- Email: [email protected]
Neonatal mouse brain exposure to mobile telephony and effect on blood-brain barrier permeability
262
CORRESPONDENCE
Neonatal mouse brain exposure to mobile telephony and effect on blood–brain barrier permeability Sir, We previously published a paper in Pathology examining vascular permeability changes in the fetal brain after whole of gestation exposure of pregnant mice to mobile communication radiofrequency (RF) fields.1 Here we evaluate blood–brain barrier (BBB) integrity in the neonatal brain after exposure to mobile telephony. Since neurogenesis is virtually complete in the mouse by the end of the third postnatal week,2 we exposed mice for the first 7 days after birth. Mobile telephone use is increasing rapidly and the developing human brain is now regularly exposed to global system for mobile communication (GSM)-type RF fields. These devices are held close to the head and the brain is exposed to relatively high specific absorption rates (SAR) compared with the rest of the body.3,4 The brain of children may be more sensitive to mobile telephony because it is still developing and conductivity is good, children’s heads absorb more RF than adults, children are known to be more sensitive to many environmental agents (chemicals, ultraviolet, and ionising radiation), and children have a longer lifetime exposure, now beginning at younger ages.4 However, a recent World Health Organization symposium5 concluded that there was insufficient research to determine whether the developing brain shows increased sensitivity to RF fields. It recommended the use of animal models to evaluate the effect of RF field exposure on the immature nervous system. Accordingly, we used the mouse as an animal model because its brain growth resembles that of humans, both species being predominantly postnatal brain developers.2 Disturbances of BBB permeability have frequently been used to study the effect of GSM-type RF fields on the brain.3,4 The BBB is a dynamic interface between blood and brain maintaining homeostasis in the central nervous system.6 The permeability properties of the BBB are largely referable to the cerebral capillary endothelium, which differs from endothelia elsewhere by possessing circumferential tight junctions and fewer caveolae for microvesicular transport. Astrocytic end-feet, which invest most of the capillary surface, also assist in the regulation of microvascular permeability.6 In mice, tight junctions appear from day 10 of embryonic development and the BBB is fully functional at day 16.7 We previously used albumin immunohistochemistry to determine whether exposure to mobile telephone-type RF fields increased vascular permeability in adult mice8–10 and fetuses after whole of gestation exposure.1 In the present study, this vascular tracer was employed in neonatal brains. Newborn BALB/c mice received a 60-min far-field, whole body exposure of 4 W/kg on 7 successive days postnatally. 4 W/kg is the internationally accepted maximum safe level of average whole body SAR.3 The RF field had similar pulsing and modulation characteristics to those used for global system for mobile communication (GSM; 900 MHz modulated at a pulse repetition
Pathology (2006), 38(3), June
frequency of 217 Hz and a pulse width of 0.6 ms). Within the exposure system, the field was homogeneous so all mice received the same exposure level. The exposure system consisted of a cylindrical parallel plate with mice restrained in clear perspex tubes arranged radially around a dipole antenna.8 The neonatal mice remained motionless in the cylinders, thus maintaining their orientation relative to the field and ensuring that the SAR could be more precisely controlled. There were three groups of animals: exposed (n510); sham-exposed (n510); and a non-exposed, freely-moving, cage control group (n510), without handling or further confinement to obviate any stress-related, exposure module confinement effects. On postnatal day 7, mice were anaesthetised (isoflurane), heads removed and immediately immersed in Bouin’s fixative. After fixation, serial coronal sections of head were cut, embedded in paraffin wax, and 6-mm sections cut and stained with H&E. In addition, a positive control group (n510) was included to confirm the ability of albumin immunohistochemistry to detect albumin extravasation. Immediately after birth, mice were given a subcutaneous injection of cadmium chloride (2 mg/kg) in saline, which produces microvascular endothelial damage, increased vascular permeability and petechial haemorrhage in the neonatal mouse brain.11 These cadmium-injected mice were killed 3 hours post-injection. Coronal brain slices containing cerebral cortex, basal ganglia, thalamus and hippocampus (Fig. 1) and cerebellum, midbrain and medulla (Fig. 2) were selected for immunohistochemistry to ensure that a wide range of neuroanatomical sites was examined. Detection of exogenous albumin was performed using goat anti-rat albumin (Cappel, USA) as the primary monoclonal antibody at a dilution of 1:20 000 and with biotinylated rabbit anti-goat
Fig. 1 Coronal section of neonatal mouse brain at postnatal day 7 showing cerebral cortex (C), thalamus (T), basal ganglia (BG) and hippocampus (H). Inset: Intraneuronal albumin staining in the cerebral cortex.
