8oxog thread (and foxo3/klotho interactions)

AlexKChen · June 4, 2025, 10:19pm

Interactions of Alpha-Klotho with FOXO3 and 8‑Oxoguanine: Mechanisms and Contexts

Introduction

Alpha-Klotho (α-Klotho) is a membrane-bound and soluble protein with anti-aging properties, first identified in mice lacking the Klotho gene who exhibited accelerated aging . It serves as a co-receptor for fibroblast growth factor 23 (FGF23) and also functions independently as a hormonal modulator of signaling pathways related to aging and stress. FOXO3 (Forkhead box O3) is a transcription factor known as a longevity and stress-resistance gene, regulating expression of antioxidant and stress-response genes. 8-oxoguanine (8-oxoG, often measured as 8-hydroxy-2′-deoxyguanosine, 8-OHdG) is a DNA lesion resulting from oxidative damage to guanine; it is a well-established marker of oxidative stress and can cause mutations or trigger cell death if not repaired. This report examines all known regulatory relationships between α-Klotho and FOXO3, and between α-Klotho and 8-oxoG, emphasizing molecular mechanisms (transcriptional regulation, protein interactions, signaling crosstalk) and relevant biological contexts such as aging, oxidative stress, and kidney function. Key findings from human studies, animal models, and cellular research are summarized, with tables highlighting representative studies.

Alpha-Klotho and FOXO3: Signaling and Regulatory Mechanisms

Signaling Crosstalk via the Insulin/IGF-1 Pathway: A major point of interaction between Klotho and FOXO3 is through the insulin/IGF-1–PI3K–Akt signaling cascade. Klotho can attenuate insulin/IGF-1 receptor signaling, leading to reduced PI3K/AKT activity . This has direct consequences for FOXO3, because active PI3K/AKT phosphorylates FOXO3, causing its export from the nucleus and functional inactivation. By inhibiting PI3K/AKT, Klotho reverses this effect – it prevents FOXO3a phosphorylation and promotes FOXO3 retention in the nucleus . In other words, Klotho signaling activates FOXO3. A recent review succinctly states that Klotho blocks IGF-1R signaling, “allowing the FOXOs to migrate into the nucleus and protect against oxidative stress” through target gene expression . This Klotho–FOXO3 crosstalk engages a conserved longevity pathway: reduced insulin/IGF signaling with subsequent FOXO activation is known to extend lifespan in multiple species . Consistent with this, overexpression of Klotho in mice extends lifespan in part by downregulating insulin/IGF-1 signaling , a mechanism that parallels the lifespan extension seen with FOXO activation. Notably, human genetic studies link both Klotho and FOXO3 to longevity – a functional variant of the Klotho gene associates with human lifespan , and common polymorphisms in FOXO3 are strongly associated with exceptional longevity in multiple populations (a well-established observation in longevity research, though the precise mechanisms are still under study).

Klotho-Activated FOXO3 and Antioxidant Gene Expression: When FOXO3 remains nuclear and active under Klotho’s influence, it drives transcription of genes that mitigate oxidative stress and cellular damage. One critical FOXO3 target is manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme. Experiments in kidney models have shown that Klotho’s activation of FOXO3 enhances FOXO3’s binding to the MnSOD gene promoter, increasing MnSOD mRNA and protein levels . For example, in a mouse model of tacrolimus-induced nephrotoxicity (Tacrolimus is a calcineurin inhibitor that causes oxidative stress in kidneys), treatment with recombinant Klotho inhibited PI3K/AKT-mediated FOXO3a phosphorylation and increased FOXO3a occupancy at the MnSOD promoter . This led to upregulation of MnSOD and markedly reduced reactive oxygen species (ROS) production and mitochondrial dysfunction in renal cells . In cell culture, Klotho added to human renal tubular cells (HK-2) likewise promoted FOXO3a binding to the MnSOD promoter and elevated MnSOD expression, an effect abolished by PI3K activation and enhanced by PI3K inhibition . Through FOXO3, Klotho thereby boosts antioxidant defenses – MnSOD detoxifies superoxide radicals in mitochondria, lowering oxidative stress within cells. Klotho has similarly been shown to increase other FOXO3-regulated antioxidants like catalase. In primary astrocytes (brain support cells), exposure to exogenous Klotho increased FOXO3a activity and raised catalase levels, thereby “shielding them from oxidative stress” . These findings illustrate a consistent mechanism: α-Klotho relieves the inhibitory phosphorylation on FOXO3, allowing FOXO3 to induce antioxidant genes (e.g. MnSOD, catalase), which in turn reduce cellular ROS and damage.

FOXO3-Dependent Protective Effects of Klotho: Beyond upregulating antioxidants, FOXO3 also mediates other Klotho-driven cytoprotective pathways. One notable example is in mitophagy, the autophagic removal of damaged mitochondria. A recent study in a contrast-induced acute kidney injury (CI-AKI) model found that Klotho’s kidney-protective effects require FOXO3-dependent activation of the mitophagy gene BNIP3. In mice and renal cells exposed to contrast media (iohexol), α-Klotho treatment upregulated nuclear FOXO3 levels and enhanced BNIP3-mediated mitophagy, leading to the clearance of damaged mitochondria and reduced mitochondrial ROS generation . When FOXO3 was knocked down, Klotho could no longer induce BNIP3 or protect the cells: FOXO3 silencing inhibited Klotho’s enhancement of mitophagy and led to increased oxidative injury and apoptosis . Thus, FOXO3 is essential for Klotho to promote mitophagy and survival in this kidney injury context. This aligns with the general model that nuclear FOXO3 (activated by Klotho) binds to promoters of stress response genes – BNIP3 in this case – to orchestrate cellular defense. In summary, many of α-Klotho’s downstream benefits (antioxidant defense, mitochondrial quality control, etc.) are FOXO3-dependent. Table 1 highlights key studies demonstrating these interactions.

