Experimental Hepatic Carcinogenesis: Oxidative Stress and Natural Antioxidants

Hepatocellular carcinoma is one of the most common cancers in the world, and it is influenced by agents such as DEN, 2-AAF, phenobarbital, alcohol, aflatoxin B1 metabolite or hepatitis viruses (B and C). Oxidative stress is becoming recognized as a key factor in the progression of hepatocarcinogenesis. Reactive oxygen species can play a leading role in initiation and promotion of hepatic carcinogenesis. The metabolites of DEN Diethylnitrosamine (DEN) mediate the binding of tumour promoters by covalently binding to the DNA with one or two oxidation-providing electrons. 2-AAF is the inducer of DEN, and it is involved in tumour formation in the bladder and liver. Reactive Oxygen species (ROS); carbohydrates, lipids, DNA and enzymes, such as affect all important structures. Additionally, an excessive amount of ROS is highly toxic to cells. Antioxidants are protects against ROS, toxic substances, carcinogens. This review focuses on the literature on studies of Hepatic Carcinogenesis, oxidative stress and antioxidant therapy.


Introduction
Hepatic carcinogenesis is the fifth most common cancer which third most common cause of cancer-related death globally and it is influenced by agents such as DE N, 2-AAF, phenobarbital (PB), alcohol, aflatoxin B 1 m etabolite or hepatitis viruses (B and C) [1][2].
Animal models are viewed as crucial tools in the study of hepatic carcinogenesis. Because of the physiologic and genetic similarities between rodents and humans, the short lifespan, the breeding capacity and the variety of manipulating methods, animal models are often used for cancer research [3]. Studies on induction of liver cancer in rats use chemic al agents such as DEN, 2-AAF, PB and aflatoxin B1 [4].
2-AAF exhibits its carcinogenic effect through the formation of DNA adducts, over production of reactive oxygen species (ROS ) and oxidative DNA damage [5]. Nitrosamines are widely recognized as carcinogenic compounds, but they require metabolic activation to exert their cytotoxic and carcinogenic activity. DEN is a nitrosamine compound that induces the formation of hepatic carcinoma. They showed that DEN increased lipid peroxidation in studies performed. This may increase the tumour [6][7].
Our aim in this study is to reveal the relationship bet ween antioxidants and oxidative stress in experimental hepatic carcinogenesis studies. And to report chemopreventive natural antioxidants used as inhibitors.

Reactive oxygen speci es (ROS)
Reactive oxygen radicals; (O 2 · -), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (O H·), which are present in small quantities during normal oxygen metabolism. Molecular oxygen (O 2 ) has two unpaired (unpaired) electrons with parallel spin states. An atom, group of atoms or molec ules containing an unpaired electron, are defined as free radicals. Transition metals such as Fe 3+ , Cu 2+ , Mn 2+ and Mo 5+ , however, are not considered free radicals even if they have unpaired electrons. But they play an important role in the formation of free radic als [8]. They are lipids that are most affected by reactive oxygen species. Since cell membranes are rich in polyunsat urat ed fatty acids (PUFAs) and cholesterol, they are easily affected by oxidant radicals. Lipid peroxidation, where the unsaturated lipids are pres ent, is a complex process that takes place with reactions involving molecular oxygen and is formed by lipid hydroperoxides. Lipid peroxidation is rather harmful as it is an autocat alytic and irreversible reaction [9][10]. Lipid peroxidation produces a wide variety of oxidation products, such as malondialdehyde (MDA), propanal, hex anal, and 4hydroxy nonanal (4-HNE). MDA appears to be the most mutagenic product of lipid peroxidation, but 4-HNE is most toxic. MDA is one of the most popular and reliable markers that det ermine oxidative stress in research [11]. Proteins are less sensitive to the effects of radic als than lipids. Protein oxidation results in the covalent modification of peptide bonds or amino acid side chains with ROS or oxidative stress products. In particular, the interaction of free radicals with unsaturated bonds and sulphide inclusion molecules is excessive. ROS may have direct or indirect effects on proteins. Amino acids such as peptide bonds, proline and lysine are quite easily affected by free radicals [12].
Protein oxidation occurs in the formation of carbonyl groups in amino acids such as histidine, tyrosine, phenylalanine. Products made by lipid peroxidation form covalent bonds with cysteine sulfhydryl groups or with lysine and histidines, leading to fragments and cross-linking of proteins. These events res ult in the deterioration of the structure and function of the proteins. Protein carbonyl levels area well-used marker for oxidative stress. [8][9][10][11][12][13][14][15].

DNA Damage and Free Radical s
The DNA molecule can undergo spontaneous chemical oxidative damage like c arbohydrates and proteins.It has been suggested that every cell DNA of the human body is exposed to oxidative damage 10 3 times a day [8]. Due to the balance between DNA damage and repair, very low levels of damage are also found in healthy individuals. Oxidative base modification (8-OHdG) has been shown even in newborn rats [16]. All changes that occur due to the effects of endogenous or exogenous factors in molecular int egration are called DNA damage. 8-OHdG indicates DNA damage [17 -18]. In recent years, base damage has frequently been analysed as an indicator of oxidative DNA damage. Since Cu 2 + ions are highly loc alized in the regions rich in G-C in DNA, the oxidative damage is the most exposed base guanine. 8-OHdG is a mutation t hat occurs in DNA, resulting from reactive oxygen species produced during normal oxidative met abolism [19]. All of the factors that lead t o increased ROS production contribut e to the formation of 8-OHdG, that is, oxidative DNA damage. The formation of 8 -OHdG by substances such as cigarette smoke, x-rays, oxidized unsaturated fatty acids, gamma rays, polyphenols, paraquat, kainic acid, diethyl butyl sterol, benzene, fecapenene, furocoumarins hydroperoxide and heavy metals has been shown in vitro [20]. For example, Cigarette smoke c ontains carcinogenic substances such as nitrosamines and polycyclic aromatic hydrocarbons and causes the increased of 8-hydroxydeoxyguanosine [19]. For this reason, the most commonly measured bas e damage is 8-OHdG. Therefore, 8-OHdG is considered as the "biologic al marker" of DNA damage [17]. Increased ROS production and oxidative DNA damage associated with hepato-carcinogenesis have been demonstrated in studies. Multivariat e analysis found that levels of 8-OHdG and fibrosis were significant risk factor for hepatocellular carcinoma, especially in patients with hepatitis C virus infection [21]. The marker of oxidative stress, such as 8-OHdG is commonly elevated in the livers of patients with chronic viral hepatitis infection, which is known to be a risk factor for HC.

