Intensity dependence of enolase activity by modulated radiofrequency radiation

179

Bioelectrochemist~ and Bioenergetics, 27 (1992) 179-189 A section of J. Electroanal. Chem., and constituting Vol. 342 (1991) Elsevier Sequoia S.A., Lausanne

JEC BB 01432

Intensity dependence of enolase activity by modulated radiofrequency radiation SK. Dutta

l, Bracie Watson, Jr. and Kaberi P. Das

Department of Botany, Department of Genetics and Human Genetics, Cancer Research Center, Howard University, Washington, DC 20059 (USA) (Received 31 July 1991)

Abstract Electromagnetic radiation (EMR) effects on calcium ion (Ca2+) efflw and acetylcholinesterase (AChE) activity in neuroblastoma cells have been studied for several years. Enhanced Ca2+ efflux and AChE activities occur at certain power density regions separated by other power density regions in which no effect is observed. AChE is generally membrane-bound. Ca 2+ ions exist both as extracellular and intracellular components that pass through the membrane to maintain the cellular physiological balance. In this study, observations are reported of EMR effects on a cytoplasmic biochemical parameter, activity of total enolase, a well-known enzyme in the glycolytic pathway of cells that is not membrane-bound. Two neuroblastoma cell lines - a human line IMR-32 and a rodent hybrid line NG-108 - were exposed for 30 min to 147 MHz radiation, amplitude-modulated (AM) at 16 Hz (80%). The power intensities used had specific absorption rate (SARI values of 0.001, 0.01, 0.02, 0.05, 0.07, 0.08, 0.09, 0.10 and 0.50 mW/g. At SAR 0.05 mW/g, there was significant enhancement in all three biochemical parameters (AChE, Ca*’ and enolase). All three biochemical activities were significantly depressed at SAR 0.01 mW/g. Enolase activity was also significantly depressed at SAR 0.08 mW/g using the NG-108 neuroblastoma cell line. Almost no effects were observed in any of these parameters at the higher intensities of 0.1 and 0.5 mW/g. The intensity dependences induced by EMR were similar, although not identical, in all three biochemical parameters tested.

INTRODUCTION

Threshold level and dose rate effect relationships and the basic mechanisms of the interactions of electromagnetic radiation (EMR) with biological systems are

To whom correspondence should be addressed Washington, DC 20059, USA. l

0302-4598/92/$05.00

at Department

of Botany, Howard University,

0 1992 - Elsevier Sequoia S.A. All rights reserved

180 Adat* t_-.-?

ChORH

A

t

Glucose

i-Phorphoglycerote

Fig. 1. Schematic diagram showing membrane-bound versus non-membrane-bound molecular nents of interest in a cell.

not well understood. In the bulk of the previous studies, calcium ion (Ca*+) efflux enhancements have been found within certain narrow power windows at various EMR frequencies. These studies were done at EMR frequencies of 50, 147 and 450 MHz [l-4]; 915 MHz [5]; and 147 MHz [61; all amplitude-modulated (AM) at 16 Hz using different cell types of neuroblastoma cells and tissues like avian brains. Calcium ions exist both as extracellular and intracellular components that pass through the cell membranes to maintain the physiological balance. Recently, another biochemical parameter, AChE (which, unlike Ca*+, is generally bound in the postsynaptic membrane in neuron cells), has been used. AChE is important for its physiological role in the acetylcholinergic neurotransmitter system of intercellular communication. Dutta et al. [71 have reported enhanced activity of AChE that has also been observed within a narrow power density window induced by 147 MHz with AM 16 Hz. This repeated phenomenon of power density windows led to the testing of another biochemical parameter in the cells, which, unlike both Ca*+ and AChE, was entirely cytoplasmic. The enolase enzyme is clinically significant and has an important role in the glycolytic pathway (see Fig. 1) of all cells, including neuron cells. Neuroblastoma cells used in these studies synthesize the gamma-gamma

181

form of enolase [8], called neuron-specific enolase (NSE). In neuron cells, NSE constitutes 3% of the total neuronal soluble protein. NSE has been used as the diagnostic marker for different kinds of cancer-like peripheral neuronal tumor, lung carcinoma [93, neuroblastoma [91, Markel cell tumor [lo], retroperitoneal tumor [ll], melanoma [12,13], etc. Figure 1 provides a schematic representation of Ca’+, AChE and enolase activities in a typical cell. Using mammalian cell lines in culture, the effects of 147-MHz, 16-I+ AM (80%) on enolase activities were studied under similar exposure conditions to those for previous Ca ‘+ flwr and AChE activity experiments. The EMR effects on all three of these biochemical parameters are compared.

