Effect of ultraviolet B radiation and 100 Hz electromagnetic fields on proliferation and DNA synthesis of Jurkat cells.

The use of ultraviolet B light (UVB) has been proven to be highly effective for treatment of various inflammatory skin diseases, but UVB phototherapy is limited by its carcinogenic side effects. It is necessary to uncover effectors that augment UVB so that similar or improved efficacy can be obtained with lower UVB doses. We found that low frequency, low intensity electromagnetic fields (EMFs) can act as such an effector and synergistically inhibit T lymphocyte proliferation. We first characterized the effects of UVB on Jurkat cells, a model for cutaneous T lymphocytes, and determined UVB's dose dependent inhibition of cell proliferation and induction of apoptosis. Cells exposed to a sublethal UVB dose retained their sensitivity to UVB, but repetitive irradiation seemed to cause accumulation of delayed DNA damage. We then exposed cells to combinations of UVB plus EMFs and found that 100 Hz, 1 mT EMFs decrease DNA synthesis of UVB-activated Jurkat cells by 34 +/- 13% compared to UVB alone. The decrease is, however, most effective when relatively high UVB doses are employed. Since EMFs alone had only a very weak inhibitory effect (10 +/- 2%), the data suggest that EMFs augment the cell killing effects of UVB in a synergistic way. These findings could provide the basis for development of new and improved clinical phototherapy protocols.

A system for simultaneous ultraviolet light and electromagnetic field exposure in in vitro experiments.

Ultraviolet light (UV) is a common treatment for skin diseases such as psoriasis, but bears the risk of carcinogenic side effects. We have biological evidence that electromagnetic fields (EMFs) can act additively with UV so that new therapeutic protocols combining UV and EMF might be developed to improve psoriasis phototherapy. In this study we report on a system that allows in vitro experiments testing this hypothesis. For simultaneous exposure of cell cultures to UVB and EMF, we built Merritt coils with an integrated UV exposure system. The coils can be operated in a sham or experimental mode (up to 1.5 mT and 20,000 Hz). Two UV bulbs were fitted inside the coils for UVB doses between 100-1000 J/m2/nm. In the exposure area the EMF is uniform within 0.0038%. For exposure, the cells are cultured in standard culture plates and placed in a specifically designed box. The box holds two plates in a top chamber covered with a Saran Wrap lid (91% UV transmission) so that cells are exposed to UVB and EMFs. The bottom chamber holds two plates, where cells are screened from UVB and only exposed to EMFs. Temperature control is maintained (+/- 1 degree C) by airflow vents on the side of the box and a fan placed 25 cm away from the cell culture box. To maintain sterility within the box the vents are covered with a bacterial filter. The box lid has additional ventilation through two air direction changes to create an additional bacterial barrier similar to that in culture plate lids.

Experiments showing that electromagnetic fields can be used to treat inflammatory diseases.

While it is well known that electromagnetic fields (EMFs) can induce repair of non-healing bone fractures, EMF therapy remains confined to orthopedic clinics mainly because the biological and physical mechanisms underlying the therapy are unknown. However, it is generally believed that non-invasive, EMF therapy might have a broad, albeit currently unrecognized clinical potential. In support of this view, we report that 0.1 mT, 60 Hz EMFs induce a 20% mean-increase in anti-CD3 binding to T cell receptors (TcRs) of Jurkat cells, a T lymphocyte cell line. Additionally, we show that 60 Hz sinusoidal EMFs and a commercial bone healing EMF modulate signal transduction pathways that regulate lymphocyte proliferation and that are normally triggered by activating the Jurkat TcR. Similar EMF effects are shown in human peripheral blood lymphocytes (hPBLs), exposed to EMFs in culture and in rat PBLs, when donor animals are exposed to a bone healing field (21 days, 4 hr/day). Although we do not yet satisfactorily understand the differences we obtain in cell and animal based experiments, our findings clearly demonstrate that EMFs can regulate lymphocyte proliferation in vitro and in vivo. Since T cells are key modulators of inflammation, the development of EMF based therapeutic devices to regulate their activity can be expected to provide important tools to treat numerous human inflammatory diseases such as psoriasis and arthritis.

Models of the uniformity of electro-magnetic fields generated for biological experiments by Merritt coils.

