Effect of Power-Frequency Magnetic Fields on Genome-Scale Gene expression on s. cerevisiae

Effect of Power-Frequency Magnetic Fields on Genome-Scale Gene expression on s....

(Parte 1 de 5)

RADIATION RESEARCH 160, 25–37 (2003) 03-7587/03 $5.0 q 2003 by Radiation Research Society. All rights of reproduction in any form reserved.

Effect of Power-Frequency Magnetic Fields on Genome-Scale Gene Expression in Saccharomyces cerevisiae

Satoshi Nakasono,a,b,1 Craig Laramee,b Hiroshi Saikia and Kenneth J. McLeodb a Bio-Science Department, Abiko Research Laboratory, Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko-City, Chiba 270- 1194, Japan; and b Program in Biomedical Engineering, State University of New York at Stony Brook, Stony Brook, New York 11794

Nakasono, S., Laramee, C., Saiki, H. and McLeod, K. J.

Effect of Power-Frequency Magnetic Fields on Genome-Scale Gene Expression in Saccharomyces cerevisiae. Radiat. Res. 160, 25–37 (2003).

To estimate the effect of 50 Hz magnetic-field exposure on genome-wide gene expression, the yeast Saccharomyces cerevisiae was used as a model for eukaryotes. 2D PAGE (about 1,0 spots) for protein and cDNA microarray (about 5,900 genes) analysis for mRNA were performed. The cells were exposed to 50 Hz vertical magnetic fields at 10, 150 or 300 mT r.m.s. for 24 h. As positive controls, the cells were exposed to aerobic conditions, heat (408C) or minimal medium. The 2D PAGE and microarray analyses for the positive controls showed high-confidence differential expression of many genes including those for known or unknown proteins and mRNAs. For magnetic-field exposure, no high-confidence changes in expression were observed for proteins or genes that were related to heat-shock response, DNA repair, respiration, protein synthesis and the cell cycle. Principal component analysis showed no statistically significant difference in principal components, with only insignificant differences between the magnetic-field intensities studied. In contrast, the principal components for the positive controls were significantly different. The results indicate that a 50 Hz magnetic field below 300 mT did not act as a general stress factor like heat shock or DNA damage, as had been reported previously by others. This study failed to find a plausible differential gene expression that would point to a possible mechanism of an effect of magnetic fields. The findings provide no evidence that the magnetic-field exposure alters the fundamental mechanism of translation and transcription in eukaryotic cells. q 2003 by Radiation Research Society

Public concern regarding the health effects of power-frequency electric and magnetic fields (EMFs) has been increasing since an epidemiological study in 1979 reported

1 Address for correspondence: Bio-Science Department, Abiko Research Laboratory, Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko-City, Chiba 270-1194, Japan; e-mail: nakasono@ criepi.denken.or.jp.

an increased cancer risk in children living near power lines (1). Since then, many studies have been conducted in an effort to explain the findings, and some recent research suggests that weak power-frequency fields (50/60 Hz) may exert biological effects. The NIEHS working group report (2) of the EMF Research and Public Information Dissemination Program stated that extremely low-frequency (ELF) EMFs may be carcinogenic to humans. However, a mechanism for the carcinogenic or biological effects of EMFs has not been forthcoming, and most laboratory results are in disagreement with this conclusion (2–6).

Several investigators have reported observations of an effect of weak magnetic fields (below 1 mT) on gene expression in bacteria (7) and on heat-shock proteins (HSPs) in yeast (8) and mammalian cells (9, 10). Additional reports suggest that the pattern of synthesis is similar to the response to general environmental stress (1). This type of response with induction of HSPs is widely reported in bacteria, yeast and human cells. In contrast, other reports indicate no such response in bacteria (12) and human cell lines (13). Some reports suggest that these discrepancies could arise from differences in exposure systems, differences in cell karyotypes between the research groups (2, 5), or a misunderstanding of experimental noise (12).

McCann and her colleagues reviewed the literature on the genotoxic potential of EMFs in 1993 and again in 1998 (3, 4). Most of the reports reviewed showed that ELF EMFs ranging from 0.15–5 mT were not genotoxic. Nevertheless, some reports were positive. An exposure to a 50 Hz, 400 mT magnetic field increased the mutation frequency in MeWo cells (14), and a 60 Hz, 0.1–0.5 mT magnetic field inhibited DNA repair in rat brain cells (15, 16) in a reaction that could be blocked by free radical scavengers, suggesting involvement of oxygen radicals.

The general stress response (elevated temperature, toxins, heavy metals, free radicals, etc.) is found in all bacteria, plant and animal cells, and it is remarkably conserved throughout evolution. The mechanism of the stress response has been widely investigated in the yeast Saccharomyces cerevisiae, whose genome has recently been fully sequenced and which provides a useful model system for studies of eukaryotes. Recently databases for yeast have

26 NAKASONO ET AL.

FIG. 1. Comparison of 2D image of yeast total protein between control and positive controls. 7: increased level of protein synthesis compared to control gel. N: decreased level of protein synthesis. The change in synthesis levels was estimated by visual observation. Protein; 1002125 mg. Control conditions: for 24 h at 258C, YPD medium. Aerobic conditions: for 12 h at 258C with 1 liter air/min, YPD medium. Heat shock: at 508C for 1 h, YPD medium. Minimal medium: for 24 h at 258C, minimal medium. Three independent experiments (control and exposure) were done for each condition. The difference in the protein profiles was estimated visually in the same experimental set. The gel images shown are typical gels for each condition.

