Spin biochemistry modulates reactive oxygen species (ROS) production by radio frequency magnetic fields

Abstract

The effects of weak magnetic fields on the biological production of reactive oxygen species (ROS) from intracellular superoxide (O2•-) and extracellular hydrogen peroxide (H2O2) were investigated in vitro with rat pulmonary arterial smooth muscle cells (rPASMC). A decrease in O2•- and an increase in H2O2 concentrations were observed in the presence of a 7 MHz radio frequency (RF) at 10 μTRMS and static 45 μT magnetic fields. We propose that O2•- and H2O2 production in some metabolic processes occur through singlet-triplet modulation of semiquinone flavin (FADH•) enzymes and O2•- spin-correlated radical pairs. Spin-radical pair products are modulated by the 7 MHz RF magnetic fields that presumably decouple flavin hyperfine interactions during spin coherence. RF flavin hyperfine decoupling results in an increase of H2O2 singlet state products, which creates cellular oxidative stress and acts as a secondary messenger that affects cellular proliferation. This study demonstrates the interplay between O2•- and H2O2 production when influenced by RF magnetic fields and underscores the subtle effects of low-frequency magnetic fields on oxidative metabolism, ROS signaling, and cellular growth.

Figure 1. The general reaction scheme involves the spin biochemistry of an enzyme-bound reduced flavin and molecular oxygen.

The RF magnetic fields modulate the interconversion rate of singlet-triplet spin correlated radical pairs. This influence disrupts ROS homeostasis, and therefore, the product distributions of H₂O₂ and O₂ ^•−, which were measured by separate spectroscopic techniques.

Figure 2. A schematic illustration demonstrates the singlet-triplet transitions that affect the kinetics of the effective intersystem crossing rate (k_ISC) in the radical pair.

The sinusoid lines represent RF-induced transitions, which influence the effective intersystem crossing rate. Radical pairs that commence in the triplet state result in an increase in singlet product yield with concomitant decrease in triplet products, vice versa.

Figure 3. A diagram is shown that represents the experimental apparatus for magnetic field exposure.

(A) Tri-dimensional representation of the tri-axial set used for controlling static and alternating electromagnetic fields. Square coil pairs in a Helmholtz configuration are geometrically aligned to control the static magnetic field (SMF) and to compensate for fluctuations in the ambient magnetic fields in the (1) horizontal (X) direction, (2) horizontal (Y) direction, and (3) vertical (Z) direction. This diagram also depicts the placement of a square coil in Helmholtz configuration for the generation of RF magnetic fields (4). A Faraday cage was also used in the RF experiments to surround the setup to minimize RF reflections, but it is not shown in this diagram for clarity. (B) This figure depicts the directions of the magnetic fields with respect to the biological samples. (1) A tri-axial set of square coils in Helmholtz configuration for SMF generation in all 3 dimensions; (2) square coils in Helmholtz configuration for RF generation in the horizontal (Y) direction; (3) an individual 6-well plate; (4) individual wells; (5) culture medium; and (6) a Faraday cage.

Figure 4. EPR signal of nitroxide free radicals that were formed by reacting cyclic hydroxylamines spin probe T-MH in the xanthine oxidase-superoxide-generating system containing xanthine oxidase 10 mU/ml, xanthine (100–400 μM), and DTPA (0.1 mM).

The efficiency of TM-H spin probe was compared to peak heights of standard TEMPO nitroxide concentrations. For the same concentrations of reacted xanthine and TEMPO, TM-H peak height slope was 1/14 as large.

Figure 5. The RF-induced cellular proliferation and hydrogen peroxide production is shown for rPASMC, and is greater compared to control samples.

After the cells reached >90% confluence, the RF electromagnetic field was turned on, Day 0. (A) RF 7 MHz magnetic fields enhanced cell growth by ∼40% on day 2 and ∼45% on day 3 as determined by direct count and trypan blue exclusion method. Data are a representative sample of 3 independent experiments. (B) H₂O₂ production was also measured to compare to cell proliferation. RF electromagnetic fields increased production of H₂O₂ by ∼50% on days 2 and 3 of exposure as determined by AUR assay. Data are normalized to SMF control.

Figure 6. RF magnetic fields increase H₂O₂ production in rPASMC independently of Paraquat and DPI.

(A) The Paraquat control (200 μM) slightly decreases H₂O₂ production, while DPI (10 μM) slightly increases H₂O₂ compared to the SMF control cells (B) RF magnetic fields show no preferential effects on Paraquat/DPI-induced H₂O₂ production. RF magnetic field overall enhances H₂O₂ production in rPASMC by 30 to 40% as seen by (B) RF vs. (A) SMF control. Data are a representation of three independent experiments.

Figure 7. EPR spectra of cyclic hydroxylamines in cell free controls and an example of RF experiments for the detection of superoxide.

(A) PEG-SOD (50 U/ml) inhibits the EPR signal by up to 40% in a cell-free xanthine/xanthine oxidase system. (B) Control and RF normalized EPR spectra. TM-H spin probe reacts with intracellular superoxide to give a nitroxide free-radical that is detectable by EPR. The RF samples have a lower EPR signal intensity compared to control, indicative of a lower intercellular superoxide concentration.

Figure 8. RF electromagnetic fields and xenobiotics had significant effects on cellular detected superoxide.

(A) RF initially decreased the amount of detected superoxide compared with SMF control by 40%. In the Paraquat (200 μM) samples, O₂ ^•− was initially suppressed by 60% compared with SMF control and the effects were enhanced by RF magnetic fields by 50% compared with the Paraquat control. (B) In the DPI (20 μM) increased O₂ ^•− production by 200% compared with SMF control, whereas RF DPI superoxide was decreased by 50% compared with DPI control. The data shows typical results observed in at least three independent experiments.