Noninvasive study of radiation-induced oxidative damage using in vivo electron spin resonance

Nitroxyl radicals injected into a whole body indicate the disappearance of signal intensity of in vivo electron spin resonance (ESR). The signal decay rates of nitroxyl have reported to be influenced by various types of oxidative stress. We examined the effect of X-irradiation on the signal decay rate of nitroxyl in the upper abdomen of mice using in vivo ESR. The signal decay rates increased 1 h after 15 Gy irradiation, and the enhancement was suppressed by preadministration of cysteamine, a radioprotector. These results suggest that the signal decay of nitroxyl in whole mice is enhanced by radiation-induced oxidative damage. The in vivo ESR system probing the signal decay of nitroxyl could provide a noninvasive technique for the study of oxidative stress caused by radiation in a living body.     

Redox evaluation in sepsis model mice by the in vivo ESR technique using acyl-protected hydroxylamine

In vivo electron spin resonance (ESR) spectroscopy is a noninvasive technique that measures the oxidative stress in living experimental animals. The rate of decay of the ESR signal right after an injection of nitroxyl radical has been measured to evaluate the oxidative stress in animals, although the probe’s disposition could also affect this rate. Because the amount of probes forming the redox pair of hydroxyl amine and its corresponding nitroxyl radical was shown to be nearly constant in most organs or tissues 10 min after the injection of 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in mice, we evaluated the oxidative stress in sepsis model mice induced by lipopolysaccharide (LPS) by intravenously injecting ACP as a precursor of redox probes. The in vivo ESR signal increased up to 7–8 min after the ACP injection and then decreased. Decay of the in vivo signal in LPS-treated mice was significantly slower than that in healthy mice, whereas no significant difference was observed in the rate of change in the total amount of redox probes in the blood and liver between these groups. ESR imaging showed that the in vivo signals observed at the chest and upper abdomen decayed slowly in LPS-treated mice. Suppression of the decay in LPS-treated mice was canceled by the administration of a combination of pegylated superoxide dismutase and catalase, or an inhibitor of nitric oxide synthase, or gadolinium chloride. These results indicate that the LPS-treated mouse is under oxidative stress and that reactive oxygen species, such as superoxide and peroxynitrite, related to macrophages are mainly involved in the oxidative stress.     

Calcium Channel Blocker Enhances Beneficial Effects of an Angiotensin II AT1 Receptor Blocker against Cerebrovascular-Renal Injury in type 2 Diabetic Mice

Recent clinical trials have demonstrated that combination therapy with renin-angiotensin system inhibitors plus calcium channel blockers (CCBs) elicits beneficial effects on cardiovascular and renal events in hypertensive patients with high cardiovascular risks. In the present study, we hypothesized that CCB enhances the protective effects of an angiotensin II type 1 receptor blocker (ARB) against diabetic cerebrovascular-renal injury. Saline-drinking type 2 diabetic KK-A^y mice developed hypertension and exhibited impaired cognitive function, blood-brain barrier (BBB) disruption, albuminuria, glomerular sclerosis and podocyte injury. These brain and renal injuries were associated with increased gene expression of NADPH oxidase components, NADPH oxidase activity and oxidative stress in brain and kidney tissues as well as systemic oxidative stress. Treatment with the ARB, olmesartan (10 mg/kg/day) reduced blood pressure in saline-drinking KK-A^y mice and attenuated cognitive decline, BBB disruption, glomerular injury and albuminuria, which were associated with a reduction of NADPH oxidase activity and oxidative stress in brain and kidney tissues as well as systemic oxidative stress. Furthermore, a suppressive dose of azelnidipine (3 mg/kg/day) exaggerated these beneficial effects of olmesartan. These data support the hypothesis that a CCB enhances ARB-associated cerebrovascular-renal protective effects through suppression of NADPH oxidase-dependent oxidative stress in type 2 diabetes.     

