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Low-frequency electromagnetic radiation enhances the induction of rat mammary tumors by nitrosomethyl urea
Cancer Let’s. 61 (1991) 75 - 79 Elsevier Scientific Publishers Ireland Ltd
75
Low-frequency electromagnetic radiation enhances the induction of rat mammary tumors by nitrosomethyl urea D.Sh.
Beniashvili.
V.G.
Bilanishvili
and
M.Z.
Menabde
Oncology Research Center, Ministry of Health and Social Security of the Republic of Georgia, Republic of Georgia (U.S.S.R.)
Lisi
Lake
380121,
Tbilisi,
(Received 3 July 1991) (Revision received 12 September 1991) (Accepted 12 September 1991)
Summary
Keywords: tumor; lowmammary; frequency electromagnetic field; nitrosomethyl urea
Low-frequency electromagnetic fields enhance the induction of mammary gland tumors in rats using nitrosomethyl urea. The incidence of tumors depended on the duration of exposure to static (dc) and oariable (ac) magnetic fields. Variable magnetic jields induced mammary gland cancer much more frequently than static ones. Apart from increasing the incidence of mammary gland tumors, household low-frequency
electromagnetic
fields
Introduction In the USSR, as well as in many developed countries, mammary gland cancer (MGC) is the predominant tumor in women by its incidence and mortality rate [l]. It is beiieved that various factors may be responsible, including genetic factors, hormones and dietary fats [2]. As epidemiological investigations show [3,4], long-term exposure to irradiation caused by the wiring in walls, household electric devices, lighting units, TV screens or computer displays may also be factors that increase the risk of oncological diseases. The development of certain tumors depends not only on the direct specific effect of carcinogenic agents but also on various modifying factors. Our knowledge about the effect of lowelectromagnetic fields on the frequency development of mammary gland tumors is very limited, and investigation of these effects may be important for prevention of MGC. In
reduced
the
mean latent period of tumor development and led to predominance of malignant tumors in the exposed animals as compared to controls. Mammary gland tumors deoeloped rarely under the effect of static or variable magnetic
fields per se, without preeliminary administration of a carcinogen. Household lowfrequency electromagnetic fields may potentially present an oncogenic hazard for animals and humans.
Correspondence
to:
Dzhemal
Sh. Beniashvili, Gamsakhurdia Republic of Georgia,
str., korp.17, kv. 11 Tbilisi 380094. U.S.S.R.
0304-3835/91/$03,50 Published and Printed
0 1991 Elsevier Scientific Publishers in Ireland
Ireland
Ltd
76
this paper we attempt to establish the significance of low-frequency static and variable magnetic fields at different exposures in the genesis of mammary gland tumors induced in rats using nitrosomethyl urea. Material
and methods
Experiments were performed on 400 virgin female rats aged from 55 to 60 days, bred at the Oncology Research Center of the Ministry of Health and Social Security of the Republic of Georgia. Animals were kept under usual light conditions and received standard laboratory diet. Nitrosomethyl urea (NMU), synthesized at the Organic Synthesis Laboratory of the N.N. Petrov Research Institute of Oncology, was diluted in 3 mM citrate buffer containing 0.8% NaCl (pH 6.0). NMU was administered at a dose of 50 mg/kg into the caudal vein of five groups of rats (groups 1 - 5) under mild ether anesthesia within 1 h after preparation of the solution. Solenoid boxes (dimensions 75 x 60 x 45 cm) manufactured at the Institute of Geophysics of the Georgian Academy of Sciences were used for our experiments and plastic cages with animals were placed inside them. Both a variable (ac) magnetic field (VMF) , at a frequency of 50 Hz, and a static (dc) magnetic field (SMF), with an intensity of 0.2 Oe, within the solenoid, were generated. Apart from carcinogen administration, rats were exposed to VMF daily for 0.5 h in group 1, and for 3 h in group 2. In group 3, rats were exposed to SMF for 0.5 h and in group 4 for 3 h daily. In groups 1 - 4 exposure to electromagnetic fields started 2 days after NMU administration. Rats in group 5 received only NMU intravenously. Animals in groups 6 - 9 were subjected only to the effect of electromagnetic fields without preliminary NMU administration. Rats in group 6 were exposed to VMF for 0.5 h and those in group 7 for 3 h daily. Rats in group 8 were kept in SMF solenoid cells for 0.5 h, and those in group 9 for 3 h daily. Rats in group 10 were not subjected to any special interventions and served as controls. Animals were under observation over a
