Biological systems are constantly exposed to electromagnetic fields (EMFs) in the form of natural geomagnetic fields and EMFs emitted from technology. While strong magnetic fields are known to change chemical reaction rates and free radical concentrations, the debate remains about whether static weak magnetic fields (WMFs; <1 mT) also produce biological effects. Using the planarian regeneration model, we show that WMFs altered stem cell proliferation and subsequent differentiation via changes in reactive oxygen species (ROS) accumulation and downstream heat shock protein 70 (Hsp70) expression. These data reveal that on the basis of field strength, WMF exposure can increase or decrease new tissue formation in vivo, suggesting WMFs as a potential therapeutic tool to manipulate mitotic activity.

In this study, we sought to determine whether WMFs could produce biological effects in vivo (in whole organisms) using the robust planarian regeneration model. Planaria are free-living flatworms that are capable of regenerating all tissues, including the central nervous system and brain, owing to a large adult stem cell (ASC) population that comprises ~25% of all cells ( 21 ). After injury, ASCs mount an animal-wide proliferative response that initially peaks at ~4 hours; this is followed by ASC migration to the wound site over the first 72 hours, when a second mitotic peak occurs ( 22 ). This activity produces the blastema, a collection of unpigmented ASC progeny that forms the core of new tissues. Full regeneration of missing structures occurs in 2 to 3 weeks through the combination of new tissue growth and the apoptotic remodeling and scaling of old tissues.

A theoretical basis exists for the effects of WMFs on the concentration of free radicals such as ROS, as outlined in ( 14 – 16 ). Traditionally viewed as harmful, ROS can trigger cell death and thus are highly regulated by antioxidant enzymes such as superoxide dismutase (SOD), but ROS are also beneficial—acting as regulatory mediators ( 17 ), assisting in muscle repair ( 18 ), and modulating cell signaling ( 19 ). More recently, ROS signaling has been shown to regulate new tissue growth, as such in zebrafish where ROS production triggers apoptosis-induced compensatory proliferation required for regeneration ( 20 ).

Exposure to electromagnetic fields (EMFs) occurs both from modern technology and Earth’s natural geomagnetic field, which averages 25 to 65 μT ( 1 ). In many circles, it is assumed that the quantum of energy associated with these weak magnetic fields (WMFs; <1 mT) is too insubstantial to be biologically important ( 2 ). Despite the fact that stronger magnetic fields are known to affect chemical reaction rates and free radical concentrations ( 3 , 4 ), initial studies of WMF effects on cell cultures produced contradictory results. While one study reported increased levels of the transcription factor c-Myc in human leukemia cells following WMF exposure, a different group failed to replicate these results ( 5 , 6 ). Another study showed that WMFs stimulated protein tyrosine kinases Lyn and Syk levels in B-lineage lymphoid cells, while two later studies found no significant differences ( 7 – 9 ). However, recent evidence indicates that WMFs can affect biological systems in multiple ways. WMF exposure increased intracellular calcium concentrations and the rate of cellular development in satellite cells, and caused embryo mortality as well as altered vertebrae development in roach embryos ( 10 , 11 ). Cell-dependent effects from WMFs were seen in rat renal versus cortical astrocyte cells, with decreased levels of apoptosis, proliferation, and necrosis in renal cells but increases in all three in astrocyte cells ( 12 ). WMFs were also found to produce transient induction of the membrane permeability transition and increased cytosolic cytochrome c levels in human amniotic cells via an increase in reactive oxygen species (ROS) ( 13 ).

Results and discussion

To determine whether WMFs affect tissue growth during planarian regeneration, we amputated animals above and below the pharynx (feeding tube) and examined blastema outgrowth at 3 days postamputation (dpa) (Fig. 1A) following WMF exposure. The setup of our magnetic field apparatus is outlined in fig. S1. We found that 200 μT WMF exposure produced blastema sizes that were significantly reduced as compared to both untreated and Earth-normal 45 μT field strength controls (Fig. 1, C and D). Temporal analyses, where regenerates were exposed for different lengths of time during the first 72 hours of regeneration (Fig. 1B), revealed that 200 μT exposure was required early and must be maintained throughout blastema formation to affect growth [24 hours postamputation (hpa) to 3 dpa]. Because shorter, single-day exposures failed to affect blastema size, these data suggest the presence of recovery mechanisms to ensure initiation of new growth. Furthermore, we found that WMFs produced field strength–dependent effects: Significant reductions of blastema size were observed from 100 to 400 μT, but conversely, a significant increase in outgrowth occurred at 500 μT (Fig. 1E).

