Food and Food Waste Antioxidants: Could They Be a Potent Defence against Parkinson's Disease?

Oxidative stress, an imbalance between reactive oxygen species (ROS) and endogenous antioxidants, plays an important role in the development of neurodegenerative diseases, including Parkinson's. The human brain is vulnerable to oxidative stress because of the high rate of oxygen that it needs and the high levels of polyunsaturated fatty acids, which are substrates of lipid peroxidation. Natural antioxidants inhibit oxidation and reduce oxidative stress, preventing cancer, inflammation, and neurodegenerative disorders. Furthermore, in the literature, it is reported that antioxidants, due to their possible neuroprotective activity, may offer an interesting option for better symptom management, even Parkinson's disease (PD). Natural antioxidants are usually found in several foods, such as fruits, vegetables, meat, fish, and oil, and in food wastes, such as seeds, peels, leaves, and skin. They can help the system of endogenous antioxidants, protect or repair cellular components from oxidative stress, and even halt lipid, protein, and DNA damage to neurons. This review will examine the extent of knowledge from the last ten years, about the neuroprotective potential effect of natural antioxidants present in food and food by-products, in in vivo and in vitro PD models. Additionally, this study will demonstrate that the pool of dietary antioxidants may be an important tool in the prevention of PD and an opportunity for cost savings in the public health area.

Review Article a Multidisciplinary Evaluation for Advanced Supportive Care of Breast Cancer Patients.

Background: Cancer cases diagnosed each year are increasing, mainly because the population is ageing and, in part, due to early detection. This implies that there are more and more persons that receive medical anticancer therapies and that are interested in maintaining their quality of life. Many oncological treatments, including chemotherapy, immunotherapy, surgery, and radiotherapy, and combined therapy are associated with cutaneous toxicity and long-term side effects to different tissues and organs. This is particularly relevant when new therapies are used since these may cause new and unexpected side effects that may be short-lived but, in some cases, may become chronic or permanent. Patients often seek advice with their oncologists on what can be done and what cannot be done. Notably, many of the cutaneous side effects can be prevented or reduced by adequate interventions. Summary: The aim of this review is to highlight how oncological patients may benefit from a closer collaboration between specialists in different branches. We will focus on women with breast cancer since we think that they may derive a special benefit from this collaboration, but we will analyse other cancers in future papers. Key Messages: The working group was created to help the medical doctor in the prevention and management of all the adverse effects of the oncological treatments, supporting patients in this phase of their life, including nutritional assessment and dietary support.

Antioxidant, Anti-Inflammatory and Anti-Proliferative Properties of Stachys circinata on HepG2 and MCF7 Cells.

According to the WHO, the overall age-standardized cancer rate keeps declining, and the number of cases diagnosed each year increases, remaining among the leading causes of death in 91 out of 172 recorded countries. In this context, novel cancer prediction and therapeutic protocols are compulsory. The effect of a Stachys circinata L'Hér dichloromethane extract (ScDME) on cell redox homeostasis and tumor proliferation was investigated. HepG2 cell feedback mechanisms to oxidative stress exposure were evaluated by determining catalase (CAT) and reduced glutathione (GSH), following the supply with ScDME (0.0-5.7 µg/µL). Cytotoxicity of ScDME against the human umbilical vein endothelial cell (HUVEC) and two human cancer cell lines (breast: MCF7; liver: HepG2) was evaluated by the MTT assay. H2O2-stressed HepG2 cells supplied with the S. circinata extracts exhibited significantly increased CAT and GSH activity as compared to unsupplied ones. The anti-inflammatory activity of the extracts was evaluated by real time-qPCR on IL-1, IL-6 and TNF-α expression. As a result, this research points out that S. circinata dichloromethane extract owns anti-inflammatory and anti-proliferative properties against MCF7 and HepG2 cells and activates CAT and GSH of the HepG2 cells' antioxidant enzyme system.

Antioxidant, Antimicrobial, and Other Biological Properties of Pompia Juice.

