Cytoprotective Effect of 120 Hz Electromagnetic Fields on Early Hepatocarcinogenesis: Experimental and Theoretical Findings

2.1. ChemCar

ChemCar is a multistep process initiated by the electron attack to nucleophilic tissues or molecules, such as DNA, producing mutations that lead normal cells to become unregulated, proliferate, and then turn malignant [8, 9, 28]. Metabolic activation of procarcinogens produces highly reactive chemical species that yield to the overproduction of reactive oxygen and nitrogen species (ROS, NOS), which cause damage to DNA and other biomolecules [29, 30]. The current mutation theory of carcinogenesis considers it to be a complicated process that broadly consists of three phases or stages: initiation, promotion, and progression (for a review see [31, 32, 33]). Chemical carcinogens may act together or in sequence to initiate or promote carcinogenesis. The initiation stage is characterized by an irreversible dose-dependent genetic change that predisposes normal cells to evolve into a malicious and immortal state [27]. During initiation, cells are induced to proliferate, but not differentiate [33], inheriting mutations and thus producing initiated daughter cells. In the promotion stage, the initiated cells are clonally selected to expand through an increase in cell proliferation or by a decrease in cell death, the so-called apoptosis process [30]. Although promoter stimuli do not interact directly with the DNA, they can be damaged indirectly by OS, and gene expression can be altered by epigenetic mechanisms. Nevertheless, it is considered that promotion is a reversible stage because the elimination of the promoter produces regression in cell proliferation maybe by apoptosis [33]. Lesions in both initiation and promotion stages are yet to be considered as preneoplastic ones or benign neoplasias [32]. However, in the irreversible third state, they are transformed into malignant lesions, i.e., progression. Cell proliferation independent of stimulus characterizes this stage: faster growth, invasion, metastasis, and morphological, biochemical, and metabolic changes in transformed cells [33].

2.2. Carcinogens

There exist a few different mediators that cause cancer, such as biological and physical types, but cancer induced by chemical agents is more frequent [34]. There are a lot of substances or their mixtures identified as carcinogens in the environment, in the diet, in workplaces, in homes, in cosmetics, in the chemicals employed for food production, and even in drugs for human therapeutic use [35]. It has been considered inclusive that cancer could be prevented just by identifying potentially carcinogenic chemicals and eliminating their subsequent human consumption [32]. However, such a practice does not avoid that patients have another kind of cancer. For this reason, search knowledge concerning the chemical and biological mechanisms of cancer development, mainly at very early stages, will contribute to our better understanding of the true nature of this perplexing sickness [8, 9]. Also, it will offer opportunities to generate better strategies to tackle neoplasia or to interrupt the process [32]. It is known that the earlier the detection and treatment of cancer, the better the probabilities of control, prevention, and cure because treatment will be more effective when cancer cells have not yet invaded nearby tissues or metastasized through the body [8, 9, 36]. Furthermore, it is best to interrupt carcinogenesis as soon as possible, maybe in the preneoplastic stages or, even more, at very early stages when carcinogens or by-products of their metabolism have not yet confronted the DNA. In this manner, it could be possible to prevent the progression of the sickness to the invasive terminal stage. The scenario to be considered to identify what mechanisms are needed to be stabilized, arrested, or even reversed [37] should be based on the correct approach to the chemoprevention field in a multidisciplinary study. Cancer research can involve physics and mathematics using physical, natural, synthetic, or biological agents, and mathematical tools of analyses and simulation in computers to reverse, suppress, or prevent either the initial phases of carcinogenesis or the progression of the premalignant cells to invasive sickness [38]. We use ELF-EMF together with quantum mechanics models to assess cancer. Chemical carcinogens are classified into two groups: (1) direct-acting or primary carcinogens and (2) indirect-acting or procarcinogens [8, 9, 39]. Direct-acting carcinogens are compounds sufficiently reactive and electrophilic that they interact directly with DNA forming adducts and chromosome breakage, 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 ROS and NOS could contribute to the prevention of cancer initiation [38].

2.3. Metabolization

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 phase II is essential for xenobiotic neutralization [45].

