Radiat Environ Biophys (2005) 44: 51–59 DOI 10.1007/s00411-005-0274-y
O R I GI N A L P A P E R
Patrizia Tarantino Æ Remigio Lanubile Giovanni Lacalandra Æ Luigi Abbro Æ Luciana Dini
Post-continuous whole body exposure of rabbits to 650 MHz electromagnetic fields: effects on liver, spleen, and brain
Received: 11 June 2004 / Accepted: 31 January 2005 / Published online: 6 April 2005 Ó Springer-Verlag 2005
Abstract This study deals with the effects of post (12 or 18 months) whole body continuous (2 years) exposure of rabbits to 650 MHz electromagnetic fields (EMF) that are characteristic of those produced by broadcasting stations, on body weight and body mass, on the morphology of liver, spleen and brain, and on apoptosis rates and glycogen distribution in the liver. Two groups of rabbits were continuously exposed for 2 years to EMF of 650 MHz followed by 12 months (group 1) or 18 months (group 2) of post-exposure; a third group (group 3) was sham exposed. It was shown that the changes in apoptosis rates were conditional during the time of exposure, but not on a specific organ and that the whole body continuous exposure gave rise to modifications whose types and intensities were related to the time of post-exposure (12 or 18 months, respectively), the type of organ, and the individual animal. A number of effects were observed only in group 1, and not in group 2, which suggests some kind of adaptive response or of long-term recovery in the rabbits following continuous exposure to 650 MHz EMF.
Introduction Since the late 1970s, there has been considerable interest in the question of whether there is a risk associated with P. Tarantino Æ R. Lanubile Æ L. Abbro Æ L. Dini (&) Department of Biological and Environmental Science and Technology, University of Lecce, Via per Monteroni, 73100 Lecce, Italy E-mail:
[email protected] Tel.: +39-0832-298614 Fax: +39-0832-298626 G. Lacalandra Department of Animal Production, University of Bari, Bari, Italy
exposure to nonionizing radiation or electric and magnetic fields (EMF) that are produced by power lines and telecommunication technologies [1– 5]. As a consequence of both the broad distribution of EMF in the environment and the fact that they are generated by natural as well as artificial sources, humans and animals are generally exposed to a complex mix of electromagnetic fields of different frequencies and intensities pertaining to the indoor and outdoor environment [6]. Epidemiological studies have made an approach to associating EMF exposure with increased incidences of cancer, but up to now there was no direct evidence for tumorigenicity or mutagenicity [7, 8]. Electromagnetic fields have also been considered as confounding factors i.e., in combination with known genotoxic and/or nongenotoxic carcinogens [9]. It has recently been hypothesized that exposure to EMF might increase the effects of known carcinogens only after exposure to both EMF and a carcinogen during an extended period of tumor development [10]. The EMF can, nonetheless, induce cellular and molecular modifications when interacting with biological materials [11]. The biological effects depend on the duration of exposure, the tissue penetration, and the heat generation of EMF that in turn are related to their intensities and frequencies. Cellular responses depend not only on the intensity and frequency of EMF, but also on their type (static or oscillatory), the form of the wave (sinusoidal, square, etc.), and the biological status of the exposed cells [12, 13], thus suggesting a very complicated interaction among the various factors. Cellular responses to EMF can be reversible or irreversible. A short classification of the biological effects due to EMF is based on the difference between acute and chronic effects of which the former are detectable immediately after and/or during the exposure. The latter are long-term effects involving subjective symptoms. They are difficult to detect, and a cause–effect relationship is difficult to determine. Some recent epidemiological studies suggest an association between lymphatic and hematopoietic cancers and residential exposure to high-frequency electro-
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magnetic fields (100 kHz–300 GHz) generated by radio and television transmitters [14]. It was the aim of this study to clarify the potential severe damage due to EMF generated by radio and television transmitters, by investigating, at the morphological level, the long-term post-exposure changes of liver, brain, and spleen in rabbits after continuous exposure to 650 MHz EMF.
