Bioelectromagnetics 20:1–4 (1999)

Effect of 50 Hz Sinusoidal ElectromagneticField on the Kinetics of 14CO2 Exhalation

After [14C]-N-NitrosodiethylamineAdministration in Mice

Satnam Singh,1 K.L. Khanduja,2 and P.K. Mittal1*1 Department of Zoology, Panjab University, Chandigarh 160014, India

2 Department of Biophysics, Postgraduate Institute of Medical Education and Research,Chandigarh 160012, India

N-Nitrosodiethylamine (NDEA) has been identified as a typical environmental carcinogen. Its metabo-lism was studied in mice under the influence of an electromagnetic field (EMF). After intraperitonealadministration of [14C]-NDEA, 0.2 mCi/100 g body weight resulted in 22.8% of the total radioactivityexhaled as 14CO2 within 1 h. Mice were exposed to a 50 Hz, 2 mT (rms) electromagnetic field, 8 h/day for 8 weeks. There was a significant increase in the metabolic turnover of [14C]-NDEA into 14CO2

at the end of both 6 and 8 weeks of field exposure, i.e., 26.9% and 37.4% respectively. The enhancedcapacity of mice to metabolize NDEA after the exposure to EMF may result in animals with a smalleramount of the bioactive carcinogen burden, thereby indicating a protective role of 2 mT EMF in awhole animal study. Bioelectromagnetics 20:1–4, 1999. q 1999 Wiley-Liss, Inc.

Key words: magnetic fields; nitrosodiethylamine; carcinogen; radiorespirometery; drugmetabolism

INTRODUCTION [Magee and Barnes, 1956] and lung tumors in mice[Clapp et al., 1971].

Experimental studies have indicated that an elec- Metabolic activation of NDEA is required to formtromagnetic field (EMF) is somehow capable of influ- reactive metabolites. This activation pathway involvesencing several cellular functions. EMF effects have a-hydroxylation leading to N-nitroso functionalitybeen demonstrated at the level of transcription [Good- [Magee and Barnes, 1967; Kroeger-Koepe et al., 1981].man and Henderson, 1991; Cridland, 1993], ion trans- The product then undergoes a fragmentation reactionport processes [Blank, 1992; Itegin et al., 1995], carci- to alkyldiazonium ion that eventually leads to alkyl-nogenesis [Beniashvili et al., 1991, Loscher et al., ation of nucleic acids and proteins, by means of carbo-1993], cell proliferation [Akamine et al., 1985; Phillips nium type intermediates [Gold and Linder, 1979].et al., 1986], and potentiation of antineoplastic drugs Motivated by conflicting reports of the effects of[Hannan et al., 1994]. Cyclophosphamide, in combina- EMF on the biological systems, including reports oftention with EMF, was found to be more toxic to bone linking EMF to cancer, this research studied the influence

of EMF on the metabolism of a potent carcinogenmarrow cells in mice [Cadossi et al., 1991], which(NDEA). This study observed the kinetics of 14CO2 exha-indicates that EMF might play a role in the toxication/lation after the administration of radiolabelled NDEA.detoxication systems in the organism.

N-Nitrosodiethylamine (NDEA) is a dialkyl ni-MATERIALS AND METHODStrosamine, known as a strong environmental carcino-Chemicalsgen. Carcinogenicity of NDEA has been reported to

[14C]-N-Nitrosodiethylamine, specific activitybe species-specific and also to depend on the route of57 mCi/mmol, was procured from Amersham (UK),administration. NDEA is carcinogenic in 22 of the ani-

mal species tested, including subhuman primates. The*Correspondence to: Dr. P. K. Mittal, Dept. of Zoology, Panjab Univer-main target organs are the trachea, lungs, nasal mucosa,sity, Chandigarh-160014, India.oesophagus, and liver [Schmahl and Habs, 1980].

NDEA induces malignant hepatic tumors in rats Received for review 10 June 1997; Final version accepted 6 April 1998

q 1999 Wiley-Liss, Inc.

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2 Singh et al.

2,5-diphenyloxazole and 1,4-bis(5-phenyl-2-oxazolyl)-benzene were obtained from Sigma Chemicals Co., St.Louis, MO. The rest of the chemicals were of analyticalgrade, procured locally from Sisco Research Labora-tories, Bombay, India.

