Mitochondria as key targets underlying hypomagnetic field-induced biological effects

Summary

With the rapid development of human deep space exploration, prolonged exposure to hypomagnetic fields (HMF, <5 μT) – an environmental parameter with significantly reduced intensity compared to Earth’s geomagnetic field (GMF) – poses unprecedented health challenges. Accumulating evidence demonstrates that HMF exposure induces negative effects on various system functions in animals, including the central nervous system, the cardiovascular system, and the musculoskeletal systems and so forth. In this review, we systematically summarize the multi-level impacts of HMF on mitochondrial quality control and function, proposing mitochondria as direct and sensitive targets of HMF, with iron-sulfur clusters potentially playing a pivotal role. We also explore possible HMF-regulated signaling pathways. In conclusion, this review provides a valuable foundation for understanding the cellular and molecular mechanisms of HMF’s potential risks to astronauts and for developing future coping strategies during spaceflight.

Graphical abstract

Space medicine; Cellular physiology; Functional aspects of cell biology

Introduction

The geomagnetic field (GMF) protects life on Earth by shielding it from intense radiation and cosmic rays and prevents the atmosphere and the water from escaping.^1,2 The origin and evolution of life occurred in the GMF.^1,2,3 However, the GMF is not static, but experiences secular variations. Palaeomagnetic studies have shown that it has frequently reversed throughout geological history. During these reversals, its intensity can drop to less than 10% of the normal level.⁴ Some research suggests a potential link between geomagnetic reversals and mass extinctions, particularly faunal extinctions and reversals of the GMF.^2,5 Furthermore, as deep space exploration advances, astronauts will inevitably be exposed to HMF environments (HMF <5 μT).⁶ For instance, the magnetic field strength in interplanetary space ranges from 2 to 8 nT, whereas the magnetic field strength on the surface of the Moon and Mars ranges from 0.2 to 300 nT and 10 nT to 5 μT, respectively.^7,8 In recent decades, the biological effects of HMF have been widely recognized.^6,9,10 HMF can affect many biological processes, including animal behaviors, metabolism, central nervous system functions, and embryonic development.^11,12 However, the mechanisms underlying the effects of HMF on organisms remain poorly understood.

The National Aeronautics and Space Administration (NASA) has identified mitochondrial dysfunction as a key regulatory nexus in space biology,⁶ and some scientists have suggested that mitochondria may be a key target of the effects of HMF.^13,14,15 According to the endosymbiotic theory, mitochondria originated from an alphaproteobacterium over a billion years ago. This bacterial cell was engulfed by an archaeal host cell. Instead of being digested, it established a symbiotic relationship inside the host. This endosymbiont eventually evolved into the mitochondrion. The acquisition of mitochondria is now considered a pivotal event that provided the host cell with a vastly more efficient energy transformation system. This was likely a major driving force behind the evolution of cellular complexity and the diversification of eukaryotic life.¹⁶ This highlights the indispensable role of mitochondria in life and their critical importance in understanding the origin of eukaryotes. Structurally, mitochondria consist of four major compartments: the outer mitochondrial membrane (OMM), the inner mitochondrial membrane (IMM), the intermembrane space (IMS), and the mitochondrial matrix.¹⁷ Functionally, mitochondria are traditionally known as the “powerhouse of the cell” due to their central role in producing adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS), representing their core catabolic function. Equally essential is their contribution to anabolic processes, whereby metabolites from the tricarboxylic acid (TCA) cycle serve as precursors for the synthesis of macromolecules, including lipids and nucleotides, respectively.¹⁸ A third key functional dimension has also been recognized: mitochondria regulate cellular fate and behavior by signal recognition and releasing diverse signaling molecules.¹⁹ Mitochondrial dysfunction, typically caused by exposure to adverse environmental conditions or noxious stimuli, can result in reduced integrity or quantity, thereby impairing their ability to perform these functions.²⁰ Mitochondrial dysfunction plays a critical role in many pathological processes, including neurodegenerative diseases, cardiovascular diseases, cancer, immune disorders, and aging.²¹ Therefore, it is of great scientific importance to study in detail how HMF affects mitochondria.

This review summarizes the effects of HMF on mitochondrial quality control (Table 1) and function (Table 2), establishing mitochondria—particularly the electron transport chain (ETC)—as the most sensitive targets of HMF’s effects. In exploring mitochondrial responses to HMF, we posit that iron-sulfur (Fe-S) clusters likely play a critical role. Furthermore, we suggest three potential signaling pathways that could mediate the impact of HMF on mitochondria.

Table 1.

The effects of HMF on mitochondrial quality control

Classification	Animal	Magnetic Intensity	Exposure Time	HMF Effects	References
Mitochondrial Number/Biogenesis	C57BL/6 mice	<500 nT	1 month	Reduced subsarcolemmal mitochondria number	Hu et al.14
	C57BL/6 mice	∼32 nT	4, 6, 8 weeks	Reduced active mitochondria and the PGC-lα gene expression level	Tian et al.22
Mitochondrial Structure/Morphology	C57BL/6 mice	<500 nT	1 month	Changed skeletal muscle mitochondrial morphology	Hu et al.14
	Pea (Pisum sativum L.) seeds	0.5–2 nT	3 days	Increased mitochondrial size and relative volume	Belyavskaya.23

Table 2.

