Breast cancer is characterized by specific metabolic changes that support tumorigenesis, highlighting the emerging appreciation of cancer as a metabolic disease. These metabolic changes are simultaneous with redox reprogramming with nuclear factor erythroid 2-related factor 2 (Nrf2) representing their master integrator. Given that interscapular brown adipose tissue (IBAT) influences whole-body metabolism, our goal was to investigate the redox-metabolic crosstalk between the tumor and the host at the systemic level by exploring Nrf2-driven metabolic changes that occur in IBAT in the orthotopic model of breast cancer in wild-type (WT) and mice lacking functional Nrf2 (Nrf2KO). We analyzed the protein expression of key enzymes involved in glucose and lipid metabolism in control groups and at different points during tumor growth (10 mg, 50 mg, 100 mg, 200 mg, and 400 mg). In both WT and Nrf2KO mice, the results indicated a transient induction of hexokinase 2 expression during the early phase of tumor growth (<100 mg). Accordingly, pyruvate dehydrogenase expression followed the same profile. In Nrf2KO mice, a general decline in glyceraldehyde 3-phosphate dehydrogenase, phosphofructokinase-1, and glucose-6-phosphate dehydrogenase expression was detected during the late phase of tumor growth (>100 mg). Since no changes in WT mice occurred, these findings are considered Nrf2-dependent. Concomitantly, a decrease in protein expression of fatty acid synthase and acetyl-CoA carboxylase in Nrf2KO mice was observed. These observations correspond to decreased levels of 5'-AMP-activated protein kinase and hypoxia-inducible factor 1 during the late-phase (>100 mg) of tumor growth in Nrf2KO mice which suggests their involvement in transcriptional regulation. Our results revealed that IBAT metabolism responds to tumor growth and underscored that this communication is Nrf2-dependent giving implications for further understanding of breast cancer in the light of systemic metabolic disease.

This research was supported by the Science Fund of the Republic of Serbia, #7750238, Exploring new avenues in breast cancer research: Redox and metabolic reprogramming of cancer and associated adipose tissue - REFRAME.

Increased damage by ROS plays a role in the development of neurodegenerative diseases, especially Alzheimer’s Disease and other dementias, and diets rich in antioxidants (high intake of fruits and vegetables) seem neuroprotective (as well as being protective against many other age-related diseases). However, attempts to treat/prevent such diseases by giving high doses of antioxidants such as vitamins E and C and carotenoids have, overall, been unsuccessful. Reasons for this will be discussed. A major focus of our work is a unique diet-derived thiol/thione with antioxidant properties, namely ergothioneine (ET). Low blood levels of ET are a risk factor for the development of neurodegenerative and cardiovascular diseases, frailty, eye disease, pre-eclampsia and age-related diseases generally. We have identified “adequate levels” of plasma ET in humans, levels below which are associated with increased disease occurrence, and the reasons leading to these low levels are under investigation. In animal studies, ET has exhibited the ability to modulate inflammation, scavenge certain ROS, protect against acute respiratory distress syndrome, decrease brain damage in models of Parkinson and Alzheimer diseases and stroke, prevent endothelial dysfunction, protect against ischemia-reperfusion injury, counteract iron dysregulation, hinder lung and liver fibrosis, and mitigate damage to the lungs, kidneys, liver, gastrointestinal tract, and testis. ET may also influence the gut microbiome. There is evidence that ET is specifically accumulated at sites of tissue injury, so we have called it an “adaptive antioxidant” that may not interfere with the normal physiological roles of ROS. But does low ET predispose to age-related diseases or is it a spurious correlation? Extensive cell and animal studies strongly suggest the former. Caveats in the use of ergothioneine supplements to prevent/ameliorate aged-related diseases include its potential to generate trimethylamine-N-oxide by the action of ergothionase enzymes in gut bacteria and its ability to be taken up by many bacteria, a few of which are pathogenic (e.g. H. pylori, M. tuberculosis). These caveats will be discussed.

