55
Atherosclerosis, 25 (1976) 55-62 @ Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
RADIO AND MICROWAVE RADIATION AND EXPERIMENTAL ATHEROSCLEROSIS
H.V. SPARKS, D.L. MOSSMAN * and C.L. SEIDEL ** Department of Physiology, 48104 (U.S.A.)
University of Michigan Medical School Ann Arbor, Mich.
(Received 20th January, 1976) (Accepted 20th February, 1976)
Summary The effect of radio and microwave radiation on dietary atherosclerosis of rabbits was tested. 16 New Zealand white rabbits were exposed to microwave (2.45 GHz) radiation at a power density of 20-30 mW/cm’ for 4 h a day, 5 days a week for 8 to 10 weeks. Irradiated animals had serum cholesterol concentrations, aortic wall cholesterol concentrations and percentage of intimal surface involved in atherosclerotic lesions which were not different from age and weight matched controls. Continuous radio frequency irradiation (1 MHz) for 8 to 11 weeks with a field strength of 30 V/cm also failed to change these indices of atherogenesis. We conclude that under the conditions of these experiments radio and microwave irradiation do not affect the course of diet induced atherogenesis.
Key words:
Aorta - Cholesterol - Hypercholesterolemia
-Rabbit
Introduction There has been some suggestion that exposure to ambient electromagnetic irradiation may affect the course of cardiovascular disease in man [l] . Since it would appear to be extremely difficult, to separate the effects of exposure to electromagnetic irradiation from other environmental influences on the course This work was supported by USPHS Grant HL-14408. * Present address: Oregon Graduate Center, 19600 N.W. Walker Road. Beaverton. Oregon 97006. U.S.A. ** Present address: Department of Physiology. University of Virginia. Charlottesville. Virginia, U.S.A.
56
of cardiovascular disease in man, we thought it would be of interest to determine whether experimentally induced atherosclerosis of rabbits is affected by exposure to electromagnetic irradiation. We chose the rabbit because this species is particularly susceptible to atherosclerosis induced by a diet high in cholesterol and therefore the disease can be produced in a relatively short time. In addition a great variety of noxious agents resulting in injury to the aortic wall have been demonstrated to enhance atherogenesis in this species [2] . We tested the hypothesis that electromagnetic irradiation causes injury to the aortic wall, either directly or indirectly, and that this in turn results in enhanced atherogenesis. Both radio frequency irradation (1 MHz) and microwave irradiation (2.45 GHz) were used since these 2 frequency ranges should have different biological effects and humans are exposed to both frequencies in normal situations. Materials and Methods Microwave irradiation Thirty-two young New Zealand albino male rabbits were divided into 2 sets of 16 (Sets 1 and 2). A diet containing 1% cholesterol (Teklad-Mills rabbit pellets with 1% by weight cholesterol added using vegetable oil as a binder [2] ) supplemented with carrots and greens was fed to all animals beginning on the first day of irradiation. Each set of 16 rabbits was divided into 2 groups, the irradiated experimental group (E) and the control group (C). The experimental groups were exposed to 2.45 GHz field with a power density of between 20 and 30 mW/cm2 for 5 days per week (exposure time 3;-4 h). The control groups were placed in a control chamber which had an interior similar to that of the irradiation chamber at the same time that the corresponding experimental group was irradiated. Experimental and control groups were paired by age and weight at the beginning of the experiment. Set 1 was exposed to microwave irradiation for 8 weeks (38 exposure days) totaling 138 h of exposure. Set 2 was exposed for 10 weeks (50 exposure days) for a total exposure time of 210 h. The microwave irradiation system employed in these experiments was fashioned after one designed by Basset and reported by McRee and Walsh [3]. The microwaves were generated by a QK-390 magnetron in a Raytheon PGM100 power generator. Microwaves were guided from the magnetron to a dualdirection coupler by RG-225/u coaxial cable with an attenuation of 0.008 decibels/100 feet. A Narda model 3022 directional coupler allowed continuous monitoring of forward line power using a 20 db attenuated thermistor mount and a Hewlett-Packard power meter. The other sampling port of the coupler was used to obtain periodic frequency readings using a Hewlett-Packard 536A frequency meter. The frequency was always within the magnetron specification of 2,450 f 25 MHz. Coaxial cable was used to connect the directional coupler to a standard gain pyramidal horn with a minimal cut off frequency of 2.3 GHz. An anechoic irradiation chamber was constructed which provided a 76 cm2 area with a uniform power density. Since the target area (the floor of the chamber) was more than 5 feet away from the horn, the target area was in the far field or Fraunhofer region. The chamber shell was made of 2”plywood and was lined with 4” hairflex. The target base was made of Emerson--Gumming
24” Eccosorb foam block microwave absorber. A blower was used to ventilate the chamber with its exhaust directed into the control chamber. The rabbits were restrained in an acrylic cage which kept each of 4 rabbits in separate quadrants of the target area. Acrylic was chosen for its resistance to destruction by the rabbits and low electromagnetic reflection properties. The acrylic polymer transmits all but 3-4s of the incident microwaves. Power density was checked daily using a Narda electromagnetic leakage monitor and probe. Irradiating power density was kept between 20-30 mw/cm*. Rectal temperatures of all rabbits were recorded before and after each irradiation period using a Yellow Springs Instrument Tele-Thermometer and probe. Radio frequency irradiation Two sets of New Zealand albino male rabbits were studied in this part of the experiment. All animals were fed the 1% cholesterol diet described previously. Each set of 18 rabbits was divided into 2 