Fig. 1 WMFs alter planarian regeneration. (A) Composite image illustrating Schmidtea mediterranea amputation scheme. (B) Temporal analyses of 200 μT WMF exposure on anterior blastema size. Each row represents an experimental group of pharynx fragments that were exposed at the indicated times and scored at 3 dpa. The length of each bar is the duration of 200 μT exposure. Red bars, blastema inhibition (Student’s t test against 45 μT; P ≤ 0.05). Gray bars, no effect. n ≥ 12 for all conditions. (C and D) Blastema size following 200 μT exposure versus untreated and 45 μT controls. Arrowheads indicate presence (solid) or lack (open) of blastema. Scale bars, 200 μm. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test; n ≥ 24. (E) Blastema size following exposure to different field strengths. Student’s t test against 45 μT; n ≥ 16. Red bars, reduced blastema size. Green bar, increased blastema size. Gray bars, no effect. For all: **P < 0.01, ***P < 0.001, and ****P < 0.0001; error bars are SEM; anterior is up; and animals scored at 3 dpa.

We hypothesized that WMF effects were due to altered ROS levels, which peak at the wound site by 1 hpa and are required for planarian blastema formation (23). Pharmacological ROS inhibition resulted in significantly reduced blastema sizes (Fig. 2, A and B), phenocopying 200 μT WMF exposure. To determine whether WMF exposure altered ROS levels, we used a cell-permeant fluorescent general oxidative stress indicator dye to examine ROS accumulation during regeneration. Our results revealed that ROS levels were significantly reduced and/or absent from the wound site after both 200 μT exposure and direct ROS inhibition, as compared to controls at 1 hpa (Fig. 2C). Furthermore, increasing ROS levels via SOD inhibition by RNA interference (RNAi) was sufficient to completely rescue regenerative outgrowth in 200 μT–exposed regenerates (Fig. 1D and fig. S2, A and B). These data suggest that WMF effects on new tissue production are largely due to manipulation of ROS levels in vivo. We also found that SOD inhibition alone was sufficient to significantly increase blastema sizes in 45 μT controls (Fig. 2D), suggesting that tissue growth is highly dose dependent on ROS levels. This is supported by measurements at 500 μT, which also resulted in increased growth, that revealed increased ROS levels (average signal intensity of 61.7 for 500 μT versus 17.7 for 45 μT controls; n = 12; P < 0.01 by Student’s t test).

Fig. 2 WMFs affect ROS levels during early regeneration. (A and B) Pharmacological ROS inhibition using 10 μM diphenyleneiodonium chloride (DPI) scored at 3 dpa. Student’s t test; n ≥ 20. Scale bars, 200 μm. DMSO, dimethyl sulfoxide. (C) Anterior ROS accumulation detection 1 hpa using the general oxidative stress indicator dye 5-(and-6)-chloromethyl-2′,7′-dicholorodihydrofluorescein diacetate (CM-H 2 DCFDA). One-way ANOVA with Tukey’s multiple comparison test; n ≥ 15. Scale bars, 200 μm. (D) RNAi of SOD imaged 3 dpa. Student’s t test against 45 μT; n ≥ 10. Scale bars, 100 μm. Red bar, reduced blastema size. Green bar, increased blastema size. Gray bar, no effect. For all: Solid arrowheads indicate normal blastemas; open arrowheads, lack of blastema; and double arrowheads, increased blastema; **P < 0.01 and ****P < 0.0001; error bars are SEM; and anterior is up.

To investigate genetic mechanisms by which ROS levels (and thus WMFs) regulate regenerative outgrowth, we examined their effects on heat shock protein 70 (Hsp70) expression. Hsp70 is a stress response protein that acts as a chaperone for protein folding during repair, promoting both normal cell survival and cancer cell growth (24). ROS have been shown to affect Hsp70 expression in cell culture (25), and cadmium exposure (which decreases SOD activity and thus increases ROS levels) alters expression of heat shock proteins in a dose-dependent manner (26). Our results demonstrate that Hsp70 inhibition by RNAi significantly reduced blastema sizes during planarian regeneration (Fig. 3, A and B), similar to 200 μT WMF exposure and direct ROS inhibition. Furthermore, Hsp70 expression was lost following both 200 μT exposure and direct ROS inhibition (Fig. 3, C and D). Consistent with these data, increasing ROS levels via SOD RNAi was sufficient to rescue Hsp70 expression in 200 μT–exposed regenerates (fig. S2C). These data suggest that increased ROS levels lead to increased Hsp70 expression during planarian regeneration and that WMFs can alter both processes in vivo.

