Hydrogen sulfide attenuates homocysteine-induced osteoblast dysfunction by inhibiting mitochondrial toxicity

Homocysteine (Hcy) is detrimental to bone health in a mouse model of diet-induced hyperhomocysteinemia (HHcy). However, little is known about Hcy-mediated osteoblast dysfunction via mitochondrial oxidative damage. Hydrogen sulfide (H₂S) has potent antioxidant, anti-inflammatory, and antiapoptotic effects. In this study, we hypothesized that the H₂S mediated recovery of osteoblast dysfunction by maintaining mitochondrial biogenesis in Hcy-treated osteoblast cultures in vitro. MC3T3-E1 osteoblastic cells were exposed to Hcy treatment in the presence or absence of an H₂S donor (NaHS). Cell viability, osteogenic differentiation, reactive oxygen species (ROS) production were determined. Mitochondrial DNA copy number, adenosine triphosphate (ATP) production, and oxygen consumption were also measured. Our results demonstrated that administration of Hcy increases the intracellular Hcy level and decreases intracellular H₂S level and expression of the cystathionine β-synthase/Cystathionine γ-lyase system, thereby inhibiting osteogenic differentiation. Pretreatment with NaHS attenuated Hcy-induced mitochondrial toxicity (production of total ROS and mito-ROS, ratio of mitochondrial fission (DRP-1)/fusion (Mfn-2)) and restored ATP production and mitochondrial DNA copy numbers as well as oxygen consumption in the osteoblast as compared with the control, indicating its protective effects against Hcy-induced mitochondrial toxicity. In addition, NaHS also decreased the release of cytochrome c from the mitochondria to the cytosol, which induces cell apoptosis. Finally, flow cytometry confirmed that NaHS can rescue cells from apoptosis induced by Hcy. Our studies strongly suggest that NaHS has beneficial effects on mitochondrial toxicity, and could be developed as a potential therapeutic agent against HHcy-induced mitochondrial dysfunction in cultured osteoblasts in vitro.     

Homocysteine-Induced Apoptosis in Endothelial Cells Coincides With Nuclear NOX2 and Peri-nuclear NOX4 Activity

Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01–2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47^phox expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ _m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ _m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47^phox, and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47^phox in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47^phox was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.     

Homocysteine-mediated activation and mitochondrial translocation of calpain regulates MMP-9 in MVEC

Hyperhomocysteinemia (HHcy) is associated with atherosclerosis, stroke, and dementia. Hcy causes extracellular matrix remodeling by the activation of matrix metalloproteinase-9 (MMP-9), in part, by inducing redox signaling and modulating the intracellular calcium dynamics. Calpains are the calcium-dependent cysteine proteases that are implicated in mitochondrial damage via oxidative burst. Mitochondrial abnormalities have been identified in HHcy. The mechanism of Hcy-induced extracellular matrix remodeling by MMP-9 activation via mitochondrial pathway is largely unknown. We report a novel role of calpains in mitochondrial-mediated MMP-9 activation by Hcy in cultured rat heart microvascular endothelial cells. Our observations suggested that calpain regulates Hcy-induced MMP-9 expression and activity. We showed that Hcy activates calpain-1, but not calpain-2, in a calcium-dependent manner. Interestingly, the enhanced calpain activity was not mirrored by the decreased levels of its endogenous inhibitor calpastatin. We presented evidence that Hcy induces the translocation of active calpain from cytosol to mitochondria, leading to MMP-9 activation, in part, by causing intramitochondrial oxidative burst. Furthermore, studies with pharmacological inhibitors of calpain (calpeptin and calpain-1 inhibitor), ERK (PD-98059) and the mitochondrial uncoupler FCCP suggested that calpain and ERK-1/2 are the major events within the Hcy/MMP-9 signal axis and that intramitochondrial oxidative stress regulates MMP-9 via ERK-1/2 signal cascade. Taken together, these findings determine the novel role of mitochondrial translocation of calpain-1 in MMP-9 activation during HHcy, in part, by increasing mitochondrial oxidative stress.     

