Magnetic field past research

In the past research, numerous epidemiological (population) studies and comprehensive reviews have evaluated magnetic field exposure and risk of cancer in people. Epidemiologic and experimental research on the potential carcinogenic effects of extremely low frequency electromagnetic fields (EMF) has now been conducted for over two decades. Cancer epidemiology studies in relation to EMF have focused primarily on brain cancer and leukemia, both from residential sources of exposure in children and adults and from occupational exposure in adult men [1]. No consistent relationship has been seen in studies of children brain tumors or cancers at other sites and residential ELF electric and magnetic fields [2].

However, these studies have generally been smaller and of lower quality Four long-term bioassays have been published in which the potential oncogenicity in experimental animals of exposure to ELF magnetic fields was evaluated in over 40 different tissues using standard chronic toxicity testing designs [1, 2]. Since the two most common cancers in children are leukemia and

A study in 1979 pointed to a possible association between living near electric power lines and childhood leukemia .The finding was strongest for childrenwho had spent their entire lives at the same address, and itappeared to be dose-related. It did not seem to be an artifactof neighborhood, street congestion, social class, or familystructure. The reason for the correlation is uncertain; possibleeffects of current in the water pipes or of AC magnetic fieldsare suggested. [3].

1.1.2 Magnetic field currently research

Among more recent studies, findings have been mixed. Some have found an association; others have not. These studies are discussed in the following paragraphs. Currently, researchers conclude that there is limited evidence that magnetic fields cause childhood leukemia, and that there is inadequate evidence that these magnetic fields cause other cancers in children [2]. Researchers have not found a consistent relationship between magnetic fields or appliances and childhood brain tumors.

1.2 Magnetic field influence with inadequate evidence

Is it harmful not to have direct evidence at present to children？ However , before research let me know the magnetic field has suspicion to the danger of the health. For adults, a Norwegian study found a risk for exposure to magnetic fields in the home [4], and a study in African-American women found that use of electric bedding devices may increase breast cancer risk [5].Some occupational studies showed very small increases in risk for leukemia and brain cancer, but these results were based on job titles and not actual measurements. More recently conducted studies that have included both job titles and individual exposure measurements have no consistent finding of an increasing risk of leukemia, brain tumors, or female breast cancer with increasing exposure to magnetic fields at work .Cases of breast cancer diagnosed during 1980–1996were identified in a cohort of women living near a high-voltage power line in Norway in 1980 or between 1986 and 1996. Women with the highest occupational exposure had an odds ratio of 1.13 (95% CI: 0.91, 1.40) when compared with those unexposed at work [4].

The findings suggest an association between exposure to magnetic fields and breast cancer in women. A total number of 608 cases and 667 controls participated. Adjusting for accepted breast cancer risk factors, we found an OR of 1.13 for lifetime occupational exposure to ELF-MF at medium or high intensities [6]. Risks were larger for exposures before age 35 (OR¼1.40), and statistically significant for exposures before 35 among cases with progesterone receptor positive tumors (OR¼1.56, 95% CI¼1.02–2.39).In a word, there appears to be a small increased risk for breast cancer among postmenopausal women exposed occupationally to ELF-MF. [7, 8]

1.2.1 Magnetic field influence in animal

Speaking of the influence that the magnetic field endangers to the health, does the magnetic field to other living thing work? In order to distinguish this, scientists learned from animal experiments about the relationship between magnetic field exposure and cancer. Studies to evaluate immune function and host resistance in animals have given negative effects for exposure to ELF electric and magnetic fields (2). In-vitro exposure of immune system cells generally did not because changes in proliferation capacity .The few animal studies on cancer-related non-genetic effects are inconclusive. Results on the effects on invitro cell proliferation and malignant transformation are inconsistent, but some studies suggest that ELF magnetic fields affect cell proliferation and modify cellular responses to other factors such as melatonin. An increase in apoptosis following exposure of various cell lines to ELF electric and magnetic fields has been reported in several studies with different exposure conditions. Numerous studies have investigated effects of ELF magnetic fields on cellular end-points associated with signal transduction, but the results are not consistent (2). The absence of animal data supporting carcinogenicity makes it biologically less likely that magnetic field exposures in humans, at home or at work, are linked to increased cancer risk [17].

1.3 Other research in magnetic field

Impact on nervous system: Some research indicate low magnetic field make the active increase of the animal often, excitability increases; And stronger magnetic field often makes the activity of the organism reduce, excitability is reduced, suppress reacting. [14]

In addition, the magnetic field also has certain function on the plant nerve, to the heartbeat, blood pressure; breathe certain influence .Impact on cardiovascular system: The magnetic field does not have obvious function on the heart under the normal condition, but to the heart under the pathology state, play certain treatment to act on. Impact on immunity[15]., internal system: Because of the difference in method, means, condition of studying,etc., have disputes to the function on the immune system of the magnetic field at present. But most researchers think, the magnetic field can improve the immune function of the organism. The magnetic field still has a secretion activity of regulating endocrine glands; strengthen the secretion of the active material of immunity.