The effects of HMF on mitochondrial functions

Function	Cell/Tissue /Animal	Magnetic Intensity	Exposure Time	HMF Effects	References
Bioenergetics (MMP and ATP Levels)	Mouse skeletal muscle cell	0.49 ± 0.031 μT	3 days	Reduced ATP content, mitochondria viability, and MMP	Fu et al.13
	Paramacro-biotus experiment-alis (tardigrade)	200-fold reduction of GMF	7,15,30 days	Decreased MMP	Nagwani et al.24
	Mouse macrophage RAW264.7 cells	∼7.4 μT	24 h	Decreased MMP	Srimai et al.25
	SH-SY5Y cells	<200 nT	24,48,72 h	Decreased MMP, Decreased ADP/ATP ratio	Wang et al.26
Biosynthesis (Glucose and Lipid Metabolism)	Mouse skeletal muscle cell	0.49 ± 0.031 μT	3 days	Reduced glucose consumption	Fu et al.13
	C57BL/6 mice	<500 nT	1 month	Reduced citric acid (TCA cycle intermediate) level	Hu et al.14
	Human spermatozoa	<150 nT	30 h	Increased glucose consumption	Truta et al.27
	SH-SY5Y cells	<200 nT	24,48,72 h	Increased cell proliferation, glucose consumption, LDH and glycolysis; Decreased OXPHOS	Wang et al.26
	Guinea pigs and white rats	100-time-weakened field	5 days	In guineapigs: Reduced cholesterol, phospholipids, triglycerides; In white rats: Increased cholesterol, phospholipids, triglycerides; Promoted shift from carbohydrate to lipid metabolism	Babych.28
	C57BL/6J mice	31.9 ± 4.5 nT	8.12 weeks	Decreased short-chain fatty acid concentrations (microbial metabolites)	Zhan et al.29
Signaling (ROS and Calcium Homeostasis)	Mouse skeletal muscle cell	0.49 ± 0.031 μT	3 days	Increased ROS level	Fu et al.13
	C57BL/6 mice	∼32 nT	4,6,8 weeks	Increased ROS levels	Tian et al.22
	C57BL/6J mice	31.1 ± 2.0 nT	8.12 weeks	Increased colonic cell proliferation and O2⋅- levels	Zhan et al.29
	C57BL/6J mice	31.9 ± 4.5 nT	8 weeks	Increased O2⋅- Levels and mRNA of pro-oxidant genes; Decreased mRNA of antioxidant genes	Tian et al.30
	Black Garden Ant	∼40 nT	14 days	Decreased brain H2O2 production; Decreased soluble cytoplasmic SOD; Increased GSR (antioxidant enzyme)	Mannino et al.31
	HT1080, AsPC-1, PAEC cells	0.2–2 μT	6,12,24 h	Suppressed H2O2 production	Martino and Castello.32
	SH-SY5Y cells	<0.2 μT	0.5, 1, 16, 24, 48 h	Decreased ROS levels, CuZn-SOD activity; Increased CuZn-SOD denaturation/aggregation	Zhang et al.33
	Mouse peritoneal neutrophils	20 nT	1.5 h	Decreased ROS levels	Novikov et al.34 Novikov et al.35
	Pea (Pisum sativum L.) seeds	0.5–2 nT	3 days	Altered Ca2+ pyroantimonate deposits	Belyavskaya.23
	Invertebrates and Fish	–	1 h	Decreased calpain activity (Ca2+-dependent protease)	Kantserova et al.36

Effects of hypomagnetic fields on mitochondrial quality control

Mitochondrial quality control is a crucial integrated system that maintains functional integrity through regulating dynamics (fission and fusion), biogenesis, and mitophagy. This system ensures a healthy population of mitochondria, whose structure is essential for their function. The OMM contains ion channels, metabolic enzymes, and other regulatory proteins.³⁷ Specifically, it contains voltage-dependent anion channels (VDACs), which mediate the flux of metabolites such as ATP, ADP, Pi, and Ca²⁺,^38,39 as well as proteins involved in signaling such as hexokinase.⁴⁰ The outer membrane (TOM) complex, also located in the OMM, is essential for importing nuclear-encoded mitochondrial proteins. The exchange of ATP and ADP through VDAC is fundamental to cellular energy provision. The electrochemical gradient (proton motive force, PMF) across the inner mitochondrial membrane, which drives ATP synthesis, is primarily established by the ETC.⁴¹ They also actively participate in cellular signaling. For example, Ca²⁺ uptake activates key metabolic enzymes and thereby influences signaling pathways that regulate processes such as cytoskeleton dynamics,³⁷ ROS act as second messengers influencing MAPK and NF-κB pathways,³⁹ and VDAC-hexokinase interactions regulate cell survival and apoptosis.^38,40 By coordinating metabolite flux and signaling events, the OMM acts as a dynamic hub that integrates metabolic and stress signals in order to regulate downstream biochemical processes and signaling pathways. The OMM also interacts with other cellular organelles to participate in lipid exchange and signal transduction.⁴² Situated between the OMM and IMM, the IMS plays a pivotal role in coordinating mitochondrial activities with other cellular processes, including the regulated initiation of apoptotic cascades and key signaling pathways.⁴³ The IMM, characterized by its highly folded cristae structures, houses the ETC (complexes I - IV), the ATP synthase, and transport proteins, thereby constituting the central site for oxidative phosphorylation (OXPHOS).^44,45 The mitochondrial matrix contains a large number of proteins, including enzymes essential for the TCA cycle, the β-oxidation of fatty acids, and amino acid catabolism.⁴⁶ Disrupting mitochondrial quality control compromises this intricate architecture, inevitably leading to functional deficits.

Substantial evidence indicates that HMF impairs mitochondrial quality control, manifesting as structural and quantitative alterations. In 2004, Belyavskaya suggested that mitochondria are highly sensitive to HMF based on her observations of HMF-induced morphological changes in mitochondria from pea root meristematic cells. She found that exposure to HMF increased the size and relative volume of the mitochondria in these cells, while decreasing cristae density.⁴⁷ These early structural alterations may be interpreted as evidence of dysfunctional mitochondrial quality control. Subsequent studies have provided further support for the idea that HMF disrupts mitochondrial quality control. In C57BL/6 mice, HMF exposure has been shown to lead to a decrease in the number of mitochondria, a reduction in their volume, and abnormal morphology in skeletal muscle,¹⁴ alongside reduced mitochondrial survival.¹³ Our laboratory has also observed a significant decrease in the number of active mitochondria in the hippocampus of mice exposed to HMF, concurrent with reduced mRNA levels encoding peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α).²² As a key regulator of mitochondrial biogenesis,⁴⁸ the downregulation of PGC-1α indicates impaired biogenesis, which may compromise the replenishment of functional mitochondria.

In summary, the available evidence demonstrates that HMF impairs the structural integrity and turnover of mitochondria, probably by disturbing the balance of mitochondrial dynamics, biogenesis, and mitophagy. By interfering with these integrated quality control mechanisms, HMF ultimately compromises mitochondrial and cellular function. Further research is required to elucidate the precise mechanisms by which HMF impacts each arm of mitochondrial quality control.