Pompia is a Citrus species belonging to Sardinian endemic biodiversity. Health benefits were attributed to its flavedo rind extracts and essential oils while the juice qualities have never been investigated. In this paper, the antioxidant, antimicrobial, and other biological properties of Pompia juice were studied. A combined LCMS/electrochemical/biological approach was used to clarify a still debated phylogeny of this species and to explain the role of its juice phenolic compounds. A closer phylogenetic relationship with lemon and citron, rather than oranges was suggested. Sensors-based electrochemical measures, together with LCMS qualitative and quantitative analyses, revealed a high contribution of ascorbic acid and phenolics with low redox potential, isorhamnetin 3-O-rutinoside, diosmin, and diosmetin 6,8-diglucoside, to antioxidant capacity. The biological assays demonstrated a marked effect of low concentration of Pompia juice against reactive oxygen species (ROS) starting from 50 µg mL-1, and a moderate capacity to reduce ROS damages on cell membrane. Treatments with Pompia juice also resulted in a significant reduction (20%) of the metabolic activity of SW48 colon cancer cells. Lastly, MIC, MBC, and MBIC antimicrobial assays demonstrated that Pompia and lemon juices have inhibitory and antibiofilm effects against the pathogenic bacteria Pseudomonas aeruginosa, Streptococcus aureus, and Enterococcus faecalis.

Synthesis and Studies of the Inhibitory Effect of Hydroxylated Phenylpropanoids and Biphenols Derivatives on Tyrosinase and Laccase Enzymes.

The impaired activity of tyrosinase and laccase can provoke serious concerns in the life cycles of mammals, insects and microorganisms. Investigation of inhibitors of these two enzymes may lead to the discovery of whitening agents, medicinal products, anti-browning substances and compounds for controlling harmful insects and bacteria. A small collection of novel reversible tyrosinase and laccase inhibitors with a phenylpropanoid and hydroxylated biphenyl core was prepared using naturally occurring compounds and their activity was measured by spectrophotometric and electrochemical assays. Biosensors based on tyrosinase and laccase enzymes were constructed and used to detect the type of protein-ligand interaction and half maximal inhibitory concentration (IC50). Most of the inhibitors showed an IC50 in a range of 20-423 nM for tyrosinase and 23-2619 nM for laccase. Due to the safety concerns of conventional tyrosinase and laccase inhibitors, the viability of the new compounds was assayed on PC12 cells, four of which showed a viability of roughly 80% at 40 µM. In silico studies on the crystal structure of laccase enzyme identified a hydroxylated biphenyl bearing a prenylated chain as the lead structure, which activated strong and effective interactions at the active site of the enzyme. These data were confirmed by in vivo experiments performed on the insect model Tenebrio molitur.

Real-time telemetry monitoring of oxygen in the central complex of freely-walking Gromphadorhina portentosa.

A new telemetric system for the electrochemical monitoring of dissolved oxygen is showed. The device, connected with two amperometric sensors, has been successfully applied to the wireless detection of the extracellular oxygen in the central complex of freely-walking Gromphadorhina portentosa. The unit was composed of a potentiostat, a two-channel sensor conditioning circuit, a microprocessor module, and a wireless serial transceiver. The amperometric signals were digitalized and sent to a notebook using a 2.4 GHz transceiver while a serial-to-USB converter was connected to a second transceiver for completing the communication bridge. The software, running on the laptop, allowed to save and graph the oxygen signals. The electronics showed excellent stability and the acquired data was linear in a range comprised between 0 and -165 nA, covering the entire range of oxygen concentrations. A series of experiments were performed to explore the dynamics of dissolved oxygen by exposing the animals to different gases (nitrogen, oxygen and carbon dioxide), to low temperature and anesthetic agents (chloroform and triethylamine). The resulting data are in agreement with previous O2 changes recorded in the brain of awake rats and mice. The proposed system, based on simple and inexpensive components, can constitute a new experimental model for the exploration of central complex neurochemistry and it can also work with oxidizing sensors and amperometric biosensors.