1.4 Caenorhabditis elegans (C.elegans)

Caenorhabditis elegans(Fig 1) is a nematode - a member of the phylum Nematoda: “Nematoda.

The roundworms and threadworms, a phylum of smooth-skinned, unsegmented worms with a long cylindrical body shape tapered at the ends; includes free-living and parasitic forms both aquatic and terrestrial” It is small, growing to about 1 mm in length, and lives in the soil - especially rotting vegetation - in many parts of the world, where it survives by feeding on microbes such as bacteria. It is of no economic importance to man. (Academic press Dictionary of Science and Technology) Around the world many hundreds of scientists are working full time investigating the biology of C. elegans. Between October, 1994 and January, 1995\ 73 scientific articles about this creature appeared in international science journals. Currently an international consortium of laboratories are collaborating on a project to sequence the entire 100,000,000 bases of DNA of the C. elegans genome.

C. elegans is about as primitive an organism that exists which nonetheless shares many of the essential biological characteristics that are central problems of human biology. The worm is conceived as a single cell which undergoes a complex process of development, starting with embryonic cleavage, proceeding through morphogenesis and growth to the adult... All 959 somatic cells of its transparent body are visible with a microscope, and its average life span is a mere 2-3 weeks. Thus C. elegans provides the researcher with the ideal compromise between complexity and tractability.

Fig 1 C.elegans anatomy

1.4.1Caenorhabditis elegans V.S. magnetic field

Change of the magnetic field, the biological behavior of the Caenorhabditis elegans is influenced. For one thing, high magnetic fields on biological process, by which alternating current magnetic stimuli as high as 1.7 T can be administered. Available evidence indicates that alternating high magnetic fields can elicit both chronic and acute biological effects but that the effects may be well tolerated or compensated for by the living organism [9]. Transgenic nematodes (Caenorhabditis elegans strain PC72), carrying a stressinducible reporter gene (escherichia coli betagalactosidase) under the control of a C. elegans hsp16 heatshock promoter, have been used to monitor toxicant responses both in water and soil[10]. For another, effect of extremely low frequency electromagnetic fields (ELFEMF) in the presence of a second stressor (mild heat shock) on the expression of a lacZ reporter gene under the control of hsp16 or hsp70 promoters in two transgenic strains of C. elegans.

[11].Exposure of Caenorhabditis elegans to extremely magnetic fields induces stress responses.

Expression of the hsp16lacZ gene was enhanced when transgenic animals were exposed to magnetic fields up to 0.5 T at 60 Hz. The hsp promoter was more efficiently expressed at the embryonic than at the postembryonic stage irrespective of exposure.Caenorhabditis elegans, revealed that intermittent exposure to the magnetic fields modestly inhibited the animal's reproduction as well as its post-embryonic development. [12]

1.4.2 C.elegans －a kind of very good research source material

C. elegans is a free-living nematode. There are two sexes: a self-fertilizing hermaphrodite and a male. The adult essentially comprises a tube, the exterior cuticle, containing two smaller tubes, the pharynx and gut, and the reproductive system. Most of the volume of the animal is taken up by the reproductive system. Of the 959 somatic cells of the hermaphrodite some 300 are neurons. Neural structures include a battery of sense organs in the head which mediate responses to taste, smell, temperature and touch - and although C.

elegans has no eyes, it might respond slightly to light. Among other neural structures is an anterior nerve ring with a ventral nerve cord running back down the body. (There is also a smaller dorsal nerve cord.) There are 81 muscle cells. C. elegans moves by means of four longitudinal bands of muscle paired sub-dorsally and sub-ventrally. Alternative flexing and relaxation generates dorsal-ventral waves along the body, propelling the animal along. The development and function of this diploid organism is encoded by an estimated 17,800 distinct genes.

http://www.wormbase.org/

http://www.cbs.umn.edu/CGC/

http://www.celeganskoconsortium.omrf.org http://www.wormatlas.org/handbook/contents.htm

Biological effect of the magnetic field: The magnetic field is influential to the organism, include the following aspects mainly:

1.5 Nanoparticles

In our experiment, we use Fe3O4 (6-10nm), Fe2O3 (30nm) and TiO2 (10-40nm).

1.5.1 Fe3O4 is nature magnet

It likes nature little magnet in the body. Most materials found in organisms are generally thought of as being nonmagnetic- for example, C.elegans[13],either diamagnetic (repelled weakly from a magnetic field, as is water and almost any fatty substance) or paramagnetic (weakly attracted to a magnetic field, as is deoxyhemoglobin in blood cells [21].

