J. Pineal Res. 2001; 31:109–113

All-night exposure to EMF does not alterurinary melatonin, 6-OHMS or immunemeasures in older men and women

Graham C, Sastre A, Cook MR, Gerkovich MM. All-night exposureto EMF does not alter urinary melatonin, 6-OHMS or immune mea-sures in older men and women. J. Pineal Res. 2001; 31:109–113.© Munksgaard, 2001

Abstract: Healthy men (n=22) and women (n=24), 40–60 years ofage, were exposed all-night (23:00–07:00 hr) to 60-Hz magnetic fieldsat an intensity (resultant flux density=28.3 microTesla [�T]) wellwithin the occupational-exposure range, or sham exposed underequivalent, counter-balanced, no-exposure (�0.2 �T) controlconditions. Concentrations of melatonin, and the major metabolite ofmelatonin, 6-hydroxymelatonin-sulfate (6-OHMS), in first-void morningurine were not altered in either gender by exposure to the magneticfield, compared to control conditions. Statistical analysis also failed toreveal any evidence for exposure-related alterations in bloodconcentrations of multiple hematologic and immune system parameters(CD3, CD4, CD8, natural killer [NK] cells). The present resultsreplicate and extend earlier negative findings based on the exposure ofyoung men to power-frequency magnetic fields.

Charles Graham, Antonio Sastre,Mary R. Cook and MaryM. GerkovichMidwest Research Institute, 425 VolkerBoulevard, Kansas City, Missouri 64110,USA

Key words: ELF – EMF – hematology –human – immunology – 60 Hz

Address reprint requests to Charles Graham,Midwest Research Institute, 425 VolkerBlvd., Kansas City, Missouri 64110, USA.E-mail: cgraham@mriresearch.org

Received June 2, 2000;accepted August 9, 2000.

Introduction

The increasing prevalence of electric power distri-bution systems in the environment has raised pub-lic health concerns. This, in turn, has led to anacceleration of research to identify possible biolog-ical effects associated with exposure to power-fre-quency (50/60 Hz) electric and magnetic fields [forreviews see Portier and Wolfe, 1998; Olden, 1999].Recent human exposure studies have examinedeffects on melatonin and its major urinarymetabolite 6-hydroxymelatonin-sulphate (6-OHMS) [for reviews see Lambrozo et al., 1996;Graham et al., 2000] and, to a limited extent, onhematologic and immune system endpoints perti-nent to physical health and well-being [e.g. Sel-maoui et al., 1996a,b]. While results to date havedone little to exacerbate public health concerns,almost all studies have limited their testing tohealthy young men. Consequently, little is knownabout possible exposure effects in older men andwomen, a sizable and perhaps more sensitive seg-ment of the population. For example, melatoninlevels are known to vary as a function of age[Waldhauser et al., 1988; Kennaway et al., 1999].Thus, it may not be appropriate to extrapolate thenegative results obtained with young men in the

laboratory to older men and women in mid-life.The paucity of available data also underscores theneed for additional information about the possibleinfluence of age and gender on field-related alter-ations in human immune activity. Our objectivewas to determine if controlled exposure to occupa-tional-intensity, 60-Hz magnetic fields, altershealth-related endpoints in men and women be-tween 40 and 60 years of age.

Materials and methods

A randomized, double-blind, cross-over designwas used in which each volunteer served as his orher own control. Research participants were 22men and 24 women between 40 and 60 years ofage (mean=46�4.8 years), who had regularsleep and dietary habits, did not work evenings ornights, and were not taking medications thatmight interfere with the study measures (e.g. betablockers, glucocorticoids). The study protocol wasapproved by the Midwest Research Institute(MRI) Institutional Review Board for HumanStudies, and written informed consent was ob-tained from each volunteer prior to participation.

Characteristics of the exposure facility at MRIhave been documented as part of the National

109Printed in Ireland—all rights reser�ed.

Graham et al.

EMF RAPID research program [Olden, 1999], andthe facility is described in Doynov et al. [1999].Half of the subjects, selected at random, wereinitially exposed over-night (23:00–07:00 hr) to acircularly polarized 60-Hz sinusoidal magnetic fieldgenerated at an intensity well within the occupa-tional-exposure range (resultant flux density=28.3microTesla [�T]). On a second control night, theywere exposed only to the ambient 60-Hz back-ground field measured in the laboratory (�0.2�T), an intensity typical of residential exposure.The remaining subjects participated in the reverseorder. The generated magnetic field was presentedintermittently over the night in an alternating 1 hrfield-on and field-off pattern, according to theprotocol described in Graham et al. [1996]. Thedecision to use circularly polarized fields, and topresent them intermittently, was based on previoushuman research with younger adults demonstrat-ing that such exposures are associated with alter-ations in physiology [Cook et al., 1992; Sastre etal., 1998], and on rodent research indicating thatcircularly polarized fields are more effective thanlinearly polarized fields in reducing nocturnalblood levels of melatonin [Kato et al., 1993].