Changes in carbohydrate metabolism are a key feature of aging which also manifest in the epidermis. Furthermore, the synthesis and distribution of epidermal lipids changes with age. Both these parameters cannot be investigated with immunohistochemistry, as neither serves as useful epitope. We developed a multimodal analytical histocytometry approach combining modalities that localize lipids and enzymatic activities with immunofluorescent imaging of the skin to localize changes that are correlated with appearance of senescent cells. The activities of key metabolic enzymes were determined on tissue sections of aged and juvenile skin with a formazan-based assay. Lipids were localized and quantified using FTICR MALDI - mass spectrometric imaging. We correlated those modalities with immunofluorescent imaging and analyzed the intensities of the respective signals at single cell level, using Strataquest tissue cytometry. We analyzed skin from donors of young (< 30 y) versus advanced (> 67 y) ages and we investigated epidermal equivalent models containing labeled UV-damaged or senescent keratinocytes. Enzymatic activities displayed specific patterns across the stratifying epidermis, and had diverging trajectories in aging, with a marked decrease in suprabasal glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD, the rate limiting enzyme of the pentose phosphate pathway was also identified as a rapid response pathway activated upon UV damage in the epidermis.  The lipid molecular imaging identified differentiation- and age-related changes of polar lipids in skin biopsies and epidermal equivalents, and pro-senescent stress dependent reactive aldehydophospholipid species in the basal epidermal layers.  While these methodologies are still in development, it is evident that correlative analytical imaging – with the aid of AI driven histocytometry – will continue to yield novel insights into skin and epidermal biology by localizing previously undetectable parameters within the epidermis in the context of aging.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that plays an essential role in the inflammatory response and various other biological effects such as activation of apoptosis and oxidative stress. Fructose-enriched diets have previously been associated with the development of low-grade inflammation leading to metabolic stress. The aim of the present study was to investigate the combined effects of deletion of the Mif gene and a 9-week 20% fructose-enriched diet on metabolic inflammation, apoptosis, and oxidative stress in the liver of wild-type (WT) and Mif knockout (MIF−/−) male C57Bl/6J mice. We analyzed liver histology and expression of pro-inflammatory genes: Tumor necrosis factor (TNF), interleukin 1β (IL-1β), and IL-6. Antioxidant activity was estimated by the protein levels of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD1), mitochondrial MnSOD (SOD2), glutathione reductase (GR) and glutathione peroxidase (GPX). The results showed that antioxidant protection was activated in the liver of MIF-deficient mice. Increased hepatic expression of the cytokines IL-6 and IL-1β was observed in the same animals. Histologic analysis confirmed the presence of apoptosis, inflammation, enlarged Kupffer cells, and regenerative changes, such as binucleated hepatocytes, anisonucleosis, and anisocytosis. In addition, confluent and focal necrosis was observed in the liver of MIF−/− mice, which was even more pronounced in the animals consuming fructose. In conclusion, MIF may play a protective role in metabolic stress, as inflammation, oxidative stress, apoptotic and necrotic changes occur in the liver in its absence.

Mitochondrial diseases are a large family of extremely heterogeneous disorders genetically determined by mutations in either the nuclear genome or the mitochondrial DNA. Most of the mitochondrial disease genes are expressed in all cell types. However, in many conditions, some cell types are more affected than others. However, the reasons for this tissue-specificity remain poorly understood. To investigate the functional basis of the striking tissue-specificity in mitochondrial diseases, we analyzed several bioenergetic parameters, including oxygen consumption rates, Q redox poise, and reactive oxygen species production in mouse brain and liver mitochondria fueled by different substrates. In addition, we determined how these functional parameters are affected by electron transport chain impairment in a tissue-specific manner using pathologically relevant mouse models lacking either Ndufs4 or Ttc19, leading to complex I or III defects, respectively. No cure is currently available for most of the mitochondrial diseases. We previously showed that the coordinated activation of autophagy, lysosomal biogenesis, and mitochondrial biogenesis by rapamycin, ameliorated the myopathic phenotype of a muscle-specific knockout mouse for Cox15 (Cox15sm), encoding an enzyme involved in heme A biosynthesis. However, the role of mitophagy has been poorly investigated. We found that urolithin A, a direct mitophagy inducer, improved motor performance and myopathy in the Cox15sm mice, without increasing the activity of the respiratory chain complexes in a 10 week-treatment. These results indicate that activation of mitophagy can be a suitable treatment to ameliorate mitochondrial myopathies.

