Mitochondria-targeted plastoquinone antioxidant SkQR1 decreases trauma-induced neurological deficit in rat

A protective effect of a mitochondria-targeted antioxidant, a cationic rhodamine derivative linked to a plastoquinone molecule (10-(6′-plastoquinonyl)decylrhodamine-19, SkQR1) was studied in the model of open focal trauma of rat brain sensorimotor cortex. It was found that daily intraperitoneal injections of SkQR1 (100 nmol/kg) for 4 days after the trauma improved performance in a test characterizing neurological deficit and decreased the volume of the damaged cortical area. Our results suggest that SkQR1 exhibits profound neuroprotective effect, which may be explained by its antioxidative activity.     

Mitochondria-targeted antioxidant SkQR1 selectively protects MDR (Pgp 170)-negative cells against oxidative stress

A conjugate of plastoquinone with decylrhodamine 19 (SkQR1) selectively accumulates in mitochondria of normal and tumor cells. SkQR1 protected the cellular pool of reduced glutathione under oxidative stress. Overexpression of P-glycoprotein (Pgp 170) multidrug resistance pump strongly suppresses accumulation of SkQR1. The inhibitors of Pgp 170 stimulate accumulation of SkQR1 in various cell lines indicating that SkQR1 is a substrate of Pgp 170. The protective effect of SkQR1 against oxidative stress is diminished in the cells overexpressing Pgp 170. It is suggested that mitochondria-targeted antioxidants could selectively protect normal (Pgp 170-negative) cells against the toxic effect of anti-cancer treatments related to oxidative stress.     

Mitochondria-targeted antioxidant SkQR1 reduces TNF-induced endothelial permeability <Emphasis Type="Italic">in vitro</Emphasis>

Prolonged or excessive increase in the circulatory level of proinflammatory tumor necrosis factor (TNF) leads to abnormal activation and subsequent damage to endothelium. TNF at high concentrations causes apoptosis of endothelial cells. Previously, using mitochondria-targeted antioxidants of SkQ family, we have shown that apoptosis of endothelial cells is dependent on the production of reactive oxygen species (ROS) in mitochondria (mito-ROS). Now we have found that TNF at low concentrations does not cause cell death but activates caspase-3 and caspase-dependent increase in endothelial permeability in vitro. This effect is probably due to the cleavage of β-catenin–an adherent junction protein localized in the cytoplasm. We have also shown that extracellular matrix metalloprotease 9 (MMP9) VE-cadherin shedding plays a major role in the TNF-induced endothelial permeability. The mechanisms of the caspase-3 and MMP9 activation are probably not related to each other since caspase inhibition did not affect VE-cadherin cleavage and MMP9 inhibition had no effect on the caspase-3 activation. Mitochondria-targeted antioxidant SkQR1 inhibited TNF-induced increase in endothelial permeability. SkQR1 also inhibited caspase-3 activation, β-catenin cleavage, and MMP9-dependent VE-cadherin shedding. The data suggest that mito-ROS are involved in the increase in endothelial permeability due to the activation of both caspase-dependent cleavage of intracellular proteins and of MMP9-dependent cleavage of the transmembrane cell-to-cell contact proteins.     

Multidrug resistance p-glycoprotein inhibits antiapoptotic action of mitochondria-targeted antioxidant SkQR1

Mitochondria-targeted antioxidants of the SkQ family that accumulate in energized mitochondria protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing cell damage induced by hydrogen peroxide. The exposure of various human transformed cell lines to SkQR1, a fluorescent member of the SkQ family, showed that SkQR1 was pumped out of the chemotherapy resistant cells by P-glycoprotein, one of the main transport proteins that determines multidrug resistance typical for many neoplastic cells. It was also shown that SkQR1 pumping is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61). In experiments on K-562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQR1 from apoptosis induced by hydrogen peroxide. The protection of resistant subline cells is only evident after the inhibition of P-glycoprotein.     

Mitochondria-targeted antioxidant SkQ1 suppresses fibrosarcoma and rhabdomyosarcoma tumour cell growth

Mitochondria are important regulators of tumour growth and progression due to their specific role in cancer metabolism and modulation of apoptotic pathways. In this paper we describe that mitochondria-targeted antioxidant SkQ1 designed as a conjugate of decyl-triphenylphosphonium cation (TPP⁺) with plastoquinone, suppressed the growth of fibrosarcoma HT1080 and rhabdomyosarcoma RD tumour cells in culture and tumour growth of RD in xenograft nude mouse model. Under the same conditions, no detrimental effect of SkQ1 on cell growth of primary human subcutaneous fibroblasts was observed. The tumour growth suppression was shown to be a result of the antioxidant action of low nanomolar concentrations of SkQ1. We have revealed significant prolongation of mitosis induced by SkQ1 in both tumour cell cultures. Prolonged mitosis and apoptosis could be responsible for growth suppression after SkQ1 treatment in RD cells. Growth suppression in HT1080 cells was accompanied by the delay of telophase and cytokinesis, followed by multinuclear cells formation. The effects of SkQ1 on the cell cycle were proved to be at least partially mediated by inactivation of Aurora family kinases.

