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AIM: Peroxisomes play a key role in lipid metabolism, and peroxisome defects have been associated with neurodegenerative diseases such as X-adrenoleukodystrophy and Alzheimer's disease. This study aims to elucidate the contribution of peroxisomes in lipid alterations of area 8 of the frontal cortex in the spectrum of TDP43-proteinopathies. Cases of frontotemporal lobar degeneration-TDP43 (FTLD-TDP), manifested as sporadic (sFTLD-TDP) or linked to mutations in various genes including expansions of the non-coding region of C9ORF72 (c9FTLD), and of sporadic amyotrophic lateral sclerosis (sALS) as the most common TDP43 proteinopathies, were analysed. METHODS: We used transcriptomics and lipidomics methods to define the steady-state levels of gene expression and lipid profiles. RESULTS: Our results show alterations in gene expression of some components of peroxisomes and related lipid pathways in frontal cortex area 8 in sALS, sFTLD-TDP and c9FTLD. Additionally, we identify a lipidomic pattern associated with the ALS-FTLD-TDP43 proteinopathy spectrum, notably characterised by down-regulation of ether lipids and acylcarnitine among other lipid species, as well as alterations in the lipidome of each phenotype of TDP43 proteinopathy, which reveals commonalities and disease-dependent differences in lipid composition. CONCLUSION: Globally, lipid alterations in the human frontal cortex of the ALS-FTLD-TDP43 proteinopathy spectrum, which involve cell membrane composition and signalling, vulnerability against cellular stress and possible glucose metabolism, are partly related to peroxisome impairment.
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Esclerosis Amiotrófica Lateral/metabolismo , Lóbulo Frontal/metabolismo , Metabolismo de los Lípidos , Peroxisomas/metabolismo , Proteinopatías TDP-43/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neuronas/metabolismoRESUMEN
Lipids are closely associated with brain structure and function. However, the potential changes in the lipidome induced by aging remain to be elucidated. In this study, we used chromatographic techniques and a mass spectrometry-based approach to evaluate age-associated changes in the lipidome of the frontal cortex and cerebellum obtained from adult male Wistar rats (8 months), aged male Wistar rats (26 months), and aged male Wistar rats submitted to a methionine restriction diet (MetR)-as an anti-aging intervention-for 8 weeks. The outcomes revealed that only small changes (about 10%) were observed in the lipidome profile in the cerebellum and frontal cortex during aging, and these changes differed, in some cases, between regions. Furthermore, a MetR diet partially reversed the effects of the aging process. Remarkably, the most affected lipid classes were ether-triacylglycerols, diacylglycerols, phosphatidylethanolamine N-methylated, plasmalogens, ceramides, and cholesterol esters. When the fatty acid profile was analyzed, we observed that the frontal cortex is highly preserved during aging and maintained under MetR, whereas in the cerebellum minor changes (increased monounsaturated and decreased polyunsaturated contents) were observed and not reversed by MetR. We conclude that the rat cerebellum and frontal cortex have efficient mechanisms to preserve the lipid profile of their cell membranes throughout their adult lifespan in order to maintain brain structure and function. A part of the small changes that take place during aging can be reversed with a MetR diet applied in old age.
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Envejecimiento/genética , Lóbulo Frontal/metabolismo , Lípidos/genética , Metionina/metabolismo , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Cerebelo/metabolismo , Cerebelo/patología , Cromatografía , Lóbulo Frontal/patología , Humanos , Lipidómica/normas , Espectrometría de Masas , Estrés Oxidativo/genética , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Lipids were determinants in the appearance and evolution of life. Recent studies disclose the existence of a link between lipids and animal longevity. Findings from both comparative studies and genetics and nutritional interventions in invertebrates, vertebrates, and exceptionally long-lived animal species-humans included-demonstrate that both the cell membrane fatty acid profile and lipidome are a species-specific optimized evolutionary adaptation and traits associated with longevity. All these emerging observations point to lipids as a key target to study the molecular mechanisms underlying differences in longevity and suggest the existence of a lipidome profile of long life.
