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Physics of biology and medicine
Reference:

Effect of Weak Alternating Magnetic Fields on Neutrophilic Granulocytes: An Analytical Review

Shaev Igor' Alexandrovich

ORCID: 0009-0006-9990-967X

Graduate student, Institute of Cell Biophysics RAS

142290, Russia, Moscow region, Pushchino, Institutskaya str., 3

algimantas444@mail.ru
Novikov Vadim Viktorovich

Doctor of Biology

Vadim V. Novikov leading Researcher, Institute of Cell Biophysics RAS

142290, Russia, Moscow region, Pushchino, Institutskaya str., 3

docmag@mail.ru

DOI:

10.7256/2730-0560.2023.1.40410.2

EDN:

SWCJQU

Review date:

10-04-2023


Publish date:

08-06-2023


Abstract: This article discusses the main achievements in recent years in the study of the biological effects of weak and super weak low-frequency magnetic fields, either variable or combined with constant ones. Considered are neutrophil granulocytes activated by chemical stimulants or intact when the magnetic fields affect isolated cells, blood, and whole organisms. The methods include recording changes in ROS concentration levels (the most noticeable effect of exposure to a weak magnetic field), priming index, calcium homeostasis, proliferative activity, immune status, and the influence of various chemical agents on these indicators. The leading methods in this field are fluorescence spectrometry and chemiluminescence analysis. The experimental results indicate the biological effectiveness of this physical factor, the specific effect of which depends on the type of biosystem, its functional status, the environment, and the parameters of the fields themselves. The data obtained can have applied significance in magnetotherapy, immune response optimization in various diseases, acceleration of tissue regeneration and repair, and increasing the body's resistance to infections. They also can have academic significance as they help identify the primary field acceptors and magnetic targets and their localization in the cell, study relationships with signal cascades, build models of biological signal amplification pathways, and find biologically significant frequencies and field amplitudes.


Keywords:

weak magnetic fields, alternating magnetic fields, combined magnetic fields, free radicals, reactive oxygen species, neutrophils, NADPH oxidase, respiratory burst, calcium homeostasis, chemiluminescence

Numerous biological effects of weak alternating magnetic fields (EMF) in the context of their impact on neutrophils have been recorded on various models, ranging from the organism to the cellular and subcellular levels. One of the most common among them in neutrophil granulocytes are changes in the activity of free radicals and in the concentration levels of reactive oxygen species (ROS), nitrogen and chlorine, and antioxidant enzymes that support the homeostasis of these forms. This is partly because neutrophils' immune functionality is associated with the ability of these cells to generate a number of biologically aggressive molecules, such as superoxide anion radical (NADPH oxidase complex), hydrogen peroxide, and hypochlorite (myeloperoxidase). In addition, like any other cell, a neutrophil granulocyte detects the use of active forms in signaling pathways, as well as the presence of some forms of ROS as a by-product in the electron transport chain (ETC) of mitochondria, the presence of a network of which in this type of cell is currently a proven fact. It should be noted that neutrophil granulocytes are the most numerous leukocytes in humans (about 2 billion in the bloodstream and 8–50 billion in the tissues) and are, in this regard, the primary sources of free radicals and other ROS in the blood and bodily tissues. A brief description of the functionally important oxidants produced by neutrophils is given in Table 1.

Table 1. Brief chemical characterization of the most important oxidizing agents in neutrophil granulocytes (adapted from Winterbourne et al.) [1].

Oxidizer

Specifications

superoxide anion

A weak one-electron oxidizer and a moderate reducing agent, ionized at neutral pH (pKa 4,8). The protonated form (HO2•) is a more active oxidizer. Low permeability for membranes. The fastest reactions with iron-sulfur centers, NO, and other radicals react slowly with thiols. Radical addition reactions give hydroperoxides.

hydrogen peroxide

A strong two-electron oxidizer, but due to the high activation energy, a small amount of biological substrates. The most rapid reactions with thiol and selenium peroxidases, with other thiols less reactive, reaction with thiolate. Reacts with centers containing transition metals to form hydroxyl radicals or initiate radical reactions. It passes through membranes, mainly through aquaporins.

hypochlorite

Strong two-electron oxidizer, has a wide range of substrates. HOCl is more reactive than OCl- (pKa 7,4) and penetrates through membranes. The fastest reactions with cysteine and methionine residues, reacts well with ionized (low pKa) thiols. Thiol oxidation products include disulfides and products with higher oxidation states. Secondary reactions include chlorination of tyrosine residues (with the formation of 3-chlorothyrosine) and nucleotides. In reactions with unsaturated lipids, chlorohydrin is formed.