Evidence for contribution of vascular NAD(P)H oxidase to increased oxidative stress in animal models of diabetes and obesity

It is well established that oxidative stress is enhanced in diabetes. However, the major in vivo source of oxidative stress is not clear. Here we show that vascular NAD(P)H oxidase may be a major source of oxidative stress in diabetic and obese models. In vivo electron spin resonance (ESR)/spin probe was used to evaluate systemic oxidative stress in vivo. The signal decay rate of the spin probe (spin clearance rate; SpCR) significantly increased in streptozotocin-induced diabetic rats 2 weeks after the onset of diabetes. This increase was completely normalized by treatment with the antioxidants alpha-tocopherol (40 mg/kg) and superoxide dismutase (5000 units/kg), and was significantly inhibited by treatment with a PKC-specific inhibitor, CGP41251 (50 mg/kg), and a NAD(P)H oxidase inhibitor, apocynin (5 mg/kg). Both obese ob/ob mice (10 weeks old) with mild hyperglycemia and Zucker fatty rats (11 weeks old) with normoglycemia exhibited significantly increased SpCR as compared with controls. Again, this increase was inhibited by treatment with both CGP41251 and apocynin. Oral administration of insulin sensitizer, pioglitazone (10 mg/kg), for 7 days also completely normalized SpCR values. These results suggest that vascular NAD(P)H oxidase may be a major source of increased oxidative stress in diabetes and obesity.     

Measurement of oxidative stress in stroke-prone spontaneously hypertensive rat brain using in vivo electron spin resonance spectroscopy

A number of researchers have reported that free radicals generated in the brain are involved in various brain dysfunctions, including ischemia-reperfusion injury, brain tumors, and neurodegenerative diseases. It has been reported that the spin probe MC-PROXYL can penetrate the blood-brain barrier and can be useful for evaluating oxidative stress in the brain. Preliminary comparisons were made by ESR imaging of the heads of live mice and isolated rat brains using the spin probe MC-PROXYL and the blood-brain-barrier impermeable probe carbamoyl-PROXYL. The results showed that MC-PROXYL, but not carbamoyl-PROXYL, was widely distributed in the brain. These methods were also applied for the imaging of brains from stroke-prone spontaneously hypertensive rats (SHRSPs). The rapid decay of 2D ESR images of MC-PROXYL in isolated SHRSP-brain was observed, compared to Wistar-Kyoto rats (WKYs), using the ESR imaging system. Furthermore, we provide evidence, by using L-band ESR non-invasively, that the decay rate of MC-PROXYL in the head region is faster in live SHRSPs than in live WKYs. Taken together, the high oxidative stress sustained by oxygen radical generation in SHRSPs may cause the alteration of MC-PROXYL metabolism in the brain. Our results suggest that in vivo ESR could be applied to the assessment of antioxidant effects on oxidative stress in the brain in animal disease models, such as the SHRSP.     

Noninvasive evaluation of in vivo free radical reactions catalyzed by iron using in vivo ESR spectroscopy.

The noninvasive, real time technique of in vivo electron spin resonance (ESR) spectroscopy was used to evaluate free radical reactions catalyzed by iron in living mice. The spectra and signal decay of a nitroxyl probe, carbamoyl-PROXYL, were observed in the upper abdomen of mice. The signal decay was significantly enhanced in mice subcutaneously loaded with ferric citrate (0.2 micromol/g body wt) and the enhancement was suppressed by pre-treatment with either desferrioxamine (DF) or the chain breaking antioxidant Trolox, but only slightly suppressed by the hydroxyl radical scavenger DMSO. To determine the catalytic form of iron, DF was administered at different times with respect to iron loading: before, simultaneously, and after 20 and 50 min. The effect of DF on signal decay, liver iron content, iron excretion, and lipid peroxidation (TBARs) depended on the time of the treatment. There was a good correlation between the signal decay, iron content, and lipid peroxidation, indicating that "chelatable iron" contributed to the enhanced signal decay. The nitroxyl probe also exhibited in vivo antioxidant activity, implying that the process responsible for the signal decay of the nitroxyl probe is involved in free radical oxidative stress reactions catalyzed by iron.     

