Lipid Peroxidation, Reduced Glutathione, and Glutathione Peroxidase Levels in Intervertebral Discs of Patients with Lumbar Degenerative Disc Disease.

BACKGROUND Either a reduction in antioxidant levels or an accumulation of reactive oxygen species can heighten susceptibility to oxidative damage in disc cells. To date, no research has investigated the levels of lipid peroxidation products (thiobarbituric acid reactive substances [TBARs]), reduced glutathione (GSH), and glutathione peroxidase (GPx) in excised human lumbar disc tissues affected by degenerative disease. Therefore, this study aimed to evaluate lipid peroxidation products in excised disc tissues from patients with degenerative disc disease. MATERIAL AND METHODS Forty-two patients were enrolled. Patients were divided into lumbar disc degeneration (LDD) and nonlumbar disc degeneration (nonLDD) groups according to Pfirrmann classification. Intervertebral discs were obtained from all patients during the operation and were homogenized for analysis. TBARs levels were measured using fluorometry. GSH levels and GPx activity were quantified spectrophotometrically using a kinetic method. RESULTS TBARs levels in excised discs from LDD patients (5.18±4.14) were significantly higher than those from nonLDD patients (2.56±1.23, P=0.008). The levels of TBARs tended to increase with the severity of degeneration according to the Pfirrmann classification. However, these 2 groups showed no significant differences in reduced glutathione levels or glutathione peroxidase activity (P>0.05). Patients with LDD exhibited a worse health-related quality of life, reflected in lower utility and EQ-VAS scores and higher Oswestry disability index scores. CONCLUSIONS There was a notable increase in lipid peroxidation products in the excised intervertebral discs of patients with LDD. This finding suggests that oxidative stress may contribute to the development of disc degeneration.

Functional Roles of Furin in Cardio-Cerebrovascular Diseases.

Furin plays a major role in post-translational modification of several biomolecules, including endogenous hormones, growth factors, and cytokines. Recent reports have demonstrated the association of furin and cardio-cerebrovascular diseases (CVDs) in humans. This review describes the possible pathogenic contribution of furin and its substrates in CVDs. Early-stage hypertension and diabetes mellitus show a negative correlation with furin. A reduction in furin might promote hypertension by decreasing maturation of B-type natriuretic peptide (BNP) or by decreasing shedding of membrane (pro)renin receptor (PRR), which facilitates activation of the renin-angiotensin-aldosterone system (RAAS). In diabetes, furin downregulation potentially leads to insulin resistance by reducing maturation of the insulin receptor. In contrast, the progression of other CVDs is associated with an increase in furin, including dyslipidemia, atherosclerosis, ischemic stroke, myocardial infarction (MI), and heart failure. Upregulation of furin might promote maturation of membrane type 1-matrix metalloproteinase (MT1-MMP), which cleaves low-density lipoprotein receptor (LDLR), contributing to dyslipidemia. In atherosclerosis, elevated levels of furin possibly enhance maturation of several substrates related to inflammation, cell proliferation, and extracellular matrix (ECM) deposition and degradation. Neuronal cell death following ischemic stroke has also been shown to involve furin substrates (e.g., MT1-MMP, hepcidin, and hemojuvelin). Moreover, furin and its substrates, including tumor necrosis factor-α (TNF-α), endothelin-1 (ET-1), and transforming growth factor-ß1 (TGF-ß1), are capable of mediating inflammation, hypertrophy, and fibrosis in MI and heart failure. Taken together, this evidence provides functional significance of furin in CVDs and might suggest a potential novel therapeutic modality for the management of CVDs.

Naturally occurring quercetin and myricetin as potent inhibitors for human ectonucleotide pyrophosphatase/phosphodiesterase 1.