CORRESPONDENCE
263
Australia; and the Australian Centre for Radiofrequency Bioeffects Research Contact Professor P. C. Blumbergs. E-mail: [email protected] ACKNOWLEDGEMENT Funding by the National Health and Medical Research Council is gratefully acknowledged.
Fig. 2 Coronal section of neonatal mouse brain at postnatal day 7 showing midbrain (MB), cerebellum (C) and medulla (M). Inset: Higher power view of albumin staining confined to the vascular lumina in the medulla.
immunoglobulin (Dako, Denmark) at a 1:500 dilution as the secondary antibody. Positive and negative controls were used in this protocol. This experimental protocol was approved (65/03) by the Animal Ethics Committee of the Institute of Medical and Veterinary Science, Adelaide, South Australia. By light microscopy, microvascular profiles were well delineated by intraluminal albumin immunostaining (Fig. 2). No albumin extravasation was detected in exposed or control (sham-exposed and freely moving caged) groups, except in leptomeningeal, choroid plexus and circumventricular organ microvessels which have no recognised BBB.6 Similarly, no BBB breakdown was found in fetal brains after whole of gestation (19 days) exposure using the same experimental paradigm.1 By contrast, in neonatal mice injected with cadmium, there was multifocal cerebellar haemorrhage and substantial attendant albumin extravasation.1 Intraneuronal albumin staining (Fig. 1) was found in many brain regions and has been implicated in neuronal development and maturation.12 Therefore, it appears that exposure of the developing mouse brain to mobile telephone-type RF fields does not produce any perturbation of BBB integrity, at least that is detectable by albumin immunohistochemistry at the light microscope level, or any fluid leakage which does occur is minimal and rapidly resolved. John W. Finnie Peter C. Blumbergs Zhao Cai Jim Manavis Timothy R. Kuchel Hanson Institute Centre for Neurological Diseases, Institute of Medical and Veterinary Science, Adelaide, South
1. Finnie JW, Blumbergs PC, Cai Z, et al. Effect of mobile telephony on blood-brain barrier permeability in the foetal mouse brain. Pathology 2006; 38: 63–5. 2. Rodier PM. Chronology of neuron development: animal studies and their clinical implications. Develop Med Child Neurol 1980; 22: 525–45. 3. Hossmann KA, Hermann DM. Effects of electromagnetic radiation of mobile phones on the central nervous system. Bioelectromagnetics 2003; 24: 49–62. 4. National Radiation Protection Board, United Kingdom. Health Effects from Radiofrequency Electromagnetic Fields. Report of an Independent Advisory Group on Non-ionising Radiation. London: National Radiation Protection Board, 2003; 78–90. 5. World Health Organization Workshop. Sensitivity of Children to Electromagnetic Fields. Istanbul, Turkey, June 9–10, 2004. 6. Ironside JW, Pickard JD. Raised intracranial pressure, oedema and hydrocephalus. In: Graham DI, Lantos PL, editors. Greenfield’s Neuropathology. 7th ed. London: Arnold, 2002; 200–3. 7. Bauer HC, Bauer H, Lametschwandtner A, et al. Neovascularisation and the appearance of morphological characteristics of the blood-brain barrier in the embryonic mouse central nervous system. Dev Brain Res 1993; 75: 269–78. 8. Finnie JW, Blumbergs PC, Manavis J, et al. Effect of global system for mobile communication (GSM)-like radiofrequency fields on vascular permeability in mouse brain. Pathology 2001; 33: 338–40. 9. Finnie JW, Blumbergs PC, Manavis J, et al. Effect of long-term mobile communication microwave exposure on vascular permeability in mouse brain. Pathology 2002; 34: 344–47. 10. Finnie JW, Blumbergs PC. Mobile telephones and brain vascular leakage. Pathology 2004; 34: 96–7. 11. Webster WS, Valois AA. The toxic effects of cadmium on the neonatal mouse CNS. J Neuropath Exp Neurol 1981; 40: 247–57. 12. Dziegielewska KM, Saunders NR. The development of the blood-brain barrier: proteins in foetal and neonatal CSF, their nature and origins. In: Meisami E, Timiras PS, editors. Handbook of Human Growth and Biological Development. Boca Raton: CRC Press, 1988; 169–91.
