3.4. Experimental results

DNA fragmentation through a calorimetric TUNEL system kit found that daily treatment with ELF-EMF did not induce apoptosis in the altered hepatocytes or caspase-3 activation, showing that ELF-EMF interferes with altered cell cycle continuity and DNA synthesis induced by ChemIndHep. This analysis also was used to detect the endogenous peroxidase activity, showing an essential diminishing in the numbers and areas of glutathione-S-transferasepositive (GST-P) liver foci and preneoplastic liver lesions in rats (Figure 1). To evaluate the effect of ELF-EMF on cell proliferation, immunohistochemical analysis of protein expression of the cell cycle such as PCN, Ki-67, and cyclin D1 were also performed. PCNA participates in replication and DNA reparation, a fundamental process for the cell cycle. Otherwise, Ki-67 is a specific replication marker of the cell cycle, which does not include the G<sup>0</sup> phase. Summarizing, the daily application of 120 Hz ELF-EMF affects the ChemIndHep process participating in the reduction of protein expression of PCNA, Ki-67, and cyclin D1. Their participation does not induce apoptosis; however, it regulates cellular homeostasis inhibiting the development of

Figure 1. Effect of ELF-EMF exposure on GGT-positive lesions and GST-P expression. (A) Representative liver sections of the NC group, CT group, and CTF group. Scale bars, 5 mm. (B) Quantification of the foci number/cm<sup>2</sup> and percent of the GGT-positive area. (C) Western blot analysis for GST-P expression. GST-P was normalized with actin expression used as the loading control. The expression of NC was adjusted to one in the densitometric unit scales. Statistically different from

Cytoprotective Effect of 120 Hz Electromagnetic Fields on Early Hepatocarcinogenesis: Experimental…

http://dx.doi.org/10.5772/intechopen.78642

49

\*NC and \*\*CT, P < 0.05. Data are expressed as the mean SEM; n = 6 for each group (of reference [10]).

preneoplastic lesions.

Cytoprotective Effect of 120 Hz Electromagnetic Fields on Early Hepatocarcinogenesis: Experimental… http://dx.doi.org/10.5772/intechopen.78642 49

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

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 evalu-

DNA fragmentation through a calorimetric TUNEL system kit found that daily treatment with ELF-EMF did not induce apoptosis in the altered hepatocytes or caspase-3 activation, showing that ELF-EMF interferes with altered cell cycle continuity and DNA synthesis induced by ChemIndHep. This analysis also was used to detect the endogenous peroxidase activity, showing an essential diminishing in the numbers and areas of glutathione-S-transferasepositive (GST-P) liver foci and preneoplastic liver lesions in rats (Figure 1). To evaluate the

knowledge concerning carcinogenesis.

ations were performed under blind conditions.

3.4. Experimental results

3.3. Experimental setup

48 Vitamin E in Health and Disease

Figure 1. Effect of ELF-EMF exposure on GGT-positive lesions and GST-P expression. (A) Representative liver sections of the NC group, CT group, and CTF group. Scale bars, 5 mm. (B) Quantification of the foci number/cm<sup>2</sup> and percent of the GGT-positive area. (C) Western blot analysis for GST-P expression. GST-P was normalized with actin expression used as the loading control. The expression of NC was adjusted to one in the densitometric unit scales. Statistically different from \*NC and \*\*CT, P < 0.05. Data are expressed as the mean SEM; n = 6 for each group (of reference [10]).

effect of ELF-EMF on cell proliferation, immunohistochemical analysis of protein expression of the cell cycle such as PCN, Ki-67, and cyclin D1 were also performed. PCNA participates in replication and DNA reparation, a fundamental process for the cell cycle. Otherwise, Ki-67 is a specific replication marker of the cell cycle, which does not include the G<sup>0</sup> phase. Summarizing, the daily application of 120 Hz ELF-EMF affects the ChemIndHep process participating in the reduction of protein expression of PCNA, Ki-67, and cyclin D1. Their participation does not induce apoptosis; however, it regulates cellular homeostasis inhibiting the development of preneoplastic lesions.