Ecto-nucleotide pyrophosphatases/phosphodiesterases 1 (ENPP1) is a key enzyme in purinergic signaling pathways responsible for cell-to-cell communications and regulation of several fundamental pathophysiological processes. In this study, Kyoto Green, a rapid chemical sensor of pyrophosphate, was employed to screen for effective ENPP1 inhibitors among five representative flavonoids (quercetin, myricetin, morin, kaempferol, and quercetin-3-glucoside), five nucleosides (adenosine, guanosine, inosine, uridine, and cytidine), and five deoxynucleosides (2'- and 3'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyinosine, and 2'-deoxyuridine). Conventional colorimetric, fluorescence, and bioluminescence assays revealed that ENPP1 was effectively inhibited by quercetin (Ki ~ 4 nM) and myricetin (Ki ~ 32 nM) when ATP was used as a substrate at pH 7.4. In silico analysis indicated that the presence of a chromone scaffold, particularly one containing a hydroxyl group at the 3' position on the B ring, may promote binding to the active site pocket of ENPP1 and enhance inhibition. This study demonstrated that the naturally derived quercetin and myricetin could effectively inhibit ENPP1 enzymatic activity and may offer health benefits in arthritis management.

Dasatinib Ointment Promotes Healing of Murine Excisional Skin Wound.

Dasatinib, a tyrosine kinase inhibitor, has been shown to produce anti-inflammatory activity and impair vascular integrity in vivo, including during skin wound healing, potentially promoting the repair process. Given that dasatinib is a lipophilic small molecule capable of penetrating skin, topical dasatinib might provide benefits in wound healing. In the present study, we investigated the impact of dasatinib ointments in skin wound healing in mice. A full thickness excisional skin wound (4 mm diameter) was generated on the shaved dorsum of eight-week-old C57BL/6 mice. Dasatinib ointment (0.1 or 0.2% w/w) or ointment base was applied twice daily (every 12 h) for 10 days. Elizabethan collars were used to prevent animal licking. The wound size was monitored daily for 14 days. The results showed that dasatinib ointments, particularly 0.1% dasatinib, promoted a 16-23% reduction in wound size (p < 0.05) during day 2 to day 6 postinjury compared to controls. Immunohistochemistry analyses demonstrated a reduction in wound neutrophils (38% reduction, p = 0.04), macrophages (47% reduction, p = 0.005), and tumor necrosis factor-α levels (73% reduction, p < 0.01), together with an induction of vascular leakage-mediated fibrin(ogen) accumulation (2.5-fold increase, p < 0.01) in the wound during day 3 postinjury (an early phase of repair) in 0.1% dasatinib-treated mice relative to control mice. The anti-inflammatory and vascular hyperpermeability activities of dasatinib were associated with an enhanced healing process, including increased keratinocyte proliferation (1.8-fold increase in Ki67+ cells, p < 0.05) and augmented angiogenesis (1.7-fold increase in CD31+ area, p < 0.05), compared to the ointment base-treated group. Following treatment with 0.2% dasatinib ointment, minor wound bleeding and scab reformation were observed during the late phase, which contributed to delayed healing. In conclusion, our data suggest that dasatinib ointment, mainly at 0.1%, promotes the repair process by reducing inflammation and producing a local and temporal vascular leakage, leading to an increase in fibrin(ogen) deposition, re-epithelialization, and angiogenesis. Therefore, topical dasatinib might be a potential novel candidate to facilitate skin wound healing.

Deferiprone, an iron chelator, alleviates platelet hyperactivity in patients with ß-thalassaemia/HbE.

