated hepatocarcinogenesis revisited: Establishing standard medium-term chemically-induced male and female models.

Guilherme Ribeiro Romualdo,Gabriel Bacil Prata,Fernando Salvador Moreno,Tereza Cristina Da Silva,Ana Angélica Henrique Fernandes,Luís Fernando Barbisan,Bruno Cogliati

doi:10.1371/journal.pone.0203879

Guilherme Ribeiro Romualdo, Gabriel Bacil Prata + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0203879

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Abstract

Hepatocellular carcinoma causes ~10% of all cancer-related deaths worldwide, usually emerging in a background of liver fibrosis/cirrhosis (70%-90% of cases). Chemically-induced mouse models for fibrosis-associated hepatocarcinogenesis are widely-applied, resembling the corresponding human disease. Nonetheless, a long time is necessary for the development of preneoplastic/neoplastic lesions. Thus, we proposed an early fibrosis-associated hepatocarcinogenesis model for male and female mice separately, focusing on reducing the experimental time for preneoplastic/neoplastic lesions development and establishing standard models for both sexes. Then, two-week old susceptible C3H/HeJ male and female mice (n = 8 animals/sex/group) received a single dose of diethylnitrosamine (DEN, 10 or 50 mg/Kg). During 2 months, mice received 3 weekly doses of carbon tetrachloride (CCl4, 10% corn oil solution, 0.25 to 1.50 μL/g b.wt.) and they were euthanized at week 17. DEN/CCl4 protocols for males and females displayed clear liver fibrosis, featuring collagen accumulation and hepatic stellate cell activation (α-SMA). In addition, liver from males displayed increased CD68+ macrophage number, COX-2 protein expression and IL-6 levels. The DEN/CCl4 models in both sexes impaired antioxidant defense as well as enhanced hepatocyte proliferation and apoptosis. Moreover, DEN/CCl4-treated male and female developed multiple preneoplastic altered hepatocyte foci and hepatocellular adenomas. As expected, the models showed clear male bias. Therefore, we established standard and suitable fibrosis-associated hepatocarcinogenesis models for male and female mice, shortening the experimental time for the development of hepatocellular preneoplastic/neoplastic lesions in comparison to other classical models.

Highlights

Hepatocellular carcinoma (HCC), the main type of primary liver cancer, is responsible for ~10% of all cancer-related deaths worldwide (~800,000 deaths/year, considering both genders) [1]
This study aimed at proposing a medium-term fibrosis-associated hepatocarcinogenesis mouse model for male and female mice separately, allying chemically-induced protocol and a susceptible mouse strain in order to reduce the experimental time to the development of hepatocellular preneoplastic and neoplastic lesions associated to liver fibrosis
Male mice submitted to DEN 50/CCl4 protocol showed a reduction in body weight throughout the experimental period (S1 Fig), but mainly at weaning (p

Summary

Introduction

Hepatocellular carcinoma (HCC), the main type of primary liver cancer, is responsible for ~10% of all cancer-related deaths worldwide (~800,000 deaths/year, considering both genders) [1]. HCC is considered a complex, multistep and multifactorial disease and it usually emerges in a background of liver fibrosis/cirrhosis (70% to 90% of all HCC cases), mainly caused by chronic hepatitis B and C virus infections, chronic ethanol abuse and nonalcoholic steatohepatitis (NASH) [2,3]. The pro-inflammatory and pro-fibrotic environment provided by these risk factors are the necessary background for the emerging of genetic and epigenetic alterations that can promote the development of dysplastic nodules and neoplastic lesions, mainly HCC [4]. Most of chemically-induced hepatocarcinogenesis mice models do not feature liver fibrosis, calling into question whether these models can reliably recapitulate key events observed during human hepatocarcinogenesis and HCC progression

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