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Advanced glycation end-products (AGEs) are known to be mutagenic, diabetogenic and vascular disease risk factors. Methylglyoxal (MG) is a dicarbonyl species that reacts with biological macromolecule (proteins, DNA and lipids) to give AGEs. Nonenzymatic glycation of MG with lysine (Lys) in the presence of copper (Cu(2+)) is reported to generate reactive oxygen species (ROS) capable of causing DNA damage. We show that DNA modification in MG-Lys-Cu(2+) system results in the generation of strand breaks, base modification, hyperchromicity and increased fluorescence intensity. Superoxide generation in the MG-Lys system was found to be significantly higher when compared with that in the MG and Lys alone. Moreover, d-penicillamine and pyridoxal phosphate significantly inhibited the formation of glycation products. The presence of a major DNA glycation adduct, N(2)-carboxyethyl-2'-deoxyguanosine (CEdG), was detected by high performance liquid chromatography (HPLC) and confirmed by nuclear magnetic resonance (NMR). As reported earlier, modified DNA (MG-Lys-Cu(2+)-DNA) was highly immunogenic in experimental animals. Furthermore, induced anti-MG-Lys-Cu(2+)-DNA antibodies were effective probe for detecting glycoxidative lesions in human genomic DNA of type I diabetes patients. Our results clearly imply that interaction of MG-Lys and Cu(2+) leads to the formation of AGEs and also the production of potent ROS, capable of causing DNA damage, thereby playing an important role in diabetes mellitus.

Copper accumulation in tissues due to a biallelic pathogenic mutation of the gene: ATP7B results in a clinical phenotype known as Wilson disease (WD). Aberrations in copper homeostasis can create favourable conditions for superoxide-yielding redox cycling and oxidative tissue damage. Drugs used in WD treatment aim to remove accumulated copper and normalise the free copper concentration in the blood. In the current study the effect of decoppering treatment on copper metabolism and systemic antioxidant capacity parameters was analyzed. Treatment naïve WD patients (TNWD) (n = 33), those treated with anti-copper drugs (TWD) (n = 99), and healthy controls (n = 99) were studied. Both TNWD and TWD patients characterised with decreased copper metabolism parameters, as well as decreased total antioxidant potential (AOP), glutathione (GSH) level, activity of catalase, glutathione peroxidase (GPx), and S-transferase glutathione, compared to controls. TWD patients had significantly lower copper metabolism parameters, higher total AOP and higher levels of GSH than TWD individuals; however, no difference was observed between these two patient groups with respect to the rest of the antioxidant capacity parameters. Patients who had undergone treatment with D-penicillamine or zinc sulphate did not differ with respect to copper metabolism or antioxidant capacity parameters, with the exception of GPx that was lower in D-penicillamine treated individuals. These data suggest that anti-copper treatment affects copper metabolism as well as improves, but does not normalize, natural antioxidant capacity in patients with WD. We propose to undertake studies aimed to evaluate the usefulness of antioxidants as well as selenium as a supplemental therapy in WD.

Purpose: (64)Cu-diacetyl-bis (N (4)-methylthiosemicarbazone) ((64)Cu-ATSM) is a promising theranostic agent that targets hypoxic regions in tumors related to malignant characteristics. Its diagnostic usefulness has been recognized in clinical studies. Internal radiotherapy (IRT) with (64)Cu-ATSM is reportedly effective in preclinical studies; however, for clinical applications, improvements to reduce radiation exposure in non-target organs, particularly the liver, are required. We developed a strategy to reduce radiation doses to critical organs while preserving tumor radiation doses by controlled administration of copper chelator penicillamine during (64)Cu-ATSM IRT., Methods: Biodistribution was evaluated in HT-29 tumor-bearing mice injected with (64)Cu-ATSM (185 kBq) with or without oral penicillamine administration. The appropriate injection interval between (64)Cu-ATSM and penicillamine was determined. Then, the optimal penicillamine administration schedule was selected from single (100, 300, and 500 mg/kg) and fractionated doses (100 mg/kg×3 at 1- or 2-h intervals from 1 h after (64)Cu-ATSM injection). PET imaging was performed to confirm the effect of penicillamine with a therapeutic (64)Cu-ATSM dose (37 MBq). Dosimetry analysis was performed to estimate human absorbed doses., Results: Penicillamine reduced (64)Cu accumulation in the liver and small intestine. Tumor uptake was not affected by penicillamine administration at 1 h after (64)Cu-ATSM injection, when radioactivity was almost cleared from the blood and tumor uptake had plateaued. Of the single doses, 300 mg/kg was most effective. Fractionated administration at 2-h intervals further decreased liver accumulation at later time points. PET indicated that penicillamine acts similarly with the therapeutic (64)Cu-ATSM dose. Dosimetry demonstrated that appropriately scheduled penicillamine administration reduced radiation doses to critical organs (liver, ovaries, and red marrow) below tolerance levels. Laxatives reduced radiation doses to the large intestine., Conclusions: We developed a novel strategy to reduce radiation exposure in critical organs during (64)Cu-ATSM IRT, thus promoting its clinical applications. This method could be beneficial for other (64)Cu-labeled compounds.

