Bary W. Wilson et al- Evidence for an Effect of ELF Electromagnetic Fields on Human Pineal Gland Function

8/3/2019 Bary W. Wilson et al- Evidence for an Effect of ELF Electromagnetic Fields on Human Pineal Gland Function

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Journal of Pineal Research 9:259-269 (1990)

Evidence for an Effect of ELFElectromagnetic Fields on Human PinealGland Function

Bary W. Wilson, Cherylyn W. Wright, James E. Morris,Raymond L. Buschbom, Donald P. Brown, Douglas L. Miller,Rita Sornmers-Flannigan, and Larry E. Anderson

Battelle, Pacific Northwes t Laborator ies, Richla nd, Was hing ton (B.W.W., C.W.W.,J.E.M., R.L.B., D.P.B., D.L.M., L.E.A.); University of Montana, Missoula, Montana (RS.-F.)

A study was carried out to determine possible effects of 60-Hz electromagnetic-fieldexposure on pineal gland function in humans. Overnight excretion of urinary 6-hydroxymelatonin sulfate (6-OHMS), a stable urinary metabolite of the pineal hor-mone melatonin, was used to assess pineal gland function in 42 volunteers who usedstandard (conventional) or modified continuous polymer wire (CPW) electric blan-kets for approximately 8 weeks. Volunteers using conventional electric blanketsshowed no variations in 6-OHMS excretion as ei ther a gro up or individuals during thestudy period. Serving as their own controls, 7 of 28 volunteers using the CPW blanketsshowed statistically significant changes in their mean nighttime 6-OHMS excret ion.The CPW blankets switched on and off approximately twice as often when in serviceand produced magnetic fields that were 50% stronger than those from the conven-tional electric blankets. On the basis of these findings,we hypothesize that periodicexposure to pulsed DC or extremely low frequency electric or magnetic fields ofsufficient intensity and duration can affect pineal gland function in certain in-dividuals.Key words: melatonin, electric blankets, electric field, magnetic field

INTRODUCTIONDuring the past two decades, interest has increased in the possibility that

exposure to static or extremely low frequency (ELF: 10-100 Hz), including 50-or 60-Hz powerline-frequency electric and magnetic fields, may cause biologi-cal effects in human populations [Savitz and Calle, 19871.Much of our work hasbeen directed toward understanding the association between ELF electric- and

Received April 24, 1990; accepted August 23, 1990.Address reprint requests to Dr. Bary W. Wilson, Battelle, Pacific Northwest Laboratories, Richland,WA 99352.


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Wi l s on et al.

Melatonin (N-acetyl-5-methoxytryptamine), the principal hormone of the

rations occur between approximately 0200 and 0400 h in humans. In allhe suprachiasmatic nuclei. The pineal is richly innervated by fibers

e superior cervical ganglia (SCG) [Moore et al., 19681 as well as by f ibersting in the hypothalamus and optic regions of the brain [Zisapel et al.,Neuronal input from th e eyes acts via the SCG as the principal regulatorn circadian rhythm in th e pineal.

Light of sufficient intensity is effective in suppressing melatonin synthesisany animals [Wurtman et al., 19631. Lewy et al. 119821 reported that the

al., 19901. Ingested19781, P-adrenergic receptor-blocking drugs such as pro-

.olol [Wetterberg, 19791, and certain kinds of stress [Troiani et al., 19871- also been reported to reduce melatonin concentrations in the pineal andtion of rats. Further, altering melatonin circadian rhythms by use of bright

effective in the t reatment of seasonal affective disorder syndromeDS) [Lewy et a]., 19871.

In the circulation, melatonin acts to suppress the function of several otherocrine glands, including the gonads. Melatonin also suppresses the growth of

tain cancers in both in vitro and in vivo models [Blask, 19901. Reduction intion has been associated with estrogen receptor-positive breast

s [Sanchez Barcelo et a]., 19881 and prostate adenocarcinoma [Buzzell et19881. Stevens [ 19871 proposed that, should there be increased cancer riskELF electromagnetic-field exposure, such risk may be a consequence ofChronic exposure to 60-Hz electric fields can reduce the normal nocturnal

n et a]., 1981, 19831. In 23-day-old rats maintained in a 60-Hz electr icfor 20 Wday from conception, the re was no difference among the pineal

in levels of animals exposed to field strengths of 10, 60, and 130 kV/m.an approximate% reduction in maximal nighttime pineal melatonin levels and an approxi-

1.4-h delay in th e occurrence of the nighttime melatonin peak [Reiter et al.,Rats first exposed at 55 days of age to a 39-kV/m electric field showed no

3 days after cessation of ELF electric-field exposure, how-strong pineal melatonin rhythms were reestablished. This effect appearedan "all-or-none" response t o electric fields between approximately 2 andkV/m [Wilson et al., 19861.

ELF FIndeed, an accumulating

netic-field exposure can affecdifferent species. Th e pineal @changes in the geomagnetic fshowed that NAT activity andsuppressed by weak ELF mamarked changes in pineal seintermittent magnetic fields aconsequence of daytime exp50-Hz electric or magnetic fieening of the circadian cycle thtemporal cues. However, we kelectromagnetic-field exposuWe have completed a smagnetic-field exposure fromtonin secretion in humans. Usure to ELF fields that normalExposure t o electric blanketsthe normal lifestyle or daily rin pineal melatonin secretiomelatonin sulfate (6-OHMS)

II MATERIALS AND METHODSI Exposure Systems1 Both conventional elect/ electric blankets were used. 1 two parallel conductors separf ing between the two conductII to temperature at any point ai for the thermal safety switchvides some degree of auto teI cause they can be safely heate

of AC and DC field effects. Oblankets should have little ostudies were completed, howDC magnetic fields can indesafety switches in the convenDC power at temperatures grunacceptable fir e hazard, anduse with DC power.

Modifications to the CPconstructed in grounded metthe bed. AC and DC power appearance or weight, and bocontrollers that t he manufacture control units were dimly


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. amine), the principal hormone of theof N-acetyltransferase (NAT) and hy-

imately 200 and 0400 h in humans. In allThe pineal is richly innervated by fibers[Moore et al., 19681 as well as by fiberspti c regions of the brain [Zisapel et al.,s via the SCG as the principal regulator

Lewy et al. [I9821 reported that the

drugs such as pro-

latonin circadian rhythms by use of brightasonal affective disorder syndrome

o suppress the function of several othern also suppresses the growth of

vo models [Blask, 19901. Reduction ind with estrogen receptor-positive breast

and prostate adenocarcinoma [Buzzell ett, should there be increased cancer risk

fields can reduce the normal nocturnalold rats maintained in a ~ O - H Z lectric

er e was no difference among the pinealof 10 ,6 0, and 130 kV/m.

e nighttime melatonin peak [Reiter et al.,to a 39-kV/m electric field showed noin melatonin secretion) after 21 days

re reestablished. This effect appearedic fields between approximately 2 and

ELF Fie ld s and Human p in e a l Gland FunctIndeed, an accumulating body of data suggests that ELF electr

netic-field exposure can affect circadian rhythms and pineal functidifferent species. The pineal glands of both pigeons and rats responchanges in the geomagnetic field [Olcese et al., 19881, and Welkershowed that NAT activity and melatonin synthesis in pinealocytesuppressed by weak ELF magnetic fields. Lerchl et al. [1990] dmarked changes in pineal serotonin metabolism in rats and miceintermittent magnetic fields at night, but no such changes were oconsequence of daytime exposure. Wever [I9 681 reported that 50-HZ lectric o r magnetic fields can act as a "zeitgeber," arrestinening of the circadian cycle that normally occurs when humans aretemporal cues. However, we know of no direct experimental evideelectromagnetic-field exposure can a ffect human pineal gland fun

We have completed a study to determine if domestic ELF magnetic-field exposure from using electric blankets could affect tonin secretion in humans. Use of electric blankets represents a pesure to ELF fields that normally occurs at night when the pineal isExposure to electric blankets, as used in this study, did not requirethe normal lifestyle or daily routine of the subjects. TO assess possin pineal melatonin secretion, we determined overnight urinarymelatonin sulfate (6-OHMS) excretion in healthy adult human vol

MATERIALS AND METHODSExposure Systems

Both conventional electric blankets and continuous polymerelectric blankets were used. The heating element of CPW blankettwo parallel conductors separated by a resistive polymer material. Cing between the two conductors through the polymer is inverselyto temperature at any point along the element. This feature eliminfor the thermal safety switches used in conventional electric blanvides some degree of auto temperature control. CPW blankets wcause they can be safely heated by either AC o r DC power, allowingof AC and DC field effects. Our original assumption was that the blankets should have little or no effect on pineal gland function.studies were completed, however, Lerchl et al. [1990] showed thatDC magnetic fields can indeed affect pineal gland function in rasafety switches in the conventional electric blankets tested tendedDC power at temperatures greater than about 140F. This arcing cunacceptable f i e hazard, and hence these blankets were deemed uuse with DC power.

