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A literal mountain of documentation generated in the past five decades showing unmistakable health hazards associated with extremely low-frequency electromagnetic fields (ELF-EMFs) exposure. However, the relation between energy mechanism and ELF-EMF exposure is poorly understood. In this study, Caenorhabditis elegans was exposed to 50 Hz ELF-EMF at intensities of 0.5, 1, 2, and 3 mT, respectively. Their metabolite variations were analyzed by GC-TOF/MS-based metabolomics. Although minimal metabolic variations and no regular pattern were observed, the contents of energy metabolism-related metabolites such as pyruvic acid, fumaric acid, and L-malic acid were elevated in all the treatments. The expressions of nineteen related genes that encode glycolytic enzymes were analyzed by using quantitative real-time PCR. Only genes encoding GAPDH were significantly upregulated (P < 0.01), and this result was further confirmed by western blot analysis. The enzyme activity of GAPDH was increased (P < 0.01), whereas the total intracellular ATP level was decreased. While no significant difference in lifespan, hatching rate and reproduction, worms exposed to ELF-EMF exhibited less food consumption compared with that of the control (P < 0.01). In conclusion, C. elegans exposed to ELF-EMF have enhanced energy metabolism and restricted dietary, which might contribute to the resistance against exogenous ELF-EMF stress.

The free-living nematode Caenorhabditis elegans has been used as a model organism to study the influences of environment conditions on human health 9 . Thus, we selected C. elegans as a model organism in this study. Previous studies proposed that ELF-EMF exposure affects the reproduction and gene expression of C. elegans 5 , 10 , 11 , 12 . However, the effects of ELF-EMF at the metabolic level remain unclear to date. The combination of C. elegans and metabolomics is a functional genomics tool that can be used to test the molecular effects of pollution/toxicant exposure 13 , metabolic pathways 14 , 15 , 16 , chemical ecology 17 , and biological variation 18 . In the present study, the effects of ELF-EMF exposure on the metabolites of C. elegans were investigated using GC-TOF/MS. Subsequently, food consumption, gene expression, and metabolite concentration in C. elegans were analyzed to investigate the relations between ELF-EMF exposure and energy metabolism.

Since the first publication of a possible link between ELF-EMF and childhood cancer 2 , numerous studies have investigated the biological effects of ELF-EMFs on humans, and most of these studies found potential harmful effects 3 , 4 . Despite the huge amount of experimental data, the molecular targets of ELF-EMF remain obscure and controversial because of the lack of clear and reproducible effects that can be easily quantified or visualized 5 . Therefore, either ELF-EMF exerts minimal biological effects to trigger major responses in the living body or organisms resist the negative effects of ELF-EMF exposure. Energy metabolism enhancement is a typical adaptive response under hypoxia-induced stress 6 and heavy metal-induced neurotoxicity 7 , 8 . As another environmental factor, ELF-EMF might also influence energy metabolism.

Magnetic fields have important functions in the origin and evolution of life; animals such as homing pigeons and sea turtles utilize magnetic fields to navigate toward a specific location 1 . However, concerns regarding the harmful effects of extremely low-frequency electromagnetic fields (ELF-EMFs) have increased with the rapid urbanization, industrialization, informatization, and the concomitant electromagnetic complexity and interference in the environment.

As an indicator of reproduction, brood size of worms exposed to ELF-EMF in various development stages was measured. No significant differences in the total progeny number were detected among the four exposure groups ( ) in this study. These results suggested that ELF-EMF exposure may not affect the reproduction of C. elegans.

The development of C. elegans from fertilization to hatching is referred to as embryogenesis. Post-embryonic development involves growth through four larval stages (L1 to L4) before the final molt to produce the adult 24 ( ). In the hatching rate analysis, the fertilized eggs in the worm's body were chosen for hatching rate analysis, because they can bear ELF-EMF exposure for a longer time than those that have been laid outside, so as to represent the effects of ELF-EMF exposure more accurately. As shown in , the number of worms hatched from the same number of eggs was nearly equal between C1 and T1. This result indicates that 50 Hz, 3 mT ELF-EMF did not affect the hatching rate of nematode eggs.

In addition, the lifespans of C. elegans exposed to 50 Hz, 3 mT ELF-EMF at the embryogenesis stage (12 h), larval stages (24, 36 and 48 h), and the whole life (WL) span were investigated. No noticeable changes were detected in all exposure groups ( and Table S2 ).

C. elegans can respond to a variety of stressors including alcohols, heavy metals, sulfhydryl-reactive compounds, salicylate, and heat, by ceasing pharyngeal pumping 23 . The effect of stressors can therefore be conveniently assayed by monitoring the decrease in the density of the bacterial food in liquid cultures of nematodes. In this study, food consumption analysis was also performed on worms exposed to 50 Hz, 3 mT ELF-EMF. Results showed that the changes in OD 600 in the exposure groups were less than those of the control groups (P < 0.01). This result indicates that food intake was restricted in the worms under 50 Hz, 3 mT ELF-EMF exposure ( ).