DOI: 10.1080/00313020600699284
Kinase domain mutation of MAP2K4 is rare in gastric, colorectal and lung carcinomas Sir, Protein kinases participate in many cellular processes which regulate cellular growth, differentiation and survival. Activation of protein kinases involved in modulating cell proliferation and survival has been implicated in oncogenic transformation. Mitogen-activated protein kinase kinase 4 (MAP2K4), also referred to as JNKK1, MAPKK4, MEK4, MKK4, PRKMK4, SEK1 and SERK1, is a serine/threonine kinase. This kinase is a component of stress and cytokine-induced signalling pathways. It has been shown to activate MAPK8/JNK1, MAPK9/JNK2, and MAPK14/p38.1 Recently, Parsons et al.2 analysed 340 serine/threonine kinase genes in 204 colorectal cancer tissues for the detection of the somatic mutations, and identified 23 mutations in eight genes. Of them, MAP2K4 gene mutations were found in six (2.9%) of the 204 colorectal cancers. Of these, four were identified in the DNA
CORRESPONDENCE
Neonatal mouse brain exposure to mobile telephony and effect on blood–brain barrier permeability Sir, We previously published a paper in Pathology examining vascular permeability changes in the fetal brain after whole of gestation exposure of pregnant mice to mobile communication radiofrequency (RF) fields.1 Here we evaluate blood–brain barrier (BBB) integrity in the neonatal brain after exposure to mobile telephony. Since neurogenesis is virtually complete in the mouse by the end of the third postnatal week,2 we exposed mice for the first 7 days after birth. Mobile telephone use is increasing rapidly and the developing human brain is now regularly exposed to global system for mobile communication (GSM)-type RF fields. These devices are held close to the head and the brain is exposed to relatively high specific absorption rates (SAR) compared with the rest of the body.3,4 The brain of children may be more sensitive to mobile telephony because it is still developing and conductivity is good, children’s heads absorb more RF than adults, children are known to be more sensitive to many environmental agents (chemicals, ultraviolet, and ionising radiation), and children have a longer lifetime exposure, now beginning at younger ages.4 However, a recent World Health Organization symposium5 concluded that there was insufficient research to determine whether the developing brain shows increased sensitivity to RF fields. It recommended the use of animal models to evaluate the effect of RF field exposure on the immature nervous system. Accordingly, we used the mouse as an animal model because its brain growth resembles that of humans, both species being predominantly postnatal brain developers.2 Disturbances of BBB permeability have frequently been used to study the effect of GSM-type RF fields on the brain.3,4 The BBB is a dynamic interface between blood and brain maintaining homeostasis in the central nervous system.6 The permeability properties of the BBB are largely referable to the cerebral capillary endothelium, which differs from endothelia elsewhere by possessing circumferential tight junctions and fewer caveolae for microvesicular transport. Astrocytic end-feet, which invest most of the capillary surface, also assist in the regulation of microvascular permeability.6 In mice, tight