Valentina Grishko

Apoptosis and mitochondrial dysfunction in human chondrocytes following exposure to lidocaine, bupivacaine, and ropivacaine.

BACKGROUND Several mechanisms have been proposed to explain toxicity of local anesthetics to chondrocytes, including the blockade of potassium channels and mitochondrial injury. The purposes of this investigation were to study the effects of lidocaine, bupivacaine, and ropivacaine on human chondrocyte viability and mitochondrial function in vitro and to characterize the type of cell death elicited following exposure. METHODS Primary chondrocyte cultures from patients with osteoarthritis undergoing knee replacement were treated with saline solution and the following concentrations of local anesthetics: 2%, 1%, and 0.5% lidocaine, 0.5% and 0.25% bupivacaine, and 0.5% and 0.2% ropivacaine for one hour. Cell viability and apoptosis were measured by flow cytometry at twenty-four hours and 120 hours after treatment. Nuclear staining and caspase 3 and 9 cleavage assays (Western blot) were used to further establish the induction of apoptosis. Mitochondrial dysfunction was evaluated by the accumulation of mitochondrial DNA damage (quantitative Southern blot), changes in adenosine triphosphate production (bioluminescence kit), and mitochondrial protein levels (Western blot analysis). RESULTS Exposure of primary human chondrocytes to a 2% concentration of lidocaine caused massive necrosis of chondrocytes after twenty-four hours, 1% lidocaine and 0.5% bupivacaine caused a detectable, but not significant, decrease in viability after twenty-four hours, while 0.5% lidocaine, 0.25% bupivacaine, and both concentrations of ropivacaine (0.5% and 0.2%) did not affect chondrocyte viability. Flow cytometry analysis of chondrocytes 120 hours after drug treatment revealed a significant decrease in viability (p < 0.05) with a concomitant increase in the number of apoptotic cells at all concentrations of lidocaine, bupivacaine, and ropivacaine analyzed, except 0.2% ropivacaine. Apoptosis was verified by observation of condensed and fragmented nuclei and a decrease in procaspase 3 and 9 levels. Local anesthetics induced mitochondrial DNA damage and a decrease in adenosine triphosphate and mitochondrial protein levels. CONCLUSIONS Lidocaine, bupivacaine, and ropivacaine cause delayed mitochondrial dysfunction and apoptosis in cultured human chondrocytes.

The Amyloid β Protein Induces Oxidative Damage of Mitochondrial DNA

Multiple lines of evidence suggest involvement of oxidative stress in the pathogenesis of Alzheimer disease (AD). The finding that amyloid beta peptide (Aβ) has neurotoxic properties and that such effects are mediated in part by free-radicals has provided an avenue to explore new therapeutic strategies. In this study, we showed that exposure of PC12 cells to an Aβ fragment induces oxidative damage of mitochondrial DNA. Cells were exposed for 24 h to 50 μM Aβ (25–35) or to 50 μM of a control peptide with a scrambled sequence. Oxidative damage of mitochondrial DNA (mtDNA) was assessed using a Southern blot technique and an mtDNA-specific probe recognizing a 13.5-kilobase restriction fragment. Treatment of DNA with NaOH was used to reveal abasic sites and single strand breaks. Treatment with endonuclease II or FAPy glycosylage was used to detect pyrimidine or purine lesions, respectively. Cells exposed to Aβ exhibited marked oxidative damage of mtDNA as evidenced by characteristic changes on Southern blots. Cells exposed to the scrambled peptide did not show such modifications. Simultaneous addition of the pineal hormone melatonin consistently prevented the Aβ-induced oxidative damage to mtDNA. Mitochondrial sysfunction in AD has been demonstrated by several laboratorie. This study provides experimental evidence supporting a causative role of Aβ in mitochondrial lesions of AD.

