Gestational diabetes causes hyperactivity of the sympathetic nervous system and hypertension in adult mice offspring.

BACKGROUND: Gestational diabetes can lead to increased blood pressure in offspring, accompanied by impaired renal sodium excretion function and vasoconstriction and diastole dysfunction. However, there are few studies on whether it is accompanied by increased sympathetic nerve activity. METHODS: Pregnant C57BL/6 mice were intraperitoneally injected with streptozotocin (35 mg/kg) or citrate buffer at day 0 of gestation. The mice of control mother offspring (CMO) and diabetic mother offspring (DMO) at 16 weeks of age were infused with vehicle (artificial cerebrospinal fluid, aCSF, 0.4 µL/h) or tempol (1 mmol/L, 0.4 µL/h) into the bilateral paraventricular nucleus (PVN) of mice for 4 weeks, respectively. RESULTS: Compared with CMO group, SBP and peripheral sympathetic nerve activity (increased heart rate, LF/HF and plasma norepinephrine and decreased SDNN and RMSSD) were increased in DMO group, which was accompanied by increased angiotensin II type-1 receptor (AT1R) expression and function in PVN. The increase in AT1R expression levels was attributed to a decrease in the methylation level of the AT1R promoter region, resulting in an increase in AT1R mRNA levels in PVN of DMO. Moreover, compared with CMO group, the levels of oxidative stress were increased and DNMT1 expression was decreased in PVN of DMO. Bilateral PVN infusion of tempol attenuated oxidative stress increased the level of DNMT1 expression and the binding of DNMT1 to the AT1R promoter region, which reduced mRNA and protein expression level of AT1R, heart rate and SBP in DMO, but not in CMO. CONCLUSIONS: The present study provides evidence for overactive sympathetic nervous systems in the pathogenesis of gestational diabetes-induced hypertension in offspring. Central antioxidant intervention in the PVN may be an important treatment strategy for fetal-programmed hypertension.

Inhibition of MMP2 activity mitigates N-omega-nitro-l-arginine-methyl ester (l-NAME)-induced right heart failure.

In rats decreased bioavailability of nitric oxide induces oxidative stress and right heart failure. Oxidative stress can activate matrix metalloproteinase-2 (MMP2). We addressed the question whether increasing oxidative defense by administration of the SOD mimetic Tempol or direct inhibition of MMP2 activity by SB-3CT mitigates right heart failure. Rats received l-NAME for four weeks and during week three and four treatment groups received either Tempol or SB-3CT in addition. After four weeks heart function was analyzed by echocardiography, organ weights and expression of NPPB and COL1A1 were analyzed, oxidative stress was monitored by DHE-staining and MMP2 activity was quantified by proteolytic auto-activation, zymography, and troponin I degradation. l-NAME induced oxidative stress and MMP2 activity stronger in the right ventricle than in the left ventricle. Troponin I, a MMP2 substrate, was degraded in right ventricles. Tempol reduced oxidative stress and preferentially affected the expression of fibrotic genes (i.e. COL1A1) and fibrosis. Tempol and SB-3CT mitigated right but not left ventricular hypertrophy. Neither SB-3CT nor Tempol alone strongly improved right ventricular function. In conclusion, both MMP2 activity and oxidative stress contribute to right ventricular failure but neither is MMP2 activation linked to oxidative stress nor does oxidative stress and MMP2 activity have common targets.

Exposure to volatile ferroptosis inhibitor, TEMPO, reduced cutaneous ischemia-reperfusion injury progression to pressure ulcer formation in a mouse model.