Homocysteine activates T cells by enhancing endoplasmic reticulum-mitochondria coupling and increasing mitochondrial respiration

Hyperhomocysteinemia (HHcy) accelerates atherosclerosis by increasing proliferation and stimulating cytokine secretioninTcells.However,whetherhomocysteine (Hcy)-mediated T cell activation is associated with metabolic reprogramming is unclear. Here, our in vivoand in vitrostudies showed that Hcy-stimulated splenic T-cell activation in mice was accompanied by increased levels of mitochondrial reactive oxygen species (ROS) and calcium, mitochondrial mass and respiration. Inhibiting mitochondrial ROS production and calcium signals or blocking mitochondrial respiration largely blunted Hcy-induced T-cell interferon γ (IFN-γ) secretion and proliferation. Hcy also enhanced endoplasmic reticulum (ER) stress in T cells, and inhibition ofERstress with 4-phenylbutyric acid blocked Hcy-induced T-cell activation. Mechanistically, Hcy increased ER-mitochondria coupling, and uncoupling ER-mitochondria by the microtubule inhibitor nocodazole attenuated Hcy-stimulated mitochondrial reprogramming, IFN-γ secretion and proliferation in T cells, suggesting that juxtaposition of ER and mitochondria is required for Hcy-promoted mitochondrial function and T-cell activation. In conclusion, Hcy promotes T-cell activation by increasing ER-mitochondria coupling and regulating metabolic reprogramming.     

Upregulation of aldose reductase by homocysteine in type II alveolar epithelial cells

Homocysteine (Hcy) has recently been recognized as an integral component of several disorders. However, the association between hyperhomocysteinemia (HHcy) and pulmonary disease is not well understood. The combination of two-dimensional electrophoresis and tandem mass spectrometry detected and identified proteins that are differentially expressed in human type II alveolar epithelial cells (A549 cells) treated by Hcy. We found that aldose reductase (AR) showed more abundant expression in the cells. Further, Hcy (100-500microM) could induce a time- and dose-dependent upregulation of AR protein levels. Immunohistochemical staining of cross-sections from HHcy mice lungs also revealed increased expression of AR protein. Intracellular levels of reactive oxygen species (ROS) were remarkably elevated in A549 cells treated with Hcy. Pretreatment of A549 cells with catalase and SOD significantly suppressed the Hcy-induced AR expression, which suggests the involvement of ROS in this process. The major signaling pathway mediating the upregulation of AR was demonstrated to be the Ras/Raf/ERK1/2 pathway. In addition, Hcy might reduce surfactant protein B (SP-B) expression in the cells, which could be significantly attenuated by Alrestatin, an AR inhibitor, indicating a damaging role of Hcy-induced AR elevation in the lung. These results show a novel and unanticipated link between HHcy and AR upregulation that may be a risk factor in pulmonary disease of patients with HHcy.     

Homocysteine-induced decrease in endothelin-1 production is initiated at the extracellular level and involves oxidative products.

The increased cardiovascular risk associated with hyperhomocysteinemia has been partly related to homocysteine (Hcy)-induced endothelial cell dysfunction. However, the intra or extracellular starting point of the interaction between Hcy and endothelial cells, leading to cellular dysfunction, has not yet been identified. We investigated the effects of both intracellular and extracellular Hcy accumulation on endothelin-1 (ET-1) synthesis by cultured human endothelial cells. Incubation of cultures with methionine (1.0 mmol x L(-1)) for 2 h induced a slight increase in cellular Hcy content but no change in ET-1 production. Incubation of cells with Hcy (0.2 mmol x L(-1)) led to a significant fall in ET-1 generation, accompanied by a significant increase in cellular Hcy content. Addition of the amino-acid transport system L substrate 2-amino-2-norbornane carboxylic acid had no effect on the Hcy-induced decrease in ET-1 production but significantly inhibited the Hcy-induced increase in the cellular Hcy content. Incubation of cells with a lower Hcy concentration (0.05 mmol x L(-1)) also reduced ET-1 production without increasing the cellular Hcy content. Co-incubation with extracellular free-radical inhibitors (superoxide dismutase, catalase and mannitol) markedly reduced the effect of Hcy on ET-1 production. Thus, it is extracellular Hcy accumulation that triggers the decrease in ET-1 production by endothelial cells through oxidative products.     