chloramines

They are generated from HOCl and amino groups of amino acids or proteins by the reaction R-NH2 + HOCl. R-NHCl + H2O. Two-electron oxidizers weaker than HOCl, and more selective with respect to sulfur-containing centers. More reactive with thiols with low pKa. Slowly decompose to form aldehydes and ammonia. They are split by transition metals with the formation of radicals. Reactions with the second HOCl molecule give more reactive dichloramines.

hypothyocyanite

The predominant form of hypothyocyanic acid (pKa 5,3) at neutral pH. Almost all reactions with cysteine residues form disulfides; reactions with thiols with low pKa are preferred. Does not react with methionine.

organic radicals

It is formed from many substrates due to the activity of myeloperoxidase. Initiates chain reactions such as lipid peroxidation. Depending on the reduction potential, they range from highly reactive (for example, NO2) to almost inert (ascorbyl).

Superoxide has a limited reactivity (the main target is iron-sulfur proteins [2]) and, in the context of the antimicrobial potential of neutrophils, plays the role of a precursor to hydrogen peroxide and other oxidants. Thus, peroxynitrite, a powerful oxidizer and microbicide [3], can be produced in the reaction of superoxide with nitrogen monoxide, provided that both are generated simultaneously. With activated neutrophils, the primary producer of superoxide is the NADPH oxidase complex NOX2, which catalyzes the one-electron reduction of oxygen during phagocytosis and in response to stimulation by bacterial components [4] and immune signals.

Hydrogen peroxide requires sufficient concentrations to manifest its bactericidal effect. Thus, in a neutrophil granulocyte, even with a myeloperoxidase deficiency, when the model predicts a peroxide concentration of about 30 microns [5], the volumes of this oxidant are not sufficient for it to manifest a toxic effect, which is realized mainly through a reaction with intracellular iron-sulfur centers with the release of iron and subsequent formation in the Fenton reaction [2] hydroxyl and other highly reactive radicals.

Hypochlorous acid (hypochlorite) produced by myeloperoxidase (MPO), even in relatively low concentrations, negatively affects a wide range of microorganisms, the destruction of which by isolated neutrophils is severely impaired in the absence or deficiency of this enzyme [6, 7]. Being a component of azurophilic granules, MPO is a heme-containing enzyme that oxidizes the chlorine anion to hypochlorite in the presence of H2O2 and makes up more than 5% of the dry mass of human neutrophils [8]. The MPO-H2O2-Cl system requires a continuous supply of chlorine ions, which causes high concentrations of anions of this element in the cytoplasm of neutrophils, from where they are transferred to phagosomes using special transporters, mainly CFTR [9, 10]. The reactions of HOCl with amino acids, peptides, and proteins have been thoroughly studied [11]. They lead to the formation of products with antimicrobial activity, including chloramines and, ultimately, aldehydes [12], which in turn leads to the formation of tyrosyl radicals capable of causing dimerization and aggregation of proteins [13]. Thus, hypochlorite is the fastest-acting and most powerful antimicrobial oxidizer neutrophils produce.

The main purpose of this review is to analyze the effects and mechanisms of biological action of weak and ultra-weak low-frequency variables and combined magnetic fields on neutrophil granulocytes. The analysis of experimental data in this area is important not only because of the significant number of "white spots" regarding understanding the effects and mechanisms of the influence of weak MP on neutrophils but also because of the unique role these cells play in the immune response. Being the most mobile fraction of immunocompetent cells, neutrophils are the first to be in the focus of inflammation, occupying the position of the initial link in the chain of protective reactions of the body. Therefore, by influencing these cells with MP, it is possible to achieve changes in the main characteristics of nonspecific immunity and general immunoreactivity and direct the vector of these changes toward optimizing the immune response.