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Membrana Celular/metabolismo , Metabolismo de los Lípidos , Longevidad , Animales , HumanosRESUMEN
AIMS/HYPOTHESIS: We aimed to investigate the potential mechanisms involved in the compromised adipogenesis of visceral (VAT) vs subcutaneous adipose tissue (SAT) using comparative metabolomics. Based on the differentially identified metabolites, we focused on the relationship between the active form of vitamin B6 (pyridoxal 5-phosphate [PLP]), known to be generated through pyridoxal kinase (PDXK), and adipogenesis. METHODS: Non-targeted metabolomics analyses were performed in paired VAT and SAT (n = 14, discovery cohort). PDXK gene expression was evaluated in two validation cohorts of paired SAT and VAT samples in relation to obesity status and insulin sensitivity, and mechanistically after weight loss in vivo and in 3T3-L1 cells in vitro. RESULTS: Comparative metabolomics showed that PLP was significantly decreased in VAT vs SAT. Concordantly, PDXK mRNA levels were significantly decreased in VAT vs SAT, specifically in adipocytes. The decrease was specially marked in obese individuals. PDXK mRNA levels showed a strong association with adipogenic, lipid-droplet-related and lipogenic genes. At a functional level, systemic insulin sensitivity positively associated with PDXK expression, and surgically-induced weight loss (improving insulin sensitivity) led to increased SAT PDXK mRNA levels in parallel with adipogenic genes. In human pre-adipocytes, PDXK mRNA levels increased during adipocyte differentiation and after administration of peroxisome proliferator-activated receptor-γ agonists, and decreased under inflammatory stimuli. Mechanistic studies in 3T3-L1 cells showed that PLP administration resulted in increased adipogenic mRNA markers during early adipogenesis, whereas the PLP antagonist 4-deoxypyridoxine exerted opposite effects. CONCLUSIONS/INTERPRETATION: Overall, these results support the notion that in situ production of PLP is required for physiological adipogenesis.
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Tejido Adiposo/metabolismo , Metabolómica/métodos , Piridoxal Quinasa/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Adipogénesis/genética , Adipogénesis/fisiología , Adulto , Animales , Femenino , Humanos , Resistencia a la Insulina , Grasa Intraabdominal/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Obesidad/metabolismo , Grasa Subcutánea/metabolismo , Vitamina B 6/metabolismoRESUMEN
Friedreich ataxia (FRDA) is a neurodegenerative disease characterized by a decreased expression of the mitochondrial protein frataxin. Major neurological symptoms of the disease are due to degeneration of dorsal root ganglion (DRG) sensory neurons. In this study we have explored the neurodegenerative events occurring by frataxin depletion on primary cultures of neurons obtained from rat DRGs. Reduction of 80% of frataxin levels in these cells was achieved by transduction with lentivirus containing shRNA silencing sequences. Frataxin depletion caused mitochondrial membrane potential decrease, neurite degeneration and apoptotic cell death. A marked increase of free intracellular Ca(2+) levels and alteration in Ca(2+)-mediated signaling pathways was also observed, thus suggesting that altered calcium homeostasis can play a pivotal role in neurodegeneration caused by frataxin deficiency. These deleterious effects were reverted by the addition of a cell-penetrant TAT peptide coupled to the BH4, the anti-apoptotic domain of Bcl-x(L). Treatment of cultured frataxin-depleted neurons with TAT-BH4 was able to restore the free intracellular Ca(2+) levels and protect the neurons from degeneration. These observations open the possibility of new therapies of FRDA based on modulating the Ca(2+) signaling and prevent apoptotic process to protect DRG neurons from neurodegeneration.