Effects of hypomagnetic fields on mitochondrial functions

Most studies on HMF have focused primarily on its effects on various mitochondrial functions, which align with the tripartite roles of mitochondria previously outlined: bioenergetics, biosynthesis, and signaling. As summarized in Table 2 and Figure 1, these effects include impaired mitochondrial membrane potential (MMP) and ATP production (bioenergetics), dysregulated glucose and lipid metabolism (spanning bioenergetics and biosynthesis), and disruptions in ROS and Ca²⁺ homeostasis (signaling). Together, these studies demonstrate that HMF impairs mitochondrial function across all three critical levels.

Figure 1.

The effects of HMF on mitochondrial function

HMF can affect MMP, ATP levels, ROS homeostasis, calcium ion concentration, glucose metabolism, and lipid metabolism. Red arrow indicates an increase after HMF exposure. Blue arrow indicates a decrease. Brown font indicates variation, with some experiments showing an increase and others showing a decrease.

MMP: Mitochondrial membrane potential; FFAs: Free fatty acids; SCFAs: Short-chain fatty acids; GSR: glutathione reductase; SOD: superoxide dismutase; Gpx3: glutathione peroxidase 3; LPO: lipid peroxidation; LDH: lactic dehydrogenase.

Effects of hypomagnetic fields on mitochondrial bioenergetics

The mitochondrial ETC consists of four transmembrane protein complexes (complexes I-IV) that are embedded in the IMM. Complexes I, III, and IV act as proton pumps.⁴¹ During oxidative respiration, the ETC converts the free energy released from electron transfer into an electrochemical potential across the IMM. This process creates an asymmetric distribution of protons (H⁺), generating the MMP. During electron transfer through the ETC, the decrease in redox potential generates the PMF (Δp), which drives ATP synthase to catalyze ATP production. Thus, the PMF consists of two interdependent components, the MMP and the transmembrane pH gradient across the IMM (ΔpH). Under physiological conditions, the MMP is the major contributor to the PMF.⁴⁹ The MMP is important for cellular homeostasis and critically influences almost all mitochondrial functions, serving as a key indicator of normal mitochondrial activity.⁵⁰

Studies on mouse skeletal muscle cells, macrophages, and the tardigrade Paramacrobiotus experimentalis have consistently shown that exposure to HMF significantly reduces cellular MMP.^13,24,25 Exposure to HMF significantly reduces ATP levels and increases the ADP/ATP ratio in mouse skeletal muscle cells. A significant decrease in the CCK-8 assay signal, which reflects the activity of the cellular dehydrogenases that generate NADH, indicates impaired activity of these enzymes under HMF conditions. This reduces the availability of NADH, which limits the supply of electrons to mitochondrial complex I (NADH: ubiquinone oxidoreductase). This suppresses the initial step of the mitochondrial electron transport chain, leading to decreased ATP synthesis.¹³ The reduction in the respiration rate of Drosophila spermatozoa induced by HMF leads to reduced ATP production. This model is supported by the finding that the effect of HMF could be counteracted by increased cAMP levels, a molecule synthesized from ATP by adenylate cyclase. Furthermore, the experiment involving ETC complex inhibitors showed that HMF specifically altered the respiration rates of Drosophila sperm by affecting complex I.⁵¹ From these hints, we propose that the reduction in MMP and ATP levels induced by HMF may be due to HMF interfering with the MMP generation process, specifically the ETC process. Furthermore, beyond ATP synthesis, the MMP is also essential for energizing other critical processes, including Ca²⁺ transport and lipid biosynthesis.⁴⁹ Therefore, the HMF-induced collapse of the MMP may further affect other MMP-consuming cellular processes. These processes are reviewed in the following sections.

Effects of hypomagnetic fields on mitochondrial biosynthesis

The mitochondrial TCA cycle supplies critical intermediates for biosynthesis, such as citrate and oxaloacetate, for the synthesis of lipids and nucleotides. Emerging evidence indicates that HMF exposure significantly disrupts these biosynthetic and metabolic functions.¹⁸ However, the effects of HMF exposure on mitochondrial biosynthesis and metabolic function appear to be complex and cell-type-specific, often disrupting these core bioenergetic and anabolic pathways.

The impact of HMF on glucose metabolism, which is a primary source of carbon for the TCA cycle, illustrates this complexity. For instance, exposure to HMF has been shown to accelerate glucose consumption in human sperm, while improving sperm motility and survival rates.²⁷ In SH-SY5Y cells, HMF was found to increase glucose consumption, lactate production, and cell proliferation. This proliferation depended on glycolysis, as it was blocked by the glycolysis inhibitor 2-deoxyglucose (2-DG). HMF also increased lactate dehydrogenase (LDH) activity and LDH inhibitors prevented HMF-induced proliferation. Additionally, HMF was found to suppress OXPHOS, likely via reduced MMP. These results demonstrate that HMF promotes glycolysis (via LDH) and proliferation while simultaneously inhibiting OXPHOS.²⁶ Interestingly, the opposite effects were observed in mouse skeletal muscle cells. Cells exposed to HMF exhibited significantly lower glucose consumption compared to GMF controls, along with enhanced glycogenesis, indicating suppressed glycolytic glucose utilization. However, exposure to HMF did not alter cellular lactate metabolism, suggesting that these changes in glucose metabolism were the result of impaired aerobic respiration.¹³ Similarly, exposure to HMF reduced citrate levels in mouse skeletal muscle. Since citrate functions not only in ATP production but also as a key precursor for cytosolic biosynthetic processes (e.g., lipid synthesis), this finding implies that HMF impairs both the bioenergetic and biosynthetic roles of the TCA cycle.¹⁴

The apparently paradoxical effects of HMF on cellular glucose metabolism, as described above, may be due to inherent differences in the patterns of cellular energy metabolism. Proliferating cells use energy more efficiently than quiescent differentiated cells. For example, HMF primarily affects glycolysis in proliferating SH-SY5Y cells, which exhibit cancer-like metabolic characteristics. Conversely, skeletal muscle cells predominantly rely on oxidative phosphorylation to meet their high ATP demand, and the effect of HMF on OXPHOS is more pronounced in these cells. The differences in these effects may be due to differences in cell types. These findings emphasize that HMF responsiveness varies depending on the cell type.