Other than its indispensable role in energy metabolism, GAPDH is also involved in several non-glycolytic processes 19 , 20 , 21 , 22 . In order to check whether the observed increase in GAPDH protein concentration is to complement the energy metabolism or to achieve any other non-glycolytic functions, intracellular GAPDH enzymatic activity and the total cellular ATP level were tested. In our results, the GAPDH enzymatic activity was increased significantly (P < 0.01); however, the total cellular ATP level was lowered approximately 1.5-fold in worms growing under ELF-EMF condition, even though this alteration showed no significance in statistics ( ).

Since the mRNA expression levels of gpd-1 and gpd-4 were increased, the endogenous GAPDH present in L4-larva stage worms was examined by western blot, with actin protein as internal control. Our results revealed that GAPDH protein concentration also increased in worms exposed to ELF-EMF compared with control ( ) (P = 0.041).

Nineteen genes encoding enzymes that regulate glycolysis and gluconeogenesis were selected for analysis to check whether or not ELF-EMF enhances energy metabolic rate. As listed in , only gpd-1 and gpd-4 exhibited significant changes in gene expression in worms under ELF-EMF exposure (P < 0.01).

As listed in , the concentrations of metabolites associated with energy metabolism (pyruvic acid, fumaric, and L-malic acids) and neurotransmission (ethanolamine, phenylethylamine, hydroxylamine, and 5-methoxytryptamine) were all increased in all the exposure groups. Moreover, the contents of some amino acids such as alanine, glycine, proline, and leucine were elevated as well. Among all the investigated metabolites, only D-glyceric acid decreased. Both multivariate statistical analysis and metabolite variation analysis showed no regular pattern with increasing magnetic strengths.

In the metabolomics analysis, six independent pair-wise comparisons were performed to eliminate false positives and negatives, thereby producing robust information on metabolite alteration under ELF-EMF exposure. All data were imported into SIMCA-P+ software (V11.0 Umetrics AB, Umea, Sweden) for processing. As shown in , unsupervised principal component analysis (PCA) revealed no noticeable separation between the exposure and control groups. The unit variance-based partial least squares discriminant analysis (PLS-DA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) as supervised principal component analyses were performed for further analysis. Cross-validation plots for the PLS-DA analyses suggest these models were reliable ( Fig. S1 ). Both PLS-DA and OPLS-DA showed a certain difference between the exposure and control groups ( and ). These results indicate that the homeostasis of C. elegans was disturbed under ELF-EMF exposure, even though the effects were not significant.

Discussion

Metabolites participate in cellular reactions, connecting different pathways that mediate and perform several cell functions; metabolite profiling shows the changes in biological functions or phenotypes in response to genetic or environmental stimuli25,26,27. With this in mind, GC-TOF/MS-based metabolomics was used in conjunction with multivariate statistics to examine the metabolite alteration induced by ELF-EMF exposure. PLS-DA and OPLS-DA analysis showed a clear separation between the groups (with exposure and without exposure), which indicates that ELF-EMF exposure affected C. elegans to a certain extent.

Among the analyzed 596 metabolites, 27 metabolites increased their concentrations in all four exposure groups, while only D-glyceric acid decreased. Their concentration variations showed no regular pattern with increasing magnetic strengths in the four groups. The main reason for these results might be that different magnetic fields have different effects on worms, and induce different response and different self-protection bio-processes in worms, which will probably affect the concentrations of related metabolites in turn28; meanwhile, different tissues might also be sensitive to different magnetic strengths. Nevertheless, the elevated concentrations of ethanolamine29, phenylethylamine30, and 5-methoxytryptamine31 in all of the treatments imply an adaptive response of C. elegans to ELF-EMF exposure, and also indicate that ELF-EMF can induce neurobiological disorder and act as a stressor for C. elegans.

Pyruvic acid, fumaric, and L-malic acids are important intermediates in energy generation; increased concentrations of these intermediates might indicate energy metabolism was enhanced. The enhanced energy metabolism contributes to a typical adaptive response under hypoxia-induced stress6 and heavy mental-induced neurotoxicity7,8. It is an ubiquitous mechanism existed in animals and plants32. Therefore, energy metabolism enhancement might be conducive to ELF-EMF-induced stress resistance.

For further confirmation of the enhanced energy metabolism, nineteen genes encoding enzymes that regulate glycolysis and gluconeogenesis were selected for gene expression analysis. However, no noticeable changes were observed in the mRNA expression of genes that encode phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase, which catalyze reactions that produce ATP. The main reason may be that glycolysis uses 10 enzymatic reactions to convert glucose into pyruvate; however, several genes are predicted to be involved in the glycolytic pathway on the basis of homology33. Thus, redundancy might contribute to the lack of a detectable gene expression alteration by qRT-PCR.