BACKGROUND: Ischemia- reperfusion (I/R) injury-induced oxidative stress is a key factor in the pathogenesis of pressure ulcer formation. Ferroptosis is an iron-dependent programmed cell death that connects oxidative stress and inflammation in various diseases. Recent studies revealed the protective effect of inhibition of ferroptosis in I/R injury. However, the role of ferroptosis in cutaneous I/R injury remains elusive. OBJECTIVE: To assess the role of ferroptosis in the progression of cutaneous I/R injury. METHODS: Cutaneous I/R injury experiments and histopathological studies were performed in wild-type mice with or without exposure to volatile ferroptosis inhibitor, TEMPO (2,2,6,6-Tetramethylpiperidine-1-oxyl). The suppressive effects of TEMPO on ferroptosis inducing cell death and oxidative stress were examined in vitro. RESULTS: Inhibition of ferroptosis with TEMPO significantly reduced ulcer formation after cutaneous I/R injury. Fluctuated ferroptosis markers, such as GPX4, ACSL4, and 4-HNE expression in the I/R skin site, were reversed by TEMPO treatment. Inhibition of ferroptosis reduced apoptosis, CD3+ infiltrating lymphocytes, and improved vascularity in the I/R skin site. Inhibition of ferroptosis also suppressed the enhancement of Nrf2 activation. In vitro, ferroptosis and the activation of ferroptosis-related gene expression by RSL3 stimulation were markedly ameliorated by TEMPO treatment in mouse fibroblasts. Inhibiting ferroptosis also suppressed the elevation of the mRNA levels of NOX2 and HO-1 caused by ferroptosis. CONCLUSION: Cutaneous I/R injury-induced ferroptosis likely promotes cell death, vascular loss, infiltration of inflammatory cells, and oxidative stress. The inhibition of ferroptosis with TEMPO might have potential clinical application as novel therapeutic agent for cutaneous I/R injury.

Anti-Tumor and Chemosensitizing Effects of the CDK Inhibitor Dinaciclib on Cholangiocarcinoma In Vitro and In Vivo.

BACKGROUND/AIM: Cholangiocarcinoma (CCA) is a highly aggressive disease. Most of CCA patients are diagnosed in an advanced stage of the disease, when it is unresectable and there is chemoresistance, resulting in poor prognosis. However, effective therapeutic regimens and molecular targets for CCA remain poor. Cyclin-dependent kinases (CDKs) are key regulatory enzymes in cell cycle progression. Aberrant CDK activation is a hallmark of cancer. Dinaciclib is a small molecule inhibitor of multiple CDKs, currently under clinical evaluation for treating advanced malignancies. The efficacy of anti-tumor activity of dinaciclib against chemotherapy resistant CCA cells was examined in vitro and in vivo. MATERIALS AND METHODS: In this study, the effect of dinaciclib on growth and cell cycle in CCA cell lines were determined using the MTT assay and cell cycle analysis. The anti-tumor activity of dinaciclib was investigated in CCA-inoculated mice. In addition, the chemosensitizing effect of dinaciclib was investigated in gemcitabine-treated CCA cell lines. RESULTS: Dinaciclib significantly suppressed cell proliferation, induced G1/S phase cell cycle arrest and apoptosis of CCA cell lines. It significantly suppressed the growth of CCA cells in xenograft mouse models. We also found that dinaciclib significantly inhibited the growth of gemcitabine-resistant CCA cell lines (KKU-213A-GemR and KKU-100-GemR). Furthermore, dinaciclib significantly enhanced the anti-tumor activity of gemcitabine in CCA cell lines. CONCLUSION: Dinaciclib has the potential to be an effective therapeutic agent to control tumor cell growth of both parental and gemcitabine-resistant CCA cells.

Tempol, a Superoxide Dismutase Mimetic, Inhibits Wallerian Degeneration Following Spinal Cord Injury by Preventing Glutathione Depletion and Aldose Reductase Activation.