Reciprocal Regulation (FOXO3 to Klotho): Current evidence for FOXO3 directly regulating Klotho expression is limited, but some studies suggest an indirect positive feedback loop may exist under stress conditions. For instance, in the tacrolimus nephrotoxicity model, the loss of Klotho protein under stress was reversed by interventions that activate FOXO3 (such as ginseng treatment) . Ginseng, known to inhibit PI3K/Akt, preserved Klotho levels in tacrolimus-treated mice via FOXO3 activation of antioxidant signaling . The data implied that FOXO3’s activity helped maintain Klotho expression and function in the face of oxidative injury . It remains unclear if FOXO3 directly binds the Klotho gene promoter; however, by reducing cellular stress and damage, FOXO3 may indirectly prevent Klotho downregulation. Moreover, Klotho and FOXO3 appear to participate in a reinforcing, anti-aging feedback cycle: Klotho activates FOXO3, which induces cytoprotective genes that help sustain Klotho levels and signaling. Both are positively influenced by certain hormonal factors (e.g. vitamin D and caloric restriction mimetics are known to upregulate Klotho and activate FOXO factors) . This cooperative relationship contributes to their joint role in healthy aging.

Table 1. Key Studies on α-Klotho and FOXO3 Interactions

Study (Year) Model / System Key Findings (Klotho–FOXO3 Relationship)

Lim et al., 2017 Mouse tacrolimus-induced kidney injury; HK-2 renal cells Klotho treatment protected kidneys from oxidative injury. Mechanism: Klotho inhibited PI3K/AKT, preventing FOXO3a phosphorylation, and thereby increased FOXO3’s binding to the MnSOD promoter. MnSOD expression rose, ROS production fell, and mitochondrial function improved . Klotho significantly reduced oxidative damage markers (8-OHdG, 4-HHE) and TUNEL+ apoptosis in the kidney , consistent with FOXO3-driven antioxidant effects.

Lim et al., 2019 Mouse tacrolimus nephropathy with ginseng intervention Ginseng (a herbal extract) activated Klotho’s anti-aging signaling via FOXO3. Tacrolimus normally lowered Klotho levels and raised oxidative stress; ginseng reversed these effects. Mechanism: Ginseng suppressed PI3K/Akt, reduced FOXO3a phosphorylation, and promoted FOXO3a binding to the MnSOD promoter . This led to higher Klotho expression and antioxidant defenses, attenuating tacrolimus-induced ROS and kidney damage.

Zhu et al., 2024 Contrast-induced AKI in mice; iohexol-treated renal cells Klotho alleviated acute kidney injury via a FOXO3–BNIP3 mitophagy pathway. Klotho-treated injured kidneys showed increased nuclear FOXO3 and upregulation of BNIP3 (mitophagy protein) with enhanced removal of damaged mitochondria . FOXO3 was required: FOXO3 knockdown blocked Klotho’s induction of BNIP3-dependent mitophagy and led to more ROS and apoptosis . Thus, FOXO3 is essential for Klotho’s mitochondrial protective effect.

Orellana et al., 2023 Mouse primary astrocytes (brain glial cells) in vitro Exogenous Klotho promoted oxidative resilience in astrocytes by modulating insulin signaling and FOXO3. Klotho treatment (1 nM) for 24 h decreased p-Akt and mTOR levels in astrocytes, which corresponded with increased FOXO3a activity and higher catalase (antioxidant enzyme) expression . The activated FOXO3-catalase axis protected astrocytes from oxidative stress. (In neurons, Klotho affected metabolism via other routes, underscoring cell-type specific effects.)

Implications for Aging and Disease: The Klotho–FOXO3 interaction emerges as a pivotal mechanism in aging-related resilience. In aging, chronic IGF-1/insulin activity and accumulated oxidative damage contribute to cellular senescence. By counteracting IGF-1 signaling and boosting FOXO3, Klotho helps maintain a youthful cellular profile of stress resistance . Both Klotho and FOXO3 individually have geroprotective effects – for example, Klotho-haplodeficient mice exhibit accelerated aging and multi-organ degeneration, while FoxO3-null mice show reduced stress tolerance – and together they form a coordinated defense against aging at the molecular level. This coordination is especially evident in high-stress organs like the kidney. The kidney is a major site of Klotho expression (especially in distal tubules), and it is exposed to constant metabolic and oxidative stress. Klotho’s renoprotective functions involve FOXO3 activation to reduce fibrosis, apoptosis, and inflammation. In models of chronic kidney disease or acute injury, raising Klotho levels correlates with increased nuclear FOXO3 in renal cells and improved outcomes . Conversely, Klotho deficiency or aging-related Klotho decline can leave FOXO3 inactivated (phosphorylated) and unable to induce protective genes, partly explaining the heightened oxidative damage and inflammation in Klotho-deficient and aged kidneys. In other contexts such as the brain, cardiovascular system, and cancer, the Klotho–FOXO pathway is also relevant. Klotho in the brain has been linked to cognitive benefits and neuroprotection, likely through metabolic reprogramming and stress resistance involving FOXO3 and other factors . In vascular cells, FOXO3 activation helps prevent oxidative damage to the endothelium; Klotho’s anti-atherosclerotic effects may utilize this route as well. In cancer biology, both Klotho and FOXO3 act as tumor suppressors – Klotho by inhibiting growth pathways (IGF-1, Wnt) and FOXO3 by enforcing cell cycle arrest and apoptosis in cancer cells – and there is emerging evidence that Klotho reactivation in tumors can lead to FOXO3-mediated growth suppression . Overall, the interplay between α-Klotho and FOXO3 constitutes a fundamental axis of anti-oxidative and anti-aging signaling across multiple systems.

