Predicting response to cancer immunotherapy using noninvasive radiomic biomarkers.

INTRODUCTION: Immunotherapy is regarded as one of the major breakthroughs in cancer treatment. Despite its success, only a subset of patients responds-urging the quest for predictive biomarkers. We hypothesize that artificial intelligence (AI) algorithms can automatically quantify radiographic characteristics that are related to and may therefore act as noninvasive radiomic biomarkers for immunotherapy response. PATIENTS AND METHODS: In this study, we analyzed 1055 primary and metastatic lesions from 203 patients with advanced melanoma and non-small-cell lung cancer (NSCLC) undergoing anti-PD1 therapy. We carried out an AI-based characterization of each lesion on the pretreatment contrast-enhanced CT imaging data to develop and validate a noninvasive machine learning biomarker capable of distinguishing between immunotherapy responding and nonresponding. To define the biological basis of the radiographic biomarker, we carried out gene set enrichment analysis in an independent dataset of 262 NSCLC patients. RESULTS: The biomarker reached significant performance on NSCLC lesions (up to 0.83 AUC, P < 0.001) and borderline significant for melanoma lymph nodes (0.64 AUC, P = 0.05). Combining these lesion-wide predictions on a patient level, immunotherapy response could be predicted with an AUC of up to 0.76 for both cancer types (P < 0.001), resulting in a 1-year survival difference of 24% (P = 0.02). We found highly significant associations with pathways involved in mitosis, indicating a relationship between increased proliferative potential and preferential response to immunotherapy. CONCLUSIONS: These results indicate that radiographic characteristics of lesions on standard-of-care imaging may function as noninvasive biomarkers for response to immunotherapy, and may show utility for improved patient stratification in both neoadjuvant and palliative settings.

Anti-inflammatory effect of afatinib (an EGFR-TKI) on OGD-induced neuroinflammation.

Activated epidermal growth factor receptor (EGFR) has been proposed in the pathophysiology of neurodegenerative diseases. In the present study, the anti-inflammatory effect of afatinib, an EGFR-tyrosine kinase inhibitor (EGFR-TKIs) was investigated using CTX-TNA2 cells and primary cultured astrocytes subjected to oxygen/glucose deprivation (OGD). We found that OGD induced EGFR phosphorylation and activated subsequent signaling pathways, including phosphorylation of AKT and extracellular signal-regulated kinases (ERK). Afatinib blocked OGD-induced phosphorylation of EGFR, AKT and ERK. At the same time, afatinib attenuated OGD-induced elevations in glial fibrillary acidic protein (a biomarker of activated astrocytes) and proliferating cell nuclear antigen expression (a cell proliferating biomarker) as well as hypoxia-induced migratory ability. Furthermore, afatinib decreased OGD-induced increases in cyclooxygenase-II and inducible nitric oxide synthase expression of the treated astrocytes as well as NO content in the culture medium. Moreover, afatinib attenuated OGD-induced caspase 1 activation (a biomarker of inflammasome activation) and interleukin-1ß levels (a pro-inflammatory cytokine). Collectively, afatinib could block OGD-induced EGFR activation and its downstream signaling pathways in astrocytes. Moreover, afatinib attenuated OGD-induced astrocyte activation, proliferation and inflammasome activation. These data support the involvement of EGFR activation in neuroinflammation. Furthermore, EGFR-TKIs may be promising in inhibiting neuroinflammation in the CNS neurodegenerative diseases.

Melatonin Ameliorates Arsenite-Induced Neurotoxicity: Involvement of Autophagy and Mitochondria.