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Apoptosis/genética , Ganglios Espinales/citología , Proteínas de Unión a Hierro/genética , Células Receptoras Sensoriales/citología , Proteína bcl-X/genética , Animales , Calcio/metabolismo , Señalización del Calcio/genética , Células Cultivadas , Ataxia de Friedreich/genética , Productos del Gen tat/genética , Homeostasis , Proteínas de Unión a Hierro/biosíntesis , Proteínas de Unión a Hierro/metabolismo , Potencial de la Membrana Mitocondrial/genética , Proteínas Mitocondriales/metabolismo , Neuritas/patología , Enfermedades Neurodegenerativas , Estructura Terciaria de Proteína/genética , Interferencia de ARN , ARN Interferente Pequeño , Ratas , FrataxinaRESUMEN
Obesity is a risk factor for highly prevalent age-related neurodegenerative diseases, the pathogenesis of whichinvolves mitochondrial dysfunction and protein oxidative damage. Lipoxidation, driven by high levels of peroxidizable unsaturated fatty acids and low antioxidant protection of the brain, stands out as a significant risk factor. To gain information on the relationship between obesity and brain molecular damage, in a porcine model of obesity we evaluated (1) the level of mitochondrial respiratory chain complexes, as the main source of free radical generation, by Western blot; (2) the fatty acid profile by gas chromatography; and (3) the oxidative modification of proteins by mass spectrometry. The results demonstrate a selectively higher amount of the lipoxidation-derived biomarker malondialdehyde-lysine (MDAL) (34% increase) in the frontal cortex, and positive correlations between MDAL and LDL levels and body weight. No changes were observed in brain fatty acid profile by the high-fat diet, and the increased lipid peroxidative modification was associated with increased levels of mitochondrial complex I (NDUFS3 and NDUFA9 subunits) and complex II (flavoprotein). Interestingly, introducing n3 fatty acids and a probiotic in the high-fat diet prevented the observed changes, suggesting that dietary components can modulate protein oxidative modification at the cerebral level and opening new possibilities in neurodegenerative diseases' prevention.
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It is assumed that the human brain is especially susceptible to oxidative stress, based on specific traits such as a higher rate of mitochondrial free radical production, a high content in peroxidizable fatty acids, and a low antioxidant defense. However, it is also evident that human neurons, although they are post-mitotic cells, survive throughout an entire lifetime. Therefore, to reduce or avoid the impact of oxidative stress on neuron functionality and survival, they must have evolved several adaptive mechanisms to cope with the deleterious effects of oxidative stress. Several of these antioxidant features are derived from lipid adaptations. At least six lipid adaptations against oxidative challenge in the healthy human brain can be discerned. In this work, we explore the idea that neurons and, by extension, the human brain is endowed with an important arsenal of non-pro-oxidant and antioxidant measures to preserve neuronal function, refuting part of the initial premise.
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One of the richest tissues in lipid content and diversity of the human body is the brain. The human brain is constitutively highly vulnerable to oxidative stress. This oxidative stress is a determinant in brain aging, as well as in the onset and progression of sporadic (late-onset) Alzheimer's disease (sAD). Glycerophospholipids are the main lipid category widely distributed in neural cell membranes, with a very significant presence for the ether lipid subclass. Ether lipids have played a key role in the evolution of the human brain compositional specificity and functionality. Ether lipids determine the neural membrane structural and functional properties, membrane trafficking, cell signaling and antioxidant defense mechanisms. Here, we explore the idea that ether lipids actively participate in the pathogenesis of sAD. Firstly, we evaluate the quantitative relevance of ether lipids in the human brain composition, as well as their role in the human brain evolution. Then, we analyze the implications of ether lipids in neural cell physiology, highlighting their inherent antioxidant properties. Finally, we discuss changes in ether lipid content associated with sAD and their physiopathological implications, and propose a mechanism that, as a vicious cycle, explains the potential significance of ether lipids in sAD.
