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Journal of the Anatomical Society of India

Cell Phone Radiation and Developing Tissues in Chick Embryo - A Light microsopic Study of Kidneys

Author(s): Ingole IV, Ghosh SK

Vol. 55, No. 2 (2006-07 - 2006-12)

Ingole IV, Ghosh SK
MGIMS, Sevagram, M.H.

Abstract:

Phenomenal increase in the number of cell phone users during late 1990s has become a matter of concern regarding the safety of the users exposed to radiation emitted from cell phone. There have been plenty of researches reportedly establishing the damaging effect of radiofrequency radiation emitted from cell phone on the biological tissues. The hazardous effects on the biological tissues as reported varied from it’s effect at the molecular level causing an increase in single and double strand DNA breakages to causing an increase in the mortality in chick embroys exposed to radiation emitted from cell phone. Conversely many have reported it to be safe to the biological tissues within the stipulated standards set. by the regulatory authorities.

Considering these discrepancies, the present work was taken up to investigate any effect caused by such exposure on the developing tissues of chick embryo at the histological level. Embryos were exposed to radiation emitted from cell phone for different durations resulting in different dose schedules. It was observed that exposure to radiation caused damage to the developing kidneys which was more extensive with longer duration of exposure inspite of discontinuing the exposure and giving an exposure free period before sacrificing.

Key words: Cell phone, radio frequency radiation, biological tissues, chick embryo, kidney.

Introduction:

In recent years rapid proliferation of wireless telecommunication industry has resulted in an increase in the number of cell phone users so much so that the radiation emitted from the cell phone has become a cause of concern for public health in general and the cell phone users in particular. Scientific researches have highlighted some extremely hazardous effects of exposure to radiation emitted from the cell phone on the human body. These effects range from those at the molecular level manifested as an increase in single and double strand DNA breakages, (Lai and Singh 1996), change in Ornithine decarboxylase activity, (Penafiel et al 1997), increased risk of brain turnours (Hardell et al 1999), to disruption of learned behavior, dysaesthesia etc (Hocking et al 1998, 2000), and an increase in chick embryo mortality (Magras and Xenos 1997).

Conversely no adverse biological effects were reported by the others. No change in peripheral blood parameters was observed on exposure to radiofrequency radiation (Dasdag et al 2000). No increase in the incidence of chromosomal aberrations and sister chromatid exchanges occurred in cultured human lymphocyte (Zeni et al 2005). On exposure to radio frequency radiation, rat testis did not show any change in the reproductive function (Dasdag et al 2003). The present work is an attempt to investigate the sensitivity of the embryonic tissue to an exposure to radiation emitted by the cell phone.

Material & Methods:

Fertile hen eggs (Gallus domesticus) were incubated in three batches.

Each batch comprising 18 eggs, out of which 9 eggs were incubated in a standard egg incubator at 37 + 0.5C and 50-55% humidity. They were treated as control.

Remaining 9 eggs were treated as exposed group. They were incubated under similar conditions of temperature and humidity as that of control group in a special incubator having a arrangement for mounting the cell phone at it’s roof. The incubator cubicle was made of a specific heat resistant nonmetallic material to avoid any internal reflection of the cell phone radiation. The meter starts adding time whenever the cell phone radiates outgoing signal. A standard cell phone hand set with a frequency bandwidth of 900 MHz, power of 2 Watt and Specific Absorption Rate (SAR) of 0.37 W/Kg was selected for radiating the embryos. The exposed groups were irradiated in sessions of half an hour each. The sessions were spaced at 12 hours interval and the first session was at 12 hours of incubation. Exposed group of 1st, 2nd and 3rd batches received 4 hrs of exposure in 8 sessions, 5 hrs of exposure in 10 sessions and 6 hrs of exposure in 12 sessions respectively. Both the control and the exposed groups of the three batches were sacrificed at the completion of 6th , 8th and 10th day of incubation respectively. The embryos wre examined for viability and congenital anomaly if any. The embryos which were in a moribund state werer discarded. The embryos which were alive and apparently healthy were chilled to death and then subjected to routine histological processing. Two embryos from control as well as exposed group of each batch wre processed for histochemistry ( alkaline phosphatase) by modified Gomori’s method using paraffin having melting point 420 – 440 C for paraffin embedding and block making (Merck). To rule out the false positive reaction, few sections were incubated simultaneously without adding substrate to the incubating medium.

