Therapeutic nuclear magnetic resonance and intermittent hypoxia trigger time dependent on/off effects in circadian clocks and confirm a central role of superoxide in cellular magnetic field effects

Abstract

Cellular magnetic field effects are assumed to base on coherent singlet-triplet interconversion of radical pairs that are sensitive to applied radiofrequency (RF) and weak magnetic fields (WEMFs), known as radical pair mechanism (RPM). As a leading model, the RPM explains how quantum effects can influence biochemical and cellular signalling. Consequently, radical pairs generate reactive oxygen species (ROS) that link the RPM to redox processes, such as the response to hypoxia and the circadian clock. Therapeutic nuclear magnetic resonance (tNMR) occupies a unique position in the RPM paradigm because of the used frequencies, which are far below the range of 0.1-100 MHz postulated for the RPM to occur. Nonetheless, tNMR was shown to induce RPM like effects, such as increased extracellular H₂O₂ levels and altered cellular bioenergetics. In this study we compared the impact of tNMR and intermittent hypoxia on the circadian clock, as well as the role of superoxide in tNMR induced ROS partitioning. We show that both, tNMR and intermittent hypoxia, exert on/off effects on cellular clocks that are dependent on the time of application (day versus night). In addition, our data provide further evidence that superoxide plays a central role in magnetic signal transduction. tNMR used in combination with scavengers, such as Vitamin C, led to strong ROS product redistributions. This discovery might represent the first indication of radical triads in biological systems.

Keywords: Circadian clock; Intermittent hypoxia; Magnetic field effects; Reactive oxygen species; Superoxide.

Graphical abstract

Fig. 1

On/off effects of hypoxia and tNMR treatments on the circadian clock according to the time of application. Bioluminescence oscillations in NIH3T3 mPer2:Luc cells. (A) 6 h of hypoxia (1% O₂) (red dots) applied during the morning (8 a.m.–2 p.m.) shifts the phase of the Per2:Luc reporter cell oscillation compared to control cells (black). (B) 6 h of hypoxia (1% O₂) applied during the first half of the subjective night (8 p.m.–2 a.m.) significantly decreases the overall level of the Per2 promoter activity and slightly shifts the phase. (C) 6 h of tNMR in the morning (dark blue dots) increases the amplitude of the Per2:Luc reporter oscillation. (D) 6 h of tNMR during the first half of the night decreases the overall promotor activity. (E) Day treatment of cells with a combination of hypoxia and tNMR (light blue dots) slightly reduces the overall promoter activity. (F) The same treatment during the night shows no differences compared to solely hypoxia treated cells. (G) Though day treatments lead to alterations in the amplitude (tNMR 21 % O₂, dark blue line) and phase (sham 1% O₂, red line) treated cells, only minor differences can be found in the overall luminescence levels (tNMR 1% O₂, light blue line). (H) The same treatments performed during the first half of the subjective night lead to significant decreases of the overall Per2:Luc promoter activities. Values are means ± SEM, n = 3 to 5; asterisks indicate significant differences, *p < 0.05, calculated by two-way ANOVA and t-test (unpaired) using Graphpad Prism 6.0), Cos wave fits were performed with the same software, as outlined in detail [25]. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Temporal differences in mitochondrial superoxide and H₂O₂ production of NIH3T3 cells after tNMR treatment in the morning hours under hypoxic conditions: (A) Fluorescence images of MitoSOX™ Green stained NIH3T3 cells for assessment of mitochondrial superoxide after 6 h treatments with hypoxia or tNMR in combination with hypoxia (1% O₂) between 8 a.m. and 2 p.m. Measuring time points were set at 0 h, 2 h, 4 h and 6 h after termination of the treatment; Menadione (100 μM f.c.) was used as a positive control, while N-acetyl l-cysteine (NAC 5 mM f.c.) served as negative control. (B) Quantification of superoxide reveals a significant increase at 4 h and 6 h after termination of the treatment; (C) Quantification of mitochondrial H₂O₂ production. Peaks occur directly (0 h) and 4 h after tNMR treatment. Values are means ± SEM, n = 6, asterisks indicate significant differences, p < 0.05, Statistics, two-way ANOVA (Graphpad Prism 6.0). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Superoxide and H₂O₂ exert opposing roles in the cellular signaling following exposure to tNMR. (A) MitoSOX™ Green staining applied before the hypoxic sham treatment (transmitted light microscopy image, fluorescence microscopy image and merged image). (B) MitoSOX™ Green staining applied before the hypoxic tNMR treatment (transmitted light microscopy image, fluorescence microscopy image and merged image). (C) Percentage of normally shaped cells and spherical cells counted in both groups. (D) Transmitted light microscopy of NAC, (E) VIT C, and (F) catalase treated cells, showing the effects of the various ROS scavengers on solely hypoxia treated sham cells (left) and hypoxia tNMR treated cells (right). n = 3–6 (10x magnification). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Catalase abolishes the harmful effects of excessive H₂O₂ caused through the scavenging of superoxide under hypoxic tNMR. (A) Sham treated cells under hypoxia pre stained with Mitosox and incubated with catalase (1500–37500 U/ml) (left: transmitted light microscopy image, right: fluorescence microscopy image, both taken directly after the treatment at 0 h). (B) tNMR treated cells under hypoxia pre stained with MitoSOX™ Green and pre-incubated with catalase (1500–37500 U/ml) (left: transmitted light microscopy image, right: fluorescence microscopy image, both taken directly after the treatment at 0 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)