Modifications to the CPW blankets consisted of power supplconstructed in grounded metal boxes that could fit near, or undethe bed. AC and DC power supply boxes could not be distinguisappearance or weight, and both types allowed use of the bedsidecontrollers that t he manufacturer supplied with the blankets. Blanture control units were dimly lit by an internal bulb that was the S


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Wilson et al.1. Measured Steady-State Magnetic Field Valuesa Generated at 10-cm Distance byuous Polymer Wire (CPW) Blanket in AC and DC Power Modes and byBlanket in AC Power Mode

Head Chest Knees0.78 0.89 0.842.4 4. 4 5.6

W (AC)~ 4.2 6.6 5.6( x ) ~ 0.56 0.56 0.57in milligauss (measured approximately 10 cm from blanket surface).were four to five times greater during warmup.

pating in the study, a larger power supply was used to accommodate thevidual temperature controllers for both sides of the bed. Subjects were not

as to whether their blankets were powered by AC o r DC at any giventional (sham) power supply boxes were provided for use with the

Volunteer subjects in the study consisted of 32 healthy, nonpregnant, pre-ausal women and 10 healthy men. Male and female participants were

o three groups. Each of the groups provided early eveningurine samples for 2 weeks (per iod 1-preexposure) before begin-

exposure. When exposure began, group 1 (n = 12 women, 2 men) sleptr 4 to 5 weeks (period 2) under AC-powered CPW blankets. Group 2= 10women, 4 men) used DC-powered blankets in the same manner. After

o 5weeks of exposure, power modes on the blankets for groups 1and 2 were5 weeks (period 3).

1 ), on e group of 14 volunteers (group 3: n =0 women, 4 men) used AC-powered, conventionally wired blankets for a total7 weeks of exposure. Urine samples were also collected from all three groups

Because of the anticipated large variation in melatonin excretion amongls, the study was designed so that volunteers would act as their own

N latitude. At this latitude,ter solstice sunrise was at 0739 h and sunset at 1613 h. To control forin the hours of[Bojkowski and Arendt, 19881, study periods 1 and 2 were contiguous

nded just before the winter solstice. Periods 3 and 4 were contiguous andjust after the winter solstice. Because of the time required t o change

3.The measure for assessing possible effects from ELF electromagnetic-fieldsu re was pineal gland function, as determined by radioimmunoassay (RIA)

y 6-OHMS. 6-OHMS s a stable metabolite of melatonin, and its levels in

ELF Fieurine reflect pineal melatonin scollection method did not allowshifts in the melatonin peak thaurine voiding before retiring an

Volunteers provided a seturine (generally around 1700 between 0600 and 0700 h), thtaken in the late afternoodeatlyvoid urine, which was used to arecorded the clock time of last well as that for the evening andated by the volunteers immediaweek, and processed in the lab wwere measured and recorded;taken, one for analysis by RIA,held for archival purposes. In tocollected and analyzed by RIA.content and to urinary volumeexpressed as nanograms of 6-Oof 6-OHMS per milligram of clent. Cretainine normalization for further statistical analyses.

I Assay for Ur i n a r y 6-HydroxymII Urinary 6-OHMS excretioI CIDtech Research Inc. [Mississ' tion of that described by Arend! using a method adapted from; (suspended in methanol) was phy plates using a butanol, w: ments in unknown samples

amounts of 6-OHMS antigen (fective working range for the 0.5 and 100 pg/ml. Within-as9.5% berween-assay varianceor three different dilutions. D250:l and nighttime urines beStatistical Analysis

Results of daytime and nfor each subject and for the statistical analyses were perfofor each group were analyzed the measured preexposure urthe delay in the start of expos

Nested analysis of variaOHMS means of preexposure


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at 10-cmDistance byModes and by

Knees

.4 5.66 5.6

surface).

accommodate theby AC or DC at any given

use with the

before begin-12 women, 2 men) sleptCPW blankets. Group 2

n the same manner. Afterfor groups 1 and 2 were

5 weeks (period 3).3: n =

blankets for a totalthree groups

latitude. At this latitude,1613 h. To control for

es in the hours of1 and 2 were contiguous

e contiguous and

ELF electromagnetic-field(RIA)

ELF Fields and Human Pineal Gland Function 263urine reflect pineal melatonin secretion over time [Arendt, 19861. The samplecollection method did not allow gathering of information on possible temporalshifts in the melatonin peak that might occur in the time span between the lasturine voiding before retiring and the first morning urination.

Volunteers provided a set of two samples, a late afternoon/early eveningurine (generally around 1700 h) and the first morning void urine (generallybetween 0600 and 0700 h), three times each week during the study. Samplestaken in the late afternoon/early evening were used as controls for the morningvoid urine, which was used to assess overnight melatonin excretion. Volunteersrecorded the clock time of last urination before retiring (urine not retained), aswell as that for the evening and morning urine samples. Samples were refriger-ated by the volunteers immediately after collection, picked up three times perweek, and processed in the lab within a few hours of pickup. Total urine volumeswere measured and recorded; three sets of aliquots ( 5 ml each) were thentaken, one for analysis by RIA, one for creatinine determination, and one to beheld for archival purposes. In total, more than 2,400 primary urine samples werecollected and analyzed by RIA. Levels of 6-OHMS were normalized to creatininecontent and to urinary volume and time. Excreted melatonin levels were thusexpressed as nanograms of 6-OHMSper milliliters u rinehour , or as nanogramsof 6-OHMS per milligram of creatinine; the measures were essentially equiva-lent. Cretainine normalization yielded lower variance and was therefore usedfor further statistical analyses.Assay for Urinary 6-Hydroxymelatonin Sulfate

Urinary 6-OHMS excretion was determined using an RIA kit supplied byCIDtech Research Inc. [Mississauga, Ontario, Canada]. The assay is a modifica-tion of that described by Arendt [ 19861 in which 6-OHMS is iodinated with '*'Iusing a method adapted from Vakkuri et al. 119841. The iodinated material(suspended in methanol) was separated on cellulose F thin-layer chromatogra-phy plates using a butanol, water, and acetic acid solvent (4:1.5:1). Measure-ments in unknown samples were based on a standard curve using knownamounts of 6-OHMS antigen (0-200 pg/ml) diluted in stripped urine. The ef-fective working range for the assay (linear portion of the curve) was between0.5 and 100 pg/ml. Within-assay variance among triplicate samples averaged9.5%; between-assay variance was 14%.Samples were run in triplicate at twoor three different dilutions. Daytime urines were diluted between 50:l and250:l and nighttime urines between 2000:l and 8000:l.StatisticalAnalysis

Results of daytime and nighttime 6-OHMS measurements were compiledfor each subject and for the three groups of subjects during the study. Allstatistical analyses were performed on overnight 6-OHMS measurements. Datafor each group were analyzed separately because of the significant difference inthe measured preexposure urinary 6-OHMS excretion of groups 1 and 2, andthe delay in the start of exposure of group 3.

Nested analysis of variance was used to test the hypothesis that the 6-OHMS means of preexposure, AC exposure, DC exposure, and postexposure


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RESULTS

Wi l s o n et al. ELF1.5

as used to test this hypothesis. A natural logarithmic transformation of ( A )each subject were analyzed independently by one-way analysis of vari- DC

nce to test t he hypothesis that the 6-OHMS means of the four periods were o^t within-period erro r term was used to 0Lest the hypothesis. Differences among means were delineated using the least- o

igniicant-difference test [Fisher, 19491. Again, a natural logarithmic transfor- V) 1.0 -ation of the data was made before the analysis to achieve homogeneity of aariances. Also, the nonparametric procedure known as the sign test [Siege], -19561 was used to evaluate the direction of the differences between pairs of V)hypotheses were tested at the 0.05 level of significance. The general linear

Esmodel (GLM) procedure from Statistical Analysis System (SAS, 1985) was em-

ployed for analysis of variance. 3g 0.5 -E l ectr i c Blanket M a g n e t i c and Electric Fields wc

Magnetic fields associated with t he CPW and conventional electric blan- 2kets were measured on three orthogonal axes using a Denol meter magnetic-ce ld measuring device. The blankets were suspended from the ceiling for these 5

:asurements. Instrument probe design obviated making actual measurementscloser than 10 cm from the blankets. Table 1 shows the steady-state magneticfields measured for both types of blankets at the human head, torso, and knee I 0 ;regions. AC magnetic fields produced in the DC power mode were approxi- I

Fi.ie. 1. (A ) Plot of current dr

mately an order of magnitude less than those measured in the AC mode and I1 o

were not distinguishable from background. 5Both the average and maximum magnetic fields associated with the CPW ZCTblankets in th e AC mode are approximately 50% higher than those for compa- Irably sized conventional electric blankets. Florig and Holburg [1990] have car- V)

ried out detai led computer simulations of both th e electr ic and magnetic fieldsassociated with conventional and CPW blankets of several sizes. Data from their

B! 5work are in general agreement with our measurements. At initial switch-on, the 3 0 . 5 -CPW blanket may draw as much as five times its steady-state current , and during 2this period pro duces a proportionally higher magnetic field. During steady-state 0

operation th e modified CPW blankets had a slightly higher current just after wswitch-on than just before switch-off. Blanket duty cycles we re characterized at 2a room temperature of 23.5"C while the blankets were maintained at approxi- L3mately 26.5"C. A current shunt and a data-logging device were used to record 0

I -Table 2 shows the group means and corresponding log-transformed data, (cpw) elect ric blankets usingAdraw during 150-sec interval fo-pressed as nanograms of 6-OHMSImg creatinine, for each exposure period.