Spinal cord contusion injury results in Wallerian degeneration of spinal cord axonal tracts, which are necessary for locomotor function. Axonal swelling and loss of axonal density at the contusion site, characteristic of Wallerian degeneration, commence within hours of injury. Tempol, a superoxide dismutase mimetic, was previously shown to reduce the loss of spinal cord white matter and improve locomotor function in an experimental model of spinal cord contusion, suggesting that tempol treatment might inhibit Wallerian degeneration of spinal cord axons. Here, we report that tempol partially inhibits Wallerian degeneration, resulting in improved locomotor recovery. We previously reported that Wallerian degeneration is reduced by inhibitors of aldose reductase (AR), which converts glucose to sorbitol in the polyol pathway. We observed that tempol inhibited sorbitol production in the injured spinal cord to the same extent as the AR inhibitor, sorbinil. Tempol also prevented post-contusion upregulation of AR (AKR1B10) protein expression within degenerating axons, as previously observed for AR inhibitors. Additionally, we hypothesized that tempol inhibits axonal degeneration by preventing loss of the glutathione pool due to polyol pathway activity. Consistent with our hypothesis, tempol treatment resulted in greater glutathione content in the injured spinal cord, which was correlated with increased expression and activity of gamma glutamyl cysteine ligase (γGCL; EC 6.3.2.2), the rate-limiting enzyme for glutathione synthesis. Administration of the γGCL inhibitor buthionine sulfoximine abolished all observed effects of tempol administration. Together, these results support a pathological role for polyol pathway activation in glutathione depletion, resulting in Wallerian degeneration after spinal cord injury (SCI). Interestingly, methylprednisolone, oxandrolone, and clenbuterol, which are known to spare axonal tracts after SCI, were equally effective in inhibiting polyol pathway activation. These results suggest that prevention of AR activation is a common target of many disparate post-SCI interventions.

Cyclic Nitroxide 4-Methoxy-Tempo May Decrease Serum Amyloid A-Mediated Renal Fibrosis and Reorganise Collagen Networks in Aortic Plaque.

Acute-phase serum amyloid A (SAA) can disrupt vascular homeostasis and is elevated in subjects with diabetes, cardiovascular disease, and rheumatoid arthritis. Cyclic nitroxides (e.g., Tempo) are a class of piperidines that inhibit oxidative stress and inflammation. This study examined whether 4-methoxy-Tempo (4-MetT) inhibits SAA-mediated vascular and renal dysfunction. Acetylcholine-mediated vascular relaxation and aortic guanosine-3',5'-cyclic monophosphate (cGMP) levels both diminished in the presence of SAA. 4-MetT dose-dependently restored vascular function with corresponding increases in cGMP. Next, male ApoE-deficient mice were administered a vehicle (control, 100 µL PBS) or recombinant SAA (100 µL, 120 µg/mL) ± 4-MetT (at 15 mg/kg body weight via i.p. injection) with the nitroxide administered before (prophylaxis) or after (therapeutic) SAA. Kidney and hearts were harvested at 4 or 16 weeks post SAA administration. Renal inflammation increased 4 weeks after SAA treatment, as judged by the upregulation of IFN-γ and concomitant increases in iNOS, p38MAPK, and matrix metalloproteinase (MMP) activities and increased renal fibrosis (Picrosirius red staining) in the same kidneys. Aortic root lesions assessed at 16 weeks revealed that SAA enhanced lesion size (vs. control; p < 0.05), with plaque presenting with a diffuse fibrous cap (compared to the corresponding aortic root from control and 4-MetT groups). The extent of renal dysfunction and aortic lesion size was largely unchanged in 4-MetT-supplemented mice, although renal fibrosis diminished at 16 weeks, and aortic lesions presented with redistributed collagen networks. These outcomes indicate that SAA stimulates renal dysfunction through promoting the IFN-γ-iNOS-p38MAPK axis, manifesting as renal damage and enhanced atherosclerotic lesions, while supplementation with 4-MetT only affected some of these pathological changes.

Evaluation of effects of Tempol on testicular ischemia/reperfusion injury.