Chronic exposure of homocysteine in mice contributes to dopamine loss by enhancing oxidative stress in nigrostriatum and produces behavioral phenotypes of Parkinson’s disease

Increased homocysteine (Hcy) level has been implicated as an independent risk factor for various neurological disorders, including Parkinson’s disease (PD). Hcy has been reported to cause dopaminergic neuronal loss in rodents and causes the behavioral abnormalities. This study is an attempt to investigate molecular mechanisms underlying Hcy-induced dopaminergic neurotoxicity after its chronic systemic administration. Male Swiss albino mice were injected with different doses of Hcy (100 and 250 mg/kg; intraperitoneal) for 60 days. Animals subjected to higher doses of Hcy, but not the lower dose, produces motor behavioral abnormalities with significant dopamine depletion in the striatum. Significant inhibition of mitochondrial complex-I activity in nigra with enhanced activity of antioxidant enzymes in the nigrostriatum have highlighted the involvement of Hcy-induced oxidative stress. While, chronic exposure to Hcy neither significantly alters the nigrostriatal glutathione level nor it causes any visible change in tyrosine hydroxylase-immunoreactivity of dopaminergic neurons. The finding set us to hypothesize that the mild oxidative stress due to prolonged Hcy exposure to mice is conducive to striatal dopamine depletion leading to behavioral abnormalities similar to that observed in PD.     

Vascular oxidative stress increases dendritic cell adhesion and transmigration induced by homocysteine

Atherosclerosis is a long-term chronic inflammatory and immunological disease. Endothelial dysfunction and the dendritic cell (DC) immune response are pivotal early events in atherogenesis. This study investigated the effects and possible mechanisms of action of homocysteine (Hcy) on DC adhesion to and transmigration between endothelial cells (ECs), and indicated a novel immunoregulatory mechanism by which Hcy induces atherogenesis. When ECs were stimulated with increasing concentrations of Hcy, immunofluorescence showed that endothelial reactive oxygen species (ROS) generation strikingly increased, while nitrite assay showed that nitric oxide (NO) release markedly decreased. Furthermore, DC adhesion and transmigration were significantly increased when ECs were activated by Hcy. However, pretreatment of ECs with antioxidant before Hcy markedly attenuated the induction of DC adhesion and transmigration, dependent on the intracellular ROS decrease and endothelial NO increase. In conclusion, DC adhesion and transmigration are significantly increased by vascular oxidative stress under conditions of elevated Hcy levels. These findings provide insight into the inflammatory processes and immune responses occurring in atherosclerosis induced by Hcy.     

Cardiac specific deletion of N-methyl-d-aspartate receptor 1 ameliorates mtMMP-9 mediated autophagy/mitophagy in hyperhomocysteinemia

Autophagy is an important process in the pathogenesis of cardiovascular diseases; however, the proximal triggers for mitochondrial autophagy were unknown. The N-methyl-d-aspartate receptor 1 (NMDA-R1) is a receptor for homocysteine (Hcy) and plays a key role in cardiac dysfunction. Cardiac-specific deletion of NMDA-R1 has been shown to ameliorate Hcy-induced myocyte contractility. Hcy activates mitochondrial matrix metalloproteinase-9 (mtMMP-9) and induces translocation of connexin-43 (Cxn-43) to the mitochondria (mtCxn-43). We sought to show cardiac-specific deletion of NMDA-R1 mitigates Hcy-induced mtCxn-43 translocation, mtMMP-9-mediated mtCxn-43 degradation, leading to mitophagy, in part, by decreasing mitochondrial permeability (MPT). Cardiac-specific knockout (KO) of NAMDA-R1 was generated using the cre/lox approach. The myocyte mitochondria were isolated from wild type (WT), WT + Hcy (1.8?g of DL-Hcy/L in the drinking water for 6 weeks), NMDA-R1 KO + Hcy, and NR1^fl/fl/Cre (NR1^fl/fl) genetic control mice. Mitochondrial respiratory capacity and MPT were measured by fluorescence-dye methods. The mitochondrial superoxide and peroxinitrite levels were detected by confocal microscopy using Mito-SOX and dihydrorhodamine-123. The mtMMP-9 activity and expression were detected by zymography and RT-PCR analyses. The mtCxn-43 translocation was detected by confocal microscopy. The degradation of mtCxn-43 and LC3-I/II (a marker of autophagy) were detected by Western blot. These results suggested that Hcy enhanced intramitochondrial nitrosative stress in myocytes. There was a robust increase in mtMMP-9 activity. An increase in translocation and degradation of mtCxn-43 was also noted. These increases led to mitophagy. The effects were ameliorated by cardiac-specific deletion of NMDA-R1. We concluded that HHcy increased mitochondrial nitrosative stress, thereby activating mtMMP-9 and inciting the degradation of mtCxn-43. This led to mitophagy, in part, by activating NMDA-R1. The findings of this study will lead to therapeutic ramifications for mitigating cardiovascular diseases by inhibiting the mitochondrial mitophagy and NMDA-R1 receptor.     