Peroxisome-generated succinate induces lipid accumulation and oxidative stress in the kidneys of diabetic mice

Diabetes normally causes lipid accumulation and oxidative stress in the kidneys, which plays a critical role in the onset of diabetic nephropathy; however, the mechanism by which dysregulated fatty acid metabolism increases lipid and reactive oxygen species (ROS) formation in the diabetic kidney is not clear. As succinate is remarkably increased in the diabetic kidney, and accumulation of succinate suppresses mitochondrial fatty acid oxidation and increases ROS formation, we hypothesized that succinate might play a role in inducing lipid and ROS accumulation in the diabetic kidney. Here we demonstrate a novel mechanism by which diabetes induces lipid and ROS accumulation in the kidney of diabetic animals. We show that enhanced oxidation of dicarboxylic acids by peroxisomes leads to lipid and ROS accumulation in the kidney of diabetic mice via the metabolite succinate. Furthermore, specific suppression of peroxisomal β-oxidation improved diabetes-induced nephropathy by reducing succinate generation and attenuating lipid and ROS accumulation in the kidneys of the diabetic mice. We suggest that peroxisome-generated succinate acts as a pathological molecule inducing lipid and ROS accumulation in kidney, and that specifically targeting peroxisomal β-oxidation might be an effective strategy in treating diabetic nephropathy and related metabolic disorders.     

ACEI/ARB 对糖尿病肺病的影响

近年来,糖尿病肺部病变越来越受到关注。研究发现糖尿病患者存在肺通气功能障碍、弥散功能障碍,易于发生肺纤维化、合 并感染等。这主要与糖尿病时炎症介质的参与,ACE 基因存在插入多态性,氧化应激损伤和抗氧化失衡等有关。目前研究发现血 管紧张素转换酶抑制剂（ACEI）/ 血管紧张素受体阻滞剂(ARB)对糖尿病肺炎、慢性阻塞性肺疾病、肺间质纤维化等肺损伤有保护 作用,这可能与ACEI具有调节免疫反应、减轻细胞因子水平、抗氧化应激损伤等机制有关。     

Flacourtia indica fruit extract modulated antioxidant gene expression,prevented oxidative stress and ameliorated kidney dysfunction in isoprenaline administered rats

This study evaluated the effect of Flacourtia indica fruit extract against isoprenaline (ISO) induced renal damage in rats. This investigation showed that ISO administration in rats increased the level oxidative stress biomarkers such as malondialdehyde (MDA), nitric oxide (NO), advanced protein oxidation product (APOP) in kidneys followed by a decrease in antioxidant enzymes functions. Flacourtia indica fruit extract, which is rich in strong antioxidants, also reduced the MDA, NO and APOP level in kidney of ISO administered rats. Inflammation and necrosis was also visible in kidney section of ISO administered rats which was significantly prevented by atenolol and Flacourtia indica fruit extract. Moreover, atenolol and Flacourtia indica fruit extract also modulated the genes expressions related to inflammation and oxidative stress in kidneys. The beneficial effects could be attributed to the presence of a number of phenolic antioxidants. This study suggests that Flacourtia indica fruit extract may prevent kidney dysfunction in ISO administered rats, probably by preventing oxidative stress and inflammation.     

Biotin amelioration of nephrotoxicity in streptozotocin-induced diabetic mice

The current study was carried out to investigate the protective role of biotin in kidney injury and oxidative stress in diabetic mice type 1. Male Swiss albino mice were randomly divided into 3 groups. Control group received saline. Diabetes type 1 was induced in second and third groups by intraperitoneal injection of streptozotocin as a single dose (150 mg/kg). Second group remained as the untreated diabetic group and the third group received 15 mg/kg daily oral dose of biotin for 12 successive days. Biochemical results showed significant elevation in blood glucose and urea levels in both diabetic groups. Also, there is an increase in glomerular areas and decrease in glomerular cellularity in both diabetic groups. Histopathological results showed severe alterations in the untreated diabetic group represented by distorted glomeruli, inflammatory cells, and giant macrophages. In addition, there was an intense immune-reaction response toward acrolein indicator of oxidative damage. Upon biotin administration of diabetic mice, the above mentioned histopathological changes were reduced and also acroline reaction of oxidative damage was diminished. Our findings prove that biotin has a protective role against streptozotocin-induced oxidative damage in kidneys of laboratory mice.     