The biosynthetic role of mitochondria is further impaired by alterations in lipid metabolism induced by HMF. However, the reported effects are inconsistent across species. For instance, exposure to HMF reduces triglyceride levels and lowers blood markers of lipid metabolism in guinea pigs. However, it has also been shown to increase triglyceride levels and promote a metabolic shift from carbohydrate to lipid utilization in rats.²⁸ These disparate phenotypic changes suggest a profound dysregulation of lipid homeostasis by HMF. The mitochondrial connection to these changes is critical. Triglycerides, which are the primary form of energy storage in adipocytes, are broken down by lipases into free fatty acids (FFAs) and glycerol. These can then be used for energy production or hepatic ketogenesis. The FFAs then enter the mitochondria via β-oxidation to produce acetyl-CoA, an essential substrate that fuels the TCA cycle. This generates the NADH/FADH₂ required for ATP synthesis by the ETC, as well as the aforementioned biosynthetic precursors. Our laboratory has observed reduced levels of short-chain fatty acids (SCFAs), which are key substrates for mitochondrial β-oxidation, in the intestines of mice exposed to HMF.²⁹ These changes in lipid metabolites following HMF exposure suggest an impact on mitochondrial β-oxidation and the TCA cycle, which could compromise both energy generation and biosynthetic output. Additionally, Belyavskaya observed that large quantities of lipid bodies were present along the cytoplasmic membrane of pea seeds after three days of exposure to HMF.²³ However, current research on the effects of HMF on lipid metabolism is limited to phenotypic observations, providing little mechanistic insight into how HMF directly influences mitochondrial biosynthetic pathways related to lipid metabolism.

In addition to supplying metabolic intermediates, a fundamental biosynthetic function of mitochondria is the production of iron-containing prosthetic groups, most notably heme and iron-sulfur clusters (Fe-S clusters). Heme, which is synthesized primarily within mitochondria, is crucial for oxygen transport and storage in hemoglobin and myoglobin, and also serves as an essential cofactor for cytochromes in the electron transport chain and numerous enzymes.⁵² Similarly, Fe-S clusters are ancient and ubiquitous cofactors found in numerous proteins and protein complexes involved in fundamental biological reactions,⁵³ representing some of the earliest catalysts in biomolecular evolution.⁵⁴ Fe-S cluster homeostasis is crucial for organisms, as these clusters play a role in regulating numerous biological processes, as well as in the replication and maintenance of the nuclear genome.⁵⁵ Disruption of Fe-S clusters can trigger a bioenergetic crisis, metabolic dysregulation, genomic instability, and disruption of iron homeostasis.⁵⁶ However, research on the effects of HMF on both heme and Fe-S clusters is currently lacking. Indirect evidence from microbiological studies suggests that an organism’s sensitivity to HMF may be linked to its reliance on Fe-S clusters. A recent study demonstrated that microorganisms that depend heavily on Fe-S clusters for core metabolism (e.g., Saccharomyces cerevisiae and Acidithiobacillus ferrooxidans) experienced significant growth inhibition upon exposure to HMF. In contrast, the growth of Lactobacillus plantarum, which does not require iron or Fe-S clusters, remained unaffected.⁵⁷ This strong correlation indicates that HMF may interfere with cellular processes involving Fe-S clusters, although this effect has yet to be demonstrated experimentally. Therefore, the effects of HMF on heme and Fe-S clusters warrant further investigation.

In summary, HMF impairs the mitochondrial biosynthetic function by disrupting the metabolic flux through the TCA cycle. At the same time, HMF disrupts lipid metabolism, likely by impairing β-oxidation and the TCA cycle. These further compromises the mitochondrial capacity to support anabolism. While the current evidence suggests significant disruption to biosynthesis, more mechanistic studies are needed to fully elucidate the pathways involved.

Effects of hypomagnetic fields on mitochondrial signaling

The function of mitochondria as signaling organelles has gained increasing recognition. Not only do mitochondria regulate cellular processes through bioenergetics and biosynthesis, but they also release signaling molecules such as ROS, TCA cycle metabolites (e.g., citrate, a precursor for cytosolic acetyl-CoA, α-ketoglutarate, and succinate), and mitochondrial DNA, and modulate the NAD+/NADH ratio and Ca²⁺ homeostasis.¹⁹ Current research on HMF primarily focuses on ROS and Ca²⁺ in the context of mitochondrial signaling.

ROS refer to oxygen-containing free radicals or compounds that are more reactive than molecular oxygen. These four main forms of intracellular ROS are the superoxide radical anion (O₂^⋅-), the hydroxyl radical (^⋅OH), hydrogen peroxide (H₂O₂), and lipid hydroperoxides (LOOH). The influence of HMF on intracellular ROS levels is the most common biological effect. ROS are considered candidate response molecules that mediate the biological effects of HMF.^{30,32,33,34,35,58}

Specifically, in vivo studies have provided evidence of both elevated and suppressed ROS levels following HMF exposure. For instance, PCR array analysis revealed that exposure to HMF significantly upregulated the expression of four pro-oxidant genes (NADPH oxidase 4 (Nox4), nitric oxide synthase 2 (Nos2), eosinophil peroxidase (Epx), and Keratin 1 (Krt1)) while downregulating two genes involved in antioxidant and cytoprotective functions (glutathione peroxidase 3 (Gpx3) and heat shock protein 1A (Hspa1a)) in the hippocampus of adult mice.³⁰ This imbalance in gene expression, particularly the marked increase in Nox4, is a major source of oxidative stress and leads to elevated ROS production in hippocampal tissue. A similar increase in O₂^⋅- was observed in the small intestines of mice exposed to HMF.²⁹ Furthermore, HMF exposure has been shown to increase lipid peroxidation (LPO) activity in guinea pigs and rats, while decreasing their antioxidant capacity.²⁸ LPO, a hallmark of oxidative stress, involves ROS-mediated attack on polyunsaturated fatty acids (PUFAs) in cellular or organellar membranes. This process disrupts lipid structures and generates oxidative end products, leading to cell damage or death.⁵⁹ By contrast, exposure to HMF reduced levels of O₂^⋅- in adult mouse hippocampal neural stem cells.⁶⁰ In black garden ants, HMF decreased H₂O₂ levels, accompanied by decreased cytoplasmic superoxide dismutase (SOD) activity and increased glutathione reductase (GSR) levels. SOD catalyzes the conversion of O₂^⋅− to molecular oxygen (O₂) and H₂O₂, which is then reduced to water by GPX in a reaction that consumes reduced glutathione (GSH). GPX activity is therefore sustained by GSR, which regenerates the essential GPX co-substrate, GSH, from its oxidized form (GSSG). In addition, the decrease in H₂O₂ content under HMF conditions may be related to an increase in melatonin content,³¹ an antioxidant that counteracts cellular oxidative stress in vertebrates.⁶¹