Disturbance of Copper Homeostasis Is a Mechanism for Homocysteine-Induced Vascular Endothelial Cell Injury

Elevation of serum homocysteine (Hcy) levels is a risk factor for cardiovascular diseases. Previous studies suggested that Hcy interferes with copper (Cu) metabolism in vascular endothelial cells. The present study was undertaken to test the hypothesis that Hcy-induced disturbance of Cu homeostasis leads to endothelial cell injury. Exposure of human umbilical vein endothelial cells (HUVECs) to concentrations of Hcy at 0.01, 0.1 or 1 mM resulted in a concentration-dependent decrease in cell viability and an increase in necrotic cell death. Pretreatment of the cells with a final concentration of 5 µM Cu in cultures prevented the effects of Hcy. Hcy decreased intracellular Cu concentrations. HPLC-ICP-MS analysis revealed that Hcy caused alterations in the distribution of intracellular Cu; more Cu was redistributed to low molecular weight fractions. ESI-Q-TOF detected the formation of Cu-Hcy complexes. Hcy also decreased the protein levels of Cu chaperone COX17, which was accompanied by a decrease in the activity of cytochrome c oxidase (CCO) and a collapse of mitochondrial membrane potential. These effects of Hcy were all preventable by Cu pretreatment. The study thus demonstrated that Hcy disturbs Cu homeostasis and limits the availability of Cu to critical molecules such as COX17 and CCO, leading to mitochondrial dysfunction and endothelial cell injury.     

Supplemental ascorbate or alpha-tocopherol induces cell death in Cu-deficient HL-60 cells

Cytochrome c oxidase (CCO) is the Cu-dependent, terminal respiratory complex of the mitochondrial electron transport chain. Inhibition of CCO can promote oxidative stress by increasing mitochondrial production of reactive oxygen species (ROS). Because mitochondria have an important role in apoptosis as both a target and source for ROS, enhanced ROS production resulting from inhibition of CCO by Cu deficiency may trigger apoptosis. The present study focuses on the mitochondrial effects of N,N'-bis(2-aminoethyl)-1,3-propanedi-amine (TET), which inhibits CCO by causing cellular Cu deficiency, and the antioxidants ascorbate and alpha-tocopherol in a human promyelocytic leukemia cell line (HL-60). The following effects were observed: (i) TET reduced both cell growth and viability only in the presence of ascorbate or alpha-tocopherol; (ii) TET reduced CCO activity and increased mitochondrial ROS production as indicated by increased expression of Mn super-oxide dismutase, but the induction of Mn superoxide dismutase was not affected by ascorbate or alpha-tocopherol; (iii) TET acted independently of ascorbate or alpha-tocopherol in disrupting mitochondrial membrane potential; (iv) TET did not increase caspase-8 activity in the absence of ascorbate or alpha-tocopherol; and (v) TET did not increase transfer of cytochrome c from mitochondria to the cytosol unless alpha-tocopherol was present. These findings indicate that reduction in CCO activity by TET-induced Cu deficiency increased oxidative stress in HL-60 cells sufficiently to disrupt the electrochemical gradient of the inner mitochondrial membrane but did not trigger cell death. Also, ascorbate and alpha-tocopherol did not alleviate oxidative stress but may have become pro-oxidants, adding to the oxidant burden sufficiently to trigger cell death in TET-treated cells.     