Targeted Therapies in Cholangiocarcinoma: Emerging Evidence from Clinical Trials.

Cholangiocarcinoma (CCA) is a highly-aggressive malignancy arising from the biliary tree, characterized by a steady increase in incidence globally and a high mortality rate. Most CCAs are diagnosed in the advanced and metastatic phases of the disease, due to the paucity of signs and symptoms in the early stages. This fact, along with the poor results of the local and systemic therapies currently employed, is responsible for the poor outcome of CCA patients and strongly supports the need for novel therapeutic agents and strategies. In recent years, the introduction of next-generation sequencing technologies has opened new horizons for a better understanding of the genetic pathophysiology of CCA and, consequently, for the identification and evaluation of new treatments tailored to the molecular features or alterations progressively elucidated. In this review article, we describe the potential targets under investigation and the current molecular therapies employed in biliary tract cancers. In addition, we summarize the main drugs against CCA under evaluation in ongoing trials and describe the preliminary data coming from these pioneering studies.

Enzyme-Based Electrochemical Biosensor for Therapeutic Drug Monitoring of Anticancer Drug Irinotecan.

Therapeutic drug monitoring (TDM) is the clinical practice of measuring pharmaceutical drug concentrations in patients' biofluids at designated intervals, thus allowing a close and timely control of their dosage. To date, TDM in oncology can only be performed by trained personnel in centralized laboratories and core facilities employing conventional analytical techniques (e.g., MS). CPT-11 is an antineoplastic drug that inhibits topoisomerase type I, causing cell death, and is widely used in the treatment of colorectal cancer. CPT-11 was also found to directly inhibit acetylcholine esterase (AChE), an enzyme involved in neuromuscular junction. In this work, we describe an enzymatic biosensor, based on AChE and choline oxidase (ChOx), which can quantify CPT-11. ACh (acetylcholine) substrate is converted to choline, which is subsequently metabolized by ChOx to give betaine aldehyde and hydrogen peroxide. The latter one is then oxidized at a suitably polarized platinum electrode, providing a current transient proportional to the amount of ACh. Such an enzymatic process is hampered by CPT-11. The biosensor showed a ∼60% maximal inhibition toward AChE activity in the clinically relevant concentration range 10-10 000 ng/mL of CPT-11 in both simple (phosphate buffer) and complex (fetal bovine serum) matrixes, while its metabolites showed negligible effects. These findings could open new routes toward a real-time TDM in oncology, thus improving the therapeutic treatments and lowering the related costs.

The role of molecular breast imaging in predicting complete tumor response to treatment and residual tumor extent following neoadjuvant therapy.

The aim of the present study was to investigate the usefulness of molecular breast imaging (MBI) in predicting complete tumor response to treatment and residual tumor extent following neoadjuvant therapy. A consecutive series of 43 female patients with large or locally advanced primary breast cancer, scheduled for surgery following neoadjuvant therapy, was retrospectively reviewed. Prior to surgery, all patients underwent MBI using a high­resolution semiconductor­based device for image acquisition. MBI data were correlated with surgical histopathological findings. Spearman's correlation coefficient was calculated to assess differences in residual tumor size with MBI and histopathological examination. From the images obtained using MBI, 7 patients were negative for residual tumors with pathological complete response (specificity, 100%) and positive in 34/36 patients with residual disease (sensitivity, 94.4%), including 26/27 patients with unifocal and 8/9 patients with multicentric/multifocal tumors, 5 of which exhibited multiple microscopic foci scattered in a fibrotic area. Overall accuracy was 95.3% and the positive predictive value (PPV) and negative predictive value (NPV) were 100 and 77.8%, respectively. MBI was false­negative in one patient with a 2.5­cm invasive ductal carcinoma located close to the chest wall and in one patient with microscopic foci of epithelial carcinoma. In the patients with unifocal residual tumors, correlation of tumor size between MBI and histopathology was r=0.981 (P<0.0001); however, MBI overestimated the number of lesions in one of these cases. In the patients with multifocal/multicentric tumors, MBI adequately assessed residual tumor extent in 5/8 positive cases, overestimating the number of lesions in one case and underestimating tumor extent in 2 further cases with microscopic foci scattered in a fibrotic area. MBI proved to be a highly accurate diagnostic tool in predicting complete tumor response to neoadjuvant therapy and residual tumor extent, correlating with surgical histopathological findings in 86% of overall cases. A positive result was always associated with the presence of residual disease and MBI tumor size was strongly correlated with histopathological analysis mainly in unifocal residual tumors.