AIM: Tempol, a synthetic antioxidant compound, has received significant attention for its potential therapeutic applications in recent years, especially against ischemia/reperfusion (I/R) injury. The aim of the present research was to assess the protective effects of Tempol on testicular I/R injury caused by testicular torsion and detorsion (T/D) in rats. METHODS: The subjects were divided into five groups: sham, testicular T/D, testicular T/D with Tempol treatment at 50 and 100 mg/kg, and healthy rats treated with Tempol at 100 mg/kg. Testicular torsion was induced by rotating the left testicles for 2 h, followed by detorsion for 24 h. Testicular tissues were evaluated for gene expression, oxidative stress markers, and histopathology, epididymal sperms were stained and analyzed, and blood serum samples were collected to measure the testosterone hormone. RESULTS: The results showed that testicular I/R caused a significant decrease in sperm velocity parameters, viability, and count, as well as an increase in abnormal sperms (p < 0.05). However, treatment with Tempol significantly improved these parameters (p < 0.05). Histopathological analysis revealed severe damage to the testicular tissues, but treatment with Tempol improved the structural integrity of the seminiferous tubules. Testicular I/R also resulted in increased oxidative stress index and decreased testosterone levels significantly (p < 0.05), but Tempol administration mitigated these effects significantly (p < 0.05). Furthermore, the expression of Bax and Bcl2, genes associated with apoptosis, were significantly altered by testicular I/R (p < 0.05), but Tempol prevented these changes significantly (p < 0.05). CONCLUSION: These findings provide strong evidence that Tempol can effectively prevent testicular I/R injury.

Pyrogallol intermediates elicit beta-amyloid secretion via radical formation and alterations in intracellular trafficking, distinct from pyrogallol-generated superoxide.

This study unveils a novel role of pyrogallol (PG), a recognized superoxide generator, in inducing beta-amyloid (Aß) secretion in an Alzheimer's disease (AD) cellular model. Contrary to expectations, the analysis of dihydroethidium fluorescence and UV-VIS spectrum scanning reveals that Aß secretion arises from PG reaction intermediates rather than superoxide or other by-products. Investigation into Aß secretion mechanisms identifies dynasore-dependent endocytosis and BFA-dependent exocytosis as independent pathways, regulated by tiron, tempol, and superoxide dismutase. Cell-type specificity is observed, with 293sw cells showing both pathways, while H4sw cells and primary astrocytes from an AD animal model exclusively exhibit the Aß exocytosis pathway. This exploration contributes to understanding PG's chemical reactions and provides insights into the interplay between environmental factors, free radicals, and AD, linking occupational PG exposure to AD risk as reported in the literature.

CDK9 inhibition as an effective therapy for small cell lung cancer.

Treatment-naïve small cell lung cancer (SCLC) is typically susceptible to standard-of-care chemotherapy consisting of cisplatin and etoposide recently combined with PD-L1 inhibitors. Yet, in most cases, SCLC patients develop resistance to first-line therapy and alternative therapies are urgently required to overcome this resistance. In this study, we tested the efficacy of dinaciclib, an FDA-orphan drug and inhibitor of the cyclin-dependent kinase (CDK) 9, among other CDKs, in SCLC. Furthermore, we report on a newly developed, highly specific CDK9 inhibitor, VC-1, with tumour-killing activity in SCLC. CDK9 inhibition displayed high killing potential in a panel of mouse and human SCLC cell lines. Mechanistically, CDK9 inhibition led to a reduction in MCL-1 and cFLIP anti-apoptotic proteins and killed cells, almost exclusively, by intrinsic apoptosis. While CDK9 inhibition did not synergise with chemotherapy, it displayed high efficacy in chemotherapy-resistant cells. In vivo, CDK9 inhibition effectively reduced tumour growth and improved survival in both autochthonous and syngeneic SCLC models. Together, this study shows that CDK9 inhibition is a promising therapeutic agent against SCLC and could be applied to chemo-refractory or resistant SCLC.

Selective but not pan-CDK inhibition abrogates 5-FU-driven tissue factor upregulation in colon cancer.