This cell-type-specific heterogeneity is further supported by in vitro studies. For instance, exposure to HMF suppressed the generation of H₂O₂ in both fibrosarcoma cancer cells and primary endothelial cells. Control experiments demonstrated that this effect could be reversed by adding catalase (CAT) or the SOD mimetic MnTBAP.³² Similarly, HMF was found to inhibit H₂O₂ production in neuroblastoma SH-SY5Y cells, which was associated with reduced CuZn-SOD (SOD1) activity. CuZn-SOD is a soluble protein found in the cytoplasm and the intermembrane space of mitochondria, where it scavenges ROS generated within the mitochondria. However, CAT and GPX activities were unaffected by HMF, indicating that HMF does not interfere with the enzymatic degradation of H₂O₂.³³ HMF also suppressed ROS production in neutrophils. This inhibitory effect was enhanced by rotenone, an inhibitor that blocks electron transfer from NADH to coenzyme Q (CoQ). The greater additional reduction in ROS caused by rotenone in HMF-treated cells compared to controls suggests that HMF acts through mechanisms involving the mitochondrial ETC.^34,35 Conversely, HMF increased ROS levels in mouse primary skeletal muscle cells.¹³

The above studies have shown that HMF affects both the generation and elimination processes of ROS. Mitochondria are the primary site of ROS generation.⁵⁰ HMF modulates ROS generation by affecting mitochondrial oxidative respiratory chain, which is closely linked to both MMP formation and ATP production. Under physiological conditions, the rate of mitochondrial ROS production is primarily regulated by the MMP.⁶² NOX is certainly also one of the sources of ROS production.⁶³ Furthermore, the aforementioned study found that HMF upregulates NOX4 mRNA expression.³⁰ Therefore, the potential influence of NOX on ROS generation cannot be ruled out. As key signaling molecules, ROS play a critical role in cellular signaling and metabolic regulation.⁶⁴ However, excessive ROS induces oxidative stress, leading to damage to DNA, proteins, and lipids^65,66,67 and is associated with cancer, cardiovascular disease, metabolic disease, depression, memory loss, and neurological disorders.^{68,69,70,71,72} Therefore, changes in ROS levels induced by HMF may act as signaling cues that modulate downstream redox-sensitive pathways such as the nuclear factor-κB (NF-κB) pathway, the phosphoinositide-3-kinase (PI3K)-Akt pathway, the mitogen-activated protein kinase (MAPK) pathway, and the Keap1-Nrf2 system. These pathways mediate crucial physiological processes, including cell proliferation, differentiation, metabolic adaptation, and the response to stress and inflammation.⁷³ Consequently, HMF-induced ROS fluctuations are poised to influence cell fate and metabolic outcomes.

The inconsistent findings regarding the effects of HMF on ROS levels across different studies may be attributed to multiple factors. Firstly, the term “ROS” refers to different species (e.g., O₂⋅^- and H₂O₂) that have distinct chemical properties and signaling roles.⁶⁷ The ROS probes used in the current study detect overall cellular and tissue levels, rather than specific sub-localizations.⁷⁴ This implies that reported “ROS” changes in the literature may reflect alterations in different ROS species. Secondly, the subcellular origin of ROS (e.g., mitochondrial ETC complexes versus NOX enzymes) varies by cell types and can dictate the response to HMF. Our previous result showing the HMF-induced upregulation of Nox4 RNA suggests the potential contribution from non-mitochondrial sources.³⁰ Current detection methods struggle to accurately distinguish between these sources. Thirdly, the net ROS level is determined by the cell’s inherent metabolic state and antioxidant capacity (e.g., proliferating or differentiated cells). Finally, the inconsistencies in experimental parameters, such as HMF exposure parameters, sample size, and sampling endpoints, and so forth, often hinder cross-study comparisons.

In addition to ROS, mitochondria are important regulators of intracellular Ca²⁺ signaling alongside the endoplasmic reticulum. Ca²⁺ serves as a critical secondary messenger that governs diverse biological processes, including cellular motility, metabolism, proliferation, and apoptosis.⁷⁵ Dysregulation of Ca²⁺ homeostasis has been identified as a critical pathological mechanism underlying neurodegenerative diseases such as Alzheimer’s and Parkinson’s.⁷⁶ The primary role of mitochondrial Ca²⁺ is to stimulate OXPHOS through the direct activation of isocitrate and α-ketoglutarate dehydrogenases, and the indirect activation of pyruvate dehydrogenase (via pyruvate dehydrogenase phosphatase 1). This coordinated upregulation of metabolic activity accelerates electron transport chain flux and increases ATP output.⁷⁷

HMF has also been found to affect Ca²⁺ homeostasis. For instance, exposure to HMF has also been shown to significantly regulate myosin phosphorylation by affecting the binding of Ca²⁺ and calmodulin.⁷⁸ In 2001, Belyavskaya first identified HMF-induced Ca²⁺ dysregulation in pea seeds and proposed that Ca²⁺ might be a key mediator of HMF responses.²³ Furthermore, exposure to HMF has also been shown to reduce calpain activity in fish and decrease the Ca²⁺ content of osteoblasts in invertebrates.^36,79 Adding the phospholipase C inhibitor U73122 reduced ROS production in the HMF condition. This indicates an association with calcium-dependent regulatory mechanisms that control the respiratory burst in neutrophils.³⁵ While studies have demonstrated that HMF disrupts intracellular Ca²⁺ homeostasis, systematic investigations into the effects of HMF on Ca²⁺ regulation remain limited.

Despite these suggestive findings, systematic investigations into the effects of HMF on Ca²⁺ regulation remain limited. Given the central role of Ca²⁺ and ROS as intertwined mitochondrial signaling molecules in regulating metabolism, cell fate, and inflammatory responses, we propose that a systematic investigation of HMF-mediated disruption to these signaling pathways would significantly advance our understanding of its pleiotropic biological effects.