In the present study, the neuroprotective effect of melatonin on arsenite-induced neurotoxicity was investigated in rat primary cultured cortical neurons. Incubation of melatonin prevented arsenite-induced neuronal cell loss in a concentration-dependent manner. Furthermore, melatonin significantly attenuated arsenite-induced elevation in microtubule-associated protein light chain 3 (LC3)-II levels, a biomarker of autophagy. Our fluorescent staining assay showed that melatonin decreased arsenite-induced elevation of co-localized fluorescent puncta of monodansylcadaverine (a specific marker of autophagic vacuoles) and lysotracker red (a specific marker of lysosomes), indicating that melatonin is capable of inhibiting arsenite-induced autophagy and autolysosome formation. Because 3-methyladenine (an autophagic inhibitor) attenuated the arsenite-reduced α-synuclein levels (a protein essential for the neurite outgrowth and synaptic plasticity), melatonin via inhibiting autophagy attenuated the arsenite-reduced α-synuclein levels. At the same time, melatonin ameliorated the arsenite-induced reduction in growth associated protein 43 (a hallmark protein of neurite outgrowth) and discontinuous neurites of rat primary cultured cortical neurons. In addition, melatonin was found to prevent arsenite-induced decreases in cytochrome c oxidase levels (a biomarker of mitochondrial mass) and elevation in co-localized fluorescent puncta of autolysosomes and cytochrome c oxidase. Moreover, melatonin prevented arsenite-induced reduction in peroxisome proliferator-activated receptor gamma co-activator 1 α, a transcriptional co-activator of mitochondrial biosynthesis. Taken together, melatonin may exert its neuroprotective action via inhibiting arsenite-induced autophagy and enhancing mitochondrial biogenesis and thus restoring α-synuclein levels, neuronal integrity, and mitochondrial mass in rat primary cultured cortical neurons.

Thiol changes during epididymal maturation: a link to flagellar angulation in mouse spermatozoa?

Caput epididymal wild-type spermatozoa and cauda epididymal spermatozoa from mice null for the adenylyl cyclase Adcy10 gene are immotile unless stimulated by a membrane-permeant cyclic AMP analogue. Both types of spermatozoa exhibit flagellar angulation where the head folds back under these conditions. As sperm proteins undergo oxidation of sulfhydryl groups and the flagellum becomes more stable to external forces during epididymal transit, we hypothesized that ADCY10 is involved in a mechanism regulating flagellar stabilization. Although no differences were observed in global sulfhydryl status between caput and cauda epididymal spermatozoa from wild-type or Adcy10-null mice, two-dimensional fluorescence difference gel electrophoresis was performed to identify specific mouse sperm proteins containing sulfhydryl groups that became oxidized during epididymal maturation. A-kinase anchor protein 4, fatty acid-binding protein 9 (FABP9), glutathione S-transferase mu 5 and voltage-dependent anion channel 2 exhibited changes in thiol status between caput and cauda epididymal spermatozoa. The level and thiol status of each of these proteins were quantified in wild-type and Adcy10-null cauda epididymal spermatozoa. No differences in the abundance of any protein were observed; however, FABP9 in Adcy10-null cauda epididymal spermatozoa contained fewer disulfide bonds than wild-type sperm cells. In caput epididymal spermatozoa, FABP9 was detected in the cytoplasmic droplet, principal piece, midpiece, and non-acrosomal area of the head. However, in cauda epididymal spermatozoa, this protein localized to the perforatorium, post-acrosomal region and principal piece. Together, these results suggest that thiol changes during epididymal maturation have a role in the stabilization of the sperm flagellum.

Role of HO-1 in the arsenite-induced neurotoxicity in primary cultured cortical neurons.

In the present study, the role of heme oxygenase (HO)-1 in sodium arsenite (arsenite)-induced neurotoxicity was investigated using primary cultured cortical neurons. Incubation with arsenite was found to cause cell death of primary cultured cortical neurons in concentration- and time-dependent manners. Furthermore, arsenite induced caspase 3 activation and decreased procaspase 12 levels, indicating that apoptosis is involved in the arsenite-induced neurotoxicity. The oxidative mechanism underlying arsenite-induced neurotoxicity was investigated. Western blot assay showed that arsenite significantly increased HO-1 levels, a redox-regulated protein. Co-incubation with glutathione (10 mM) attenuated arsenite-induced HO-1 elevation and caspase 3 activation, suggesting that oxidative stress is involved in the arsenite-induced neurotoxicity. The neurotoxic effects of inorganic arsenics were compared; arsenite was more potent than arsenate in inducing HO-1 expression and caspase 3 activation. Moreover, the cell viabilities of arsenite and arsenate were 60 ± 2 and 99 ± 2 % of control, respectively. HO-1 siRNA transfection was employed to prevent arsenite-induced HO-1 elevation. At the same time, arsenite-induced caspase 3 activation and neuronal death were attenuated in the HO-1 siRNA-transfected cells. Taken together, HO-1 appears to be neuroprotective in the arsenite-induced neurotoxicity in primary cultured cortical neurons. In addition to antioxidants, HO-1 elevation may be a neuroprotective strategy for arsenite-induced neurotoxicity.