Mitochondrial DNA damage is involved in apoptosis caused by pro-inflammatory cytokines in human OA chondrocytes

OBJECTIVE Pro-inflammatory cytokines play a pivotal role in cartilage destruction during the progression of osteoarthritis (OA). Additionally, these cytokines are capable to generate reactive oxygen and nitrogen species within chondrocytes. Mitochondrion is a prime target of oxidative damage and an important player in aging and degenerative processes. The purpose of the present study was to investigate whether these cytokines will alter the mitochondrial DNA (mtDNA) integrity and mitochondrial function in both normal and osteoarthritic human chondrocytes. DESIGN Primary normal and osteoarthritic human chondrocyte cultures were exposed to various concentrations of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) for different time. Following exposure, chondrocytes were evaluated for mitochondrial DNA damage, ATP production, changes in mitochondrial transcription, and apoptosis. Adenoviral vectors were used to deliver DNA repair enzyme hOGG1 to mitochondria. RESULTS Pro-inflammatory cytokines IL-1beta and TNF-alpha disturb mitochondrial function in human chondrocytes by inducing mitochondrial DNA damage, decreasing energy production and mitochondrial transcription, which correlated with the induction of apoptosis. Increased NO production was the key factor responsible for accumulation of mtDNA damage after cytokine exposure. Mitochondrial superoxide production was also enhanced following pro-inflammatory cytokine exposure. OA chondrocyte mitochondria were more susceptible to damage induced by pro-inflammatory cytokines then mitochondria from normal chondrocytes. Protection of human chondrocytes from mtDNA damage by the mitochondria-targeted DNA repair enzyme hOGG1 rescued mtDNA integrity, preserved ATP levels, reestablished mitochondrial transcription, and significantly diminished apoptosis following IL-1beta and TNF-alpha exposure. CONCLUSION Mitochondrion is an important target in pro-inflammatory cytokine toxicity, maintaining of mitochondrial DNA integrity is necessary to prevent chondrocytes from apoptosis induced by IL-1beta and TNF-alpha.

Hypoxia promotes oxidative base modifications in the pulmonary artery endothelial cell VEGF gene

Hypoxia, a stimulus for angiogenesis and vascular remodeling, has been proposed to use reactive oxygen species as second messengers in signal transduction. This contention remains controversial, in part because of vagaries associated with fluorescence‐based methods of free‐radical detection. We took a different approach. Rat main pulmonary artery endothelial cells (PAECs) were cultured in hypoxia for up to 48 h, and, with dichlorofluorescein fluorescence to detect free‐radical production, we used quantitative Southern blot and ligation‐mediated PCR analyses to search for oxidative modifications in the mitochondrial genome and in the nuclear vascular endothelial cell growth factor (VEGF) gene. In accord with previous studies in other cell types, we found that acute hypoxic exposure promoted time‐dependent dichlorofluorescein fluorescence in PAECs. Quantitative Southern blot analysis showed that although hypoxia failed to alter mitochondrial DNA integrity, prominent oxidative lesions occurred in a 5.0‐kb sequence of the VEGF promoter. Using ligation‐mediated PCR to map the modifications at single nucleotide resolution, we found clusters of oxidized bases in a VEGF promoter sequence that included the AP‐1 and HIF‐1 response elements. These actions of hypoxia differed from exogenous xanthine oxidase, which obliterated the mitochondrial genome but failed to erode integrity of the VEGF promoter. Our observations indicate that hypoxia promotes an oxidant stress in main PAECs as detected by oxidative base modifications in the nuclear VEGF gene. The presence of hypoxia‐induced, oxidative base modifications in functionally significant sequences within the VEGF promoter suggests new concepts for mechanisms by which reactive oxygen species participate in hypoxic signal transduction.