Materials and methods Animals and magnetic field application Adult male (n=12) and female (n=12) rabbits, New Zealand and California strains, were used. Animals were housed in rooms maintained on a 12-h light-dark cycle (light on from 7:00 a.m. to 7:00 p.m.) and at an ambient temperature of 22°C and a relative humidity of 65%. Animals were fed laboratory diet specific for reproducing rabbits and had free access to water. In the present study, electromagnetic fields of 650 MHz, similar to those used for broadcasting, were generated by a linear polarization antenna with help of transmitting panels (frequency field 650 MHz, average intensity of EMF 10 V/m). Figure 1 presents a schematic illustration of the exposure room, which was shielded with aluminum to avoid external environmental disturbances. A double-tubed cell of aluminum (180 cm height, 300 cm length, 150 cm depth) has been built to maintain EMF confined to a defined space. The aluminum cage functions as a resonant multimode cavity. To maintain a homogenous average intensity of EMF in the cells, these were supplied with a mode mixer (MM-UV NIS-SMA).Taking into account that rabbits are very sensitive to movements, noises, and various other environmental disturbances, the mode has been scrambled by using a frequency modulation of about 10 MHz. In the middle of the aluminum cell, there were 24 cages for rabbits (38 cm height, 60 cm length, 50 cm depth), one cage for one rabbit, made of wood and plastic to avoid any perturbation to the EMF. The average absorption rate (SAR) of the whole body was 3.8 W/kg. In the exposure room the background flux density was less than 1 lT (50 Hz). The adult rabbits were continuously exposed for 2 years. They were divided into 3 groups of 8 animals
each. Each group was equilibrated in terms of male and female animals and of strains. Group 1 was continuously exposed for 2 years and sacrificed after 12 months after the end of treatment (i.e., total of 3 years). Group 2 was continuously exposed for 2 years and sacrificed 18 months after the end of treatment (i.e., total of 3.5 years). Group 3 included the controls, nontreated and maintained for comparison. The animals of the control group were housed in a room next to the exposure room, and they were treated in the same way as the study animals. During the post-exposure period, all animals, including the controls, were kept together in the same room. Animals were treated according to the national animal protection standards. Local geomagnetic flux was about 46 lT. Changes in the body weight and mass of the rabbits during these series were recorded to evaluate the degree of stress induced by the 650 MHz EMF. Light and electron microscopy After removal of the organs (liver, spleen, brain), small samples were immediately fixed in Bouin’s solution and embedded in paraffin; 3-lm-thick sections were stained with hematoxylin and eosin. Silver staining of sections by using a mixture of silver nitrate (3 mg/ml) and esamethylentetramine (75 mg/ml) was also done for evaluation of apoptotic nuclei. Liver slides were stained with the PAS reaction for the detection of glycogen. Toluidine bleu– stained slides were obtained by Spurr resin embedded samples. The liver ultrastructure was determined by transmission electron microscopy. Samples were fixed with 2.5% glutaraldehyde in cacodylate buffer, pH 7.4, for 1 hr at ice temperature, postfixed with 1% OsO4 in the same buffer, dehydrated, embedded in Epon 812 resin and examined under a Philips CM12 TEM. Quantification of apoptosis and mitosis Hematoxylin–eosin or silver stained liver sections were examined for the scoring of cells in mitosis or undergoing apoptosis. The number of mitotic and apoptotic cells was randomly counted in 50 to 100 high-power microscopic fields (x40) per organ per animal. The number of apoptotic cells was expressed as number per microscopic field (at x40). Two separate counts were done. Statistical analyses were performed using one-way analysis of variance (ANOVA) with 95% confidence limits. Data are presented in text and figures as mean ± ES. A value of p0.5; n=8 for each group
depleted of cytoplasm but filled with granules, also surrounding the nuclear membrane; and frequent binuclear hepatocytes with less condensed chromatin and enlarged space between the inner and outer nuclear membranes. A significant numerical drop in Kupffer cells was observed in group 1 and a drop in endothelial cells in both groups. The number of Kupffer cells in group 2 was similar to that of group 3 (Table 1 and Fig. 4a). Apoptotic cells in the hepatic parenchyma were quite scarce in all groups. Apoptotic cells inside the sinusoids, which are normally rare in group 3 animals, initially fell further (group 1) during the post-exposure period, and subsequently increased to four times above control values (group 2). Apoptotic sinusoidal cells were abundant only in group 2 (Table 2 and Fig. 4b). Glycogen, in the form of rosettes, was homogeneously distributed all over the lobule inside the hepatocytes of group 3 (Fig. 5). A significant drop in hepatic glycogen in group 1 was followed by a substantial increase in group 2. In the livers showing severe micromorphological alterations, glycogen, mainly concentrated in a few hepatocytes (Fig. 5, arrows), was recognizable by intense reaction staining. Individual modifications (i.e., type I or type II) were found only for animals of group 2.