Anti-inflammatory effects of pioglitazone on iron-induced oxidative injury in the nigrostriatal dopaminergic system.

AIMS: Transition metals, oxidative stress and neuroinflammation have been proposed as part of a vicious cycle in central nervous system neurodegeneration. Our aim was to study the anti-inflammatory effect of pioglitazone, a peroxisome proliferative activated receptor-γ agonist, on iron-induced oxidative injury in rat brain. METHODS: Intranigral infusion of ferrous citrate (iron) was performed on anaesthetized rats. Pioglitazone (20 mg/kg) was orally administered. Oxidative injury was investigated by measuring lipid peroxidation in the substantia nigra (SN) and dopamine content in the striatum. Western blot assay and DNA fragmentation were employed to study the involvement of α-synuclein aggregation, neuroinflammation as well as activation of endoplasmic reticulum (ER) and mitochondrial pathways in iron-induced apoptosis. RESULTS: Intranigral infusion of iron time-dependently increased α-synuclein aggregation and haem oxygenase-1 levels. Furthermore, apoptosis was demonstrated by TUNEL-positive cells and DNA fragmentation in the iron-infused SN. Systemic pioglitazone was found to potentiate iron-induced elevation in nuclear peroxisome proliferative activated receptor-γ levels. However, pioglitazone inhibited iron-induced α-synuclein aggregation, elevations in interleukin-1ß and interleukin-6 mRNA levels as well as increases in oxygenase-1, cyclo-oxygenase II, nitric oxide synthase and ED-1 protein levels, an indicator of activated microglia. Moreover, iron-induced DNA laddering as well as activation of ER and mitochondrial pathways were attenuated by pioglitazone. In addition, pioglitazone decreased iron-induced elevation in lipid peroxidation in the infused SN and depletion in striatal dopamine level. CONCLUSIONS: Our results suggest that pioglitazone prevents iron-induced apoptosis via both ER and mitochondrial pathways. Furthermore, inhibition of α-synuclein aggregation and neuroinflammation may contribute to the pioglitazone-induced neuroprotection in central nervous system.

Role of autophagy in protection afforded by hypoxic preconditioning against MPP+-induced neurotoxicity in SH-SY5Y cells.

A sublethal preconditioning has been proposed as a neuroprotective strategy against several CNS neurodegenerative diseases. In this study, the involvement of autophagy in the protection provided by hypoxic preconditioning against 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity was studied in SH-SY5Y neuroblastoma cells. In contrast to the cytotoxicity of 0.1% oxygen, 1% oxygen hypoxia for 24h did not cause significant cell death. A transient increase in LC3-II level, a biomarker of autophagy, was demonstrated during hypoxic treatment. At the same time, 8-h hypoxia increased fluorescence due to monodansylcadaverine, a specific dye for autophagosomes, in the treated cells. Co-incubation with bafilomycin A1 (10 nM) further increased hypoxia-induced LC3-II levels but 3-methyladenine (3-MA; 10 mM) reduced the elevation in LC3-II levels induced by 8-h hypoxia. Moreover, 8-h hypoxia increased free radical formation and nuclear HIF-1alpha level. Glutathione was found to diminish hypoxia-induced LC3-II elevation. In contrast to the elevated LC3-II level, 8-h hypoxia significantly decreased mitochondrial mass. Furthermore, a rebound elevation in mitochondrial mass was observed under 8-h hypoxia and subsequent 12-h normoxia. Prior hypoxia attenuated the MPP(+)-induced elevation in LC3-II levels and cell death. Moreover, hypoxic pretreatment inhibited MPP(+)-induced activation of caspase-3 and DNA fragmentation. Co-incubation with 3-MA during hypoxia prevented the protection afforded by hypoxic preconditioning against MPP(+)-induced increases in LC3-II levels and neurotoxicity. Taken together, our results suggest that sublethal hypoxia induces autophagy that is mediated by oxidative stress. Furthermore, autophagy may be involved in the protection provided by hypoxic preconditioning against MPP(+)-induced neurotoxicity, indicating a neuroprotective role of autophagy in hypoxic preconditioning.

Iron-generated hydroxyl radicals kill retinal cells in vivo: effect of ferulic acid.