Animals

Experiments were carried out in 6- to 7-week-oldmale Swiss inbred mice of 22 to 24 g of body weight(b.w.), attaining 26 to 28 g of b.w. by the end of theexperiment at 14–15 weeks of age.

ELECTROMAGNETIC FIELD EXPOSUREFig. 1. Pattern of 14CO2 exhaled before and after (8 weeks) EMFA sinusoidal EMF was generated in a solenoid exposure of 2 mT after IP administration of 14C-NDEA.

coil as described earlier [Singh et al., 1997]. The ani-mals were exposed to the electromagnetic field in plas-tic cages that were positioned in the region of uniform

of total radioactivity administered per 10-min intervalfield of the solenoid, which was alligned in the North-by the formula:South direction of the earth’s magnetic field. The mice

had free access to food and water in the solenoid.14CO2 exhaled/10 min (DPM)

DPM of 14C-NDEA administered IP1 100

RADIORESPIROMETRY

The area under the curve (AUC 0–60 min) wasSix mice were exposed to 2 mT EMF, 8 h/daydetermined by taking sum (mean { SD) of the totalfor 8 weeks. Exhalation of 14CO2 after intraperitonealradioactive CO2 (%) exhaled by all six animals during(IP) administration of [14C]-NDEA was recorded be-60 min. The time interval of the peak 14CO2 exhaledfore exposure, at 4, 6, and after 8 weeks of the experi-was recorded for interpretation of the experiment.ment. At 0 week, i.e., 1 day before EMF exposure, and

Statistical analysis of the results was done byat the end of the 4th, 6th, and 8th weeks of exposure,using Student’s t test.each animal was injected intraperitoneally 14C-NDEA

at a dose of 0.2 mCi/100 g of b.w. in 0.2 ml of sterilenormal saline. Another group of six mice (of the same RESULTSage) were sham-exposed to EMF and received identicaldoses of radioactive NDEA at the same intervals. Expiration of 14CO2 was detectable soon after an

IP dose of [14C]-NDEA. Peak exhalation of 14CO2 be-After the injection, each animal was immediatelyput in an airtight Perspex chamber with an inlet and tween 0 and 10 min was followed by a gradual decline

in the release of 14CO2 every 10 min, which was re-outlet for air flow. Atmospheric air was pumped intothe chamber through the inlet at a steady flow rate of corded for 60 min. Figure 1 shows the sequence of

14CO2 exhaled (percentage of administered [14C]-1.36 l/min. The expired air was passed through differ-ent test tubes, for each 10 min interval. Each tube NDEA) per 10 min interval, measured before EMF

exposure and at the end of the 8th week of the experi-contained 2 ml of a mixture of ethanolamine:ethyl gly-col monomethylether:Triton X-100 (3:1:2 V/V). A so- ment. In the parallel group of control animals receiving

sham-exposure, exhalation of 14CO2 remained un-lution of 0.2 ml of saturated NaOH was added to eachtube. The total trapping procedure continued for 60 min. changed throughout the experiment, i.e., 0-, 4-, 6-, and

8-week intervals (data not shown). The values wereThe trapping mixture was then transferred to vials con-taining 10 ml of toluene-based scintillation fluid; not significantly different than those at 0 week in the

experimental group (Table 1). Therefore, to avoid any2 to 3 ml of methanol was added to remove any tur-bidity. Radioactivity was measured in a liquid scintilla- unnecessory confusion in the interpretation of results,

the six exposed mice were used as their own controls;tion counter having an inbuilt provision for conversionof counts per minute (CPM) to disintegrations per mi- their exhalation of 14CO2 at 0 week was used as the

control value.nute (DPM).Exhalation of 14CO2 was calculated as percentage At 0 week, during the control measurement, there

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EMF Influences NDEA Metabolism 3

TABLE 1. Effect of 2 mT, 50 Hz EMF on the Metabolism of [14C]-NDEA Administered IPin Mice Exposed for 8 Weeks (8 h/day)†

14CO2 Exhaled (% of administered radioactivity)