The variety of effects of PEMP in relation to neutrophils is mainly reduced to their activation. In Cuppen et al. [14], it has been shown that short-term exposure to low-frequency EMF in vivo has a significant activating effect on neutrophils of the peripheral blood of humans. As a marker of activation, the degree of degranulation was considered, which is a critical effector function in these cells, triggered at an early stage of neutrophil recruitment. Thus, a decrease in the number of granules corresponds to a higher state of neutrophil activation [15]. In the experiment, the induction coil was positioned so that the field generated by 5 µT in the center was almost parallel to the lines of force of the geomagnetic field, the magnitude of which was 47 µT. The total field was formed at frequencies of 320, 730, 880, and 2600 Hz, and the exposure time was 30 minutes. The achieved effect was manifested in a decrease in the number of granules in neutrophils (a shift of 0.7 compared to the baseline level of 140), which, despite a relatively small value, may imply a further strengthening of the immune signal, as was demonstrated earlier in experiments on animal objects, the results of which also confirm the biological relevance of fields with these induction values. Thus, an in vitro study on such objects as phagocytes of the kidneys of common carp showed that exposure to a low-frequency electromagnetic field (LF EMF) with parameters of 200–5,000 Hz at 5 µT and 1.5 MT (30 min exposure) led to an increase in immune activity relative to the control by 42 and 33%, respectively, and with preliminary chemical stimulation phagocyte activity after exposure to low-frequency EMF increased by 18% (5 µT) and 22% (1.5 MT). In addition, experiments on whole organisms (goldfish and broiler chickens) demonstrated a significant reduction in mortality of infected animals, reduced tissue damage, increased viability and enhanced functional response of immune cells after infection (also in infectious models with LPS in experiments with cell cultures in vitro), which revealed the biological activity of fields in the range of from 0.15 to 50 MCT, which in the case of fish was expressed in a decrease in mortality from 50% to 20% 18 days after exposure to fields of 5 MCT, and in the case of chickens (6.5 MCT) led to a decrease in intestinal damage caused by coccidiosis by 40% [16]. The effect was observed uniformly over the entire studied range of magnetic induction values, falling to zero at values below 0.01 µT. According to the authors, these results indicate the ability of fields with these characteristics to cause moderate immune stress, leading to a more optimal launch of the immune response with minimal negative consequences. The nature of the immune signal enhancement demonstrated in these studies may be explained by the fact that the body contains a large number of neutrophils, of which only a few need sufficient activation. Having the ability to trigger amplification processes with positive feedback, these cells contribute to effective migration along the chemotactic gradient, induce multiple waves of recruitment to the site of inflammation and infection, and contribute to the implementation by other cells of various effector functions to ensure antimicrobial immunity [17].

In the case of body subsystems containing neutrophils, such as, for example, blood, PEMPS also demonstrate their biological effectiveness. In the work with the use of combined magnetic fields (CMF) [18, 19] under the influence of this physical factor (a constant magnetic field [PMF] of 42 µT; PeMP of 1 Hz, 600 Nt; 4.4 Hz, 100 Nt; 16.5 Hz, 160 Nt) on human blood, a significant increase in the luminol-, as well as lucigenin-dependent chemiluminescence. Theoretical constructions [20] made it possible to consider as possible causes of this effect the generation of singlet oxygen at the primary reception of the energy of the CMP by protons of the aquacomplexes of the medium, as well as the paramagnetism of deoxyhemoglobin (experiments were conducted on venous blood), but the leveling of this effect by apocynin (NADPH oxidase inhibitor) and its reproducibility with lucigenin (selective probe for superoxide anion radical) pointed to the determining role of Nox proteins. At the same time, experiments with different values of the induction of a variable component in the range of 108–3440 NT demonstrated a linear dependence of the magnitude of the effect on the amplitude. Concerning singlet oxygen and its role in the bioeffects of CMP, experiments with histidine [21], which is an interceptor of this ROS in concentrations up to 1 mM, demonstrated a negligible effect on the priming effect of CMP: the priming index was 2.3 in the control and 2.8 in the experiment. The increase in the index in samples with histidine is explained by the more pronounced inhibitory effect of this agent on control samples.

Subsequent experiments [22] with activated formylated peptide (fMLF) or phorbol ether (PMA) neutrophils, both human and mouse, showed the significance of weak CMR (variable component 0.86 µT) as a priming factor (approximately 10-fold increase in chemiluminescence in experimental samples). The intensity of this effect, which is almost the same with a tenfold difference in the concentration of the added fMLF, together with the fact of enhanced response to stimulation with phorbol ether (direct activator of protein kinase C), indicated that the nature of the priming mechanism is most likely not related to the activation of receptors. The absence of a significant signal of luminol-dependent chemiluminescence of neutrophils during the pretreatment of CMP and its considerable growth in the suspension of activated neutrophils may indicate, in the latter case, the participation of the main oxidizer of luminol-hypochlorite [23] or its precursors (hydrogen peroxide). Also, the nonreceptive nature of the biological response to the effects of CMP was confirmed in an experiment with partial degassing of a neutrophil suspension (the oxygen content was 412 ng-atom O/ml), in which the priming ability of a weak CMP was significantly reduced (approximately 4 times) [24]. Taking into account the fact that the degassing procedure does not affect the ability of neutrophils to be functionally activated under the action of a formylated peptide in the absence of field exposure, the results obtained may indicate a connection between the oxygen level in the medium and the reception of weak CMR.