Abbreviations: TPP⁺: Triphenylphosphonium cation; ROS: Reactive oxygen species; mtROS: Mitochondrial reactive oxygen species; NAC: N-acetyl-L-cysteine; DCFH-DA: Dichlorodihydrofluorescein diacetate; APC: Anaphase promoting complex; ABPs: Actin-binding proteins; DMEM: Dulbecco’s modified Eagle media; SDS: sodium dodecyl sulfate; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

Multidrug resistance p-glycoprotein inhibits the antiapoptotic action of mitochondria-targeted antioxidant SkQR1

Mitochondria-targeted antioxidants of the SkQRI family, being accumulated in energized mitochondria, protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing the cell-damaging effect induced by hydrogen peroxide. Using various human transformed cell lines and SkQR1 (a fluorescent member of the SkQ family), we show that SkQR1 is ejected from chemotherapy-resistant cells by P-glycoprotein--one of the main transport proteins determining multidrug resistance typical for many neoplastic cells. It is also shown that SkQR1 ejection is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61). In experiments on K562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQR1 from apoptotic action of hydrogen peroxide. Protection of the resistant subline occurs only after inhibition of P-glycoprotein.     

Multidrug resistance P-glycoprotein inhibits the antiapoptotic action of mitochondria-targeted antioxidant SkQR1

Mitochondria-targeted antioxidants of the SkQR1 family, being accumulated in energized mitochondria, protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing the cell-damaging effect induced by hydrogen peroxide. Using various human transformed cell lines and SkQR1 (a fluorescent member of the SkQ family), we show that SkQRI is ejected from chemotherapy-resistant cells by P-glycoprotein - one of the main transport proteins determining multidrug resistance typical for many neoplastic cells. It is also shown that SkQR1 ejection is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61). In experiments on K562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQRI from apoptotic action of hydrogen peroxide. Protection of the resistant subline occurs only after inhibition of P-glycoprotein.     

Effect of liposomes on energy-dependent uptake of the antioxidant SkQR1 by isolated mitochondria

The mitochondria-targeted antioxidant SkQR1 composed of a plastoquinone part covalently bound to a cationic rhodamine 19 moiety via a decane linker was previously shown to effectively protect brain and kidney from ischemia injury accompanying generation of reactive oxygen species. In the present paper the energy-dependent SkQR1 uptake by isolated rat liver mitochondria was studied by fluorescence correlation spectroscopy peak intensity analysis (FCS PIA). This approach can be used to measure the number of fluorescent molecules per single mitochondrion. A large portion of SkQR1 appeared to be taken up by mitochondria in an energy-independent fashion because of its high affinity to membranes. Liposomes were found to compete effectively with mitochondria for the energy-independent SkQR1 binding, thereby facilitating, as an "SkQR1-buffer", observation of energy-dependent SkQR1 accumulation in mitochondria. The rate of energy-dependent SkQR1 uptake by mitochondria observed in the presence of liposomes was rather low (minutes) which was apparently due to slow redistribution of SkQR1 between liposomal and mitochondrial membranes. This can explain the low rate of staining of mitochondria by SkQR1 in living cells containing, besides mitochondria, other membrane components (endoplasmic reticulum, Golgi membranes, endosomes, lysosomes, etc.) which can compete with mitochondria for the energy-independent SkQR1 binding.     

Mitochondria-targeted antioxidant SkQ1 accelerates maturation in Campbell dwarf hamsters (Phodopus campbelli)

We tested two hypotheses. 1) SkQ1 positively affects postnatal development of hamsters in litters born to parents receiving long-term SkQ1 treatment. 2) SkQ1 accelerates maturation of juvenile females receiving the antioxidant treatment from 10 days of age. Parental pairs were kept in an outdoor vivarium under conditions close to natural. At the age of 25 days, juvenile males in litters born to parents treated daily with SkQ1 (50 nmol/kg per os) had higher epididymis mass. Both the size of a litter and SkQ1 affected epididymis mass in young males. Both the litter size and SkQ1 affected uterus mass in 25-day-old females. Juvenile females who received SkQ1 treatment from 10 days of age demonstrated earlier opening of the vagina. This experiment was replicated with the same result. At the age of 2.5 months, virgin females treated with SkQ1 from the early age demonstrated higher ovary mass.     

