**3. Most commonly used models for the study of CLD**

CLD is associated with a wide pathological spectrum of alterations, from inactive liver fat storage, associated with an asymptomatic benign clinical course, to progressive cardiovascular, metabolic, and/or liver and kidney diseases with higher cancer risks [25–27]. CLD results from the persistent action of various harmful agents on the liver tissue, exceeding the liver's capacity for defense and repair. The perpetuation of the damage and the liver's response to the damage can produce fat hepatic accumulation without inflammation—an excessive lipid accumulation that induces lipotoxicity accompanied by necrosis and fibrosis development. A constant liver injury might lead more easily to chronicity, as well as a gradual decrease in the hepatocellular mass with subsequent progressive anatomical and functional distortion. From an anatomical point of view, this results in cirrhosis; from a functional point of view, it leads to chronic liver failure. CLD is histologically characterized by the presence of inflammation, steatosis, fibrosis, cirrhosis, and the development of carcinoma (**Table 1**). Therefore, most animal models for the study of liver disease mimic one or more of these histopathological features. We will now address some of the histopathological and functional characteristics of CLD and the animal models that best mimic them.

#### **3.1 Models for the study of inflammation, steatosis, and liver fibrosis**

An imbalance between lipid deposition and elimination may lead to hepatic steatosis. There are several cardinal features of hepatic steatosis such as synthesis of triglycerides (TG) and accumulation of free fatty acid (FFA) in the liver. FFA can be processed *via* two different pathways: The first pathway is β-oxidation to generate ATP, and the second is esterification of FFA to produce TG that are either stored within hepatic cells or incorporated into very low-density lipoprotein (VLDL) for release from hepatic cells, thus stimulating an imbalance in fat input/fat output that in turn leads to hepatic steatosis [58].

Histologically, the accumulation of fat in the liver is combined with lobular and periportal inflammation and cell damage (necrosis and ballooning) [59]. Hepatocytes are the main cells involved in the metabolism and mobilization of lipids in the liver. When there is lipid accumulation in hepatocytes, mitochondrial function is overloaded, leading to mitochondrial and peroxisomal dysfunction and increased oxidative stress. Uncontrolled and incomplete lipid oxidation generates toxic lipid products that cause hepatocyte damage and ultimately lead to lipoapoptosis [60, 61]. Apoptosis of hepatocytes can be considered a major cause of liver inflammation, persistent liver damage, and liver regeneration [61, 62]. Chronic inflammation of the liver can induce activation, expression, and signaling of growth factors [63, 64]. As a consequence, the hepatic stellate cells (HSCs) are activated and differentiate from the quiescent phenotype to proliferative and contractile myofibroblasts. Additionally, cells will have the ability to synthesize extracellular matrix [65, 66]. Therefore, a complex network of cytokineinduced pathways arises to coordinate the pro-fibrogenic cell interactions leading to the progression of fibrosis [67–69]. Liver fibrosis is considered a highly complex tissue repair process that appears in the face of sustained hepatocellular damage and that will go through the stages of steatosis, inflammation, regeneration, and liver fibrosis.

Several cells, growth factors, interleukins, receptors, and their signaling pathways participate in the development of CLD and must therefore be analyzed. The study of inflammation, steatosis, and fibrosis can be currently carried out using models that allow for the identification of the typical characteristics of said processes. Models developed for this purpose include diet-induced models, chemical/pharmacologically induced models, and genetic models. Some of the most important features of these models are described below.

#### *3.1.1 Diet-induced models*

The diet-induced models include a variety of dietary regimens that range from the administration of substances and fat supplies to caloric intake and additional supplements to facilitate the development of a NASH-like phenotype animal model.


*Models of Hepatotoxicity for the Study of Chronic Liver Disease DOI: http://dx.doi.org/10.5772/intechopen.106219*

*HFD, high-fat diet; CD, choline deficiency; MCD, methionine and choline deficiency; CCL4, AS, albumin serum; PS, porcine serum; carbon tetrachloride; D-GH, D-galactosamine hydrochloride; LPS, lipopolysaccharide; TAA, thioacetamide; DEN, diethylnitrosamine; PB, phenobarbital; BDL, bile duct ligation; HCC, hepatocellular carcinoma.*