Diminished mitochondrial DNA integrity and repair capacity in OA chondrocytes

OBJECTIVES Osteoarthritis (OA) is characterized by the failure of chondrocytes to respond to injury and perform the cartilage remodeling process. Human articular chondrocytes actively produce reactive oxygen and nitrogen species (ROS and RNS) capable of causing cellular dysfunction and death. A growing body of evidence indicates that mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage play a causal role in disorders linked to excessive generation of oxygen free radicals. The aim of this study was to determine whether mtDNA damage was present in OA chondrocytes, and whether mtDNA repair capacity is compromised in OA chondrocytes following oxidative stress, leading to chondrocyte death. METHODS Human articular cartilage was isolated from knee joints of cadavers available through the Anatomical Gifts Program at the University of South Alabama (normal donors) or OA patients undergoing total knee replacement surgeries (OA patients). Total DNA was isolated from either chondrocytes released following collagenase digestion, or from first passage chondrocytes grown in culture and exposed to ROS or RNS. mtDNA integrity and repair capacity were analyzed by quantitative Southern blot analysis, using a mtDNA-specific radioactive probe. Cell viability was determined by the trypan blue exclusion method. RESULTS mtDNA damage was found in chondrocytes from OA patients compared to normal donors. It was accompanied with reduced mtDNA repair capacity, cell viability, and increased apoptosis in OA chondrocytes following exposure to ROS and RNS. CONCLUSIONS These results indicate that mtDNA damage and poor mtDNA repair capacity for removing damage caused by oxidative stress may contribute to the pathogenesis of OA.

Effects of Hyaluronic Acid on Mitochondrial Function and Mitochondria-driven Apoptosis following Oxidative Stress in Human Chondrocytes

Hyaluronic acid is widely used in the treatment of osteoarthritis and exerts significant chondroprotective effects. The exact mechanisms of its chondroprotective action are not yet fully elucidated. Human articular chondrocytes actively produce reactive oxygen and nitrogen species capable of causing cellular dysfunction and death. A growing body of evidence indicates that mitochondrial dysfunction and mitochondrial DNA damage play a causal role in disorders linked to excessive generation of oxygen free radicals. We hypothesized that the chondroprotective effects of hyaluronic acid on oxidatively stressed chondrocytes are due to preservation of mitochondrial function and amelioration of mitochondria-driven apoptosis. When primary human chondrocyte cultures were exposed to reactive oxygen or nitrogen species generators, mitochondrial DNA damage along with mitochondrial dysfunction and mitochondria-driven apoptosis accumulated as a consequence. In addition, cytokine-treated primary human chondrocytes showed increased levels of mitochondrial DNA damage. Pretreatment of chondrocytes with hyaluronic acid caused a decrease of mitochondrial DNA damage, enhanced mitochondrial DNA repair capacity and cell viability, preservation of ATP levels, and amelioration of apoptosis. The results of these studies demonstrate that enhanced chondrocyte survival and improved mitochondrial function under conditions of oxidative injury are probably important therapeutic mechanisms for the actions of hyaluronic acid in osteoarthritis.

Oxidants in signal transduction: impact on DNA integrity and gene expression

Physiological stimuli using reactive oxygen species (ROS) as second messengers caused nucleotide‐specific base modifications in the hypoxic response element of the VEGF gene in lung vascular cells, with the 3′ guanine of the HIF‐1 DNA recognition sequence uniformly targeted. Modeling this effect by replacing the targeted guanine with an abasic site increased incorporation of HIF‐1 and the bi‐functional DNA repair enzyme and transcriptional coactivator, Ref‐1/Ape1, into the transcriptional complex and engendered more robust reporter gene expression. Oxidants generated in the context of physiological signaling thus affect nuclear DNA integrity and may facilitate gene expression by optimizing DNA‐protein interactions.—Ziel, K. A., Grishko, V., Campbell, C. C., Breit, J. F., Wilson, G. L., Gillespie, M. N. Oxidants in signal transduction: impact on DNA integrity and gene expression. FASEB J. 19, 387‐394 (2005)

