Repeated exposure to nanosecond high power pulsed microwaves increases cancer incidence in rat

High-power microwaves are used to inhibit electronics of threatening military or civilian vehicles. This work aims to assess health hazards of high-power microwaves and helps to define hazard threshold levels of modulated radiofrequency exposures such as those emitted by the first generations of mobile phones. Rats were exposed to the highest possible field levels, under single acute or repetitive exposures for eight weeks. Intense microwave electric fields at 1 MV m-1 of nanoseconds duration were applied from two sources at different carrier frequencies of 10 and 3.7 GHz. The repetition rate was 100 pps, and the duration of train pulses lasted from 10 s to twice 8 min. The effects on the central nervous system were evaluated, by labelling brain inflammation marker GFAP and by performing different behavioural tests: rotarod, T-maze, beam-walking, open-field, and avoidance test. Long-time survival was measured in animals repeatedly exposed, and anatomopathological analysis was performed on animals sacrificed at two years of life or earlier in case of precocious death. Control groups were sham exposed. Few effects were observed on behaviour. With acute exposure, an avoidance reflex was shown at very high thermal level (22 W kg-1); GFAP was increased some days after exposure. Most importantly, with repeated exposures, survival time was 4-months shorter in the exposed group, with eleven animals exhibiting a large sub-cutaneous tumour, compared to two in the sham group. A residual X-ray exposure was also present in the beam (0.8 Gy), which is probably not a bias for the observed result. High power microwaves below thermal level in average, can increase cancer prevalence and decrease survival time in rats, without clear effects on behaviour. The parameters of this effect need to be further explored, and a more precise dosimetry to be performed.


Introduction
High power microwaves (HPM) are used to inhibit the electronic systems of threatening vehicles. 26 Concern has arisen as to whether HPM could lead to health hazards for operators of emitting 27 systems and for personnel exposed in targeted vehicles. The health effects of HPM have been 28 studied since the discovery of radar in the middle of the past century. Many experiments have been 29 performed with microsecond pulses at levels of several hundred kilovolts per meter. Some studies 30 have been published, but many others have been presented only as reports or at scientific meetings. 31 Studies performed with a specific absorption rate (SAR) above the thermal threshold of 4 W kg -1 32 have shown biological effects. Below 4 W kg -1 , for studies showing effects, the pulse duration of 33 single pulses was between 40 ns and 10 µs, and peak-SAR was from 5 to 20 MW kg -1 . Half of the 34 studies on HPM addressed behavioral endpoints, reviewed by D'Andrea [1]. Others bear on the 35 cardio-vascular [2,3], visual [4], and auditory systems [5]. Only sparse work concerned blood-36 brain-barrier permeability [6,7], DNA damage [8,9], carcinogenesis [10-12], or cellular or sub-37 cellular mechanisms [13]. Concerning cancer, Zhang [14] and Devyatkov et al. [10] reported 38 protective effects at levels above thermal threshold, with smaller tumors and a 30% increase of 39 survival rate in exposed animals. Several years after Seaman' article [15], a recent review by 40 Schunck reported only one new paper in 2009, and no other effects on cancer were reported [16]. 41 However, durations of exposure in those studies were often short. 48 We looked at the scientific and medical literature (NCBI-PubMed, Current Contents and Science 49 Direct, more recently Web of Science) and at specialized databases of papers, scientific meetings 50 and reports (EMF Database, IEEE ICES EMF Literature Database and WHO-EMF-Portal). The 51 following keywords were used: HPM, high power microwave, high peak, electromagnetic pulse, 52 microwave radiation, high exposure microwave, HPPP, EHPP, high intensity microwave. 53 Exposure systems 54 The sources of high-power microwaves (HPM) were two superradiance generators, one in X-band 55 at 10 GHz (SRX) with pulses of 1 ns, the other in S-band at 3.7 GHz (SRS) with pulses of 2.5 ns. 56 The strongest possible microwave electric fields were applied, of about 1 MV m-1, at a repetition 57 rate of 100 pps ( Table 1). The "SINUS type" electron accelerator of this system is made of a Tesla 58 generator and a continuous formation line. A great advantage of this system is its small size. The 59 superradiance source is derived from the back-wave oscillator, with the following characteristic 60 features: ultrashort microwave pulses, and very high peak power.