Background: Hyperfunctional platelets play important roles in thromboembolism in patients with ß-thalassaemia/ haemoglobin E (ß-thal/HbE). Our previous study revealed ex vivo inhibitory effects of deferiprone on normal platelets. Herein, we aimed to investigate the in vivo effects on platelets in patients with ß-thal/HbE. Methods: A prospective, self-controlled clinical study on 30 patients with ß-thal/HbE who had received therapeutic deferiprone (20.8-94.5 mg/kg/day) was conducted. The study included a 4-week washout period followed by 4 and 12 weeks of deferiprone treatment. Platelet aggregation was performed by a turbidimetric method. Levels of deferiprone and soluble platelet (sP)-selectin in serum were measured by high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) kit, respectively. Results: The washout period significantly enhanced platelet hyperactivity both in patients who had undergone splenectomy and in those who had not. At 2 hours following the administration of a single dose of deferiprone, platelet sensitivity to ADP and arachidonic acid was significantly reduced. The inhibitory effects of deferiprone were gradually increased over the period of 4 and 12 weeks. Deferiprone also depressed sP-selectin levels, but the effect was stable over longer follow-up periods. Correlation analysis demonstrated the relationship between serum levels of deferiprone, sP-selectin, and platelet activities induced by ADP and arachidonic acid. Conclusion: We first demonstrated the in vivo antiplatelet effect and benefit of short-term treatment of deferiprone in patients with ß-thal/HbE. The impact on thrombotic outcomes deserves further study.

Sustained AT1R stimulation induces upregulation of growth factors in human cardiac fibroblasts via Gαq/TGF-ß/ERK signaling that influences myocyte hypertrophy.

Stimulation of angiotensin II receptor (ATR) with angiotensin II (Ang II) accelerates cardiac fibroblast activation, resulting in upregulation of cytokines and growth factors. Growth factors were strongly upregulated in animal models of myocardial fibrosis and hypertrophy as well as patients with heart failure. Nevertheless, the signal transduction of ATR for upregulation of growth factors in human cardiac fibroblasts contributing to myocyte hypertrophy have not fully understood. Long-term Ang II treatment of human cardiac fibroblasts provokes the synthesis and secretion of connective tissue growth factor (CTGF), transforming growth factor beta1 (TGF-ß1), and vascular endothelial growth factor (VEGF) through the AT1R subtype. Blockade of Gαq, not Gαi or Gα12/13, protein signaling inhibited AT1R-mediated upregulation of CTGF, TGF-ß1, and VEGF. In addition, AT1R overstimulation induced upregulation of growth factors via the TGF-ß-dependent and ERK1/2-dependent pathways. Growth factors secreted from cardiac fibroblasts are necessary for the induction of hypertrophic markers, atrial natriuretic peptide (ANP) and ß-myosin heavy chain (ß-MHC), resulting in myocyte hypertrophy. Candesartan, irbesartan, and valsartan had greater effects than losartan for blockade of fibrotic and hypertrophic effects of Ang II. Our data support the concept whereby sustained AT1R stimulation contributes to the development of myocardial fibrosis and hypertrophy, and advances understanding of this complex AT1R signaling, including fibroblasts-myocytes communication during pathological conditions.

Iron chelation therapy with deferiprone improves oxidative status and red blood cell quality and reduces redox-active iron in ß-thalassemia/hemoglobin E patients.

The oxidative status of twenty-three ß-thalassemia/hemoglobin E patients was evaluated after administration of 75 mg/kg deferiprone (GPO-L-ONE®) divided into 3 doses daily for 12 months. Serum ferritin was significantly decreased; the median value at the initial and final assessments was 2842 and 1719 ng/mL, respectively. Progressive improvement with significant changes in antioxidant enzyme activity, including plasma paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH), and in antioxidant enzymes in red blood cells (glutathione peroxidase (GPx), catalase and superoxide dismutase (SOD)) were observed at 3-6 months of treatment. The levels of total GSH in red blood cells were significantly increased at the end of the study. Improved red blood cell membrane integrity was also demonstrated using the EPR spin labeling technique. Membrane fluidity at the surface and hydrophobic regions of the red blood cell membrane was significantly changed after 12 months of treatment. In addition, a significant increase in hemoglobin content was observed (6.6 ± 0.7 and 7.5 ± 1.3 g/dL at the initial assessment and at 6 months, respectively). Correlations were observed between hemoglobin content, membrane fluidity and antioxidant enzymes in red blood cells. The antioxidant activity of deferiprone may partly be explained by progressive reduction of redox active iron that catalyzes free radical reactions, as demonstrated by the EPR spin trapping technique. In conclusion, iron chelation therapy with deferiprone notably improved the oxidative status in thalassemia, consequently reducing the risk of oxidative-related complications. Furthermore, the improvement in red blood cell quality may improve the anemia situation in patients.