Spleen The spleen has the consistency of a dense sponge formed by a red pulp, a connective tissue framework, and a very rich vascular supply that filters blood, surveys it for antigens or circulating pathogens, and removes unwanted platelets and erythrocytes with recovery of iron. 25% of the organ is formed by scattered lymphoid follicles, which form the white pulp. The spatial distribution of the white and red pulp changed progressively during the post-exposure period (Fig. 6): the network of the reticular fibers of the red pulp enlarged. Apoptotic cells, abundant in the red pulp of group 3, progressively decreased (from 15% in group 3 to 2% in group 2) (Fig. 6b–d) in groups 1 and 2. Macrophages, recognizable by iron storage in their cytoplasm in the form of brown yellowish granules, derived from the phagocytosis of old damaged red blood cells, were extremely scarce in group 1. The number of macrophages and the number and size of iron-filled vacuoles increased during the postexposure period, especially in group 2 (Fig. 6h, l).
54 Fig. 3a–k Light micrographs of livers from control (group 3) and exposed rabbits, 2 years plus 12 (group 1) or 18 months (group 2) of post-exposure. a Control normal liver lobule; b apoptotic cells in the parenchyma (arrow) and circulating in the sinusoids (arrowheads). Group 1: c slightly enlarged sinusoids; d Kupffer cell (arrow) morphologically unchanged; e apoptotic cell (arrow) inside the sinusoid. Group 2: individual responses to EMF exposure: f,i liver without or g,l liver with morphological modifications; l arrows indicate apoptotic hepatocytes; h,k liver with highly damaged hepatocytes, enlarged sinusoid with rare sinusoidal cells. Types I and II refer to individual modifications found only for animals of group 2
Brain The brain consists of gray and white matter. The gray matter is composed of neuron cell bodies, their dendrites and axons, neuroglial cells and blood vessels. Observations of the gray matter revealed for both groups 1 and 2 a general enlargement and vacuolization of the tissue, whose intensity was related to the individual animal. During the post-exposure period, the space between neuron cell bodies and neuroglial cells increased and vessels were progressively lost. Apoptosis was lower in
the gray matter of the brain tissue of control rabbits (group 1); its rate increased in group 1 and especially in group 2 (Fig. 7).