Iron-sulfur clusters may be potential hypomagnetic fields-sensitive elements in mitochondria

Two principal, non-mutually exclusive biophysical mechanisms have been proposed to explain the bioeffects of HMF: the radical pair mechanism (RPM) and direct influence on Fe-S clusters. Both offer a plausible framework for understanding the effects of HMF, albeit with distinct molecular premises. It is important to note that these two mechanisms are not necessarily mutually exclusive. Future research could even reveal that they interact synergistically or overlap; for example, certain Fe-S cluster proteins could potentially participate in or be regulated by radical pair-based processes. A key challenge for the field is designing experiments that can distinguish the contributions of these potential mechanisms to the pleiotropic biological effects of HMF.

The RPM is a well-established quantum biological hypothesis, best known for its role in avian magnetoreception. It posits that magnetic fields can influence the spin state of pairs of transient radicals generated photochemically, for instance, within cryptochrome proteins, thereby affecting subsequent chemical reactions and potentially leading to a cellular signal.^80,81 The nature of the radical pair is crucial. Although a flavin-superoxide (FAD^⋅-/O₂^⋅-) pair has been theorized in the context of HMF,^82,83,84 the rapid spin relaxation of superoxide poses a challenge to this specific pair.⁸⁵ However, it is important to recognize that the RPM is a general mechanism that does not rely on any single pair. Other, more stable radical pairs (e.g., flavin-tryptophan) could potentially transduce HMF effects, and exploring their relevance is an important ongoing area of research.^86,87

In parallel, the potential of Fe-S clusters as primary magnetosensitive targets has been gaining traction.^57,88 Building upon this idea, our review summarizes the substantial functional evidence from HMF studies and puts forward a coherent argument that mitochondria, and specifically their Fe-S clusters, could represent sites for HMF sensing. We hypothesize that mitochondrial dysfunction upon HMF exposure may largely arise from the disruption of electron transfer processes and/or electron cloud distribution within Fe-S clusters (Figure 2).

Figure 2.

Fe-S clusters as a potential key HMF-sensitive element in the mitochondria ETC

HMF may affect electron transport, MMP maintenance, ROS production, and ATP synthesis primarily by targeting Fe-S clusters, particularly in complex I, thereby influencing downstream biological effects. The blue arrows indicate the direction of electron transfer within the Fe-S cluster.

OMM: Outer membrane; IMS: intermembrane space; and IMM: inner membrane.

Several lines of evidence suggest an association between HMF-induced mitochondrial defects and the potential disruption of Fe-S clusters. Complexes I and III are primary sites of ROS production. Notably, eight of the 12 Fe-S clusters identified in ETC complexes are found in Complex I, three in Complex II, and one in Complex III.⁸⁹ Previous studies using ETC complex inhibitors revealed that HMF has the most pronounced effect on Complex I.^34,35,51 A previous study has found that HMF reduces citrate levels, suggesting an impairment of the TCA cycle. We speculate that this may be due to the influence of enzymes containing Fe-S clusters, such as aconitase, which catalyzes the conversion of citrate to isocitrate.^14,90 However, theoretically, impaired aconitase activity would be expected to increase citrate levels, whereas the literature reports a decrease. This suggests that the reduction in citrate observed under HMF treatment reflects a more complex metabolic disruption. Initial damage to aconitase and the TCA cycle may trigger compensatory cellular responses. For example, cells may reduce citrate synthesis at its source. Notably, complexes I-III of the mitochondrial respiratory chain contain Fe-S clusters and are likely to be impaired. Dysfunction of the ETC would suppress the entire TCA cycle by hindering NAD⁺ regeneration and creating a buildup of NADH, thereby reducing the flux of citrate synthesis at its source. These explanations need experimental verification in the future. Therefore, we propose that Fe-S clusters could be among the most direct and plausible targets for the bioeffects of HMF.

So, what is the exact mechanism by which HMF affects Fe-S clusters? Some researchers have suggested an association with iron metabolism. For example, exposure to HMF was found to increase iron content in MC3T3-E1 osteoblasts with the increased expression of transferrin receptor 1 (TFR1), a gene that is key to iron metabolism.⁹¹ Similarly, HMF exposure led to elevated serum iron concentrations in rats.⁹² Furthermore, analogous effects have been observed in plants: Arabidopsis thaliana grown under HMF conditions exhibited altered iron uptake and homeostasis.⁸⁸ Taken together, these studies demonstrate that HMF significantly impacts iron homeostasis in various organisms. However, based on the synthesis of evidence in this review, we prefer that HMF most plausibly affects Fe-S clusters by disturbing their electron transfer processes and/or electron cloud distribution. This is because the processes of MMP and ATP production, as well as ROS generation, all involve electron transport within the ETC. The most crucial role of Fe-S clusters in mitochondria is therefore to mediate this electron transfer. For enzymes in the TCA cycle (such as aconitase), the distribution of electron clouds is also involved. Nevertheless, we do not exclude the possibility that HMF could directly modulate ions/ion channels as an alternative mechanism. The aforementioned studies have found that HMF can disrupt iron and calcium ion homeostasis. Furthermore, studies in electromagnetic and high-gradient magnetic fields have reported the direct modulation of ion channels, along with related hypotheses.^93,94,95 Additionally, we cannot rule out the possibility that HMF may affect the Fe-S cluster assembly process, as demonstrated by the magnetosensory protein MagR.

Interestingly, it is crucial to distinguish between the potential role of Fe-S clusters in random HMF bioeffects and their proposed role in magnetoreception. The recently discovered magnetoreceptor protein MagR is a member of the IscA family and functions as a scaffold for Fe-S cluster assembly.⁹⁶ It contains conserved Fe-S clusters and forms rod-like polymers that may align with the GMF.^97,98,99 MagR is critical for magnetosensation in insects such as the oriental armyworm.¹⁰⁰ However, the proposed mechanism for MagR in navigation involves directional magnetoreception, potentially through physical torque or alignment. By contrast, we hypothesize that the mechanism underlying random HMF bioeffects is fundamentally different, involving the chemical disruption of cluster stability, electron transfer efficiency, or cluster-protein interactions within core metabolic enzymes, such as those in the ETC and TCA cycles. This bioenergetic disruption represents a passive, outcome rather than an active sensory mechanism.