Dasatinib induces loss of vascular integrity and promotes cutaneous wound repair in mice.

BACKGROUND: Inflammatory bleeding due to depletion of platelet glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) has been proposed as a potential novel mechanism to promote skin wound healing. Dasatinib inhibits a broad range of tyrosine kinases, including Src and Syk, the signaling molecules downstream of GPVI and CLEC-2. OBJECTIVES: To investigate whether dasatinib affects skin wound healing. METHODS: A single (4-mm diameter) full-thickness excisional skin wound was generated in mice. Dasatinib (5 or 10 mg/kg) or dimethyl sulfoxide (DMSO) vehicle was intraperitoneally injected daily during the first 4 days. The wound was monitored over 9 days post injury. RESULTS: Dasatinib induced loss of vascular integrity during the inflammatory phase of wound repair (day 1 to day 3 post injury), which was associated with the inhibition of platelet function stimulated by collagen and rhodocytin, the ligands for GPVI and CLEC-2, respectively. Dasatinib-treated mice, particularly at 5 mg/kg, exhibited accelerated wound closure compared to DMSO-treated controls. Transient bleeding into the wound during the inflammatory phase in dasatinib-treated mice allowed for extravasation of fibrinogen. The increased deposition of fibrinogen and fibrin in the wound on day 3 post injury was associated with the augmented progression of re-epithelialization and angiogenesis, attenuated infiltration of neutrophils and macrophages, and decreased levels of tumor necrosis factor-α (TNF-α). CONCLUSIONS: Our data show that dasatinib promotes skin wound healing, and the mechanisms include blocking GPVI- and CLEC-2-mediated platelet activation, leading to self-limited inflammatory bleeding and fibrinogen/fibrin deposition, in association with reduced inflammation, increased re-epithelialization, and enhanced angiogenesis.

Anti-inflammatory effect of photodynamic therapy using guaiazulene and red lasers on peripheral blood mononuclear cells.

INTRODUCTION: Photodynamic therapy improves oral mucositis treatment. The reactive oxygen species (ROS) generated from this reaction could contribute to an anti-inflammatory effect by suppressing inflammatory cells. OBJECTIVE: To evaluate the anti-inflammatory effect of photodynamic therapy using guaiazulene and a red laser in peripheral blood mononuclear cells (PBMCs). METHODS: Guaiazulene solutions (1, 2, 5, 25, 35, and 100 µM in 99.8 % methanol) were irradiated with red laser light (625 nm, 146.2 mW/cm2) in continuous mode at 0, 4, and 8 J/cm2 in black 96-well plates. ROS were measured using spin trapping technique with electron spin resonance (ESR) spectroscopy and fluorescence. The two highest concentrations were tested using cell viability (PrestoBlue®) and anti-inflammation (RANTES and PGE2 ELISA) assay kits. Kruskal-Wallis and Dunn Bonferroni tests were used for statistical analyses with significant differences at p-value < 0.05. RESULTS: Guaiazulene solutions between 2 and 5 µM exposed to red laser light at 4-8 J/cm2 generated significantly more singlet oxygen compared to the no guaiazulene group (p < 0.01) and reduced RANTES and PGE2 levels in TNF-α-inflamed peripheral blood mononuclear cells without affecting cell viability. CONCLUSION: Photodynamic activation of guaiazulene generated singlet oxygen and suppressed inflammatory markers in PBMCs.

Antiplatelet activity of deferiprone through cyclooxygenase-1 inhibition.

