Associations Of 5-HTR Gene Polymorphisms With Cognitive Alterations After Electromagnetic Radiation

Objective: With the increased applications of modern technologies, the effect of electromagnetic field has become a public health concern. The nervous system is a sensitive target of electromagnetic radiation（EMR）, though the impact of EMR on central nervous system and the machanisms remain unconcluded. 5-HT system regulates the brain development, cognition and emotion. Genetic polymorphisms lead to individual differences. In this study, we evaluated the alterations of cognitive function after EMR and if any, the involvement of 5-HT system and wheather the single nucleotide polymorphisms（SNP） of 5-HT receptors were associated with the process. The aim of this study was to provide new evidences for understanding genetic machanisms of EMR effects. Materials and methods:（1） In the in vivo study, Wistar rats were sham exposed or exposed to 2.856 GHz microwaves with the average power density of 5, 10, 20 or 30 m W/cm₂ respectively for 6 min three times a week up to 6 weeks. In the in vitro study, PC12 cell line and 293 T cell line were sham exposed or exposed to 30 m W/cm₂ microwaves for 5 min up to three times with an interval of 5 min.（2） Morris water maze test was implemented to assess spatial learning and memory of rats.（3） The electroencephalogram（EEG） signals were obtained through a BIOPAC MP-150 system and power spectral analyses were performed.（4） Morphology structures of rats’ hippocampi were observed through H&E staining.（5） Neurotransmitters of the hippocampus and cerebrospinal fluid（CSF） were measured by high performance liquid chromatography.（6） Monoamine oxidase（MAO） activities in rats’ hippocampi were measured using a detection kit.（7） Expressions of tryptophan hydroxylase 1（TPH1） in rats’ hippocampi and 5-HT_1A/2A/2CR in rats’ hippocampi and cerebral cortices were measured by western blot.（8） 5-HT_1A/2A/2CR in rats’ hippocampi and cerebral cortices were identified by immunohistochemistry reactions.（9） The promoter region of 5-HT_1A/2A/2CR was amplified by PCR after the genomic DNA was extracted from the whole blood. Following that, SNPs were detected by direct sequencing.（10） Promoter activities of candidate SNPs were determined using 9 luciferase reporter gene assay and electrophoretic mobility shift assay.（11） 5-HT1 AR m RNA expression was detected by Real-Time PCR.（12） Basic informations of workers from power plant were obtained with questionnaires and the mental symptoms were evaluated with scales.（13） Blood samples were collected, and polymerase chain reaction-restriction fragment length polymorphism technique was used to genotype rs6311、rs6313、rs6314、rs6318、rs6295 polymorphisms of 5-HTR in all workers.（14） Routine blood indices, serum biochemical parameters, BNP, LENK and NPY were tested. ECG and ERPs were recorded.Results: 1. Role of 5-HT system in the process of microwave induced cognitive alterations.（1） 2.856 GHz, with the average power density of 5, 10, 20 and 30 m W/cm₂ could induce deficit of spatial learning and memory.（2） 14 days after exposure, the amplitude was increased and the mean frequency was decreased with the delta band relative power being significantly increased in EEG.（3） Karyopyknosis occurred and the nuclei shrank into blue pieces in exposure groups at 14 days, 28 days and 2 months after exposure, among which the most serious injury occurred in 30 m W/cm₂ group at 28 days after microwave exposure.（4） Derangement of neurotransmitter contents in the hippocampus and CSF was observed. Among which 5-HT was increased in 10, 20 and 30 m W/cm₂ groups at 28 days after exposure, and 5-HT was increased while 5-HIAA was decreased in all exposure groups at 2 months after exposure.（5） Above results raised our interest in 5-HT system. TPH1 and MAO, two important rate-limiting enzymes in 5-HT synthesis and metabolic process, were detected respectively. TPH1 expressions in the hippocampus did not vary after 30 m W/cm₂ microwave exposure while MAO activity was decreased at 28 days and 2 months after 30 m W/cm₂ microwave exposure.（6） 5-HT1 AR were significantly increased in CA1, CA2 and CA3 regions of rats’ hippocampi at 14 days after microwave exposure, increased in CA1 and CA2 regions significantly at 28 days after exposure, and only increased in CA3 region at 2 months after exposure. 5-HT2 CR was increased in CA3 region at 14 days and 2 months after exposure but decreased significantly in the CA2 region at 28 days after exposure. As for the cerebral cortex region, expressions of 5-HT1 AR and 5-HT2 CR were increased at 28 days after exposure. There were no alterations of 5-HT2 AR expression. 2. Associations of 5-HTR gene SNPs with cognitive alterations of rats after microwave exposure.（1） Two SNPs were screened in rat 5-HT_1A/2A/2CR promoter region, including 5-HT₁AR-215 T>C（rs198585630） and 5-HT2 AR 74₇5 ins G. 5-HT1 AR rs198585630 10 genotypes in rats were TT, TC and CC.（2） The function of SNP rs198585630 was studied. Transcriptional activity of 5-HT1 AR promoter including C allele was higher than that including T allele. Transcriptional activity of 5-HT1 AR promoter was stimulated by 30 m W/cm₂ microwave exposure, and C allele was more susceptible to microwave with a stronger transcriptional activation being induced.（3） Associations of 5-HT1 AR rs198585630 with cognitive alterations of rats after microwave exposure were studied. ? Rats carring TT genotype performed lower spatial learning abilities than those carring TC or CC genotype. Spatial learning abilities were enhanced in TT-genotypic rats while decreased in TC and CC-genotypic rats after 30 m W/cm₂ microwave exposure. Spatial memories of TC and CC-genotypic rats were decreased after the exposure. ? 14 days after exposure, the amplitude was increased, the mean frequency was decreased and the delta band relative power was significantly increased presenting higher increasement in TC and CC-genotypic than TT-genotypic rats. ? The ratio of 5-HIAA/5-HT declined in TT-genotypic rats after exposure. ? 5-HT1 AR m RNA and protein expression were up-regulated in TC and CC-genotypic rats after 30 m W/cm₂ microwave exposure. 3. Associations of 5-HTR gene polymorphisms with human susceptibility to electromagnetic radiation.（1） Cardiac and brain functions were related to radiation dosage. Higher incidence of abnormal ECG happened in operating group and patrolling group than control group. The latency of N2 wave of workers in patrolling group was prolonged than those in operating group and control group. BNP content of workers in operating group was increased compared to those in control group. LENK content of workers in patrolling group was increased than those in operating group.（2） Latencies of N2 and P3 waves were prolonged with the increase of radiation dosage in workers with rs6311 TT genotype. A difference existed between TT-genotypic workers and CC-genotypic workers. The latency of N2 wave and LENK content were increased with the augment of radiation dosage in workers with rs6295 GG genotype. Workers who carried rs6313 TT or rs6295 GG were more sensitive to electromagnetic radiation. Conclusions:（1） Chronic exposure to microwave（2.856 GHz, with the average power density of 5, 10, 20 and 30 m W/cm₂） could induce deficit of cognitive functions in rats and 5-HT system was involved in this process.（2） Transcriptional activity of 5-HT1 AR promoter including-215 C allele was higher than that including T allele. C allele was more susceptible to 30 m W/cm₂ microwave with a stronger transcriptional activation.（3） Electromagnetic radiation from working in power plant induced the decrease of workers’ cardiac and brain functions.（4） 5-HT1 AR SNP rs6295 and 5-HT2 AR SNP rs6313 could be biomarkers being potentially used in screening workers who would be occupationally exposed to electromagnetic radiation.