3.1. Hepatocarcinogenesis

fusion, deletion, missegregation, or nondisjunction that lead to genomic damage. Conversely, procarcinogens do not interact directly with the genome until they are metabolically activated, producing genotoxic electrophilic metabolites or ROS. We consider that an essential critical point is the process of activation of chemical procarcinogens in which the proper enzymatic reaction of the substrate produces electrophiles and ROS [8, 9]. CYP450 is responsible for these enzymatic reactions. CYP450 is a family of proteins that share common mechanisms of activation of procarcinogens that result in OS [30, 40]. A particular event in ChemCar is the very origin of reactions in cascades, intermediates, and products, which constitute the insult to cells after procarcinogens have been incorporated into the organism [8, 9]. Thus, downregulation of

Usually, drug metabolism is the process of biochemical transformation using drug metabolism enzymes (DME) of xenobiotics, i.e., exogenous compounds introduced into organisms. The organs and tissues of animals have a diversity of DME that protects the body against both potential harmful substances from the environment (xenobiotics) and certain substances produced by the organism itself (endobiotics) [41]. Detoxification reactions comprise three phases and enzymes: phases I and II consist of metabolization reactions and enzymes, and phase III consists of transporters involved in efflux mechanisms [42]. Enzymes of phase I participate in reactions of conversion, mainly involving oxidation, reduction, and hydrolysis. These are classified as oxidoreductases (such as CYP450 monooxygenases, flavine monooxygenases, cyclooxygenases, and alcohol dehydrogenase) and hydrolases [33, 43]. Conversely, enzymes of phase II participate in reactions of conjugation and inactivation of chemical carcinogens and include transferases. Altogether, enzymes of both phases occurring independently, sequentially, or simultaneously transform xenobiotics into polar water-soluble and excretable metabolites [41]. In mammals, the metabolism of exogenous chemicals is carried out mainly in the liver, the primary site in which CYP450 enzymes are present [44]. Although procarcinogens tend to be chemically stable, they are metabolized in the liver by CYP450, a phase I enzyme, which detoxifies typically xenobiotic compounds. The activity of detoxifying enzymes in phase I is critical for carcinogenic activation of xenobiotics, while the activity of enzymes of

During the catalytic cycle of procarcinogen activation carried on by CYP450, short-lived spincorrelated RP intermediates are produced, which can either recombine or continue the catalytic process. The cycle initiates at resting state with the enzyme in the ferric form where a water molecule is implemented as the sixth ligand to iron in the active site. The ferric state of the resting enzyme has its five valence electrons occupying its orbitals, equilibrating the lowspin and high-spin state [46]. CYP450 at the beginning has ferric iron predominantly in the low-spin state. The substrate induces conformational changes displacing the water molecule from the distal axial coordinate position of the heme iron. This fact results in iron displacement from the porphyrin plane, which makes the heme a better electron sink and triggers electron

ROS and NOS could contribute to the prevention of cancer initiation [38].

phase II is essential for xenobiotic neutralization [45].

2.4. The catalytic cycle of CYP450

2.3. Metabolization

46 Vitamin E in Health and Disease

Hepatocellular carcinoma (HCC) is one of the public health enemies in low-income countries like Mexico and Brazil. It represents the fifth cause of death in the economic stage of man and ninth in women [48]. Therefore, it is important that HCC is properly understood. HCC is induced by several factors: environmental, infectious, nutrimental, metabolic, and endocrine. Certain factors such as chronic infection with hepatitis B and C, aflatoxin exposure, excessive consumption of alcohol, tobacco, and polysaturated meat consumption can also increase risk [49]. In fact, HCC is associated with liver cirrhosis and chronic hepatitis. Hepatocarcinogenesis is a complex multifactorial phenomenon that appears with loss of heterozygosity, somatic mutation, methylation, and functional inactivation [49]. The disease has a poor prognosis despite the pathophysiological advances and treatments. Chronic liver disease has an initiation point with intrahepatic inflammation promoting the dysregulation of cellular signaling pathways, triggering cell proliferation, and expanding malignant cells [50]. During this stage OS and metabolic disorders appear.

### 3.2. Nrf2 transcriptional factor + ELF-EMF

The Nrf2 (nuclear factor erythroid 2-related factor 2) transcription factor offers essential protection to cells against OS, binding antioxidant response elements and detoxifying enzymes such as glutathione S-transferase A2 and NADPH quinone oxidoreductase [51, 52]. In normal conditions, Nrf2 is found in cytoskeletal protein Keap1 (Kelch-like ECHassociated protein 1). However, when ROS and electrophiles are present, it is dissociated from Keap1, translocating Nrf2 to the nucleus, activating cytoprotective genes that participate in the electrophile conjugation and the excretion of xenobiotics. Since Nrf2 activates phase I and II enzymes, it can be considered as a target for cancer chemoprotection [53]. However, in recent studies, the beneficial antioxidant activity of Nrf2 has been extended to protect cancer cells, since excessive Nrf2 activity provokes mutations in NFE2L2 or Keap1, avoiding chemotherapy efficiency [52, 54, 55]. Another study gave evidence of Nrf2 activation in cancer [56]. Despite the enormous benefits and therapeutic chances offered by the use of Nrf2, however, the regulatory mechanisms involved at the molecular level are still not completely clear. Furthermore, ELF-EMF induces the activation of the antioxidant pathway of Nrf2, associated with the protective effect induced by the administration of 3 nitropropionic acid, which causes neurotoxicity [57], showing that ELF-EMF mitigates oxidative damage [58, 59]. For this reason, it is crucial to implement other useful tools to gain knowledge concerning carcinogenesis.

### 3.3. Experimental setup

We induce experimental hepatocarcinogenesis through the use of diethylnitrosamine (DEN) when it is hydroxylated by the CYP450 isozymes in the liver, employing an alkylation mechanism to bioactivate it, and reacting with DNA, causing ethylation in their bases, which are called DNA adducts. When those interrupt the base pairing, they provoke mutations and activation of proto-oncogenes such as ras and inhibition tumor suppressor genes such as p53, generating HCC [60]. In our experimental setup in Ref. [10], where MRHM was implemented to generate ChemIndHep, it was found that provoking the rapid proliferation of altered hepatocytes formed preneoplastic lesions in the rat liver to assess the effects of ELF-EMF on hepatocarcinogenesis. The results indicate that applying periodically an ELF-EMF was possible for achieving the modulation of the magnetic-sensitive short-lived RP intermediaries produced during the catalytic cycle. Such daily treatment with ELF-EMF (4.5 mT (120 Hz)) inhibits more than 50% of the number and area of preneoplastic lesions in rats through reduction of cell proliferation and without altering the apoptosis process. The general idea is straightforward: modulate, applying ELF-EMF, the potentially harmful products yield of the reaction by competitive kinetics of RP selective reactions [8], which give the role of a molecular motor to the enzymatic protein. Whose provision of catalyzing electrons to the reaction, is executed when through the substrate of CYP450 are metabolized the xenobiotics that participate in the ChemIndHep [9]. During the implementation of MRHM, the rats were administered a single necrotic dose of DEN (200 mg/kg b.w., i.p.). Seven days later, over 3 consecutive days, the rats were administered 2 acetylaminofluorene (20 mg/kg b.w., orally), after which they were subjected to a two-thirds partial hepatectomy. We employed three groups of six rats (normal control group (NC), shamexposure group (CT), 120 Hz ELF-EMF group (CTF)) and the biochemical and molecular evaluations were performed under blind conditions.