1.5.2 TiO2, Fe2O3 and Fe3O4 toxicity

Fe3O4, Fe2O3, and TiO2 had no measurable effect on the cells toxicity. Toxicity of iron oxides and metabolites of benzo apyrene alone or in combination in cells culture and identification by laser microprobe mass spectrometry.[22] These results suggest that Fe2O3 and Fe3O4 alone are not very toxic but the association of one of these compounds with BaP increases the toxicity of the latter. On the other hand, TOF-LMMS seems to show a metabolization of iron oxide into reduced form. But, it is necessary to raise the ambiguity about the iron which is always in the cells. \TiO2 had no measurable effect on the cells until the concentrations reached greater than 200 microg/mL [23].

LDH leakage significantly increased in the cells exposed to ZnO (50 to 100 microg/mL), while other nanoparticles tested displayed LDH leakage only at higher doses (>200 microg/mL).

1.6 Magnetic field and Gene expression

Exposure to magnetic field (5 mT at 60 Hz) does not affect cell growth and c-myc gene expression designed and manufactured equipments for long-term and low-density (0 to 9 mT) exposures of cultured cells to extremely low frequency magnetic fields (ELF-MF), and examined the effects of ELF-MF on cell growth and c-myc mRNA expression in Chinese hamster ovary (CHO) cells [16].

The wide-spread induction of stress-related genes and transcription factors, and a depression of genes associated with cell wall metabolism, are prominent examples. Magnetic Field-Responsive Sequences in hsp70. [18] HSP70 gene expression is induced by a wide range of environmental stimuli, including 60-Hz electromagnetic fields. In an earlier report we showed that the induction of HSP70 gene expression by magnetic fields is effected at the level of transcription and is mediated through c-myc protein binding at two nCTCTn sequences at 2230 and 2160. In the human HSP70 promoter. report on the identification of a third c-myc binding site (between 2158 and 2162) that is an important regulator of magnetic field-induced HSP70 expression.We also show that the heat shock element (HSE), lying between 2180 and 2203, is required for induction of HSP70 gene expression by magnetic fields[20]. High magnetic field induced changes of gene expression in

Arabidopsis[19) Field strengths in excess of about 15 Tesla induce expression of the Adh/GUS transgene in the roots and leaves. From the microarray analyses that surveyed 8000 genes, 114 genes were differentially expressed to a degree greater than 2.5 fold over the control. These results werequantitatively corroborated by qRT-PCR examination of 4 of the 114 genes [19].

1.7 Apoptosis and gene

Apoptosis is mediated by a family of cysteine-aspases (caspases), which are expressed as inactive zymogens and are prototypically processed to an active state following an apoptotic stimulus. Many important human diseases are caused by abnormalities in the control of cellular apoptosis (programmed cell death). These abnormalities can result in either a pathological increase in cell number (e.g. cancer) or a damaging loss of cells (e.g. degenerative diseases).

1.7.1 Human apoptosis related gene and apoptosis pathway

The intrinsic pathway (Fig 2) requires disruption of the mitochondrial membrane and the release of mitochondrial proteins including Smac/DIABLO, HtRA2, and cytochrome c. Cytochrome c functions with Apaf-1 to induce activation of caspase-9, thereby initiating the apoptotic caspase cascade, while Smac/DIABLO and HtrA2 bind to and antagonize IAPs. Mitochondrial membrane permeabilization is regulated by the opposing actions of pro- and antiapoptotic Bcl-2 family members. Multidomain proapoptotic Bcl-2 proteins (e.g., Bak and Bax) can be activated directly following interaction with the BH3-only Bcl-2 protein Bid. Alternatively, binding of other BH3-only proteins (e.g., Noxa, Puma, Bad, and Bim) to antiapoptotic Bcl-2 proteins (e.g., Bcl-2 and Bcl-XL) results in activation of Bax and Bak. The regulated release of proapoptotic factors from the mitochondria cause induction of downstream caspases, and potential loss of mitochondrial function [28].

Fig 2.Apoptosis pathway

Cells have a discrete cell death pathway defined by a specific set of genes. These genes encode proteins that form the biochemical cascade that ultimately leads to cell death (Fig. 2). The key genes that control the cell death process are the cell death effectors of the CED-3/ICE ("caspase") family and the cell death inhibitors of the BCL2 family [29].

We probe into CED-3 and CED-6 gene expression in apoptosis pathway. In our experiment,

We suppose the magnetic field induction to apoptosis.

1.7.2 C.elegans apoptosis and cancer related gene in wormbase

Form wormbase website (Fig 3), we can find thousands of kinds of genes about the C.elegans.

Among which are about apoptosis, cancer and nerve, etc.