Synthesis of magnolol and honokiol derivatives and their effect against hepatocarcinoma cells.

The hepatocellular carcinoma is one of the most common malignant tumour with high level of mortality rate due to its rapid progression and high resistance to conventional chemotherapies. Thus, the search for novel therapeutic leads is of global interest. Herein, a small set of derivatives of magnolol 1 and honokiol 2, the main components of Magnolia grandiflora and Magnolia obovata, were evaluated in in vitro assay using tumoral hepatocytes. The pro-drug approach was applied as versatile strategy to the improve bioactivity of the compounds by careful transformation of the hydroxyl groups of magnolol 1 and honokiol 2 in suitable ester derivatives. Compounds 10 and 11 resulted to be more potent than the parental honokiol 2 at concentration down to 1 µM with complete viability of treated fibroblast cells up to concentrations of 80 µM. The combination of a butyrate ester and a bare phenol-OH group in the honokiol structure seemed to play a significant role in the antiproliferative activity identifying an interesting pharmacological clue against hepatocellular carcinoma.

Microdialysis as a New Technique for Extracting Phenolic Compounds from Extra Virgin Olive Oil.

The amount and composition of the phenolic components play a major role in determining the quality of olive oil. The traditional liquid-liquid extraction (LLE) method requires a time-consuming sample preparation to obtain the "phenolic profile" of extra virgin olive oil (EVOO). This study aimed to develop a microdialysis extraction (MDE) as an alternative to the LLE method to evaluate the phenolic components of EVOO. To this purpose, a microdialysis device and dialysis procedure were developed. "Dynamic-oil" microdialysis was performed using an extracting solution (80:20 methanol/water) flow rate of 2 µL min-1 and a constant EVOO stream of 4 µL min-1. The results indicated a strong positive correlation between MDE and the LLE method, providing a very similar phenolic profile obtained with traditional LLE. In conclusion, the MDE approach, easier and quicker in comparison to LLE, provided a reliable procedure to determine the phenolic components used as a marker of the quality and traceability of EVOO.

Real-time monitoring of glucose and phenols intestinal absorption through an integrated Caco-2TC7cells/biosensors telemetric device: Hypoglycemic effect of fruit phytochemicals.

An integrated device for real-time monitoring of glucose and phenols absorption, that consists of a sensors/biosensors system (SB) and a Caco-2TC7 human intestinal cell culture, is described in this study. The SB is composed of a glucose oxidase-based biosensor, a sentinel platinum sensor, a laccase/tyrosinase-based biosensor and a sentinel carbon sensor, all located in the basolateral compartment (BC) of a cell culture plate. Caco-2TC7 cells, differentiated on culture inserts, separated the apical compartment that simulates the intestinal lumen, from the BC which represented the bloodstream. The system recorded currents relative to glucose (1mM) absorption, obtaining bioavailability values (5.1%) comparable to HPLC analysis (4.8%). Phloridzin and phloretin, specific phenolic inhibitors of SGLT1 and GLUT2 glucose transporters, reduced the glucose transport of almost 10 times. They were minimally absorbed in the BC with a bioavailability of 0.13% and 0.49% respectively. The hypoglycemic potential of blueberry and pomegranate juices was also studied. In particular, the amount of glucose absorbed through the Caco-2TC7 monolayer was 8‰ for pomegranate and 1.7‰ for blueberry, demonstrating the potential hypoglycemic effect of the juices. Polyphenols absorption was also monitored by the SB and an increase was recorded during the first 50min in presence of both blueberry and pomegranate juices, then a constant decrease occurred. The proposed device has been developed as innovative tool for the dynamic monitoring of natural compounds effects on glucose absorption, in order to manage postprandial hyperglycemia.

