Impact of Static Magnetic Field Exposure on the Development and Transcriptome of Medaka Fish Embryos

The impact of static magnetic field (SMF) exposure on the medaka fish has been investigated recently elucidating the interaction mechanisms of SMF exposure to human cells. A recent study of the in-vivo medaka fish embryo experiment has suggested that there was no obvious impact on the developmental progress of the embryos growth rate under prolonged SMF exposure; however, an increase in percentage of abnormal fish embryos was observed. This paper focuses on transcriptomic analysis of SMFaffected medaka fish embryos at various stages of development following prolonged SMF exposure. This paper verified that under prolonged exposure of SMF there was no significant impact on the embryo growth rate based on the examination of a total of 150 fish samples. However, there was an observable difference in the number of abnormal embryos between the treated and control groups at developmental stage 22 to stage 39 of the embryos. Transcriptomic analysis of biological processes by RNA sequencing were hence carried out; the outcomes of the functional annotation of the KEGG pathways revealed differential expression of biological processes related to ribosomal diseases, and to digestion and absorption of fat in the treated groups when compared to the control groups with p-values of less than 0.05. Received: October 23, 2020


Introduction
There are unintentional static magnetic fields (SMFs) with magnitudes hundreds of times larger than that of the earth's magnetic field existing in our recent environments; and is causing potentially serious public health and safety concerns. Electrified railway or transportation systems, e-vehicles and several major technologies involving DC power energy conversion are considered as common unintentional man-made sources that expose humans to prolonged SMF exposures. For example, some models of hybrid electric vehicles are reporting a 1mT SMF intensity [1]; while US and European transportation systems have recorded SMF levels of 2 mT in the passenger cabins of trains [2][3][4]. An average of 10 mT was measured in electrolytic processing plants, a maximum to 50 mT has also been reported at some accessible locations in superconducting systems for DC power energy conversion technology, and up to 100 mT has been measured in aluminum production plants [5]. Long term SMF exposures have raised public health concerns in recent decades; ICNIRP [6] has been investigating the potential ill effects of SMF exposure by using cell or animal models to assess the possible health risk. There are so far insufficient in-vitro studies of human cells to determine any health effects caused by electromagnetic radiation. In the absence of any conclusive evidence from in-vivo studies, there has been insufficient corroborating evidence linking SMF exposure human health risks.
It is reported that SMF will affect some endpoints of in-vitro cellular changes at a low intensity magnetic field [7] at mT range.
When rat lymphocytes were exposed to a SMF intensity of 7mT in DOI: 10.26717/BJSTR.2020.31.005158 24485 the presence of ferrous chloride, the number of damaged cells was significantly increased [8]. When Reina et al. [9] exposed cellular membrane seeds to magnetic fields of 0 to 10 mT, it was observed that there were changes in the magnitude of the current density of ions across the cell membrane and in the ionic concentration with an increased dosage of the field intensity. Hirai [10] investigated the gene expression in hippocampal neurons of rats; a brief exposure of only 15 minutes SMF of 100 mT would lead to a transient potent increase of DNA immaturity. Amara [11] investigated the effects of SMF to 128 mT with 1 hour per day for a consecutive 30 days exposure together with anti-oxidative enzymes activity in a male rat brain; the study indicated that the exposure to SMF will induce oxidative stress in the rat's hippocampus and frontal cortex. Sun [12] has adopted medaka fish to investigate the embryo development as a pilot in-vivo study with no observable impact on the embryo development rate under prolonged SMF exposure.
However, unusual abnormal growth was observed in some embryo samples of the treated groups. It is the aim of this paper to carry out a second-round experiment on a larger sample size for further evidential data on the observation of the edema growth together with transcriptome analysis by RNA sequencing.

Methodology
The experiment was carried out with a sample size of 150 embryos, with 75 embryos of the treated groups and with the same number of 75 embryos of the control groups. 15 embryos were placed in each petri dish for the experiment. SMF exposure for the treated groups was adopted to a full hatching period of 19 days. The SMF was setup by two paralleled NdFeB magnets, the petri dishes of the treatment group were placed between the magnets, and the 15 embryos in each dish were placed in the middle of the dish as the exposure region with water droplets extruded from a dropper.
The magnetic flux density within the embryo exposure region was between 80-100 mT measured by a GM05 Gaussmeter. Details of the experimental set up is according to the pilot study as previously reported [12]. The setup is illustrated in (Figures 1-3).   (Tables 1 & 2), respectively. Abnormal edema in the fish embryo is also illustrated in Figure 3, with the normal embryo appearance, that is without edema in the experiment, as a comparison -the forehead and the atrium of the heart should have had a clear outline in the yolk of the egg after stage 23 for the normal embryos with a relatively small space around the heart; blurred atrium of the heart and cuvierian duct were considered as edema growth in our experiment. The appearance of the unusual edema growth of the embryo as compared to the normal embryo is illustrated in Figure 3.
2. Severe pericardial edema Same as above.
3. Severe blood clot Severe blood clot in the left ducts of Cuvier 4. Small blood clots Small blood clots inside the developing brain (possibly in the primordial midbrain channel)

Over-pigment
Over-pigmented in the cranial region. The left eye is darkened.

deformed head (tapered) & eye cup
The cranial roof round the forebrain appears collapsed.
Final hatched abnormal Abnormal fish that is unable to swim normally with tingled tail.    (Tables 3 & 4); the protein binding and transporter activity were identified to be the main significant molecular functions. The KEGG pathway was also analyzed to identify any genomic information to the gene function; the outcomes of the KEGG pathway are shown in (Table 5). Genes related to functions of ribosome, Huntington's disease, pyrimidine metabolism, and the RNA polymerase from the treated groups were identified to be significantly different as compared to the controlled groups after a 5 day exposure; genes related to function of fat digestion and absorption for the 7 days exposure.

Conclusion
This paper presents an in-vivo medaka fish embryo development experiment under static magnetic field exposure with additional transcriptome analysis. It was concluded that there is no impact of SMF to the embryo growth rate, however abnormal embryo growth was observed with further functional annotation and KEGG pathway analysis. This study provides evidential data of an in-vivo experiment to supplement and clarify the ambiguity of the SMF impacts on human exposure by inference from the outcomes of the medaka fish model, for the possible health risk assessment for a human model.