period of 2 years after the carcinogen injection. All animals that survived until that time were sacrificed using ether vapor. Autopsy examinations were performed on these animals as well as in animals that had died earlier. Tumor pieces were fixed in neutral formalin. Celloidin sections were stained with hematoxylin-eosin and picrofuchsin. To investigate a possible effect of electromagnetic fields on the incidence of mammary gland tumors, statistical analysis was performed using empirical distribution analysis (x2 criterion) and the correlation ratio with evaluation of its significance (P value). Results As Table I shows, the incidence of mammary gland cancer in rats depended on the duration of exposure of these animals to VMF and SMF. Rats in groups 2 and 4 (daily exposure to VMF and SMF for 3 h ) had a statistically significant increase in the incidence of mammary gland tumors (x2 = 13.5 for group 2 compared to group 5, P < 0.05, and x2 = 6.5 for group 4 compared to group 5, P < 0.05). By contrast, no such dependence was observed for rats in groups 1 and 3 (daily exposure to VMF and SMF during 0.5 h) (x2 = 1.1 for group 1 compared to group 5, P > 0.05, and x 2 = 0.9 for group 3 compared to group 5, P > 0.05). Some mammary gland tumors also appeared in rats from groups 6, 7 and 9 which were exposed only to VMF or SMF without preliminary carcinogen administration, and VMF produced mammary gland tumors much more frequently than SMF (P < 0.05). Low-frequency electromagnetic fields also decreased the latent period of development of mammary gland neoplasms: the tumors emerged much earlier in rats from groups 2 and 4 (3-h exposure to VMF or SMF) than in the other groups of animals. The electromagnetic fields also affected the morphological spectrum of NMU-induced mammary gland tumors. In animals exposed daily to VMF or SMF during 3 h , malignant
77
Table 1. Group No.
1 2 3 4 5 6 7 8 9 10
Effect of low-frequency Modifying factors
NMU + VMF (0.5 h) NMU + VMF (3 h) NMU + SMF (0.5 h) NMU + SMF (3 h) NMU VMF (0.5 h) VMF (3 h) SMF (0.5 h) SMF (3 h) Control
electromagnetic
Initial number of rats
fields on the development No. of rats with mamma-
50
49
33
40
32.4
64.8
+ 10.5
50
46
43
75
136.5
45.5
f
50
48
32
36
32.7
65.4
+ 18.2
50
45
39
43
158.4
52.8
ztz 17.11
50 25 25 25
46 24 23 24
27 1 7 _
31 1 8 -
-
74.4 158 141.7 _
It 14.9
79 425.1 -
25 50
23 48
-
561 -
187 _
ry tumors
ry tumors
Time of exposure to electromagnetic fields
tumors
No. of rats survived till first tumor
giand tumors mammary prevailed (57 adenocarcinomas and 18 fibroadenomas in group 2 and 28 adenocarcinomas and 15 fibroadenomas in group 4) as compared to other groups (13 adenocarcinomas and 27 fibroadenomas in group 1; 15 adenocarcinemas and 21 fibroadenomas in group 3; 16 adenocarcinomas and 15 fibroadenomas in group 5; one fibroadenoma in group 6; 2 adenocarcinomas and 6 fibroadenomas in group 7 and one fibroadenoma in group 9). The main location of tumors in animals given NMU and exposed to VMF or SMF was the mammary gland (Fig. 1) but neoplasms were also found occasionally in the kidneys, large intestine, uterus and thyroid ovary, gland. Mammary gland tumors emerged as small nodules which gradually increased in size, the skin over them got thinner and later ulcerated. Sometimes tumors grew to a large size (up to 9 x 7 x 5 cm), being light-grey at
No. of mamma-
of rat mammary
(h)
1
1 -
Mean latent period of mammary tumor development (days)
11.7
A 27.5
section and with necrotic sites in the center. Small tumors were predominantly benign (adenomas and fibroadenomas), large tumors in most cases were malignant (high- and lowdifferentiated adenocarcinomas) . Discussion