Intervals (weeks) of 14C NDEA administrationExhalationtime (min) 0 4 6 8

10 9.3 { 0.8 10.4 { 1.0 12.3 { 1.3 14.3 { 2.020 5.2 { 1.0 4.7 { 1.9 4.9 { 1.2 7.8 { 1.630 3.0 { 0.6 3.5 { 1.4 3.2 { 1.2 5.9 { 0.540 2.2 { 0.6 2.2 { 0.5 2.3 { 0.9 4.0 { 0.650 1.6 { 0.4 1.6 { 0.7 2.2 { 0.6 2.8 { 0.660 1.4 { 0.4 1.4 { 0.5 1.9 { 0.7 2.4 { 0.6AUC (%) 22.8 { 0.6 23.8 { 1.0 26.9 { 1.0* 37.4 { 1.0***†Values are expressed as mean { SD (n Å 6); AUC (%) (0–60 min), area under the curve is meanof total radioactive CO2 (percentage of administered radioactivity) exhaled in 60 min by all the sixmice (***P õ .001; *P õ .05); EMF, electromagnetic field; NDEA, N-nitrosodiethylamine.

was a decrease in the exhalation of 14CO2 from a peak [Magee and Barnes, 1967; Appel et al., 1979; Kroeger-Koepka et al., 1981], with the possible involvement ofof 9.3 { 0.8% at 10 min to 5.2 { 1.0, 3 { 0.6, 2.2 {

0.6, 1.6 { 0.4, 1.4 { 0.4% at 20, 30, 40, 50, and other nonenzymatic factors responsible for degradationof NDEA. In a separate study, we observed that, after60 min, respectively. At the end of 8 weeks of exposure

to EMF, the peak level of 14CO2 released was signifi- protracted EMF exposure of the same intensity andduration, the carcinogen detoxifing enzyme system incantly enhanced (P õ .001) to 14.3 { 2.0% at 10 min,

which gradually decreased to 7.8 { 1.6, 5.9 { 0.5, 4.0 mice was affected favourably (Singh et al., 1998). Theinduction of hepatic cytochrome P-450 in our earlier{ 0.6, 2.8 { 0.6, 2.4 { 0.6 at 20, 30, 40, 50, and 60

min, respectively (Table 1). study, therefore, lends support to the accelerated me-tabolism of 14C-NDEA.Cumulative 14CO2 exhaled in 60 min (percentage

of 14C-NDEA administered dose) at 2 mT EMF did There has been no report so far on the effects ofEMF on NDEA metabolism, but several workers havenot increase significantly from 22.8 { 0.6 (0 week) to

23.8 { 1.0% at 4 weeks of EMF exposure. Significant found an influence of EMF on carcinogenesis. McLeanet al. [1991] reported induction of mouse skin tumorsincreases to 26.9 { 1.0% (P õ .05) was observed at

6 weeks and to 37.4 { 1.0% (P õ .001), at the end of using the carcinogen 7, 12-dimethylbenz(a)anthracenewith coexposure to 60 Hz magnetic fields. Cain et al.8 weeks (Fig. 2). The maximal amount of radioactive

CO2 recovered was 37.4% of the total intraperitoneally [1993] reported that 60 Hz EMF could enhance celltransformation and act as copromoter. However, sev-administered [14C]-NDEA (0.2 mCi/100 g of b.w.) in

the form of expired air during 60 min of the observationperiod.

DISCUSSION

The end product of 14C-labelled NDEA metabo-lism is 14CO2. Therefore, the exhalation of 14CO2 wastaken as an index of the carcinogen metabolism. Intra-peritoneal administration of [14C]-NDEA was followedby its rapid metabolism in the liver, resulting in thepeak exhalation of 14CO2 within first 10 min of theinjection. The rapid exhalation of 14CO2 in mice showsthat a highly efficient mechanism to metabolize NDEAexists in rodents. This mechanism was further harn-essed by EMF exposure of 2 mT after 6 and 8 weeksof study. Fig. 2. Total 14CO2 exhaled in 60 min after IP administration of

NDEA is metabolized by a-hydroxylation and 14C-NDEA in mice exposed to 2 mT EMF for 8 weeks. *P õ .05;***P õ .001.denitrosation by cytochrome P-450 dependent enzymes

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4 Singh et al.

of human fibroblasts exposed to ELF magnetic fields. In: Blankeral other studies [Cridland et al., 1993; Paile et al.,M, editor. Electricity and Magnetism in Biology and Medicine.1995] did not find any influence of 50 Hz, 2 mT EMFSan Francisco: San Francisco Press, Inc. pp 628–632.

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