groups, the irradiated experimental group (E) and the control group (C). The experimental groups were exposed to a radio frequency electric field by placing them in stainless steel cages with dimensions of 60 X 30 X 46 cm. A 50 X 25 cm steel sheet covered with a polyurethane resin was placed in the top of the cage and the steel grid upon which the rabbits rested served as the opposite plate allowing development of an electric field of 30 V/m oscillating at a frequency of 1 MHz. A small dipole probe was used to measure the near electric field strength in the cage which was uniform to within 2 cm of the cage walls where it dropped off markedly. The top plate and floor grid were attached to a solid state oscillator designed to drive up to 10 irridation chambers in parallel. The voltage across the cages as well as the field strength within the cages was checked on approximately a weekly basis. Three rabbits from Set 1 and 2 from Set 2 died before sacrificing due to causes unrelated to the experiment (ear mite infestation and pneumonia). As a result group 1 had 15 rabbits (7 experiment&) and group 2 had 16 rabbits at the time of sacrifice. The experimental group of Set 1 was continuously exposed to radio frequency irradiation for 55 days and the experimental group of Set 2 was continuously exposed for 81 days. Control animals were kept for the same length of time in the same type of stainless steel cages except for the irradiation equipment. They ate the same diet. Experimental and control groups were paired by age and weight at the beginning of the experiment. Measurements A 3 ml blood sample was drawn from an ear vein of each rabbit before initiating the high cholesterol diet and electromagnetic field irradiation. Serum cholesterol was determined using the method of Abel1 et al. [4] . At the end of the experimental period, animals were sacrificed by cervical dislocation and blood was drawn from the heart for determining serum cholesterol. The aorta from the aortic valve to the celiac artery was removed and placed in a 0.9% saline solution. Fat was then removed from adventitia and the vessel was sliced lengthwise to expose the intima. The fraction of the intima containing macroscopic atherosclerotic lesions was determined by placing a transparent grid (2.5 mm2 squares) over the intimal surface of the aorta and counting (by 3 individuals) the number of squares containing any evidence of a lesion. Although
58
histologic preparations of aortas were made and inspected, the well known variability due to sampling problems precluded use of this type of information in assessing the extent of atherosclerosis. After obtaining the wet weight of the aortas they were lyophilized and reweighed. The dried aortas were weighed and then cut into small pieces with scissors and placed in glass homogenizing tubes. The aortas were homogenized (approximately 5 min) in a 2 : 2 : 1.8 mixture of chloroform, methanol and water until no tissue pieces could be observed. The homogenized solution was then centrifuged at lO,OOO-15,000 rpm for 20 min. The chloroform layer was recovered and evaporated to dryness. The residue was reconstituted in l-2 ml of chloroform depending on the expected cholesterol concentration and a small aliquot of this analyzed for cholesterol concentration [4]. Using this method the recovery of cholesterol added to fresh aortas was 97.8 f 4.4%. Means plus or minus one standard error of the mean are reported. Student’s t-test was performed to evaluate the statistical significance of difference. The Michigan Interactive Data Analysis System was used for all statistical computations. Results Microwave experiments
Table 1 summarizes the results of exposure of each of the sets of animals to microwave irradiation. In both experimental and control groups the 1% cholesterol diet caused a marked elevation in serum cholesterol as would be expected from many previous studies [ 21. The younger animals (2 months old at onset of experiment) exposed for a longer period of time (Set 2) had higher serum cholesterols at the termination of the experiment than those in Set 1 (3 months old at onset). All groups developed atherosclerotic lesions. Aortas of rabbits not fed the cholesterol containing diet had no visible lesions. Wall cholesterol content was also markedly elevated in all groups as compared to animals which were not exposed to a high cholesterol diet. Wall cholesterol content of control animals with no added cholesterol in diet averaged 5 t 0.4 mg/g. When experimental groups are compared to control groups few significant differences emerge. The prediet serum cholesterol concentration of control animals in Set 2 (C,) was significantly higher than the prediet serum cholesterol of the experimental animals of Set 2 (E2). A relatively small fraction of the total intimal area was covered with lesions in the first set (C, and E,) and a larger fraction of the area was covered in Set 2 (C, and E,). In both cases there was not a statistically significant difference between non-irradiated control and irradiated experimental animals. This was also true of the cholesterol content of the aortas. Another index of injury of the aorta might be the accumulation mucopolysaccharide and thus a change in the ratio of wet to dry weight of the aortas. Neither set showed a significant difference in this ratio when irradiated and non-irradiated aortas are compared. The irradiated animals had a significant increase in body temperature immediately following the irradiation period. Radiowave experiments
Table 2 summarizes similar data for the animals exposed to radio frequency irradiation and the unexposed controls. Although the average serum cholesterol
1
18.4
-5 138
Each v&e
+0.60 f
expos. (OC) Change in weight (g) Exposure time (h)
(8)
(8)
(8)
(7)
(El
f
0
44*
-0.07
f
39.17 f
4.48 +
23.6
6.5
(8)
(8)
(8)
(7)
86
(7)
0.04 (8)
0.03 (8)
0.82
0.001
0.10
0.73
0.58
0.94
0.49
0.41
P
NON-IRRADIATED
0.20 (8)
7.6
9.1
+ 215
f
E2) AND
23.03 +
1905
85.4
Cl
AND
is mean f standard error. P = P value for Student’s t-test, ( ) = number of aninxik.