How could we use wormbase? Wormbase is database of an introduction C.elegans, include data of nematode genome inside, main function

(1) sequencing, genome of C.elegans has already been solved out, can find the materials of the array on this website.

(2) Mapping can find out the gene location is in C. elegans which of one chromosome at the position (3) Phenotypic information has database which lists RNAi to C. elegans

If you want to experiment as RNAi of model organism, you can consult this website.I find some genes, as apoptosis related gene, unusual function with magnetic field in C. elegans

Fig 3. Wormbase website

Chapter 2

Research experiment and design 2.1 C.elegans culture

The nematode Caenorhabditis elegans has proven to be an important model organism for.

aging-related research, providing a well characterized paradigm for NGM + 50 μM FUDR (DR-FD media) culture at 20^oC

2.2 Magnetic field design

Fig 4 . Magnetic field for experiment

What we work is research that under the function of the magnetic field C.elegans biological behavior and physiological mechanism enable denying changing?

Yes .Change in our experiment.

We have designed a series of experiments and inferred for this the gene expression.

First, we buy to the strongest and steady magnet NdFeB N38 (Fig 5) on the market.

Fig 5. Manufacturer's information

Force the nematode to grow under stabilizing and covering on the function of the magnetic field with my design of the experiment. We use 6cm plate and cover and invite 3mm agar. Because the magnetic field is inversely proportional to the square of distance strongly Make use of this best magnetic field which we can obtain of method (Fig 4) .Our magnetic field that quantity examine is 0.5T on.

2.3 Video record system

We combines against Sony video cassette recorder three-dimensionally and microscope notebook computer (Fig 6). This kind of method lowers costs, and the result is very good too. In me experiment, I 8design many device that not been used before this.

Fig 6.Video record system

2.4 Experiment flow chart

Fig 7. Experiment flow chart

Magnetic design

Behavior different biochemical action

protein

2.4.1Flow chart of gene analysis

Fig 8.Flow chart of gene analysis chart

2.5 SDS-PAGE GEL

SDS-PAGE、officially sodium dodecyl sulfate polyacrylamide gel electrophoresis、is a technique used in biochemistry、genetics and molecular biology to separate proteins according to their electrophoresis mobility (a function of length of polypeptide chain or molecular weight as well as higher order protein folding、posttranslational modifications and other factors).

Fig 9.SDS-PAGE device ＆ sample

2.5.1 SDS-PAGE protocol Materials

30% acrylamide 10% SDS

10% APS (make fresh each time) TEMED

0.006% (w/v) Coomassie Blue dye 90% ddH2O

Isopropanol Fixing Solution 10% (v/v) acetic acid 25% (v/v) isopropanol 65% ddH2O

SDS sample loading buffer (40 ml) ddH2O 16 ml

0.5 M Tris, pH 6.8 5 ml 50% Glycerol 8 ml 10% SDS 8 ml

2- mercaptoethanol 2 ml (add immediately before use)　 bromophenol blue

10% (v/v) acetic acid Protocol

1. Prepare polyacrylamide gel according to standard protocol.

2. Load samples and run gel @ 25 mA (2 gels run @ 50 mA) in 1x SDS Running Buffer.

3. At this point, the gel can either be transferred to a membrane (see Western protocol) or stained with Coomassie (see below).

4. Place gel in a plastic container. Cover with isopropanol fixing solution and shake at room temperature. For 0.75 mm-thick gels, shake 10 to 15 min; for 1.5 mmthick gels, shake 30 to 60 min.

5. Pour off fixing solution. Cover with Coomassie blue staining solution and shake at RT for 2 hr.

6. Pour off staining solution. Wash gel with 10% acetic acid to destain, shaking at RT ON.

2.6 Primer design

The gene of interest usually has to be amplified from genomic or vector DNA by PCR (polymerase chain reaction) before it can be cloned into an expression vector. The first step is the design of the necessary primers.

Important features are:

Primer sequence. Especially the 3'-end of the primer molecule is critical for the specificity and sensitivity of PCR. It is recommemded not to have:

Primer pairs should be checked for complementarity at the 3'-end. This often leads to primer-dimer formation . Bases at the 5'-end of the primer are less critical for primer annealing. Therefore, it is possible to add sequence elements, like restriction sites, to the 5'-end of the primer molecule.

We use a primer length of 18-23 bases is optimal for our PCR applications Find out the gene that we are interested in from wormbase . Make using of knowning gene sequence to design the primers.

..

2.6.1 Gene primer designed

We look for the suitable gene correlated with our design environment as much as possible

With the behavior and observation of C.elegans, we roughly sum up into the gene that four groups are correlated with and form a cluster.Among them it is UNC family correlated with the behavior ,

With the behavior and observation of C.elegans, we roughly sum up into the gene that four groups are correlated with and form a cluster.Among them it is UNC family correlated with the behavior ,