Inhibition of DNA methyltransferase aberrations reinstates antioxidant aging suppressors and ameliorates renal aging

DNA methylation alterations play mechanistic roles in aging; however, the epigenetic regulators/mediators causally involved in renal aging remain elusive. Here, we report that natural and D‐galactose (D‐gal)‐induced aging kidneys display marked suppression of antiaging factor NRF2 (nuclear factor erythroid‐derived 2‐like 2) and KLOTHO, accompanied by upregulations of DNA methyltransferase (DNMT) 1/3a/3b and NRF2/KLOTHO gene promoter hypermethylations. Administration of a DNMT inhibitor SGI‐1072 effectively hypomethylated the promoters, derepressed NRF2/KLOTHO, and mitigated the structural and functional alterations of renal aging in D‐gal mice. Moreover, oleuropein (OLP), an olive‐derived polyphenol, also displayed similar epigenetic modulation and antiaging effects. OLP inhibited the epigenetic NRF2/KLOTHO suppressions in a gain of DNMT‐sensitive manner in cultured renal cells, demonstrating a strong DNA‐demethylating capacity. In NRF2 knockout and KLOTHO knockdown D‐gal mice, OLP exhibited reduced antiaging effects with KLOTHO displaying a prominent gene effect and effect size; consistently in KLOTHO knockdown mice, the antiaging effects of SGI‐1027 were largely abrogated. Therefore, the KLOTHO recovery is critical for the antiaging effects of DNA demethylation. Collectively, our data indicate that aberrant DNMT1/3a/3b elevations and the resultant suppression of antiaging factors contribute significantly to epigenetic renal aging, which might be targeted for epigenetic intervention by synthetic or natural DNA‐demethylating agents.     

Geldanamycin derivative ameliorates high fat diet-induced renal failure in diabetes

Diabetic nephropathy is a serious complication of longstanding diabetes and its pathogenesis remains unclear. Oxidative stress may play a critical role in the pathogenesis and progression of diabetic nephropathy. Our previous studies have demonstrated that polyunsaturated fatty acids (PUFA) induce peroxynitrite generation in primary human kidney mesangial cells and heat shock protein 90β1 (hsp90β1) is indispensable for the PUFA action. Here we investigated the effects of high fat diet (HFD) on kidney function and structure of db/db mice, a widely used rodent model of type 2 diabetes. Our results indicated that HFD dramatically increased the 24 h-urine output and worsened albuminuria in db/db mice. Discontinuation of HFD reversed the exacerbated albuminuria but not the increased urine output. Prolonged HFD feeding resulted in early death of db/db mice, which was associated with oliguria and anuria. Treatment with the geldanamycin derivative, 17-(dimethylaminoehtylamino)-17-demethoxygeldanamycin (17-DMAG), an hsp90 inhibitor, preserved kidney function, and ameliorated glomerular and tubular damage by HFD. 17-DMAG also significantly extended survival of the animals and protected them from the high mortality associated with renal failure. The benefit effect of 17-DMAG on renal function and structure was associated with a decreased level of kidney nitrotyrosine and a diminished kidney mitochondrial Ca(2+) efflux in HFD-fed db/db mice. These results suggest that hsp90β1 is a potential target for the treatment of nephropathy and renal failure in diabetes.     

Therapeutic effects of orally administrated antioxidant drugs on acute noise-induced hearing loss

Abstract

Objective. The objective of this study was to investigate the dose-dependent therapeutic effect of the orally administrated antioxidant drugs [4-hydroxy alpha-phenyl-tert-butylnitrone (4-OHPBN) and N-acetyl-L-cysteine (NAC)] on acute noise-induced hearing loss because oral administration is the most commonly used method of drug administration due to its convenience, safety, and economical efficiency. Methods. Thirty chinchilla were exposed to a 105 dB octave band noise centered at 4 kHz for 6 h and randomly assigned to a control group (saline only) and three experimental groups [4-OHPBN (10 mg/kg) plus NAC (20 mg/kg), 4-OHPBN (20 mg/kg) plus NAC (50 mg/kg), and 4-OHPBN (50 mg/kg) plus NAC (100 mg/kg)]. The drugs were orally administrated beginning 4 h after noise exposure and then administered twice daily for the next 2 days. Permanent auditory brainstem response threshold shifts, distortion product otoacoustic emission threshold shifts, and the percentage of missing outer hair cell were determined. Results. The oral administration significantly reduced permanent hearing threshold shift, distortion product otoacoustic emission threshold shift, and the percentage of missing outer hair cell in a dose-dependent manner. Discussion. This result demonstrates that orally administered drugs can treat acute noise-induced hearing loss in a dose-dependent manner. This suggests that oral administration was effective in treating acute noise-induced hearing loss as in intraperitoneal administration.     

