Extremely low frequency electromagnetic fields activate the ERK cascade, increase hsp70 protein levels and promote regeneration in Planaria

Abstract

Abstract Purpose To use regenerating Planaria Dugesia dorotocethala as a model to determine whether an intermittent modulated extremely low frequency electro-magnetic field (ELF-EMF) produces elevated levels of the heat shock protein hsp70 and stimulates intracellular pathways known to be involved in injury and repair. We focused on serum response element (SRE) binding through the extra-cellular signal-regulated kinase (ERK) cascade. Materials and methods Planaria were transected equidistant between the tip of the head and the tip of the tail. Individual head and tail portions from the same worm were exposed to a 60 Hertz 80 milliGauss ELF- EMF for one hour twice daily for 15 days post transection under carefully controlled exposure conditions. The regenerating heads and tails were photographed and the lengths measured at 3-day intervals. In other experiments, the timing of the appearance of pigmented eyes was monitored in the tail portion at 12 hour intervals following transection in both ELF-EMF exposed and sham control. In some experiments protein lysates were analyzed for hsp70 levels, doubly phosphorylated (pp)-ERK, Elk-1 kinase activity and serum response factor (SRF) -SRE binding. Results ELF-EMF exposure during the initial 3-days post surgery caused a significant increase in regeneration for both heads and tails, but especially tails. The first appearance of eyes occurred at day seven post-transection in tail portions exposed to ELF-EMF. In the sham control tail samples the initial appearance of eyes occurred 48 hours later. Concurrently, ELF-EMF-exposed heads and tails exhibited an elevation in the level of hsp70 protein, an activation of an ERK cascade, and an increase in SRF-SRE binding. Conclusion Exposures to a modulated sinusoidal ELF-EMF were delivered by a Helmholtz configuration at a frequency of 60Hz and 80mG twice a day for one hour. This is accompanied by an increase in hsp70 protein levels, activation of specific kinases and up-regulation of transcription factors that are generally associated with repair processes. Keywords: Extremely low frequency-electromagnetic fields, hsp70, serum response element, ERKMAPK, transcription factor, regeneration Introduction Extremely low frequency electro-magnetic fields (ELF-EMF) have been used in clinical settings to prevent ischemia reperfusion injury (Albertini et al. 1999; DiCarlo, et al. 1999; Shallom et al. 2002; George et al. 2008), accelerate bone and wound healing, and to promote neural regeneration following injury (reviewed in Bassett 1995). Previous reports showed that exposure of cells to an ELF-EMF up-regulates the heat shock gene HSP70 and induces elevated levels of hsp70 protein (Goodman and Henderson 1988; Goodman et al. 1994; Goodman and Blank, 1998; Lin et al. 1998a; 1998b; 1999a; 2001; Han et al. 1998; Carmody et al. 2000). Activation of the HSP70 occurs through the binding of heat shock factor 1 (HSF 1) to a heat shock element (HSE) in a region of the HSP70 promoter that contains three nCTCTn consensus sequences (referred to as electromagnetic field response elements; EMRE). The EMRE have a different consensus sequence from that which responds to elevated temperature in the heat shock domain (Lin et al. 1997; Morimoto et al. 1998). The energy of ELF-EMF required to induce elevated hsp70 protein levels is approximately fourteen orders of magnitude lower than the energy required to induce elevated hsp70 by thermal stress (Blank and Goodman 1997; Goodman and Blank 1998; Goodman and Blank 2002; Lin et al. 1997). Unlike thermal stress, ELF-EMF induction of hsp70 is neither sensitive to increased temperature nor does it turn off baseline protein synthesis as occurs with heat shock (Goodman and Henderson 1988; Goodman and Blank 1998). The hsp70 protein is cytoprotective and has been shown to act as a ‘chaperone’ to refold and restore the function of cellular proteins damaged by the stress of injury or inflammation (Morimoto 1998; Kultz 2005). The efficacy of applied ELF-EMF in regeneration protocols is probably due, at least in part, to increased levels of this cytoprotective stress response protein. There is convincing evidence that ELF-EMF elevate the levels of hsp70 protein via the activation of the extracellular signal regulated kinases, ERK, members of the mitogen-activated protein kinase (MAPK) family at radio-frequency as well as at ELF-EMF (Jin et al. 2000; Leszczynski et al. 2002; Weisbrot et al. 2003; Friedman et al, 2007). Studies using a variety of cell types have shown that activated ERK can phosphorylate the transcription factor Elk-1 and that this ultimately leads to increased serum response factor- serum response element (SRF-SRE) binding and the initiation of transcription. This cascade, which is triggered by injury, has been shown to occur following transection of peripheral nerves in both invertebrates and rodents (Ambron et al. 1995; Lin et al. 2003). Given the ability of an ELF-EMF to upregulate HSP70 gene, levels of