The rest of the embryos were subjected to routine paraffin embedding. Blocks were made in such a way that the whole embryos were cut in coronal sections. 5 μ thick paraffin sections were cut. Sections were stained with H & E and studied under light microscope.

Observations:

It was observed that in the control group of the 1st batch sacrificed at the completion of 6th day all the embryos were alive whereas in the exposed group of the same batch, one embryo was dead. In the second batch sacrificed at the completion of 8th day, one embryo was dead in the control group and two embryos were dead in the exposed group. In the third batch sacrificed at the completion of l0th day, control group had no mortality whereas in the exposed group, two embryos were dead. Thus exposed group showed a higher mortality as compared to that in the control group in each batch. There was one interesting observation that the embryo placed just below the antenna was dead in the exposed groups of all the three batches. H&E stained sections from control group Under low magnification, in the coronal section the outline of the kidney was seen on the posterior abdominal wall.

6 days old embryos of control group showed well developed mesonephric kidneys with mesonephric duct dorsolaterally, lined by columnar epithelium and gonad on the medial side. Renal corpuscles were seen along the medial side of the kidney (fig.1). Kidney tissue showed two types of tubules. One type of tubules were lined by tall columnar cells with large rounded vesicular nuclei. Cytoplasm of these cells was eosinophilic. Other type of tubules were lined by simple cuboidal epithelium with cells having large nuclei and dense basophilic cytoplasm. Bowman’s capsule was clearly seen with it’s parietal layer of simple squamous epithelium. Glomerular epithelium (visceral layer) of Bowman’s capsule showed large cells with the cell bodies (podocytes) protruding into the Bowman’s space. Glomerular capillary network was well differentiated (fig. 2). Endothelial lining of glomerular capillaries could be made out at places. Capillaries showed presence of nucleated red blood cells.

In the sections from 8 days old embryo, the number of renal corpuscles seen in the field was more and they were not limited to the medial margin only but were seen in the deeper part also. The size of the renal corpuscles was also larger with a large glomerular capillary network (fig.4) with nucleated red blood cells. The sections from 10 days old control embryo showed similar histological features.

Fig. 1: Coronal section of 6 days old control embryo showing kidney, gonad (G) and Mesonephric duct (MN). H&E, X40

Fig. 2: Kidney of 6 days old control embryo showing tubules lined by tall columnar cells (T), cuboidal cells©, Bowman’s capsule with squamous parietal epithelium (E), podocytes (P) and glomerular capillaries (G), H&E, X400

Fig. 3: Kidney of 6 days old exposed embryo showing cytoplasmic vacuolation (V), disruption of luminal border (B) and pyknotic nuclei (N) in tubules lined by tall columnar cells. H&E, X100.

Fig. 4: Kidney of 8 days old exposed embryo showing exaggerated cellular damage i.e. cytoplasmic vacuolation (V), disruption of luminal border (B) and pyknotic nuclei (N) in tubules lined by tall columnar cells and glomerulus seen as a clump of cells. H&E, X100.

H&E stained sections from exposed groups. General histological features of exposed group showed similar organization as seen in the control group. But in detailed histological study, several changes were noted in the kidneys. Sections from 6 days old embryos showed mild to moderate degree of degenerative changes. In these sections tubules lined by tall columnar epithelium were affected while those lined by cuboidal epithelium were normal. Some of the cells lining the affected tubules showed cytoplasmic vacuolation, disruption of luminal border and pyknotic nuclei. Some of the renal corpuscles were normal with distinct and intact epithelial lining of the parietal layer of Bowman’s capsule. Some of the renal corpuscles showed narrowed Bowman’s space. The glomerular capillaries were normal and the cells of the visceral layer of Bowman’ s capsule (podocytes) were normal (fig.3). Sections of 8 days old embryos showed extensive damage in the form of cytoplasmic vacuoles, densely stained pyknotic nuclei and disruption of the luminal border of the epithelium in majority of the tubules lined by tall columnar epithelium. The glomerular capillaries were not distinctly seen and at places the glomerulus was seen as a crowded mass of cells (fig. 4). Bowman’s space was seen to be reduced at places in some renal corpuscles.

Sections of 10 days old embryos showed more extensive changes mentioned above.

Sections stained for alkaline phosphatase activity (modified Gomori’s method).