MATERIALS AND METHODS

Cell cultures IMR-32 Human neuroblastoma cells (IMR-32, obtained from American Type Culture Collection) were seeded with identical aliquots in 25 cm* culture flasks for surface tissue growth and grown to a confluent monolayer in 5 ml of Eagle’s minimum essential medium (MEM) supplemented with 10% fetal calf serum, glutamine (0.02 mMI and gentamicin (1%) containing potassium and necessary elements. NGlO&15 cell line NG108-15 cells were obtained from M. Nirenberg of the National Institutes of Health, Bethesda, MD. This cell line resulted from the fusion of N18TG-2 mouse neuroblastoma with C6BU-1 rat glioma [14]. NGlO8-15 cells were grown at 37°C to a confluent monolayer of 25 cm* growth surface in tissue culture flasks containing 5 ml of Dulbecco’s modified Eagle medium supplemented with 1% gentamicin (Whittaker M.A. Bioproducts, Walkersville, MD), 1% aminopterin (5.65 ml from 0.01 mM stock solution), 1% hypoxanthine/thymidine (5.65 ml 0.01 H/1.6 mM T stock solution) and 10% fetal bovine serum. Cell growth and optimum enolase activities in cells The NG-108 cell line was subcultured in a 25 cm* tissue culture flask. The numbers of cells were counted with the corresponding estimation of enolase specific activity at 24-h intervals starting from 0 h. Figure 2a shows that steady increases in cell numbers were observed for 168 h. The specific activity of enolase (from Fig. 2b) increased steadily for 144 h starting from 0 h. At 144 h of culture growth, enolase specific activity was optimum. Similar observations were made for the IMR-32 cell line.

01 0 24

46

72

96 126 144 166 192

Time in culture/h

0

24

40

72

96 126 144 166 192

Time in culture/h

Fig. 2. (a) Growth curve of the NG-108 neuroblastoma cells from 0 to 168 h (O-7 days) in culture. (b) Specific activity of enolase in NG-108 cells according to the growth rate of the cells.

Cellular exposure The Crawford EMR exposure system is basically similar to that described by Dutta et al. [5]. Theoretical considerations regarding the equation for SAR are described by Dutta et al. [15]. Monolayer cells in each experimental flask were placed inside the TEM Crawford chamber and irradiated for 30 min with 147 MHz radiation sinusoidally amplitude-modulated to 80% of peak amplitudes at 16 Hz. The entire exposure chamber was placed in an incubator kept at 37°C. Two identical control flasks were kept at 37°C outside the exposure chamber within the same incubator. Similarly, sham flasks (i.e. flasks kept inside the exposure chamber .but without any EMR) were set up to be compared with respective controls. A series of experiments were done at SAR values of 0.001, 0.01,0.02,0.05, 0.07,0.08, 0.09, 0.1, 0.5 and 1.0 mW/g. After irradiation, cells were gently scraped from the surfaces of the two culture flasks, pooled in a centrifuge tube and then centrifuged at 500 g for 5 min at 4°C. The supernatant was discarded. The control and experimental cells were homogenized separately following the method of Marangos et al. 1161.The cell pellet was homogenized in 1 ml of 10 mM Tris-HCl (pH 7.4) and 1 mM MgSO,. The homogenate was centrifuged at 13000 g for 10 min at 4°C and the supernatant was collected as the enzyme source.

183 TABLE 1 Typical sample data generated by 147-MHz, AM 16-Hz (SAR 0.01 mW/g) RFR on enolase activities of human neuroblastoma cell line IMR-32 a Expt. No.