the stress response protein hsp70 and ERK were examined during regeneration of Planaria Dugesia dorotocethala. Because their tissues regenerate from pluripotent stem cells (neoblasts), Planaria are particularly well suited for studies of regeneration following injury. Additionally, many of the genes that govern events during regeneration, neural development, and specific stem cell regulation have been identified (Agata et al, 1998; Cebria et al. 2002a; 2002b; 2002c; Agata 2003;Sanchez Alvarado et al. 1999; 2002; Agata and Umesono 2008.). Successful regeneration was accompanied by increased levels of hsp70 protein, activation of ERK, and an increase in the formation of a transcription complex that binds to the SRE. These results indicate that ELF-EMF can accelerate repair by activating pathways that normally participate in regeneration (Sanchez Alavarado, 2003; Newmark and Sanchez Alvarado, 2001). Materials and methods Planaria Dugesia dorotocethala (Carolina Biological Supply Company; cat. # 132950) were shipped overnight and allowed to ‘recover’ for at least 24 hours in fresh oxygenated pond water (Carolina Biological Supply Co., Charlotte, NC, USA). Planaria were maintained in near darkness at 22–24°C (Precision Scientific incubator; Fisher Scientific, NJ, USA) throughout the experiments. Electromagnetic field exposure system The ELF-EMF exposure system uses Helmholtz coils (22 × 24cm with 10.5cm spacing) (designed and made by R. Cangialosi, Electro-Biology Inc, Parsippany, NJ, USA). The coils are composed of 19G copper wire bundles wound 164 times around a Plexiglas form and positioned vertically so that the oscillating magnetic field is generated in the horizontal plane inducing a relatively uniform electric field in the conductive medium. To minimize stray fields during ELF-EM field exposures, all Helmholtz coils containing samples were enclosed in individual 30cm high, 15cm diameter cylindrical mu metal containers (0.040″ thick, Amuneal Corp. Philadelphia, PA, USA) inside the incubator. The 60Hz shielding factor is Min. 90.1= 39.08dB. Experimental exposures were performed simultaneously with unexposed sham control samples in identical mu metal containers. All ELF-EMF field strength measurements were monitored with a Sypris triaxial magnetic field meter (Model 4080, Bell Laboratories, Orlando, FL, USA) before, during and after activation of the ELF-EMF within the mu metal containers in the incubator. ELF-EM field conditions were produced with a function generator (BK Precision 4011A 5MHz, Yorba Linda, CA, USA) and monitored with a digital multimeter (BK Precision 2706A, Yorba Linda, CA, USA). Field parameters were monitored with a Hitachi V-1065 100MHz oscilloscope and a calibrated (according to the National Institute of Standards and Technology) inductive 25X search coil. Parameters of the 60Hz field at a power setting of 5uV induced electric field and the estimated electric field measurements are described in Goodman et al. (1992). Detailed measurements of background magnetic fields in the incubator, harmonic distortion, DC magnetic fields and mean static magnetic fields in the incubators have been determined (Jin et al. 1997). The DC in these experiments was 0.95G (measured by Dr. Arthur Pilla). Exposure protocol The 60Hz frequency and 80mG field strength were selected based on previous experiments in which modulated sine waves were tested at a variety of different frequencies and field strengths (Goodman et al, 1989; Goodman and Bumann, 1991; Goodman and Blank 1998). In the experiments described here, Planaria were transected equidistant between the tip of the tail and the head (Figure 1). Each head and tail portion was photographed using a Nikon digital camera mounted on a Wilde dissecting microscope. Images were stored for subsequent measurements using ImageJ (see section on Quantitation). Head and tail portions were placed in separate Petri dishes (Falcon 351007 60 × 15mm; Fisher Scientific, NJ, USA) containing pond water approximately 0.6cm deep. Dishes were labeled so that heads and tails of the same worm could be identified for measurements. Dishes were placed on a Plexiglas stand between the Helmholtz coils so that the entire area of the dish was exposed to a uniform field. All exposures took place within an incubator and exposures were for 1 hour twice a day with a 4 hour interval between exposures. Control dishes were sham exposed. The temperature in the incubator and within the coils was maintained at 22–24°C and monitored with a thermocouple probe, with a sensitivity +/− 0.01°C (Physitemp, model BAT12, Hackensack, NJ, USA). Growth was assessed at three-day intervals from day 0 (immediately following transection and the onset of ELF-EMF exposure) to day 15 post-transection. In a series of separate experiments pigmented eye spot development was monitored at 12 hour intervals in transected tails exposed to ELF-EMF and sham control immediately following transection. In some experiments protein was extracted from the transected heads and tails, both experimental and sham , for analyses of hsp70 levels, Elk1 kinase activity and SRE-binding.