Salicylates and reversible hearing loss

Skin and soft tissue infections were studied in 21 seriously ill narcotic addicts who had been admitted to hospital. Subcutaneous abscesses were present in 14 patients; cellulitis was noted in 3, pyomyositis in 2 and necrotizing fasciitis in 2. In four patients there was septicemia. Infections in 14 patients (66.6 percent) were associated with anaerobic bacteria, which were the exclusive isolates in 6 patients. In seven patients (33.3 percent) isolates were exclusively aerobic bacteria and in eight both aerobes and anaerobes were present. The anaerobic isolates were clostridia (six), peptostreptococci (five), bacteroides (five), peptococci (three), and one of each of Veillonella, Propionibacterium, Eubacterium, Fusobacterium and Actinomyces. Staphylococcus aureus, generally thought to be the most common cause of subcutaneous infections in addicts, was found only in four (19 percent) patients. The other aerobic isolates were Klebsiella (five) and Enterobacter (four) species. When clinical features or the Gram stain of pus suggest that anaerobic bacteria may be present, antibiotic therapy should be directed against both aerobic and anaerobic bacteria until culture results are available.     

Zinc bone loss in chronic renal failure and chronic metabolic acidosis

The effects of chronic metabolic acidosis (CMA) on zinc (Zn) bone content and urinary excretion were examined in the presence of normal or reduced renal function together with some aspects of calcium (Ca) metabolism. Four groups of rats were compared. All were fed a 30% protein and 9 mg Zn/100 g diet. Two were uremic (U): The first developed acidosis (UA), which was suppressed in the other (UNA) by NaHCO₃ supplement. Two other groups had normal renal function: One was normal (CNA), and the other had NH₄Cl in the drinking water and acidosis (CA). Femur total Zn and Ca content was markedly reduced by CMA and was not affected by uremia. Zn urinary excretion was increased by CMA and unaltered by uremia. Ca urinary excretion was markedly reduced in uremic rats, but was enhanced in both acidotic conditions. Urinary Ca and Zn showed a strong correlation in uremic and in control rats. Plasma parathormone and 1,25(OH)₂D₃ were unchanged by CMA. These data are in agreement with a direct primary effect of CMA on bone in releasing buffers. CMA induces bone resorption and a parallel decrease of mineral bone components, such as Ca and Zn, with little or no role of PTH, 1,25(OH)₂D₃ and of uremia itself.     

Effect of SOD1 overexpression on age- and noise-related hearing loss

Reactive oxygen species (ROS) have been implicated in hearing loss associated with aging and noise exposure. Superoxide dismutases (SODs) form a first line of defense against damage mediated by the superoxide anion, the most common ROS. Absence of Cu/Zn SOD (SOD1) has been shown to potentiate hearing loss related to noise exposure and age. Conversely, overexpression of SOD1 may be hypothesized to afford a protection from age- and noise-related hearing loss. This hypothesis may be tested using a transgenic mouse model carrying the human SOD1 gene. Contrary to expectations, here, we report that no protection against age-related hearing loss was observed in mice up to 7 months of age or from noise-induced hearing loss when 8 week old mice were exposed to broadband noise (4-45 kHz, 110 dB for 1 h). Mitochondrial DNA deletion, an index of aging, was elevated in the acoustic nerve of transgenic mice compared to nontransgenic littermates. The results indicate the complexity of oxidative metabolism in the cochlea is greater than previously hypothesized.     

Lack of a functional alternative complement pathway ameliorates ischemic acute renal failure in mice

Ischemia/reperfusion (I/R) injury of the kidney is a common cause of acute renal failure (ARF) and is associated with high morbidity and mortality in the intensive care unit. The mechanisms underlying I/R injury are complex. Studies have shown that complement activation contributes to the pathogenesis of I/R injury in the kidney, but the exact mechanisms of complement activation have not been defined. We hypothesized that complement activation in this setting occurs via the alternative pathway and that mice deficient in complement factor B, an essential component of the alternative pathway, would be protected from ischemic ARF. Wild-type mice suffered from a decline in renal function and had significant tubular injury, particularly in the outer medulla, after I/R. We found that factor B-deficient mice (fB(-/-)) developed substantially less functional and morphologic renal injury after I/R. Furthermore, control wild-type mice had an increase in tubulointerstitial complement C3 deposition and neutrophil infiltration in the outer medulla after I/R, whereas fB(-/-) mice demonstrated virtually no C3 deposition or neutrophil infiltration. Our results demonstrate that complement activation in the kidney after I/R occurs exclusively via the alternative pathway, and that selective inhibition of this pathway provides protection to the kidneys from ischemic ARF.     