(8)
0.10 (8)
0.04 (8)
0.21 (8)
5.4
+ 137
39.08 f
before expos. (“C) Temperature during
4.37 f
f
aorta (6) Mean temperature
(mrk) Wet/dry weight
10.1
22.0
lesions WaII cholesterol
f
1702 + 185
(mUlO ml), sacrifice % of intimal area with
18.0
68.9
f
IRRADIATED
(mg/lOO mu. pre-diet Serum cholesterol
RI
Set 1
OF MICROWAVE
Serum cholesterol
Variable
COMPARISON
TABLE (CI
(8)
(8)
(8)
(8)
0.04 (8)
0.05 (8)
0.14 (8)
t 114
+0.61 f
39.21 f
1252 210
1.3
87
5.48 (8)
?: 13.8
f
f
3.88 f
84.0
94.7
2332
54.4
E2 f
C2) RABBITS
Set 2
AND
2
f
+
f
f
1239 0
-0.14
(8)
(8)
(8)
(8)
(8)
0.06 (8)
0.07 (8)
0.15 (8)
14.0
1.6
98
8.3
f 198
f
39.35 *
4.0
93.8
92.2
2534
78.2
C2
0.96
0.001
0.15
0.57
0.63
0.24
0.15
0.03
P
2
657 55
(7)
(7)
(7)
f 111
(7)
0.12 (7)
+_ 12.6
3.391’
67.1
14.9
k239.0
f
IRRADIATED
(El
48.2
0
k
338
(8)
(8)
(8)
(8)
0.23 (8)
10.6
* 144
3.81 +_
43.0
8.5
0.11 -
0.14
0.17
-
0.62
0.26
P
NON-IRRADIATED
? 215.4
+_
E2) AND
1332.3
Cl
AND
(C,
Each value is mean ?:standard error, P = P value for Student’s t-test. ( ) = number of animals.
aorta (g) Change in weight (g) Temperature (OC!) Exposure time (days)
(mglg) Wet/dry weight
-
1170.0
(mg/lOO ml). sacrifice % of intimal area with lesions Wall cholesterol
67.7
(mg/lOO ml). pre-diet Serum cholesterol
Rl
set 1
OF RADIOWAVE
Serum cholestexol
Variable
COMPARISON
TABLE
7.1
f
0.11 (8)
(8)
(8)
(7)
(8)
488 f 242 (8) 38.16 f 0.33 (7) 81
4.57 f
19.5
12.3
?r144.0
113.97 +
64.1
697.4
44.4
R2 f
C2) RABBITS
set 2
AND
0
7.6
f
0.08 (8)
(8)
(8)
(8)
(8)
8.8 f 103 (8) 39.13 + 0.19 (8)
4.67 f
19.5
6.0
f 120.2
f
112.73 f
88.6
866.2
39.9
C2
0.08 0.02
0.46
0.46
0.10
0.38
0.67
P
O-2 0
61
of animals in Set 2 was lower at sacrifice than any other set, the average wall cholesterol concentration was highest. Perhaps the relatively high wall cholesterol is due to the longer duration of this experiment (81 days). There were no significant differences in any of the variables measured at the end of the experiment including serum cholesterol, area of the intima involved in atherosclerotic lesions, wall cholesterol content of the aortas or the ratio of wet to dry weights. The temperature of the animals in Set 2 exposed to radio frequency irradiation was not higher than that of the unexposed animals and in fact was significantly lower. We do not have an explanation for this and can only say that the animals were in the same room and were exposed to the same ambient temperature. The temperature of the animals in Set 1 was not recorded. Discussion We wished to test the possibility that exposure to electromagnetic irradiation could influence the extent of diet-induced atherosclerosis of rabbits. Results reported above indicate that irradiation in radio frequency and microwave frequency ranges does not increase cholesterol content or intimal surface involvement in this species. We doubt that this negative result is due to inadequate exposure of the animals to electromagnetic energy. Both groups were exposed to microwave and radio frequency irradiation at far greater than defined allowable ambient exposure levels for humans [ 5,6]. Atherogenesis in rabbits is very susceptible to the effects of aortic wall injury. A wide variety of noxious agents including x-ray irradiation [ 71, cold [ 81, and various pharmacologic interventions [2] result in accelerated atherogenesis. For this reason we feel that if either type of electromagnetic irradiation had caused significant injury to the aortic wall by direct or indirect means, it would have resulted in enhanced atherogenesis. We have no way of knowing from these experiments whether longer term exposure to electromagnetic irradiation would yield a different result. The development of atherosclerosis in rabbits as induced by dietary cholesterol is extremely variable so that measurements of cholesterol content, serum cholesterol and intimal area involved in atherogenesis all exhibit large variation in both control and experimental groups. This raises the possibility that some rather subtle change