Our findings suggest that the use of lowfrequency variable and static magnetic fields contribute to the development of rat mammary tumors induced by NMU. The duration of exposure to low-frequency VMF and SMF had a large effect on the induction of mammary gland tumors; the tumors developed under the influence of both VMF and SMF but earlier and more frequently in animals exposed to VMF daily for 3 h. There have been various reports in the literature recently suggesting that lowfrequency electromagnetic fields may play a
Fig. 1. Left mammary gland tumor of rat (a) after exposure to VMF (group 7). Right mammary gland tumor (b) of rat after NMU administration and exposure to VMF (group 2) (c) Fibroadenoma of the mammary gland of rat after NMU administration and exposure to SMF (group 4). stained with hematoxylin-eosin, magnification x 100. (d) Cystoadenocarcinoma of the mammary gland of rat after NMU administration and exposure to VMF (group 2). stained x 100. with hematoxylin-eosin, magnification
79
major role in the etiology of various tumors. Some authors [5,6] attach importance in the development of mammary gland tumors to the effect of low-frequency electromagnetic fields They propose that the on the epiphysis. observed decrease in melatonin stimulates mammary cancer formation induced with DMBA or NMU [7 - 91. That the ability of lowfrequency VMF and SMF to decrease melatonin level may be the first step for the subsequent development of mammary gland cancer is suggested by data of Wilson et al. [lo]; these authors subjected 55-day old rats to constant electric fields with 60 Hz frequency for 20 h daily for 1 month and noted a decrease in melatonin level. Tamarkin [l l] and Shah [12] also observed a decrease in melatonin concentration and a rise in the incidence of mammary gland cancer induced by DMBA in pinealectomy. Stevens [5] suggested that the decrease in melatonin level as a result of the effect of low-frequency magnetic fields in rats affected the ovary function, causing a rise in estrogen content and increasing the risk of development of mammary gland tumors. Our investigations point to the potential oncogenic hazard of using household lowfrequency electromagnetic radiation. References 1
Napalkov, N P. and Merabishvili, V.M. (1989) Main characteristics of cancer mortality in the USSR. Vopr. Onkol
6. 649-657.
Gregorio. D., Emrich. L. and Graham, S (1985) Dietary fat consumption and survival among women with breast cancer. J. Natl. Cancer Inst., 75, 37 - 41, Cartwright. R.A. (1989) Low frequency alternating electromagnetic fields and leukaemia. Cancer, 60. 649 - 651. Wertheimer. N. and Leeper, E (1979) Electrical wiring configuration and childhood cancer. Am. J Epidemiol.. 109. 273 - 284 Stevens, R. (1988) Electric power, melatomn and breast cancer In: The Pineal Gland and Cancer, pp. 233 - 244 Editors: D. Gupta. A. Attanasio and R.J Reiter Bram 6
7 8
9 10
11
12
Research Promotion, London, Tubingen Wilson, B.. Anderson, L.. Hi1ton.D and Phillips, R. (1981) Chronic exposure to 60 Hz electric fields: effects on pineal function in the rat. Bioelectromagnetics. 2, 371-380. Anisimov. V.N and Reiter. R. (1990) Pineal gland function in cancer and aging. Vopr. Onkol.. 3. 259 - 268 Blask. D. and Hill. S.M (1988) Melatonin and cancer: basic and clinical aspects. In: Melatonin. Clinical Perspectives, pp. 128- 173. Editors: A. Miles and D R S. Philbrick. Oxford University Press, Oxford Stevens. R. (1987) Electric power use and breast cancer Am. J. Epidemiol 125. 556-561 Wilson. R.. Leung. F., Buschbom. R.. Stevens, R G.. Anderson. L E. and Reiter. R.J. (1988) Electric fields, the pineal gland and cancer In: The Pineal Gland and Cancer, pp. 245-259. Editors: D. Gupta. A Attanasio and R. Reiter. Brain Research Promotton. London, Tubingen Tamarkin. L Cohen, M and Roselle. D (1981) Melatomn inhibition and pinealectomy enhancement of 7.12.dimethylbenz(a)anthracene-Induced mammary tumors in the rat. Cancer Res.. 41. 4432 -4436 Shah. P Mhatre. M. and Kotheari. L. (1984) Effect of melatonin on mammary carcmogenesis in intact and pinealectomized rats in varying Res., 44. 3403 - 3407
photopenods.