Animals
Six-weeks-old Sprague Dawley male rats were purchased from Charles River, L'Arbresle, France. 66 They were either exposed or sham-exposed. Two types of acute exposures were carried out. The SRX exposure lasted 10 s every 5 min for one 75 hour, and the SRS exposure lasted 2 x 8 min with 10 min interval (26 min total). For acute 76 exposures, rats were exposed one by one directly at the horn output (168 animals in total, 12 per 77 group). Besides, one protocol of repeated exposures was used with SRS source. The 26 minutes-78 exposure was repeated each day, 5 days/week for 8 weeks. When performing mean term repetitive 79 exposures every day with a realistic source that cannot easily be duplicated, there is a need for 80 optimization of the design to expose many animals at the same time. The circular beam produced 81 by the TM01 mode of the waves was adapted to this goal, with a beam width large enough to 82 simultaneously expose 12 animals at 2.5 m from the SRS output. Animals were exposed 2 by cage 83 in six cages placed each day at different positions on the circle. Then every day, 2 series of 12 84 animals were exposed, alternating with 2 series of sham exposure in-between to allow time for the 85 equipment to cool down between two successive exposure sessions. In total, two groups of 24 rats 86 received a repeated exposure, either real, either sham ( After the end of repeated exposure, the animals were observed and followed up to 2 years of age. 94 Lifespan was recorded, and anatomo-pathological examination was performed at the animal death. 95 For each test, a group of 12 exposed animals was compared to a similar-sized group of sham-96 exposed animals, put in the same place and under the same ambient conditions than the exposed 97 animals, but without emission from the source.

Investigations on the central nervous system
After one acute or the last repetitive exposure, different behavioural tests were performed: beam-100 walking, rotarod, T-maze, open-field. An avoidance test was also performed during an acute SRS 101 exposure, applied continuously for 14 minutes (Table 2). In the avoidance test, animals can choose 102 to spend time in two parts of a box. One part is protected against the beam (shielded), the other is 103 not. The time spent in the non-protected side is recorded. 104 After the behavioural tests were performed, animals were sacrificed, and an immunohisto-105 chemical labelling of the brain inflammation marker GFAP was achieved on 40 µm thick slices 106 for 5 areas of the brain: frontal cortex, gyrus dentate, putamen, pallidum and cerebellar cortex, 2 107 days after the SRX and the SRS exposures, and 7 days after the SRX exposure. (Table 2). 108 The global design of this study and the sample size in each test are summarized in Table 2. 109

110
After the end of repeated exposures, the animals were followed up to 2 years of age. For animals 111 needing an ethical sacrifice, the lifespan was recorded. Either at this time or at 104 weeks, animals 112 were sacrificed with a lethal overdose of pentobarbital (5.0 ml kg-1 IP), organs were collected and 113 fixed in 4% isotone buffered formalin for 48 to 72h. Organs larger than 5 mm were cut for an 114 optimized fixation and all tissue samples were included in paraffine blocks. 115 Five µm slices were cut with a microtome and 6 slides per organ were prepared. An anatomo-116 pathological examination was performed on two slices per organ. One slide was coloured with 117 haematoxylin-eosin stain (HES), the second was stored in case of need for any other specific 118 labelling. 119

Dosimetry
Electric field has been measured at the actual exposure distance of 2.2 m from the source output 121 with a germanium detector and calculated for closer distances. As numerical computation of 122 specific absorption rate (SAR) by FDTD has not been available, the whole-body specific 123 absorption rates (SAR) (defined as electromagnetic power absorbed per unit of tissue mass) were 124 calculated for each condition from the rat's position and size as described by Gandhi  Percentages of time spent in the exposed or in the blinded box were compared by two-way 132 ANOVA with two factors: exposure (exposed or sham) and period (habituation or exposure). 133 Percentages of labelled areas for GFAP were compared by two-way ANOVA with two factors: 134 exposure (exposed or sham) and localisation (brain area). Survival rates of repetitively exposed performance was significantly enhanced in exposed animals: 41/72 exposed animals succeeded, 146 compared to 23/72 sham animals -p < 0.001. Also, during an acute SRS exposure, with a choice 147 for rats to stay in an exposed or a shielded compartment (avoidance reflex), exposed animals spent 148 3.7% of time on the exposed side, compared to 21.9% for the sham group -p < 0.001.