( B )A

I

'Deno is a registered trademark of Electric Field Measurements Co., West Stockbridge, M A .

current draw. Current levels and the on-off cycle for a queen-size CPW blanket 0with one side operating are shown in Figure 1A . Comparable data from a con- 1ventional queen-size electric blanket are shown in Figure 1B. 0


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subject within-period error- >mogeneity of variances., one-way analysis of vari-

iod error term was used toleast-

ventional electric blan-magnetic-

rom the ceiling for thesethe steady-state magnetic

ated with the CPWthan those for compa-

[19901 have car-tric and magnetic fields

ral sizes. Data from theirt initial switch-on, the

current, and duringic field. During steady-statecharacterized at

to recorda queen-size CPW blanket

Figure 1B .

log-transformed data,, for each exposure period.

Co., West Stockbridge, MA.

ELF Fields and Human Pineal Gland Function 2651.5

TIME (sec)Fig. 1. (A) Plot of current draw during typical 150-sec interval for continuous polymer wire(CPW) electric blankets using AC power (thick line) and DC power (thin line). ( B ) Plot of currentdraw during 150-sec interval for conventional electric blanket using AC power.


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6 Wilson et al.2. Gr o u p Me a ns a fo r 6 -Hy d r o x yme la ton in S u lb t e (6-OHMS) E x c r e t io n During

Exposure Period1 4

(preexposure) 2 3 (postexposure)AC DC

1 (CPW) 21.84 2 3.74 23.462 3.22 20.73 & 3.41b 24.53 2 3.26b( n = 14) 2.8 820 .17 2.92k0.18 2.7720.1 8 3.01 2 0.15

DC AC2 (CFW) 14.1321.83 17.8622.10 13.97k1.55 1 8 . 2 7 2 . 8 9 ~( n = 14) 2.49 2 0.14 2.71 C 0.13 2.48 k 0.12 2.69* 0.16

AC3 (conventional) 18.892 2.89 18.46f .95 - 9.58 2 3.49( n = 14 ) 2.682 0.21 2.60k 0.19 - .682 0.19& Values are standard error of the mean.different from previou s exposure period by the sign test.ransformed ( log e) values are listed beneath their respective means.tre was no statistically significant difference in 6-OHMS excretion between, C and DC exposure periods as determined by analysis of variance of the

p means. However, as determined by the nonparametric sign test, there was2 and 3, and

3 and 4 in group 1, as well as between periods 3 and 4 in2.Comparison of mean 6-OHMS excretion for individual subjects among the

periods showed that seven CPW users ( 6 women and 1 man) hadfferences in the mean levels of 6-OHMS excretion as determined by

s of variance. That is, there was a statistically significant difference be- [the levels of 6-OHMS excretion among at least two of the latter three test Ire periods ranged between P < 0.04and P < 1

1Figure 2 is a plot of nightly 6-OHMS excretion from a CPW blanket user.n values for each exposure period are denoted by the height of the shaded

s a significant decrease (P c0.05) uring exposure period 3 as2 and a rebound to higher values after the ces-(P


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Excretion During

eriod

test.

6-OHMS excretion between

2 and 3, and3 and 4 ints among the

6 women and 1 man) hadrcretion as determined by

nt difference be-o of the latter three test

between P < 0 . 0 4 and P < from a CPW blanket user.y the height of the shaded3 as

exhibiting differences2 populations in

e and after either 3ria in group 3.

ELF Fields and Human Pineal Gland Function 267

Height of each shaded area representsthe average 6-OHMS excretion for that

.-exposure period. .

$ 3 0

1 Pre- ( 1 ) 1 Dc (2) I AC (3) (post- (4 ) 1DAYS (EXPOSURE PERIOD)Fig. 2. Nightly 6-hydroxymelatonin sulfate (6-OHMS) excretion for continuous polymer wireblanket user. Height of shaded area represents period mean. Note increased 6-OHMS excretionimmediately after onset and cessation of exposure.

DISCUSSIONData on individual subjects serving as their own controls provided evi-

dence to suggest that exposure to either o r both intermittent DC, and 60 -HzAC,electric or magnetic fields of sufficient magnitude or duration may give rise tochanges in melatonin excretion in some individuals. From the pattern of 6-OHMS excretion observed for those volunteers who showed a response to thefields, it appeared that there was a transient increase in 6-OHMS excretion inresponse to onset of exposure and a similar increase, of greater magnitude, atcessation of exposure.

DuringAC operation, the CPW blankets produced a magnetic field approx-imately 50% higher than did the conventional electric blankets. Owing to theirduty cycle, CPW blankets switched on and off approximately twice as often asdid the conventional blankets. Other possible factors that may have affected theoutcome of the study include the combined effects of AC and DC exposure,differences in the switching transients of the two types of blankets, and thepresence of operating shielded transformers in the bedrooms of the CPW vol-unteers. It is also possible that there were temporal shifts in the nighttimemelatonin peak for the conventional electric blanket users that were not de-tected in the urinary 6-OHMS assay.

It should be noted that there was no group in the study wherein blanketheatingwas present without either an AC or a DC electric field. In the literature,however, we could find no evidence that warmth generated by a heated blankethas a physiological effect different from that achieved by using more or heavier


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68 Wilson et al. ELF Fts. In addition, the conventional electric blanket users showed n o changes

n 6-OHMS levels, lending strength to the hypothesis that the electromagneticated with the CPW blankets, and not the heat that they generate, can

ffect human pineal function.In further studies, it would be of interest to determine what, if any, phys-

ological or genetic factors may be common to those individuals who exhibitedure. The report of McIntyre et al . [I9901 cited earlier illustrated large varia-ions in pineal gland sensitivity among individuals. Further work will be re-

quired to determine more precisely those electromagnetic-field characteristicsthat may be responsible for the observed changes in 6-OHMS excretion forcertain individuals in the study.

This work was sponsored by the.Electric Power Research Institute underContract RP-799-1 with Battelle, Pacific Northwest Laboratories.

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A review of the evidence supporting melatonin'srole as an antioxidant 23

Reiter RJ,Melchiorri D, Sewerynek E, Poeggeler B, Barlow-Walden L, Chuang I,Ortiz GG, Acuiia-Castroviejo D. A review of the evidence supporting melatonin'srole as an antioxidant. J. Pineal Res. 1995;18: 1-1 1.Abstract: This survey summ arizes the findings, accumulated within the last 2years, concerning melatonin's role in defending against toxic free radicals. Freeradicals are chemical constituents that have an unpaired electron in their outer or-bital and, because of this feature, are highly reactive. Inspired oxygen, which sus-tains life, also is harmful because up to 5% of the oxygen (02) taken in isconverted to oxygen-free radicals. The addition of a single electron to 0 2 pro-duces the superoxide anion radical ( 0 2 7 ) ; Of is catalytic-reduced by superoxidedismutase, to hydrogen peroxide (H2 02). Although Hz02 is not itself a free radi-cal, it can be toxic at high conc entrations and, m ore importantly, it can be reducedto the hydroxyl radical (.OH). Th e .O H is the most toxic of the oxygen-based radi-cals and it wreaks havoc within cells, particularly with m acromolecules; n rec2ntj;-?itro studies, e a t o n i n was shown to be a very efficient neutralizer of the .OH;indeed, in thd system used to test its free radical scavenging ability it was found tobe significantly more effective than the well known antioxidant, glutathione(GSH ), in doing so. Likewise,.melatonin has been shown to timulate glutathioneperoxidase (GSH -Px) activity in neural tissue; GSH-PX metabo lizes reduced glu-tathione td its oxidized form and In doing so it & =~ ~0 2 to H20, thereby re-- the .OH by eliminating its precu_rsor. ~ b r e cent studies--m elaton in is ; efficien t sca ve nge r of the pe roxyl rad i-cal than is vitamin E. The peroxyl radical is generated during lipid peroxidationthe chain reaction that leads to massive $id destruction in cellmembranzs. In v p o studies have dem onstrated that mei'atonin is remarkably go-


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Reiter et al.(GSH), melatonin proved five times better in neutralizing- -the .O H and, when compared to vitamin E, melatonin was

effective in inactivating the-0.. GS H (Me ister,'1992) and vitamin E (Packer, 1994) are considered to bepremier antioxidants within the cell. Besides these directantioxidative actions of melatonin, there are indirect ef-fects as well. Thus, melatonin .stjmulates klg athi on e per-,y----oxidase (GSH-Px) a& ( ~ ar lo w -W a l de n etl., 1955)

rand inhibits nitric oxide synthase (NOS) (Pozo et al.,1994). GSH-PX is an important antioxidative enzymebecause it metabolizes hydroperoxides including hydro-gen peroxide (H202), thereby reducing the formation of thehighly toxic .OH (Liochev and Frido vich, 1994). By inhib-iting NOS, melatonin reduces the formation of the freeradical nitric oxide (NO.) (Palmer et al., 1988). AlthoughN O performs a variety of important functions in organ-isms (Moncada and Higgs, 1993), it also interacts withother radicals to produce the toxic peroxynitrite anion(ONOO -), which can generate reactive oxygen-based radi-cals by way of its interaction with the superoxide anionradical (027) (Beckman, 1991; Radi et al., 1991).