Fig. 3 WMF effects on new tissue growth are caused by changes in both Hsp70 expression and proliferation. (A and B) Hsp70 RNAi scored at 3 dpa. Student’s t test; n ≥ 15. Arrowheads indicate presence (solid) or lack (open) of blastema. Control RNA: Venus-GFP. Scale bars, 200 μm. (C) Untreated intact animal whole-mount in situ hybridization (WISH) with the Hsp70 probe (n = 13). Scale bar, 200 μm. (D) Effects on Hsp70 expression visualized by WISH at 3 dpa. The anterior region is shown (n ≥ 5). Scale bars, 100 μm. (E) Phospho–histone H3 (pH3) staining of whole regenerates at 4 hpa. Only the anterior region is shown in the images. One-way ANOVA with Tukey’s multiple comparison test; n ≥ 6. Scale bars, 50 μm. For all: DPI used at 10 μM; **P < 0.01, ***P < 0.001, and ****P < 0.0001; error bars are SEM; and anterior is up.

To determine whether the observed changes in blastema size were due to changes in proliferation, we examined mitotic activity via phospho–histone H3 (pH3) staining at the wound site at 4 hpa. Our data revealed that 200 μT WMF exposure, direct ROS inhibition, and direct Hsp70 inhibition all resulted in significantly reduced mitotic activity as compared to control conditions (Fig. 3E). In planarians, ASCs are the only mitotically active cells, suggesting that WMFs (through ROS and Hsp70) affect stem cell activity. We used a planarian ASC marker (Piwi) to examine stem cell population levels during regeneration, as well as a late-progeny marker (AGAT) to examine stem cell differentiation. We found that 200 μT WMF exposure, direct ROS inhibition, and direct Hsp70 inhibition all resulted in significantly reduced ASC levels and stem cell differentiation near the blastema at 3 dpa (Fig. 4, A and B). Together, these data suggest that WMFs are able to alter stem cell regulation during regeneration via changes in ROS signaling.

Fig. 4 WMFs affect stem cell regulation during early regeneration. (A and B) Fluorescence in situ hybridization at 3 dpa to examine (A) the stem cell population (Piwi probe; n ≥ 6) and (B) stem cell differentiation (AGAT probe; n ≥ 5). The anterior region is shown. DPI used at 10 μM. Top panels are significantly different from bottom panels (Student’s t test; P ≤ 0.01). Scale bars, 50 μm. (C) Model for WMF effects on radical pair recombination. (D) Proposed pathway for 200 μT WMF effects on planarian regeneration.

Our data confirm that WMFs affect biological systems and establish a nascent mechanistic pathway by which this occurs. Currently, the main hypothesis for how magnetic fields interact with biological systems is through radical pair recombination (Fig. 4C) (1, 3, 27). In this model, components of a parent molecule can dissociate into a radical pair. Each unpaired electron will have opposing valence spin directions but may undergo a shift in spin direction. Antiparallel valence electron spins (singlet state) allow quick recombination of radicals back into the parent molecule. Alternatively, parallel spin states (triplet state) prevent recombination, providing sufficient time for the pair to diffuse away from one another, creating free radicals (3). Our data suggest that WMF exposure promotes singlet or triplet states depending on field strength, which results in decreased or increased ROS concentrations, respectively.

Our data reveal an underlying pathway by which WMFs affect planarian regeneration (Fig. 4D). WMF exposure alters ROS levels, which lead to changes in Hsp70 expression, which has consequences on stem cell proliferation and subsequent differentiation regulating blastema formation. It is likely that the effects on differentiation are the result of reduced numbers of proliferating stem cells, although direct effects cannot be ruled out. These findings are consistent with recent research highlighting the importance of ROS signaling in the cell in general (13, 17–19) and in regeneration specifically (20, 23, 28, 29). In addition, these data are consistent with studies that have linked EMF exposure to both increased Hsp70 expression and increased regeneration (30–32). Previous studies have also shown the importance of ROS signaling in initiating apoptotic-induced compensatory proliferation during regeneration (20). While our data demonstrate a link between WMFs and ROS-mediated stem cell proliferation, it is possible that effects on stem cell migration to the wound site and/or on apoptosis are also involved. Thus, future studies should investigate these mechanisms as possibilities for WMF effects on stem cell activity.

The ability of WMFs to modulate regenerative outgrowth in vivo suggests that WMFs could be a potential therapeutic tool. In support of this, our investigations with mouse fibroblast cells revealed that WMF exposure caused reduced growth of fibrosarcoma cell cultures but had no effect on noncancerous fibroblast controls (33). Together, these data suggest that highly proliferative cell populations may be specifically targeted during WMF exposure. If true, this would suggest novel possibilities for cancer treatments, where improved methods are needed to inhibit tumor growth while leaving surrounding cells unaffected.