Thalassemia patients are susceptible to both iron overload and thromboembolism. Deferiprone is an iron chelator that shows an antiplatelet activity and thus may alleviate platelet hyperactivation in thalassemia. Therefore, this study aimed to characterize the inhibitory effects and mechanisms of deferiprone on normal human platelets. The results illustrated that deferiprone inhibited platelet aggregation at the iron chelating concentrations (0.08-0.25 mmol/l). Deferiprone inhibited human platelet aggregation stimulated by arachidonic acid and ADP more potently than epinephrine and collagen, with the IC50 of 0.24 mmol/l and 0.25 mmol/l vs. 3.36 mmol/l and 3.73 mmol/l, respectively. Interestingly, deferiprone significantly inhibited COX-1 activity, with the IC50 of 0.33 mmol/l, and slightly increased cAMP level at the high concentration of 4 mmol/l. Moreover, the results from molecular docking showed that deferiprone interacted closely with key residues in the peroxidase active site of COX-1. These results suggested that deferiprone possessed antiplatelet activity mainly through the inhibition of COX-1 activity.

MRI imaging and histopathological study of brain iron overload of ß-thalassemic mice.

Brain iron overload is chronic and slow progressing and plays an important role in the pathogenesis of neurodegenerative disorders. Magnetic resonance imaging (MRI) is a useful noninvasive tool for determining liver iron content, but it has not been proven to be adequate for evaluating brain iron overload. We evaluated the usefulness of MRI-derived parameters to determine brain iron concentration in ß-thalassemic mice and the effects of the membrane permeable iron chelator, deferiprone. Sixteen ß-thalassemic mice underwent 1.5T MRI of the brain that included a multiecho T2*-weighted sequence. Brain T2* values ranged from 28 to 31ms for thalassemic mice. For the iron overloaded thalassemic mice, brain T2* values decreased, ranging from 8 to 12ms, which correlated with the iron overload status of the animals. In addition, brain T2* values increased in the group with the treatment of deferiprone, ranging from 18 to 24ms. Our results may be useful to understand brain pathology in iron overload. Moreover, data could lead to an earlier diagnosis, assist in following disease progression, and demonstrate the benefits of iron chelation therapy.

Photodynamic therapy of Curcuma longa extract stimulated with blue light against Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Curcumin, one of an established curcuminoid substances extracted from Curcuma longa, has been used as a photosensitizer (PS) in photodynamic therapy (PDT). Curcuminoid substances has been reported to have benefits in treating dental chronic infection and inflammation diseases, such as chronic periodontitis. The purpose of this study was to find the optimum concentration of Curcuma longa (CL) extract, containing all curcuminoid substances, and the power density of blue light (BL) in photodynamic therapy against periodontally pathogenic bacteria, A. actinomycetemcomitans. METHODS: Antibacterial activity of various concentrations of CL extract against A. actinomycetemcomitans was determined. Exponentially growing bacteria were combined with 2-fold dilution of CL extract solution ranging from 25 to 0.098 µg/ml. Co-culture bacteria treated with 0.12% chlorhexidine (CHX) served as the positive control. The effect of photostimulation with light emitting diode (LED) 420-480 nm at 16.8 J/cm2 for 1 min on the selected concentration of CL extract was examined. Bacteria viability was determined by plate counting technique. In addition, production of free radicals was tested by electron spin resonance spectroscope (ESR) with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). RESULTS: The antibacterial activity of CL extract was dose dependent. Without BL, 25 µg/ml CL extract showed 6.03 ±â€¯0.39 log10A. actinomycetemcomitans. Interestingly, the combination of BL and 0.78 µg/ml CL extract solution showed complete absence of A. actinomycetemcomitans. Peak signal intensity of hydroxyl radical production was also detected with the combination of BL and CL. CONCLUSIONS: CL extract not only had antimicrobial activity but also could be used as an effective PS when stimulated with BL in PDT. The optimal antibacterial effect of CL extract with BL was equal to the standard oral disinfectant, 0.12% CHX.