The purpose of this brief review is to summarize thenewly discovered intracellular functions of melatonin thatrelate to free radical generation. Other reviews discuss the,poten tial implications of these new findings for aging(Reiter, 1994a; Reiter et al., 1994a) and age-related dis-eases (Poeggeler et al., 1993; Reiter et al., 1993, 1994b,1 9 9 4 ~ ) .

Free radical generation and antioxidative defensemechanisms,A free radical is an atom o r a molecule that contains anunpaired electron. Usually, electrons associated with at-om s or molecules are paired; pairing of electrons makes

molecules relatively stable and unreactive. C onversely, theloss of or addition of an electron leaves the atom or-nstable and, relatively mo re hig hly reactive than

%s non-radical gu nte rpa rt. The chemical reactivity of ftee-' ~ r a d E & v aries w T T h e s im ple st fr ee rad ical i s th ehydrogen radical (which is identical to the hydrogenatom ); it contains a single proton an d on e unpaired elec-tron. Removal of a hydr%en radical (or alom) from 9p o l y u ~ a c i d ( ~ ~ ~ ~ )n a cell m e m b ~ s b y/=B30ng reducing a g e n z a n ini tia te radical cha in reaction sQch as in lipid p er zd at io n )_ _. _ (Kan ner el al., 1987). whichare highly destructwe to cellular mqrphology and function.Although there are a variety o f e d i c a l s producedin organisms, those that are by~ rod uc ts f-molecular oxy-gen (dioxygen or 02) have received a great deal of inves-tigative interest and they exkrt esensive damage,particularly over tims(Harman, 1994). Altho ugh estima tesvary som ewhat, it is believed that up to 5 % of the 0 2 akenin by organisms may eventually end up as damaging

fez+e- SOD d3+

0 2---42' ---4 z02---------+ OHFig. 1. The three-electron reduction of molecular oxygen ( 0 2 ) tothe hydroxyl radical (.O H). The addition of a single e- to 0 2produces the superoxideanion radical ( 0 2 3 , which is catalyticallyconverted by superoxide disrnutase (SOD) to hydrogen peroxide(H202).H20 2 can bemetabolized to nontoxic products (see Fig.3 below) or, in the presence of a transition metal, usually Fez+, tis reduced to the highly toxic .OH.

oxygen-based radicals. In a human, this means that therecould be the equivalent of 2 kg of 0 2 7 produced each year(Halliwell and Gutteridge, 1989). 0 2 : is generated by theaddition of a sing le electron to 0 2 Fig. 1); he 0 2 7 is ratherunreactive (Liochev and Fridovich, 1994). 0 2 7 is usuallyclassified as being generated accidentally, as in followingthe leakage of electrons from the mitochondria1 electrontransport chain and by the direct interaction of certainmolecules, e.g., catecholamines, with 0 2 . On the otherhand, 027s also deliberately formed by a variety of acti-vated phagocy es, e.g., eo sinophils, macrophages, mond-T t e s , a n n h i l s , ~ ~ ~ u r ~a z r i at- -and other foreign organisms (Babior and Woodman,

'1990). In chronic inflamm hory disease, the normal pro-duction of 0 2 7 may induce dam age to normal tissue. Otherfindings suggest that under certain conditions, low levelsof free radical production are important because they mayac t as intracellular second messengers. For example, theresponse of cytosolic NF-KB to tumor necrosis factor,which acts via membrane receptors, relies on intracellu-larly produced oxygen radicals as second messengers(Schreck and Baeuerle, 1991; Schreck et al., 1991) (Fig.2). O2'is enzymatically reduced to H20 2 in the presence ofa ubiquitous enzyme, superoxide dismutase (SOD)(McCo rd and Fridovich, 1969). SOD, usually classified asan antioxidative enzyme that affords protection againstfree radical damage, in so me cases ca n be associated withincreased oxidative stress. Thus, the over-expression ofSO D, suc h a s oc curs in t r i s p ~ ~ , 2 1Down syndrome), may-be responsible for many of the neur&egenerative changesand cataracts these i n d i ~ i x ~ r i e n c et an early age(Kedziora and Bartosz, 1988). 7

H20 2 does not possess an unpaired electron and, there-fore, is not a radical per se. Thus , it is usually classifled asa reactive oxygen intermediate o r species. H 20 2 can dif-fuse through membranes and it has a half-life much longerthan that of 027. Hz02 has several fates intracellularly. Itcan be metabolized by on e of two antioxidative enzymes,i.e., GSH-PX or catalase, and, in the worst case scenario,in th e p resen ce of th e tra ns itio n m etals ~ e ~ +r C ul+, it isreduced to the .OH via the Fenton reaction (Fig. 1) (Me-


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1 CytosolNF.&.l.%

Fig. 2. Oxygen-based radicals may act as physiological secondmessengers, as illustrated in this figure . Thus, interleukin 1 (IL- 1)and tumor necrosis factor (TNF) via their respective receptongenerate oxygen radicals intracellularly; this is also the case forprotein k inaseC and hydrogen peroxide (H2 02 ).Oxygen radicalscause the dissociation of NPKB,allowing NF-KB to translocateto the nucleus and to bind DNA. Phorbol ester PMA (phorbol12-myristate 13-acetate). Modified from Schreck and Baeuerle(1991).

Melatonin as an antioxidant

Radicals, ho wever, ca n also interact w ith another radl-cal to form a stable molecule. In this case, the unpairedelectrons in each radical fon n a covalent bond. This is whathappens when a 0 2 7 encounters NO- with the resultantformation of peroxynitrite anion.

0 2 ' + NO- -3 ONOO-ONOO- by itself can damage proteins and can alsodecompose into toxic produ cts including nitrogen dioxide

gas (NO2.), -OH, and the nitronium ion NO^+). Thus, bothONOO -, as well as the products it generates, are toxic tocellular elements.

The phrase given to describe the damage done by freeradicals in oxidative stress (Sies, 1991). The degree ofoxidative stress a cell endures may determine whether itremains healthy or becomes d iseased. Under conditions ofsevere oxidative da2a ge, many cel ls u nd er ~ o i ther ne-

osis or apoptasis. There a re a variety of conditions thath v e tress, including ingestion of toxins,excessive exercise, ionizing radiation, infection, is-chem idreper fusion, and thermal dama ge (Farrington et al.,1973; Freeman et al., 1 987; Keizer et al., 1990; Aust et al. 71993; Zimmerm an and Granger, 1994 ). Th e accumulatedsubcellular damage cau sed by a lifetime of oxidative stressmay also be related to the degenerative diseases of aging,and to aging itself (Subborao e t al., 1990; Taylor e t al.,1993; Harman, 1994; Reiter, 1994b, 19 94 ~ ).

Fortunately, cells have means to resist free radicalabuse. Collectively, this is referred to as the a ntioxidativedefense system (Sies, 1991). Enzymatic antioxidants,which have already been mentioned, include SODneghini and Martins, 1993). (McCord a nd Fridovich, 19 69), GS H-P X (M aiorino et al.,- y r eac tiv e a n d h i g h _ l y ~ i c . t 199 ), and catalase (Chance et al., 1979). These enzymaticindiscriminately reacts with any molecule it encounters. antioxidants catalytically met ab ol~ ze ither a free radicalAmong radicals, it could be classified as the radical's (02T in the case of SOD) or a reactiveoxygen intermediateradical. Because of their large size and ele ct r~ re ac tiv ity ~l t (H202 in the case of GSH-PX and catalase) tois no tunco3monor -O H interact and damage macro- less toxic o r non-toxic p roduc ts (Fig. 3) . Since SOD re-'ho lec ule s such as DXA, =ins, carbohydrates, and lip- duces 02: o ~ ~ 0 ~ ,hich can be converted to the highlyids (Kehrer, 1993). Oxidative da&age to m ~c ro m ol & ile sAis especially noticeable because, compare d to the smaller

-L)Y molecules in cells, they are present in limited n u m b e r s 2h*he case of DNA, dama ge inflicted b y the O H San lead to'c anc er ( ~ i z d a r o ~ l u , '993). .OH are also ~ m t h i n\% hep they are exposed to ionizing rad iat~ on; n this

i cas the electromagnetic radiation splits water molecules' to produce the highly toxic -O H (Littlefield et al., 1988).1F _ -Th e reactions of radicals with non-radicals, which mostmole cules in an organism are, result, by necessity, in theform ation of a new radical; thus, radica ls beget radicals. Insom e cases, these newly formed radicals may also be rather

I toxic and, in fact, they may lnrtiate other damaging freeradical reactions. An exam ple of this type of chain rea ctionis lipid peroxidatlon, w here the RO O-, on ce produced,. abstracts a hydrogen atom from another PUFA (Girotti,1985).

glutathioneHz0 2 + 2GSH 2 H 2 0 + GSSGglutathioneGSSG + NADPH + H+ NADP+ + 2GSHreductrse

Fig. 3. Hydrogen peroxide can be metabolized to nontoxic prod-ucts by the enzymes catalase and gluta thione peroxidase. In theprocess glutathione peroxidase also oxidizes glutathione (GSH)to its disulfide form (GSSG). GSSG is recycled back to GSH inthe presence of the enzyme glutathione reductase.