Cancer
75
Low-frequency electromagnetic radiation enhances the induction of rat mammary tumors by nitrosomethyl urea D.Sh.
Beniashvili.
V.G.
Bilanishvili
and
M.Z.
Menabde
Oncology Research Center, Ministry of Health and Social Security of the Republic of Georgia, Republic of Georgia (U.S.S.R.)
Lisi
Lake
380121,
Tbilisi,
(Received 3 July 1991) (Revision received 12 September 1991) (Accepted 12 September 1991)
Summary
Keywords: tumor; lowmammary; frequency electromagnetic field; nitrosomethyl urea
Low-frequency electromagnetic fields enhance the induction of mammary gland tumors in rats using nitrosomethyl urea. The incidence of tumors depended on the duration of exposure to static (dc) and oariable (ac) magnetic fields. Variable magnetic jields induced mammary gland cancer much more frequently than static ones. Apart from increasing the incidence of mammary gland tumors, household low-frequency
electromagnetic
fields
Introduction In the USSR, as well as in many developed countries, mammary gland cancer (MGC) is the predominant tumor in women by its incidence and mortality rate [l]. It is beiieved that various factors may be responsible, including genetic factors, hormones and dietary fats [2]. As epidemiological investigations show [3,4], long-term exposure to irradiation caused by the wiring in walls, household electric devices, lighting units, TV screens or computer displays may also be factors that increase the risk of oncological diseases. The development of certain tumors depends not only on the direct specific effect of carcinogenic agents but also on various modifying factors. Our knowledge about the effect of lowelectromagnetic fields on the frequency development of mammary gland tumors is very limited, and investigation of these effects may be important for prevention of MGC. In
reduced
the
mean latent period of tumor development and led to predominance of malignant tumors in the exposed animals as compared to controls. Mammary gland tumors deoeloped rarely under the effect of static or variable magnetic
fields per se, without preeliminary administration of a carcinogen. Household lowfrequency electromagnetic fields may potentially present an oncogenic hazard for animals and humans.
Correspondence
to:
Dzhemal
Sh. Beniashvili, Gamsakhurdia Republic of Georgia,
str., korp.17, kv. 11 Tbilisi 380094. U.S.S.R.
0304-3835/91/$03,50 Published and Printed
0 1991 Elsevier Scientific Publishers in Ireland
Ireland
Ltd
76
this paper we attempt to establish the significance of low-frequency static and variable magnetic fields at different exposures in the genesis of mammary gland tumors induced in rats using nitrosomethyl urea. Material
and methods
Experiments were performed on 400 virgin female rats aged from 55 to 60 days, bred at the Oncology Research Center of the Ministry of Health and Social Security of the Republic of Georgia. Animals were kept under usual light conditions and received standard laboratory diet. Nitrosomethyl urea (NMU), synthesized at the Organic Synthesis Laboratory of the N.N. Petrov Research Institute of Oncology, was diluted in 3 mM citrate buffer containing 0.8% NaCl (pH 6.0). NMU was administered at a dose of 50 mg/kg into the caudal vein of five groups of rats (groups 1 - 5) under mild ether anesthesia within 1 h after preparation of the solution. Solenoid boxes (dimensions 75 x 60 x 45 cm) manufactured at the Institute of Geophysics of the Georgian Academy of Sciences were used for our experiments and plastic cages with animals were placed inside them. Both a variable (ac) magnetic field (VMF) , at a frequency of 50 Hz, and a static (dc) magnetic field (SMF), with an intensity of 0.2 Oe, within the solenoid, were generated. Apart from carcinogen administration, rats were exposed to VMF daily for 0.5 h in group 1, and for 3 h in group 2. In group 3, rats were exposed to SMF for 0.5 h and in group 4 for 3 h daily. In groups 1 - 4 exposure to electromagnetic fields started 2 days after NMU administration. Rats in group 5 received only NMU intravenously. Animals in groups 6 - 9 were subjected only to the effect of electromagnetic fields without preliminary NMU administration. Rats in group 6 were exposed to VMF for 0.5 h and those in group 7 for 3 h daily. Rats in group 8 were kept in SMF solenoid cells for 0.5 h, and those in group 9 for 3 h daily. Rats in group 10 were not subjected to any special interventions and served as controls. Animals were under observation over a