27POWER-FREQUENCY MAGNETIC FIELDS AND GENE EXPRESSION IN YEAST

FIG. 2. Comparison of 2D images of yeast total protein in control and magnetic-field-exposed cells. The changes in synthesis levels were observed visually. Protein: 100–125 mg. Control conditions: for 24 h at 258C, YPD medium. Magnetic-field exposure: 50 Hz, 10 mT, sinusoidal, vertical, for 24ha t2 58C, YPD medium.

been constructed: The SWISS-2DPAGE database2 for protein and the YPD (17), MIPS3 and Stanford Genomic Resources4 databases for mRNA.

Genome-scale screening methods such as 2D PAGE and cDNA microarray analyses are powerful methods for identifying global responses to various environmental perturbations, such as heat shock (18), different carbon sources (19, 20), UV radiation (21), mutagens (2), osmotic shock, radicals (20) and EMFs (18). Protein 2D PAGE provides a protein profile that is related to the cell and reflects differences in protein function. This method can identify a large number of proteins (about one-sixth of the total proteins in yeast). cDNA microarray provides an mRNA synthesis profile that reflects the cellular response to environmental changes rather than the cell type. Thus functional analysis is easier, since all of the genes on the array have already been characterized. Thus using both 2D PAGE and cDNA microarrays should be one of the most powerful approaches for identifying both the effect(s) and mechanism(s) of environmental perturbation.

If the magnetic-field exposure causes differential gene expression that is related to an unknown or known process in eukaryotic cells, such as the heat-shock response, DNA repair, respiration, protein synthesis and the cell cycle, a response in yeast also should be found after exposure to a

2 SWISS-2DPAGE (http://expasy.hcuge.ch/ch2d/ch2d-top.html). 3 Munich Information Centre for Protein Sequences (MIPS, http:// mips.gsf.de/proj/yeast/). 4 Stanford Genomic Resources (http://genome-w.stanford.edu/).

high-intensity magnetic field using genome-scale gene expression analysis. The aim of this study is to clarify the existence of an effect of high-intensity (up to 300 mT) 50 Hz magnetic fields on genome-wide gene expression in yeast by analyses using 2D PAGE and cDNA microarrays (containing over 95% of the total expected genes). The magnetic-field intensities in this study are at least several hundred times higher than the intensities found in homes or intensities in occupational settings, and these are at least tens of thousands of times higher than the intensity that caused the health effects reported in some epidemiological studies (1), but they will provide a reference point for further studies.

Yeast Strain, Culture Vessels and Growth Conditions

The yeast S. cerevisiae (IAM 4206) was obtained from the IAM Culture Collection (The University of Tokyo, Japan). The cells were precultured at 25.0 6 0.28C at 160 rpm for 12 h in 5 ml of YPD medium. The medium was made from 10 g of yeast extract, 20 g of peptone, and 20 g of dextrose in 1 liter of deionized water. For 10 mT magnetic-fieldexposed cultures and positive controls (aerobic, heat shock, minimal medium), 50 ml of the preculture was inoculated into 300 ml of medium (final cell density about 104 cells/ml) in an annular-shaped culture vessel (external diameter 20 cm, internal diameter 15 cm) (12). For 150 mT and 300 mT magnetic fields, 17 ml of preculture was inoculated into 100 ml of YPD medium (final cell density about 104 cells/ml) in an annularshaped culture vessel (external diameter 9.5 cm, internal diameter 6.0 cm). The control cells in each experiment were cultured at the same time in an identical culture vessel in the sham-exposure system. The conditions

28 NAKASONO ET AL.

TABLE 1 Comparison of Changes in Expression between Proteins and mRNAs

ORF Gene name

Protein name on

2D gel Process Function Aerobic

Protein mRNA ratio CL

YJR121W YPL106C YFL039C YAL005C

YLL024C

ATP2 SSE1 ACT1 SSA1

SSA2

ATPB HS78 ACT HS71

HS72

ATP synthesis calmodulin signaling cytoskeleton ER and mitochondrial translocation

ER and mitochondrial translocation

F1F0-ATPase complex, F1 beta subunit HSP70 family actin cytosolic HSP70 cytosolic HSP70

YPR035W YBR221C YDR050C YFR053C YGL253W

GLN1 PDB1 TPI1 HXK1 HXK2 glutamine biosynthesis glycolysis glycolysis glycolysis glycolysis glutamine synthetase pyruvate dehydrogenase triosephosphate isomerase hexokinase I hexokinase I

— YGR192C YHR174W YJL052W YKL152C YLR044C

TDH3 ENO2 TDH1 GPM1 PDC1

G3P3 ENO2 G3P1 PMG1 DCP1 glycolysis glycolysis glycolysis glycolysis glycolysis glyceraldehyde-3-phosphate dehydrogenase 3 enolase I glyceraldehyde-3-phosphate dehydrogenase 1 phosphoglycerate mutase pyruvate decarboxylase

— A (UP) YLL026W

YLR355C

YCL018W YIL051C

YDR226W

HSP104 ILV5

LEU2 MMD1

ADK1

(Parte 1 de 5)

Comentários