might be hidden in the variability. We are somewhat reassured that this is not the case by the fact that trends of the means went in different directions in different experiments with the control sometimes having a higher wall cholesterol or intimal area involvement and other times having a lower one. The expected effect on atherogenesis should depend on the way in which the microwave or radiowave exerts its effect on the animal. Both the 1 MHz and the 2.45 GHz waves should penetrate the rabbit to some extent [5] so that the aortas are exposed directly to at least some energy dissipation. If this is so, either microwave or radiowave irradiation could have resulted in a direct effect on the aorta, for example by heating. The possibility of significant direct heating of the aorta seems remote since the aortic blood flow should dissipate any heat produced. Either frequency of irradiation could have a direct effect on some other organ (e.g. skin) leading to an indirect effect on the aorta. For
62
example Gordon has shown that exposure to microwave frequencies results in a transient increase in blood pressure followed after several weeks by a decrease [9]. At radio frequencies the decreased blood pressure predominates after a very short initial increase. Since elevated blood pressure is known to enhance atherogenesis [lo], increased atherosclerosis might be expected in our experiments. Apparently any effects on blood pressure in our experiments were not sufficient to result in a measurably different rate of atherogenesis in the animals we have studied. Perovsky et al. reports the microwave irradiation causes diminished atherogenesis in rabbits [ll] . They found a decrease in serum cholesterol and aortic cholesterol content. We cannot support the results of that study based on the current findings. In summary it appears that if electromagnetic irradiation of the frequencies investigated in this study has an effect on atherogenesis, it is too subtle to be uncovered using the number of rabbits and intensity of exposures used in this experiment. Acknowledgement We wish to thank Dr. John M. Schwartz providing many helpful ideas.
for suggesting
the study to us and
References Medvedyev, V.P. Diseases of the cardiovascular system in persons exposed in the past to the action of a super high frequency electromagnetic field, Gigiena Truda, 17 (1973) 6-9. Constantlnldes. P. Experimental Atherosclerosis, Elssvier. Amsterdam, 1965. McRee. D. and Walsh, P.. Microwave exposure system for biological specimens, Rev. Sci. Instr., 42 (1971) 1860-1864. AbelI, L.L., Levy. B.B., Brodie. B.B. and Kendal, F.E.. A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity, .I. Biol. Chem., 195 (1952) 357. Schwan, H.P. Microwave radiation - Biophysical considerations and standards criteria, IEEE Trans. Biomed. Engng.. BME-19 (1972) 304-312. Van de Grlek, A. and Britain. R., Amendments to the U.S. Department of Health, Education and Welfare Microwave Oven Performance Standard. J. Microwave Power, 9 (1974) 3-11. Gold, H.. Production of atherosclerosis in the rat -Effect of X-ray and high fat diet, Arch. Path., 71 (1961) 268-273. Kelly. F.B.. Taylor, C.B. and Hass. G.M., Experimental atheroarteriosclerosis -Localization of Lipids in experimental arterial lesions of rabbits with hypercholesterolemia, Arch. Path., 53 (1952) 419436. 9 Gordon, Z.V., Biological Effects of Microwave in Occupational Hygiene. Published for the Natl. Aero. and Space Admin. and Natl. Sci. Foundation, Washington, D.C.. by the Israel Program for Sci. Trsnslations, 1970, p. 40. 10 BronteSteward, B. and Hepinstall. R.H., The relationship between experimental hypertension and cholesterol-induced atheroma in rabbits, J. Path. Bact.. 68 (1954) 407-417. 11 Perovslty, A.I., Ya Trotsenko, S.. Ya Guz, S., Volkov. E.S., Barko. A.J. and Koshlyak, T.N.. The effect of superhigh frequency electromagnetic field on the course of experimental atherosclerosis, Pat. Fiziol. Eksp. Ter., 13 (1969) 64-66.