NDUFV1 attenuates renal ischemia–reperfusion injury by improving mitochondrial homeostasis

Impaired mitochondrial function and dysregulated energy metabolism have been shown to be involved in the pathological progression of kidney diseases such as acute kidney injury (AKI) and diabetic nephropathy. Hence, improving mitochondrial function is a promising strategy for treating renal dysfunction. NADH: ubiquinone oxidoreductase core subunit V1 (NDUFV1) is an important subunit of mitochondrial complex I. In the present study, we found that NDUFV1 was reduced in kidneys of renal ischemia/reperfusion (I/R) mice. Meanwhile, renal I/R induced kidney dysfunction as evidenced by increases in BUN and serum creatinine, severe injury of proximal renal tubules, oxidative stress, and cell apoptosis. All these detrimental outcomes were attenuated by increased expression of NDUFV1 in kidneys. Moreover, knockdown of Ndufv1 aggravated cell insults induced by H₂O₂ in TCMK-1 cells, which further confirmed the renoprotective roles of NDUFV1. Mechanistically, NDUFV1 improved the integrity and function of mitochondria, leading to reduced oxidative stress and cell apoptosis. Overall, our data indicate that NDUFV1 has an ability to maintain mitochondrial homeostasis in AKI, suggesting therapies by targeting mitochondria are useful approaches for dealing with mitochondrial dysfunction associated renal diseases such as AKI.     

Quantitative mass spectrometry of diabetic kidney tubules identifies GRAP as a novel regulator of TGF-β signaling

The aim of this study was to define novel mediators of tubule injury in diabetic kidney disease. For this, we used state-of-the-art proteomic methods combined with a label-free quantitative strategy to define protein expression differences in kidney tubules from transgenic OVE26 type 1 diabetic and control mice. The analysis was performed with diabetic samples that displayed a pro-fibrotic phenotype. We have identified 476 differentially expressed proteins. Bioinformatic analysis indicated several clusters of regulated proteins in relevant functional groups such as TGF-β signaling, tight junction maintenance, oxidative stress, and glucose metabolism. Mass spectrometry detected expression changes of four physiologically relevant proteins were confirmed by immunoblot analysis. Of these, the Grb2-related adaptor protein (GRAP) was up-regulated in kidney tubules from diabetic mice and fibrotic kidneys from diabetic patients, and subsequently confirmed as a novel component of TGF-β signaling in cultured human renal tubule cells. Thus, indicating a potential novel role for GRAP in TGF-β-induced tubule injury in diabetic kidney disease. Although we targeted a specific disease, this approach offers a robust, high-sensitivity methodology that can be applied to the discovery of novel mediators for any experimental or disease condition.     

Acute knockdown of uncoupling protein-2 increases uncoupling via the adenine nucleotide transporter and decreases oxidative stress in diabetic kidneys

Increased O(2) metabolism resulting in chronic hypoxia is common in models of endstage renal disease. Mitochondrial uncoupling increases O(2) consumption but the ensuing reduction in mitochondrial membrane potential may limit excessive oxidative stress. The present study addressed the hypothesis that mitochondrial uncoupling regulates mitochondria function and oxidative stress in the diabetic kidney. Isolated mitochondria from kidney cortex of control and streptozotocin-induced diabetic rats were studied before and after siRNA knockdown of uncoupling protein-2 (UCP-2). Diabetes resulted in increased UCP-2 protein expression and UCP-2-mediated uncoupling, but normal mitochondria membrane potential. This uncoupling was inhibited by GDP, which also increased the membrane potential. siRNA reduced UCP-2 protein expression in controls and diabetics (-30-50%), but paradoxically further increased uncoupling and markedly reduced the membrane potential. This siRNA mediated uncoupling was unaffected by GDP but was blocked by ADP and carboxyatractylate (CAT). Mitochondria membrane potential after UCP-2 siRNA was unaffected by GDP but increased by CAT. This demonstrated that further increased mitochondria uncoupling after siRNA towards UCP-2 is mediated through the adenine nucleotide transporter (ANT). The increased oxidative stress in the diabetic kidney, manifested as increased thiobarbituric acids, was reduced by knocking down UCP-2 whereas whole-body oxidative stress, manifested as increased circulating malondialdehyde, remained unaffected. All parameters investigated were unaffected by scrambled siRNA. In conclusion, mitochondrial uncoupling via UCP-2 regulates mitochondria membrane potential in diabetes. However, blockade of the diabetes-induced upregulation of UCP- 2 results in excessive uncoupling and reduced oxidative stress in the kidney via activation of ANT.     