In vitro studies show that even a minor effect of LF MP can affect cellular mechanisms [25]. Indeed, the cells were found to respond to the induction of a field of only ~0.15 µT [26]. In vitro, neutrophils demonstrate increased ROS production after 15 minutes of exposure to MP [27]. In this experiment, the values of PEMP induction were 10, 40 and 60 µT, the frequency was 180–195 Hz (the cyclotron frequency of the calcium ion was also present as modulation), the geomagnetic field (GMP) was 50 Mt, the activator was PMA, and the fluorescent probes were 2'7'-dichlorofluorescein-diacetate and dihydrorhodamine 123 (detection was carried out by flow cytometry). It has been shown that only MP-containing modulation tuned to the frequency of cyclotron resonance of calcium ions can affect the production of ROS in neutrophils, and statistical analysis has demonstrated the dependence of this effect on the magnitude of the magnetic induction of the field. At the same time, unstimulated neutrophils slightly reduced ROS production in response to incubation in this CMC, while a more pronounced effect of fields on cells was observed in samples in which dichlorofluorescein was used to detect ROS. Although this probe is less sensitive than dihydrorhodamine 123, it is probably more selective as an indicator of altered ROS production after exposure to MP [28, 29].

The authors' opinion regarding the explanation of the decrease in ROS levels in unstimulated neutrophils is based on the assumption of a change in the biological activity of enzymes that catalyze reactions associated with the formation of ROS, for example, peroxidase [30] or glutathione-S-transferase [31]. Considering a similar effect for inducible nitric oxide synthase [32], such results could be viewed as a general method of MP exposure to resting unstimulated cells. However, it has been shown that other MP parameters cause opposite (stimulating) effects [33].

As for the increased production in stimulated neutrophils, in this case, an increase in the activity of one of the enzymes that catalyze the cascade of reactions during a respiratory explosion is assumed. The most likely candidate is NADPH oxidase [34], the activation of which leads to the formation of ROS.

The "window effect" associated with the effectiveness of certain EMF frequencies is confirmed by earlier studies, in particular, Gamalei et al. An increase in the enzymatic activity of NADPH oxidase in mice was reported in the EMF range of 15-99 Hz with a maximum of 50 Hz [35]. "Windows" can also be observed in the ranges of magnetic induction since, for example, the occurrence of a reaction of the "cyclotron resonance" type depends on several parameters—the frequency and intensity of the variable and constant components of the applied field. This was confirmed in experiments on the effect of pulsed magnetic fields on the activity of NOS [36], where the action of the enzyme increased only at 0.1 Mt, and in experiments with monoamine oxidases (50 Hz, 10–340 Mt) [37], which demonstrated the potentiation of MAO-B activity at 100 Mt. It should be noted that an important condition for the occurrence of a reaction of the type of induced cyclotron resonance is the parallelism of the applied static magnetic (the role of which can be played by a geomagnetic field) and alternating magnetic fields [38, 39, 40, 41, 42].

The relevance of variable fields with frequencies tuned to the resonance of Ca2+ ions with respect to modulating the levels of neutrophilic ROS production was also confirmed in the study of Belova et al. [43], where it was shown on activated cells that the sign of the effect demonstrates dependence not only on the characteristics of the physical factor itself but also on the type of chemical stimulator. Thus, neutrophils activated with phorbol ether and placed in a field with parameters 74.7 MCT, 31.0 Hz (constant component, also known as GMP, 40.6 MCT) demonstrated a decrease in the luminol-dependent chemiluminescence signal by 23% against the control. At the same time, the cells activated by the bacterial peptide fMLF showed an increase in the rate of ROS production by 21%. The fact that the type of chemical activator is not decisive in relation to the sign of the effect was shown by the example of a pulsed magnetic field (1500 µT, 15 Hz, angle with respect to the GMP vector 30°) in the case of which a 20% increase in ROS was observed in PMA-activated neutrophils. The authors try to explain the inhibitory effect of CMR through the mechanism of parametric resonance of the double-charged Ca2+ ion (primary magnetomishen), the time of which in the binding center of kinase enzymes (calmodulin-dependent and protein kinase C) changes, in their opinion, under the influence of these fields, which affects the phosphorylation of the protein p47phox NADPH oxidase complex. In the case of stimulation with a bacterial peptide, the effects obtained can be explained by negative feedback between the activities of phospholipase C and protein kinase C in peptide-activated neutrophils [44], as well as the involvement of NADPH oxidase activation pathways by phospholipid hydrolysis products phospholipases [45]. The result with an IMP, the primary target for which is not apparent, can be explained based on the similarity of its effect, which consists in the sign of the latter, with the impact of the KMP tuned to the frequency of the K+ ion [46]. Ultimately, the enzymatic activity of the aforementioned kinases changes with a response to the final link – NADPH oxidase.