Atherosclerosis, 25 (1976) 55-62 @ Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
RADIO AND MICROWAVE RADIATION AND EXPERIMENTAL ATHEROSCLEROSIS
H.V. SPARKS, D.L. MOSSMAN * and C.L. SEIDEL ** Department of Physiology, 48104 (U.S.A.)
University of Michigan Medical School Ann Arbor, Mich.
(Received 20th January, 1976) (Accepted 20th February, 1976)
Summary The effect of radio and microwave radiation on dietary atherosclerosis of rabbits was tested. 16 New Zealand white rabbits were exposed to microwave (2.45 GHz) radiation at a power density of 20-30 mW/cm’ for 4 h a day, 5 days a week for 8 to 10 weeks. Irradiated animals had serum cholesterol concentrations, aortic wall cholesterol concentrations and percentage of intimal surface involved in atherosclerotic lesions which were not different from age and weight matched controls. Continuous radio frequency irradiation (1 MHz) for 8 to 11 weeks with a field strength of 30 V/cm also failed to change these indices of atherogenesis. We conclude that under the conditions of these experiments radio and microwave irradiation do not affect the course of diet induced atherogenesis.
Key words:
Aorta - Cholesterol - Hypercholesterolemia
-Rabbit
Introduction There has been some suggestion that exposure to ambient electromagnetic irradiation may affect the course of cardiovascular disease in man [l] . Since it would appear to be extremely difficult, to separate the effects of exposure to electromagnetic irradiation from other environmental influences on the course This work was supported by USPHS Grant HL-14408. * Present address: Oregon Graduate Center, 19600 N.W. Walker Road. Beaverton. Oregon 97006. U.S.A. ** Present address: Department of Physiology. University of Virginia. Charlottesville. Virginia, U.S.A.
56
of cardiovascular disease in man, we thought it would be of interest to determine whether experimentally induced atherosclerosis of rabbits is affected by exposure to electromagnetic irradiation. We chose the rabbit because this species is particularly susceptible to atherosclerosis induced by a diet high in cholesterol and therefore the disease can be produced in a relatively short time. In addition a great variety of noxious agents resulting in injury to the aortic wall have been demonstrated to enhance atherogenesis in this species [2] . We tested the hypothesis that electromagnetic irradiation causes injury to the aortic wall, either directly or indirectly, and that this in turn results in enhanced atherogenesis. Both radio frequency irradation (1 MHz) and microwave irradiation (2.45 GHz) were used since these 2 frequency ranges should have different biological effects and humans are exposed to both frequencies in normal situations. Materials and Methods Microwave irradiation Thirty-two young New Zealand albino male rabbits were divided into 2 sets of 16 (Sets 1 and 2). A diet containing 1% cholesterol (Teklad-Mills rabbit pellets with 1% by weight cholesterol added using vegetable oil as a binder [2] ) supplemented with carrots and greens was fed to all animals beginning on the first day of irradiation. Each set of 16 rabbits was divided into 2 groups, the irradiated experimental group (E) and the control group (C). The experimental groups were exposed to 2.45 GHz field with a power density of between 20 and 30 mW/cm2 for 5 days per week (exposure time 3;-4 h). The control groups were placed in a control chamber which had an interior similar to that of the irradiation chamber at the same time that the corresponding experimental group was irradiated. Experimental and control groups were paired by age and weight at the beginning of the experiment. Set 1 was exposed to microwave irradiation for 8 weeks (38 exposure days) totaling 138 h of exposure. Set 2 was exposed for 10 weeks (50 exposure days) for a total exposure time of 210 h. The microwave irradiation system employed in these experiments was fashioned after one designed by Basset and reported by McRee and Walsh [3]. The microwaves were generated by a QK-390 magnetron in a Raytheon PGM100 power generator. Microwaves were guided from the magnetron to a dualdirection coupler by RG-225/u coaxial cable with an attenuation of 0.008 decibels/100 feet. A Narda model 3022 directional coupler allowed continuous monitoring of forward line power using a 20 db attenuated thermistor mount and a Hewlett-Packard power meter. The other sampling port of the coupler was used to obtain periodic frequency readings using a Hewlett-Packard 536A frequency meter. The frequency was always within the magnetron specification of 2,450 f 25 MHz. Coaxial cable was used to connect the directional coupler to a standard gain pyramidal horn with a minimal cut off frequency of 2.3 GHz. An anechoic irradiation chamber was constructed which provided a 76 cm2 area with a uniform power density. Since the target area (the floor of the chamber) was more than 5 feet away from the horn, the target area was in the far field or Fraunhofer region. The chamber shell was made of 2”plywood and was lined with 4” hairflex. The target base was made of Emerson--Gumming
24” Eccosorb foam block microwave absorber. A blower was used to ventilate the chamber with its exhaust directed into the control chamber. The rabbits were restrained in an acrylic cage which kept each of 4 rabbits in separate quadrants of the target area. Acrylic was chosen for its resistance to destruction by the rabbits and low electromagnetic reflection properties. The acrylic polymer transmits all but 3-4s of the incident microwaves. Power density was checked daily using a Narda electromagnetic leakage monitor and probe. Irradiating power density was kept between 20-30 mw/cm*. Rectal temperatures of all rabbits were recorded before and after each irradiation period using a Yellow Springs Instrument Tele-Thermometer and probe. Radio frequency irradiation Two sets of New Zealand albino male rabbits were studied in this part of the experiment. All animals were fed the 1% cholesterol diet described previously. Each set of 18 rabbits was divided into 2 