Canine hyperadrenocorticism (HAC) or Cushing’s syndrome is a multisystemic clinical condition caused by chronic exposure to elevated concentrations of glucocorticoids. It has been considered that oxidative stress is implicated in pathophysiology of HAC. The exact impact of uric acid (UA) on oxidative stress in hyperadrenocorticism remains unclear, given its ability to act as both an antioxidant and a pro-oxidant. In addition, increased UA levels are related to the development of hypertension, dyslipidemia, and type II diabetes in humans with HAC. For this purpose, we aimed to investigate the association of UA with the components of oxidative stress in dogs with HAC. This study included 12 dogs with newly diagnosed HAC and 12 healthy controls. The oxidative stress in serum samples was assessed by advanced oxidation protein product (AOPP) and thiobarbituric acid–reactive substances (TBARS), and antioxidative status by total antioxidant capacity (TAC), reduced glutathione (GSH) and paraoxonase-1 (PON-1). Uric acid was compared between two groups and correlated with oxidative stress parameters. The results showed that dogs with HAC exerted markedly higher level of UA compared to healthy controls (p<0.001). Additionally, higher levels of AOPP and TBARS (p=0.001; p =0.043) were observed in the HAC group, indicating oxidative damage compared to the controls. Among antioxidants, only GSH exhibited a difference between groups (p=0.001). Correlation analysis of UA revealed strong association with TBARS (r=0.615; p=0.037), which implies that UA is linked to an increase of oxidative stress in canine Cushing’s syndrome. The results of this study indicate a possible pro-oxidant role of UA in dogs with HAC. 

Rett syndrome (RTT), a devastating neurodevelopmental disorder, is caused in 95% of the cases by mutations in the X-chromosome-localized MECP2 gene. RTT manifests as a range of multisystem disturbances including altered lipid profile, subclinical inflammation, and overall OxInflammatory status in which mitochondrial dysfunction acts as central player. To decipher the molecular mechanisms underlying the pathophysiological manifestations affecting patients, we investigated whether mitochondria may play a role in the aberrant immune and oxidative responses of RTT. Recent findings from our and other labs unraveled several abnormalities in RTT mitochondria including atypical mitochondrial structure, deregulated expression of genes encoding oxidative phosphorylation factors and mitochondrial organization factors, impaired mitochondrial quality control, depressed energetic profile, and augmented mt-ROS production. In other brain diseases, mitochondrial dysfunction is a vital event during the activation of NLPR3 inflammasome, a multi-protein complex involved in innate immune response, that represents a common denominator in the crosstalk between inflammation and oxidative stress. Interestingly, using primary fibroblasts and lympho-monocytes isolated from RTT patients, we found a constitutive hyperactivation of NLRP3:ASC inflammasome associated with increased levels of nuclear p65 and ASC proteins, and pro-IL-1β mRNA, without the ability to further respond to the LPS + ATP stimuli. Furthermore, increased circulating levels of ASC, interleukin (IL)-18, and 1β were found in RTT individuals, thus corroborating the aforementioned cellular findings. In order to evaluate NLRP3 involvement in the transition from pre-symptomatic to symptomatic phase of RTT, we detected higher serum levels of IL-1β and IL-18 in symptomatic Het mice compared to WT. Of note, increased gene expression of Il-1b, Nlrp3, and ASC was observed in Het brains at the pre-symptomatic stage, suggesting a likely role of NLRP3 impairment in the early stages of the disease. Preliminary data showed that treatment with resveratrol, known to improve mitochondrial function, ameliorated the RTT mouse phenotype by restoring levels of some NLRP3-related components. Furthermore, mitochondrial dysfunction can result in ferroptosis, a form of cell death characterized by iron-dependent lipid peroxidation and accumulation of reactive oxygen species.  After treatment with two ferroptosis inducers, erastin (GPX4 inhibitor) or RSL3 (inhibitor of the cystine/glutamate antiporter), we found changes in GPx and GR activity, alteration in GPX4 protein levels and increased formation of 4HNE protein adducts. Mitochondrial ROS production and lipid peroxidation levels were higher in RTT after ferroptosis induction, while co-treatment with ferrostatin-1, a well-known inhibitor of ferroptosis, significantly prevented these processes. Interestingly, co-treatment with mito-TEMPO, a mitochondria-targeted superoxide dismutase mimetic, mitigated mitochondrial oxidative burden and prevented ferroptosis cell death in RTT cells. Overall, our results demonstrate the decisive role of mitochondrial dysfunction in RTT OxInflammation. Thus, we can speculate that exposure of RTT cells to any condition affecting the already compromised mitochondrial function could not only hyperactivate the inflammatory status but also precipitate ferroptosis cell death. Targeting mitochondria in RTT could represent a strategic coadjuvant therapy to improve the quality of life of the affected patients.