OD of enzyme at 240 nm Initial

OD at 280 nm

Change

Final

Protein

Enzyme

Specific

cont./

activity

activity

mg ml-’

/pmol min-’ mg-’

1c E

0.270 0.264

0.375 0.360

0.105 0.096

0.170 0.165

0.257 0.249

1.582 1.446

0.307 0.290

2c E

0.260 0.273

0.365 0.377

0.105 0.104

0.158 0.171

0.239 0.259

1.582 1.566

0.330 0.302

3c E

0.264 0.264

0.368 0.352

0.104 0.088

0.165 0.165

0.249 0.249

1.566 1.325

0.314 0.266

4c E

0.265 0.260

0.374 0.355

0.109 0.095

0.159 0.162

0.240 0.245

1.642 1.431

0.342 0.292

a C, control; E, experimental; OD, optical density.

Assay of enzyme from the sample

The specific activity of enolase was determined according to Marangos et al. 1171 by measuring the velocity of reaction against a blank at 240 nm using a Bausch & Lomb Spectromic 601. The sample (0.05 ml) was mixed with 0.950 ml of assay buffer [50 mM Tris-HCI, pH 7.0; 1.5 mM MgSO,; 0.15 M KCl; and substrate 1 mM 2-phosphoglyceric acid (2-PGA)]. The reaction was allowed to continue for 3 min at 25°C. A linear absorbance increase of 0.100 was interpreted to be equivalent to the formation of 0.226 PM phosphoenolpyruvate [18]. The concentration of protein from the same sample was determined at 280 nm against a blank using the same Fisher/Milton Roy spectrophotometer 601 [19]. The specific activities of the enolase enzyme were expressed in units of pmol/min per mg of protein at 25°C. Table 1 presents sample data obtained at SAR 0.01 mW/g. These results are from four independent experiments. Two flasks (containing the same quantity of monolayer cells) were placed in the same isothermal incubator at 37 + 0.2”C for controls and two similar flasks were placed in the Crawford exposure chamber, which was kept in the same incubator. As an example for estimating enolase specific activity: enzyme activity =

increase in OD in 3 min x 2.26 x 20 3

pmol/min

per ml

where OD is the optical density at 240 nm. Using the data from experiment No. 1, control, Table 1, (0.105 X 2.26 X 20)/3 = 1.582 fimol/min per ml. The factor of 2.26 pmol of phosphoenolpyruvate was adopted from a previous procedure [17]. The dilution factor was 20 in this experiment, because 50 ~1 of the

184

extract was added to 0.95 ml of assay buffer containing 4.0 ~1 (1 mM) of the substrate 2-PGA. The formula for estimating the amount of protein is 1 OD at 280 nm = 1.515 mg protein/ml [19]. Therefore, 0.170 OD = 0.257 mg protein in 50 ,ul of the extract. Hence, the protein concentration = 0.257 x 20 = 5.14 mg/ml. Specific activity of enolase =

(activity of enzyme/ml) (mg of protein/ml)

= 0.307

1.582 = -5.140

pm01 min-’ mg-‘.

RESULTS

Growth patterns of neuroblastoma cell line NG-108 The cells increased in number as expected from 0 to 8 days, at each counting. The doubling time was determined to be approximately 3.5 days (84 h), as shown in Fig. 2. These findings are close to the doubling time of 3.0 days reported by Tumilowicz et al. [20]. In these cultures, confluence was reached in 4-5 days. After approximately 5 days (120 h), numerous cells became detached and were suspended in the medium. The floaters were a potential source of error during exposure because dosimetry was calibrated on the basis of an attached confluent monolayer of cells. Both attached and suspended cells were tested with trypan blue. The number of cells that absorbed the dye ranged from 0 to never more than 10. This range indicated greater than 99% cellular viability. The growth patterns of the IMR-32 neuroblastoma cell line were similar. Specific activities of enolase correlated closely with the growth patterns. Depending on the confluence of cells, the maximum enolase activities were thus noticed within 4-5 days. Control versus sham experiments These tests were carried out to determine whether under this experimental conditions there was a significant difference between activities of the controls and shams. The results obtained are shown as (Fig. 3). The means of the specific activities of enolase of the sham

21 0.2 C .z ti .-” 0.1 )c: cn” 0.0l-MllJl

1

2

3

laboratory’s the specific a histogram and control

4

Experiment number

Fig. 3. Comparison of total enolase specific activity of shams (ml and controls (0) using the IMR-32 human neuroblastoma cell line.