Experimental Hepatic Carcinogenesi s
Hepatic carcinogenesis can be created experimentally in experimental animals by the application of various chemicals such as aromatic amines, nitrogen containing dyes, nitrosamines and aflatoxins [22]. Xenobiotics are carcinogenic to animals s uch as DE N, 2-AAF and phenobarbital, mouse, rat, hamster, rabbit, dog, pig and monkey. DEN and 2-AAF are the chemic als that cause tumours to form in the biological system. [23-24-25]. Coadministration of DE N and 2-AAF initiates hepatocarcinogenesis in rodents and causes preneoplastic initiation in hepat ocytes. [26-27-28]. In a study with FB acting as a promoter, such as DEN and 2-AAF, it was reported that they caused a mutation in codon 61 of H-Ras [29].

Role of DEN in tumorigenesi s
DEN is mostly used as tumour inducer in cancer res earches [30][31]. In the structure of DE N; Amide, urease and carbon containing compounds are available [32]. It has been reported that DENs are composed of intoxicates, from agroc hemicals and nitrattan, from those in cigarette smoke, as well as the formation of nutrients and nutrient nitrat es [27][28][29][30][31][32][33]. DEN has a direct effect on cancer formation. This means t hat DEN spontaneously hy drolyzes, regardless of the enzymes. This biological activation of the active DEN by two hydroxylation reactions is catalysed by cytochrome p450. One strong mechanistic link between c ancer is through the increased production of free radicals at the site of the resulting molecular changes, which include lipid peroxidation and oxidative DNA damage [34][35].

The role of 2-AAF in tumorigenesi s.
2-AAF occurs as a result of the acetylation of the 2-amino floran in the synthetic arylamine structure. 2-AAF acts in the second phase of the det oxification reactions and after the first step of DE N, it binds to guanine base for the s econd time in DNA and creates a toxic effect This toxic effect occurs in the form of preneoplastic, neoplastic, benign neoplasm and malignant neoplasm, respectively, resulting in mutations [36][37][38][39]. If the levels of DE N and 2 -AAF chemical tumour inducing agents increase in cells, the smooth endoplasmic reticulum enzymes are synthesized and detoxified [40][41][42].

Antioxidant System s Against Reactive oxygen Species
İn the cells and extracellular fluid there are antioxidant defence mec hanisms that try to bring the reactive oxygen radicals to a harmless state, Antioxidant enzyme systems, which convert ROS into less toxic products: Superoxide dismutase (SOD), catalas e (CA T) and glutathione redox cycle enzymes (such as glutathione peroxidase (GSH-Px), glutathione reductase, etc.). SOD enzymatically converts superoxide anion t o hydrogen peroxide and molecular oxygen. Hydrogen peroxide is reduced by water and oxygen with two important intracellular enzymes, catalase and glutathione peroxidase [43][44][45][46].
Antioxidants that catch and neutralize the radicals: Alpha Tocopherol (E vitamin) and Ascorbic acid (C vitamin) function as antioxidants. Vitamin E prevents lipid peroxidation in the cell membrane. Ascorbic acid shows antioxidant activity in the cytoplasm and extracellular fluids and inhibits the inactivation of antiproteases with oxidants. Additionally, Glutathione is a multifunctional intracellular antioxidant, α-Lipoic acid (ALA), whic h is a sulfur-containing antioxidant with metal-chelating and antiglycation capabilities. N-acetyl-L-cysteine is a thiol cont aining an antioxidant that has been us ed to decrease conditions of oxidative stress. The most reported activity of flavonoids is protection against oxidative stress. Thus flavonoids can help scavenger ROS and are effective inhibit ors of lipid peroxidation [47].

System s that prevent the formation of ROS and prevent the formation of ROS
Structures such as ceruloplasmin, ferritin, transferrin, lactoferrin, zinc, selenium, cytochrome oxidase reduce ROS. For example; Zi nc has been serving as a metal that prevents lipid peroxidation and DNA damage [48][49].

Experimental Investigations
Most of the factors that influence tumour fo rmation cause radical production in the cell. These factors also induce tumour formation and development by affecting the initiation, development and progression stages of carcinogenesis. Various animal model studies have been done on this subject. As seen in Table 1 [60] In conclusion, the relationship between HC and oxidative stress is a researc h area. ROS contribut es to the initiation and progression of HC. In current clinical trials, the mec hanisms of HC treatment of drugs or compounds may be partly due to antioxidative ability, especially the effect originating from ROS. Therefore, antioxidant therapeutics play an important role in the t reatment of HC. Time, effective doses and reliable doses require further investigation of antioxidant absorption and bioavailability.