In summary, given the widespread impact of HMF on mitochondrial function (affecting ETC, TCA cycle, and ROS), we hypothesize that Fe-S clusters could act as key magnetosensitive elements that mediate the earliest response to HMF exposure. Their perturbation provides a potential mechanistic explanation for the observed bioenergetic deficits, which subsequently could contribute to the wide range of downstream mitochondrial dysfunctions (Figure 2).

Potential downstream signaling pathways in iron-sulfur-induced mitochondrial abnormalities

Based on the aforementioned proposal that Fe-S clusters may be a potential target of the effects of HMF, we posit that HMF acts directly on intracellular Fe-S clusters as a physical stimulus, which serve as potential “magnetic receptors.” Dysfunction of these clusters triggers immediate biochemical consequences. Building on the observed mitochondrial effects of HMF, we further propose that HMF-induced mitochondrial dysfunction propagates to and activates three key stress-response signaling pathways: the AMPK pathway, the mammalian target of rapamycin (mTOR) pathway, and the sirtuin pathway (Figure 3). We hypothesize that the primary disruption of mitochondrial Fe-S clusters by HMF leads to a bioenergetic and redox crisis (e.g., decreased ATP and altered NAD+/NADH levels), which triggers the activation of these pathways as a compensatory, pro-survival mechanism. The activation of AMPK, mTOR, and sirtuin signaling, and the crosstalk between these pathways, represent the cell’s attempt to restore energy homeostasis, manage metabolic flux, and enhance stress resilience. However, this cellular rescue effort may be unsuccessful; under prolonged or intense HMF stress, the compensatory mechanisms may become overwhelmed or dysregulated, ultimately resulting in the observed functional deficits. In addition, we also hypothesize the potential regulatory proteins within these signaling pathways that mediate the effects of HMF on mitochondria.

Figure 3.

Potential HMF-regulated signaling pathways

This schematic representation depicts the hypothesis that HMF-induced mitochondrial dysfunction affects potential signaling pathways. HMF primarily targets Fe-S clusters, causing core mitochondrial deficits (e.g., decreased ATP and MMP and increased ROS). It is proposed that these deficits deregulate the integrated AMPK, mTOR, and sirtuin signaling networks. Black text represents biological effects of HMF that have been experimentally documented. Red text indicates specific genes or proteins whose modulation is hypothesized to occur downstream of HMF exposure, but which have not been directly verified. The black circles highlight the three key signaling pathways that are proposed to play a central role in translating initial mitochondrial damage into broader cellular outcomes.

AKT: Protein kinase B; HIF-1α: hypoxia-inducible factor 1-alpha; LCAD: long-chain acyl-CoA dehydrogenase; GDH: glutamate dehydrogenase; SOD: superoxide dismutase; IDH2: isocitrate dehydrogenase 2; ROS: reactive oxygen species; PKM2: pyruvate kinase M2; ANT2: adenine nucleotide translocator 2; MCD: malonyl-CoA decarboxylase; PDH: pyruvate dehydrogenase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; NADPH: nicotinamide adenine dinucleotide phosphate.

AMPK signaling pathway

As demonstrated in previous studies, HMF affects ATP levels and mitochondrial biogenesis regulator PGC-1α.^13,51 The serine/threonine kinase AMPK signaling pathway is a central energy sensor in cellular metabolism that regulates energy homeostasis by monitoring intracellular ATP levels through the detection of the AMP/ATP ratio. An increased intracellular AMP/ATP ratio (indicating reduced ATP levels) activates AMPK.¹⁰¹ Therefore, we hypothesize that the HMF-induced bioenergetic crisis (i.e., decreased ATP) serves as the primary signal that triggers the activation of the AMPK pathway. Activation of the AMPK pathway is a compensatory stress response aimed at restoring energy balance. Activated AMPK then promotes the expression of PGC-1α through phosphorylation. This regulates mitochondrial DNA replication and the expression of genes related to ETC to enhance mitochondrial biogenesis. Therefore, it is likely that the activation of the AMPK pathway is a protective response to counteract the energy deficiency induced by HMF by stimulating the production of new, functional mitochondria. Further experimental validation is essential to confirm the role of the AMPK pathway in HMF-induced mitochondrial alterations.

Mammalian target of rapamycin signaling pathway

Current evidence indicates that HMF modulates cellular metabolism, particularly processes involved in glycolysis, OXPHOS, and lipid metabolism.^{13,14,23,26,28,29} Furthermore, our recent research demonstrates that the HMF-induced suppression of cell proliferation is reversible upon re-exposure to GMF. This process is mediated by activated mTOR phosphorylation.¹⁰² mTOR is a highly conserved serine/threonine protein kinase belonging to the PI3K-related kinase family. It plays a central role in regulating mitochondrial metabolism. mTOR forms two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2).¹⁰³ mTORC1 enhances the translation of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α), promoting a shift in cellular metabolism from OXPHOS to glycolysis.¹⁰⁴ In addition, mTORC1 regulates AKT-dependent lipid synthesis.¹⁰⁵ Conversely, mTORC2 is involved in glycolysis, lipid synthesis, and cell migration via AKT-dependent mechanisms that differ from those of mTORC1.¹⁰⁶ It has been proposed that the AMPK-dependent activation of mTORC2 aims to engage the anti-apoptotic AKT signaling pathway, thereby promoting cell survival during acute energy stress.¹⁰⁷ Metabolic disturbances induced by HMF are likely to be sensed by the mTOR signaling pathway network. We hypothesize that the modulation of mTOR activity in the presence of HMF represents a complex stress adaptation. The exact role of mTOR in HMF-mediated effects requires further investigation to establish causality.

Sirtuin signaling pathway

The review found that, in addition to reducing MMP and ATP content and affecting cellular metabolism, HMF can disrupt ROS homeostasis (Table 1). Research indicates that the sirtuin signaling pathway plays a pivotal role in the regulation of ETC activity, ATP levels, cellular metabolism, and ROS homeostasis.¹⁰⁸ The sirtuin protein family is a class of highly conserved NAD⁺-dependent deacetylases whose activity is closely linked to the NAD⁺/NADH ratio. Sirtuins play crucial regulatory roles in various physiological processes, including glucose and lipid metabolism, the response to oxidative stress, cellular senescence, apoptosis, and autophagy.¹⁰⁹ Given that exposure to HMF impacts both the energy charge (ATP/ADP/AMP) and, due to ETC dysfunction, likely the NAD⁺/NADH ratio, we propose that these changes serve as key activating signals for sirtuin signaling pathways. The subsequent modulation of sirtuin signaling activity (e.g., SIRT3 and SIRT5) is therefore a coordinated stress response that aims to fine-tune mitochondrial metabolism, enhance antioxidant defense, and promote metabolic adaptation in order to cope with HMF-induced challenges.