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P<0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P<0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P>0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P>0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P>0.05) in the presence of heparin and PHE (P<0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development.

Previous studies have shown that both 3-amino-1,2,4-triazole (AT), which inhibits metabolism of ethanol (EtOH) to acetaldehyde by inhibiting catalase, and D-penicillamine (D-P), an acetaldehyde-sequestering agent, modulate EtOH-conditioned place preference (CPP) in male albino Swiss (IOPS Orl) mice. These studies followed a reference-dose-like procedure, which involves comparing cues that have both been paired with EtOH. However, the role of EtOH-derived acetaldehyde has not been examined using a standard CPP method, and efficacy of these treatments could be different under the two circumstances. In the present investigation, we manipulated the strength of CPP across five separate studies and evaluated the effect of D-P and AT on EtOH-induced CPP following a standard unbiased CPP procedure. Mice received pairings with vehicle-saline injections with one cue and, alternatively, with AT- and D-P-EtOH with another cue. Our studies indicate that AT and D-P only disrupt CPP induced by EtOH in mice when the number of conditioning sessions and the dose of EtOH are low. These findings suggest that acquisition of EtOH-induced CPP may depend on the levels of acetaldehyde available during memory acquisition and the strength of the memory. Therefore, we propose that, at least when the memory processes are labile, brain acetaldehyde could participate in the formation of Pavlovian learning elicited by EtOH.

Glioblastoma is a highly vascularized brain tumor, and antiangiogenic therapy improves its progression-free survival. However, current antiangiogenic therapy induces serious adverse effects including neuronal cytotoxicity and tumor invasiveness and resistance to therapy. Although it has been suggested that the physical microenvironment has a key role in tumor angiogenesis and progression, the mechanism by which physical properties of extracellular matrix control tumor angiogenesis and glioblastoma progression is not completely understood. Herein we show that physical compaction (the process in which cells gather and pack together and cause associated changes in cell shape and size) of human glioblastoma cell lines U87MG, U251, and LN229 induces expression of collagen types IV and VI and the collagen crosslinking enzyme lysyl oxidase and up-regulates in vitro expression of the angiogenic factor vascular endothelial growth factor. The lysyl oxidase inhibitor β-aminopropionitrile disrupts collagen structure in the tumor and inhibits tumor angiogenesis and glioblastoma multiforme growth in a mouse orthotopic brain tumor model. Similarly, d-penicillamine, which inhibits lysyl oxidase enzymatic activity by depleting intracerebral copper, also exhibits antiangiogenic effects on brain tumor growth in mice. These findings suggest that tumor microenvironment controlled by collagen structure is important in tumor angiogenesis and brain tumor progression., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Low molecular weight thiols (LMWTs) like N-acetyl cysteine, D-penicillamine, captopril, Disulfiram and Amifostine, etc. have been used as chemo-preventive agents. Recent studies have reported cell growth inhibition and cytotoxicity in several different types of cancer cells following treatment with several LMWTs. Cytotoxic and cytostatic effects of LMWTs may involve interaction of the thiol group with cellular lipids, proteins, intermediates or enzymes. Some of the mechanisms that have been proposed include a p53 mediated apoptosis, thiyl radical induced DNA damage, membrane damage through lipid peroxidation, anti-angiogenic effects induced by inhibition of matrix metalloproteinase enzymes and angiostatin generation. LMWTs are strong chelators of transition metals like copper, nickel, zinc, iron and cobalt and may cause metal co-factor depletion resulting in cytotoxicity. Oxidation of thiol group can also generate cytotoxic reactive oxygen species (ROS)., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Rationale: Nowadays, very few approved anti-relapse treatments for alcoholism exist, and their overall efficacy can be considered moderate. An exciting rationale drug development opportunity for the treatment of chronic alcoholism is the use of acetaldehyde sequestering agents. Although these compounds are able to attenuate or prevent most of the behavioral and neurochemical effects of ethanol, the efficacy of acetaldehyde sequestration, by using agents such as D-penicillamine (DP), in relapse prevention has not been studied yet., Objectives: The aim of this study was to analyze the effects of DP treatment on the alcohol deprivation effect (ADE) in long-term ethanol-experienced rats as a model of relapse behavior and measure drug plasma and brain levels during treatment., Methods: Rats were subcutaneously implanted with mini-osmotic pumps delivering 0, 0.25, or 1 mg/h of DP during 1 week. The efficacy to prevent ADE was determined. DP plasma and brain levels achieved during its subcutaneous administration were measured. In a second experiment, animals received bilateral infusions of 0 or 1.5 μg/h of DP directly into pVTA, and the appearance of ADE was evaluated., Results: One milligram per hour, but not 0.25 mg/h, DP infusion prevented ADE and reduced the total ethanol preference in animals. DP plasma concentrations associated with ADE suppression were around 3-4 μg/ml, and brain DP levels in these conditions were about 2-3 % of those found in plasma. Intra-pVTA DP administration also suppressed ADE., Conclusion: DP is able to prevent alcohol-relapse-like drinking in rats suggesting that this drug may be a useful new tool in the management of relapse in alcohol-dependent patients.