In conclusion, the study's authors emphasize the significance of the field parameters for the sign of the effect. While Ca2+-KMP (74.7 MCT, 31.0 Hz) reduces the ROS concentration, in the works of Roy et al. and Noda et al. CPM with resonance on hydrogen nuclei (141 MCT, 60 Hz) causes an increase in the production of these metabolites in PMA-activated neutrophils (by 12.4%) [47, 48]. Experiments on planaria, where their regenerative ability was used as a marker, also revealed a multidirectional effect for fields with resonance on calcium and hydrogen (stimulation of regeneration and its inhibition, respectively) [49].

An interesting hypothesis explaining the effect of EMF on NADPH oxidase in activated neutrophils is the assumption that the activity of this enzyme can be coordinated with the work of potential-dependent selective proton channels detected in neutrophil granulocytes [50]. The activity of NADPH oxidase during a respiratory explosion leads to the release of protons into the cytoplasm, which causes rapid depolarization of the cell membrane and a change in pH in the cell; both of these mechanisms regulate the opening of potential-dependent proton channels. Thus, a study using a proton channel blocker VSOP/HV1—zinc sulfate (100 mM) [51] demonstrated a weakening of the respiratory explosion in neutrophils from experimental samples more substantial than in control (a drop in the priming index by 16%), which may indicate the role of these channels in maintaining the membrane potential and intracellular pH at values, favoring the regular operation of superoxide-producing enzymatic complexes, which, due to their electrogenicity, can shift these values, forming negative feedback.

An increase in the production of ROS by the NADPH oxidase complex in response to EMF stimulation can serve as a prologue to a change in the immune status by, for example, enhancing the formation of neutrophil traps initiated by chemical stimulants [52]. Of particular interest is the role of calcium homeostasis in these events, while this role, as shown by these experiments, turned out to be insignificant or zero. Thus, studies using human promyelocyte lines differentiated into neutrophils have demonstrated that in vitro exposure to a 50 Hz sinusoidal field at 5 µT (peak value of 500 µT) did not affect the transmission of signals along calcium pathways [53]. The expression of genes associated with calcium-dependent signaling pathways, cellular morphology, and the presence of intracellular microvilli also did not change under the influence of low-frequency fields in either HL-60 or PLB-985 cell lines [54]. Thus, under these specific experimental conditions, the nature of exposure to fields, and the type of analyzed cell lines, the effect of CNF MP does not seem to affect the homeostasis of calcium in neutrophils. At the same time, a study conducted by Novikov et al. [55] demonstrated, using a luminol-dependent chemiluminescent method, the blocking of intracellular calcium chelator BAPTA AM (acetoxymethyl ether 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) priming effect of KMP (PMP 42 MCT; PeMP 1.0; 4.4 and 16.5 Hz; 0.86 MCT) on neutrophils activated by formylated peptide. A similar result with a chelating agent was achieved in experiments on whole blood. In the same experiment with the use of a hydroxyl radical interceptor (dimethyl sulfoxide (DMSO) at concentrations of 0.025–1.0 mM), the hypothesis about the role of lipid peroxidation in the cell membrane was tested, the possible initiation of which under the action of CMR would lead to replenishment of the pool of calcium ions in neutrophils from the extracellular medium. The results obtained (the absence of the DMSO effect in the indicated concentration range) showed favor of the process of calcium recruitment from intracellular depots as a key condition for the development of neutrophil priming under the influence of CMR. As for the role of the hydroxyl radical in the effects of CMP, it can be said that since the use of another interceptor of this ROS—ethanol [52] at 0.45% concentration—showed the significance of this chemical agent in relation to a decrease in the priming index (by 27%) of neutrophils activated with formylated peptide and treated with CMP, in the light of previously obtained experiments with DMSO The results may indicate both the debatable nature of the question of the role of the hydroxyl radical in the effects of CMR and the possible conditionality of this result by the special physicochemical properties of ethanol.