Alpha-Klotho and 8‑Oxoguanine (8-oxoG): Oxidative DNA Damage and Repair

While Klotho and FOXO3 operate at the level of signaling and gene regulation, their impact extends to DNA integrity, notably in the prevention and repair of oxidative DNA lesions like 8-oxoguanine. 8-oxoG is one of the most common DNA base lesions caused by reactive oxygen species; if not repaired, 8-oxoG can pair erroneously during replication (causing G→T mutations) or trigger apoptosis. The relationship between α-Klotho and 8-oxoguanine is primarily an indirect but crucial one: Klotho reduces the accumulation of 8-oxoG in cells and tissues by lowering oxidative stress and by supporting DNA repair mechanisms.

Klotho’s Role in Reducing Oxidative DNA Damage: Multiple studies have observed that raising Klotho levels correlates with decreased markers of oxidative DNA damage, including 8-oxoG/8-OHdG. For example, in the tacrolimus-induced kidney injury model, mice given recombinant Klotho had markedly lower 8-OHdG levels in kidney tissue (by immunostaining) and reduced urinary excretion of 8-OHdG, compared to untreated mice . Tacrolimus caused a spike in 8-oxoG lesions (reflecting high ROS damage), but co-administration of Klotho “dramatically downregulated” these oxidative DNA damage markers . Consistently, Klotho-treated kidneys also showed fewer TUNEL-positive apoptotic cells, indicating that preventing DNA damage helped reduce cell death . In the ginseng study (tacrolimus + ginseng, which boosts endogenous Klotho), immunohistochemistry likewise showed that 8-OHdG levels were elevated by tacrolimus but significantly blunted by the Klotho-preserving ginseng treatment . These findings underscore that Klotho’s antioxidant effects translate into genomic protection: by curbing ROS (through FOXO3 and other pathways), Klotho lowers the generation of 8-oxoG in DNA.

Regulation of 8-oxoG Repair (OGG1 Pathway): Beyond preventing damage, evidence suggests α-Klotho influences the DNA repair machinery that recognizes and fixes 8-oxoguanine lesions. The primary repair enzyme for 8-oxoG in DNA is 8-oxoguanine DNA glycosylase (OGG1), which initiates base excision repair. Notably, Klotho deficiency has been linked to impaired OGG1 function. In a study of diabetic nephropathy (high-glucose-induced oxidative stress in kidneys), Klotho-knockout or Klotho-deficient mice exhibited increased 8-OHdG accumulation and decreased OGG1 expression in glomerular podocytes . The absence of Klotho significantly aggravated DNA damage in diabetic kidneys: 8-OHdG levels were higher and OGG1 protein levels were much lower compared to Klotho-sufficient controls . The researchers concluded that “Klotho deficiency may cause loss of regulation in OGG1 expression,” thereby allowing oxidative DNA lesions to build up . Indeed, immunostaining confirmed that diabetic mice lacking Klotho had weak OGG1 signals in podocytes, and in vitro high-glucose treatment of podocytes also reduced OGG1; however, adding recombinant Klotho protein restored OGG1 levels . Klotho or the antioxidant N-acetylcysteine was able to “inhibit the reduction” of OGG1 in high-glucose conditions . These data reveal a regulatory relationship: α-Klotho helps maintain OGG1 expression (and possibly activity), enabling efficient repair of 8-oxoG lesions, whereas Klotho deficiency compromises OGG1 and thus DNA repair capacity . The mechanism by which Klotho influences OGG1 is not entirely elucidated. It may be indirect – for example, chronic oxidative stress can lead to OGG1 inactivation or degradation (via calpain protease) , and Klotho’s ROS-lowering effect could preserve OGG1. There may also be transcriptional regulation at play, where Klotho signaling (or Klotho-induced FOXO3/Nrf2 activity) upregulates DNA repair genes. Regardless, the outcome is clear: in the presence of Klotho, cells are better equipped to fix oxidative DNA damage.

Biological Contexts of Klotho–8-oxoG Interactions: The interplay between Klotho and 8-oxoguanine is especially relevant in conditions of high oxidative stress and during aging. In aging, accumulation of 8-oxoG in DNA is a well-documented phenomenon contributing to genomic instability. Klotho, known to decline with age in serum and tissues, likely mitigates age-associated DNA damage when it is sufficiently expressed. Conversely, low Klotho levels (as in aged individuals or Klotho-knockout models) correspond with increased oxidative DNA damage. The diabetic kidney study described above illustrates this: older or diabetic animals with Klotho deficiency had worse 8-oxoG burdens in their DNA . In chronic kidney disease (CKD) patients, who often have reduced Klotho, urinary 8-OHdG is elevated as a marker of systemic oxidative stress , potentially reflecting insufficient Klotho-mediated protection. Enhancing Klotho in such contexts is being explored as a therapeutic strategy to reduce oxidative damage. In renal pathologies more broadly, Klotho’s protective effect has a significant DNA component. Acute injuries (toxin-induced, ischemic, etc.) often involve bursts of ROS that inflict DNA damage; Klotho can ameliorate these. For instance, exogenous Klotho in ischemia–reperfusion injury models improved outcomes and was associated with lower tubular cell DNA damage (less 8-oxoG and apoptosis) . In neurodegenerative contexts, oxidative DNA damage including 8-oxoG in neurons is linked to cognitive decline. While less studied than in kidney, brain Klotho might similarly guard against neuronal DNA damage by boosting antioxidant defenses. Klotho’s ability to activate the transcription factor Nrf2 in addition to FOXO3 could be relevant here, since Nrf2 upregulates certain DNA repair and antioxidant genes.