Subjects refrained from consuming alcohol for24 hr prior to a test session, and had no caffeineafter 17:00 hr on the day of a session. On arrival atthe laboratory, they emptied their bladders andchanged into sleepwear. A pre-exposure bloodsample was drawn via venipuncture from an an-ticubital vein, and a post-exposure blood samplewas drawn at 07:00 hr the next morning. Thesesamples were assayed for possible field-related ef-fects on hematological and immune systemparameters. The lights in the exposure test roomwere turned off at 23:00 hr and the subject re-mained in bed until morning. If a subject needed touse the bathroom during the night, he or she worefluoroscopic goggles (Model 502300, Cone Instru-ments, Solon, OH, USA) similar to those used byX-ray technicians. The goggles provided a controlfor possible light-induced interference with noctur-nal melatonin secretion, as they allowed nightvision to be maintained while limiting by 97% thephotopic transmittance of the incident light. First-void morning urine samples (these included allurine voided after 23:00 hr) were collected. Thesesamples were aliquoted, and frozen at −20°C forlater assay of melatonin and 6-OHMS.

Melatonin concentrations in urine were assayedwith the Buhlmann RIA kit (ALPCO, Ltd., Wind-ham, NH, USA). This assay has a detection limitof 0.3 pg/mL and, in our hands, the inter- andintra-assay CVs are routinely below 10% through-out the range of the assay [Graham et al., 1998;

Cook et al., 2000]. 6-OHMS was assayed using theStockgrand RIA kit (Guildford, Surrey, UK). Theinter- and intra-assay coefficients of variation (CV)for this assay are also routinely below 10% and thelower limit of quantitation is 3.1 pg/mL. To correctfor individual differences in urine volume, urinarycreatinine was also assayed using a Cobas Mira™chemistry analyzer. 6-OHMS results were normal-ized to creatinine concentration and expressed asng 6-OHMS per mg creatinine. All assays wereperformed blind.

Pre- and post-session blood samples were storedin EDTA tubes at room temperature until pro-cessed. Sample preparation for hematology andflow cytometry was completed before noon afterthe 07:00 hr collection. Aliquots of whole bloodwere assayed for clinical blood counts, hematologyand differential counts using a Coulter STKRcounter (Coulter Corporation, Hialeah, FL, USA).Endpoints included: hemoglobin concentration,hematocrit, platelets, red blood cells (RBC), totalleukocytes, total lymphocytes, monocytes, andgranulocytes. A Coulter EPICS 752 Flow Cytome-ter equipped with an argon laser emitting at 488 nmwas used for lymphocyte immunophenotyping.Two-color flow cytometry was performed usingbivariate histogram data collection and analysiswith fluorescein isothiocyanate (FITC)- and phyco-eryrthrin (PE)-labeled antibodies. Flourescent sig-nals for all FITC and PE antibody histograms werecollected from a FALS versus 90° side scattergating histogram with the elliptical gate set aroundthe lymphocyte population. Isotypic controls weremouse IgG2a. Endpoints included the followinglymphocyte subtypes: Total T-cells (CD3), helperT-cells (CD4), suppressor T-cells (CD8), the CD4/CD8 ratio, and natural killer (NK) cells (CD56).The assays were performed blind.

Analysis of variance (ANOVA) was the primarystatistical technique used to test for differencesbetween exposed and control conditions. Analysisvariables included: field (exposed vs. control), or-der (exposed/control vs. control/exposed), gender,and pre- versus post-session measures. Probabilityvalues were corrected for lack of sphericity usingthe Huynh–Feldt epsilon technique [Huynh andFeldt, 1976]. Significant main effects or interac-tions were followed up with simple effects analyses.Results were considered statistically significant ifP�0.05.