Siderosis bulbi is vision threatening. An investigation into its mechanisms and management is crucial. Experimental siderosis was established by intravitreous administration of an iron particle (chronic) or FeSO(4) (acute). After siderosis, there was a significant dose-responsive reduction in eletroretinogram (a/b-wave) amplitude, and an increase in OH level, greater when caused by 24 mM FeSO(4) than that by 8 mM FeSO(4). Furthermore, the FeSO(4)-induced oxidative stress was significantly blunted by 100 microM ferulic acid (FA). Siderosis also resulted in an excessive glutamate release, increased [Ca(++)](i), and enhanced superoxide dismutase immunoreactivity. The latter finding was consistent with the Western blot result. Obvious disorganization including loss of photoreceptor outer segments and cholinergic amacrines together with a wide-spreading ferric distribution across the retina was present, which were related to the eletro-retinographic and pathologic dysfunctions. Furthermore, b-wave reduction and amacrine damage were respectively, significantly, dose-dependently, and clearly ameliorated by FA. Thus, siderosis stimulates oxidative stress, and possibly, subsequent excitotoxicity, and calcium influx, which explains why the retina is impaired electro-physiologically and pathologically. Importantly, FA protects iron toxicity perhaps by acting as a free radical scavenger. This provides an approach to the study and treatment of the iron-related disorders such as retained intraocular iron and Alzheimer disease.

Arsenite-induced cytotoxicity in dorsal root ganglion explants.

Peripheral neuropathy is common in people chronically overexposed to arsenic. We studied sodium arsenite (arsenite)-induced cytotoxicity in dorsal root ganglion (DRG) explants. Incubation with arsenite concentration- and time-dependently increased the expression of stress proteins, heat shock protein 70, and heme oxygenase-1 in DRG explants. Furthermore, apoptosis was involved in the arsenite-induced cytotoxicity in the treated DRG. Elevation in cytosolic cytochrome c levels and reduction in procaspase 3 levels suggested an involvement of the mitochondrial pathway in arsenite-induced apoptosis in this preparation. At the same time, increases in the activating transcription factor-4 and C/EBP homologous protein and reduction in procaspase 12 levels indicated activation of the endoplasmic reticulum (ER) pathway in the arsenite-induced cytotoxicity in DRG explants. Salubrinal (30 microM), an ER inhibitor, was found to attenuate arsenite-induced DNA fragmentation and reduction in procaspase 12 in DRG explants. Cytotoxic effects by arsenite, sodium arsenate (arsenate), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were compared, and the potency was as follows: arsenite >>> arsenate>MMA and DMA. Recombinant adenovirus vectors encoding glial-cell-derived neurotrophic factor (AdGDNF) genes allowed a stable delivery of GDNF genes to the infected cells in DRG explants. Applied in this manner, AdGDNF was found to inhibit arsenite-induced DNA fragmentation in DRG explants. Moreover, AdGDNF attenuated the arsenite-induced reduction in procaspases 3 and 12 levels. Taken together, our study demonstrates that arsenite is capable of inducing cytotoxicity in DRG explants. Both ER and mitochondria pathways are involved in the arsenite-induced apoptosis in DRG explants. Glial-cell-derived neurotrophic factor appears to be protective against arsenite-induced peripheral neuropathy.

Hypoxic preconditioning attenuated in kainic acid-induced neurotoxicity in rat hippocampus.

The neuroprotective effect of hypoxic preconditioning on kainate (KA)-induced neurotoxicity, including apoptosis and necrosis, was investigated in rat hippocampus. Female Wistar-Kyoto rats were subjected to 380 mm Hg in an altitude chamber for 15 h/day for 28 days. Intrahippocampal infusion of KA was performed in chloral hydrate anesthetized rats, which acutely elevated 2,3-dihydroxybenzoic acid levels in normoxic rats. Seven days after the infusion, KA increased lipid peroxidation in the infused hippocampus and resulted in hippocampal CA3 neuronal loss. A 4-week hypoxic preconditioning attenuated KA-induced elevation in hydroxyl radical formation and lipid peroxidation as well as KA-induced neuronal loss. The effects of hypoxic preconditioning on KA-induced apoptosis and necrosis were investigated further. Two hours after KA infusion, cytosolic cytochrome c content was increased in the infused hippocampus. Twenty-four hours after KA infusion, pyknotic nuclei, cellular shrinkage, and cytoplasmic disintegration, but not TUNEL-positive staining, were observed in the CA3 region of hippocampus. Forty-eight hours after KA infusion, both DNA smear and DNA fragmentation were demonstrated in the infused hippocampus. Furthermore, TUNEL-positive cells, indicative of apoptosis, in the infused hippocampus were detected 72 h after KA infusion. Hypoxic pretreatment significantly reduced necrotic-like events in the KA-infused hippocampus. Moreover, hypoxic preconditioning attenuated apoptosis induced by KA infusion, including elevation in cytosolic cytochrome c content, TUNEL-positive cells, and DNA fragmentation. Our data suggest that hypoxic preconditioning may exert its neuroprotection of KA-induced oxidative injuries via attenuating both apoptosis and necrosis in rat hippocampus.

Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation.

The ability to culture CD34+ stem cells, while maintaining their pluripotency, is essential for manipulations such as gene transfection for therapeutic trials. Human peripheral blood (PB) CD34+ cells (> or = 90% purity) were cultured for up to 4 days in serum-free culture medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), with or without PIXY321 (IL-3/GM-CSF fusion protein) and human serum. The CD34 mean fluorescence intensity (MFI) and cell cycle status were evaluated daily using flow cytometry and hypotonic propidium iodide. Prior to culture (day 0), 97.0 +/- 0.9%, 1.9 +/- 0.3% and 1.0 +/- 0.6% of the selected CD34+ cells were in G0-G1, S-phase, or G2-M, respectively. After 2-4 days in culture with TPO/SCF/Flt-3L, there was an increase in the percent of cells in S-phase to 26.4 +/- 0.1% without significant loss of CD34 MFI. The addition of PIXY321 increased.the percentage of CD34+ cells in S-phase to 36.3 +/- 4.0%, but the CD34 MFI and numbers of CFU (colony-forming units) were significantly decreased at day 3 when cultured with PIXY321 or various recombinant cytokine combinations that included IL-3 and IL-6. There is an increase from day 0 to day 4 in the percentages of CD34+ with CD38-, HLA-DR-, and c-kit(low), but not Thy-1+ cells. Electroporation with EGFP reporter gene showed that 1-2 days of pre-stimulation in X-VIVO 10 supplemented with TPO/SCF/Flt-3L was necessary and sufficient for efficient transfection. Flow cytometry analysis demonstrated that 22% of the viable cells are CD34+/EGFP+ 48 h post electroporation. The introduced reporter gene appears to be stable as determined by EGFP+/LTC-IC (long-term colony-initiating cells), at 30-40 positive colonies (16 +/- 7%) per 1 x 10(5) electroporated CD34+ cells.

Lack of protective effect by intermittent hypoxia on MPTP-induced neurotoxicity in mice.

In the study we report here, several lines of evidence support the preventive action of intermittent hypoxia against oxidative injuries in CNS. Our in vitro data showed that autooxidation and iron-induced lipid peroxidation were attenuated in cortical homogenates of intermittent hypoxia-treated animals. Furthermore, our preliminary study found that iron induced oxidative injuries were abolished in rat brain after intermittent hypoxic treatment (paper submitted). Several antioxidative defensive systems improve in response to intermittent hypoxia. Since attenuation of autooxidation and iron-induced lipid peroxidation were observed in cortical homogenates of intermittent hypoxia-treated mice, the lack of prevention by intermittent hypoxia of MPTP-induced neurotoxicity may be due to the MPTP action that is not oxidative related. Together with our previous studies, in which several antioxidants were shown to successfully prevent oxidative injuries, our data here suggest that intermittent hypoxia may offer a potential treatment for preventing CNS degenerative diseases.

Coexistence of zinc and iron augmented oxidative injuries in the nigrostriatal dopaminergic system of SD rats.

Clinical studies have demonstrated an excess of transition metals, including zinc and iron, in the substantia nigra (SN) of Parkinson's patients. In the present study, the neurotoxic effect of zinc was investigated using iron as a positive control. Addition of zinc or iron to brain homogenates increased lipid peroxidation. Zinc was less potent than iron in inducing lipid peroxidation. Coincubation with desferrioxamine prevented zinc- and iron-induced lipid peroxidation. Furthermore, glutathione (GSH), S-nitroso-N-acetylpenicillamine, or melatonin inhibited zinc-induced lipid peroxidation. The oxidative effect of zinc was further investigated in anesthetized rats. Seven days after intranigral infusion of zinc, lipid peroxidation was elevated in the infused SN, and dopamine content and tyrosine hydroxylase-positive axons were decreased in the ipsilateral striatum. Zinc was less potent than iron in inducing neurodegeneration in vivo. L-Buthionine-[S,R]-sulfoximine pretreatment (i.c.v.), which depletes cellular GSH levels, enhanced zinc-induced oxidative injuries in the nigrostriatal dopaminergic system. Moreover, simultaneous infusion of zinc and iron appeared to augment oxidative injuries in rat brain. Taken together, our results demonstrate that intranigral infusion of zinc caused degeneration of the nigrostriatal dopaminergic system in rat brain. Furthermore, coexistence of zinc and iron augmented oxidative injuries in rat brain. These findings indicate that both zinc and iron contribute to the etiology of Parkinsonism.