#### **Table 1.**

*Models for the study of chronic liver disease.*

1.*High-fat animal models.* This group includes the diets characterized by fat intake (up to 70%) with some other combinations such as a diet rich in fat but deficient in choline; a diet rich in fructose intake; a diet rich in cholesterol; a diet rich in cholesterol and fructose; a diet rich in cholesterol, fructose, and trans fats; or a diet rich in fat plus chemicals [28–34, 70]. The advantages are an oral route of

administration, small specimens, and an induction disease time that generally ranges between 12 and 30 weeks. However, the development of some models can be extended for longer periods. This includes a diet deficient in methionine and choline (up to 84 weeks), the high-cholesterol diet (up to 52 weeks), or the chemical diet (up to 52 weeks). The presence of cholesterol in these diets has been reported to increase the severity of the disease [35]. These diets generally lead to the development of steatosis (grade +++), the presence of inflammation (grade ++ or +++), and the development of metabolic syndrome; they are usually employed to address drug discovery or omics data. One advantage of these models is that most can reach the fibrosis stage (grade ++) and some, such as the high-fat diet combined with chemicals, achieve more advanced fibrosis (grade +++) [71, 72]. The development of hepatocellular carcinoma has only been reported with the choline-deficient diet, the high-fat, choline-deficient diet, and the high-fat, high-cholesterol diet [30–32, 70].

2.*The Western/cafeteria diet model.* Other diets try to imitate the bad eating habits that take place among people from big urban centers and with fast food ingestion [73]. These diets are made from a mixture of fat, sugar, and cholesterol. The disadvantage is that they are not standardized and vary greatly across different studies, which makes comparison and reproducibility difficult. There are several combinations that have produced NAFLD using the Western diet method. For example, rats have been fed a 40% fat, 40% sugar, and 2% cholesterol diet for 16 weeks. This diet induced the development of periportal steatosis and inflammation, but there was no development of fibrosis [74]. Another study using hamsters employed a diet containing 40.8% fat, 0.5% cholesterol, and sugar water for 12–16 weeks, leading to the development of microvesicular steatosis, fibrosis, obesity, and insulin resistance. A third study reported a diet based on 40% calories from fat, added sucrose/fructose, and 0.15–2% cholesterol (with and without sugar water) for 24–50 weeks and administered to mice. This study found the presence of steatosis, inflammation, and hepatic fibrosis, as well as obesity and insulin resistance [75]. Although these westernized diets show that the histopathological characteristics of NAFLD can be induced, an analysis of histological patterns, cytokine profile, activation of metabolic pathways, and the degree of damage shows differences in the metabolic profile and hepatic histopathological phenotype [76–78]. Therefore, the extrapolation of these findings to humans is difficult, and these models are not considered adequate for the study of new drugs or for obtaining omics data.

### *3.1.2 Chemical-induced models*

Several studies have reported the use of hepatotoxic agents such as CCL4, thioacetamide, and diethylnitrosamine to induce NAFLD in rodents. The degree of liver damage varies with the type of agent, the route, and the time of administration. However, said agents have been used for this purpose for over 50 years. CCl4 is one of the toxic agents that has been widely employed in the research of the liver disease. It can be used by itself or in combination with other agents, either intraperitoneally, orally, by inhalation, or subcutaneously. Its administration ranges from 6 to 12 weeks. The advantage of using this agent is that the histopathological and functional characteristics it produces in experimental animals are already well established. This is one of the models that produces a high degree of steatosis (grade +), as well as a high

### *Models of Hepatotoxicity for the Study of Chronic Liver Disease DOI: http://dx.doi.org/10.5772/intechopen.106219*

grade of inflammation and fibrosis (++ or +++), and can even cause portal hypertension and ascites in rodents [38, 39]. In combination with other hepatotoxic, it can lead to hepatocellular carcinoma. Differences in route of administration and dose are associated with variations between research groups. Thioacetamide is a toxic agent that is administered intraperitoneally or in drinking water, achieving liver damage in 10–12 weeks. This agent does not attain a high degree of steatosis, but it produces a high degree of inflammation and fibrosis (grade +++), and even portal hypertension in rodents. However, there have been discrepancies regarding the characteristics of the resulting liver injury. Hepatocellular carcinoma may develop if combined with other agents [40, 41]. Diethylnitrosamine (DEN) is a highly hepatotoxic agent that can be administered orally, intraperitoneally, and in drinking water. It achieves liver damage in 7–18 weeks. The degree of steatosis and fibrosis is very low, but there is a significant inflammatory response [42–44]. Due to its carcinogenic potential, it can lead to hepatocellular carcinoma. These toxic agents can be used as steatosis models for the study of new drugs and to obtain omics data.