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Celiac disease (CD) is included in the group of complex or multifactorial diseases, i.e., those caused by the interaction of genetic and environmental factors. Despite a growing understanding of the pathophysiological mechanisms of the disease, diagnosis is still often delayed and there are no effective biomarkers for early diagnosis. The only current treatment, a gluten-free diet (GFD), can alleviate symptoms and restore intestinal villi, but its cellular effects remain poorly understood. To gain a comprehensive understanding of CD's progression, it is crucial to advance knowledge across various scientific disciplines and explore what transpires after disease onset. Metabolomics studies hold particular significance in unravelling the complexities of multifactorial and multisystemic disorders, where environmental factors play a significant role in disease manifestation and progression. By analyzing metabolites, we can gain insights into the reasons behind CD's occurrence, as well as better comprehend the impact of treatment initiation on patients. In this review, we present a collection of articles that showcase the latest breakthroughs in the field of metabolomics in pediatric CD, with the aim of trying to identify CD biomarkers for both early diagnosis and treatment monitoring. These advancements shed light on the potential of metabolomic analysis in enhancing our understanding of the disease and improving diagnostic and therapeutic strategies. More studies need to be designed to cover metabolic profiles in subjects at risk of developing the disease, as well as those analyzing biomarkers for follow-up treatment with a GFD.
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Enfermedad Celíaca , Humanos , Niño , Dieta Sin Gluten , Mucosa Intestinal , Metabolómica , Biomarcadores , GlútenesRESUMEN
Non-targeted LC-MS/MS-based lipidomic analysis was conducted in post-mortem human grey matter frontal cortex area 8 (GM) and white matter of the frontal lobe centrum semi-ovale (WM) to identify lipidome fingerprints in middle-aged individuals with no neurofibrillary tangles and senile plaques, and cases at progressive stages of sporadic Alzheimer's disease (sAD). Complementary data were obtained using RT-qPCR and immunohistochemistry. The results showed that WM presents an adaptive lipid phenotype resistant to lipid peroxidation, characterized by a lower fatty acid unsaturation, peroxidizability index, and higher ether lipid content than the GM. Changes in the lipidomic profile are more marked in the WM than in GM in AD with disease progression. Four functional categories are associated with the different lipid classes affected in sAD: membrane structural composition, bioenergetics, antioxidant protection, and bioactive lipids, with deleterious consequences affecting both neurons and glial cells favoring disease progression.
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Aging biology entails a cell/tissue deregulated metabolism that affects all levels of biological organization. Therefore, the application of "omic" techniques that are closer to phenotype, such as metabolomics, to the study of the aging process should be a turning point in the definition of cellular processes involved. The main objective of the present study was to describe the changes in plasma metabolome associated with biological aging and the role of sex in the metabolic regulation during aging. A high-throughput untargeted metabolomic analysis was applied in plasma samples to detect hub metabolites and biomarkers of aging incorporating a sex/gender perspective. A cohort of 1030 healthy human adults (45.9% females, and 54.1% males) from 50 to 98 years of age was used. Results were validated using two independent cohorts (1: n = 146, 53% females, 30-100 years old; 2: n = 68, 70% females, 19-107 years old). Metabolites related to lipid and aromatic amino acid (AAA) metabolisms arose as the main metabolic pathways affected by age, with a high influence of sex. Globally, we describe changes in bioenergetic pathways that point to a decrease in mitochondrial ß-oxidation and an accumulation of unsaturated fatty acids and acylcarnitines that could be responsible for the increment of oxidative damage and inflammation characteristic of this physiological process. Furthermore, we describe for the first time the importance of gut-derived AAA catabolites in the aging process describing novel biomarkers that could contribute to better understand this physiological process but also age-related diseases.
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Aminoácidos Aromáticos , Metaboloma , Masculino , Femenino , Humanos , Adulto , Persona de Mediana Edad , Anciano , Anciano de 80 o más Años , Adulto Joven , Aminoácidos Aromáticos/metabolismo , Envejecimiento/metabolismo , Metabolómica/métodos , Biomarcadores/metabolismoRESUMEN
Methionine constitutes a central hub of intracellular metabolic adaptations leading to an extended longevity (maximum lifespan). The present study follows a comparative approach analyzing methionine and related metabolite and amino acid profiles using an LC-MS/MS platform in the hearts of seven mammalian species with a longevity ranging from 3.8 to 57 years. Our findings demonstrate the existence of species-specific heart phenotypes associated with high longevity characterized by: (i) low concentration of methionine and its related sulphur-containing metabolites; (ii) low amino acid pool; and (iii) low choline concentration. Our results support the existence of heart metabotypes characterized by a down-regulation in long-lived species, supporting the idea that in longevity, less is more.