Melatonin suppresses iron-induced neurodegeneration in rat brain.

The antioxidative action of melatonin on iron-induced neurodegeneration in the nigrostriatal dopaminergic system was evaluated in vivo. Intranigral infusion of iron chronically degenerated the dopaminergic transmission of the nigrostriatal system. An increase in lipid peroxidation in the infused substantia nigra and reductions in dopamine levels and dopaminergic terminals in the ipsilateral striatum were observed 7 d after iron infusion. Whereas local infusion of melatonin (60 microg/microl, 1 microl) alone did not alter dopaminergic transmission, coinfusion of melatonin with iron suppressed iron-induced oxidative damages. Systemic infusion of melatonin via osmotic pumps had no effect on iron-induced neurodegeneration. However, repetitive intraperitoneal injections of melatonin (10 mg/kg) prevented iron-induced oxidative injuries. The ratio of glutathione (GSH)/oxidized glutathione (GSSG) was moderately increased in the lesioned substantia nigra of the melatonin-treated rats compared to that of the lesioned group in control rats. The antioxidative effect of melatonin was verified in cortical homogenates. Melatonin dose-dependently suppressed autoxidation and iron-induced lipid peroxidation. Melatonin was as effective as GSH and was less effective than Trolox (a water-soluble analogue of vitamin E) in inhibiting iron-elevated lipid peroxidation of brain homogenates. Our data suggest that melatonin is capable of at least partially preventing the iron-induced neurodegeneration in the nigrostriatal dopaminergic system.

Recovery by NO of the iron-attenuated dopamine dynamics in nigrostriatal system of rat brain.

In vivo electrochemical detection was employed to study the chronic effect of nitric oxide on iron-induced alterations in dopamine dynamics, including K+ -evoked dopamine overflows and the clearance of exogenous dopamine in rat striatum. Intranigral infusion of fresh S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, did not alter either dopamine dynamics in the striatum or the lipid peroxidation in substantia nigra 7 days after the infusion, indicating that nitric oxide is not neurodestructive. By contrast, infusion of iron in substantia nigra chronically degenerated the nigrostriatal dopaminergic system. Co-infusion of iron and fresh SNAP, but not aged SNAP, prevented the iron-induced reductions in K+ -evoked dopamine overflows. Furthermore, the clearance of exogenous dopamine was attenuated in the striatum ipsilateral to the substantia nigra infused with iron. An improvement by fresh SNAP of iron-induced reduction in dopamine clearance was observed in rats co-infused with fresh SNAP and iron mixture compared to iron-lesioned group. Taken together, our in vivo electrochemical study suggests that nitric oxide does not alter dopamine dynamics in nigrostriatal dopaminergic system. Rather, nitric oxide appears to protect dopamine dynamics from iron-induced oxidative stress in rat brain.

Carboxyfullerene prevents iron-induced oxidative stress in rat brain.

Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, was investigated as a protective agent against iron-induced oxidative stress in the nigrostriatal dopaminergic system of anesthetized rats. Intranigral infusion of exclusive carboxyfullerene did not increase lipid peroxidation in substantia nigra or deplete dopamine content in striatum. Infusion of ferrous citrate (iron II) induced degeneration of the nigrostriatal dopaminergic system. An increase in lipid peroxidation in substantia nigra as well as decreases in K+-evoked dopamine overflow and dopamine content in striatum were observed 7 days after the infusion. Co-infusion of carboxyfullerene prevented iron-induced oxidative injury. Furthermore, tyrosine hydroxylase-immunoreactive staining showed that carboxyfullerene inhibited the iron-induced loss of the dopaminergic nerve terminals in striatum. The antioxidative action of carboxyfullerene was verified by in vitro studies. Incubation of brain homogenates increased the formation of the Schiff base fluorescent products of malonaldehyde, an indicator of lipid peroxidation. Both autooxidation (without exogenous iron) and iron-induced elevation of lipid peroxidation of brain homogenates were suppressed by carboxyfullerene in a dose-dependent manner. Our results suggest that intranigral infusion of carboxyfullerene appears to be nontoxic to the nigrostriatal dopaminergic system. Furthermore, the potent antioxidative action of carboxyfullerene protects the nigrostriatal dopaminergic system from iron-induced oxidative injury.

