Bioelectromagnetics15377-383 (1994)

Frequency-Dependent Alterations in
Enolase Activity in Escherichia coli
Caused by Exposure to Electric and
Magnetic Fields
S.K. Dutta, M. Verma, and C.F. Blackman
Departments of Biology, Genetics, and Human Genetics, and Cancer Research
Center, Howard University, Washington, DC (S.K. D., M. V.); Health Effects Research
Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North
Carolina (C.F.B.)

Some neurochemical effects of low-intensity electric and magnetic fields have been shown
to be nonlinear functions of exposure parameters. These effects occurred within narrow
ranges of frequency and intensity. Previous studies on membrane-associated endpoints
in cell culture preparations demonstrated changes in calcium efflux and in acetylcho-
linesterase activity following exposure to radiofrequency radiation, amplitude modulated
(AM) at 16 and at 60 Hz, at a specific absorption rate of 0.05 W/kg. In this study, these
modulation frequencies were tested for their influence on the activity of a cytoplasmic
enzyme, enolase, which is being tested clinically for detection of neoplasia. Escherichia
coli cultures containing a plasmid with a mammalian gene for enolase were exposed for
30 min, and cell extracts were assayed for enolase activity by measuring absorbance at
240 nm. The enolase activity in exposed cultures was compared to the activity in paired
control cultures. Exposure to 147 MHz carrier waves at 0.05 Wlkg, AM at 16 Hz showed
enolase activity enhanced by 62%, and AM at 60 Hz showed enolase activity reduced
by 28%. Similarly, exposure to 16 Hz fields alone, at 21.2 V/mrms(electric) and 97 nTrm,
(magnetic), showed enhancement in enolase activity by 59%, whereas exposure to 60
Hz fields alone, at 14.1 V/mrms(electric) and 65 nTrms(magnetic), showed reduction in
activity by 24%. Sham exposures as well as exposure to continuous-wave 147 MHz
radiation at 0.05 W/kg showed no change in enolase activity. Although the underlying
basis for these field effects in the cytoplasmic compartment has not been established,
differential sensitivities to 16 Hz and to 60 Hz signals provide a clear focus for addi-
tional research to determine the responsible mechanism. 01994 Wiley-Liss, Inc.

Key words: radiofrequency radiation, amplitude modulation, ELF, 16 Hz,60 Hz,modulation,

enolase activity

Received for review January 25, 1993; revision received February 25, 1994.

Address reprint requests to Dr. S.K. Dutta, Department of Biology, Howard University, Washington,
DC 20059.

Q 1994 Wiley-Liss, Inc.

378 Dutta et al.

INTRODUCTION
Extremely-low frequency (ELF) electric and magnetic fields have been shown
to alter calcium ion efflux from central nervous system (CNS) or CNS-innervated
systems when used directly or as amplitude modulation (AM) of radiofrequency
(RF) carrier waves [Dutta et al., 1984, 1989, 1992; Blackman et al., 1991; Blackman,
1992; Adey, 1989, 1992; Schwartz et al., 19901. These alterations in calcium ion
efflux have unusual exposure-intensity and frequency dependencies that have been
associated with field-induced changes at the level of the cell plasma membrane [Adey,
1981; Blackman et al., 1988b; Blackman, 19891. It was therefore desirable to test
the influence of fields on a cytoplasmic enzyme activity under the exposure con-
ditions that have been shown to be effective on the calcium ion-release endpoint.
We selected enolase (2-phospho-D-glycerate hydrolase), which is responsible for
the dehydration of 2-phosphoglycerate to enolpyruvic acid, because it is the most
extensively studied enzyme of the ten that make up the glycolytic pathway. This
enzyme activity is also clinically important because it is being tested for detection
of neoplasia and monitoring of therapy for cancer [Mercer, 1990; Nutini et al., 1990;
Jorgensen et al., 1991; Viallard et al., 1990; Tarle and Rados, 19911. Escherichia
coli was selected as the biological test system because of its wide utilization in
molecular biological studies.
A comparison of the relative influence of ELF-modulated RF radiation to ELF
waves alone on the alteration of calcium ion efflux has yielded conflicting results;
Bawin and colleagues have reported enhancement for modulated RF radiation and
inhibition for ELF fields alone [Bawin et al., 1975; Bawin and Adey, 19761, whereas
Blackman et al. have reported enhancement for both exposure regimes [Blackman
et al., 1979, 19821. Blackman et al. [1991] have shown that the temperature of the
sample directly before and during exposure may be a critical factor that can account
for enhancement and inhibition of calcium efflux. Because it is generally believed
that the calcium efflux changes occur due to changes at the plasma membrane [Adey,
19811, we decided to test the relative influence of ELF signals alone and as AM of
a 147 MHz RF radiation on a cytoplasmic enzyme activity.
The logic of choosing the 16 Hz and 60 Hz frequencies is that data for these
frequencies are available that correlate alterations in several biological functions,
such as calcium release [Dutta et al., 1984, 1989; Blackman et al., 1979, 1982, 1985,
1988a,b; Blackman, 1989; Lee et al., 1987; Schwartz et al., 19901 and acetylcho-
linesterase activity [Dutta et al., 19921. However, we do not know whether changes
in these endpoints involving the plasma membrane are causally related to the changes
in cytoplasmic enolase activity reported here. In addition, since 60 Hz electric fields
are used for the transmission and utilization of electric power in the United States,
we were interested to see whether low-intensity fields at that frequency would alter
the activity of a cytoplasmic enzyme that is elevated in clinically ill patients.

