Part1: Protective Effects Of Flavonoids Against Mitochondriopathies And Associated Pathologies: Focus On The Predictive Approach And Personalized Prevention

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Abstract: Multi-factorial mitochondrial damage exhibits a"vicious circle" that leads to a progression of mitochondrial dysfunction and multi-organ adverse effects. Mitochondrial impairments (mitochondriopathies) are associated with severe pathologies including but not restricted to cancers, cardiovascular diseases, and neurodegeneration. However, the type and level of cascading pathologies are highly individual. Consequently, patient stratification, risk assessment, and mitigating measures are instrumental for cost-effective individualized protection. Therefore, the paradigm shift from reactive to predictive, preventive, and personalized medicine (3 PM)is unavoidable in advanced healthcare. Flavonoids demonstrate evident antioxidant and scavenging activity is of great therapeutic utility against mitochondrial damage and cascading pathologies. In the context of 3 PM, this review focuses on preclinical and clinical research data evaluating the efficacy of flavonoids as a potent protector against mitochondriopathies and associated pathologies.

Keywords: natural substances; phytochemicals; flavonoids; anti-oxidant activity;genoprotection;stress; mitochondrial impairment; mitochondriopathy;mitochondrial function; dysfunction; injury;tumorigenesis; cancer; cardiovascular disease; neurodegeneration; predictive preventive personalized medicine (PPPM/3PM); patient stratification

1. Introduction

The terms mitochondrial function and dysfunction are widely employed in bioenergetics and cell biology. Abnormalities in mitochondrial processes, including adenosine triphosphate(ATP)generation, apoptosis, cytoplasmic and mitochondrial matrix calcium regulation, reactive oxygen species (ROS)generation and detoxification, metabolite synthesis, and intracellular transport, can be termed mitochondrial dysfunction [1. Mitochondrial dysfunction affects various organs and tissues, including the brain, muscle, retina, cochlea, liver, and kidney, which are most susceptible to oxidative phosphorylation (OXPHOS) defects. Patients with mitochondrial disorders (mitochondriopathies)exhibit various symptoms, including deafness, visual impairment, heart, liver, and kidney problems, stroke, migraines, diabetes, epilepsy, ataxia, delayed motor and mental development, and failure to thrive, all of which are frequently observed in several non-mitochondrial disorders [2]. Therefore, the effective management of mitochondriopathies is a major challenge in medicine.

Currently, mitochondrial disorders are diagnosed based on functional studies, clinical, biochemical, and histopathologic examinations, and molecular genetic testing [3]. However, diagnostic techniques utilizing cell-free nucleic acids or biofluids such as blood, urine, saliva, cerebrospinal fluid, sweat, or tears could replace invasive tissue biopsies [4-7]. The paradigm shift from reactive to predictive, preventive, and personalized medicine(3 PM)is based on healthcare approaches leveraging targeted preventive measures that account for chronic diseases and ethical as well as economic aspects of medical services [8,9].3 PM also involves individualized patient profiling, which is important for patient stratification, characterization of individual predisposition, and personalized treatments [10]. Moreover, multi-level diagnostic approaches include molecular biological characterization, novel eHealth-based diagnostic tools, questionnaires, and medical imaging [9].

In recent years, the beneficial health effects of flavonoids, naturally occurring polyphenolic compounds, have attracted medical research, including their utilization in pathologies associated with mitochondrial impairments [11]such as cancers, cardiovascular and neurodegenerative diseases[12]. The efficacy of flavonoids is supported by extensive pre-clinical evidence that represents the basis for further research into the potential future use of these compounds in specific targeted and personalized therapy of mitochondriopathies according to the 3 PM approach [13-19].

This review discusses the efficacy of flavonoids in mitochondriopathies such as cancer, cardiovascular diseases (CVDs), and neurodegenerative disorders, highlighting the need for advanced implementation of 3 PM.

2. Mitochondrial Damage and Associated Impairments

In eukaryotic organisms, mitochondria have an essential role in cellular functions such as energy metabolism, biosynthesis, ionic regulation, oxidation and/or reduction, and signaling pathways associated with cell communication, aging, immune responses, apoptosis, survival, and death [12]. The principal functions of mitochondria are ATP synthesis through OXPHOS, metabolite oxidation by the Krebs cycle, and fatty acid β-oxidation [20]. The mitochondrial genome encodes key electron transport chain (ETC) proteins that play an essential role in energy production in aerobic organisms [21]. Human mitochondrial DNA (mtDNA) is a double-stranded circular molecule consisting of 16,569 base pairs [22]. Under normal conditions, mitochondria contain multiple copies(100 to 10,000 per cell) of their DNA [23].