The antioxidative effect of carboxyfullerenes (C3/D3) on iron-induced oxidative injury in CNS.

Carboxyfullerenes, including two regioisomers C3 and D3, were investigated as antioxidants against iron-induced oxidative stress in vivo and in vitro. Both C3 and D3 dose-dependently inhibited autoxidation and iron-elevated lipid peroxidation in cortical homogenates. The antioxidative property of C3 was compared to Trolox (a water-soluble analogue of vitamin E) and glutathione. C3 was more effective than glutathione but was less effective than Trolox in inhibiting iron-induced elevation in lipid peroxidation. In urethane-anesthetized rats, intranigral infusion of iron degenerated the nigrostriatal dopaminergic system, including as elevation in lipid peroxidation in the infused substantia nigra (SN) and reductions in K(+)-evoked dopamine overflow and dopamine content in the ipsilateral striatum 7 days after the infusion. Local application of iron with C3 or D3 prevented iron-induced oxidative injuries. Our data suggest that carboxyfullerenes have a neuroprotective effect in preventing iron-induced oxidative injury in CNS.

Role of dopamine uptake in NMDA-modulated K(+)-evoked dopamine overflow in rat striatum: an in vivo electrochemical study.

An involvement of dopamine uptake in the N-methyl-D-aspartate (NMDA)-modulated dopaminergic transmission in rat striatum was studied using the technique of in vivo electrochemical detection. Microinjection of potassium (K+) evoked dopamine overflows from the dopamine-containing nerve terminals in the striatum. While application of NMDA did not evoke any dopamine overflow, co-application of NMDA and K+ induced larger dopamine overflows than those by K+ alone. Furthermore, dynamic analysis showed that the rate of clearance (Tc) was reduced by NMDA. Indeed, our uptake study demonstrated an NMDA-induced inhibition of dopamine clearance. The time course of electrochemical signals evoked by microinjection of exogenous dopamine was increased and Tc was reduced following NMDA application. In order to delineate the effects of NMDA on K(+)-evoked dopamine overflows and/or on dopamine uptake, nomifensine, a dopamine uptake inhibitor was used. Application of nomifensine potentiated K(+)-evoked dopamine overflows. Co-administration of NMDA further augmented dopamine overflows by the K+ and nomifensine mixture. Taken together, our data suggest that NMDA concomitantly potentiated dopamine overflows in response to depolarizing stimuli and attenuated dopamine uptake. The increment by NMDA of K(+)-evoked dopamine overflows may partially result from an attenuated dopamine uptake in rat striatum.

Striatal dopamine dynamics are altered following an intranigral infusion of iron in adult rats.

In vivo electrochemical detection was employed to examine iron-induced oxidative injury on dopamine dynamics in the nigrostriatal system of urethane-anesthetized rats. Seven days after an intranigral infusion of iron, oxidative stress was confirmed by an elevated lipid peroxidation in lesioned substantia nigra (SN). Local application of potassium (K+) evoked dopamine releases from the dopamine-containing nerve terminals in the striatum. Both amplitude and rate of clearance (Tc) of evoked dopamine releases were lower in striatum with lesioned SN; however, the time course parameters of dopamine release in the lesioned group were not different from those of the intact group, indicating a reduction in dopamine clearance. Indeed, iron-induced oxidative stress attenuated the effect of nomifensine, a high-affinity dopamine uptake blocker, on dopamine clearance. In striatum with intact SN, the amplitude and time course parameters of signals by exogenous dopamine were increased and Tc was decreased by nomifensine. In contrast, nomifensine did not significantly alter the dopamine signals of the lesioned group. Taken together, in addition to the increased lipid peroxidation in SN, our in vivo electrochemical data demonstrates that iron-induced oxidative injury attenuates K+-evoked dopamine release and dopamine uptake in the ipsilateral striatum. The diminished nomifensine effect implies a lack of high-affinity dopamine uptake sites.