Results

ANOVA failed to reveal any differences in meanconcentrations of melatonin or 6-OHMS in first-

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EMF effects in older volunteers

Fig. 1. All-night exposure to 60-Hz magnetic fields (28.3 �T)did not alter mean (�S.E.) levels of melatonin or 6-OHMS inthe first-void morning urine of men and women 40–60 yearsof age, compared to control conditions.

within typical clinical ranges, indicating that thesubject sample was normal and healthy. As ex-pected, many parameters showed statistically sig-nificant diurnal variation between evening andmorning collections in both men and women. Theabsolute number of leukocytes (WBC) in circula-tion declined over the night, with correspondingdecreases seen in the lymphocyte sub-populationand particularly in the CD4 fraction of that popu-lation. Little variation, however, was observed inthe CD8 cell fraction. The number and percentageof NK cells in circulation also increased over thenight in both genders, but were observed morestrongly in the men. While many of these diurnalchanges achieved traditional levels of statisticalsignificance, none was particularly large in termsof biological magnitude. Of more relevance to theaims of this paper, analysis failed to reveal anymagnetic field-related effects on the hematologicaland immune system parameters listed in Table 1.Values obtained on the field-exposure night didnot differ from values obtained on the no-expo-sure control night.

Discussion

Overnight exposure of older men and women topower-frequency magnetic fields, at an intensitywell within the range of occupational exposures,had no apparent effect on melatonin or its majormetabolite 6-OHMS. These results replicate andextend earlier negative findings from laboratory-

void morning urine as a function of magnetic fieldexposure, compared to equivalent no-exposurecontrol conditions (melatonin: F [1,42]=1.31,P=0.26; 6-OHMS: F [1,41]=2.42, P=0.13).These results are presented graphically in Fig. 1.Mean (�S.E.) concentrations of melatonin and6-OHMS in first-void morning urine are plottedfor all subjects in the control and exposure testconditions.

Table 1 compares hematological and immuneparameters assessed at 23:00 hr and at 07:00 hrunder control and exposure test conditions for themale and female volunteers. Parameter values are

Table 1. All-night exposure to 60-Hz magnetic fields at an occupational intensity (28.3 �T) did not alter hematologic or immune systemmeasures in men and women 40–60 years of age, compared to equivalent, no-exposure control conditions

Women (n=24)Men (n=22)

Control ControlExposure Exposure

07:00 hr 23:00 hr 07:00 hr 23:00 hr 07:00 hr 23:00 hrParameter 07:00 hr23:00 hr

16�0.2 15�0.2 16�0.2 13�0.2 13�0.2Hemoglobin (g/dL) 13�0.2 13�0.215�0.239�0.638�0.639�0.637�0.745�0.543�0.545�0.743�0.6Hematocrit

(% PRBCV)225�9.5 228�9.7 224�9.3 250�7.3Platelets (103/�L) 246�7.0 251�7.9 248�7.4229�9.5

RBC (106/�L) 4.8�0.1 5.0�0.1 4.7�0.1 5.0�0.1 4.2�0.1 4.3�0.1 4.2�0.1 4.3�0.16.4�0.37.7�0.36.1�0.37.6�0.37.2�0.57.8�0.46.9�0.57.9�0.5WBC (103/�L)

Monocytes (103/�L) 0.8�0.1 0.5�0.1 0.8�0.1 0.5�0.1 0.7�0.0 0.4�0.0 0.7�0.0 0.4�0.04.1�0.3 4.1�0.4 4.0�0.3 4.3�0.4Granulocytes 4.0�0.2 3.5�0.2 4.1�0.2 3.7�0.2

(103/�L)Lymphocytes 2.3�0.12.8�0.2 2.3�0.1 2.9�0.2 2.3�0.1 2.9�0.1 2.2�0.1 2.9�0.1

(103/�L)

Lymphocyte (LY) subsets:CD3 cells (% LY) 72�1.5 68�1.6 73�1.7 68�1.6 73�1.5 69�1.9 74�1.6 70�2.0

42�1.646�1.341�1.446�1.2 46�2.050�1.945�1.7CD4 cells (% CD3) 49�1.520�1.921�1.6CD8 cells (% CD3) 21�1.9 20�1.8 19�1.2 18�1.2 18�1.0 18�1.1

2.9�0.22.7�0.22.9�0.22.6�0.32.8�0.32.5�0.32.7�0.3CD4/CD8 ratio 2.7�0.218�1.711�1.018�1.310�0.8NK cells (% LY) 16�1.411�0.8 11�0.9 17�1.5

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Graham et al.

based exposure studies conducted primarily withyoung men [e.g. Graham et al., 1996, 2000; Sel-maoui et al., 1996a; Akerstedt et al., 1999]. Itshould be noted that these results are based solelyon measures derived from samples of first-voidmorning urine. Melatonin and 6-OHMS concen-trations in such samples, however, are known toaccurately reflect nocturnal plasma concentrationsof melatonin, both in terms of area under thecurve and peak values [Lushington et al., 1996;Graham et al., 1998; Cook et al., 2000]. Otherresearch has also indicated that use of creatinineas a reference parameter for 6-OHMS monitoringis appropriate and provides additional confidencein the reliability of the results obtained [Klante etal., 1997]. Such a correction is not necessary forurinary melatonin, however, since plasma mela-tonin and kidney tubule melatonin readily equili-brate with one another because of the rapiddiffusion of melatonin across lipid membranestructures. The urinary measures, of course, donot provide information about the timing of peakplasma values. In young men, however, no effectson peak plasma values as a function of exposureto 28.3 �T magnetic fields have been observed [e.g.Graham et al., 2000].