185 TABLE 2 Effects of 147-MHz, 16-Hz AM (80%) low-level RFR on total enolase activity in IMR-32 cells at different dose rates SAR/mW g-’

0.001 0.01 0.02 0.05 0.07 0.1 0.5

Specific activity/pm01 min-’ mg-’ Control (mean f SEMI

Exposed (mean f SEMI

Change of specific activities/%

0.231 f 0.02 0.323 f 0.007 0.276 + 0.004 0.190*0.01 0.200*0.01 0.135 f 0.003 0.152kO.01

0.212 + 0.02 0.287 f 0.007 0.269 f 0.004 0.242kO.01 0.188 f 0.005 0.139&-0.002 0.159*0.01

-8 -lla -3 -1-27’ -6 +3 +5

a P < 0.01. Results are the means of four separate exposure experiments.

cells were 0.157 f 0.003 and 0.153 f 0.002 ~mol/min per mg, respectively. The t value was 1.655 (0.10

Table 2 presents the results of several independent experiments conducted at increasing dose rates beginning at SAR 0.001 mW/g and ending at SAR 0.5 mW/g in the human cell line IMR-32. The data indicate a depression of enolase specific activity at SAR 0.001, 0.01 and 0.02 mW/g, and an enhancement at SAR 0.05 mW/g relative to the control values. Enhancement of enolase specific activities is not significant above SAR 0.1 mW/g. The maximum significant decrease (11%) was at SAR 0.01 mW/g; the maximum significant increase (27%) was at SAR 0.05 mW/g. [At 147-MHz EMR when tested without 16-Hz amplitude modulation (80%), enolase specific activities remained at control values.] Table 3 presents results of several independent experiments using the NG-108 cell line conducted under conditions similar to those for IMR-32. The largest significant depressions (21 and 13%) were at SAR 0.01 and 0.08 mW/g, respectively and the largest significant enhancement (19%) was at SAR 0.05 mW/g. Comparison of dose-dependent effects of enolase with other parameters

Figure 4 shows composite results in histograms generated from numerous experiments already published [6]. Histograms obtained from the AChE experiments have been submitted for publication. Comparisons of the histograms show significant enhancements in activities of all the biochemical parameters tested (Ca*+, AChE and enolase). On the other hand, for all the biochemical parameters used, the activities were depressed at SAR 0.01 mW/g. At SAR 0.08 mW/g, another decrease using the neuroblastoma cell line NG108-15 was observed.

186 TABLE 3 Effect of 147-MHz, 16-Hz AM (80%) RFR on total enolase activity from NG10815 at different dose rates SAR/mW g-i

0.001 0.01 b 0.01 0.02 0.05 0.07 0.08 0.09 0.1 0.5

Specific activity/pm01 mitt-’ mg-’ Control (mean f SEM) (N) a

Exposed (mean f SEM) (N)

Change of specific activities/%

0.351 f 0.04(4) 0.266 f O.Ol(3) 0.345 f O.Ol(4) 0.424 f O.Ol(5) 0.464 + 0.03(71 0.567 + 0.05(7) 0.211 f 0.007(41 0.368 f 0.006(4) 0.466 f O.w(5) 0.274 f 0.03(5)

0.340 f 0.05(4) 0.215 f 0.008(3) 0.274 f 0.01(4) 0.406 f O.Ol(5) 0.552 f 0.02(7) 0.537 f 0.04(7) 0.184 f 0.003(4) 0.363 f 0.005(4) 0.482 + 0.05(5) 0.255 rtO.2(5)

-3 -18’ -2ld -4 19 e -5 -13’ -1 4 7

a N, number of tests. b Independent tests at SAR 0.01 mW/g conducted by two separate persons. Statistical analyses were done using Student’s t-test; ’ P < 0.02; d P < 0.002; e P < 0.05; ’ P < 0.01.

t-q

0.001

0.01

0.02

0.05

0.07

0.08

0.09

0.1

0.5

SAR /mW g-1 ql4.05,

r.p<.oi,

ypc.02,

~~pdol

Fig. 4. Comparison of EMR effects using histograms for different biochemical parameters expressed as functions of increasing dose rate levels. Data on Ca ‘+ efflux studies are taken from Dutta et al. [5,6]. AChE data are taken from Dutta et al. [7]. (0) Calcium in IMR-32; (~1 acetylcholinesterase in NG-108; (gx) enolase in IMR-32; (@I enolase in NG-108. l P < 0.05; l * P < 0.01; t P < 0.02; 17 P < 0.001.