Lifespan
Most strikingly, six exposed animals deceased early between 33 and 47 weeks, leading to a 4-162 months decrease of lifespan in the repetitively exposed group (n=24) compared to the sham group 163 (n=24) (Fig 2). The median lifespan was 590 days for the exposed group, compared to 722 days 164 for the sham group -p < 0.0001. One sham rat was used as sentinel for sanitary control, eleven 165 sham animals survived at the end of the experiment, whereas only two animals survived in the  Left column: exposed group (#=1-24); right column: sham group (#=25-48). a /: no macroscopic 177 abnormality; b italic: internal masses found at death or at 24 months; c bold: large external masses 178 leading to ethical sacrifice. Only two exposed rats survived at the end of the experiment at 103 One of the exposed animals with an external tumour also had a pituitary tumour, and at death, six 185 other exposed animals had abdominal masses and one had a pituitary tumour. Tumour types and 186 lifespan are detailed in Table 3.  [16].
No effect was seen in learning experiments, but a positive effect was found in the rotarod test, 198 which mainly addresses a sensori-motor activity. This could be due to a slight heating at the SAR 199 of 4.7 W kg -1 of the SRS source. Such a heating effect has been hypothesized by Preece who 200 observed an increased reactivity (shorter reaction time) in human volunteers exposed to mobile 201 phones at a SAR of 1.7 W kg -1 [20]. Avoidance of the SRS beam was significant in exposed 202 animals subjected to a thermal SAR of 22 W kg -1 , which is high above the thermal threshold of More importantly, an increased and early rate of sarcomas and fibrosarcomas and higher associated 216 mortality were observed in animals exposed to repetitive sessions at an average SAR of 0.8 W 217 kg -1 , five times below the thermal threshold ( increased incidence of heart schwannoma and glioma in whole-body exposed rats to phone-type 227 microwaves at much higher SARs than those used in humans [26]. Although experiments with 228 newer extremely short pulses (a few ns long) have been performed, HPM had only been used in 229 acute experiments, and most studies reporting an effect looked at physiological reactions, without 230 addressing genotoxicity or carcinogenicity endpoints. This work therefore corresponds to the first 231 report with in vivo exposure to extremely short duration peak pulses, with a high repetition rate, 232 and with a design of repeated exposure for eight weeks. 233 Tinkey showed that very high doses of X-rays (> 46 Gy) were needed to induce sarcomas in 234 Sprague-Dawley rats [29]. then the low 0.8 Gy residual X-ray level of this study cannot explain 235 the observed early tumour increase. Therefore, this study shows that the observed tumours and 236 decreased lifespans were due to repeated exposures at a SAR below the known health threshold of 237 with a liver carcinosarcoma and exposed to 10 ns pulses at 9 GHz, which paradoxically was 241 beneficial [10]. The peak power was 100 MW, but the electric field or SAR was not specified. 242 More recently, after 16 -1000 ns ultra-wide band pulses (UWB) with a frequency of 0.6 -1.0 243 GHz, a duration of 4 -25 nanosecond, an amplitude of 0.1 -36 kV cm -1 , and a pulse repetition 244 rate of 13 pulses per second (pps), Zharkova also found an inhibition of mitochondrial activity 245 which has been interpreted rather as an anti-tumoral activity [30]. 246 Other studies bring some mechanistic explanation that would support a cancerogenic effect. Observed tumours were mostly subcutaneous, but were also ubiquitous, which is not indicative of 251 a specific mechanism or sensitivity of a given tissue or organ. This means that inflammatory 252 processes or genotoxic effects should be investigated in the different target tissues where tumours 253 appeared: connective tissue, muscle, fat, vessels, pituitary gland, lymph nodes, etc. To check if 254 this is a general phenomenon or a strain/specie specific effect, this experiment should be repeated 255 with other rat strains and different animal species (e.g. mice, and/or rabbits) which are usual 256 models for human toxicology. 257 The actual corner stones of guidelines for RF exposures consider behavioral effects as the most 258 sensitive biological endpoint that had yet been observed as a deleterious effect on health. Up to 259 date, this decreased behavioral performance is today attributed to the temperature elevation 260 produced in rodents or primates, consecutive to the above-mentioned dielectric absorption, only 261 linked to the average absorbed power (rms SAR). This study shows that extremely high intensity microwave pulses, around one million volts per meter (1 MV.m-1), comparable to those that have 263 in part been used in the Gulf War, produce a clear increased incidence of cancer in exposed 264 animals. Furthermore, it tells that even an aggressive damage such as cancer can occur without so 265 much decreased cognitive performance, even at a level below the known thermal threshold of 266 whole-body SAR (4W/kg). Then the peak SAR should be re-considered in the definition of 267 guidelines. 268 The original hypothesis was: is there an effect of high-power microwaves? In which conditions? 269 If yes, does it obey to a classical thermal mechanism or a mechanism other than thermal? This 270 study showed: i) few behavioural effects from either acute or repeated exposure; ii) an 271 inflammatory effect of acute exposure to HPM; and iii) a surprising increase of lethal cutaneous 272 or subcutaneous tumour incidence of sarcoma or fibrosarcoma type, in the repetitively exposed 273 group (46% versus 8% in the sham-control group). This increased cancer incidence was associated 274 with decreased lifespan in rats exposed to HPM with an average SAR level below the thermal 275 threshold of 4 W kg -1 . Furthermore, this effect was not associated with clear effects on behaviour, 276 as could have been expected from previous knowledge. The underlying mechanisms are likely to 277 be different from thermal effects and need to be further explored. Also, the thresholds or dose-278 responses in SAR level, duration and number of exposure sessions need to be defined. 280 We thank A. Braun for review and editing the paper. As the following contributors could not be 281 contacted for formal approval of the paper, they are acknowledged for their active participation to