Iron distribution and histopathological study of the effects of deferoxamine and deferiprone in the kidneys of iron overloaded ß-thalassemic mice.

Renal glomerular and tubular dysfunctions have been reported with high prevalence in ß-thalassemia. Iron toxicity is implicated in the kidney damage, which may be reversed by iron chelation therapy. To mimic heavy iron overload and evaluate the efficacy of iron chelators in the patients, iron dextran (180mg iron/mouse) was intraperitoneally (i.p.) injected in heterozygous ß-globin knockout mice ((mußth-3/+), BKO) and wild type mice (C57BL/6J, WT) over a period of 2 weeks, followed by daily i.p. injection of deferoxamine (DFO) or deferiprone (L1) for 1 week. In BKO mice, iron preferentially accumulated in the proximal tubule with a grading score of 0-1 and increased to grade 3 after iron loading. In contrast, iron mainly deposited in the glomerulus and interstitial space in iron overloaded WT mice. Increased levels of kidney lipid peroxidation, glomerular and medullar damage and fibrosis in iron overloaded mice were reversed by treatment with iron chelators. L1 showed higher efficacy than DFO in reduction of glomerular iron, which was supported by a significantly decreased the amount of glomerular damage. Notably, DFO and L1 demonstrated a distinct pattern of iron distribution in the proximal tubule of BKO mice. In conclusion, chelation therapy has beneficial effects in iron-overloaded kidneys. However, the defect of kidney iron metabolism in thalassemia may be a determining factor of the treatment outcome in individual patients.

Effects of Iron Chelators on Pulmonary Iron Overload and Oxidative Stress in ß-Thalassemic Mice.

AIM: To evaluate the effect of iron chelators on iron-related pulmonary pathology and oxidative stress in an animal model of ß-thalassemia. METHODS: Pulmonary iron overload was induced in heterozygous ß-globin knockout mice (mußth-3/+, BKO). Over a period of 2 weeks, 180 mg of iron/mouse was loaded by intraperitoneal injection of iron dextran, and subsequently treated daily via intraperitoneal with either deferoxamine (DF) or deferiprone (L1) at an equimolar concentration of iron binding (0.2 and 0.6 µmol/g body weight, respectively) for 7 days. RESULTS: Iron loading resulted in iron deposition in peribronchial regions, septa and also in alveolar macrophages with a grading score of 3. This iron burden resulted in lung epithelial injuries, fibrosis and corresponded with increased lipid peroxidation and decreased tissue catalase activity. Treatment with DF or L1 resulted in a reduction of iron-laden alveolar macrophages and decreased oxidative stress and tissue damage, showing the iron mobilizing ability of both compounds. CONCLUSION: Iron chelation therapy, with DF and L1, may protect against pulmonary damage by sequestering catalytic iron and improving oxidative status. It may be beneficial in the prevention of pulmonary complications in thalassemia.

Effect of iron overload on furin expression in wild-type and ß-thalassemic mice.

Furin is a proprotein convertase enzyme. In the liver, it cleaves prohepcidin to form active hepcidin-25, which regulates systemic iron homeostasis. Hepcidin deficiency is a component of several iron overload disorders, including ß-thalassemia. Several studies have identified factors that repress hepcidin gene transcription in iron overload. However, the effect of iron overload on furin, a post-translational regulator of hepcidin, has never been evaluated. The present study aimed to investigate the changes in furin and related factors in parenteral iron-overloaded mice, including those with ß-thalassemia. Wild-type (WT) and ß-thalassemia intermedia (th3/+) C57BL/6 mice were intraperitoneally injected with 9 doses of iron dextran (1 g iron/kg body weight) over 2 weeks. In the iron overload condition, our data demonstrated a significant Furin mRNA reduction in WT and th3/+ mice. In addition, the liver furin protein level in iron-overloaded WT mice was significantly reduced by 70% compared to control WT mice. However, the liver furin protein in iron-overloaded th3/+ mice did not show a significant reduction compared to control th3/+ mice. The hepcidin gene (hepcidin antimicrobial peptide gene, Hamp1) expression was increased in iron-overloaded WT and th3/+ mice. Surprisingly, the liver hepcidin protein level and total serum hepcidin were not increased in both WT and th3/+ mice with iron overload, regardless of the increase in Hamp1 mRNA. In conclusion, we demonstrate furin downregulation in conjunction with Hamp1 mRNA-unrelated pattern of hepcidin protein expression in iron-overloaded mice, particularly the WT mice, suggesting that, not only the amount of hepcidin but also the furin-mediated physiological activity may be decreased in severe iron overload condition.