187 DISCUSSION

Low-dose rate RFR at a frequency of 147 MHz sinusoidally amplitude-modulated (80%) at 16 Hz can induce a significant alteration in the total enolase specific activity in human IMR-32 and rodent hybrid NG-108 neuroblastoma cells in culture at narrow power windows. In both cell lines, this RFR depresses enolase specific activities at SAR 0.01 mW/g and enhances them at 0.05 mW/g (Tables 2 and 3). Interestingly, similar effects were noticed (Fig. 4) when the other biochemical parameters, Ca*+ efflux and AChE activity, were used. In the absence of 16-Hz amplitude modulation, no significant changes in the total enolase specific activity were noticed. The following experimental conditions are emphasized: (i) The human neuroblastoma cell line used was IMR-32. Its growth and gross morphological characteristics were consistent with those reported by Tumilowicz et al. [20] and those identified by the American Type Culture Collection. (ii) The data demonstrated that there was no significant difference between shams and controls (Fig. 3), confirming that there was no significant leakage, at the time of exposure, of low-intensity RFR that would be a potential source of error. (iii> When the culture medium alone was tested for total enolase specific activity after O-5 days in culture, no significant total enolase specific activity was detected using the standard assay procedure. Although some enolase appearing in the medium might be expected because of cell death, it did not occur during the O-5 day time period. In addition, this finding is consistent with a report by Evans and Marangos [21] which indicated, in considering whether the level of enolase (specifically NSE) in serum was caused by tumor breakdown or an active transport system, that little NSE is present in the media of actively dividing neuroblastoma cell cultures. The finding of no enolase specific activity excludes native enolase as a possible source of error. On the basis of these conditions, it is concluded that any change in the total enolase specific activity is the result of exposure of the IMR-32 and NG108 cells to the low-intensity RFR specified in these experiments. Exposure experiments using the frequencies 147 and 915 MHz, 16 Hz sinusoidally amplitude-modulated (80%) have shown that low-intensity RFR affects Ca*+ efflux. Dutta et al. [5,6] have reported both dose rate and frequency-dependent significant Ca*+ efflux enhancement in IMR-32 cells at 0.0005, 0.0007, 0.05 and 1.0 mW/g. The findings of Dutta et al. [5,6] corroborated those of Bawin and Adey [22] and Blackman et al. [l-3]. They studied Ca*+ efflux in chick and cat cerebral tissue at 147 MHz. They found that the Ca*+ efflux response is elicited in a rather abrupt manner at particular SAR values. This response has been called a “window effect” [23-251. This window effect is taken to mean that there exists limiting combinations of frequency and amplitude outside of which the perceived effect disappears. This idea is somewhat controversial. The disappearance of the effect does not necessarily mean that it will not reappear at either lower or higher values of SAR or frequency [241. However, this window effect fits the data gathered by Dutta et al. [5,6] and independently by Blackman et al. [26,27] and Bawin and Adey [22].

188

The data shown in Fig. 4 provide evidence that all three biochemical parameters follow an oscillatory pattern of effects at low intensities. The patterns of dose-response rate effects are similar irrespective of whether the biochemical parameters tested so far are membrane-bound or not. Recently, Litovitz et al. 1281proposed a model in which biological effects increased and tapered off depending on the time and intensity of exposure, but their model does not predict any depression of activities such as those evident from the data presented here. From the present study, it is concluded that the intensity relations induced by RFR are similar, but not identical, in all three biochemical parameters tested.

ACKNOWLEDGEMENTS

This work was supported in part by grants received from the US Environmental Protection Agency (No. R814126-01-O) and from an institutional grant (No. 2S06GM08016) from the NIGMS-NIH and by the Collaborative Core Unit, Graduate School of Arts and Sciences, Howard University. We appreciate the participation of Ms. Tonya Touchstone and Mr. A. Cyril Spiro in a few aspects of these studies. We are grateful to Dr. D.F. Minner for editorial help.

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