Thromboembolic events are complications in cancer patients and hypercoagulability has been linked to the tissue factor (TF) pathway, making this an attractive target. Here, we investigated the effects of chemotherapeutics and CDK inhibitors (CDKI) abemaciclib/palbociclib (CDK4/6), THZ-1 (CDK7/12/13), and dinaciclib (CDK1/2/5/9) alone and in combination regimens on TF abundance and coagulation. The human colorectal cancer (CRC) cell line HROC173 was treated with 5-FU or gemcitabine to stimulate TF expression. TF+ cells were sorted, recultured, and re-analyzed. The effect of treatment alone or in combination was assessed by functional assays. Low-dose chemotherapy induced a hypercoagulable state and significantly upregulated TF, even after reculture without treatment. Cells exhibited characteristics of epithelial-mesenchymal transition, including high expression of vimentin and mucin. Dinaciclib and THZ-1 also upregulated TF, while abemaciclib and palbociclib downregulated it. Similar results were observed in coagulation assays. The same anticoagulant activity of abemaciclib was seen after incubation with peripheral immune cells from healthy donors and CRC patients. Abemaciclib reversed 5-FU-induced TF upregulation and prolonged clotting times in second-line treatment. Effects were independent of cytotoxicity, senescence, and p27kip1 induction. TF-antibody blocking experiments confirmed the importance of TF in plasma coagulation, with Factor XII playing a minor role. Short-term abemaciclib counteracts 5-FU-induced hypercoagulation and eventually even prevents thromboembolic events.

Mito-Tempo alleviates ox-LDL-provoked foam cell formation by regulating Nrf2/NLRP3 signaling.

Our previous studies have demonstrated that Mito-Tempol (also known as 4-hydroxy-Tempo), a mitochondrial reactive oxygen species scavenger, alleviates oxidized low-density lipoprotein (ox-LDL)-triggered foam cell formation. Given the effect of oxidative stress on activating the NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) inflammasome, which promotes foam cell formation, we aimed to explore whether Mito-Tempo inhibits ox-LDL-triggered foam cell formation by regulating NLRP3 inflammasome. The results revealed that Mito-Tempo re-activated Nrf2 and alleviated macrophage foam cell formation induced by ox-LDL, whereas the effects were reversed by ML385 (a specific Nrf2 inhibitor). Mito-Tempo restored the expression and nuclear translocation of Nrf2 by decreasing ox-LDL-induced ubiquitination. Furthermore, Mito-Tempo suppressed ox-LDL-triggered NLRP3 inflammasome activation and subsequent pyroptosis, whereas the changes were blocked by ML385. Mito-Tempo decreased lipoprotein uptake by inhibiting CD36 expression and suppressed foam cell formation by regulating the NLRP3 inflammasome. Taken together, Mito-Tempo exhibits potent anti-atherosclerotic effects by regulating Nrf2/NLRP3 signaling.

The evaluation effect of nanoliposome-loaded Mito-Tempo on sperm parameters during human sperm cryopreservation.

AIM: The aim of this study is the evaluation effect of nanoliposome-loaded Mito-Tempo on sperm parameters during human sperm cryopreservation. METHODS: Semen samples of 50 Asthenoteratozoospermia men (random) were collected. Sperm parameters were analyzed based on World Health Organization (WHO, 2010) criteria (2021) and each sample was divided into 5 groups (E1-E5). E1 (control group): the sperm was cryopreserved without nanoliposome, and Mito-Tempo. E2: sperm cryopreservation with Mito-Tempo-loaded nanoliposome (Mito-Tempo 0.1 mM) + freezing medium. E3: sperm cryopreservation with Mito-Tempo-loaded nanoliposome (Mito-Tempo 0.2 mM) + freezing medium. E4: in this group, the cryopreservation sperm with Mito-Tempo 0.3 mM + freezing medium. E5: the cryopreservation sperm with Mito-Tempo 0.2 mM + freezing medium. RESULTS: The result of this study indicated that sperm parameters and total antioxidant capacity (TAC) significantly increase in E3 and E4 groups, compared to E1, E2, and E5 groups respectively (P < 0.05). The percentage of abnormal morphology, DNA fragmentation index (DFI), malondialdehyde (MDA), and the levels of ROS significantly decrease in E3 and E4 groups, compared to E1, E2, and E5 groups (P < 0.05). In addition, the sperm parameters and stress oxidative factors significantly improve in E3 group compared to other groups (P < 0.05). CONCLUSIONS: In conclusion, the combination of Mito-Tempo with nanoliposome due to its ability to cooperate with lipid layers may lead to significant performance in reducing oxidative stress damage and increasing the quality of sperm parameters.