2.4. The catalytic cycle of CYP450

During the catalytic cycle of procarcinogen activation carried on by CYP450, short-lived spin-correlated 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 low-spin 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 transference [46], changing the oxidation/reduction potential in the spin state of the heme iron from the low-spin form towards the high-spin form, which is a better electron acceptor. Thus, the electron donor, which could be an iron/sulfur protein or a flavoprotein, reduces the high-spin iron(III) heme to the high-spin iron(II) state. This intermediate compound has a singlet ground state and is a resonating mixture of the ferrous and ferric forms. This conjugation requires that the dioxygen will be in a singlet state so that the empty orbital of the oxygen can mix with the second occupied orbital of the iron [46]. It is transferred in the oxygen from the activated CYP450, that is, compound I, to the heteroatom of the substrate. It has been described in the general catalytic mechanism as an odd-electron process, which involves the transfer of one electron or hydrogen atom to generate an intermediate complex that collapses by recombination [47].

3.1. Hepatocarcinogenesis

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 ECH-associated 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), sham-exposure group (CT), 120 Hz ELF-EMF group (CTF)) and the biochemical and molecular evaluations were performed under blind conditions.

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-transferase-positive (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 G0phase. 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.

4.1. Fundamental facts

Facing our limited knowledge and using the available information, we reported the cytoprotective effect of 120 Hz ELF-EMF on early ChemIndCar during the enzymatic procarcinogen activation of CYP450 by quantum measurements in Refs. [9, 79]. We proposed that when CYP450 metabolizes the xenobiotics used in the experimental setup to ChemIndHep [10], the enzymatic proteins act as a molecular motor providing a catalyzing electron that interacts with the RP formed during the OS generated when the substrate of the enzyme is oxygenated [9]. Since metabolization is carried on in the liver, the hepatocytes are in contact with the enzymatic protein as in a thermal bath and with a Gibbsian distribution, interacting with the RP as a harmonic oscillator [9]. Employing quantum measurements concepts, we argue the way in which the MF modulates the singlet spin population to diminish the preneoplastic lesion observed during ChemIndHep. The completely formed system between RP and electronic configuration of hepatocytes interacting through the HypInt alters the quantum spin state removing the spin prohibition and giving rise to the appearance of new reaction products. These products in our case result in spin selectivity plus HypInt, affecting the magnetic properties, which impact on the so-called initiated hepatocytes that later become the preneoplastic lesion to study.

We explained that on the formation of the OS was involved through the administration of xenobiotics in the MRHM, an electron transfer, and the MF modulates those in the current Haberkorn approach [9, 79, 80]. The allowed electrophilic reactions that appear in the enzymatic reactions do not require a change of spin because the spin total is zero. Those spin-forbidden reactions, involving paramagnetic participants, can combine their spins freely in any electronic configuration, but it does not mean that all configurations have a chemical reaction [9, 79]. The electron spin gives origin to the MFE, the magnetic isotope effect, and induces nuclear chemical polarization. We use the fact that according to angular momentum conservation, which is a fundamental and universal principle, all ChemReac are spin selective. This means that only those ChemReac that satisfied such a rule are allowed in the product formation. Magnetic interaction is the masterpiece that controls and accelerates the ChemReac. Nevertheless, it is many orders of magnitude less than coulomb energy. However, it has the responsibility of changing the electron spin state through the interchange energy of the channels of spin allowed and prohibited, controlling chemical reactivity [81]. The MFE act over the enzymatic DNA synthesis killing cancer cells [76]. We use the RPM because it controls life at the molecular level [9, 82, 83, 84]. We use the fact that a combination of weak static and pulsating EMF can affect radical concentration in a ChemReac [85], modifying the population of nuclear and electron spin states, their energy levels, and the RP alignment of their magnetic moments. Such changes can modify the BS [9]. Also, we use the experimental findings concerning the significant modulation of catalase, CYP450, and inducible nitric oxide synthase activity in myelogenous leukemia cells [86] to reaffirm our idea with respect to the effect of ELF-EMF on the enzymological system. A more critical step is the activation of xenobiotics whose interaction produces OS in the form of electrophiles and ROS [30, 40]. The main protagonist in our approach are the so-called RP, short-lived intermediates that participate in almost all reactions in solution in a correlated way. The RP can recombine or participate in other ChemReac. They are responsible for a few phenomena such as chemical polarization of electrons and nuclei, and the influence of static and pulsating MF [9, 79]. An RP can decay by recombination, or pull apart the radical by diffusion, or react with other radicals. One of the properties of RP is that recombination probability depends on spin multiplicity, and it varies during RP lifetime. Such variations, as an interesting detail, are manifested as dynamic quantum oscillations, the so-called quantum beats between (S, T) spin states of the RP. The quantum beats modulate the probability of appearance of some reaction channels of the RP that at the time affect the MFE. By studying these quantum beats, one can reveal valuable information concerning the structure, reaction, molecular, and spin dynamics of RP [9, 63, 79, 87]. The RP spin correlation is formed in the coherent state, which oscillates between theSand Tspin state, an oscillation that depends on the spin Hamiltonian operator parameters (see references [8, 9] for details), in particular that of the HypInt. The period of the oscillation on organic radicals is in the range of nanoseconds, making RP recombination a plausible test that suggests that small EMF affects BS [9, 88].