Importantly, 8-oxoguanine is not just a marker of damage but can also trigger inflammatory signaling: oxidized DNA fragments or unrepaired 8-oxoG can engage DNA-sensing pathways and promote inflammation. By reducing 8-oxoG load, Klotho may also indirectly quell inflammation. This complements Klotho’s known anti-inflammatory actions (e.g. inhibiting NF-κB and the NLRP3 inflammasome) . Interestingly, the OGG1 enzyme itself, when bound to 8-oxoG, has been found to act as a sensor that can recruit inflammatory mediators like NF-κB to DNA . Klotho’s maintenance of OGG1 levels might ensure proper repair rather than pathological signaling. In summary, α-Klotho helps preserve genomic stability by both preventing the formation of 8-oxoguanine lesions (via ROS suppression) and facilitating their repair (via OGG1 regulation). Table 2 summarizes key findings on Klotho’s relationship with 8-oxoG.

Table 2. Key Studies Linking α-Klotho to 8-oxoguanine (Oxidative DNA Damage)

Study (Year) Model / Context Key Findings (Klotho–8-oxoG Relationship)

Lim et al., 2017 Tacrolimus-induced oxidative stress in mouse kidney Tacrolimus (Tac) caused high oxidative DNA damage in kidneys (elevated 8-OHdG in renal tissue and urine). Recombinant Klotho co-treatment greatly reduced 8-oxo-dG levels, as shown by decreased 8-OHdG staining in kidneys and lower urinary 8-OHdG excretion . Klotho-treated mice also had fewer TUNEL-positive apoptotic cells. These results indicate Klotho can prevent DNA damage and cell death caused by Tac-induced ROS .

Chen et al., 2020 Diabetic nephropathy (streptozotocin-diabetic mice); high-glucose podocyte culture Klotho deficiency exacerbated DNA damage in diabetic kidneys. Klotho-knockout diabetic mice showed significantly higher 8-OHdG levels in podocyte nuclei alongside lower OGG1 expression, compared to diabetic mice with normal Klotho . In vitro, high glucose reduced OGG1 in podocytes, but adding recombinant Klotho restored OGG1 levels . Conclusion: Klotho deficiency impairs OGG1-mediated repair of 8-oxoG, leading to accumulation of DNA damage, whereas Klotho sufficiency preserves OGG1 and limits 8-oxoG lesions .

Orellana et al., 2023 (Sci. Rep.) Klotho-treated neuronal cells under metabolic stress Klotho’s antioxidative signaling (via FOXO3/Nrf2) in the brain may similarly reduce oxidative DNA damage. While this study focused on metabolic changes, it showed Klotho enhancing antioxidant responses in astrocytes . By inference, prolonged Klotho treatment in the CNS or other tissues is expected to lower 8-oxoG accrual over time, though direct DNA damage measures were not reported here. (This highlights a need for further research on Klotho’s genomic protection in neural cells.)

Clinical CKD context Chronic kidney disease patients vs. controls Patients with CKD (who typically have low Klotho levels) showed significantly elevated urinary 8-OHdG compared to healthy controls . This suggests higher systemic oxidative DNA damage in low-Klotho states. Therapeutic strategies to increase Klotho in CKD are hypothesized to reduce such damage and are under investigation.

Conclusion: The interactions between α-Klotho, FOXO3, and 8-oxoguanine form an interconnected network central to oxidative stress resistance and longevity. Klotho serves as an upstream enabler of FOXO3, unleashing FOXO3’s transcriptional program of antioxidant and repair genes. Through this and other pathways, Klotho minimizes the occurrence of oxidative DNA lesions like 8-oxoG and ensures their repair via OGG1. In high-stress organs (kidney, brain, etc.), and in the context of aging or disease, this triad (Klotho–FOXO3–DNA repair) is crucial for maintaining cellular homeostasis. Reduced Klotho or FOXO3 function leads to unchecked oxidative damage – including accumulation of 8-oxoG – which contributes to aging phenotypes and organ dysfunction. Conversely, boosting Klotho or FOXO3 activity fortifies cells against oxidative injury, helping to preserve genomic integrity and function. Ongoing research is exploring Klotho mimetics, FOXO3 activators, and OGG1 enhancers as potential interventions to combat aging and oxidative-stress-related diseases . In summary, α-Klotho and FOXO3 act in concert to protect cells from oxidative stress, and a key outcome of this protective crosstalk is the reduction of 8-oxoguanine DNA damage – a molecular safeguard that underlies their roles in healthy aging and disease prevention.

AlexKChen · June 4, 2025, 10:19pm

Investigating FOXO3 Variants and 8‑oxo-7,8-dihydroguanine (8-oxoG) Levels

Human Studies: FOXO3 Longevity Alleles and Oxidative Damage Markers

Longevity-associated FOXO3 polymorphisms (most notably rs2802292, G allele) have been reproducibly linked to exceptional human longevity . Direct measurements of 8‑oxoG or its derivative 8‑oxo-2′-deoxyguanosine (8‑oxodG, often measured as 8‑OHdG) in humans by FOXO3 genotype are scarce. However, several indirect findings suggest that carriers of “superior” FOXO3 variants experience lower oxidative DNA damage:

Telomere Maintenance: A recent study of older adults (55+ years) in Okinawa found that carriers of the FOXO3 rs2802292 G allele had significantly slower telomere shortening in blood cells . Shorter telomeres are associated with oxidative damage to DNA; thus, the longevity allele’s protection against telomere attrition implies reduced cumulative oxidative DNA damage in these individuals . In the same cohort, G-allele carriers showed slightly higher FOXO3 mRNA expression with age and favorable inflammatory cytokine profiles, consistent with improved stress response .