MATERIALS AND METHODS

Exposure Systems and Treatment

RF radiation. A transmission-line exposure system housed in a large in-
cubator maintained at 37 "C, as described by Dutta et al. [1984], was used to treat
the cells for 30 min with 147 MHz radiation, AM at 16 Hz sine wave (80% modu-
 Enolase Activity Changes 379

lation), at a specific absorption rate (SAR) of 0.05 W/kg. Unmodulated (CW) ex-
posure conditions were also examined. The vector of the local static magnetic field
(Bd,) at the Washington, DC, site was uniform over the exposure volume at a flux
density of 16 pT, inclined at 53" to the horizontal plane. The orthogonal sinusoi-
dal electric and magnetic components were horizontal. Identically handled flasks
of cells were placed in the incubator outside the transmission line to serve as the
control samples. One trial at a given exposure condition consisted of two exposed
and two control flasks containing cells in 5 ml of growth medium.
ELF signals. A transmission-line exposure system described by Blackman
et al. [1982] was used. Exposures were for 30 min either to 16 Hz electric (21.2 V/
mrm,)and magnetic (97 nTrm,)sine wave fields or to 60 Hz electric (14.1 V/mrms)
and magnetic (65 nTr,,> sine wave fields. The vector of the local static magnetic
field (Bd,) at the Research Triangle Park, NC, site was uniform over the exposure
volume at a flux density of 38 pT, and inclined at 85" to the horizontal plane, which
contained the orthogonal/sinusoidal electric and magnetic components. Circulat-
ing air maintained the flasks of cells at 37 "C. Flasks of cells that served as con-
trol samples were placed in an air incubator at 37 "C during the exposure period
but were otherwise treated identically. All other conditions and handling procedures
were the same as for samples exposed to RF radiation. Sham exposure tests were
also performed, in which the field generation devices were not connected to the
transmission-line exposure system, to examine the cell responses in the exposed
vs. control locations without the imposed fields.
Cell Preparation
The E. coli strain used in these studies contained a plasmid coding for mam-
malian neuron-specific enolase (NSE). The preparation and characterization of this
strain have been described by Dutta et al. [1990].We have shown the activity of enolase
in this clone to be responsive to modulated RF fields [Verma and Dutta, 19931.
A small inoculum of E. coli cells was grown overnight in Luria broth medium
at 37 "C under shaking aeration. Five milliliters of the exponentially growing cultures
(optical density at 600 nm of 0.6-0.8) were placed in each of four 25 cm2 tissue
culture flasks. Two flasks were used as control samples and two for exposed samples.
Sampling and Analysis
The activity of total cellular enolase (E.C.4.2.1.11) was determined spectro-
photometrically within 3 min after the exposure period. Cells from each flask in a
trial were collected by centrifugation at 4 "C. The pellets were resuspended in 1
ml of homogenizing buffer (50 mM Tris, 1 mM EDTA, 84 mM 13-mercaptoethanol,
pH of final solution 7.5) at 4 "C, and the cells were broken open by 20 rotations in
a mortar and pestle containing acid-washed sea sand (Fisher). The sample was then
transferred to a 1.5 ml microfuge tube and centrifuged at lO0OOg for 10 rnin at 4
OC.A 50 p1 aliquot of the supernatant was then added to 0.95 ml of the reaction
buffer (0.15 mM KC1, 1.5 mM MgSO,, 1 mM 2-phospho-D-glycerate, 50 mM Tris
HC1, pH 7.0) at 25 "C to start the reaction. The sample absorbance at 240 nm was
recorded immediately and again 3 min later to measure the amount of phospho-
enolpyruvate formed. A change in absorbance of 0.1 optical density units (OD) was
taken as equivalent to the formation of 0.226 ymol of the product [Baranowski et
al., 19681. We used the 3 min reaction time as suggested in the original method.
380 Dutta et al.