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The primary function of frataxin, a mitochondrial protein involved in iron homeostasis, remains controversial. Using a yeast model of conditional expression of the frataxin homologue YFH1, we analyzed the primary effects of YFH1 depletion. The main conclusion unambiguously points to the up-regulation of iron transport systems as a primary effect of YFH1 down-regulation. We observed that inactivation of aconitase, an iron-sulfur enzyme, occurs long after the iron uptake system has been activated. Decreased aconitase activity should be considered part of a group of secondary events promoted by iron overloading, which includes decreased superoxide dismutase activity and increased protein carbonyl formation. Impaired manganese uptake, which contributes to superoxide dismutase deficiency, has also been observed in YFH1-deficient cells. This low manganese content can be attributed to the down-regulation of the metal ion transporter Smf2. Low Smf2 levels were not observed in AFT1/YFH1 double mutants, indicating that high iron levels could be responsible for the Smf2 decline. In summary, the results presented here indicate that decreased iron-sulfur enzyme activities in YFH1-deficient cells are the consequence of the oxidative stress conditions suffered by these cells.
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Regulación Fúngica de la Expresión Génica , Proteínas de Unión a Hierro/genética , Proteínas Hierro-Azufre/química , Hierro/metabolismo , Saccharomyces cerevisiae/metabolismo , Aconitato Hidratasa/metabolismo , Proteínas de Transporte de Catión/metabolismo , Iones , Proteínas de Unión a Hierro/fisiología , Manganeso/química , Manganeso/metabolismo , Modelos Biológicos , Estrés Oxidativo , Consumo de Oxígeno , Plásmidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fracciones Subcelulares/metabolismo , Regulación hacia Arriba , FrataxinaRESUMEN
Available evidences point to methionine metabolism as a key target to study the molecular adaptive mechanisms underlying differences in longevity. The plasma methionine metabolic profile was determined using a LC-MS/MS platform to systematically define specific phenotypic patterns associated with genotypes of human extreme longevity (centenarians). Our findings demonstrate the presence of a specific plasma profile associated with human longevity characterized by an enhanced transsulfuration pathway and tricarboxylic acid (TCA) cycle intermediates, as well as a reduced content of specific amino acids. Furthermore, our work reveals that centenarians maintain a strongly correlated methionine metabolism, suggesting an improved network integrity, homeostasis and more tightly regulated metabolism. We have discovered a particular methionine signature related to the condition of extreme longevity, allowing the identification of potential mechanisms and biomarkers of healthy aging.
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Metionina , Espectrometría de Masas en Tándem , Anciano de 80 o más Años , Cromatografía Liquida , Humanos , Longevidad/genética , Metaboloma , Metionina/metabolismoRESUMEN
Friedreich ataxia is a genetic disease caused by the deficiency of frataxin, a mitochondrial protein. Frataxin deficiency impacts in the cell physiology at several levels. One of them is oxidative stress with consequences in terms of protein dysfunctions and metabolic alterations. Among others, alterations in lipid metabolism have been observed in several models of the disease. In this review we summarize the current knowledge of the molecular basis of the disease, the relevance of oxidative stress and the therapeutic strategies based on reduction of mitochondrial reactive oxygen species production. Finally, we will focus the interest in alterations of lipid metabolism as a consequence of mitochondrial dysfunction and describe the therapeutic approaches based on targeting lipid metabolism.
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Ataxia de Friedreich/metabolismo , Metabolismo de los Lípidos , Mitocondrias/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Animales , Modelos Animales de Enfermedad , HumanosRESUMEN
Brain neurons offer diverse responses to stresses and detrimental factors during development and aging, and as a result of both neurodegenerative and neuropsychiatric disorders. This multiplicity of responses can be ascribed to the great diversity among neuronal populations. Here we have determined the metabolomic profile of three healthy adult human brain regions-entorhinal cortex, hippocampus, and frontal cortex-using mass spectrometry-based technologies. Our results show the existence of a lessened energy demand, mitochondrial stress, and lower one-carbon metabolism (particularly restricted to the methionine cycle) specifically in frontal cortex. These findings, along with the better antioxidant capacity and lower mTOR signaling also seen in frontal cortex, suggest that this brain region is especially resistant to stress compared to the entorhinal cortex and hippocampus, which are more vulnerable regions. Globally, our results show the presence of specific metabolomics adaptations in three mature, healthy human brain regions, confirming the existence of cross-regional differences in cell vulnerability in the human cerebral cortex.