In the future, the absence of a determining role of lipid peroxidation (POL) relative to the priming effect of CMP was confirmed in experiments with the use of an inhibitor of lipid peroxidation-ionol (10 microns) [22]. The slight increase in POL detected in these experiments, recorded by the rise in the content of malondialdehyde by 10.2% after pretreatment of the CMP (the parameters are the same as in the previous study), is most likely not associated with functional preactivation of activated bacterial peptide and field-treated neutrophils, since the addition of ionol (2,6-Di-tert-butyl-4-methylphenol) did not significantly affect the priming index. Thus, in these experiments, the role of endogenous calcium in the biological effect of CMC was confirmed, which is consistent with the results of Bertagna et al. [56] obtained on HEK 293 cells, for which the influence of alternating radio frequency and constant magnetic fields (induction of 0.4 and 1 MT, respectively) on intracellular calcium levels was assessed. The effect was to increase the level of Ca2+ in both cases, but was blocked by the endoplasmic reticulum Ca2+-ATPASE inhibitor tapsigargin (10 microns), which indicates the role of this organoid in the reaction to the MP of Ca2+ homeostasis. In addition, in a recent study by Ye et al. [57], the effect of shallow frequency (50 Hz) fields on the proliferation of human amniotic epithelial cells was demonstrated by the impact of field-induced intensification of cell division, which was completely blocked by the intracellular calcium chelator BAPTA, as well as partially by the L-type calcium channel inhibitor NIF. In addition, inhibition of field-activated sphingosine kinase 1 (SK1) by its specific SKI II inhibitor eliminated the field-induced proliferative effect. Further analysis of the role of certain components (ERK, PKCa, and Akt kinases) of signaling pathways in this effect showed that only Akt (serine/threonine-specific protein kinase, also referred to as protein kinase B) is activated in response to exposure to the field and that the use of an inhibitor (2-morpholine-4-il-8-phenylchrome-4-oh, LY294002) of this phosphoinositide-3-kinase led to the elimination of the effect of MP. Based on these data, it can be concluded that the proliferative effect is associated with releasing intracellular calcium from the depot due to the activation of the Akt-SK1 signaling pathway.

These and other works confirming the change of calcium homeostasis under the influence of both variable and static magnetic fields [58; 59; 60; 61], as well as studies in which these changes have not been proven [62], indicate the extreme variability of the effects caused by the magnetic field, which, as it is believed, they depend on the frequency and amplitude of the area, the exposure time and the studied model [63].

In addition to the NADPH oxidase complex, myeloperoxidase is another candidate for the role of an enzyme involved in the effects of alternating magnetic fields on neutrophils. This is supported by the results obtained using sodium azide [22]. The addition of this myeloperoxidase inhibitor in concentrations up to 1 mM leads to a decrease in the chemiluminescence signal of neutrophils activated by the formylated peptide by approximately 5 times, both in control and in experimental samples, and the priming effect of CMP (42 MCT; 1.0, 4.4 and 16.5 Hz; 860 Nt) does not develop (the index drop from 3.75 to 1). In another experiment with CMR, the variable component of which is close to environmentally significant parameters (industrial frequencies), the inhibitory effect of the field on the respiratory explosion in fMLF-activated neutrophils was demonstrated [64]. 40-minute processing by a field with parameters of PMP 60 µT; PeMP 49.5 Hz, 80-180 Nt caused a decrease in the luminol-dependent chemiluminescent signal at all values of this induction range, the maximum at the point of 120 Nt (60%). Beyond the boundaries of the range (outside the amplitude "window"), the inhibition effect is manifested to a much lesser extent and, at the point of 480 NT, does not manifest at all. The frequencies bordering with the studied one have a lower inhibition potential (20% each for 46.0 and 48.5 Hz with an amplitude of 120 Nt), and 33 Hz is not valid, indicating a frequency "window." The use of a multi-frequency signal (33.0 Hz, 46.0 Hz; 48.5 Hz, 49.5 Hz, the total amplitude of 480 NT) did not reveal an additive nature in terms of biological effect, but on the contrary, demonstrated an inverse synergy, which consisted in reducing the intensity of the respiratory explosion by 30% (2 times less impact than in the case of 120 NT, 49.5 Hz), which can be associated with going beyond the boundaries of the amplitude "window." Since the value of 49.5 Hz corresponds to the resonant frequency of the Fe3+ ion, and the luminol-dependent signal suggests the presence of hypochlorite in the tested object, it seems possible to link the biological efficiency of the field with the effect on myeloperoxidase, as an iron-containing enzyme.