Neurological morphofunctional differentiation induced by REAC technology in PC12. A neuro protective model for Parkinson's disease.

Research for the use of physical means, in order to induce cell differentiation for new therapeutic strategies, is one of the most interesting challenges in the field of regenerative medicine, and then in the treatment of neurodegenerative diseases, Parkinson's disease (PD) included. The aim of this work is to verify the effect of the radio electric asymmetric conveyer (REAC) technology on the PC12 rat adrenal pheochromocytoma cell line, as they display metabolic features of PD. PC12 cells were cultured with a REAC regenerative tissue optimization treatment (TO-RGN) for a period ranging between 24 and 192 hours. Gene expression analysis of specific neurogenic genes, as neurogenin-1, beta3-tubulin and Nerve growth factor, together with the immunostaining analysis of the specific neuronal protein beta3-tubulin and tyrosine hydroxylase, shows that the number of cells committed toward the neurogenic phenotype was significantly higher in REAC treated cultures, as compared to control untreated cells. Moreover, MTT and Trypan blue proliferation assays highlighted that cell proliferation was significantly reduced in REAC TO-RGN treated cells. These results open new perspectives in neurodegenerative diseases treatment, particularly in PD. Further studies will be needed to better address the therapeutic potential of the REAC technology.

Simultaneous amperometric detection of ascorbic acid and antioxidant capacity in orange, blueberry and kiwi juice, by a telemetric system coupled with a fullerene- or nanotubes-modified ascorbate subtractive biosensor.

Four fullerenes- or nanotubes-modified graphite sensor-biosensor systems (SBs), coupled with a dual-channel telemetric device, based on an ascorbate oxidase (AOx) biosensor, were developed for on line simultaneous amperometric detection of ascorbic acid (AA) and antioxidant capacity in blueberry, kiwi and orange juice. Fullerene C60 (FC60), fullerene C70 (FC70), single-walled carbon nanotubes (SWCN) and multi-walled carbon nanotubes (MWCN) increased the sensitivity of graphite toward AA and phenols 1.2, 1.5, 5.1 and 5.1 times respectively. Fullerenes combined with AOx improved the selectivity toward AA more than nanotubes, being able to hold a higher number of AOx molecules on the biosensor surface. The SBs work at an applied potential of +500 mV, in a concentration range between the LOD and 20 µM, with a response time of two minutes. The LOD is 0.10, 0.13, 0.20 and 0.22 µM for SBs modified with FC60, FC70, SWCN and MWCN respectively. Biosensors register lower AA currents than the sensors due to the enzyme capability to oxidize AA before it reaches the transductor surface. Phenols currents registered by sensors and biosensors did not differ. Based on the difference between sensor and biosensor recorded currents a AA selectivity index was developed as an indicator of specificity toward AA and of the capacity to distinguish between AA and phenols contribution to the antioxidant capacity. This value is almost zero for fullerene-modified SBs, 0.13 and 0.22 for SWCN- and MWCN-modified SBs respectively. The results of juices analysis performed with SBs were in accordance with reference methods.

Development and characterization of an ascorbate oxidase-based sensor-biosensor system for telemetric detection of AA and antioxidant capacity in fresh orange juice.