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Friedreich ataxia (FRDA) is a hereditary disease caused by deficient frataxin expression. This mitochondrial protein has been related to iron homeostasis, energy metabolism, and oxidative stress. Patients with FRDA experience neurologic alterations and cardiomyopathy, which is the leading cause of death. The specific effects of frataxin depletion on cardiomyocytes are poorly understood because no appropriate cardiac cellular model is available to researchers. To address this research need, we present a model based on primary cultures of neonatal rat ventricular myocytes (NRVMs) and short-hairpin RNA interference. Using this approach, frataxin was reduced down to 5 to 30% of control protein levels after 7 days of transduction. At this stage the activity and amount of the iron-sulfur protein aconitase, in vitro activities of several OXPHOS components, levels of iron-regulated mRNAs, and the ATP/ADP ratio were comparable to controls. However, NRVMs exhibited markers of oxidative stress and a disorganized mitochondrial network with enlarged mitochondria. Lipids, the main energy source of heart cells, also underwent a clear metabolic change, indicated by the increased presence of lipid droplets and induction of medium-chain acyl-CoA dehydrogenase. These results indicate that mitochondria and lipid metabolism are primary targets of frataxin deficiency in NRVMs. Therefore, they contribute to the understanding of cardiac-specific mechanisms occurring in FRDA and give clues for the design of cardiac-specific treatment strategies for FRDA.
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Cardiomiopatías/patología , Proteínas de Unión a Hierro/genética , Metabolismo de los Lípidos/genética , Mitocondrias Cardíacas/patología , Miocitos Cardíacos/metabolismo , Aconitato Hidratasa/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Ataxia de Friedreich/patología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/citología , Estrés Oxidativo/fisiología , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Ratas Sprague-Dawley , FrataxinaRESUMEN
Protein carbonyl detection has been commonly used to analyze the degree of damage to proteins under oxidative stress conditions. Most laboratories rely on derivatization of carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this approach, Western blot and immunodetection are no longer needed, shortening the procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy hydrazide allows easy matching of the spots of interest and those obtained by general fluorescent protein staining methods, which facilitates excising target proteins from the gels and identifying them. This method is effective for detecting protein carbonylation in samples of proteins submitted to metal-catalyzed oxidation "in vitro" and assessing the effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast cells.
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Colorantes Fluorescentes/química , Hidrazinas/química , Estrés Oxidativo/fisiología , Carbonilación Proteica/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Electroforesis Bidimensional Diferencial en Gel/métodos , Espectrometría de Fluorescencia/métodosRESUMEN
Iron overload is involved in several pathological conditions, including Friedreich ataxia, a disease caused by decreased expression of the mitochondrial protein frataxin. In a previous study, we identified 14 proteins selectively oxidized in yeast cells lacking Yfh1, the yeast frataxin homolog. Most of these were magnesium-binding proteins. Decreased Mn-SOD activity, oxidative damage to CuZn-SOD, and increased levels of chelatable iron were also observed in this model. This study explores the relationship between low SOD activity, the presence of chelatable iron, and protein damage. We observed that addition of copper and manganese to the culture medium restored SOD activity and prevented both oxidative damage and inactivation of magnesium-binding proteins. This protection was compartment specific: recovery of mitochondrial enzymes required the addition of manganese, whereas cytosolic enzymes were recovered by adding copper. Copper treatment also decreased Deltayfh1 sensitivity to menadione. Finally, a Deltasod1 mutant showed high levels of chelatable iron and inactivation of magnesium-binding enzymes. These results suggest that reduced superoxide dismutase activity contributes to the toxic effects of iron overloading. This would also apply to pathologies involving iron accumulation.