Homocysteine induces VCAM-1 gene expression through NF-kappaB and NAD(P)H oxidase activation: protective role of Mediterranean diet polyphenolic antioxidants

Hyperhomocysteinemia is a recognized risk factor for vascular disease, but pathogenetic mechanisms involved in its vascular actions are largely unknown. Because VCAM-1 expression is crucial in monocyte adhesion and early atherogenesis, we evaluated the NF-kappaB-related induction of VCAM-1 by homocysteine (Hcy) and the possible inhibitory effect of dietary polyphenolic antioxidants, such as trans-resveratrol (RSV) and hydroxytyrosol (HT), which are known inhibitors of NF-kappaB-mediated VCAM-1 induction. In human umbilical vein endothelial cells (HUVEC), Hcy, at 100 micromol/l, but not cysteine, induced VCAM-1 expression at the protein and mRNA levels, as shown by enzyme immunoassay and Northern analysis, respectively. Transfection studies with deletional VCAM-1 promoter constructs demonstrated that the two tandem NF-kappaB motifs in the VCAM-1 promoter are necessary for Hcy-induced VCAM-1 gene expression. Hcy-induced NF-kappaB activation was confirmed by EMSA, as shown by the nuclear translocation of its p65 (RelA) subunit and the degradation of the inhibitors IkappaB-alpha and IkappaB-beta by Western analysis. Hcy also increased intracellular reactive oxygen species by NAD(P)H oxidase activation, as shown by the membrane translocation of its p47(phox) subunit. NF-kappaB inhibitors decreased Hcy-induced intracellular reactive oxygen species and VCAM-1 expression. Finally, we found that nutritionally relevant concentrations of RSV and HT, but not folate and vitamin B6, reduce (by >60% at 10(-6) mol/l) Hcy-induced VCAM-1 expression and monocytoid cell adhesion to the endothelium. These data indicate that pathophysiologically relevant Hcy concentrations induce VCAM-1 expression through a prooxidant mechanism involving NF-kappaB. Natural Mediterranean diet antioxidants can inhibit such activation, suggesting their possible therapeutic role in Hcy-induced vascular damage.     

Resveratrol protects cardiomyocytes from oxidative stress through SIRT1 and mitochondrial biogenesis signaling pathways

Reactive oxygen species (ROS) is generated by oxidative stress and plays an important role in various cardiac pathologies. The SIRT1 signaling pathway and mitochondrial biogenesis play essential roles in mediating the production of ROS. SIRT1 activated by resveratrol protects cardiomyocytes from oxidative stress, but the exact mechanisms by which SIRT1 prevents oxidative stress, and its relationship with mitochondrial biogenesis, remain unclear. In this study, it was observed that after stimulation with 50 μM H₂O₂ for 6 h, H9C2 cells produced excessive ROS and downregulated SIRT1. The mitochondrial protein NDUFA13 was also downregulated by ROS mediated by SIRT1. Resveratrol induced the expression of SIRT1 and mitochondrial genes NDUFA1, NDUFA2, NDUFA13 and Mn-SOD. However, the production of these genes was reversed by SIRT1 inhibitor nicotinamide. These results suggest that resveratrol inhibits ROS generation in cardiomyocytes via SIRT1 and mitochondrial biogenesis signaling pathways.     

Regulatory role of thioredoxin in homocysteine-induced monocyte chemoattractant protein-1 secretion in monocytes/macrophages

We have previously shown that homocysteine (Hcy) can induce monocyte chemoattractant protein-1 (MCP-1) secretion via reactive oxygen species (ROS) in human monocytes. Here, we show that Hcy upregulates expression of an important antioxidative protein, thioredoxin (Trx), via NADPH oxidase in human monocytes in vitro. The increase of Trx expression and activity inhibited Hcy-induced ROS production and MCP-1 secretion. Of note, 2-week hyperhomocysteinemia (HHcy) ApoE^−/− mice showed accelerated lesion formation and parallel lower Trx expression in macrophages than ApoE^−/− mice, suggesting that HHcy-induced sustained oxidative stress in vivo might account for impaired Trx and hence increased ROS production and MCP-1 secretion from macrophages, and subsequently accelerated atherogenesis.     