The total protein present was measured as OD at 280 nm, using the conversion 1
OD = 1.515 mg/ml, according to standard methods [Ausubel et al., 19901. This assay
measures total enolase activity and does not select for NSE activity. Enolase en-
zyme activity is expressed as enzyme units/mg protein (pmol phosphoenolpyruvate
formed/min/mg of protein at 25 "C). For each four-flask exposure trial, mean ac-
tivities were calculated for exposed and for control flasks.
The ratios of the mean enzyme activities for each trial were calculated as ex-
posed (flask 1 + flask 2) divided by control (flask 1 + flask 2). Between five and eight
trials were conducted for each EMF exposure condition. All ratio data were log trans-
formed to normalize the distributions and to eliminate heterogeneity of variances among
groups being tested, and a one-way analysis of variance (GLM; SAS Institute) was
used to test this data for each exposure condition. For each treatment condition, the
data are presented as mean control and exposed enolase activities. Mean ratios were
calculated from all trials and are presented as mean percentage difference [(E - C)/
C] x 100, and the P value was calculated comparing the transformed ratios to the null
result and were considered significantly different at P < 0.05.

RESULTS
The results are shown in Table 1. Both ELF fields and ELF-AM RF radiation
produced changes in enolase activity ratios in E. coli cells exposed for 30 min. No
changes in activity ratios were observed in sham exposure or unmodulated RF
radiation (CW) exposure conditions.
These results also demonstrate that 16 Hz, whether alone or as modulation
of a carrier wave, caused approximately a 60% enhancement in enolase activity over
control values, whereas 60 Hz caused approximately a 25% reduction in activity
compared to control values. All these results were statistically different from con-
trols at the P 5 .01 level.

TABLE 1. Influence of EMF Exposure on Enolase Activity i n E. eoli Cells

Enolase activity Mean

( p o l e s productlminlmg protein; percent
Exposure mean f SEM) differenceb
conditions Control (C) Exposed [E) Na f SEM P (lee)'
Sham 0.135 rt 0.014 0.138 f 0.015 5 2.7 f 5.1 .7 14
147 MHzd
cw 0.069 k 0.023 0.078 f 0.031 5 3.6 rt 6.7 ,673
16 Hz 0.054 f 0.005 0.088 f 0.010 8 61.6 f 9.1 ,0001***
60 Hz 0.220 f 0.084 0.147 rt 0.053 8 -28.5 f 4.1 .0007**
ELF alone
16 Hz 0.148 f 0.027 0.240 f 0.057 7 *
59.3 13.1 .0011**
60 Hz 0.371 f 0.114 0.299 rt 0.102 8 -23.8 f 2.8 ,000 1* **
"Number of trials (mean of two exposed and control flasks in each trial; see text for details).
bRatio of exposed and control means for each trial was used to calculate the mean percent difference
[(E - C)/C] x 100 for each treatment condition.
' P value comparing log-transformed ratios to the null result: .05, ** .01, *** .001.
dSAR if 0.05 W k g .
'Sixteen Hertz fields at 21.2 Vlmm\(electric) and 97 nTYm5(magnetic); 60 Hz fields at 14.1 V/mrms(electric)
and 65 nTpm5 (magnetic).
 Enolase Activity Changes 381