The ETC is also a source of ROS and reactive nitrogen species (RNS), byproducts of OXPHOS that cause DNA, RNA, and protein damage [24]. The inability of base excision repair (BER) to repair the damaged mtDNA leads to ETC disruption associated with ROS production (shown in Figure 1). Further, the activity of ETC can also act as a predictor and target of drug (venetoclax)sensitivity in multiple myeloma patients [25]. Moreover, oxidative stress and insufficient DNA damage repair could increase DNA damage resulting in mitochondrial dysfunction in patients with depression. Therefore, a marker 8-oxoguanine of oxidative DNA damage obtained from fluid biopsies (blood, urine) could be beneficial for the prevention and prediction of neurodegenerative disorders such as mito chondriopathies [26]. Subsequently, extensive oxidative mtDNA damage manifests in several mitochondrial dysfunctions and diseases [27]. Mitochondrial dysfunctions can also be caused by mtDNA mutations, deletions, and impaired DNA replication (shown in Figure 1)[28]. For example, the mtDNA m.3243A>G mutation can lead to clinical phenotypes related to two clinical syndromes: maternally inherited diabetes and deafness (MDD), and mitochondrial encephalomyopathy, lactic acidosis, and strokelike episode (MELAS)syndrome [29]. Moreover, some clinical features of mitochondrial syndromes associated with mtDNA mutations include a maternal family history due to the maternal pattern of mitochondrial inheritance. The simplicity of analysis of mitochondrial genome sequencing due to the availability of consensus human sequence could help to recognize mtDNA disorders in terms of heredity. Other mtDNA mutations associated with mitochondrial dysfunction are acquired during life by the aging process. These acquired mtDNA mutations are often connected to age-related diseases such as diabetes. Therefore, the progress in the understanding of basic mitochondrial genetics is considered an important tool for analysis of the relationship between inherited mitochondrial mutations and disease phenotypes through the identification of acquired mtDNA mutations[30,31].Moreover, mitochondrial dysfunction can be caused by pathogenic variants in nuclear genes associated with mtDNA maintenance, including those encoding mtDNA replication enzymes, proteins that function in the maintenance of the mitochondrial nucleotide pool, and proteins that participate in mitochondrial fusion (shown in Figure 1)[32]. Besides, the aging process is connected to a decrease in mitochondrial biogenesis(fusion and fission)and also in a critical process of eliminating dysfunctional mitochondria characterized as mitophagy [20]. Moreover, the incidence and frequency of mtDNA mutations increase markedly with age, contributing to cellular senescence [33].

As discussed above, mitochondrial impairments are associated with various highly heterogeneous diseases in terms of their different clinical features and genetic etiology. Therefore, the analysis and/or elucidation of molecular mechanisms associated with mitochondrial impairments can represent the challenges for diagnosis and further clinical management [34]. Finally, mitochondrial dysfunction is a hallmark of many diseases known as mitochondriopathies, including malignancies, CVDs, and neurodegeneration. Therefore, it is imperative to find novel therapies targeting mitochondrial disease mechanisms.

2.1.Mitochondiopaties Are Involved in Cancer Development

Mitochondria have essential functions in apoptotic pathways and the mechanisms of the Warburg phenotype, processes that are closely related to cancer. Mitochondria play an essential role in the intrinsic pathway of apoptotic cell death associated with mitochondrial outer membrane permeabilization, cytochrome c release, apoptosome formation, caspase activation, and cell death [35]. Apoptosis evasion is a hallmark of human cancer development. Cancer cells leverage several survival strategies, including the activation of anti-apoptotic and pro-survival signaling through the inhibition of mitochondrial apoptosis. Therefore, intrinsic (mitochondrial) apoptotic pathways represent a promising target for anticancer strategies [36].

