*3.2.1 Chemical-induced models*

CCl4 in combination with D-galactosamine hydrochloride (GaIN) with or without lipopolysaccharides (LPS) is one of the more often employed models for developing liver cirrhosis. LPS is added to activate Kupffer cells and stimulate TNF-α, as well as an immune response *via* NF-κB pathway activation. D-galactosamine is used to potentiate this response by depleting the uridine nucleotides and interfering in protein synthesis, leading to acute lesions as a precipitating event [87]. Liver cirrhosis is developed in 10 weeks. In this model, we see an increase in aspartate amino transferase (AST) and alanine amino transferase (ALT), as well as the presence of necrosis, and excessive fibrosis and regenerative nodules. Thioacetamide combined with LPS has been used to induce decompensated cirrhosis. After 10 weeks of its administration in the drinking water, increases in liver enzymes, portal hypertension, fibrosis, and cirrhosis can be observed.

#### **Figure 2.**

*Models of cirrhosis and hepatocellular carcinoma. Examples of animal models to induce cirrhosis and hepatocellular carcinoma: chemicals, surgery, genetic engineering, and humanized mice. CCl4: carbon tetrachloride; GaIN: D-galactosamine hydrochloride; LPS: lipopolysaccharides; AS: albumin serum; PS; porcine serum; DEN: diethylnitrosamine.*

Albumin serum (AS) and porcine serum (PS) administrations are two other potential models for inducing immune cirrhosis. Albumin becomes hepatotoxic when there is an imbalance of it in the liver [88]. Albumin or PS may induce cirrhosis when interacting with LPS or when undergoing irreversible alkalization *via* drug metabolization. The administration of albumin or PS twice a week for 6 weeks leads to CLD decompensation and subsequent histological and functional liver changes. There is also an increase in several inflammatory markers, such as IL-6, IL-18, and HMGB-1 [89]. The PS model may be more adequate for the induction of immune cirrhosis. It induces a response that is closer to human liver disease, is easier to manipulate, and results in lower mortality rates for animals when compared with the other mentioned models.

DEN is the most important and widely used toxic agent for the development of chemically induced HCC in rodents. It is a carcinogen, and DEN bioactivation occurs in the liver *via* cytochrome P450 (CYP 2E1) [90]. DEN is metabolized *via* alphahydroxylation and dealkylation reactions, producing an unstable ethyldiazonium hydroxide molecule that can generate highly reactive carbon, oxygen (ROS), and nitrogen (RNS) ion species. These reactive molecules bind to DNA and proteins and can contribute to genomic instability, DNA damage, mutation, and tumor initiation [91]. DEN "initiates" the hepatocarcinogenesis process and produces a stable and heritable mutational change in hepatocytes, inducing genomic alterations in oncogenes and tumor suppressor genes. HCC can clonally expand from this single DEN-"initiated" hepatocyte and result in HCC.

There are different protocols where DEN is used to induce HCC in rodents. For example, it can be administered alone and in single intraperitoneal doses ranging from 1 to 5 mg, resulting in adenomas (0–20%) in 24 weeks or carcinoma in 36 to 52 weeks [92–94]. When administered in multiple doses, it is used in doses of 25 to 50 mg for 4–8 weeks, resulting in preneoplastic lesions, adenoma, or carcinoma depending on the total administration time. DEN may be used in single or multiple doses in combination with CCl4 0.5 mg/mL to obtain preneoplastic lesions, adenomas, or carcinomas, also depending on the total time of administration [95, 96]. In another rat protocol, a single dose of DEN plus 0.03% thioacetamide is administered, leading to adenomas or carcinomas between 24 and 52 weeks [97, 98]. A rat model employs DEN in a single dose (80–200 mg) in combination with phenobarbital (PB) (0.025–0.1%) to obtain the desired preneoplastic lesions, adenomas, or carcinomas [99]. DEN has also been combined with 2-acetylaminofluorene (0.02%) and PB (67%) to cause carcinoma and metastasis [45].

#### *3.2.2 Surgery model*

Common bile duct ligation is a model of secondary biliary cirrhosis. The surgical resection of the bile duct induces bile accumulation in the liver and thus leads to damage, inflammation, fibrosis, portal inflammation, and cirrhosis after 2 weeks [46, 55]. An increase in AST, ALT, and total bilirubin levels can be observed after surgery in rats and mice. Recent reports suggest that the use of a single dose of LPS can induce decompensated disease [56]. This model is used for drug discovery and omics data.