Stress-Induced FOXO3 Activation: The rs2802292 G allele has been shown to enhance FOXO3’s activation under stress via a mechanistic enhancer effect. Grossi et al. (2018) demonstrated that the G allele creates a binding site for Heat Shock Factor 1 (HSF1) within an intronic enhancer. Under oxidative or heat stress conditions, HSF1 binds this region in G/G cells and triggers a chromatin loop that upregulates FOXO3 expression . As a result, homozygous G-carriers mount a stronger FOXO3-driven transcriptional response – upregulating antioxidant, metabolic, and DNA repair genes – compared to non-carriers . This stress-responsive boost in FOXO3 activity would be expected to mitigate reactive oxygen species (ROS) and enhance repair of oxidative lesions like 8‑oxoG.

Epidemiological Interactions: The FOXO3 longevity genotype appears most beneficial under high-stress conditions, consistent with a role in oxidative stress resistance. For example, one study found that the FOXO3 “G” allele mitigated the mortality risk in individuals with cardiometabolic disease, presumably by bolstering stress defense pathways . Another report noted that lifestyle factors (e.g. antioxidant-rich diets such as green tea intake) conferred cognitive benefits only in those carrying favorable FOXO genotypes . Such gene–environment interactions align with the idea that the longevity allele’s advantage lies in coping with oxidative/inflammatory stress.

Limitations: To date, no published human study has directly quantified DNA 8‑oxoG/8‑oxodG levels or urinary 8‑OHdG stratified by FOXO3 genotypes. The evidence above is indirect – telomere length, gene expression, and population outcomes. Thus, while it is plausible that superior FOXO3 alleles confer a lower 8‑oxoG burden, this has not been causally proven in humans. Ongoing research (e.g. biomarker studies in long-lived populations) is needed to confirm whether FOXO3 “longevity” variants correlate with lower 8‑oxodG (or other oxidative damage markers like F2-isoprostanes) in vivo.

Animal Model Evidence: FOXO3 and 8‑oxoG in Tissues

A wealth of animal studies demonstrate that FOXO3 is critical for limiting oxidative DNA damage. In mice, genetic manipulations of Foxo3 lead to measurable changes in 8‑oxoG/8‑oxodG levels across multiple tissues:

Hematopoietic Stem/Progenitor Cells (HSPCs): Foxo3-knockout (Foxo3⁻/⁻) mice accumulate excess oxidative DNA lesions in the stem cell compartment. Bigarella et al. (2017) showed that Foxo3⁻/⁻ HSPCs have higher baseline DNA damage and specifically more 8‑oxodG in their DNA . By flow cytometry with an 8‑OHdG antibody, Foxo3⁻/⁻ Lin⁻Sca1⁺cKit⁺ cells exhibited significantly elevated 8‑oxoG content compared to wild-type . Consistently, comet assays revealed increased DNA strand breaks in Foxo3⁻/⁻ HSPCs, especially when using the FLARE modification (hOGG1 digestion) that converts 8‑oxoG lesions into breaks . This indicates Foxo3-deficient cells fail to adequately repair 8‑oxoG. Indeed, Foxo3⁻/⁻ HSPCs showed compromised base-excision repair (BER): mRNA levels of key BER genes (DNA polymerase β, XRCC1, Ligase 1) were significantly down-regulated in Foxo3⁻/⁻ stem cells . Notably, OGG1 glycosylase activity (the enzyme that excises 8‑oxoG) was reduced in Foxo3⁻/⁻ cells despite unchanged Ogg1 transcript levels . This suggests FOXO3 may regulate OGG1 post-translationally or via co-factors. Treating Foxo3⁻/⁻ mice with the antioxidant N-acetylcysteine partly rescued the HSPC DNA damage phenotype , reinforcing that elevated ROS (due to loss of FOXO3) caused the excess 8‑oxoG lesions. Overall, FOXO3 is essential in HSCs to prevent oxidative DNA damage, maintaining genomic stability .

Erythrocytes (RBCs): Foxo3 also protects red blood cells from oxidative damage. Foxo3⁻/⁻ mice have been shown to suffer hemolytic stress under oxidant challenge. Marinkovic et al. (2007) reported that Foxo3-deficient erythroid cells have lower expression of ROS-scavenging enzymes and accumulate more oxidative damage byproducts . Consequently, Foxo3⁻/⁻ erythrocytes exhibit a shortened lifespan in circulation due to ROS-mediated injury . The Foxo3⁻/⁻ mice were extremely vulnerable to induced oxidative stress (phenylhydrazine injection), with high mortality and hemolysis compared to wild-types . This phenotype underscores FOXO3’s role in upregulating antioxidant defenses in RBCs, indirectly preventing oxidative DNA/RNA lesions (8‑oxoG in RBC genomes or oxidized heme). While 8‑oxoG in RBC DNA was not directly measured (mature erythrocytes lack nuclei), elevated ROS in developing Foxo3⁻/⁻ erythroid cells likely causes DNA damage during erythropoiesis. This is evidenced by increased oxidative damage markers and cell-cycle arrest (p21^CIP1 upregulation) in Foxo3⁻/⁻ erythroid precursors .