Another possible source of ROS in neutrophils is associated with mitochondrial ETCS. However, this compartment does not seem to play a significant role in the effects of variable MP since an attempt to influence the deployment of priming in neutrophils with rotenone (an inhibitor of the first ETC complex) demonstrated its inefficiency, which may indirectly indicate the absence of a link between the effector potential of variable fields (specifically KMP) with processes in mitochondrial ETCS [52].

Localization of the discussed ROS, the increased concentration of activated neutrophils following EMF treatment, is intracellular. In Roy et al. on rat peritoneal neutrophils, it was shown that when PMA neutrophils were started at a concentration of 50 nM, the effect of an alternating magnetic field of 0.1 Mt (60 Hz) consisted in a 12.4% increase in the DCFH fluorescence signal compared with the control, while the fluorescence of the buffer directly adjacent to the neutrophil (within one cell diameter) was similar to background fluorescence up to stimulation of PMA, which indicated the intracellular origin of ROS [48]. An additional experiment with hydrogen peroxide showed that the latter's reaction with PMA is not decisive for the manifestation of the magnetic field effect. The range of possible explanations for this effect includes both physical hypotheses (an increase in the lifetime of radicals demonstrated in chemical systems, for example, with respect to the tocopheroxyl radical [65]) and biological (the effect on the activation of the respiratory explosion of PMA). These results are consistent with the assessment of intracellular ROS production using dichlorofluorescein diacetate and Dihydrorhodamine 123 in the work of Novikov et al., where the effects of CMP (variable component 0.86 µT) were demonstrated, consisting in an increase in ROS production and a significant potentiation of this growth when neutrophils are activated by chemical stimulants [34]. At the same time, priming in the case of using phorbol ether was more pronounced and was recorded on both probes, while the result of the action of the formylated peptide manifested itself when using the dichlorofluorescein method.

The obtained cumulative data indicate mainly the stimulating effect of variable magnetic fields of the nano- and microtesl range on neutrophil granulocytes, which in the vast majority of cases manifests itself in the form of changes in the intensity of intracellular ROS production, while, as a rule, the activity of the NADPH oxidase complex, as well as myeloperoxidase, is modulated. The mitochondrial apparatus does not take a significant part in these effects of PeMP and CMP. The range of ROS data includes superoxide, hydrogen peroxide, and hypochlorite. The roles of singlet oxygen and hydroxyl radicals are debatable. Variable fields have priming ability, which is based on mechanisms unrelated to the involvement of receptors or the development in the membranes of the POL. The bioeffect's dependence on the field's amplitude can be linear or with extremes at certain induction values. The presence of "windows" of efficiency is also observed in the frequency range. The role of calcium ions as secondary messengers in developing the effect is ambiguous. The presence of changes in calcium homeostasis depends on the parameters of the field.

In conclusion, it should be noted that the range of up-to-date information on the biological action of weak PEMPS and CMPs indicates that the heterogeneous effects of this physical factor demonstrate dependence on the type of biosystem (in particular, cells), their functional status, their environment and the parameters of the fields themselves. The revealed experimental dependences of the effects of PeMP on frequency do not exclude the possibility of the realization of resonant effects, theoretical approaches to which have been developed for a long time [66], and despite the apparent progress [67, 68, 69, 70] are still far from being fully understood. It is still unclear to what extent the widely discussed mechanism of radical pairs [71, 72] and other general physical mechanisms [73] can be applied to the analysis of these effects.

Tables 2 and 3 summarize the biological effects of weak variables and combined magnetic fields and the impact of various chemical inhibitors on these effects.