period of 2 years after the carcinogen injection. All animals that survived until that time were sacrificed using ether vapor. Autopsy examinations were performed on these animals as well as in animals that had died earlier. Tumor pieces were fixed in neutral formalin. Celloidin sections were stained with hematoxylin-eosin and picrofuchsin. To investigate a possible effect of electromagnetic fields on the incidence of mammary gland tumors, statistical analysis was performed using empirical distribution analysis (x2 criterion) and the correlation ratio with evaluation of its significance (P value). Results As Table I shows, the incidence of mammary gland cancer in rats depended on the duration of exposure of these animals to VMF and SMF. Rats in groups 2 and 4 (daily exposure to VMF and SMF for 3 h ) had a statistically significant increase in the incidence of mammary gland tumors (x2 = 13.5 for group 2 compared to group 5, P < 0.05, and x2 = 6.5 for group 4 compared to group 5, P < 0.05). By contrast, no such dependence was observed for rats in groups 1 and 3 (daily exposure to VMF and SMF during 0.5 h) (x2 = 1.1 for group 1 compared to group 5, P > 0.05, and x 2 = 0.9 for group 3 compared to group 5, P > 0.05). Some mammary gland tumors also appeared in rats from groups 6, 7 and 9 which were exposed only to VMF or SMF without preliminary carcinogen administration, and VMF produced mammary gland tumors much more frequently than SMF (P < 0.05). Low-frequency electromagnetic fields also decreased the latent period of development of mammary gland neoplasms: the tumors emerged much earlier in rats from groups 2 and 4 (3-h exposure to VMF or SMF) than in the other groups of animals. The electromagnetic fields also affected the morphological spectrum of NMU-induced mammary gland tumors. In animals exposed daily to VMF or SMF during 3 h , malignant
77
Table 1. Group No.
1 2 3 4 5 6 7 8 9 10
Effect of low-frequency Modifying factors
NMU + VMF (0.5 h) NMU + VMF (3 h) NMU + SMF (0.5 h) NMU + SMF (3 h) NMU VMF (0.5 h) VMF (3 h) SMF (0.5 h) SMF (3 h) Control
electromagnetic
Initial number of rats
fields on the development No. of rats with mamma-
50
49
33
40
32.4
64.8
+ 10.5
50
46
43
75
136.5
45.5
f
50
48
32
36
32.7
65.4
+ 18.2
50
45
39
43
158.4
52.8
ztz 17.11
50 25 25 25
46 24 23 24
27 1 7 _
31 1 8 -
-
74.4 158 141.7 _
It 14.9
79 425.1 -
25 50
23 48
-
561 -
187 _
ry tumors
ry tumors
Time of exposure to electromagnetic fields
tumors
No. of rats survived till first tumor
giand tumors mammary prevailed (57 adenocarcinomas and 18 fibroadenomas in group 2 and 28 adenocarcinomas and 15 fibroadenomas in group 4) as compared to other groups (13 adenocarcinomas and 27 fibroadenomas in group 1; 15 adenocarcinemas and 21 fibroadenomas in group 3; 16 adenocarcinomas and 15 fibroadenomas in group 5; one fibroadenoma in group 6; 2 adenocarcinomas and 6 fibroadenomas in group 7 and one fibroadenoma in group 9). The main location of tumors in animals given NMU and exposed to VMF or SMF was the mammary gland (Fig. 1) but neoplasms were also found occasionally in the kidneys, large intestine, uterus and thyroid ovary, gland. Mammary gland tumors emerged as small nodules which gradually increased in size, the skin over them got thinner and later ulcerated. Sometimes tumors grew to a large size (up to 9 x 7 x 5 cm), being light-grey at
No. of mamma-
of rat mammary
(h)
1
1 -
Mean latent period of mammary tumor development (days)
11.7
A 27.5
section and with necrotic sites in the center. Small tumors were predominantly benign (adenomas and fibroadenomas), large tumors in most cases were malignant (high- and lowdifferentiated adenocarcinomas) . Discussion