A new carbon ascorbate oxidase-based sensor-biosensor system (SB) was coupled to a dual-channel telemetric device for online simultaneous electrochemical detection of ascorbic acid (AA) and antioxidant capacity in Hamlin, Sanguinello, and Moro orange varieties. The electrocatalytic performances of the SB were investigated by cyclic voltammetry and amperometric techniques. The phenol composition of orange juice of each variety, and the cyclic voltammetries of the most represented phenols, were provided. The in vitro calibrations were performed in PBS (pH 5.6), applying a constant potential of +500 mV. A standard mixture of phenols, based on orange juice composition, was used as reference material for studying SB behavior. SB works at an applied potential of +500 mV, in a concentration range comprised between the LOD 0.26 µM and 20 µM. In this concentration range, limiting the data acquisition time to 2 min, the problems of electrode passivation due to phenols polymerization were overcome. AA calibration showed that the biosensor registered statistically lower currents than the sensor since the enzyme oxidized AA before it reached the electrode surface. Standard mixture calibration showed that currents registered by sensor and biosensor did not statistically differ. The difference between sensor and biosensor AA registered currents was used to calculate an AA selectivity index and, consequently, to determine the AA content and the antioxidant capacity in the juices. The novelty of the SB is its ability to distinguish between AA and phenols contribution to antioxidant capacity. The obtained results were in accordance with reference methods.

LRRK2 affects vesicle trafficking, neurotransmitter extracellular level and membrane receptor localization.

The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2(G2019S) pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2(G2019S) shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.

The MPTP mouse model: cues on DA release and neural stem cell restorative role.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to cause parkinsonism in humans and this fact is a major incentive for using this toxin as an animal model to study the pathogenesis of Parkinson's disease (PD). Although the monkey MPTP model remains the best, most studies have been performed in mice. The so-called acute and sub-acute regimens are commonly used. Both induce tissue striatal dopamine (DA) depletion and nigral neuron death. Tissue striatal DA depletion does not necessarily correlate with impairment of striatal dopaminergic functioning. In freely moving mice, systemic acute or sub-acute MPTP directly induces prolonged release of striatal DA. Such DA release may be considered the first step in MPTP-induced striatal DA depletion. Reportedly, neural stem cells improve symptoms in the MPTP model of PD by interacting with the MPTP-induced pathological nigrostriatal milieu.

Endogenous melatonin protects L-DOPA from autoxidation in the striatal extracellular compartment of the freely moving rat: potential implication for long-term L-DOPA therapy in Parkinson's disease.

We previously showed, using microdialysis, that autoxidation of exogenous L-dihydroxyphenylalanine (L-DOPA) occurs in vivo in the extracellular compartment of the freely moving rat, with a consequent formation of L-DOPA semiquinone (L-DOPA-SQ). In the present study, intrastriatal infusion of L-DOPA (1.0 microm for 200 min) increased dialysate L-DOPA concentrations (maximum increases up to 116-fold baseline values); moreover, L-DOPA-SQ was detected in dialysates. Individual dialysate concentrations of L-DOPA were negatively correlated with those of L-DOPA-SQ. Co-infusion of N-acetylcysteine (100 microm) or melatonin (50 microm) increased L-DOPA (up to 151- and 246-fold, respectively) and decreased L-DOPA-SQ (by about 53% and 87%, respectively) dialysate concentrations. Systemic L-DOPA [25 mg/kg intraperitoneally (i.p.) twice in a 12-h interval] significantly increased striatal baseline dialysate concentrations of L-DOPA and decreased dopamine (DA) and ascorbic acid (AsAc) concentrations, when compared with controls. Following systemic L-DOPA, L-DOPA-SQ was detected in dialysates. Endogenous melatonin was depleted in rats maintained on a 24-h light cycle for 1 wk. In melatonin-depleted rats, systemic L-DOPA induced a smaller increase in dialysate L-DOPA, a greater increase in L-DOPA-SQ formation, and a greater reduction in DA and AsAc dialysate concentrations. Co-administration of melatonin (5.0 mg/kg, i.p., twice in a 12-h interval) with L-DOPA, in control as well as in light-exposed rats, significantly increased dialysate L-DOPA concentrations, greatly inhibited L-DOPA-SQ formation, and restored up to the control values dialysate DA and AsAc concentrations. These findings demonstrate that endogenous melatonin protects exogenous L-DOPA from autoxidation in the extracellular compartment of the striatum of freely moving rats; moreover, systemic co-administration of melatonin with L-DOPA markedly increases striatal L-DOPA bioavailability in control as well as in melatonin-depleted rats. These results may be of relevance to the long-term L-DOPA therapy of Parkinson's disease.