Discussion The wide spread of broadcasting stations in recent years has brought about research activities to determine the potential consequences of exposure to electromagnetic fields of high intensity. In particular, the direct biologi-
55 Table 2 One-way ANOVA of the number of apoptotic cells in sinusoids, of apoptotic hepatocytes and apoptotic sinusoidal cells per microscopic field (x40) in livers from control, group 1, and group 2 rabbits. SS Sum of squares, DF degrees of freedom, MS mean square, F variance ratio, P significance; 6.45E 17 = 0.0000000000000000645; 2.57E 08 = 0.00000000257 Variability source
SS
DF
Apoptotic cells in the sinusoids Among groups 36.4 2 Between groups 7.4 42 Total 43.8 44 Apoptotic hepatocytes Among groups 0.4 2 Between groups 1.7 42 Total 2.0 44 Apoptotic sinusoidal cells Among groups 7.9 2 Between groups 6.1 42 Total 14.1 44
Fig. 4 a Number of endothelial and Kupffer cells per microscopic field (x40) in rabbit livers from control (Ctrl, group 3), 2 years of exposure plus 12 (2 years + 12 months = group 1) or 18 (2 years + 18 months = group 2) of post-exposure. b Number of apoptotic cells per microscopic field (x40) circulating in the sinusoids (white columns) and number of apoptotic hepatocytes (brushed columns) or sinusoidal cells (dotted columns) per microscopic field (x40) in rabbit livers from control (Ctrl, group 3), 2 years of exposure plus 12 months (group 1) or 18 months (group 2) of post-exposure. The data are mean ± ES. Significances are shown in Tables 1 and 2
cal (thermal) effects of exposure to EMF in some organs, such as liver, spleen, and brain (gray matter), need to be conclusively validated. Although the biological effects of exposure to EMF have been extensively investigated, the data obtained are still contradictory due to a variety of experimental conditions [12, 13] (e.g., in vivo or in vitro, time of exposure, frequency, intensity, type of field and of waveform) and of applied parameters (cellular or histological, e.g., plasma membrane, enzymes, genes, Table 1 One-way ANOVA of the number of endothelial and Kupffer cells per microscopic field (x40) in livers from control, group 1, and group 2. SS Sum of squares, DF degrees of freedom, MS mean square, F variance ratio, P significance; 9.9E 11 = 0.000000000099; 1.07E 07 = 0.000000107 Variability source
SS
Endothelial liver cells Among groups 33.5 Between groups 16.8 Total 50.3 Kupffer cells Among groups 8.8 Between groups 7.7 Total 16.5
DF
MS
F
P
2 42 44
16.7 0.4
41.9
9.9E 11
2 42 44
4.4 0.2
24.1
1.07E 07
MS
F
P
18.2 0.2
102.9
6.45E 17
0.2 0.04
4.6
0.016
3.9 0.1
27.3
2.57E 08
etc.) [15–21]. The duration of exposure and the time of post-exposure are crucial factors: on the one hand, because the exact duration of exposure is different from individual to individual and, on the other hand, because setting the duration for experimental conditions is a complicated matter. The subject of a recent report [22] was the possible development of biological abnormalities due to exposure to moderate-intensity static MF (6 mT) in in vitro experiments (e.g., cell shape, cell surface, cytoskeleton, and apoptotic rate alteration) that may lead to serious diseases including cancer. The present paper deals with the investigation of the in vivo system. The data obtained derive from whole body exposure to electromagnetic fields of an intensity that is comparable to that of people who are living in the vicinity of broadcasting stations. Results are presented on the effects on the micromorphology of liver, spleen, and brain in rabbits that have, for up to 2 years, been continuously exposed to 650 Mhz EMF and have been analyzed 12 or 18 months later. There is evidence of biological effects due to continuous exposure of animals to EMF; however, without pathological manifestations or clear diseases, not even during the period of recovery. Indeed, up to now, experimental data have failed to demonstrate EMF to be tumorigenic or mutagenic per se [7, 9]; however, their possible role as cofactors for the development of cancer, in combination with carcinogenic stimuli, has been hypothesized [10]. The results indicate that the response to the long-term post-exposure after continuous exposure to 650 MHz EMF varies with the individual animal. Therefore, the biological condition of the exposed organs (in terms of metabolic activity and genetic pattern) is a crucial factor for the type and the extent of the response. If the intensity of the response to the continuous exposure to EMF is related to the individual animal, the macroscopical response to EMF exposure has its own hierar-