groups, the irradiated experimental group (E) and the control group (C). The experimental groups were exposed to a radio frequency electric field by placing them in stainless steel cages with dimensions of 60 X 30 X 46 cm. A 50 X 25 cm steel sheet covered with a polyurethane resin was placed in the top of the cage and the steel grid upon which the rabbits rested served as the opposite plate allowing development of an electric field of 30 V/m oscillating at a frequency of 1 MHz. A small dipole probe was used to measure the near electric field strength in the cage which was uniform to within 2 cm of the cage walls where it dropped off markedly. The top plate and floor grid were attached to a solid state oscillator designed to drive up to 10 irridation chambers in parallel. The voltage across the cages as well as the field strength within the cages was checked on approximately a weekly basis. Three rabbits from Set 1 and 2 from Set 2 died before sacrificing due to causes unrelated to the experiment (ear mite infestation and pneumonia). As a result group 1 had 15 rabbits (7 experiment&) and group 2 had 16 rabbits at the time of sacrifice. The experimental group of Set 1 was continuously exposed to radio frequency irradiation for 55 days and the experimental group of Set 2 was continuously exposed for 81 days. Control animals were kept for the same length of time in the same type of stainless steel cages except for the irradiation equipment. They ate the same diet. Experimental and control groups were paired by age and weight at the beginning of the experiment. Measurements A 3 ml blood sample was drawn from an ear vein of each rabbit before initiating the high cholesterol diet and electromagnetic field irradiation. Serum cholesterol was determined using the method of Abel1 et al. [4] . At the end of the experimental period, animals were sacrificed by cervical dislocation and blood was drawn from the heart for determining serum cholesterol. The aorta from the aortic valve to the celiac artery was removed and placed in a 0.9% saline solution. Fat was then removed from adventitia and the vessel was sliced lengthwise to expose the intima. The fraction of the intima containing macroscopic atherosclerotic lesions was determined by placing a transparent grid (2.5 mm2 squares) over the intimal surface of the aorta and counting (by 3 individuals) the number of squares containing any evidence of a lesion. Although
58
histologic preparations of aortas were made and inspected, the well known variability due to sampling problems precluded use of this type of information in assessing the extent of atherosclerosis. After obtaining the wet weight of the aortas they were lyophilized and reweighed. The dried aortas were weighed and then cut into small pieces with scissors and placed in glass homogenizing tubes. The aortas were homogenized (approximately 5 min) in a 2 : 2 : 1.8 mixture of chloroform, methanol and water until no tissue pieces could be observed. The homogenized solution was then centrifuged at lO,OOO-15,000 rpm for 20 min. The chloroform layer was recovered and evaporated to dryness. The residue was reconstituted in l-2 ml of chloroform depending on the expected cholesterol concentration and a small aliquot of this analyzed for cholesterol concentration [4]. Using this method the recovery of cholesterol added to fresh aortas was 97.8 f 4.4%. Means plus or minus one standard error of the mean are reported. Student’s t-test was performed to evaluate the statistical significance of difference. The Michigan Interactive Data Analysis System was used for all statistical computations. Results Microwave experiments
Table 1 summarizes the results of exposure of each of the sets of animals to microwave irradiation. In both experimental and control groups the 1% cholesterol diet caused a marked elevation in serum cholesterol as would be expected from many previous studies [ 21. The younger animals (2 months old at onset of experiment) exposed for a longer period of time (Set 2) had higher serum cholesterols at the termination of the experiment than those in Set 1 (3 months old at onset). All groups developed atherosclerotic lesions. Aortas of rabbits not fed the cholesterol containing diet had no visible lesions. Wall cholesterol content was also markedly elevated in all groups as compared to animals which were not exposed to a high cholesterol diet. Wall cholesterol content of control animals with no added cholesterol in diet averaged 5 t 0.4 mg/g. When experimental groups are compared to control groups few significant differences emerge. The prediet serum cholesterol concentration of control animals in Set 2 (C,) was significantly higher than the prediet serum cholesterol of the experimental animals of Set 2 (E2). A relatively small fraction of the total intimal area was covered with lesions in the first set (C, and E,) and a larger fraction of the area was covered in Set 2 (C, and E,). In both cases there was not a statistically significant difference between non-irradiated control and irradiated experimental animals. This was also true of the cholesterol content of the aortas. Another index of injury of the aorta might be the accumulation mucopolysaccharide and thus a change in the ratio of wet to dry weight of the aortas. Neither set showed a significant difference in this ratio when irradiated and non-irradiated aortas are compared. The irradiated animals had a significant increase in body temperature immediately following the irradiation period. Radiowave experiments
Table 2 summarizes similar data for the animals exposed to radio frequency irradiation and the unexposed controls. Although the average serum cholesterol
1
18.4
-5 138
Each v&e
+0.60 f
expos. (OC) Change in weight (g) Exposure time (h)
(8)
(8)
(8)
(7)
(El
f
0
44*
-0.07
f
39.17 f
4.48 +
23.6
6.5
(8)
(8)
(8)
(7)
86
(7)
0.04 (8)
0.03 (8)
0.82
0.001
0.10
0.73
0.58
0.94
0.49
0.41
P
NON-IRRADIATED
0.20 (8)
7.6
9.1
+ 215
f
E2) AND
23.03 +
1905
85.4
Cl
AND
is mean f standard error. P = P value for Student’s t-test, ( ) = number of aninxik.