junctions appear from day 10 of embryonic development and the BBB is fully functional at day 16.7 We previously used albumin immunohistochemistry to determine whether exposure to mobile telephone-type RF fields increased vascular permeability in adult mice8–10 and fetuses after whole of gestation exposure.1 In the present study, this vascular tracer was employed in neonatal brains. Newborn BALB/c mice received a 60-min far-field, whole body exposure of 4 W/kg on 7 successive days postnatally. 4 W/kg is the internationally accepted maximum safe level of average whole body SAR.3 The RF field had similar pulsing and modulation characteristics to those used for global system for mobile communication (GSM; 900 MHz modulated at a pulse repetition
Pathology (2006), 38(3), June
frequency of 217 Hz and a pulse width of 0.6 ms). Within the exposure system, the field was homogeneous so all mice received the same exposure level. The exposure system consisted of a cylindrical parallel plate with mice restrained in clear perspex tubes arranged radially around a dipole antenna.8 The neonatal mice remained motionless in the cylinders, thus maintaining their orientation relative to the field and ensuring that the SAR could be more precisely controlled. There were three groups of animals: exposed (n510); sham-exposed (n510); and a non-exposed, freely-moving, cage control group (n510), without handling or further confinement to obviate any stress-related, exposure module confinement effects. On postnatal day 7, mice were anaesthetised (isoflurane), heads removed and immediately immersed in Bouin’s fixative. After fixation, serial coronal sections of head were cut, embedded in paraffin wax, and 6-mm sections cut and stained with H&E. In addition, a positive control group (n510) was included to confirm the ability of albumin immunohistochemistry to detect albumin extravasation. Immediately after birth, mice were given a subcutaneous injection of cadmium chloride (2 mg/kg) in saline, which produces microvascular endothelial damage, increased vascular permeability and petechial haemorrhage in the neonatal mouse brain.11 These cadmium-injected mice were killed 3 hours post-injection. Coronal brain slices containing cerebral cortex, basal ganglia, thalamus and hippocampus (Fig. 1) and cerebellum, midbrain and medulla (Fig. 2) were selected for immunohistochemistry to ensure that a wide range of neuroanatomical sites was examined. Detection of exogenous albumin was performed using goat anti-rat albumin (Cappel, USA) as the primary monoclonal antibody at a dilution of 1:20 000 and with biotinylated rabbit anti-goat
Fig. 1 Coronal section of neonatal mouse brain at postnatal day 7 showing cerebral cortex (C), thalamus (T), basal ganglia (BG) and hippocampus (H). Inset: Intraneuronal albumin staining in the cerebral cortex.
CORRESPONDENCE
263
Australia; and the Australian Centre for Radiofrequency Bioeffects Research Contact Professor P. C. Blumbergs. E-mail: [email protected] ACKNOWLEDGEMENT Funding by the National Health and Medical Research Council is gratefully acknowledged.
Fig. 2 Coronal section of neonatal mouse brain at postnatal day 7 showing midbrain (MB), cerebellum (C) and medulla (M). Inset: Higher power view of albumin staining confined to the vascular lumina in the medulla.