A cell chip is a valuable tool to evaluate the effects of anticancer drugs, antibiotics and environmental toxicants on various kinds of cells. In this study, a conductive composite material composed of multi-walled carbon nanotubes (CNTs) and RGD-MAP-C peptide was fabricated on gold electrode surface for enhancing electrochemical signals from HEK293T cells. The topological characteristics and electrochemical performance of composite materials with different concentrations of CNTs were analyzed by scanning electron microscopy and cyclic voltammetry (CV), respectively. CNTs/RGD peptide composites (CP) electrode containing 20 microg/ml CNT was found to be excellent for improving the sensitivity of cell chip compared with that of bare gold or RGD peptide modified electrode. Finally, two kinds of nephrotoxic antibiotics were treated to HEK293T cells and their toxicity were successfully monitored by CV. Our CP composites can be used as a suitable conducting material for the fabrication of various kinds of cell-based chips.

We determined whether pretreatment with (1) the μ-/δ-opioid receptor (μ-/δ-OR) antagonist, naloxone, (2) the δ1,2-OR antagonist, naltrindole, or (3) the peroxynitrite scavenger, d-penicillamine, affects the development of tolerance to the ventilatory depressant effects of morphine in rats. The injection of morphine in vehicle-pretreated rats decreased minute ventilation predominantly via decreases in tidal volume. Pretreatment with naloxone blunted the responses to morphine whereas pretreatment with naltrindole or d-penicillamine did not. A second injection of morphine, given one day later, elicited markedly smaller responses in vehicle rats whereas it elicited pronounced ventilatory depression in rats that were pretreated with naloxone, naltrindole or d-penicillamine (prior to morphine) the day before. Moreover, the ventilatory responses elicited by subsequent exposure to a hypoxic-hypercapnic challenge were markedly depressed in naloxone- or d-penicillamine-pretreated rats compared to vehicle-pretreated rats. These findings suggest that activation of μ- and δ-ORs causes tolerance to the ventilatory depressant effects of morphine at least partly via the generation of peroxynitrite., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Neuroinflammation and oxidative stress are involved in the pathogenesis of neurodegenerative diseases such as Alzheimer's diseases and Parkinson's disease. Naturally derived kavalactones isolated from Piper methysticum (Piperaceae) have been shown to exhibit neuroprotective effects. We have previously reported that a chemically synthesized kavalactone derivative, 2',6'-dichloro-5-methoxymethyl-5,6-dehydrokawain (compound 1) protects against oxidative stress-induced neuronal cell death through activation of Nrf2 signaling. In the present study, we examined the effect of compound 1 on neuroinflammation. In BV2 microglial cells, compound 1 strongly inhibited LPS-stimulated iNOS induction and NO production, but did not affect LPS-stimulated induction of COX2. At 6h after LPS challenge, when iNOS induction was not clearly seen, treatment with LPS or compound 1 alone increased expression of heme oxygenase 1 (HO-1) whose transcription is regulated by Nrf2. When treated with both, compound 1 enhanced LPS-stimulated HO-1 induction, which was more evident at 24h after LPS treatment. Furthermore, LPS-stimulated activation of Nrf2 signaling and nuclear translocation of Nrf2 were potentiated by compound 1. The mechanism by which compound 1 activated Nrf2 signaling was supposed to be a covalent modification of the sulfhydryl groups of Keap1 by an α,β-unsaturated carbonyl group present in the compound 1. Treatment with hemin, a HO-1 inducer, and with [Ru(CO)₃Cl₂]₂, a CO donor, decreased LPS-stimulated iNOS induction and NO production. In contrast, siRNA-mediated knockdown of HO-1 expression reduced the inhibitory effect of compound 1 on LPS-stimulated iNOS induction and NO production. The compound 1 inhibited LPS-stimulated ERK phosphorylation after LPS treatment. Finally, compound 1 suppressed LPS/IFN-γ-stimulated NO production in primary microglial cells. These results suggest that compound 1 is capable of inhibiting LPS-stimulated iNOS induction and NO production via activation of Nrf2 signaling and HO-1 induction in microglial cells. Taken together, compound 1 has a potential to reduce neuroinflammation as well as oxidative stress in neurodegenerative diseases through activation of Nrf2 signaling., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Rationale: Animal studies indicate that central acetaldehyde, dependent on catalase metabolism of ethanol (EtOH), modulates ethanol reinforcement. Brain catalase activity and acetaldehyde (ACD) production are significantly higher in rat pups compare d with adults. Interestingly, infant rats show high EtOH affinity for alcohol consumption and are particularly sensitive to the drug's reinforcing effects., Objectives: We tested whether central ACD is necessary and sufficient to induce appetitive conditioning in newborn rats through the artificial nipple technique., Methods: Vehicle, EtOH (100 mg%), and acetaldehyde (0.35 μmol) were administered into the cisterna magna (1 μl). Half of the animals also received a central administration of 75 μg (experiment 1) or 40 μg of D-penicillamine (experiment 2). Afterwards, pups were exposed to an olfactory cue (conditioned stimulus). One hour later, neonates were tested with an artificial nipple in the presence of the conditioned cue. Nipple attachment duration, mean grasp duration, and number of nipple disengagements served as dependent variables., Results: Positive responses to the scented nipple occurred in neonates conditioned with EtOH or ACD (experiments 1 and 2). In experiment 1, there were indications that D-penicillamine weakened the reinforcing effects of EtOH and ACD. In experiment 2, D-penicillamine (40 μg) significantly inhibited appetitive conditioned responses dependent upon EtOH or ACD., Conclusions: Appetitive conditioning was observed when employing either central EtOH or ACD as unconditioned stimuli. Central abduction of ACD inhibited conditioned appetitive responsiveness to the surrogate nipple. Central ACD is involved in the determination or modulation of EtOH's motivational properties during early stages in development.