Iron distribution and histopathological characterization of the liver and heart of ß-thalassemic mice with parenteral iron overload: Effects of deferoxamine and deferiprone.

The liver and heart are the major target organs for iron accumulation and iron toxicity in ß-thalassemia. To mimic the phenomenon of heavy iron overload resulting from repeated blood transfusions, a total of 180 mg of iron dextran was intraperitoneally injected into C57BL/6J mice (WT) and heterozygous ß-globin knockout mice ((mu)ß(th-3/+), BKO). The effects of deferiprone and deferoxamine in this model were investigated. The iron was distributed homogenously throughout the 4 liver lobes (left, caudate, right and median) and was present in hepatocytes, Kupffer cells and the sinusoidal space. Iron accumulation in phagocytic macrophages, recruitment of hepatic lymphocytes and nucleus membrane degeneration were observed as a result of iron overload in the WT and BKO mice. However, the expansion of hepatic extramedullary hematopoiesis was observed only in the BKO mice with iron overload. In the heart, the iron accumulated in the cardiac interstitium and myocytes, and moderate hypertrophy of the myocardial fibers and cardiac myocyte degeneration were observed. Although the total liver iron was not significantly altered by iron chelation therapy, image analysis demonstrated a difference in the efficacies of two iron chelators. The major site of chelation was the extracellular compartment, but treatment with deferiprone also resulted in intracellular iron chelation. Interestingly, iron chelators reversed the pathological changes resulting from iron overload in WT and BKO mice despite being used for only a short treatment period. We suggest that some of these effects may be secondary to the anti-inflammatory activity of the chelators.

Curcumin I mediates neuroprotective effect through attenuation of quinoprotein formation, p-p38 MAPK expression, and caspase-3 activation in 6-hydroxydopamine treated SH-SY5Y cells.

6-Hydroxydopamine (6-OHDA) selectively enters dopaminergic neurons and undergoes auto-oxidation resulting in the generation of reactive oxygen species and dopamine quinones, subsequently leading to apoptosis. This mechanism mimics the pathogenesis of Parkinson's disease and has been used to induce experimental Parkinsonism in both in vitro and in vivo systems. In this study, we investigated the effects of curcumin I (diferuloylmethane) purified from Curcuma longa on quinoprotein production, phosphorylation of p38 MAPK (p-p38), and caspase-3 activation in 6-OHDA-treated SH-SY5Y dopaminergic cells. Pretreatment of SH-SY5Y with curcumin I at concentrations of 1, 5, 10, and 20 µM, significantly decreased the formation of quinoprotein and reduced the levels of p-p38 and cleaved caspase-3 in a dose-dependent manner. Moreover, the levels of the dopaminergic neuron marker, phospho-tyrosine hydroxylase (p-TH), were also dose-dependently increased upon treatment with curcumin I. Our results clearly demonstrated that curcumin I protects neurons against oxidative damage, as shown by attenuation of p-p38 expression, caspase-3-activation, and toxic quinoprotein formation, together with the restoration of p-TH levels. This study provides evidence for the therapeutic potential of curcumin I in the chemoprevention of oxidative stress-related neurodegeneration.