Specifically, SIRT3 primarily maintains intracellular ATP levels by enhancing the activities of ETC complexes I, II, and V.^110,111 Notably, SIRT3 interacts with ATP synthase to modulate MMP.¹¹² It promotes ROS scavenging by activating isocitrate dehydrogenase 2 (IDH2), glutamate dehydrogenase (GDH), and SOD2.¹¹³ It also regulates fatty acid β-oxidation by activating long-chain specific acyl-CoA dehydrogenase (LCAD).¹¹⁴ SIRT4 reduces ATP production by uncoupling the mitochondria via the adenine nucleotide translocator 2 (ANT2) protein and by downregulating GDH activity (102). SIRT5 increases glycolysis by activating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase M2 (PKM2).^115,116 It modulates the TCA cycle through the STAT3-mediated inhibition of PDC and suppression of succinate dehydrogenase (SDH) activity.^117,118 Additionally, SIRT5 activates IDH2 to promote NADPH generation and regulates redox balance via the activation of Cu/Zn superoxide dismutase (SOD1).^117,119

Given the central role of the sirtuin signaling pathway in regulating ETC activity, ATP levels, cellular metabolism, and ROS homeostasis, we propose that disruption to the sirtuin signaling pathway is a critical downstream mechanism through which HMF exerts its broad effects on mitochondrial function. However, this proposition is speculative and necessitates dedicated studies to verify the functional involvement of sirtuins in HMF-induced mitochondrial responses.

Connections between signaling pathways

It is important to note that these three signaling pathways do not function in isolation; rather, they exhibit extensive crosstalk in various biological contexts. Based on this, we hypothesize that they may form an integrated network in response to HMF-induced energetic stress that coordinately dictates the ultimate cellular outcome. For example, reduced ATP production elevates the intracellular AMP/ATP and ADP/ATP ratios, thereby activating AMPK. Subsequently, AMPK activation can inhibit mTORC1 by either promoting TSC complex activity or by directly phosphorylating RAPTOR. Conversely, mTORC1 reciprocally phosphorylates AMPK to limit its activity.¹²⁰ AMPK also enhances mTORC2 activity independently of mTORC1-mediated negative feedback by specifically phosphorylating mTOR at Ser1261 within the mTORC2 complex.¹⁰⁷ Additionally, SIRT1 activates AMPK via NAD⁺ sensing,¹²¹ while AMPK phosphorylates NAMPT (the rate-limiting enzyme in NAD⁺ biosynthesis), establishing a positive feedback loop that increases NAD⁺ production.¹²² There is a notable antagonistic relationship exists between SIRT1 and mTOR signaling. SIRT1 represses mTORC1 via TSC2, thereby stimulating autophagy and mitochondrial turnover. Conversely, mTOR activation reciprocally inhibits SIRT1, impairing cellular stress adaptation.¹²³ Interestingly, under SIRT4-deficient conditions, AMPK activation increases the expression of PGC-1α and its downstream targets involved in β-oxidation and mitochondrial biogenesis.¹²⁴ While the interactions between these pathways are biologically plausible, the manner in which they are integrated and function within the context of HMF exposure remains an open and unvalidated question. We hypothesize that the primary disruption of mitochondrial Fe-S clusters by HMF leads to the deregulation of this integrated signaling network. The resulting net imbalance, manifested as incoherent communication between AMPK, mTOR, and sirtuins pathways, leads to a failure to fine-tune energy homeostasis, metabolic flux, and stress resilience. This signaling dysfunction is a key molecular basis for the wide-ranging functional deficits observed in mitochondria under HMF exposure. Validating these crosstalk mechanisms under HMF conditions represents a significant and necessary future research endeavor.

However, it is important to emphasize that the involvement of these pathways remains hypothetical, and their validation is a crucial direction for future research rather than an established mechanism. This provides novel insights into investigating the molecular mechanisms underlying the effects of HMF on mitochondrial structure and function.

Conclusion

In summary, this review provides a systematic synthesis of the research into the effects of HMF on mitochondrial quality control and function. Specifically, our analysis reveals that exposure to HMF significantly alters the mitochondrial structure and impairs essential physiological processes, including energy production, cellular metabolism, ROS homeostasis, and Ca²⁺ buffering capacity. Further in-depth analysis suggests that the effects of HMF may originate from its interference with electron transfer processes and/or electron cloud distribution within Fe-S clusters. We propose that these primary defects trigger a bioenergetic crisis characterized by decreased ATP and MMP and increased ROS. This crisis activates key stress-response signaling pathways—AMPK, mTOR, and sirtuin pathways—as a compensatory, pro-survival mechanism. These pathways work together to try to restore energy homeostasis (via AMPK/PGC-1α), adapt metabolism (via mTOR/HIF-1α and sirtuins/TCA cycle enzymes), and boost antioxidant defenses (via sirtuins/SOD). However, under prolonged or intense HMF stress, these compensatory efforts may become overwhelmed or dysregulated, ultimately failing to prevent the observed mitochondrial and cellular dysfunction. In conclusion, future studies should focus on elucidating the precise regulatory role of HMF in mitochondrial quality control and function, and on investigating the influence of HMF on electron transfer and distribution in Fe-S clusters from biophysical and quantum mechanical perspectives. This will be crucial for clarifying the intrinsic mechanisms underlying the biological effects of HMF and may contribute to understanding the secrets of biological magnetoreception.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant 42388101) and the CAS Project for Young Scientists in Basic Research (No. YSBR-097).

Author contributions

Conceptualization, R.J.; investigation, R.J., W.M.L., and Z.C.C.; validation, R.J., W.M.L., Z.C.C., and L.Y.K., writing – original draft, R.J.; writing – review and editing, R.J., and W.M.L.; supervision, R.J. and T.L.X.

Declaration of interests

The authors declare no conflicts of interest.