A facile synthesis of chiral penicillamine protected Au nanoclusters with different optical properties has been reported. We have for the first time observed the reversal of CD signals after the scaffolds adsorbed onto the surface of Au NCs. Such Au NCs are utilized for bioimaging due to their low cytotoxicity and stable fluorescence emission.

Unlabelled: Hepatic methionine metabolism may play an essential role in regulating methylation status and liver injury in Wilson's disease (WD) through the inhibition of S-adenosylhomocysteine hydrolase (SAHH) by copper (Cu) and the consequent accumulation of S-adenosylhomocysteine (SAH). We studied the transcript levels of selected genes related to liver injury, levels of SAHH, SAH, DNA methyltransferases genes (Dnmt1, Dnmt3a, Dnmt3b), and global DNA methylation in the tx-j mouse (tx-j), an animal model of WD. Findings were compared to those in control C3H mice, and in response to Cu chelation by penicillamine (PCA) and dietary supplementation of the methyl donor betaine to modulate inflammatory and methylation status. Transcript levels of selected genes related to endoplasmic reticulum stress, lipid synthesis, and fatty acid oxidation were down-regulated at baseline in tx-j mice, further down-regulated in response to PCA, and showed little to no response to betaine. Hepatic Sahh transcript and protein levels were reduced in tx-j mice with consequent increase of SAH levels. Hepatic Cu accumulation was associated with inflammation, as indicated by histopathology and elevated serum alanine aminotransferase (ALT) and liver tumor necrosis factor alpha (Tnf-α) levels. Dnmt3b was down-regulated in tx-j mice together with global DNA hypomethylation. PCA treatment of tx-j mice reduced Tnf-α and ALT levels, betaine treatment increased S-adenosylmethionine and up-regulated Dnmt3b levels, and both treatments restored global DNA methylation levels., Conclusion: Reduced hepatic Sahh expression was associated with increased liver SAH levels in the tx-j model of WD, with consequent global DNA hypomethylation. Increased global DNA methylation was achieved by reducing inflammation by Cu chelation or by providing methyl groups. We propose that increased SAH levels and inflammation affect widespread epigenetic regulation of gene expression in WD., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Different in vitro models have been developed to understand the interaction of gametes and embryos with the maternal reproductive tract. We recently showed that bovine oviductal monolayers three-dimensionally cultured in Gray's medium on collagen-coated microporous polycarbonate inserts under liquid-air interface conditions are well polarized, develop cilia, remain viable for at least 3 weeks postconfluence, and mantain the viability of bound spermatozoa significantly better than bidimensionally cultured monolayers. Herein, we used these culture conditions to understand whether: (1) spermatozoa adhering to three-dimensionally cultured oviductal monolayers can be released by heparin or penicillamine as previously shown with bidimensionally cultured oviductal monolayers and explants; and (2) media conditioned by three-dimensionally cultured oviductal monolayers were able to release spermatozoa adhering to oviductal explants. Findings demonstrated that (1) spermatozoa adhering to three-dimensionally cultured oviductal monolayers are readily released by heparin and penicillamine, (2) media conditioned by three-dimensionally cultured oviductal monolayers are able to release spermatozoa bound to oviductal explants, (3) do not depress sperm motility and viability, (4) they improve sperm kinetics, and (5) promote binding to the zona pellucida. In conclusion, in vitro data suggest that the release of spermatozoa adhering to the oviductal reservoir in vivo can be triggered by factors secreted by the oviduct itself that induce sperm capacitation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Wilson's disease is a rare autosomal recessive disease characterised by the deposition of copper in the brain, liver; cornea, and other organs. The overload of copper inevitably leads to progressive liver and neurological dysfunction. Copper overload in patients with Wilson's disease is caused by impairment to the biliary route for excretion of dietary copper A combination of neurological, psychiatric and hepatic symptoms can make the diagnosis of Wilson's disease challenging. Most symptoms appear in the second and third decades of life. The disease affects between one in 30,000 and one in 100,000 individuals, and is fatal if left untreated. Five drugs are currently available to treat Wilson's disease: British Anti-Lewisite; D-penicillamine; trientine; zinc sulfate or acetate; and ammonium tetrathiomolybdate. Each drug can reduce copper levels and/or transform copper into a metabolically inert and unavailable form in the patient. The discovery and introduction of these five drugs owes more to the inspiration of a few dedicated physicians and agricultural scientists than to the resources of the pharmaceutical industry.