In assessing field effects on immune activity, wefocused on the T lymphocytes because of theirrecognized importance and wide-spread distribu-tion within the body [for review see Haynes andFauci, 1998]. T-cells are the primary regulatorsand mediators of cellular immune reactions. Afterbeing processed into immunologically competentcells in the thymus gland, T-cells circulate freelythroughout the body, cycling back and forth be-tween tissues and blood. They constitute about75% of the lymphocyte population found in theperipheral blood supply. Given the age range ofour volunteers, it was also of interest that thethymus atrophies with age, being reduced to about15% of its maximum size by 50 years of age. Ourresults indicate that acute nocturnal exposure tooccupational-intensity power-frequency magneticfields is not associated with abnormal up- ordown-regulation of circulating quantities of im-portant fractions of the T lymphocyte population.This is consistent with previous negative findingsfrom exposure studies with young men [e.g. Gra-ham et al., 1990; Selmaoui et al., 1996b]. Thepresent results are based on a comparison of pre-versus post-exposure values in immune cell counts.Further research would be needed to determine ifexposure alters the functional capability of specificimmune cell types to respond appropriately whenstimulated or challenged. The likelihood of sucheffects being found, however, is not enhanced by

the negative results reported in recent animal stud-ies of exposure effects on cell-mediated immunefunction as measured in blood, spleen, and lymphnodes [e.g. Thun-Battersby et al., 1999].

Identification of a plausible biophysical mecha-nism linking ambient EMF exposure to alterationsin human physiology is a central research issue. ByFaraday’s law, exposure to a time varying mag-netic field will induce an electric field inside aconducting body. If the induced field is of suffi-cient intensity, it could provide a plausible mecha-nism to modulate cellular activity or function. Inrecent comprehensive reviews [Gailey et al., 1997;Portier and Wolfe, 1998] the intensity of 1 mV/mwas identified as the threshold required for docu-mented electric field-induced alterations in cellularactivity. Mature T-cells leave the thymus via thecirculation and ‘‘home to’’ such secondarylymphoid tissues as the spleen and the thoracicduct. Detailed dosimetric analyses with anatomi-cally correct models [Dawson et al., 1997, 1999]indicate that our exposure intensity (28.3 �T)would induce peak electric fields at intensitiesgreater than 2.2 mV/m in the circulation and thesecondary tissues. This intensity is above the bio-logical response threshold identified previouslyand could provide a plausible biological mecha-nism. Despite our present negative results in re-gard to field effects on immune activity,performance of additional mechanistic studieswould seem prudent.

Synthesis and release of melatonin from thepineal gland are under control of the suprachias-matic nucleus (SCN) and the superior cervicalganglion (SCG). The pineal gland and the SCNare located near the anatomical center of thebrain. According to the above dosimetric model, itwould require an exposure to 60-Hz magneticfields at an intensity almost 10 times higher thanthat used here (�300 �T) to induce a 1 mV/melectric field in this central region of the brain.Higher values than those used here would also berequired to induce electric fields of 1 mV/m in theSCG, which is the main relay station for mela-tonin release. There would appear to be little bio-physical support for the hypothesis that melatoninmay be suppressed in humans by nocturnal expo-sure to power-frequency magnetic fields. Recentfield studies of people exposed to EMF while athome or at work, however, do report some evi-dence for suppression [for review see Graham etal., 2000]. Thus, the possibility exists that distur-bances of the melatonin rhythm may be related toas yet unknown aspects of the more complex mag-netic fields found in the man-made environment.

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Acknowledgments

Research funding was provided by the National Instituteof Environmental Health Sciences (Grant ES07053), andby the U.S. Department of Energy/Oak Ridge NationalLaboratory (Contract No. 85X-SN602C). Hematology andflow cytometry were performed at the University of Kan-sas Medical Center under the direction of Dr Bruce Kim-ler. We thank Donald W. Riffle, Dr Michael Gibertini,Deborah I.L.Dozier, Bill Justice and Brian E. Peterson fortheir valuable help in data collection and analysis.

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