In 1956, Otto Warburg described the process by which cancer cells sustain rapid proliferation; this process, known as the Warburg effect, is characterized by increased glucose uptake and lactate secretion (aerobic glycolysis) even under normoxic conditions, suggesting that defects in mitochondrial respiration can promote tumorigenesis [37,38]. In mammals, modulation of protein kinases (PK), such as PKL, PKR, PKM1, and PKM2, augments the Warburg effect in cancer cells [39]. Moreover, mtDNA depletion leads to alterations in mitochondrial function in breast, renal, prostate, and other cancers, and age-related diseases, underscores the role of mitochondria in tumorigenesis [40-43]. Furthermore, various mutations in Krebs cycle enzymes, including succinate dehydrogenase (SDH), fumarate hydratase (FH), and isocitrate dehydrogenase 1(IDH1) and 2(IDH2), are described in cancer cells [44]. SDH mutations are associated with hypoxia pathway activation, which can alter mitochondrial fusion and fission, mitophagy, and OXPHOS. In addition, FH and IDH mutations lead to tumor initiation through the repression of cellular differentiation, and IDH1 and DH2 mutations cause an energy shift in cancer cells [45,46]. Abnormalities in the mentioned Krebs cycle enzymes promote carcinogenesis through the production of one-metabolites, including 2-hydroxyglutarate and citrate, increased fatty acid β-oxidation, and epithelial-mesenchymal transition (EMT) induction [47].

Moreover, mutations in mtDNA, especially in genes for complexes I, IⅢ, IV, and V, which are closely associated with OXPHOS and redox regulation, were observed in endometrial, cervical, breast, and epithelial ovarian cancer cells [44,46,48]. Specifically, mutations in complex I am associated with a higher α-ketoglutarate/succinate ratio, which promotes tumorigenesis through hypoxia-inducible factor 1α(HIF1α) destabilization [49]. Although mutations in mtDNA lead to mitochondrial dysfunction and the potential for cancer development, these mutations also affect nuclear gene expression through retrograde signaling [44].

2.2. Mitochondrial Dysfunction in Cardiovascular Diseases

CVDs are the leading cause of global mortality and morbidity [50]. Mitochondria have a pivotal role in the homeostasis of the heart. Mitochondrial morphology is responsive to changes in cardiomyocytes [51]. Mitochondrial diseases that preferentially affect the heart are associated with mitochondrial dysfunctions, such as disruptions in OXPHOS or the ETC[52]. Structural and functional alterations in mitochondrial organelles cause ischemic cardiomyopathy, heart failure, and stroke [53].

Furthermore, disruptions in mitochondrial dynamics, including mitochondrial fusion, fission, biogenesis, and mitophagy, lead to the development and progression of CVDs such as diabetic cardiomyopathy, atherosclerosis, damage from ischemia-reperfusion, cardiac hypertrophy, and decompensated heart failure [54]. Several nuclear genes regulating mtDNA maintenance and replication, including mitochondrial transcription factor A (TFAM), mtDNA polymerase γ （POLG）， and PEO1 （Twinkle）， are altered in CVDs 【55】. Besides，mtDNA mutations that dysregulate mtDNA gene expression promote the pathogenesis of stroke and myocardial infarction [56]. Moreover, hypoxia causes changes in cellular mechanisms that lead to oxidative stress and subsequent mitochondrial dysfunction [57].