The prolonged incubation of human spermatozoa in vitro was found to induce a loss of motility associated with the activation of mitochondrial reactive oxygen species generation in the absence of any change in mitochondrial membrane potential. The increase in mitochondrial free radical production was paralleled by a loss of protein thiols and a concomitant rise in the formation of 4-hydroxynonenal, an electrophilic product of lipid peroxidation that was found to directly suppress sperm movement. These results prompted a search for nucleophiles that could counteract the action of such cytotoxic aldehydes, as a means of ensuring the long-term survival of spermatozoa in vitro. Four nucleophilic compounds were consequently assessed (penicillamine, homocysteine, N-acetylcysteine, and mercaptosuccinate) in three species (human, rat, and horse). The results of this analysis revealed drug and species specificity in the manner in which these compounds affected sperm function, with penicillamine conferring the most consistent, effective support. This prosurvival effect was achieved downstream of mitochondrial reactive oxygen species generation and was associated with the stabilization of 4-hydroxynonenal generation, the preservation of sperm thiols, and a reduction in 8-hydroxy-2'-deoxyguanosine formation. Theoretical calculations of Fe-S and Cu-S bond distances and corresponding binding energies suggested that the particular effectiveness of penicillamine may, in part, reflect the ability of this nucleophile to form stable complexes with transition metals that catalyze lipid peroxidation. The practical implications of these findings were indicated by the effective preservation of equine spermatozoa for 8 days at ambient temperature when the culture medium was supplemented with penicillamine.

D-Penicillamine (3,3-dimethyl-D-cysteine; DP) is an FDA-approved redox-active D-cysteine-derivative with antioxidant, disulfide-reducing, and metal chelating properties used therapeutically for the control of copper-related pathology in Wilson's disease and reductive cystine-solubilization in cystinuria. Based on the established sensitivity of metastatic melanoma cells to pharmacological modulation of cellular oxidative stress, we tested feasibility of using DP for chemotherapeutic intervention targeting human A375 melanoma cells in vitro and in vivo. DP treatment induced caspase-dependent cell death in cultured human metastatic melanoma cells (A375, G361) without compromising viability of primary epidermal melanocytes, an effect not observed with the thiol-antioxidants N-acetyl-L-cysteine (NAC) and dithiothreitol. Focused gene expression array analysis followed by immunoblot detection revealed that DP rapidly activates the cytotoxic unfolded protein response (UPR; involving phospho-PERK, phospho-eIF2α, Grp78, CHOP, and Hsp70) and the mitochondrial pathway of apoptosis with p53 upregulation and modulation of Bcl-2 family members (involving Noxa, Mcl-1, and Bcl-2). DP (but not NAC) induced oxidative stress with early impairment of glutathione homeostasis and mitochondrial transmembrane potential. SiRNA-based antagonism of PMAIP1 expression blocked DP-induced upregulation of the proapoptotic BH3-only effector Noxa and prevented downregulation of the Noxa-antagonist Mcl-1, rescuing melanoma cells from DP-induced apoptosis. Intraperitoneal administration of DP displayed significant antimelanoma activity in a murine A375 xenograft model. It remains to be seen if melanoma cell-directed induction of UPR and apoptosis using DP or improved DP-derivatives can be harnessed for future chemotherapeutic intervention.

Wilson's disease is an orphan disease due to copper homeostasis dysfunction. Mutations of the ATP7B gene induces an impaired functioning of a Cu-ATPase, impaired Cu detoxification in the liver and copper overload in the body. Indeed, even though copper is an essential element, which is used as cofactor by many enzymes playing vital roles, it becomes toxic when in excess as it promotes cytotoxic reactions leading to oxidative stress. In this perspective, human copper homeostasis is first described in order to explain the mechanisms promoting copper overload in Wilson's disease. We will see that the liver is the main organ for copper distribution and detoxification in the body. Nowadays this disease is treated life-long by systemic chelation therapy, which is not satisfactory in many cases. Therefore the design of more selective and efficient drugs is of great interest. A strategy to design more specific chelators to treat localized copper accumulation in the liver will then be presented. In particular we will show how bioinorganic chemistry may help in the design of such novel chelators by taking inspiration from the biological copper cell transporters.