(8)
0.10 (8)
0.04 (8)
0.21 (8)
5.4
+ 137
39.08 f
before expos. (“C) Temperature during
4.37 f
f
aorta (6) Mean temperature
(mrk) Wet/dry weight
10.1
22.0
lesions WaII cholesterol
f
1702 + 185
(mUlO ml), sacrifice % of intimal area with
18.0
68.9
f
IRRADIATED
(mg/lOO mu. pre-diet Serum cholesterol
RI
Set 1
OF MICROWAVE
Serum cholesterol
Variable
COMPARISON
TABLE (CI
(8)
(8)
(8)
(8)
0.04 (8)
0.05 (8)
0.14 (8)
t 114
+0.61 f
39.21 f
1252 210
1.3
87
5.48 (8)
?: 13.8
f
f
3.88 f
84.0
94.7
2332
54.4
E2 f
C2) RABBITS
Set 2
AND
2
f
+
f
f
1239 0
-0.14
(8)
(8)
(8)
(8)
(8)
0.06 (8)
0.07 (8)
0.15 (8)
14.0
1.6
98
8.3
f 198
f
39.35 *
4.0
93.8
92.2
2534
78.2
C2
0.96
0.001
0.15
0.57
0.63
0.24
0.15
0.03
P
2
657 55
(7)
(7)
(7)
f 111
(7)
0.12 (7)
+_ 12.6
3.391’
67.1
14.9
k239.0
f
IRRADIATED
(El
48.2
0
k
338
(8)
(8)
(8)
(8)
0.23 (8)
10.6
* 144
3.81 +_
43.0
8.5
0.11 -
0.14
0.17
-
0.62
0.26
P
NON-IRRADIATED
? 215.4
+_
E2) AND
1332.3
Cl
AND
(C,
Each value is mean ?:standard error, P = P value for Student’s t-test. ( ) = number of animals.
aorta (g) Change in weight (g) Temperature (OC!) Exposure time (days)
(mglg) Wet/dry weight
-
1170.0
(mg/lOO ml). sacrifice % of intimal area with lesions Wall cholesterol
67.7
(mg/lOO ml). pre-diet Serum cholesterol
Rl
set 1
OF RADIOWAVE
Serum cholestexol
Variable
COMPARISON
TABLE
7.1
f
0.11 (8)
(8)
(8)
(7)
(8)
488 f 242 (8) 38.16 f 0.33 (7) 81
4.57 f
19.5
12.3
?r144.0
113.97 +
64.1
697.4
44.4
R2 f
C2) RABBITS
set 2
AND
0
7.6
f
0.08 (8)
(8)
(8)
(8)
(8)
8.8 f 103 (8) 39.13 + 0.19 (8)
4.67 f
19.5
6.0
f 120.2
f
112.73 f
88.6
866.2
39.9
C2
0.08 0.02
0.46
0.46
0.10
0.38
0.67
P
O-2 0
61
of animals in Set 2 was lower at sacrifice than any other set, the average wall cholesterol concentration was highest. Perhaps the relatively high wall cholesterol is due to the longer duration of this experiment (81 days). There were no significant differences in any of the variables measured at the end of the experiment including serum cholesterol, area of the intima involved in atherosclerotic lesions, wall cholesterol content of the aortas or the ratio of wet to dry weights. The temperature of the animals in Set 2 exposed to radio frequency irradiation was not higher than that of the unexposed animals and in fact was significantly lower. We do not have an explanation for this and can only say that the animals were in the same room and were exposed to the same ambient temperature. The temperature of the animals in Set 1 was not recorded. Discussion We wished to test the possibility that exposure to electromagnetic irradiation could influence the extent of diet-induced atherosclerosis of rabbits. Results reported above indicate that irradiation in radio frequency and microwave frequency ranges does not increase cholesterol content or intimal surface involvement in this species. We doubt that this negative result is due to inadequate exposure of the animals to electromagnetic energy. Both groups were exposed to microwave and radio frequency irradiation at far greater than defined allowable ambient exposure levels for humans [ 5,6]. Atherogenesis in rabbits is very susceptible to the effects of aortic wall injury. A wide variety of noxious agents including x-ray irradiation [ 71, cold [ 81, and various pharmacologic interventions [2] result in accelerated atherogenesis. For this reason we feel that if either type of electromagnetic irradiation had caused significant injury to the aortic wall by direct or indirect means, it would have resulted in enhanced atherogenesis. We have no way of knowing from these experiments whether longer term exposure to electromagnetic irradiation would yield a different result. The development of atherosclerosis in rabbits as induced by dietary cholesterol is extremely variable so that measurements of cholesterol content, serum cholesterol and intimal area involved in atherogenesis all exhibit large variation in both control and experimental groups. This raises the possibility that some rather subtle change