Role of endogenous melatonin in the oxidative homeostasis of the extracellular striatal compartment: a microdialysis study in PC12 cells in vitro and in the striatum of freely moving rats.

A capillary apparatus for in vitro microdialysis was used to investigate melatonin and ascorbic acid effects on dopamine (DA) autoxidation or nitric oxide (NO)-mediated oxidation in suspended PC12 cells. Following high K+ (KCl 75 mm) infusion, secreted DA underwent a partial autoxidation or peroxynitrite-mediated oxidation when the potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1, 1.0 mm) was co-infused with KCl. Ascorbic acid was supplied to the medium by means of intracellular reduction of infused dehydroascorbic acid (DHAA) (5.0 mm). Melatonin (50 microm) and DHAA showed a synergistic effect in inhibiting DA autoxidation and peroxynitrite-mediated DA oxidation. Moreover, melatonin increased dialysate recovery of ascorbic acid released from PC12 cells. Endogenous melatonin was depleted in rats maintained on a 24-hr light cycle for 1 wk. In melatonin-depleted rats, baseline levels of dialysate ascorbic acid were lower than controls, while those of DA were unaffected. In these rats, intrastriatal infusion of 5.0 mm SIN-1 induced DA increases significantly lower than in controls; in addition, dialysate ascorbic acid concentrations exhibited significant decreases. Melatonin co-infusion restored SIN-1 effects on dialysate DA and antagonized SIN-1-induced ascorbic acid decreases. Melatonin-depleted rats were allowed to recover. In these rats, striatal baseline ascorbic acid, as well as SIN-1-induced increases in dialysate DA did not differ from controls. Taken together, these findings suggest that endogenous melatonin is an active component of the striatal extracellular antioxidant pool, as it maintains endogenous ascorbic acid in its reduced status and co-operates with ascorbic acid in protecting extracellular DA from exogenous NO-mediated oxidation.

Signaling pathways in the nitric oxide and iron-induced dopamine release in the striatum of freely moving rats: role of extracellular Ca2+ and L-type Ca2+ channels.

We showed previously that exogenous iron potentiated nitric oxide (NO) donor-induced release of striatal dopamine (DA) in freely moving rats, using microdialysis. In this study, the increase in dialysate DA induced by intrastriatal infusion of the NO-donor 3-morpholinosydnonimine (SIN-1, 1.0 mM for 180 min) was scarcely affected by Ca2+ omission. N-methyl-d-glucamine dithiocarbamate (MGD) is a thiol compound whose NO trapping activity is potentiated by iron(II). Intrastriatal co-infusion of MGD either alone or associated with iron(II), however, potentiated SIN-1-induced increases in dialysate DA. In contrast, co-infusion of the NO trapper 4-(carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO) significantly attenuated the increase in dialysate DA induced by SIN-1 (5.0 mM for 180 min). SIN-1+MGD+iron(II)-induced increases in dialysate DA were inhibited by Ca2+ omission or co-infusion of either deferoxamine or the L-type (Ca(v) 1.1-1.3) Ca2+ channel inhibitor nifedipine; in contrast, the increase was scarcely affected by co-infusion of the N-type (Ca(v) 2.2) Ca2+ channel inhibitor omega-conotoxin GVIA. These results demonstrate that exogenous NO-induced release of striatal DA is independent on extracellular Ca2+; however, in presence of the NO trapper MGD, NO may preferentially react with either endogenous or exogenous iron to form a complex which releases striatal DA with an extracellular Ca2+-dependent and nifedipine-sensitive mechanism.