Cardiac Tissue: FOXO transcription factors safeguard the heart from oxidative DNA damage, especially under stress. In a cardiomyocyte-specific Foxo1/Foxo3 double knockout, baseline 8‑OHdG levels in the myocardium were modestly elevated, and after ischemic injury these mice showed significantly increased 8‑oxodG immunoreactivity in cardiomyocyte nuclei . Similarly, cardiac-specific Foxo3 deletion exacerbates oxidative damage upon toxin exposure. Mice with Foxo3 ablated in heart were challenged with the pro-oxidant paraquat, which induces ROS and mimics aging. In Foxo3-cKO hearts, paraquat caused an exaggerated accumulation of 8‑OHdG-positive cells in cardiac tissue compared to paraquat-treated controls . In other words, without FOXO3, the heart could not restrain oxidative DNA lesions under stress. Functionally, the Foxo3-deficient hearts had worse contractile performance after paraquat, whereas wild-type hearts (with FOXO3 intact) were partially protected . These results show that FOXO3 activation in cardiomyocytes is cardioprotective, upregulating defenses (e.g. autophagy and antioxidants) that limit 8‑oxoG accumulation in DNA during oxidative stress.

Other Tissues: FOXO3’s protective effect extends to other organs and contexts. For example, brain and neural cells: FoxO factors (Foxo1/3/4) are required to maintain neural stem cell pools by preventing oxidative stress–driven attrition. Triple-FoxO-knockout neural progenitors show elevated ROS and DNA damage, leading to premature stem cell depletion . In peripheral tissues like kidney, FOXO3 activation via Klotho (an aging-suppressor hormone) has been linked to lower oxidative DNA damage. A study of tacrolimus-induced nephropathy in mice found that treatment with a Klotho-activating ginseng extract upregulated FoxO3 and MnSOD, and concomitantly reduced 8‑OHdG levels in the kidney and blood* . Tacrolimus normally raised 8‑OHdG (indicating DNA oxidation) in renal cells, but FoxO3 activation reversed this, lowering 8‑OHdG to near-baseline . This aligns with FOXO3’s general role in suppressing ROS across many cell types.

Note: In most models FOXO3 is protective, but context matters. Constitutively forcing FOXO3 activity in certain settings can have complex effects. For instance, one cancer study in mice expressing a liver-specific constitutively-active Foxo3 reported increased markers of oxidative damage (8‑OHdG and γH2AX) in the liver . The authors suggested chronic FOXO3 activation might cause metabolic stress or a senescence response in hepatocytes, thereby paradoxically elevating ROS. This outlier finding highlights that FOXO3’s effect on oxidative damage is context-dependent – tightly regulated activation is beneficial, whereas unregulated or excessive FOXO3 activity might induce other stress pathways. Nonetheless, under physiological conditions or moderate stress, FOXO3 clearly acts to minimize oxidative DNA lesions.

Cellular and Molecular Mechanisms: FOXO3, Antioxidant Defense, and DNA Repair

At the cellular level, FOXO3 functions as a transcriptional switch for oxidative stress resistance, directly regulating many genes that can influence 8‑oxoG levels. Key mechanisms include:

Upregulation of Antioxidant Enzymes: FOXO3 drives expression of enzymes that neutralize ROS before they attack DNA. Notably, FOXO3 (and its FOXO family homologs) induce manganese superoxide dismutase (MnSOD/SOD2) in mitochondria, catalase in peroxisomes, and peroxiredoxin-III, among others . By increasing ROS scavengers, FOXO3 lowers steady-state levels of superoxide and hydrogen peroxide, thus reducing the ongoing oxidative assault on the genome. In many cell types, FOXO3 is activated by oxidative stimuli (via JNK, AMPK, or ATM signaling) and in turn limits further ROS accumulation . For example, in human fibroblasts and cardiac cells, FOXO3 activation correlates with decreased intracellular ROS and protection from oxidative injury . This antioxidant gene program is a first line of defense to prevent the formation of 8‑oxoG lesions.

DNA Repair of 8‑oxoG: When oxidative lesions do occur, FOXO3 helps ensure their prompt repair. FOXO3 has been implicated in maintaining the efficiency of the base-excision repair (BER) pathway that removes 8‑oxoG. As described above, Foxo3⁻/⁻ HSPCs had lower expression of Polβ, XRCC1, Lig1 – all essential for BER – and exhibited deficient repair of 8‑oxoG (detected by persistence of lesions in FLARE assays) . Interestingly, FOXO3 does not markedly change the mRNA of OGG1 glycosylase itself , but Foxo3-null cells showed reduced OGG1 enzyme activity . This hints that FOXO3 may regulate post-translational modifications or accessory factors for OGG1. One possibility is via sirtuin deacetylases: FOXO3 is known to cooperate with SIRT1 and SIRT3 during stress responses . SIRT3, for instance, can deacetylate and stabilize OGG1, enhancing its DNA repair capacity in mitochondria . FOXO3 activation (which can occur under caloric restriction or AMPK activation) might promote such interactions – e.g. an AMPK–FOXO3–SIRT3 axis has been described that improves mitochondrial stress resistance . Thus, through direct transcriptional control of BER components and indirect support of repair enzyme function, FOXO3 helps remove 8‑oxoG lesions more effectively, preserving genome integrity.

Autophagy and Mitochondrial Quality Control: FOXO3 is a known inducer of autophagy genes (e.g. LC3, Bnip3) that remove damaged proteins and organelles. By promoting turnover of dysfunctional mitochondria (mitophagy), FOXO3 prevents excess ROS production at the source. In chronically stressed cells, FOXO3-mediated autophagy reduces oxidative burden . For instance, in hypoxic kidney cells FOXO3 upregulated Atg genes to drive autophagy, lowering oxidative stress . Fewer ROS translates to fewer 8‑oxoG lesions in both nuclear and mitochondrial DNA. This indirect protection via quality control is another mechanism linking FOXO3 to reduced 8‑oxoG. Consistently, experiments activating FOXO3 show less oxidative damage: in cultured thyrocytes, FOXO3-overexpressing clones had a significantly lower DNA damage score after UV exposure compared to controls . Even though UV induces other lesions too, FOXO3’s activation likely accelerated repair and mitigated oxidative base damage resulting from UV-generated ROS. In summary, FOXO3 orchestrates a multi-pronged cellular defense – antioxidant production, DNA repair, and damage removal – all of which converge to minimize 8‑oxoG accumulation.