4.2. The model

ChemReac can be seen as a physical process involving a set of regrouping atoms with the rearrangement of electronic shells of reacting participants, giving place to the generation of new molecular structures called reaction products. The new ways of controlling ChemReac have their basis on the selectivity of spin, a process involving the spins of molecules, electrons, and nuclei of all participants. For this reason, the rate of spin-selective processes is dependent on MF, which alters the spins of the participants, changing partially or wholly the spin selectivity [9, 89, 90]. Thus, to reveal the interaction to explain the cytoprotective effect of ELF-EMF in CYP450, we must define the conditions where the quantum measurement is performed. The first condition is that all quantum states participating in the hepatocytes-RP system in the enzymatic reaction are singlets, because of their high reactivity. The second condition is during the enzymatic procarcinogen activation of CYP450 when the xenobiotics are metabolized, in which appear the RP when is generated the OS. The RP intermediaries are produced in this step, and they are responsible for the insult to hepatocytes, which become the future preneoplastic lesions after to finish the ChemIndHep. The third condition is daily MF stimulation during all ChemIndHep. Nonetheless, the spin evolution of RP is driven by the MF through the HypInt and their reactivity is controlled by spin dynamics, converting non-reactive triplets into reactive singlets through quantum measurement. We showed the way in which the MF modulates the charges in migration evaluating the recombination probability to exemplify. In this respect, when the RP interacts with another electrons’ spin, this interaction acts as a catalyst, increasing the recombination probability and accelerating the S⟷Tinterconversion [9, 90, 91].

4.2.3. Results on hepatocytes

We described the effect through the following dynamical mapping [9, 79] ∥S>∥0>Hep→η1∥P>∥σ>Hep+η2∥S>∥0>Hep, ∥T0>∥0>Hep→∥T0>∥0>Hep.When we normalize it, measurement of the generation of some reaction product on the hepatocytes ∥P>is represented according to [9, 79]:

ρ̂0=∥AS∥2ASAT0∗AT0AS∗∥AT0∥2→ρ̂n=β∥η1∥2∥AS∥2η2ASAT0∗η2∗AT0AS∗∥AT0∥2,β=11−∥η1AS∥2.E4

This fact means that the hepatocytes measure the spin nature of the RP that is participating. The spin state of the hepatocyte changes according to the spin nature of the RP that is interacting. During the measurement process, the Sand T0components do not change, but the hepatocytes change their spin states according to the RP spin character following the dynamical mapping [9, 79] ∥S>∥0>Hep→β1∥S>∥σ>Hep+β2∥S>∥0>Hep,∥T0>∥0>Hep→β3∥T0>∥χ>Hep+β4∥T0>∥0>Hep,instituted by the interaction [9, 79]:

Ĥin=ηS∥S><S∥⨂∥σ>Hep<0∥+∥0>Hep<σ∥+ηT0∥T0><T0∥⨂∥χ>Hep<0∥+∥0>Hep<χ∥E5

whereηSand ηT0give us the strength of the interaction of the hepatocytes with the RP spin character, appearing in the new term, β3∥T0>∥χ>Hep, without ∥P>states. In this case, the probabilities ∥β1∥2or ∥β3∥2express the appearance of ∥S>or ∥T0>spin states, represented by ∥σ>and ∥χ>[9]. They measure the fraction of singlets transformed into a reaction product or transformed cell. It is precisely this kind of product formation that changes the electronic configuration of the hepatocytes, as we claim. The result is evident from Figure 2, where we note that by increasing the hyperfine coupling constant, A, the singlet population is diminished. Thus, once the hepatocytes interact during δtwith the CYP450, they change their spin states through the application of the selective spin operator. For each time interval, δtwill apply some dynamical mapping to each new healthy hepatocyte in the tissue ∥0>en,incorporating them into the enzymatic reaction that provides catalyzing electrons during the metabolization of xenobiotics [9], ∥Ψδt>sen=Uδt∥Ψδt>0=e−iĤinδt∥ϕ0>s∥0>en.Also, neglecting the memory effects due to the previous results with other singlets, which are diminishing as is evidenced by the quantum measurement ∥β1∥2=kSδt, changes the electronic configuration of the hepatocytes when the RP is converted in a reaction product by the recombination kinetics [9, 79].