In patients with atherosclerosis and associated CVDs dysfunctional mitochondria affect cellular respiration and energy production and also act as dangerous ROS generators leading to the induction of apoptosis [58]. The accumulation of ROS and RNS in the heart by dysfunctional mitochondria is associated with several CVDs, including cardiomyopathies and heart failure [59,60]. Interestingly, ROS production caused by TFAM dysfunction is related to mtDNA damage and consequent cardiomyocyte cell cycle arrest resulting in lethal cardiomyopathy [61]. Moreover, the prognosis of cardiomyopathy is poor in children with mitochondrial diseases, especially those with mtDNA defects, including the m.3243A>G mutation in mitochondrially encoded tRNA-Leu(UUA/G)1(MT-TL1), the m.13513G>A mutation in mitochondrially encoded NADH: Ubiquinone oxidoreductase core subunit 5(MT-ND5), the m.8528T>Cmutation in the overlapping region of mitochondrially encoded ATP synthase membrane subunits 6(MT-ATP6) and 8(MT-ATP8), the m.3302A>G mutation in MT-ND1, the m.1644G>A mutation in mitochondrially encoded tRNA valine (MT-TV), and pathogenic mutations in BolA family member 3(BOLA3) and tafazzin TAZ. Children with mentioned mitochondrial mutations have a higher risk of cardiomyopathy and associated mortality. Therefore, the genetic analysis with detailed phenotyping of mitochondrial impairments could be useful for the prognosis of cardiomyopathy [62]. Moreover, several nuclear gene mutations can directly affect the mitochondrial respiratory chain and its components. The alterations in genes of complex I (NDuFS1, NDuFS2, NDUFS3), complex IV(SURF1, SCO1, SCO2, COX10, COX15), complex V(ATP12, TMEM70), mitochondrial translation (TACO1, EFG1), and cardiolipin biosynthesis (TAZ) are associated with cardiomyopathy [59]. Furthermore, intermyofibrillar mitochondria represent a well-organized network of long and dense organelles and contractile myofilaments. In heart failure, a disturbance in the physical and chemical interactions between intermyofibrillar mitochondria and sarcoplasmic reticular reduces cardiomyocyte contractility and induces cell death[63]. Moreover, heart failure can be characterized by mitochondrial calcium overload, higher ROS release, and reduced ATP production [64]. During heart failure, calcium overload commonly increases mitochondrial fission and dysfunction. Subsequently, these processes lead to a decrease in the activity of the heart that is characterized by a reduced ability to fill the left ventricle and eject blood to match the body's demands. This metabolic demand of the heart could be associated with alterations in heart rate, myocardial inotropic state, and myocardial wall tension that in conclusion promote heart injury. The calcium accumulation is also associated with a reduction in mitochondrial energetics(ATP production) that leads to negative changes in ETC and OXPHOS associated with the generation of cell-damaging ROS and apoptosis induction [65]. Furthermore, cardiolipin is a key mitochondrial phospholipid in the inner mitochondrial membrane required for the activity of the ETC. The loss of cardiolipin causes ROS production associated with the disruption of cardiolipin peroxidation and cytochrome c release leading to cardiomyocyte apoptosis. In heart failure, this vicious cycle leads to mitochondrial dysfunction and subsequent cardiomyocyte death [66].

2.3. Mitochondriopathies in the Neurodegeneration

Normal mitochondrial dynamics are important for maintaining polarity in highly polarized neurons [67,68]. Neuronal cell death in brain disorders (neurodegeneration) and injury (neurotoxicity and ischemia) is connected to various changes in mitochondrial homeostasis and/or function that include traffic, quality control, turnover, bioenergetics, electron transport, and signaling [69]. Neurons depend more on OXPHOS to fulfill their energy demands than other cell types [70]. Neurodegenerative disorders are also characterized by the gradual accumulation of mtDNA mutations that can potentially decrease ETC and ATP production efficiency and increase ROS production [71]. A higher ROS level could cause further mtDNA mutations in a"vicious circle" that leads to cell death [72]. Moreover, abnormalities of the microtubule-associated protein tau (tau) were observed in various neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and Pick's disease [73]. Mitochondrial dysfunction is closely associated with tau pathology in AD; overexpression of hyperphosphorylated and aggregated tau is suggested to damage axonal transport and cause the abnormal distribution of mitochondria [74].

Mitochondrial dysfunction and oxidative stress contribute to AD and PD, the two most common age-related neurodegenerative diseases [71]. AD, a form of senile dementia, is characterized by the accumulation of damaged mitochondria during aging. Extracellular deposition of amyloid β-peptide(Aβ) plaques and the intracellular formation of neurofibrillary tangles (NFIs)occur in the cerebral cortex of AD patients [75]. In AD, oligomers of Aβ with hyperphosphorylated pTau cause the loss of synaptic function and cognitive impairment [76,77]. Several mutations are closely associated with mitochondrial function, including those in the genes encoding β-amyloid precursor protein (APP), presenilin 1(PSEN1)and 2(PSEN2), and apolipoprotein E(APOE4), lead to AD development. Various missense or deletion mutations of mitochondrial APP cause the inherited form of AD[73]. In addition to APP mutations, mutations in PSENI and PSEN2 are observed in early-onset familial AD [78]. Moreover, the contribution of APOE4 to AD pathogenesis is related to APOE4-mediated alterations of Aβ aggregation and clearance. APOE4 mutations constitute one of the major genetic risk factors for late-onset sporadic AD [79].