56 Fig. 5a–h Light micrographs of PAS-stained rabbit livers from control (group 3) and 2 years plus 12 (group 1) or 18 (group 2) months of post-exposure rabbits (first and second row). Differences in the glycogen distribution are evidenced by the differences of staining. Individual modifications (i.e., type I or type II) were found only for animals of group 2. Transmission electron micrographs of livers from group 3 (a–c), group 1 (d,e) or group 2 (f–h), showing desmosomes (a, arrows), glycogen rosettes (a, arrowheads; d,e, arrows), bile duct (b, arrow), mitochondria (c, arrow), a Kupffer cell with large phagosomes (f), and large aggregates of glycogen (g,h, arrows). Bars (a–h) = 2 lm
chy: i.e., the type of modification is dependent on the specific organ considered and, within the organ, on the type of cells that were analyzed. However, the modulation of apoptotic rates does not appear to be an individual event for the different organs. Our in vivo experiments showing a reduced apoptotic rate in liver and spleen after EMF exposure are in agreement with literature reporting that apoptosis, spontaneous or drug-induced, is modulated by exposure
to static MF as well as to EMF [18, 22, 23]. The modulation of apoptotic rates as exerted by EMF could be one factor that might help explain the development of cancer and confirm the co-carcinogenic and co-tumorgenic action of EMF. Conversely, in the brain there is a progressive increase of apoptosis, which could be the expression of sublethal modifications to the gray matter, slowly eliminated through apoptosis from the brain tissue.
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Fig. 6a–j Light micrographs of rabbit spleens from control (group 3), 2 years of exposure plus 12 (group 1) or 18 (group 2) months of post-exposure. Arrows show iron storage vacuoles; arrowheads show apoptotic cells. Bars = 5 lm
Under our experimental conditions (650 MHz of chronical exposure plus long-term post-exposure) all investigated organs underwent morphological modifications to various degrees. Liver modifications can be summarized as a progressive rounding up of the hepatocytes, that leads to an alteration of the space of Disse`, and to progressive dilatation/contraction of the lumen of sinusoids and depletion of Kupffer cells. The fact that liver macrophages were depleted is not surprising. Indeed, depletion of Kupffer cells achieved by heavy metals such as gadolinium chloride or unfavorable microenvironmental conditions has been reported previously [24–26]. Further studies are necessary to learn which sublethal damage due to EMF may trigger such macrophage depletion. Morphological modifications of the spleen were mainly characterized by extra storage for hemosiderite in the splenic macrophages probably due to an
increased process of hemolysis. One can hypothesize that the storage might be due either to an increased depletion of red blood cells or to an inhibition of the mechanism of iron recycling. Given the fact that blood catabolism occurs in the liver and bone marrow (only under particular pathological conditions does it also occur in other organs, such as the lungs) the question arises why this process may primarily be confined to the spleen. The brain was the most sensitive organ among those that have been studied. Tissue damage increased dramatically in group 2, in particular with reference to the alteration of apoptotic rates and to the intense dilatation of space among the neurons. Also, similar morphological alterations in brain of rats exposed to 1,439 MHz TDMA (time division multiple access) field have been reported by Tsurita et al. [27]. Therefore, it may be assumed that EMF continuous exposure followed by a period of post-exposure can induce some alteration of certain brain functions. In conclusion, the morphological alterations induced by 650 MHz EMF vary according to the duration of exposure. The type and the extent of alterations are
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Fig. 7a–f Light micrographs of gray matter brain tissue from control (group 3), 2 years of exposure plus 12 (group 1) or 18 (group 2) months of post-exposure rabbits. Progressive enlargement of spaces among neurons and increase of apoptotic cells are observed. Bars = 10 lm
different in different organs and—with regard to the same organs—they vary from animal to animal.
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