Restaurant noise,hearing loss,and hearing aids.

Our multidisciplinary team obtained noise data in 27 San Francisco Bay Area restaurants. These data included typical minimum, peak, and average sound pressure levels; digital tape recordings; subjective noise ratings; and on-site unaided and aided speech discrimination tests. We report the details and implications of these noise measurements and provide basic information on selecting hearing aids and suggestions for coping with restaurant noise.     

Endothelin-1 in chronic renal failure and hypertension

Investigation into the role of endothelin-1 (ET-1) in renal function has revealed two major direct actions leading to the control of extracellular volume and blood pressure. These are the regulation of renal hemodynamics and glomerular filtration rate and the modulation of sodium and water excretion. In the rat remnant kidney model of chronic renal failure, ET-1 production is increased in blood vessels and renal tissues. These changes are related to an increase in preproET-1 expression and correlate with the rise in blood pressure, the development of cardiovascular hypertrophy, and the degree of renal insufficiency and injury. Selective ETA receptor blockade prevents the progression of hypertension and the vascular and renal damage, supporting a role for ET-1 in chronic renal failure progression. The increase in ET-1 production can be associated with other local mediators, including angiotensin II, transforming growth factor-beta1 and nitric oxide, the local production of which is also altered in chronic renal failure. In human patients with essential hypertension, atherosclerosis, and nephrosclerosis, plasma ET-1 levels are increased compared with patients with uncomplicated essential hypertension. Similarly, plasma ET-1 concentrations are markedly increased in patients with end-stage renal disease undergoing dialysis, and this correlates with blood pressure, suggesting that ET-1 may contribute to hypertension in these patients. The treatment of anemia in patients with renal failure with human recombinant erythropoietin increases blood pressure by accentuating the underlying endothelial dysfunction and the elevated vascular ET-1 production. Overall, these results support a role for ET-1 in hypertension and the end-organ damage associated with chronic renal failure. ETA receptor blockade may then represent a potential target for the management of hypertension and cardiovascular and renal protection.     

Profound urinary protein loss and acute renal failure caused by cyclooxygenase-2 inhibitor

Cumulative evidence has shown that nonsteroidal anti-inflammatory drugs (NSAIDs) can induce acute renal failure and nephrotic-range proteinuria. Cyclooxygenase-2 (COX-2) inhibitors have less nephrotoxicity; however, recent data indicate that they may cause the same renal problems as NSAIDs do. Herein, we present a case of celecoxib-associated minimal change disease (MCD) with profound urinary protein loss and acute renal failure. Renal function and nephrotic syndrome in this patient resolved completely after discontinuation of celecoxib and treatment with methylprednisolone. Clinicians should keep high index of suspicions in patients developing nephrotic syndrome and acute renal failure after taking COX-2 inhibitors since secondary MCD responds well to timely cessation of COX-2 inhibitors and administration of steroid therapy.     

14-Aminotetradecanoic acid exhibits antioxidant activity and ameliorates xenobiotics-induced cytotoxicity

Natural compounds with free-radical scavenging activity have potential role in maintaining human health and preventing diseases. In this study, we report the antioxidant and cytoprotective properties of 14-aminotetradecanoic acid (ATDA) isolated from the Decalepis hamiltonii roots. ATDA is a potent scavenger of superoxide (O(2) (?-)), hydroxyl ((?)OH), nitric oxide ((?)NO), and lipid peroxide (LOO(?)) physiologically relevant free radicals with IC(50) values in nM (36-323) range. ATDA also exhibits concentration-dependent secondary antioxidant activities like reducing power, metal-chelating activity, and inhibition of protein carbonylation. Further, ATDA at nM concentration prevented CuSO(4)-induced human LDL oxidation. ATDA demonstrated cytoprotective activity in primary hepatocytes and Ehrlich ascites tumor cells against oxidative stress inducing xenobiotics apart from the in vitro free-radical scavenging activity. The mechanism of cytoprotective action involved maintaining the intracellular glutathione, scavenging of reactive oxygen species, and inhibition of lipid peroxidation. It is suggested that ATDA is a novel bioactive molecule with potential health implications.