Furthermore, the pathological hallmarks of PD include the loss of dopaminergic neurons in the substantia nigra and the presence of misfolded α-synuclein (c-syn) in intra-cytoplasmic inclusions known as Lewy bodies [80]. PD arises from various mitochondrial dysfunctions, including bioenergetic and transcriptional defects, and alterations in dynamics (fusion or fission), size, morphology, trafficking, transport, and movement. Undoubtedly, mutations in mtDNA, nuclear DNA, and mitochondrial proteins are well described in PD[81]. Therefore, mutations or disturbances in E3 ubiquitin ligase (Parkin), c-syn, a parkin-associated protein involved with oxidative stress(DJ1), ubiquitin carboxy-terminal hydrolase L1(UCHL1), auxilin (DNAJC6), putative serine-threonine kinase(PINKT), synaptojanin1(SYN1), serine peptidase 2(HTRA2), and endophilin A1 (SH3GL2) disrupt several mitochondrial functions and may cause PD development [12].

3. Flavonoids Classification and Functions

Flavonoids represent an important class of natural substances. All flavonoids are synthesized in plants as bioactive secondary metabolites and contain a basic flavan skeleton that consists of a 15-carbon phenylpropanoid chain(C6-C3-C6 system) with a characteristic polyphenolic structure consisting of two phenyl rings and a heterocyclic pyran ring [82,83]. Flavonoids can be divided into six major groups: isoflavonoids, flavanones, flavanols, flavanols, flavones, and anthocyanidins [84]. Additional minor classes of flavonoids include chalcones, dihydrochalcones, and aurones are categorized into minor flavonoids [85,86]. Moreover, flavonoids are abundant in plant-based foods and are thus consumed through fruits, vegetables, nuts, seeds, grains, bark, roots, stems, flowers, tea, and wine [84]. The general chemical structures [83] and key representatives of the six major flavonoid classes [87,88] are presented in Figure 2.

Flavonoids have many beneficial properties, such as antioxidant, free radical scavenging, hepatoprotective, cardioprotective, anti-inflammatory, immunomodulatory, antiangiogenic, antiviral, anticancer activities, and antidepressant-like effects [82,89-91]. Various flavonoids (vitexin, and baicalin) and other phytochemical compounds such as curcumin (diarylheptanoid), lycopene(carotene), and ginsenoside(triterpenes), have neuroprotective effects against ischemic-induced injury [92]. Moreover, flavonoids can modulate several key mitochondrial enzymatic pathways [93]. Redox potentials associated with flavonoids' chemical structure allow these compounds to thermodynamically scavenge ROS, including hydroxyl, superoxide, alkoxyl, alkyl-peroxyl, and nitric oxide radicals [94]. On the other hand, the oxidized reactive byproducts of the redox and scavenging mechanisms of flavonoids chemically destabilize these compounds [95]. Notably, the redox properties of flavonoids vary with the cellular conditions, dosage, treatment time, experimental model, tumorigenic state, and other factors. Under specific cellular conditions such as the occurrence of environmental factors or stressors, the antioxidants can act also as prooxidants. The pro-oxidant activity of flavonoids,e.g., luteolin and fisetin, can be characterized by the ability to undergo autoxidation catalyzed by the transition metals to produce superoxide anions [96,97]. For the determination of prooxidant status is important to evaluate various reductant-oxidant markers such as glutathione(GSH) to GSSG, NADPH to NAPD-, and NADH to NAD-[98]. Prooxidant properties of flavonoids can cause oxidative damage through reactions with different biomolecules, such as lipids, proteins, and DNA [99,100].

Flavonoids generally exhibit low oral bioavailability due to their poor aqueous solubility. The composition of their sources can also affect their bioavailability. Therefore, the gut microbiome is crucial for the absorption and metabolism of flavonoids [101]. Anthocyanidins and pro-anthocyanidins have the lowest bioavailability, while quercetin glucosides, catechin, flavanones, isoflavones, and gallic acid have the highest one [102]. This is the issue that has to be considered from a biotechnological point of view for increasing their bioavailability and facilitating clinical implementation.

Flavonoids provide a valuable contribution to the framework of 3 PM. The role of 3 PM is to introduce predictive analytical approaches by cost-effective targeted prevention and personalization of medical services. Predispositions and early diagnostics, targeting high-risk individuals, individualized patient profiling, and patient stratification could significantly improve the therapeutic strategies for various diseases [12]. Despite the above-mentioned limitations, flavonoids represent environmentally friendly and cost-effective substances with minimal side effects during long-term administration. Health beneficial effects of flavonoids are promising for 3 PM concepts including predictive approaches, targeted prevention, and personalization of medical services, that can positively influence preventive and therapeutic strategies, eg., anti-cancer effects of flavonoids that can inhibit the initiation of metastasis and their spread in high-risk individuals [86].