DISCUSSION
We asked whether field conditions similar to those that elicit changes in cal-
cium flux [Blackman, 19891 and acetylcholinesterase activity [Dutta et al., 19921,
which presumably occur at the cell membrane [Adey, 1989, 19921, could cause
changes in cytoplasmic enzyme activity. We selected the activity of enolase, an enzyme
in the glycolytic pathway, as our endpoint. Our assay was for total enolase activ-
ity. The results show that exposure to both ELF fields and ELF-AM RF radiation
for 30 min produced changes in enolase activity in E. coli cells. We do not know
whether the reported changes in calcium flux or acetylcholinesterase activity are
causally related to the enolase activity changes observed here.
Unmodulated RF modulation radiation (CW) caused no change in enolase
activity, in agreement with findings of Ward et al. [1975]. These results are also
consistent with the reports of ELF field and AM RF radiation-induced changes in
the flux of calcium ions from CNS and CNS-derived samples; in those reports, the
ELF signal was the essential component. Furthermore, these results demonstrate
that very-low-intensity fields [ELF-modulated RF fields at an SAR of 0.05 W/kg
and ELF signals under 20 V/mmsin air (electric) and 100 nTms(magnetic)}can induce
changes in a cytoplasmic enzyme activity after 30 min of exposure. The physiological
consequences of this effect remain to be determined.
We also tested whether the ELF fields alone and ELF-amplitude modulation
of an RF carrier wave would produce the same effect. The results for 16 Hz and
for 60 Hz signals were consistent, causing the same changes in enolase activity
whether applied directly or as modulation of a carrier wave. The surprising result
was that the 16 Hz component stimulates an increase in enolase activity, whereas
the 60 Hz component causes a reduction in that activity. We do not have an expla-
nation for these results. It is unlikely that laboratory-site-specific influences cause
this, because comparable results were obtained in two different exposure systems,
one at Howard University, where the RF exposures were conducted, and the other
at the U.S. EPA, where the ELF exposures were performed.
We selected a 30 min exposure time and immediate analysis for our assay of
total enolase activity to be consistent with the conditions used in our calcium ion
flux and acetylcholinesterase studies.A more thorough examination of enzyme activity
would include a dependence of enzyme activity as a function of various times of
exposure and for various times after the exposure is terminated. This information
would provide some clues to the underlying regulatory mechanisms, e.g., transcription,
post-transcription, translation, or post-translation mechanisms that are sensitive to
field exposure. Those studies are planned. Furthermore, selective examination of
NSE activity would provide more detailed information on field-induced changes
in the expression of the NSE mammalian gene. Pilot data (not shown) indicate that
NSE activity expression is altered by exposure in a manner similar to total eno-
lase activity described here and that comparisons with trust stocks of E. coli show
that the plasmid is somewhat unstable with time in culture. This instability appears
to be the cause of the variation in total enolase activity exhibited by control cells
under the various exposure conditions, because the tests were performed at differ-
ent times (sometimes separated by many months).
Recent reports have demonstrated elevated levels of NSE in serum or body
fluid in conjunction with the diagnosis and monitoring of therapy for cancer [Mercer,
1990; Nutini et al., 1990; Jorgensen et al., 1991; Viallard et al., 1990; Tarle and
382 Dutta et at.

Rados, 19911. For noncancerous diseases, a pathological rise in plasma NSE lev-
els, indicating ischemic brain damage, occurs in nonsurviving patients after car-
diac arrest and resuscitation [Dauberschmidt et al., 19911 and a rise in NSE levels
occurs in the pleural fluid of patients with nonmalignant lung disease [Plavec et
al., 19901. Shambaugh et al. [1990] have shown that serum factors in blood from
pregnant rats have a gestational-age-dependent effect on enolase activity in fetal
rat brain cell cultures. The relationship of our results of field-induced enolase activity
changes to physiologically significant changes in NSE activity should be investi-
gated further.

ACKNOWLEDGMENTS
We acknowledge partial support from an NIGMS-NIH Institutional Training
Grant (8S06GM08016) and from EPA grants CR-812100 and R-813126-01-0 to
Howard University (S.K.D.), while C.F.B. acknowledges partial support from DOE,
Office of Energy Management, IAG DE-A101-89CE34024. The research described
in this article has been reviewed by the Health Effects Research Laboratory, U.S.
Environmental Protection Agency, and is approved for publication. Approval does
not signify that the contents necessarily reflect the views and policies of the Agency,
nor does mention of trade names or commercial products constitute endorsement
or recommendation for use.

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