The in vivo determination of oxidative stress always remains a great challenge. Our approach in Liège CHU consists of simultaneously measuring in blood samples four different kinds of biomarkers: enzymatic and non-enzymatic antioxidants, trace elements, markers of oxidative damage to lipids, and identification of sources leading to increased reactive oxygen species (ROS) production. All these biomarkers (n = 16) have been investigated in patients: 1) with Abdominal Aortic Aneurysm (AAA)¹ or operated for Thoracic Abdominal Dissection (TAD)², 2) suffering from Chronic Obstructive Pulmonary Disease (COPD)³ or FacioScapuloHumeral Myopathy (FSHM)⁴, 3) with COVID-19^5,6 and 4) with delirium⁷. When compared to our internal reference values, depletion in non-enzymatic antioxidants (vitamin C, β-carotene, vitamin C/vitamin E ratio, thiol proteins) and trace elements (zinc, selenium) was observed in the majority of these pathologies. By contrast, increased levels in glutathione peroxidase, copper/zinc ratio, lipid peroxides (ROOH), and myeloperoxidase are common in all these diseases.

Mechanisms and factors that lead to the formation of lipomas, benign tumors of adipose tissue, are still insufficiently elucidated. Mesenchymal stem cells (MSCs) isolated from lipomas have some similar characteristics to MSCs isolated from white adipose tissue but differ at the molecular level and in their differentiation potential. Considering histological appearance of lipomas, it is not clear to what extent lipomas share common characteristics with other adipose tissue type, brown adipose tissue. Therefore, the aim of this study was to examine the level of uncoupling protein 1 (UCP1), a marker of brown adipose tissue, expression in lipoma tissue as well as in MSCs isolated from lipomas, i.e. lipoma-derived mesenchymal stem cells (LDSCs). LDSCs were grown in standard cell culture conditions and subjected to adipogenic differentiation. UCP1 expression was examined at the RNA level, using Real-Time PCR, and at the protein level, using immunohistochemistry and immunogold staining. Expression of UCP1 in lipoma tissue and LDSCs was compared with the expression of UCP1 in subcutaneous white adipose tissue (scWAT) and adipose-derived mesenchymal stem cells (ADSCs) grown and differentiated in the same cell culture conditions. Differences were observed in UCP1 expression at both RNA and protein levels in lipomas compared to scWAT directing the future research towards the potential of browning mechanisms of adipose tissue involved in lipoma tissue formation.

This research was financially supported by the Science Fund of the Republic of Serbia, PROMIS, #6066747, WARMED and the Ministry of Science, Technological Development and Innovations of the Republic of Serbia, Contract No. 451-03-65/2024-03/200113.