Previous studies on animals have revealed that garlic (Allium sativum) is effective in reducing blood and tissue lead concentrations. The aim of this study was to investigate therapeutic effects of garlic and compare it with d-penicillamine in patients with chronic lead poisoning. After coordination and obtaining informed consent, clinical examinations and blood lead concentration (BLC) of 117 workers at a car battery industry were investigated. BLC was determined by heated graphite atomization technique of an atomic absorption spectrometer. The workers were randomly assigned into two groups of garlic (1200 μg allicin, three times daily) and d-penicillamine (250 mg, three times daily) and treated for 4 weeks. BLC was determined again 10days post-treatment. Clinical signs and symptoms of lead poisoning were also investigated and compared with the initial findings. Clinical improvement was significant in a number of clinical manifestations including irritability (p = 0.031), headache (p = 0.028), decreased deep tendon reflex (p=0.019) and mean systolic blood pressure (0.021) after treatment with garlic, but not d-penicillamine. BLCs were reduced significantly (p=0.002 and p=0.025) from 426.32±185.128 to 347.34±121.056 μg/L and from 417.47±192.54 to 315.76±140.00μg/L in the garlic and d-penicillamine groups, respectively, with no significant difference (p=0.892) between the two groups. The frequency of side effects was significantly (p=0.023) higher in d-penicillamine than in the garlic group. Thus, garlic seems safer clinically and as effective as d-penicillamine. Therefore, garlic can be recommended for the treatment of mild-to-moderate lead poisoning., (© 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.)

Peroxynitrite (PN) worsens pathological conditions associated with oxidative stress. However, beneficial effects have also been reported. PN has been shown to demonstrate vasodilator as well as vasoconstrictor properties that are dependent upon the experimental conditions and the vascular bed studied. PN-induced vascular smooth muscle relaxation may involve the formation of nitric oxide (NO) donors. The present results show that PN has significant vasodilator activity in the pulmonary and systemic vascular beds, and that responses to PN were not attenuated by L-penicillamine (L-PEN), a PN scavenger, whereas responses to sodium nitroprusside (SNP) were decreased. PN had a small inhibitory effect on decreases in arterial pressure in response to the NO donors diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) and S-nitrosoglutathione (GSNO). PN partially reversed hypoxic pulmonary vasoconstriction. PN responses were attenuated by the soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and responses to PN and the PN precursor, 3-morpholinosydnonimine (SIN-1), were different. These data show that PN has potent pulmonary vasodilator activity in the rat, and provide evidence that a PN interaction with S-nitrosothiols is not the major mechanism mediating the response. These data suggest that responses to PN are mediated by the activation of sGC, and that PN has a small inhibitory effect on NO responses.

Wilson disease (WD) is characterized by the accumulation of copper arising from a mutation in the ATP7B gene. Penicillamine (PA) makes 10-50% of the patients with neurologic symptoms neurologically worse at the early stage of administration. The aim of this study was to determine how the copper metabolism changes and whether the change impairs the brain of toxic milk (tx) mice, an animal model of WD, during the PA administration. The free copper and protein-bound copper concentrations in the serum, cortex and basal ganglia of tx mice with PA administration for 3 days, 10 days and 14 days, respectively, were investigated. The expression of copper transporters, ATP7A and CTR1,was analyzed by real-time quantitative PCR, immunofluorescence and Western blot. Then SOD, MDA and GSH/GSSG were detected to determine whether the oxidative stress changed correspondingly. The results revealed the elevated free copper concentrations in the serum and brain, and declined protein-bound copper concentrations in the brain of tx mice during PA administration. Meanwhile, transiently increased expression of ATP7A and CTR1 was observed generally in the brain parenchyma by immunofluorescence, real-time quantitative PCR and Western blot. Additionally, ATP7A and CTR1 were observed to locate mainly at Golgi apparatus and cellular membrane respectively. Intense staining of ATP7A in the choroid plexus was found in tx mice on the 3rd and 10th day of PA treatment, but rare staining of ATP7A and CTR1 in the blood-brain barrier (BBB). Decreased GSH/GSSG and increased MDA concentrations were also viewed in the cortex and basal ganglia. Our results suggested the elevated free copper concentrations in the brain might lead to the enhanced oxidative stress during PA administration. The increased free copper in the brain might come from the copper mobilized from brain parenchyma cells but not from the serum according to the ATP7A and CTR1 expression analysis.