Glial cell-specific differences in response to alkylation damage

Oligodendrocytes are preferentially sensitive to the toxic, carcinogenic, and teratogenic effects of methylnitrosourea (MNU). The mechanisms responsible for this enhanced sensitivity have not been fully elucidated. One of the most vulnerable cellular targets for this chemical is mitochondrial DNA (mtDNA). To determine if differences in mtDNA damage and repair capacity exist among the different CNS glial cell types, the effects of MNU exposure on oligodendroglia, astroglia, and microglia cultured separately from neonatal rat brain were compared. Quantitative determinations of mtDNA initial break frequencies and repair efficiencies showed that whereas no cell type‐specific differences in initial mtDNA damage were detected, mtDNA repair in oligodendrocytes, oligodendrocyte progenitors, and microglia was significantly reduced compared to that of astrocytes. In astrocytes, and all other cell types previously evaluated in our laboratory, >60% of N‐methylpurines were removed from the mtDNA by 24 hr. In contrast, only 35% of lesions were removed from mtDNA of oligodendrocytes, oligodendrocyte progenitors, and microglia during the same time period. Mitochondrial perturbations by a variety of xenobiotics have been linked to apoptosis. In the present study, apoptosis, as determined by DNA laddering and ultrastructural analysis, was clearly induced by MNU treatment of cultured oligodendrocyte progenitors and microglia, but not in astroglia. These data demonstrate a correlation between diminished mtDNA repair capacity and the induction of apoptosis. However, further experimentation is necessary to determine if a causal relationship exists and contributes to the vulnerability of oligodendroglia following exposure to N‐nitroso compounds in the environment or in chemotherapeutic regimen. GLIA 24:304–312, 1998.

The role of mitochondrial reactive oxygen species in cartilage matrix destruction

Upregulation of matrix metalloproteinases (MMPs) is a hallmark of osteoarthritis progression; along with the role reactive oxygen species (ROS) may play in this process. Moreover, mitochondrial DNA damage and dysfunction are also present in osteoarthritic chondrocytes. However, there are no studies published investigating the direct relationship between mitochondrial ROS, mitochondrial DNA damage, and MMP expression. Therefore, the purpose of the present study was to evaluate whether mitochondrial DNA damage and mitochondrial-originated oxidative stress modulates matrix destruction through the upregulation of MMP protein levels. MitoSox red was utilized to observe mitochondrial ROS production while a Quantitative Southern blot technique was conducted to analyze mitochondrial DNA damage. Additionally, Western blot analysis was used to determine MMP protein levels. The results of the present study show that menadione augmented mitochondrial-generated ROS and increased mitochondrial DNA damage. This increase in mitochondrial-generated ROS led to an increase in MMP levels. When a mitochondrial ROS scavenger was added, there was a subsequent reduction in MMP levels. These studies reveal that mitochondrial integrity is essential for maintaining the cartilage matrix by altering MMP levels. This provides new and important insights into the role of mitochondria in chondrocyte function and its potential importance in therapeutic approaches.

Catalytic activity of poly(ADP-ribose) polymerase is necessary for repair of N-methylpurines in nontranscribed, but not in transcribed, nuclear DNA sequences

The role of poly(ADP-ribose) polymerase (PADPRP) in nuclear DNA repair and other nuclear processes has been intensely studied and debated for decades. Recent studies have begun to shed new light on these arguments with firm experimental data for its role, primarily, as a remodeler of chromatin structure. Those studies imply that PADPRP plays an indirect role in DNA repair, serving to expose DNA to repair enzymes through chromatin remodeling. Only DNA that is tightly packaged would require PADPRP activity for its repair; while DNA in an open conformation would be accessible to DNA repair enzymes and not require PADPRP activity. The purpose of the current studies was to address the above hypothesis directly. Using quantitative Southern blot analysis, we studied repair in transcribed and nontranscribed nuclear DNA sequences in ADPRT 351 cells 95% deficient in PADPRP activity. Cells were exposed to methylnitrosourea (MNU) for 1 h and allowed to repair for 8 or 24 h. Densitometric scans of autoradiographs revealed that, when compared to their parental V79 cell line, ADPRT 351 cells 95% deficient in PADPRP activity were equally as efficient in repair of N-methylpurines in the transcribed sequence containing the dihydrofolate reductase gene. However, the ADPRT 351 cells were deficient in the ability to repair these lesions in the nontranscribed sequence containing the IgE gene compared to repair of the same sequence in the parental V79 cells. Nucleoid sedimentation assays demonstrated that the ADPRT 351 cells are deficient in repair across the entire genome when compared to the parental V79 cells. These studies indicate that PADPRP activity is not required for repair of N-methylpurines in transcribed nuclear DNA sequences but is necessary for the repair of these lesions in nontranscribed nuclear DNA sequences as well as across the entire genome since the DNA in a given cell is predominantly nontranscribed.