Cell Cycle and Apoptosis Control: FOXO3 also transiently arrests the cell cycle (through p21^CIP1, Gadd45α, etc.) in response to oxidative stress . This gives cells time to repair DNA lesions like 8‑oxoG before replication, preventing fixation of mutations. In Foxo3⁻/⁻ cells, an inability to properly induce cell-cycle arrest can lead to replication of damaged DNA or to cell death. In HSPCs, FOXO3 loss caused only a mild increase in apoptosis despite high DNA damage, suggesting Foxo3⁻/⁻ cells were attempting to endure oxidative stress with incomplete repair . This could lead to error-prone survival. Normally, FOXO3 helps balance DNA repair and apoptosis – if damage is too great, FOXO3 can trigger apoptosis, culling heavily damaged cells. Thus, FOXO3 safeguards the genomic integrity of cell populations over time by multiple routes.

Aging, Caloric Restriction, and Oxidative Stress Contexts

Aging: During aging, oxidative DNA lesions (like 8‑oxoG) tend to accumulate, and FOXO3’s activity/status can change with age. Recent work in mice showed that vascular aging is accompanied by rising 8‑oxo-dG levels and declining expression of Foxo3a and OGG1 in arteries . Aged mouse aortas had less OGG1 (including reduced active, acetylation-regulated OGG1) and lower FOXO3, correlating with more DNA damage and stiffer arteries . This finding suggests that the age-related drop in FOXO3 may be one factor allowing 8‑oxoG lesions to accumulate in older tissues. Conversely, long-lived individuals often maintain effective stress responses. It is notable that centenarians (enriched for FOXO3 longevity alleles) generally show lower markers of oxidative damage and inflammation relative to their age peers . While many pathways are involved in human aging, FOXO3’s role in sustaining genomic maintenance is likely important. Its ability to activate DNA repair and antioxidant genes might slow the accumulation of oxidative DNA hits over a lifetime. Indeed, FOXO3 is often dubbed a “guardian” against aging: it is required for the extraordinary lifespan of Hydra (a non-senescence model) and extends lifespan when overexpressed in Drosophila and mice . Aging-associated FOXO3 decline could remove this guardian, accelerating oxidative DNA damage. This provides a plausible causal link between high FOXO3 function (as in longevity alleles) and reduced 8‑oxoG with age.

Caloric Restriction (CR): Dietary restriction is a classic intervention that delays aging and lowers oxidative damage, and FOXO3 is a known mediator of CR’s benefits. CR (30–40% reduced calories) consistently decreases 8‑oxodG levels in tissues of rodents. For example, aged mice on CR have significantly less 8‑oxodG in the heart DNA compared to ad libitum-fed mice . Multiple studies cited in reviews show CR reduces oxidative DNA and protein damage in the brain, liver, and kidney as well . Mechanistically, CR activates Sirtuin deacetylases (SIRT1/SIRT3) and upregulates FOXO factors . Shimokawa et al. (2015) demonstrated that the life-extending effect of dietary restriction requires FOXO3: Foxo3-knockout mice failed to reap the full longevity and tumor-suppression benefits of CR . Thus, under CR, FOXO3 is activated (partly via lowered insulin/IGF signaling and NAD⁺-dependent sirtuins) and in turn FOXO3 induces the stress resistance programs described above. This is a strong indication that FOXO3 causally contributes to lower oxidative damage under CR, as CR animals with functional FOXO3 accumulate fewer 8‑oxoG lesions (and live longer) than those without FOXO3 . In essence, CR “supercharges” FOXO3, which then keeps DNA oxidation in check.

Oxidative Stress Exposure: In acute or chronic oxidative stress scenarios (e.g. toxin exposure, hypoxia, radiation), FOXO3 activity is dynamically modulated to meet the challenge. Mild oxidative stress (e.g. low-dose H₂O₂) can activate FOXO3 by JNK phosphorylation or ATM signaling, causing FOXO3 to translocate to the nucleus and induce defense genes . This preemptive activation often prevents excessive DNA damage. For example, human thyrocytes with higher FOXO3 showed less DNA breakage after UV/H₂O₂, as noted earlier . In contrast, severe or sustained oxidative stress can overwhelm FOXO3 or even inactivate it (excess AKT activity in some diseases keeps FOXO3 cytosolic). In pathological states like diabetes or neurodegeneration, FOXO3 may be dysregulated, contributing to higher 8‑oxoG levels observed in those conditions . The FOXO3 rs2802292 G allele discussed above is particularly relevant in stress: it essentially gives cells a “fast response” enhancer to ramp up FOXO3 when stress hits . This could be life-saving in scenarios like heat waves, infections, or oxidative insults – consistent with the observed survival advantage in high-risk groups . Overall, whether the context is aging, CR, or acute stress, higher FOXO3 activity correlates with lower 8‑oxoG burden, whereas FOXO3 insufficiency or inactivity permits oxidative genome damage to rise.

Conclusion: Do Longevity-Associated FOXO3 Variants Lower 8‑oxoG?