DL-penicillamine (DL-P) and Prussian blue (PB) given alone or in combination were tested as possible treatments against acute thallium toxicity. Rats were intoxicated by i.p. injection of thallium (I) acetate at LD(50) (32 mg/kg). A day later, pharmacological treatment was administered until day 4 as follows: (1) vehicles, (2) PB 50mg/kg, by oral route, twice a day, (3) DL-P 25mg/kg i.p. route, twice daily and (4) PB+DL-P. The Estimated Probability Survival (EPS) was recorded during the experiment for each treatment. DL-P alone did not show a significant effect on survival. However, when it was used in combination with PB, it increased the survival significantly (EPS=0.8, P<0.05) as compared to the control group (EPS=0.4). In a different experiment, using 16 mg/kg of Thallium I acetate, the metal levels were analyzed in blood, body organs and brain regions after treatments. DL-P given alone decreased slightly the thallium content in blood, organs and brain. Meanwhile, its administration in combination with PB diminished the thallium levels significantly (P<0.05) in the majority of tissues, at levels lower than those achieved in the PB group. Those results indicate that DL-P administered alone did not prevent the mortality nor accumulation of the metal in body tissues. Its combination with PB could be considered an alternative antidotal treatment in thallium toxicity, because this chelating agent given alone did not cause thallium redistribution to the brain. When given in combination with PB it has an additive effect in the treatment of acute thallotoxicosis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Purpose: To determine the effect of intraperitoneal and intravitreal D-penicillamine (DPA) on retinal neovascularization in a murine model of oxygen-induced retinopathy., Methods: On postnatal day 7, 16 mice were injected intraperitoneally with 300 mg/kg/day DPA for 3 days followed by 50 mg/kg/day for 7 days. A different group of 7 mice were injected intraperitoneally with 600 mg/kg/day DPA for 3 days followed by 100 mg/kg/day for 7 days. A third group of 14 mice were injected with 1,500 mg/kg/day DPA for 2 days; a control cohort of 17 mice received intraperitoneal phosphate-buffered saline (PBS). An additional 15 mice underwent intravitreal injection of 1 μL of 100 mg/mL DPA in the right eye and 1 μL PBS intravitreally in the left eye as a control. All groups were placed in a 75% oxygen chamber for 7 days then room air for 3 days before being sacrificed and enucleated. The retinas were stained and flat-mounted to determine the severity of retinal neovascularization by quantifying neovascular buds., Results: After intraperitoneal injection, the mean number of glomeruli and tubules was similar in the DPA and PBS groups (P = 1.0), regardless of DPA dosage. The dosage of 1,500 mg/kg/day proved to be uniformly lethal. After intravitreal injections, the mean number of glomeruli (P = 0.16) and tubules (P = 0.7) were similar in the DPA and PBS groups., Conclusions: Neither intraperitoneal nor intravitreal injection of DPA inhibits retinal neovascularization in a murine model of oxygen-induced retinopathy., (Copyright © 2011 American Association for Pediatric Ophthalmology and Strabismus. Published by Mosby, Inc. All rights reserved.)

D-penicillamine is a melanogenesis inhibitor. This in vivo study on ten black guinea pigs using a 5% D-penicillamine ointment showed its lack of any skin-lightening effect. The potential reasons for this ineffectiveness are discussed in the paper, which could be very helpful for researchers exploring new skin-lightening agents.

We propose a new nonparametric test to compare treatment effects using a composite endpoint comprising a longitudinal continuous measurement and multiple time-to-event outcomes. The composite endpoint incorporates the severity of each outcome as measured against the whole cohort in the entire study. This new proposed test is conceptually simple and computationally easy to implement. Unlike many currently available methods, our strategy is very flexible and can be applied to many types of clinical settings including the situation where we have multiple time-to-event outcomes. We apply our method to reanalyze two clinical trials for Scleroderma patients and also use simulation studies to show that the performance of our method is compatible with the results from a popular method proposed by Finkelstein and Schoenfeld (Statist. Med. 1999; 18:1341-1354)., (Copyright © 2011 John Wiley & Sons, Ltd.)