Design, synthesis, and biological evaluation of dinaciclib and CAN508 hybrids as CDK inhibitors.

The scaffolds of two known CDK inhibitors (CAN508 and dinaciclib) were the starting point for synthesizing two series of pyarazolo[1,5-a]pyrimidines to obtain potent inhibitors with proper selectivity. The study presented four promising compounds; 10d, 10e, 16a, and 16c based on cytotoxic studies. Compound 16a revealed superior activity in the preliminary anticancer screening with GI % = 79.02-99.13 against 15 cancer cell lines at 10 µM from NCI full panel 60 cancer cell lines and was then selected for further investigation. Furthermore, the four compounds revealed good safety profile toward the normal cell lines WI-38. These four compounds were subjected to CDK inhibitory activity against four different isoforms. All of them showed potent inhibition against CDK5/P25 and CDK9/CYCLINT. Compound 10d revealed the best activity against CDK5/P25 (IC50 = 0.063 µM) with proper selectivity index against CDK1 and CDK2. Compound 16c exhibited the highest inhibitory activity against CDK9/CYCLINT (IC50 = 0.074 µM) with good selectivity index against other isoforms. Finally, docking simulations were performed for compounds 10e and 16c accompanied by molecular dynamic simulations to understand their behavior in the active site of the two CDKs with respect to both CAN508 and dinaciclib.

Differential tempol effects in prostatic cancer: angiogenesis and short- and long-term treatments.

Prostate cancer (PCa) is the second cause of cancer death among men worldwide. Several processes are involved in the development and progression of PCa such as angiogenesis, inflammation and oxidative stress. The present study investigated the effect of short- or long-term Tempol treatment at different stages of prostate adenocarcinoma progression, focusing on angiogenic, proliferative, and stromal remodeling processes in TRAMP mice. The dorsolateral lobe of the prostate of TRAMP mice were evaluated at two different stages of PCa progression; early and late stages. Early stage was again divided into, short- or long-term. 50 mg/kg Tempol dose was administered orally. The results demonstrated that Tempol mitigated the prostate histopathological lesion progressions in the TRAMP mice in all treated groups. However, Tempol increased molecules involved in the angiogenic process such as CD31 and VEGFR2 relative frequencies, particularly in long-term treatment. In addition, Tempol upregulated molecule levels involved in angiogenesis and stromal remodeling process VEGF, TGF-ß1, VE-cadherin and vimentin, particularly, in T8-16 group. Thus, it was concluded that Tempol treatment delayed prostatic lesion progression in the dorsolateral lobe of the TRAMP mice. However, Tempol also led to pro-angiogenic effects and glandular stromal microenvironment imbalance, especially, in the long-term treatment.

Algal growth inhibition test with TEMPO-oxidized cellulose nanofibers.