immunoglobulin (Dako, Denmark) at a 1:500 dilution as the secondary antibody. Positive and negative controls were used in this protocol. This experimental protocol was approved (65/03) by the Animal Ethics Committee of the Institute of Medical and Veterinary Science, Adelaide, South Australia. By light microscopy, microvascular profiles were well delineated by intraluminal albumin immunostaining (Fig. 2). No albumin extravasation was detected in exposed or control (sham-exposed and freely moving caged) groups, except in leptomeningeal, choroid plexus and circumventricular organ microvessels which have no recognised BBB.6 Similarly, no BBB breakdown was found in fetal brains after whole of gestation (19 days) exposure using the same experimental paradigm.1 By contrast, in neonatal mice injected with cadmium, there was multifocal cerebellar haemorrhage and substantial attendant albumin extravasation.1 Intraneuronal albumin staining (Fig. 1) was found in many brain regions and has been implicated in neuronal development and maturation.12 Therefore, it appears that exposure of the developing mouse brain to mobile telephone-type RF fields does not produce any perturbation of BBB integrity, at least that is detectable by albumin immunohistochemistry at the light microscope level, or any fluid leakage which does occur is minimal and rapidly resolved. John W. Finnie Peter C. Blumbergs Zhao Cai Jim Manavis Timothy R. Kuchel Hanson Institute Centre for Neurological Diseases, Institute of Medical and Veterinary Science, Adelaide, South
1. Finnie JW, Blumbergs PC, Cai Z, et al. Effect of mobile telephony on blood-brain barrier permeability in the foetal mouse brain. Pathology 2006; 38: 63–5. 2. Rodier PM. Chronology of neuron development: animal studies and their clinical implications. Develop Med Child Neurol 1980; 22: 525–45. 3. Hossmann KA, Hermann DM. Effects of electromagnetic radiation of mobile phones on the central nervous system. Bioelectromagnetics 2003; 24: 49–62. 4. National Radiation Protection Board, United Kingdom. Health Effects from Radiofrequency Electromagnetic Fields. Report of an Independent Advisory Group on Non-ionising Radiation. London: National Radiation Protection Board, 2003; 78–90. 5. World Health Organization Workshop. Sensitivity of Children to Electromagnetic Fields. Istanbul, Turkey, June 9–10, 2004. 6. Ironside JW, Pickard JD. Raised intracranial pressure, oedema and hydrocephalus. In: Graham DI, Lantos PL, editors. Greenfield’s Neuropathology. 7th ed. London: Arnold, 2002; 200–3. 7. Bauer HC, Bauer H, Lametschwandtner A, et al. Neovascularisation and the appearance of morphological characteristics of the blood-brain barrier in the embryonic mouse central nervous system. Dev Brain Res 1993; 75: 269–78. 8. Finnie JW, Blumbergs PC, Manavis J, et al. Effect of global system for mobile communication (GSM)-like radiofrequency fields on vascular permeability in mouse brain. Pathology 2001; 33: 338–40. 9. Finnie JW, Blumbergs PC, Manavis J, et al. Effect of long-term mobile communication microwave exposure on vascular permeability in mouse brain. Pathology 2002; 34: 344–47. 10. Finnie JW, Blumbergs PC. Mobile telephones and brain vascular leakage. Pathology 2004; 34: 96–7. 11. Webster WS, Valois AA. The toxic effects of cadmium on the neonatal mouse CNS. J Neuropath Exp Neurol 1981; 40: 247–57. 12. Dziegielewska KM, Saunders NR. The development of the blood-brain barrier: proteins in foetal and neonatal CSF, their nature and origins. In: Meisami E, Timiras PS, editors. Handbook of Human Growth and Biological Development. Boca Raton: CRC Press, 1988; 169–91.
DOI: 10.1080/00313020600699284
Kinase domain mutation of MAP2K4 is rare in gastric, colorectal and lung carcinomas Sir, Protein kinases participate in many cellular processes which regulate cellular growth, differentiation and survival. Activation of protein kinases involved in modulating cell proliferation and survival has been implicated in oncogenic transformation. Mitogen-activated protein kinase kinase 4 (MAP2K4), also referred to as JNKK1, MAPKK4, MEK4, MKK4, PRKMK4, SEK1 and SERK1, is a serine/threonine kinase. This kinase is a component of stress and cytokine-induced signalling pathways. It has been shown to activate MAPK8/JNK1, MAPK9/JNK2, and MAPK14/p38.1 Recently, Parsons et al.2 analysed 340 serine/threonine kinase genes in 204 colorectal cancer tissues for the detection of the somatic mutations, and identified 23 mutations in eight genes. Of them, MAP2K4 gene mutations were found in six (2.9%) of the 204 colorectal cancers. Of these, four were identified in the DNA