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Reiter et al.toxic -OH, it is important that the antioxidative enzymesGSH-PX and catalase, both of which metabolize H202.work in concert with SOD (Chance et al., 1979).

In the process of the conversion of H202 o water byGSH-PX, the tripeptide GSH is converted to its disulfideoxidized form, GSSG (Fig. 3). GSH is an important anti-oxidant itself. It is found in millimolar concentrationswithin cells and it has important roles in xenobiotic meta-bolism and leukotriene synthesis (Chance et al., 1979).GSH-PX, which removes H202, is a selenium containingmolecule; a related enzyme removes lipid hydroperoxides.which are formed during lipid peroxidation, from cellularmembranes (Maiorino et al., 1991 .As shown in Figure 1, the reduction of H202 to .OHrequires a transition metal, usually ~ e ~ +ut occasionallyCul+.Because of this, it is important that these metals arenot in the free state in cells and any' molecule that bindsthem and renders them incapable of interacting with H202is classified as part of the antioxidative defense system. Acommon storage-form of iron in serum is transferrin (Win-terboum and Sutton, 1984), whereas-co~mr s often se-

- - ---questered by ceruloqmin (Goldstein et al., 1979). Inthese forms, the transition metals cannot promote freeradical reactions. Besides those mentioned here, there area wide variety of other antioxidative enzymes, free radialscavengers, and transition metal binders that contribute tothe total antioxidant capacity of the organism.The role of melatonin in the antioxidative defense systemFor the last decade, some reports related to the actions ofmelatonin on metabolic processes have been consideredinconsistent with the rather limited distribution of mem-brane receptors in cells (Reiter, 1991). It seemed likely that

&+& certain actions of melatonin, e.g., those related to there ulation of reproduction (Reiter, 1980) and those con-jj LY*; ;->----------.-------.cemed with circadian regulation (Armstrong, 1989), willprove to be mediated by membrane receptors on specificcells related to these functions (Vanecek et al., 1987;Morgan and Williams, 1989). However, the existence ofmelatonin in unicellular organisms (Poeggeler et al.,1991), as well as its widespread actions, described else-where (Reiter, 199 I) , in multicellular organisms !gd-ustospeculate that melatonin performed functions within cellsthat did not require an interaction with a receptor, particu-larly not a receptor located on the limiting membrane ofthe cell. ~ur th ei or e,he high li&olubility of the indolea it ready acce&- to the 9 o ~ o fall cells, also-indicating that the melatonin's actions may not be limitedto actions at the cell membrane level. Interestingly, therecent demonstration that melatonin is also quite solublein aqueous media is consistent with the intracellular ac-tions of melatonin (Shida et al., 1994). Finally, the recentfinding that melatonin-intracellularlymay be r a t he r highconcentrations i? the nu$ei (Mennenga et al., 199 1; Me--

nendez-Pelaez and Reiter, 1993; Menendez-Pelaez et al.,1993) and that there may be specific binding sites formelatonin associated with nucleoproteins (AcuiiaCastroviejo et al., 1993, 1994), suggest the possibility thatmelatonin may function like some other hormones, e.g.,steroid and thyroid hormones, on molecular events in thenuclei of cells.The initial studies from which we deduced that mela-tonin may alter the !edox state of the cell were those ofChen et al. (1993). In this investigation ca2+-stimulated+~ ~ ~ + - d e ~ e n d e n tTPase (~ a~ +- ~ u rn p )ctivity in the heartwas found to be influenced by the pineal gland and mela-tonin. Initially, a daylnight difference in ca2'-pump activ-ity was noted with highest levels at night. When animalswere pinealectomized, the nighttime rise in the activity ofthe pump did not occur, so it was assumed that the rise wasprobably mediated by melatonin. When cardiomyocytemembranes were in fact incubated with melatonin, ca2+-ATPase activity increased in a dose-dependent manner

(Chen et al., 1993). Since the activity of this enzyme isnormally depressed in a high free radical atmosphere(Kaneko et al., 1989), wespeculated that melatonin alteredthe redox state of the cell by neutralizing toxic free radi-cals, which then allowed ca2+-pump ctivity to rise pas-sively. This idea is also supported by more recent studieswherein rats were treated with alloxan, which is known togenerate free radials. This treatment significantly reduced~ a ~ + - ~ u m ~ctivity, which was again reversed by concur-rent melatonin treatment (Chen et al., 1994). Although theevidence is indirect, both studies indicated a potentialinvolvement of melatonin with the oxidative status ofcardiac cells.These initial studies were followed by a series of inves-tigations that were designed to specifically examine theability of melatonin to function as a free radical scavengerand antioxidant. Of specific interest was the interaction ofmelatonin with the highly toxic -OH. To check this, wedeveloped a simple in vitro system in which H202 wasexposed to 254 nm ultraviolet light to generate the .OH(Tan et al., 1993a). However, because of their extremelyshort half-life (1 x sec at 37OC), .OH are difficult tomeasure directly. To overcome this, a spin trapping agent,53-dimethylpyrroline N-oxide, or DMPO, was added tothe mixture. DMPO forms an adduct with the -OH and,

since the adducts have a much longer half-life, they can bequantitated as an index of .OH generation. The adducts(DMPO--OH) were qualitatively and quantitatively evalu-ated using both high pressure liquid chromatography withelectrochemical detection and electron spin resonancespectroscopy (Tan et al., 1993a).By also adding melatonin(or other known scavengers) o the mixture, it was possibleto estimate the -OH scavenging capacity of the compoundsof interest. In this system, melatonin proved to be verysignificantly more efficient than either GSH or mannitol


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TABLE 1. Concentration of various constituents required to scavenge50%(ICs) of the .OH generated in v itm following the exposure of Hf12to ultraviolet ghlScavenger -- ~CSOMelatonin(N-acefyl-5-methoxytryptamine) 21pMReduced glutathione 123pMMannitol -- 183pM

in scavenging the -OH (Table 1). This finding generatedconsiderable interest because both GSH and mannitol arevery effective intracellular free radical scavengers, sug-gesting that melatonin may well have a physiologicallysignificant role as an antioxidant. More importantly, of allthe radicals produced in the organism, the .OH is consid-ered the most toxic; thus, any compound that neutralizesthis radical could play an important role in the antioxida-tive defense system.

The free radical scavenging capacity of melatonin mayextend to other radicals as well. A year following ourreported demonstration of melatonin as a neutralizer of the.OH (Tan et al., 1993a), Pieri and colleagues (1994)claimed that the indole exhibits a similar action in refer-ence to the peroxyl radical (ROO.). Using a well estab-lished in vitro system for evaluating the radical scavengingcapachy of a compound (Cao et al., 1993), Pieri et al.(1994) claimed that melatonin was better than vitamin Ein scavenging the ROO-, which is a consequence of lipid

I peroxidation (Table 2). Clearly, in this system melatoninwas twice as effective as vitamin E, a well known andimportant chain-breaking antioxidant (Packer, 1994). inhalting llpid peroxidation. Thus, melatonin would be ex-pected to be highly effective against lipid peroxidation in

I vivo for several reasons: 1 ) melatonin is highly lipophilicand should, therefore, normally be found in rather high

I concentrations in cellular membranes; 2) melatonin, likeI

I vitamin E, is an effective chain breaking antioxidant and,thus, it would reduce oxidation of lipids; and 3) melatonin,I by virtue of its ability to scavenge the .OH, would alsoI reduce the initiation of lipid peroxidation. The .OH is oneI of the radicals that is sufficiently toxic to abstract a hydro-, gen atom, i.e., initiate lipid peroxidation, from a PUFAI (Niki et al., 1993).