Comparison of pharmacokinetics and urinary iron excretion of two single doses of deferiprone in ß-thalassemia/hemoglobin E patients.

Dose-related pharmacokinetics and urinary iron excretion (UIE) of an orally active iron chelator, deferiprone (L1), was investigated in 12 severe ß-thalassemia/hemoglobin E patients. The patients received two single doses of 25 and 50 mg/kg with a 2-week washout period. Deferiprone was rapidly absorbed and reached maximum concentration (C(max)) within 1 h after administration. Pharmacokinetic parameters including C(max) and area under concentration time curve from time zero to infinity (AUC(0-∞)) as well as urinary excretion of non-conjugated and glucuronide-conjugated deferiprone (L1 and L1-G) increased proportionally with the dose of deferiprone. A constant ratio of AUC(0-∞) of L1-G to L1 and a percentage of urinary excretion of L1-G indicated that increasing the dosage does not influence deferiprone biotransformation. Longer terminal elimination half-lifeand higher volume of distribution of L1 were observed with the high dose and correlated with deferiprone-chelated iron in serum. Unexpectedly, UIE did not show a linear relationship with the increased dose of deferiprone. The correlation between UIE and creatinine clearance suggested the possibility of L1-iron complex redistribution in patients with renal impairment treated with high-dose deferiprone.

Hepatic reduction of carbamoyl-PROXYL in ferric nitrilotriacetate induced iron overloaded mice: an in vivo ESR study.

Reduction of a nitroxyl radical, carbamoyl-PROXYL in association of free radical production and hepatic glutathione (GSH) was investigated in iron overloaded mice using an in vivo L-band electron spin resonance (ESR) spectrometer. Significant increases in hepatic iron, lipid peroxidation and decrease in hepatic GSH were observed in mice intraperitoneally (i.p.) administrated with ferric nitrilotriacetate (Fe(III)-NTA, a total 45 µmol/mouse over a period of 3 weeks). Free radical production in iron overloaded mice was evidenced by significantly enhanced rate constant of ESR signal decay of carbamoyl-PROXYL, which was slightly reduced by treatment with iron chelator, deferoxamine. Moreover, the rate constant of ESR signal decay was negatively correlated with hepatic GSH level (r=-0.586, p<0.001). On the other hand, hepatic GSH-depletion (>80%) in mice through daily i.p. injection and drinking water supplementation of L-buthionine-[S,R]-sulfoximine (BSO) significantly retarded ESR signal decay, while there were no changes in serum aspartate aminotransferase and liver thiobarbituric acid-reactive substances levels. In conclusion, GSH plays two distinguish roles on ESR signal decay of carbamoyl-PROXYL, as an antioxidant and as a reducing agent, dependently on its concentration. Therefore, it should be taken into account in the interpretation of free radical production in each specific experimental setting.

Caffeine potentiates methamphetamine-induced toxicity both in vitro and in vivo.

Ya-Ba, a combination of the two potent psychostimulants methamphetamine (METH) and caffeine (CAF), is commonly used by drug abusers in Thailand and neighboring countries. While the neurotoxic effects of METH are well documented, the toxicity of this combination is mostly unknown. This study aimed to elucidate the effects of this particular drug combination using both in vitro and in vivo models. We found that combined treatment of METH and CAF at individually non-toxic concentrations significantly decreased viability of human neuroblastoma SK-N-SH cells. The reduction in cell survival was accompanied by an increase in reactive oxygen species (ROS) formation and the Bax/Bcl-2 ratio. In vivo data showed that combined administration of METH and CAF increased the mortality rate of rats, with an increase in the level of thiobarbituric acid reactive substances (TBARS), the indicator of oxidative stress, in striatal tissues. The results indicate that caffeine potentiates the toxic effects of methamphetamine, possibly via a mechanism involving an increase in dopamine release and excess ROS generation.