might be hidden in the variability. We are somewhat reassured that this is not the case by the fact that trends of the means went in different directions in different experiments with the control sometimes having a higher wall cholesterol or intimal area involvement and other times having a lower one. The expected effect on atherogenesis should depend on the way in which the microwave or radiowave exerts its effect on the animal. Both the 1 MHz and the 2.45 GHz waves should penetrate the rabbit to some extent [5] so that the aortas are exposed directly to at least some energy dissipation. If this is so, either microwave or radiowave irradiation could have resulted in a direct effect on the aorta, for example by heating. The possibility of significant direct heating of the aorta seems remote since the aortic blood flow should dissipate any heat produced. Either frequency of irradiation could have a direct effect on some other organ (e.g. skin) leading to an indirect effect on the aorta. For
62
example Gordon has shown that exposure to microwave frequencies results in a transient increase in blood pressure followed after several weeks by a decrease [9]. At radio frequencies the decreased blood pressure predominates after a very short initial increase. Since elevated blood pressure is known to enhance atherogenesis [lo], increased atherosclerosis might be expected in our experiments. Apparently any effects on blood pressure in our experiments were not sufficient to result in a measurably different rate of atherogenesis in the animals we have studied. Perovsky et al. reports the microwave irradiation causes diminished atherogenesis in rabbits [ll] . They found a decrease in serum cholesterol and aortic cholesterol content. We cannot support the results of that study based on the current findings. In summary it appears that if electromagnetic irradiation of the frequencies investigated in this study has an effect on atherogenesis, it is too subtle to be uncovered using the number of rabbits and intensity of exposures used in this experiment. Acknowledgement We wish to thank Dr. John M. Schwartz providing many helpful ideas.
for suggesting
the study to us and
References Medvedyev, V.P. Diseases of the cardiovascular system in persons exposed in the past to the action of a super high frequency electromagnetic field, Gigiena Truda, 17 (1973) 6-9. Constantlnldes. P. Experimental Atherosclerosis, Elssvier. Amsterdam, 1965. McRee. D. and Walsh, P.. Microwave exposure system for biological specimens, Rev. Sci. Instr., 42 (1971) 1860-1864. AbelI, L.L., Levy. B.B., Brodie. B.B. and Kendal, F.E.. A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity, .I. Biol. Chem., 195 (1952) 357. Schwan, H.P. Microwave radiation - Biophysical considerations and standards criteria, IEEE Trans. Biomed. Engng.. BME-19 (1972) 304-312. Van de Grlek, A. and Britain. R., Amendments to the U.S. Department of Health, Education and Welfare Microwave Oven Performance Standard. J. Microwave Power, 9 (1974) 3-11. Gold, H.. Production of atherosclerosis in the rat -Effect of X-ray and high fat diet, Arch. Path., 71 (1961) 268-273. Kelly. F.B.. Taylor, C.B. and Hass. G.M., Experimental atheroarteriosclerosis -Localization of Lipids in experimental arterial lesions of rabbits with hypercholesterolemia, Arch. Path., 53 (1952) 419436. 9 Gordon, Z.V., Biological Effects of Microwave in Occupational Hygiene. Published for the Natl. Aero. and Space Admin. and Natl. Sci. Foundation, Washington, D.C.. by the Israel Program for Sci. Trsnslations, 1970, p. 40. 10 BronteSteward, B. and Hepinstall. R.H., The relationship between experimental hypertension and cholesterol-induced atheroma in rabbits, J. Path. Bact.. 68 (1954) 407-417. 11 Perovslty, A.I., Ya Trotsenko, S.. Ya Guz, S., Volkov. E.S., Barko. A.J. and Koshlyak, T.N.. The effect of superhigh frequency electromagnetic field on the course of experimental atherosclerosis, Pat. Fiziol. Eksp. Ter., 13 (1969) 64-66.