Bisphenol A induces oxidative stress and mitochondrial dysfunction in lymphoblasts from children with autism and unaffected siblings

Autism is a behaviorally defined neurodevelopmental disorder. Although there is no single identifiable cause for autism, roles for genetic and environmental factors have been implicated in autism. Extensive evidence suggests increased oxidative stress and mitochondrial dysfunction in autism. In this study, we examined whether bisphenol A (BPA) is an environmental risk factor for autism by studying its effects on oxidative stress and mitochondrial function in the lymphoblasts. When lymphoblastoid cells from autistic subjects and age-matched unaffected sibling controls were exposed to BPA, there was an increase in the generation of reactive oxygen species (ROS) and a decrease in mitochondrial membrane potential in both groups. A further subdivision of the control group into two subgroups—unaffected nontwin siblings and twin siblings—showed significantly higher ROS levels without any exposure to BPA in the unaffected twin siblings compared to the unaffected nontwin siblings. ROS levels were also significantly higher in the autism vs the unaffected nontwin siblings group. The effect of BPA on three important mtDNA genes—NADH dehydrogenase 1, NADH dehydrogenase 4, and cytochrome b—was analyzed to observe any changes in the mitochondria after BPA exposure. BPA induced a significant increase in the mtDNA copy number in the lymphoblasts from the unaffected siblings group and in the unaffected twin siblings group vs the unaffected nontwin siblings. In all three genes, the mtDNA increase was seen in 70% of the subjects. These results suggest that BPA exposure results in increased oxidative stress and mitochondrial dysfunction in the autistic subjects as well as the age-matched sibling control subjects, particularly unaffected twin siblings. Therefore, BPA may act as an environmental risk factor for autism in genetically susceptible children by inducing oxidative stress and mitochondrial dysfunction.     

Increase in mitochondrial mass in human fibroblasts under oxidative stress and during replicative cell senescence

Abnormal proliferation of mitochondria generally occurs in muscle of aged individuals and patients with mitochondrial myopathy. An increase in the mitochondrial DNA (mtDNA) copy number has also been observed in aging human tissues. However, the molecular mechanism underlying the increase in mitochondrial mass and mtDNA is still unclear. In a previous study, we demonstrated that sublethal levels of oxidative stress caused an increase in mitochondrial mass in human lung cells. In this communication, we report our recent findings that the mitochondrial mass in human lung fibroblasts (MRC-5) in a later proliferation stage is significantly increased compared to that in the early stages of proliferation. The extent of the increase in mitochondrial mass in the senescent cells was similar to that in cells in the early stages of proliferation that had been treated with low concentrations ( 180 µM) of hydrogen peroxide (H₂O₂). Moreover, we found that the rate of reactive oxygen species (ROS) production was higher in cells in the later proliferation stage compared to cells in the early proliferation stages. A similar phenomenon was also observed in cells in the early proliferation stages under low levels of oxidative stress. On the other hand, the mRNA levels of many nuclear DNA-encoded proteins involved in mitochondrial biogenesis, particularly nuclear respiratory factor-1, were found to increase in cells in later proliferation stages and in cells in early proliferation stages that had been treated with 180 µM H₂O₂. Interestingly, the increase in mitochondrial mass in the cells under oxidative stress could be repressed by treatment with cycloheximide orm-chlorocarbonyl cyanide phenylhydrazone but not by chloramphenicol. Furthermore, the mitochondrial mass of mtDNA-less ° cells was also significantly increased by exposure to low concentrations (e.g. 180 µM) of H₂O₂. These results suggest that the increase in mitochondrial mass in replicative senescent cells may result from an increase in ROS production, and that it is dependent on both de novo synthesis of nuclear DNA-encoded proteins and their import into mitochondria, dictated by the membrane potential of mitochondria.