In C. elegans, the highly conserved enzyme GAPDH, which is predicted to reversibly catalyze the oxidation and phosphorylation of glyceraldehyde-3-phosphate to 1, 3-diphosphoglycerate during glycolysis, was encoded by four homologous genes. GPD-1 and GPD-4 are required for embryogenesis and larva development, while GPD-2 and GPD-3 mainly play a role in adulthood33,34,35,36. In the present study, GPD-1 and GPD-4 were chosen for gene expression analysis because the samples we studied were L4 larva stage worms. Both of their gene expressions were upregulated significantly (P < 0.01) and data from western blot analysis ( ) further confirmed the upregulated expression level of GAPDH (P < 0.05). However, there is only a minimal increase observed in the fold of protein increase, which does not reflect the increased mRNA levels of gpd-1 and gpd-4. One explanation may be that gpd-2 and gpd-3 still have background expressions, which obscured the effect of elevated gpd-1 and gpd-4 transcription on GAPDH protein expression level. In addition, the regulation mechanisms during translation and modification process might also resulted in disproportionate variation between gpd-1 and gpd-4 mRNA and GAPDH protein expression level.

Theoretically, increased GAPDH expression indicates increased ratios of 1, 3-bisphosphoglycerate to glyceraldehyde-3-phosphate33. Glyceraldehyde-3-phosphate accumulation can glycate proteins, leading to deleterious effects within cells37. The concentration of glyceraldehyde-3-phosphate available to glycate proteins was lower in the exposure groups than in the control groups. The lower level of such altered protein benefitted the worms exposed to ELF-EMF.

Given that the mRNA expression of most genes involved in glycolysis did not show a significant alteration, we wondered whether the observed increase in GAPDH level in the current study is to complement the energy metabolism or to achieve any other non-glycolytic functions. The elevated enzyme activity indicates that the increased GAPDH expression is probably utilized for complementing the energy metabolism because enzymatic function of GAPDH is contributed to its role in metabolism rather than non-metabolic functions. Moreover, the higher concentration of pyruvic acid also implies enhanced glycolysis, for pyruvic acid is a main product of glycolysis pathway. Taken together, the increased gene/protein expression and the elevated enzyme activity of GAPDH promoted glycolysis pathway in worms exposed to ELF-EMF.

Interestingly, the intracellular ATP level decreased in worms under ELF-EMF exposure, even though the concentration of intermediates involved in glycolysis pathway and TCA cycle were elevated. Given that the food intake was reduced in worms exposed to ELF-EMF, we speculated that GAPDH activity is increased to compensate for the depletion of ATP (due to stress tolerance) but it is still not enough to counteract the effect of diminished food intake and elevated ATP consumption in stress response process. Previous study demonstrated that reduced ATP level was related with increased stress tolerance against high temperature, starvation, or mitochondrial toxicity38. Thus, the change of ATP intracellular amount implies a response to the stress caused by ELF-EMF exposure.

Both metabolomics analysis and gene expression analysis showed that the rate of energy metabolism was enhanced in the worms exposed to ELF-EMF. To maintain a balance between ATP demand and supply in energy metabolism with reduced level of substrate, various pathways should be activated to produce energy. To date, numerous works have reported that dietary restriction (DR) nematodes increased their oxygen consumption39, and that decreased available nutrients activated nutrient-sensing pathways40. A switch in fuel utilization from carbohydrates to short chain fatty acids41 and/or a more efficient utilization of ATP42 may be responsible for energy metabolism and fundamentally contributes to DR. Moreover, DR has been associated with elevated protein turnover43 and higher mRNA levels of GAPDH44,45, which are in accordance with our results of metabolomics and gene expression analysis, respectively. DR also exhibits many changes that may contribute to physiological benefits, such as reduced oxidative damage, slowed aging-associated decline in DNA repair, altered contents of hormones and induced metabolic changes46,47. Thus, DR might also contribute to the adaptive response of C. elegans to ELF-EMF exposure.

Although variations were found in the level of molecular biology, no significant changes were detected in lifespan, hatching rate and brood size of C. elegans under ELF-EMF exposure. A plausible explanation is that the effects of ELF-EMF on C. elegans were too tiny that they were covered by worms' adapting system48. Furthermore, the relationship between ATP level and phenotype is complicated and still remains unclear. For example, in some nematode mutations, lifespan has a positive correlation with ATP levels49, whereas uncoupled relationship also have been observed in some other C. elegans mutants50,51.

On the basis of these findings, we propose a biological adaptation model ( ). Under ELF-EMF stress, self-protective processes expend lots of ATP, and promote the enhancement of energy metabolism, including glycolysis and TCA. DR is also involved in the response to ELF-EMF exposure as an adaptive mechanism. In addition, elevated amino acids concentration suggest that protein metabolism might be enhanced to maintain the concentrations of substrates involved in energy metabolism due to the less food intake in worms under ELF-EMF exposure41.