Beneficial effects of angiotensin II receptor blocker,olmesartan, in limiting the cardiotoxic effect of daunorubicin in rats

Abstract

The aim was to evaluate the role of the combination of olmesartan, an angiotensin II (Ang II) receptor blocker (ARB), with daunorubicin (DNR) in reducing cardiac toxicity in rats. DNR was administered at a dose of 3 mg/kg/day every other day for 12 days. Olmesartan was administered orally every day for 12 days. Rats treated with DNR alone showed cardiac toxicity as evidenced by worsening cardiac function, elevation of malondialdehyde level in heart tissue and decreased in the level of total glutathione peroxidase activity; treatment with ARB reversed these changes. Furthermore, ARB treatment down-regulated matrix metalloproteinase-2 expression, myocardial expression of Ang II, attenuated the increased protein expressions of p67^phox and Nox4 and reduced oxidative stress-induced DNA damage evaluated by expression of 8-hydroxydeoxyguanosine. In conclusion, the result demonstrated that Ang II and oxidative stress play a key role in anthracycline-induced cardiotoxicity and that treatment with ARB will be beneficial against DNR-induced cardiotoxicity.     

Determination of reactive oxygen species associated with the degeneration of dopaminergic neurons during dopamine metabolism

Abstract

Oxidative stress is believed to be an important mechanism underlying dopamine-induced neuronal damage. This study provides X-band electron spin resonance (ESR) spectroscopic evidence for reactive oxygen species (ROS) generation during dopamine metabolism. The authors induced excess dopamine metabolism in the mouse striatum by bathing it in tyramine-containing perfusate using microdialysis. The addition of tyramine to the perfusate raised the levels of extracellular dopamine and hydrogen peroxide significantly. The ESR signal from hydroxy-TEMPO decayed during tyramine perfusion and treatment with a monoamine-oxidase inhibitor or radical scavenger suppressed the signal decay. Decreases in the number of tyrosine hydroxylase-immunopositive fibres and in dopamine concentration after tyramine perfusion were observed. Moreover, the tyramine-perfused mice showed a marked methamphetamine-induced rotational response. Notably, these effects of tyramine were suppressed by the simultaneous perfusion of hydroxy-TEMPO. These findings indicate that the ROS generation, which was monitored by hydroxy-TEMPO, caused oxidative damage to the dopaminergic neurons.     

Statin attenuates high glucose-induced and diabetes-induced oxidative stress in vitro and in vivo evaluated by electron spin resonance measurement

An increased oxidative stress may contribute to the accelerated atherosclerosis in diabetic patients. Here we show that 3-hydroxy-3-methylglutaryl CoA reductase inhibitor (statin) attenuates a high glucose-induced and a diabetes-induced oxidative stress through inhibition of vascular NAD(P)H oxidase. Exposure of cultured aortic endothelial cells and smooth muscle cells to a high glucose level (450 mg/dl) for 3 days significantly increased oxidative stress compared with a normal glucose level (100 mg/dl), as evaluated by the staining with 2',7'-dichlorofluorescein diacetate and electron spin resonance (ESR) measurement. This increase was completely blocked by the treatment with pitavastatin (5 x 10(-7)M) as well as a NAD(P)H oxidase inhibitor (diphenylene iodonium) or a PKC inhibitor (calphostin C) in parallel with the change of small GTPase Rac-1 activity, a cytosolic regulatory component of NAD(P)H oxidase. Next, using streptozotocin-induced diabetic rats, the effect of pitavastatin on oxidative stress was evaluated by in vivo ESR measurements, which is a sensitive, noninvasive method. Administration of pitavastatin (5 mg/kg/day) for 4 days attenuated the increased oxidative stress in diabetic rats to control levels. In conclusion, pitavastatin attenuated a high glucose-induced and a diabetes-induced oxidative stress in vitro and in vivo. Thus, our data may provide a new insight into antioxidative therapy in diabetes.     