Table 2. Biological effects of weak variable and combined magnetic fields

PMP Parameters

Type and object of the experiment

Marker

Biological effect

5 mkTl, 320, 730, 880, and 2600 Hz, GMP 47 mkTl (collinearly), 30 min [14]

In vivo

Peripheral blood of people

Degranulation

Activation

5 MCT and 1.5 MT, 250-5000 Hz, 30 min [16]

In vitro

Phagocytes of the kidneys of common carp

Growth of the spectrophotometric signal of nitrosine tetrazolium oxidation products

Activation

From 0.15 to 50 µT, 250-5000 Hz, 30 min [16]

In vivo

Goldfish; broiler chickens

Reduction of mortality; reduction of intestinal damage caused by coccidiosis

Optimization of the immune response

1 Hz, 600 Nt; 4.4 Hz, 100 Nt; 16.5 Hz, 160 Nt; GMP 42 MCT [18, 19]

In vitro

Human blood

signal the growth of luminol- and lucigenin-dependent chemiluminescence

Activation of NADPH oxidase, generation of ROS

0.86 MCT, 1; 4.4 and 16.5 Hz, GMP 42 MCT (collinearly), 60 min [22]

In vitro

Mouse and human neutrophils activated with formylated peptide or forbol ether

Enhancement of chemiluminescence

Activation Growth

10, 40 and 60 MCT, 180 – 195 Hz, GMP 50 MT [27]

In vitro

Activated PMA neutrophils

Amplification of the fluorescent signal

AFC generation

74.7 µT, 31.0 Hz, GMP collinearly 40.6 µT (parametric resonance for Ca2+), 0 – 60 min [43]

In vitro

Activated mouse PMA and FMLP neutrophils

Decrease or increase of the luminol-dependent chemiluminescence signal depending on the type of activator

Generation of ROS by NADPH oxidase activated via different signaling pathways

Table 3. Influence of various chemical inhibitors on the biological effects of weak variable and combined magnetic fields

Experimental object, field characteristics

Field effect

Inhibitor and its function

Inhibitor effect

Human venous blood, PMP 42 MCL; PeMP 1 Hz, 600 Nt; 4.4 Hz, 100 Nt; 16.5 Hz, 160 Nt [18, 19]

Increase in luminol- and lucigenin-dependent chemiluminescence

Apocinin; inhibition of NADPH oxidase

Canceling the field effect

Neutrophils, PMP 42 MCT; PeMP 1 Hz, 600 Nt; 4.4 Hz, 100 Nt; 16.5 Hz, 160 Nt [21]

Priming

Histidine; interception of singlet oxygen

Negligible effect on the priming effect of the KMP

Neutrophils, PMP 42 MCT; PeMP 1 Hz, 600 Nt; 4.4 Hz, 100 Nt; 16.5 Hz, 160 Nt [51]

Priming

Zinc sulfate; blocking of proton channels VSOP/HV1

Attenuation of respiratory explosion

Activated fMLP neutrophils, whole blood, PMP 42 MCT; PeMP 1.0; 4.4 and 16.5 Hz; 0.86 MCT [55]

Priming, enhancement of luminol-dependent chemiluminescence

BAPTA AM; chelation of intracellular calcium

Canceling the field effect

- // -

- // -

Dimethyl sulfoxide; interception of hydroxyl radical

No DMSO effect

Activated fMLP neutrophils, PMP 42 MCT; PeMP 1.0; 4.4 and 16.5 Hz; 0.86 MCT [52]

Priming

Ethanol; interception of hydroxyl radical

Decrease in the priming index

Activated fMLP neutrophils, PMP 42 MCT; PeMP 1.0; 4.4 and 16.5 Hz; 0.86 MCT [22]

Priming

Ionol; inhibition of POL

No significant impact

HEK 293, RF and PMP cells, 0.4 and 1 MT, respectively [56]

increasing the level of Ca2+

Thapsigargin; inhibition of Ca2+-ATPASE of the endoplasmic reticulum

Canceling the field effect

Human amniotic epithelial cells, MP 50 Hz [57]

Increased proliferative activity

BAPTA; chelation of intracellular calcium.

NIF; inhibition of L-type calcium channels.

SKI II; inhibition of sphingosine kinase 1.

LY294002; inhibition of protein kinase in

Complete cancellation of the field effect in the case of BAPTA, SKI II, and LY294002; partial in the case of NIF

Activated fMLP neutrophils, PMP 42 MCT; PeMP 1.0; 4.4 and 16.5 Hz; 0.86 MCT [22]

Priming

Sodium azide; inhibition of myeloperoxidase

Canceling the field effect

Activated fMLP neutrophils, PMP 42 MCT; PeMP 1.0; 4.4 and 16.5 Hz; 0.86 MCT [52]

Priming

Rotenone; blocking of complex I of mitochondrial ETC

No influence



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