I The demonstration that melatonin affords protectionagainst oxidative stress in vivo followed soon after the invitro studies indicating?hat melatonin is a potent scaven-ger of both the -OH (Tan et al., 1993a) and ROO. (Pieri etal., 1994). In reference to oxidative damage to nuclearDNA, Tan and co-workers (199313,1994) in a series of tworeports found that-hepatic DNA damage inflicted by sa-frole, a chemlcal carcinogen, Gas highly significantly re--duced when the rats also r e c c d y melatoijn.%&oleedamages DNA at least in part because i

TABLE 2. Peroxyl radical (ROO ) scavengingcapacity, as measured noxygen rad~cal bsorbing capacity (ORAC) units, of the four compoundsindicatedaScavenger - ORACperoqiMelatonin (N-acetyl-5-methoxylryptamne) 2.04Vitamin C (ascorbate) 1.12Trolox (water soluble vitamin E analogue) 1OOReduced glutathione 0.68aThe findings suggest hat, of the four ROO- scavengers checked,melatonin is the most efficient.

production of toxic-free radicals (Boberg et al., 1983).w-.'perhaps the most remarkable feature of melatonin's pro-tection against safrole-induced DNA damage was thAitwas effective at verv low concentrations relative to the.---v e ~ ~ i s t e r e d T ; ' T h u s ,ven whenthe amount of melatonin administered was 1,000-foldlower than the dose of safrole, most of the DNA damagewas prevented. Furthermore, when safrole was giveneither during the day or at night, in the latter case DNAdamage was less. The implication of this obse-rvaiion i_sthat- ven the nighttimsd n s e i n o g e n o u s melatonin is

- -sufficient to provide protection against oxygen toxicity%iting from xenobiotic administration (Tan et al., 1994).- -The protective effect of melatonin against oxygen radi-cal damage to DN A was also observed in another modelsystem (Vijayalaxmi et al., 1995). In this case, we incu-bated human lymphocytes and subjected them to 150 cGy- ionizing radiation with and w i t E t concurrent treatment

-the cells with thenc ~ e n e t i c a l l yvaluated by an investigator who was un-aware of the experimental design of the study. Melaton&in a dose-response manner, significantly reduced the num-ber of micronuclei, thi number of cells with exchange

%errations (both of which are indices of genomlc dam-- -. ---_---.-. _-__-age), and the total number of cell with any type of ~ P Oiiiosomal damage (Fig. 4). At a concentration of 2 mM /melatonin reduced ionizing radiation-induced da%e by' ~ ~ Y 1 s ~ l f o x i d eDMSO), a known ra- /dioprotective agent (Littlefield et al., 1988), to provide asimilar level ofDNA protection adose of 1 M was required(Fig. 4) (Vijayalaxmi et al., 1995). Thus, in this systemmelatonin seemed to be on the order of 500 times moreeffective than DMSO as a radioprotecto Free radicalsinduced by ionizing radiation3re the causative fact01 indamage to the genomic material (Okada et al., 1983).

Melatonin as a general protector against ionizing radia-tion is certainly also suggested by the observations oflinke en staff and co-workers (1994). This group found thatalmost 50% of mice treated with melatonin prior to expo-sure to 950 cGy ionizing radiation survived at least 30days, whereas within the same time frame all irradiated


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Reiter et al.Total Number of Cells with Chromosomal Damage

50 100 150 200 250 300

Mel(0.5mM)Me1 (0.5mM) + 150cGy 37.6%

Met (l.OmM)Mel (1.OmM) + 150cGy 51.5%

Met (2.0mM)Mel(2.0mM) + 150cGy 69.1%

OMS0 (1 OM)OMS0 (1 OM) + 150cGy 73.036

Fig.4. Percentage reductionofthenumber of human lymphocytesexhibiting chromosomal damage after their exposure to 150 cGyionizing radiation.At aconcentrationof 2.0 mM in the incubationmediu;, melatonin reduced the percentage of damaged cells by69.1%. For the knownradio~rotectordimethvlsu~foxideDMSO)to reduce chromosomal daAage by roughly he same percentage(73%), its concentration had to be 1 M. Modified from Vijay-alaxmi et al. (1994).

mice that did not receive melatonin died.The protection of macromolecules from oxidativestress by melatonin is not restricted to nuclear DNA. In astudy where oxidative damage to the lens of the eye wasassessed, we found that melatonin also provided signifi-cant protection against lenticular degeneration (Abe et al.,1994). Cataractogenesis s known to be a free radical-me-diated condition where the lens becomes cloudy followingoxidative attack on lenticular protein and other macro-molecules (Spector, 1991). One of the major antioxidativedefense constituents in the lens is GSH (Pau et al., 1990).One model in which to investigate the importance of GSHin protecting the lens from oxygen radical-based cataractsis to inject newborn rats with a drug (buthionine sulfoxi-mine or BSO) that depletes the organisms of this keyantioxidant; BSO acts by inhibiting y-glutamylcysteinesynthaqe, which regulates GSH formation (Martensson etal., 1989; Meister, 1992). When BSO is given shortly afterbirth, rats typically have cataracts at the time their eyesopen (around 2 weeks of age). Interestingly, the pinealgland of newborn rats also produces only small amountsof melatonin during the first 2 weeks of life (Reiter, 1991).Thus in reality, following BSO administration, the new-born animals are really deficient in two important antioxi-dants, i.e., GSH and melatonin.

Considering this, we treated BSO-injected (to depletetheir GSH levels) newborn rats with melatonin for the first2 weeks of life to determine if the indole would altercataractogenesis (Abe et al., 1994). The animals receivingBSO only exhibited the usual high incidence of cataracts,whereas those treated with BSO and melatonin had a very

low incidenceof cataracts (Table 3). In these animals, BSOhad indeed highly significantly reduced lenticular GSHlevels whether or not they had been given melatonin. Theclear implication is that melatonin was the active agent inreducing oxidative damage and suppressing cataract for-mation. Furthermore, although the evidence is obviouslyindirect it seems likely melatonin was effective in thismodel system because it reduced oxidative damage toprotein (Spector, 1991).There is, of course, a great deal of interest in lipidperoxidation because it is devastating to cell membranesand it either disrupts the functions of these critical cellularorganelles or, in the worst case scenario, it leads to thedeath of the cell (Ursini et al., 1991). As mentioned pre-viously, the best known lipid antioxidant is vitamin E,usually represented by a-tocopherol (Packer, 1994).How-ever, Pieri and colleagues' demonstration (1994) showingthat, at least in an in vitro situation, melatonin is a moreefficient scavenger of the ROO than is vitamin E itself,led us to examine melatonin's ability to reduce perox~da-tion of lipid in the lungs of rats treated with the highly toxicherbicide paraquat. Although the mechanisms by whichparaquat inflicts its damage to lipid membranes is com-plex, the damage is believed in part to be a consequence ofthe induction of oxygen-free radicals (Ogata and Manobe,1990). Thus, we administered paraquat to rats with andwithout concurrent melatonin treatment and biochemicallyevaluated the degree of oxidative damage in the lungsusing three indices, i.e., the concentration of malondialde-hyde (MDA) and 4-hydroxyalkenals, total glutathione ev-els, and the ratio of oxidized glutathione (GSSG) to totalglutathione (Melchiorri et ai., 1994). MDA and 4-hy-droxyalkenals are degraded lipid products in cell mem-branes that are taken as an index of oxidative damage(Ursini et al., 1990). In this experimental system, as in theothers where it has been tested, melatonin provided re-markably potent protection against lipid peroxidation (Fig.5). All indices of oxidative stress were returned to normallevels when paraquat-treated rats were also given mela--tonin. Furthermore, in yet-unpublished findings we havefound that the lethal dose of paraquat rgquired to kill 50%of the&1mals ( ~ ~ 5 i jncreases m d n melatoninpretkatedrats (D. MelcGorri and R.S. Reiter, unpublished0ljz iGzG).

TABLE 3. lncidence o cataractsin newborn rats after varioustreatmentsIncidence o Percent o ratsTreatment--- cataracts with cataracts----one (controls) 0117 0

Buthionine sulfoximine 18118 100Buthimine sulfoximine+Melatonin 1/15 7


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Mela tonin a s an ant ioxidantMD A + HDA (nmoYmg protein )

Mel Mel1Fig. 5. Lipid peroxidation products (MDA + HDA) in lungs ofparaquat (PQ)-treated rats. One of two doses of paraquat (LoPQ

= 20 mglkg and HiPQ = 70 m&g) was given to rats with orwithout concurrent melatonin (Me1= I0 m a g ) reatment. Mela-tonin cotreatment overcame the effects of paraquat. Modifiedfrom Melchiorri et al. (1994).

1 This remarkably potent protection against paraquat tox-icity by melatonin certainly exceeded the most optimisticexpectations. Seem ingly, the results cannot be exp lainedby the mere ability of melatonin to interrupt propagationof lipid peroxida tion by scavenging the ROO . (Pieri et al.,1994). Protection is also likely afforded by melatonin'sability to scavenge the .OH (Tan et al., 1993 a), which i scertainly a sufficiently toxic radical to initiate lipid peroxi-dation. Even th ese two mechanisms alone may not accountfor the remarkable ability of melatonin to curtail the per-oxidative processes in the lungs of paraquat-treated rats.Several other potential mechanisms are currently beinginvestigated. Pierrefiche and colleagues (1993), using anm vitro system and brain homogenates, also report thatmelatonin may prevent lipid peroxidation in the brain butthe protection in this organ was reportedly not as great asthat provid ed by its metabolite, 6-hydroxymelatonin. Thisleaves open th e possibility that som e of melatonin's anti-oxidative protection in vivo may follow its hepatic meta-bolism to its hydroxylated metabolite.