Effect of anaesthesia-induced alterations in haemodynamics on in vivo kinetics of nitroxyl probes in electron spin resonance spectroscopy

Although the advent of in vivo electron spin resonance (ESR) spectroscopy has allowed analysis of the redox status of living animals, whether the haemodynamic condition affects the signal decay rate remains unknown. Three kinds of haemodynamic conditions were generated by changing the anaesthetic dosage in mice. Haemodynamics was analysed (n=6 each) and in vivo ESR was performed to measure the signal decay rates of three nitroxyl spin probes (carbamoyl-, carboxy- and methoxycarbonyl-PROXYL) at the chest and head regions (n=6 for each condition and probe). Haemodynamic analysis revealed negative inotropic and chronotropic effects on the cardiovascular system depending on the depth of anaesthesia. Although signal decay rates differed among three probes, they were not affected by heart rate alteration. In this study we report the haemodynamics-independent signal decay rate of nitoxyl probes.     

Effects of Stimulation of Soluble Guanylate Cyclase on Diabetic Nephropathy in Diabetic eNOS Knockout Mice on Top of Angiotensin II Receptor Blockade

The prevalence of diabetes mellitus and its complications, such as diabetic nephropathy (DN), is rising worldwide and prevention and treatment are therefore becoming increasingly important. Therapy of DN is particularly important for patients who do not adequately respond to angiotensin receptor blocker (ARB) treatment. Novel approaches include the stimulation of soluble guanylate cyclase (sGC) as it is reported to have beneficial effects on cardiac and renal damage. We aimed to investigate the effects of the sGC stimulator riociguat and ARB telmisartan on kidney function and structure in a hypertensive model of diabetic nephropathy.Seventy-six diabetic male eNOS knockout C57BL/6J mice were randomly divided after having received streptozotocin: telmisartan (1 mg/kg/d), riociguat (3 mg/kg/d), riociguat+telmisartan (3+1 mg/kg/d), and vehicle. Fourteen mice were used as non-diabetic controls. Treatment duration was 11 weeks.Glucose concentrations were increased and similar in all diabetic groups. Telmisartan insignificantly reduced blood pressure by 5.9 mmHg compared with diabetic controls (111.2±2.3 mmHg vs. 117.1±2.2 mmHg; p = 0.071). Treatment with riociguat both alone and in combination with telmisartan led to a significant reduction of blood pressure towards diabetic vehicle (105.2±2.5 mmHg and 105.0±3.2 mmHg, respectively, vs. 117.1±2.2 mmHg). Combined treatment also significantly decreased albuminuria compared with diabetic controls (47.3±9.6 µg/24 h vs. 170.8±34.2 µg/24 h; p = 0.002) reaching levels similar to those of non-diabetic controls (34.4±10.6 µg/24 h), whereas the reduction by single treatment with either telmisartan (97.8±26.4 µg/24 h) or riociguat (97.1±15.7 µg/24 h) was not statistically significant. The combination treatment led to a significant (p<0.01) decrease of tissue immunoreactivity of malondialdehyde, as consequence of reduced oxidative stress.In conclusion, stimulation of sGC significantly reduced urinary albumin excretion in diabetic eNOS knockout mice treated already with ARB. Thus, this new drug class on top of standard ARBs administration may offer a new therapeutic approach for patients resistant to ARB treatment.     

富硒发酵毛头鬼伞菌丝对四氧嘧啶致糖尿病小鼠抗氧化和降血糖的影响

本文研究了富硒发酵毛头鬼伞(鸡腿菇)Coprinus comatus菌丝体对四氧嘧啶致糖尿病小鼠抗氧化和降血糖的影响。四氧嘧啶建立糖尿病小鼠模型后,将富硒发酵毛头鬼伞菌丝均匀混入饲料中,由小鼠自由取食进行治疗,3周后观察富硒毛头鬼伞菌丝对糖尿病小鼠血糖、MDA、SOD和GSH-Px的影响。结果发现,采用富硒发酵毛头鬼伞菌丝进行治疗后,糖尿病小鼠血糖明显降低;血清和组织中MDA含量显著下降;血清和组织中的SOD和GSH-Px的活力明显的增加。由此推断,富硒发酵毛头鬼伞菌丝对四氧嘧啶致糖尿病小鼠的降血糖效果可能是通过提高机体抗氧化能力来实现的。