AI evidence extraction

Main findings

Exposure to 60 Hz, 80 mG ELF-EMF for 1 hour twice daily significantly increased regeneration rates in Planaria, especially in tail portions, advanced the appearance of pigmented eyes by 48 hours, elevated hsp70 protein levels, activated the ERK cascade, and increased SRF-SRE binding compared to sham controls.

Outcomes measured

• hsp70 protein levels
• ERK cascade activation
• serum response element (SRE) binding
• regeneration rate
• appearance of pigmented eyes

Limitations

• Study conducted on Planaria, an invertebrate model, limiting direct extrapolation to humans
• Sample size not specified
• No long-term effects assessed
• Exposure parameters specific and may not generalize to other ELF-EMF conditions

Suggested hubs

• occupational-exposure (0.3)
  Study involves ELF-EMF exposure relevant to occupational or experimental settings.

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    "study_type": "randomized_trial",
    "exposure": {
        "band": "ELF",
        "source": null,
        "frequency_mhz": 0.059999999999999997779553950749686919152736663818359375,
        "sar_wkg": null,
        "duration": "1 hour twice daily for 15 days post-transection"
    },
    "population": "Planaria Dugesia dorotocethala",
    "sample_size": null,
    "outcomes": [
        "hsp70 protein levels",
        "ERK cascade activation",
        "serum response element (SRE) binding",
        "regeneration rate",
        "appearance of pigmented eyes"
    ],
    "main_findings": "Exposure to 60 Hz, 80 mG ELF-EMF for 1 hour twice daily significantly increased regeneration rates in Planaria, especially in tail portions, advanced the appearance of pigmented eyes by 48 hours, elevated hsp70 protein levels, activated the ERK cascade, and increased SRF-SRE binding compared to sham controls.",
    "effect_direction": "benefit",
    "limitations": [
        "Study conducted on Planaria, an invertebrate model, limiting direct extrapolation to humans",
        "Sample size not specified",
        "No long-term effects assessed",
        "Exposure parameters specific and may not generalize to other ELF-EMF conditions"
    ],
    "evidence_strength": "moderate",
    "confidence": 0.6999999999999999555910790149937383830547332763671875,
    "peer_reviewed_likely": "yes",
    "keywords": [
        "extremely low frequency electromagnetic fields",
        "hsp70",
        "ERK cascade",
        "serum response element",
        "regeneration",
        "Planaria"
    ],
    "suggested_hubs": [
        {
            "slug": "occupational-exposure",
            "weight": 0.299999999999999988897769753748434595763683319091796875,
            "reason": "Study involves ELF-EMF exposure relevant to occupational or experimental settings."
        }
    ]
}

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