The sections from the exposed groups in all the three batches showed exaggerated reaction in the damaged tubules in the form of intense staining at their luminal borders (fig.6) as compared to that of the control groups which showed mild to moderate degree of reaction (fig.5).

Fig. 5: Kidney of 8 days old control embryo showing mild reaction for alkaline phosphatase activity at the luminal border of tubules (^). (Modified Gomori’s method) X100

Fig. 6: Kidney of 8 days old exposed embryo showing intense reaction for alkaline phosphatase activity at the luminal border of tubules (^) and extensively damaged tubules. (Modified Gomori’s method) X100.

Discussion:

In recent years there have been many reports on the biological effects of electromagnetic fields on cells Goodman (1993), tissues (Bernhardt (1992) Dovrat et al (2005) organs (Milin 1998) and embryonic development (Martin 1988) and (Berman et al 1990). On the other hand several studies dealing with RFR of various frequencies and power intensities revealed no direct genotoxic, mutagenic or cytotoxic effect, (Elder- 2003).

To date such studies reporting hazardous as well as no hazardous effects have used different experimental animal models like rat (Pyrpasopoulou et al. 2004), mice (Magras and Xenos 1997) chick (Espinar et al 1997) etc. For our study the chick embryo was selected as an experimental animal, since the experiment was easily reproducible and the embryos were not compromised by changes in the mother’s biological system. Saito et (1991) justified the use of chick embryo in preference to mammals on the basis that it was easy to control the temperature and Specific Absorption Ratio (SAR) could be accurately estimated.

The deleterious effects which had been reported were attributed to hyperthennia of the tissues in animal species, Warkany (1986),as well as in humans, Edwards (1993) caused by high frequency radiation. In our study the source of radiation used for exposure was a cell phone with a frequency of 900MHz, a lower range in the radio frequency bandwidth (300-3000MHz) to minimize the possibility of heating of embryos, Tofani et al (1986), power 2W and SAR 0.37 W /Kg. It was observed that mesonephric kidneys proceeded in development equally in control and exp?sed embryos. But subsequently degenerative changes were noted in case of exposed embryos predominantly in the tubules lined by tall c,ells. In spite of discontinuing the exposure to radiation and allowing the incubation to progress further i.e. 8th and lOth day, the degei1erative changes were extensive involving even the epithelium of Bowman’s capsule and glomerular .capillaries. An interesting feature was of intense reaction showing increased alkaline pho’sphatase activity in case of damaged tubules, suggesting biological activation of catabolic enzymes, nonnally present in the cytoplasm. Many of the reports have described the effects at the molecular level in the context of cellular proliferation. It was reported to be genotoxic by d’ Ambrosio et al (2002). Change in the morphology and in the inhibition of proliferation of human astrocytoma cells has been reported after exposure to radiation at 835MHz frequency by French et al (2001). Many of the reports from studies on the effects of Radio Frequency Electromagnetic Field on the development of chicks have highlighted the parameters like hatchability, hatching weight, viability and immunohistochemistry studies (Maya & Meshevich 2003) etc. with variable results. Few of the reports available on histogenesis have stated serious developmental alterations in cerebellar morphogenesis in chick embryo on RFR exposure for 20 minutes (Espinar et al 1997). It was inferred to be the result of an effect on histogenesis and synaptogeriesis. Aberrant expression of bone morphogenetic protein and it’s receptors were observed in the kidney of newborn rats whose mother animals were exposed to GSM like RFR during early pregnancy (Pyrpasopoulou et al 2004). Our results seem to be much in . conformity with the reports of the above researchers who have stated that whatever changes were induced at the level of regulation of gene expression during the first week of intrauterine opment seem to be persistent or affect development beyond the removal of the RFR emitting source.

Conclusion:

It has been postulated that persistent and delayed effects of GSM like RFR exposure are more pronounced when exposure takes place during embryogenesis rather than early organogenesis (Pyrpasopoulou et al 2004). Our study showed that RFR can induce changes in development. The effects and degenerative patterns are different in different cells. The degenerative changes seem to be persistent beyond the removal of source of RFR radiation. Discrepancies in the results of different experiments are possibly due to usage of different experimental animals, different parameters used, different exposure fields in respect of power densities. frequencies, duration of exposure and shape of pulse as stated by (Berman et al 1990).

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