More recently, we have used another model system toexamin e melaton in's protective actions against peroxida-tive damage. Bacterial lipopolysaccharide (LPS) is ahighly toxic endotoxin that induces extensive cellulardamage in many organs (Ghezzi et al., 1986; Peavy andFairchild, 1986 ) because of its ability to generate free

radicals (Gram et al., 1986). W e hav e recently found thatmelatonin highly reslsts the peroxidative effects of LPS(Sewerynek et al., 1995) with the degree of efficiencybeing equal to that when paraquat is used as the freeradical-generating molecule. T he s ignificance of the find-ings relates to the fact that LPS causes w idespread oxida-tive damage in a num ber of organs, all of which are negatedby melatonin treatment (Sewery nek et al., 1995); thus, theprotection against free radical attack by m elatonin is obvi-ously not confined to a single organ but probably extendsto every organ and cell in an organism.

There are also several important enzym es that are partof the antioxidative defense system of animals that areinfluenced by melatonin. In the brain, GSH-PX is a pre-mier enzyme in warding off oxidative attack since thisenzyme metabolizes H202 o water, thereby reducing theformation of the toxic .OH (Halliwell and Gutteridge,1989). Indeed, GSH-PX activity is considered possibly themost important means by which neural tissue protectsitself from the devastating actions of free radicals. In aseries of studies, we have shown that melatonin greatlypromotes GSH-PX activity in the brain (Fig. 6) (Barlow-Walden et al., 19 95). This correlates with the rapid uptakeof melatonin by the brain when it is administered to ani-mals (Menen dez-Pelaezet al., 1 993). Th e clear implicationof the findings of Barlow-Walden an d co-workers (1995)is that besides its direct scavenging ability, melatoninstimulates the most important antioxidative enzym e in thebrain, GSH-PX, and thus provides indirect as well as directprotection against free radical attack.

We have also found that the activity of NOS, whichcontrols the quantity of NO. produced (Mayer et al., 1990),is suppressed in the cerebellum by physiological concen-trations of melatonin (Pozo et al., 1 994). This finding hasnum erous implications in terms of melat onin regulation ofneural as well as cardiovascular physio logy, but also couldbe another mechanism by which the indole quells freeradical generation. NO., itself a free radical, can, in thepresence of 0 2 7 induce the formatio n of ONOO-, which,althoug h not a free radical itself, is rathe r toxic within cellsand can also degrade to the .OH via peroxynitrous acid(Beckman et al., 1990). Thus, by virtue of melatonin'sability to reduce NO- formation by limiting NOS activity,free radical production from this source would be limited(Pozo et al., 1994) thereby reducing the likelihood ofoxidative destruction.

Finally, another enzyme closely related to the antioxi-dative defense system of any organism is cytochromeP450. This microsomal comp lex enzym e often is involvedin the metabolism of xenob iotics with the resultan t produc-tion of free radicals (Gram et al., 1986; Coo n et al., 1992).Kothari and Subramanian (1992 ) have recently found thatthe activity of cytochrome P450 is reduced in the presenceof melatonin; we have confirmed this findin g by showing


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Reiter et al.B R A I N GSH Peroxidase A c t i v i t y

51 0.01 Afler 30 minulea.a c kfter 180 minutesa -a0 4t 00. 0.05 :, 0.0,6"\ 0.04

E"\'i c-\ E 0.021 0.0) \".d II-g 0.02 3X O 0.01n 2n2 0.01 Pz


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the reac t ive ini tia t ing and propagat ing -O H and R OO -, byelectron donation and the relatively inert 0 2 - by adductformation in a two-step process (Ha rdeland e t al ., 1993).In the f irs t s tep, the indolyl ca t ion radica l i s form ed whenmelatonin don ates an electron; thereafter, the indolyl cat-ion radica l i s quickly oxidized by the omnipresent 0 2 - toform 5-methoxy-N-acetyl-N-formyl-kynuramine. Thus.mela tonin i s i r reversibly oxidized a nd cannot be regener-ated as i s the case w i th som e other ant ioxidants.

Consider ing the la rge number of radica ls produced inan organism i t seems tha t there may be an insufficientnumber of mela tonin m olecules produced endogenous ly toprovide a significant radical-sc aveng ing action. Howe ver,the multiplicity of melato nin's action as both a free radicalscaven ger (Hardeland et al . , 199 3; Reiter et al ., 1993; Tanet al ., 1993); Pieri et al. , 1994 ) an d as an ant ioxidant(Poeg geler et al. , 1993, 1994; Reiter et al. , 1993; 1994 a;19 94 ~ : ozo e t a l. , 1994; Barlow-W alden e t al ., 1995)greatly increases the l ikelihood th at the quantity of endo -genously produced mela tonin provides a significant de-fense against oxidat ive a t tack (Tan et al . , 1994 ); thispossibil i ty is suppo rted by the findings that melatonin maybe produced in organs in addi t ion t o the pineal gland.However, even if melatonin is only pharmacologicallyrelevant as an antioxidant i ts therapeutic value and poten-tial , considering i ts virtual lack of toxicity, would beseem ingly almo st l imitless.AcknowledgmentsWork by the authors was supported by NSF grant no. 9 1-21263.E.S. was supported by a Fogarty International Fellowship fromNIH; B.P. was su pported by a Feodo r Lynen fellowship from theAlexander von Humboldt Foundation.Literature CitedAB E, M., R.J. REITER , .B. O RH II,M. HARA,B. POEGGELER(1994) Inhibitory effect of melatonin on cataract formation inaewbom rats: Evidence for an antioxidative role for melatonin.J. Pinea l Res. 17:94-100.ACURA-CASTROVIEJO,., M.I. PABLOS, . MENENDEZ-PELA EZ,R.J. REITER (1993) Melatonin receptors in purlfled cell nucleiof liver. Res. Com mun. Chem. Pathol. P harm acol. 82:253-256.ACUNA-CASTROVIEJO,., R.J. REITER, . MEN END U-PELA EZ,M.I. PA BLO S, . BURG OS1994 ) Char acterizatio n of high-af-finity melatonin binding sites in purified ce ll nuclei of rat liver.J. Pineal Res. 16:lOO-113.ARMSTRONG,. (1989) Melatonin: The internal Zei~geber fmam mals? Pineal Res. Rev. 7: 158-202.AUST,S.D., C.F. CHIGNELL,.M. BRAY, . KALYA NARA MAN ,R.P. MASON199 3) Free radicals in toxicolo gy. Toxicol. Appl.Phmacol. 120: 168-178.BAB IOR, .M., R.C. WO ODM AN1990) C hronic granulornatousdisease. Semin. H ematol. 27:247-259.BARLOW-WALDEN,.R., R.J. REITER, . ABE,M.1. P AB LO S, .MENENDEZ-PELAEZ,.D. CHEN, . POEGGELER1995) M ela-tonin stimulates brain g lutathione peroxidase act~ vity.Neuro-chern. Int.. in press.BECKMAN,.S. (1991) The double-edged role of nitric oxide ~n