Electromagnetic field (EMF) producing wire coils were described by Merritt et al, Rev. Sci. Instrum. 54 (7), 1983. Merritt coils produce large volume EMFs in which statistical numbers of biological experiments are performed. We build and use Merritt coils for cell/animal studies and are developing therapeutic EMF systems. Here we present models illustrating the EMFs produced by our coils and discuss the criteria that should be applied to the use of Merritt and other coils to achieve valid experimental results. In a companion paper at this meeting Nindl et al, describe biological experiments, using these Merritt coils, showing that EMFs may be useful in treating many inflammatory disease states. Although the large-volume EMFs produced by Merritt coils are convenient for biological experiments the EMFs are not perfectly uniform and the deviations can be a significant source of experimental error. The orientation and size of experimental objects are key contributors to these deviations. To evaluate our Merritt coils we solved the Biot-Savart law explicitly for ideal 3-coil and 4-coil Merritt systems and compared these theoretical EMFs with those of our systems. We present a detailed examination of deviations in magnetic field amplitude, as well as magnetic field direction, as a function of location within the coils. We find that spherically shaped experimental sets minimize these deviations. We developed simple formulae for accurately predicting deviations associated with Merritt coils.

Growth stage dependent effects of electromagnetic fields on DNA synthesis of Jurkat cells.

A 1.8 mT, bone healing, electromagnetic field (EMF) and power frequency EMFs of 0.1 and 0.4 mT significantly inhibit DNA synthesis in otherwise unstimulated Jurkat (E 6.1) cells. Inhibition is generally most prominent in cells from mid log phase growth. In complete medium the bone healing EMF inhibits [3H] thymidine uptake of the latter cells by almost 50% vs. 20-25% inhibition by 60 Hz fields. Cells in conditioned medium are even more sensitive to EMFs with inhibition up to ca. 60%. The effects of the 0.1 and 0.4 mT power frequency EMFs were very similar suggesting saturation at 0.1 mT or lower.

A mechanism for action of extremely low frequency electromagnetic fields on biological systems.

This report outlines a simple mechanism, based on the Hall Effect, by which static and low frequency (50-60 Hz) pulsed electromagnetic fields (PEMFs) can modify cation flow across biological membranes and alter cell metabolism. We show that magnetic fields commonly found in the environment can be expected to cause biologically significant interactions between transported cations and basic domains of cation channel proteins. We calculate that these interactions generate forces of a magnitude similar to those created by normal transmembrane voltage changes known to gate cation channels. Thus PEMFs are shown to have the potential of regulating flow through cation channels, changing the steady state concentrations of cellular cations and thus the metabolic processes dependent on cation concentrations.

Changes in mitochondrial DNA during aging.

Mitochondrial DNA (mtDNA) was isolated from liver mitochondria of rats between 2 and 24 months of age. The mtDNA was purified by cesium chloride--ethidium bromide isopycnic density gradient centrifugation. In the gradients, in addition to the two expected bands of ethidium--DNA complex, there was observed a third, more dense band (d = 1.69 g/cm3). This novel band, rarely observed in preparations from younger animals, was present in most preparations from older animals. The latter was characterized using the diphenylamine assay(s) and ascertained to contain DNA and carbohydrate components. Agarose gel electrophoresis revealed the DNA of the novel band to have a migration identical to form I mtDNA. Digestion of the novel band with the restriction endonuclease Bam HI yielded products identical to those obtained upon treatment of form I mtDNA with Bam HI. The observation of mtDNA at a density of 1.69 g/cm3 indicates the presence, predominantly in older animals, of a subclass of mtDNA molecules with altered ethidium binding properties. The significance of this mtDNA and its position in the gradient is unclear at this time.

Reaction of malonaldehyde with mitochondrial membranes.

Malonaldehyde formed by lipid oxidation is regarded as a main crosslinker in the formation of natural age pigment. To elucidate the mechanism of pigment formation the reaction of malonaldehyde with biomembranes using fluorescence spectroscopy has been studied. Rat liver mitochondrial ghosts or bovine serum albumin were reacted with malonaldehyde. In both cases two main fluorescence changes were observed: protein fluorescence decreased to 50% of its initial value in about two hours; aminoiminopropene fluorescence reached a maximum at a much slower rate. The kinetics support a two-step reaction hypothesis. First, malonaldehyde reacts with protein quenching its fluorescence. Next fluorescent interprotein aminoiminopropene (AIP) crosslinks are formed. The fluorescence lifetime value of the induced AIP fluorophore was shown to be similar to the lifetime of naturally occurring age pigment previously reported for mitochondrial ghosts prepared from aged animals (5.4 ns +/- 0.3 and 5.9 ns +/- 0.6, respectively).