Ecotoxicity data on cellulose nanofibers (CNFs) are limited despite their wide potential applications prospects, such as structural and packaging materials, filters, coatings, foods, and cosmetics. In this study, toxicity tests of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized CNFs (TEMPO-CNFs), which are one of the major CNF products commercially available in Japan, on the green alga Raphidocelis subcapitata were conducted. As nanomaterials are considered difficult-to-test substances, the Organisation for Economic Co-operation and Development has released a guidance document that provides considerations regarding ecotoxicity tests of nanomaterials. In the algal growth inhibition tests of TEMPO-CNFs, there were specific issues to be examined, including the effects of medium components on the characteristics of TEMPO-CNFs, CNF interference with algal density measurements, algal interference with CNF measurements, and the effects of ion concentration changes in the test medium by the addition of CNFs on algal growth. To examine these issues, we conducted preliminary studies and established a suitable test method for algal growth inhibition tests of TEMPO-CNFs. We confirmed that the components in the medium for algal growth inhibition tests had negligible effects on the characteristics (zeta-potential, viscosity, and morphology) and concentration stability of TEMPO-CNFs and that in vitro and in vivo fluorescence measurements were applicable for estimating the algal densities, without interference by TEMPO-CNFs. In contrast, we observed that the grown algae interfered with the CNF concentration measurements. Therefore, we established a method to correct the measured CNF concentrations by estimating the algal contribution. Furthermore, we found that the nutrient salt concentrations in the medium changed due to interactions with CNFs; however, this change did not affect algal growth. Based on the results of the preliminary studies, algal growth inhibition tests of TEMPO-CNFs were conducted using in vitro and in vivo fluorescence measurements, along with measurements of CNFs and ion concentrations in the test dispersions. The test results showed that no growth inhibition was observed on growth rate or yield even at the maximum CNF concentration of 100 mg/L, suggesting that the ecological effect of TEMPO-CNFs on algae was relatively low. The results of this study will be valuable for conducting ecotoxicity assessments on additional CNFs and comparable nanomaterials in future studies.

The Cellular and Organismal Effects of Nitroxides and Nitroxide-Containing Nanoparticles.

Nitroxides are stable free radicals that have antioxidant properties. They react with many types of radicals, including alkyl and peroxyl radicals. They act as mimics of superoxide dismutase and stimulate the catalase activity of hemoproteins. In some situations, they may exhibit pro-oxidant activity, mainly due to the formation of oxoammonium cations as products of their oxidation. In this review, the cellular effects of nitroxides and their effects in animal experiments and clinical trials are discussed, including the beneficial effects in various pathological situations involving oxidative stress, protective effects against UV and ionizing radiation, and prolongation of the life span of cancer-prone mice. Nitroxides were used as active components of various types of nanoparticles. The application of these nanoparticles in cellular and animal experiments is also discussed.

Hair Follicle Damage after 100 mGy Low-Dose Fractionated X-Ray Irradiation and the Protective Effects of TEMPOL, a Stable Nitroxide Radical, against Radiation.

The effects of long-term low-dose X-ray irradiation on the outer root sheath (ORS) cells of C3H/He mice were investigated. Mice were irradiated with a regime of 100 mGy/day, 5 days/week, for 12 weeks (Group X) and the results obtained were compared to those in a non-irradiated control (Group C). Potential protection against ORS cells damage induced by this exposure was investigated by adding the stable nitroxide radical 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) at 1 mM to the drinking water of mice (Group X + TEMPOL). The results obtained were compared with Group C and a non-irradiated group treated with TEMPOL (Group C + TEMPOL). After fractionated X-ray irradiation, skin was removed and ORS cells were examined by hematoxylin and eosin staining and electron microscopy for an abnormal nuclear morphology and nuclear condensation changes. Fractionated X-irradiated mice had an increased number of ORS cells with an abnormal nuclear morphology as well as nuclear condensation changes. Sections were also immunohistochemically examined for the presence of TdT-mediated dUTP nick-end labeling (TUNEL), 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE), vascular endothelial growth factor (VEGF), nitrotyrosine, heme oxygenase 1 (HO-1), and protein gene product 9.5 (PGP 9.5). Significant increases were observed in TUNEL, 8-OHdG, and 4-HNE levels in ORS cells from mice in Group X. Electron microscopy also showed irregular shrunken ORS cells in Group X. These changes were prevented by the presence of TEMPOL in the drinking water of the irradiated mice. TEMPOL alone had no significant effects. These results suggest that fractionated doses of radiation induced oxidative damage in ORS cells; however, TEMPOL provided protection against this damage, possibly as a result of the rapid reaction of this nitroxide radical with the reactive oxidants generated by fractionated X-ray irradiation.