Considering all evidence, there is a compelling biological plausibility that superior FOXO3 gene variants (like rs2802292 G) lead to a decreased burden of 8‑oxo-guanine in DNA. Human longevity alleles of FOXO3 are associated with enhanced stress resistance – for instance, better maintenance of telomeres and inducible FOXO3 upregulation under stress – which would logically reduce long-term oxidative damage. Direct proof in humans is still pending, but animal and cellular studies strongly support a causal link: when Foxo3 is knocked out in mice, cells accumulate more 8‑oxoG , and when FoxO3 is activated, oxidative lesions diminish . FOXO3 coordinates a network of antioxidants and DNA repair enzymes that specifically counteract the formation and persistence of 8‑oxoG in the genome. Therefore, it is very likely that individuals with higher FOXO3 function (due to genetic variation or other factors) experience lower levels of 8‑oxoG and 8‑oxodG over time, contributing to their longevity. The current evidence stops short of an absolute demonstration in humans, and one must acknowledge that longevity is multifactorial. Nevertheless, the trend across human, animal, and cell data points to the same conclusion: FOXO3’s activity is inversely linked with oxidative DNA damage, and the “better” FOXO3 variants tilt this balance in favor of genomic preservation. In summary, while more research is needed for direct confirmation, the superior FOXO3 genotype is highly likely to causally impart a lower 8‑oxoG burden, thereby promoting cellular longevity and organismal healthspan.

References: Key sources supporting these findings include human genetic studies , mouse model experiments , and molecular analyses of FOXO3’s role in oxidative stress and DNA repair , as detailed above. All evidence consistently underscores FOXO3 as a pivotal protector against oxidative DNA damage.

Study (Year)	Model / System	Key Findings (Klotho–FOXO3 Relationship)
Lim et al., 2017	Mouse tacrolimus-induced kidney injury; HK-2 renal cells	Klotho treatment protected kidneys from oxidative injury. Mechanism: Klotho inhibited PI3K/AKT, preventing FOXO3a phosphorylation, and thereby increased FOXO3’s binding to the MnSOD promoter. MnSOD expression rose, ROS production fell, and mitochondrial function improved . Klotho significantly reduced oxidative damage markers (8-OHdG, 4-HHE) and TUNEL+ apoptosis in the kidney , consistent with FOXO3-driven antioxidant effects.
Lim et al., 2019	Mouse tacrolimus nephropathy with ginseng intervention	Ginseng (a herbal extract) activated Klotho’s anti-aging signaling via FOXO3. Tacrolimus normally lowered Klotho levels and raised oxidative stress; ginseng reversed these effects. Mechanism: Ginseng suppressed PI3K/Akt, reduced FOXO3a phosphorylation, and promoted FOXO3a binding to the MnSOD promoter . This led to higher Klotho expression and antioxidant defenses, attenuating tacrolimus-induced ROS and kidney damage.
Zhu et al., 2024	Contrast-induced AKI in mice; iohexol-treated renal cells	Klotho alleviated acute kidney injury via a FOXO3–BNIP3 mitophagy pathway. Klotho-treated injured kidneys showed increased nuclear FOXO3 and upregulation of BNIP3 (mitophagy protein) with enhanced removal of damaged mitochondria . FOXO3 was required: FOXO3 knockdown blocked Klotho’s induction of BNIP3-dependent mitophagy and led to more ROS and apoptosis . Thus, FOXO3 is essential for Klotho’s mitochondrial protective effect.
Orellana et al., 2023	Mouse primary astrocytes (brain glial cells) in vitro	Exogenous Klotho promoted oxidative resilience in astrocytes by modulating insulin signaling and FOXO3. Klotho treatment (1 nM) for 24 h decreased p-Akt and mTOR levels in astrocytes, which corresponded with increased FOXO3a activity and higher catalase (antioxidant enzyme) expression . The activated FOXO3-catalase axis protected astrocytes from oxidative stress. (In neurons, Klotho affected metabolism via other routes, underscoring cell-type specific effects.)

Study (Year)	Model / Context	Key Findings (Klotho–8-oxoG Relationship)
Lim et al., 2017	Tacrolimus-induced oxidative stress in mouse kidney	Tacrolimus (Tac) caused high oxidative DNA damage in kidneys (elevated 8-OHdG in renal tissue and urine). Recombinant Klotho co-treatment greatly reduced 8-oxo-dG levels, as shown by decreased 8-OHdG staining in kidneys and lower urinary 8-OHdG excretion . Klotho-treated mice also had fewer TUNEL-positive apoptotic cells. These results indicate Klotho can prevent DNA damage and cell death caused by Tac-induced ROS .
Chen et al., 2020	Diabetic nephropathy (streptozotocin-diabetic mice); high-glucose podocyte culture	Klotho deficiency exacerbated DNA damage in diabetic kidneys. Klotho-knockout diabetic mice showed significantly higher 8-OHdG levels in podocyte nuclei alongside lower OGG1 expression, compared to diabetic mice with normal Klotho . In vitro, high glucose reduced OGG1 in podocytes, but adding recombinant Klotho restored OGG1 levels . Conclusion: Klotho deficiency impairs OGG1-mediated repair of 8-oxoG, leading to accumulation of DNA damage, whereas Klotho sufficiency preserves OGG1 and limits 8-oxoG lesions .
Orellana et al., 2023 (Sci. Rep.)	Klotho-treated neuronal cells under metabolic stress	Klotho’s antioxidative signaling (via FOXO3/Nrf2) in the brain may similarly reduce oxidative DNA damage. While this study focused on metabolic changes, it showed Klotho enhancing antioxidant responses in astrocytes . By inference, prolonged Klotho treatment in the CNS or other tissues is expected to lower 8-oxoG accrual over time, though direct DNA damage measures were not reported here. (This highlights a need for further research on Klotho’s genomic protection in neural cells.)
Clinical CKD context	Chronic kidney disease patients vs. controls	Patients with CKD (who typically have low Klotho levels) showed significantly elevated urinary 8-OHdG compared to healthy controls . This suggests higher systemic oxidative DNA damage in low-Klotho states. Therapeutic strategies to increase Klotho in CKD are hypothesized to reduce such damage and are under investigation.