brain function and superoxide mediated injury. J. Dev. Physiol.1 5 5 9 4 9 .BECKMAN,.S., T.W. BECK MA N,. CHEN, .A. MARS HALL,.A.FREEMAN1990) Apparent hydroxyl radical production byperoxynitrite: Implication for endothelial injury from nitricoxide and superoxide. Proc. Natl. Acad. Sci. USA 87:1620-1624.BLINKENSTAFF,.T., S.M. BRAN DSTAD TER,. REDDY, . W IT-(1994) Potential radioprotective agents. I . Hom ologs of mela-tonin. J. P harm. Sci. 83:21&218.BOBERG,.W., E.C. M ELER, .A. MILL ER, . POLAND. . LIE M(1983) Strong evidence from studies with brachyrnorphic miceand pentachlorophenol that 1'-sulfoxysafrole is the major ulti-mate electrophilic and carcinogenic metabolite of 1'-hy-droxy safrole in mous e liver. Can cer Res. 43:s 163-5 173.CAO,G. , K.M. ALESSIO,R.G. CUTLER 1993) Oxygen-radialabsorbance capacity assay fo r an tioxidants. Free Radical Biol.Med. 14:303-3 11.CHAN CE, ., H. SIES,A. BOVERIS1979) Hydroperoxide meta-bolism in mam malian organs. Physiol. Rev. 62:527-605.CH EN , .D., D.X. TA N , .J. REITER, . YAGA, . POEGGELERK U M A R ,.C. MANCHESTER,.P. CHAMB ERS1993) In vivo andin vitro effects of the pineal gland and melatonin on [C2++Mg2+] ependent ATPase in cardiac sarcolemma. J. Pineal Res.14: 178-183.CHEN, .D., P. KUMAR,.J. REITER, .X. TA N, .C. MANCHESTER, J.P. CHAMBERS,. POEGGELER,. SAARELA1994) Mela-tonin prevents the suppression of cardiac Caz+-stimulatedATP ase activity induced by alloxan. Am. J. Physiol. 267:E57-E62.COON,M.J., X. DING,A.D.N. VAZ (1992) C ytochrome P450:Progress and predictions. FASEB J.6:6 694 73.DIZDAROGLU,. (1993) Chemistry of free radical damage toDNA and nucleoproteins. In: DNA and Free Radicals, B.Halliwell, 0.1. Aruoma, eds. Ellis Hawood, Chichester, pp.19-39.FARRINGTON,.A., M. EBE RT, .J. L AN D,K. FLETCIIBR1973)Bipyridylium qu aternary salts and related compounds. V. P ulseradioly sis of the reaction of paraqu at radical with oxygen.Biochem. Biophys. Acta 314:372-381.FREEMA N, .L . , N.C. SCIDMORE, . W. MALCOLM,M.J.MEREDITH1987) Diamide exposure, thermal resistance andsynthesis of stress (heat shock) p roteins. Biochem. P harmacol.36:21-28.GIIEZZI, ., B. SACCARDO,. BIANCHI1986) Role of reactiveoxygen intermediates in the hepatotoxicity of end otoxin. Im-munop harmacology l2:241-244.G I R O ~ I ,.W. (19 85) Mecha nism s of lipid peroxidation. FreeRadic. Biol. Med. 13 7-95.GOLDSTEIN,.M., H.B. KA PLAN ,.S. EDELSON,.R. WEISSMA(1979) Cemloplasmin: A scavenger of superoxide anion radi-cal. J. B iol. C hem . 254:[email protected] R A M ,.E., L.K. OK IN E, .A. GR AM 1986) The metabolism ofxenobiotics by certain extrahepatic organs and its relation totoxicity. Annu. Rev. Pharmacol. Toxicol. 26:259-276.HALLIWELL,., J.M.C. GUITERIDGE1989) Free Radicals inBiology and M edicine, 2nd Ed., Clarendon Press, Oxford.HARDELAND,., R.J. REIT ER , . POEG GELE R,.X. TAN 1993)The significance of the metabolism of the neurohorm one mela-tonin: Antio xidative protection and form ation of bioactive sub-stances. Neurosci. Biobehav. Rev. 17:347-357.H A N N A N ,. (1994) Free radical theory of aging: Increasing thefunctional life span. A nn. N.Y . Acad. Sci. 717: 1-15.KANEKO,., R.E. BEAM ISH,.S. DHALLA1989) Depression ofheart sarcolernmaC z'-pumping A TPase activity by oxygen free


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tects human blood lymphocytes from radiation-induced ch ro-osome damage. Mutat. Res., in press.WINTERBOUM,.C., H.C. SUTTON1984) H ydroxyl radical pro-duction from hydrogen peroxide and enzymatically generatedparaquat radicals: Catalytic requirements and oxy gen dep end-enc e. Arch . Bio chem. Biophys. 23 5: 116-126.Z I M M E R M A N ,.J., D.N. GRANGER1994) Oxygen free radicalsand the gastrointestinal tract: Role in ischemia-reperfusioninjury. Hepato-Gastroenterology 41:337-342.


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/ P i n e a l R;. ; 1996; ?1:200-213Prrnred In :,i? L'nired Srores ojdr neric n-dl1 r i ~h rs eserred

~ 1 7 U S ~Mini ReviewMelatonin in relation to physiology inadult humans

Cagnacci A. Melatonin in relation to ph ysiology in adult hum ans. J . PinealRes. 1996; 2 1 :3-00-213. O Munksgaard, CopenhagenAbstract: The role exerted by melatonin in human physiology has not beencompletzly ascertained. Melatonin levels have been measured in differentphysiopathological conditions, but the effects induced by melatoninadministration or withdrawal have been tested only recently. Some effectshave been clearly documen ted. Melatonin has hypothermic properties, andits nocturnal secretion generates about 40% of the amplitude of thecircadian body temp erature rhythm. M elatonin has sleep inducingproperties, and exerts important activities in the regulation of circadia nrhythms. M elatonin is capable of phase shifting hliman circadian rh ythms,of entraining free-running circadian rhythms, and of antago nizing ph aseshifts induced by nighttime exposure to light. Its effect on humanreproduction is not completely clear, but stimulatory effects ongonadotropin secretion have been reported in the follicular phase of themenstrual cycle. Direct actions on ovarian cells and spermatozoa have beenalso documented. Beside these, new im portant actions for melatonin m aybe proved. Melatonin may exert protective effects on the cardiovascu larsystem, by reducing the risk of atherosclerosis and hypertension, and m ayinfluence immune responses. Finally, by acting as an antioxidant,melatonin could be important in slowing the processes of ageing.

Cop?rr.ehr0 Murrks~yoord. 996Journal 3 f Pineal ResearchISSN 0742-3098

Angelo CagnacciInstitute of Obstetrics an d Gynecology.University of Modena. 41 100Modena, Italy

Key wolds: Melatonin -humans -reproduction -temperature - sleep -ageing -circadian rhythmsAddress reprint requests toDr Angelo Cagnacci. lstituto diFisiopatologia della Riproduz ione Umana.via del Pouo 71, 41 10Modena, Italy.Received Ju ly 31 . 1996; acceptedSeptember 16, 1996.

Introduction Reiter, 19911. Tryptophan is taken up by the pine-Physiology of melatonin has been extensively stud-ied in animals. For y ears data obtained in animalshave been extrapolated to humans without criticalevaluation. Indeed, only more recent studies havetried to investigate the mechanisms of synthesis,regulation, and action of melatonin in humans. Thepresent review will focus almost entirely on dataobtained in humans that have defined mechanismsof melatonin production by the pineal gland, and theeffects of melatonin on biological and endocrinefunctions.Metatonin synthesis

cr -Studies performed in vitro and in animals have clari-fied the mechanisrns involved in the regulation ofmelatonin synthesis by the pineal gland [Cardinalim d Vacas, 198 7; Krause and Dubocovich, 1990;

alocyte, is transformed to serotonin,-and seroioninis finally converted into melatonin by a two-stepprocess that involves the sequential activities of twoenzymes, N-acetyltransferase (NAT), which is be-lieved to be the limiting enzyme for the synthesisof mela tonin, and hydroxy indo le -0 -methy l t rans -ferase (HIOM T). The synthesis of melatonin is ini-tiated by the binding of norepinephrine to adrenerg icDl receptors, subsequent activation of pineal ade-nyla te cyclase , increase in cycl ic A M P (CAM P),bindin g, and d e novo synthesis of N.4T or of its ac-tivator. Th e potent CAMP-induced ge ne transcrip-tion repressor (ICER) is ac t iva ted in conjunct ionwith NAT and represents a mechanism that limitsthe nocturnal production of melatonin [Stehle et al.,19931. The Dl adrenergic receptor stimulus is en-hanced by al- adrenoceptors, via calcium (ca2')-phospholipid-dependent protein kinase C (PKC) an dby prostaglandins, whose synthesis is activated by


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Melatonin in humansthe influx of ~ a "nto the pinealocyte that followsa1 adrenergic action [Cardinali and Vacas, 1987;Krause and Dobocovich. 19901.Additional stimuli to melatonin synthesis derivefrom VIPergic neurons that reach the pineal glandthrough the pineal stalk [Cardinali and Vacas, 19871by oploids that bind to o receptors [Jansen et al.,19901 and by pituitary adenylate cyclase-activatingpolypeptide [Chik and Ho, 1995; Yuw~ler t al,19951. By contrast, GABA (and benzodiazepines),dopamine, ,ohtamate, and delta-sleep-inducing pep-tide seem to inhibit melatonin production [Krauseand Dubocovich, 19901.Whether all the above mechanisms are relevantto melatonin secretion in humans is not completelyknown. As in animals, in humans rnelatonin synthesisalso depends upon tryptophan availability and is re-duced by acute tryptophan depletion [Zimmermann etal., 19931. Evidence indicated that also i n humansthe adrenergic stimulus is important for rnelatoninsecretion. Beta 1-adrenergic blockers suppress thenocturnal secretion of melatonin [Cowen et al.,1983; Arendt at al., 1985; Brismar et al., 1987;Demitrack et a]., 1990, Cagnacci et al., 19941, withan effect that seems to be inversely related to noc-turnal levels of the hormone [Cagnacci et al., 19941.Similarly, a reduction of nocturnal melatonin secretioncan be obtained with the administration of eitherclonidine, which reduces the endogenous adrenergictonus [Lewy et al., 19861, or alpha-methyl-para-ty-rosine, which reduces presynaptic catecholarnine syn-thesis [Zimmermann et al., 19941. Conversely,melatonin secretion is increased by the administrationof drugs capable of augmenting catecholarnine avail-ability, such as MA0 inhibitors or tricyclic antidepres-sants [Murphy et al., 1986; Skene et al., 19941. Theimportance of intracellular calcium is supported, al-though not conclusively, by the capability ofdihydropyridine calcium antagonists

Bary W. Wilson et al- Evidence for an Effect of ELF Electromagnetic Fields on Human Pineal Gland Function

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