Tempol improves aortic mechanics in a mouse model of hypertension.

Hypertension-induced arterial remodeling is thought to be a response to increases in both mechanical stress and oxidative stress. The superoxide dismutase mimetic Tempol has been shown to reduce adverse aortic remodeling in multiple murine models of hypertension but in the absence of a detailed assessment of the biaxial biomechanics. We show that concurrent treatment with Tempol in a common mouse model of systemic hypertension results in modest reductions in both wall thickening and circumferential material stiffness that yet work together to achieve a significant reduction in calculated aortic pulse wave velocity. Reducing elevated values of pulse wave velocity engenders multiple benefits to cardiovascular function.

Effect of Low Concentration of Nitroxides on SH-SY5Y Cells Transfected with the Tau Protein.

Nitroxides, stable synthetic free radicals, are promising antioxidants, showing many beneficial effects both at the cellular level and in animal studies. However, the cells are usually treated with high millimolar concentrations of nitroxides which are not relevant to the concentrations that could be attained in vivo. This paper aimed to examine the effects of low (≤10 µM) concentrations of three nitroxides, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO), 4-hydroxy-TEMPO (TEMPOL) and 4-amino-TEMPO (TEMPAMINE), in pure chemical systems and on SH-SY5Y cells transfected with the human tau protein (TAU cells), a model of chronic cellular oxidative stress, and transfected with the empty plasmid (EP cells). All nitroxides were active in antioxidant-activity tests except for the 2,2'-azinobis-(3-ethylbenzthiazolin-6-sulfonate) radical (ABTS•) decolorization assay and reduced Fe3+, inhibited autoxidation of adrenalin and pyrogallol and oxidation of dihydrorhodamine123 by 3-morpholino-sydnonimine SIN-1. TEMPO protected against fluorescein bleaching from hypochlorite, but TEMPAMINE enhanced the bleaching. Nitroxides showed no cytotoxicity and were reduced by the cells to non-paramagnetic derivatives. They decreased the level of reactive oxygen species, depleted glutathione, and increased mitochondrial-membrane potential in both types of cells, and increased lipid peroxidation in TAU cells. These results demonstrate that even at low micromolar concentrations nitroxides can affect the cellular redox equilibrium and other biochemical parameters.

The Antioxidant TEMPOL Protects Human Hematopoietic Stem Cells From Culture-Mediated Loss of Functions.

In a steady state, hematopoietic stem cells (HSC) exhibit very low levels of reactive oxygen species (ROS). Upon stress, HSC get activated and enter into proliferation and differentiation process to ensure blood cell regeneration. Once activated, their levels of ROS increase, as messengers to mediate their proliferation and differentiation programs. However, at the end of the stress episode, ROS levels need to return to normal to avoid HSC exhaustion. It was shown that antioxidants can prevent loss of HSC self-renewal potential in several contexts such as aging or after exposure to low doses of irradiation suggesting that antioxidants can be used to maintain HSC functional properties upon culture-induced stress. Indeed, in humans, HSC are increasingly used for cell and gene therapy approaches, requiring them to be cultured for several days. As expected, we show that a short culture period leads to drastic defects in HSC functional properties. Moreover, a switch of HSC transcriptional program from stemness to differentiation was evidenced in cultured HSC. Interestingly, cultured-HSC treated with 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-hydroxy-TEMPO or Tempol) exhibited a higher clonogenic potential in secondary colony forming unit cell (CFU-C) assay and higher reconstitution potential in xenograft model, compared to untreated cultured-HSC. By transcriptomic analyses combined with serial CFU-C assays, we show that Tempol, which mimics superoxide dismutase, protects HSC from culture-induced stress partly through VEGFα signaling. Thus, we demonstrate that adding Tempol leads to the protection of HSC functional properties during ex vivo culture.