Our findings suggest that the use of lowfrequency variable and static magnetic fields contribute to the development of rat mammary tumors induced by NMU. The duration of exposure to low-frequency VMF and SMF had a large effect on the induction of mammary gland tumors; the tumors developed under the influence of both VMF and SMF but earlier and more frequently in animals exposed to VMF daily for 3 h. There have been various reports in the literature recently suggesting that lowfrequency electromagnetic fields may play a
Fig. 1. Left mammary gland tumor of rat (a) after exposure to VMF (group 7). Right mammary gland tumor (b) of rat after NMU administration and exposure to VMF (group 2) (c) Fibroadenoma of the mammary gland of rat after NMU administration and exposure to SMF (group 4). stained with hematoxylin-eosin, magnification x 100. (d) Cystoadenocarcinoma of the mammary gland of rat after NMU administration and exposure to VMF (group 2). stained x 100. with hematoxylin-eosin, magnification
79
major role in the etiology of various tumors. Some authors [5,6] attach importance in the development of mammary gland tumors to the effect of low-frequency electromagnetic fields They propose that the on the epiphysis. observed decrease in melatonin stimulates mammary cancer formation induced with DMBA or NMU [7 - 91. That the ability of lowfrequency VMF and SMF to decrease melatonin level may be the first step for the subsequent development of mammary gland cancer is suggested by data of Wilson et al. [lo]; these authors subjected 55-day old rats to constant electric fields with 60 Hz frequency for 20 h daily for 1 month and noted a decrease in melatonin level. Tamarkin [l l] and Shah [12] also observed a decrease in melatonin concentration and a rise in the incidence of mammary gland cancer induced by DMBA in pinealectomy. Stevens [5] suggested that the decrease in melatonin level as a result of the effect of low-frequency magnetic fields in rats affected the ovary function, causing a rise in estrogen content and increasing the risk of development of mammary gland tumors. Our investigations point to the potential oncogenic hazard of using household lowfrequency electromagnetic radiation. References 1
Napalkov, N P. and Merabishvili, V.M. (1989) Main characteristics of cancer mortality in the USSR. Vopr. Onkol
6. 649-657.
Gregorio. D., Emrich. L. and Graham, S (1985) Dietary fat consumption and survival among women with breast cancer. J. Natl. Cancer Inst., 75, 37 - 41, Cartwright. R.A. (1989) Low frequency alternating electromagnetic fields and leukaemia. Cancer, 60. 649 - 651. Wertheimer. N. and Leeper, E (1979) Electrical wiring configuration and childhood cancer. Am. J Epidemiol.. 109. 273 - 284 Stevens, R. (1988) Electric power, melatomn and breast cancer In: The Pineal Gland and Cancer, pp. 233 - 244 Editors: D. Gupta. A. Attanasio and R.J Reiter Bram 6
7 8
9 10
11
12
Research Promotion, London, Tubingen Wilson, B.. Anderson, L.. Hi1ton.D and Phillips, R. (1981) Chronic exposure to 60 Hz electric fields: effects on pineal function in the rat. Bioelectromagnetics. 2, 371-380. Anisimov. V.N and Reiter. R. (1990) Pineal gland function in cancer and aging. Vopr. Onkol.. 3. 259 - 268 Blask. D. and Hill. S.M (1988) Melatonin and cancer: basic and clinical aspects. In: Melatonin. Clinical Perspectives, pp. 128- 173. Editors: A. Miles and D R S. Philbrick. Oxford University Press, Oxford Stevens. R. (1987) Electric power use and breast cancer Am. J. Epidemiol 125. 556-561 Wilson. R.. Leung. F., Buschbom. R.. Stevens, R G.. Anderson. L E. and Reiter. R.J. (1988) Electric fields, the pineal gland and cancer In: The Pineal Gland and Cancer, pp. 245-259. Editors: D. Gupta. A Attanasio and R. Reiter. Brain Research Promotton. London, Tubingen Tamarkin. L Cohen, M and Roselle. D (1981) Melatomn inhibition and pinealectomy enhancement of 7.12.dimethylbenz(a)anthracene-Induced mammary tumors in the rat. Cancer Res.. 41. 4432 -4436 Shah. P Mhatre. M. and Kotheari. L. (1984) Effect of melatonin on mammary carcmogenesis in intact and pinealectomized rats in varying Res., 44. 3403 - 3407
photopenods.
Cancer