Although recently published studies seem to confirm the important role displayed by acetaldehyde (ACH), the main metabolite of ethanol, in the behavioral effects of ethanol, the origin of ACH is still a matter of debate. While some authors confer more importance to the central (brain metabolism) origin of ACH, others indicate that the hepatic origin could be more relevant. In this study we have addressed this topic using an experimental approach that combines local microinjections of ethanol into the ventral tegmental area (VTA) (which guarantees the brain origin of the ACH) to induce motor activation in rats together with systemic administration (i.p.) of several doses (0, 12.5, 25 and 50 mg/kg) of D-penicillamine (DP), a sequestering agent of ACH with contrasted efficiency to abolish the behavioral effects of the drug. Our results clearly show that DP prevented in a dose-dependent manner the motor activation induced by intra-VTA ethanol, being the 50 mg/kg dose the most efficient. DP per se did not affect the basal activity of the rats. In order to determine the specificity of the DP action, we also studied the effects of DP 50 mg/kg on the DAMGO-induced motor activation after the intra-VTA administration of this mu-opioid receptors agonist. DP did not significantly modify the motor activation induced by DAMGO thus confirming the specificity of the DP effects. Our results clearly suggest that the brain-derived ACH is necessary to manifest the activating effects resulting from ethanol administration., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Nitric oxide (NO) was proposed to be an important molecule against some microorganisms. In this study, we investigated the inhibitory effect of NO on the infection by porcine reproductive and respiratory syndrome virus (PRRSV) in vitro and the role of NO in the defense against PRRSV. Our results indicated that exogenous NO did not inhibit PRRSV infection. Unexpectedly, N-acetylpenicillamine (NAP), a commonly used compound as negative control for NO-producing reagents, inhibited PRRSV replication. Thus, the inhibition effect of NAP on PRRSV replication was further explored. We found that the maximal inhibition effect of NAP on PRRSV replication was achieved upon treatment 1 h after virus infection and the virus yield was reduced by approximately 50 fold in the presence of 400 muM NAP. An obvious inhibitory effect on viral RNA and protein synthesis was also observed. However, the inhibitory effect was only achieved at early phase of virus infection. The normal virus yield could be restored upon the removal of NAP treatment. The inhibitory effect might be caused by sulfhydryl-reducing capacity and metal chelating properties of NAP. These studies suggested that (i) NO production or NO synthase (NOS) expression profiling may not be a reliable index for the immune response to PRRSV; (ii) NAP could inhibit the replication of PRRSV.

Compound 1 ((-)-gossypol) has been long known as a chemical anticancer agent. With its low water solubility and toxicity, it is not widely used as a commercial drug. To overcome these disadvantages, several novel derivatives of gossypol were designed, synthesized, and analyzed. One of the derivatives, compound 7 (6-aminopenicillanic acid sodium-gossypolone), was identified with great water solubility and anticancer property, suggested by inducing a dramatically decrease in Bcl-2 and Bcl-xL protein expression level found in vitro and growth inhibition of murine colon tumor in vivo. Furthermore, it was also recognized with less toxicity than compound 1 in vivo and significantly increased chemotherapeutic sensitivity against colon cancer in combination with traditional chemotherapeutic agent 5-fluorouracil. Therefore, it is concluded that compound 7 is superior to parent compound 1, and further preclinical studies of compound 7 is necessary for colon cancer therapy.

Neuropathic pain states and tolerance to opioids can result from system changes in the CNS, such as up-regulation of the NK1 receptor and substance P, lead to antiopioid effects in ascending or descending pain-signaling pathways. Bifunctional compounds, possessing both the NK1 antagonist pharmacophore and the opioid agonist pharmacophore with delta-selectivity, could counteract these system changes to have significant analgesic efficacy without undesirable side effects. As a result of the introduction of cyclic and topological constraints with penicillamines, 2 (Tyr-cyclo[d-Pen-Gly-Phe-Pen]-Pro-Leu-Trp-NH-[3',5'-(CF(3))(2)-Bzl]) was found as the best bifunctional compound with effective NK1 antagonist and potent opioid agonist activities, and 1400-fold delta-selectivity over the mu-receptor. The NMR structural analysis of 2 revealed that the relative positioning of the two connected pharmacophores as well as its cyclic and topological constraints might be responsible for its excellent bifunctional activities as well as its significant delta-opioid selectivity. Together with the observed high metabolic stability, 2 could be considered as a valuable research tool and possibly a promising candidate for a novel analgesic drug.

Asthma is characterized by accumulation of eosinophils in the lungs and delayed apoptosis may be one mechanism leading to eosinophilia. Nitric oxide (NO), present in inflamed lungs, has been shown to possess both anti- and proeosinophilic properties. We previously showed that NO induces apoptosis in the presence of survival prolonging cytokine IL-5 in human eosinophils. In the present study, we examined the intracellular mechanisms of NO-induced apoptosis in granulocyte macrophage-colony stimulating factor (GM-CSF)-treated eosinophils concentrating on the role of caspases and calpains. Eosinophils were isolated from human blood and apoptosis was determined by relative DNA fragmentation assay, morphological analysis and/or Annexin-V FITC assay. We showed that NO-donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) induced apoptosis in GM-CSF-treated eosinophils. SNAP-induced DNA fragmentation was totally prevented by an inhibitor of caspase-6 (Z-VEID-FMK). Decreased levels of caspase-6 proenzyme and increased amounts of cleaved lamin A/C in SNAP-treated cells indicated activation of caspase-6. Furthermore, SNAP-induced lamin A/C and B fragmentation was totally abolished by an inhibitor of caspase-6. According to our results, caspase-6 mediates lamin and DNA fragmentation also in spontaneously dying eosinophils. Inhibitor of calpains prevented most of DNA fragmentation related to spontaneous apoptosis but had no effect in eosinophils undergoing NO-induced apoptosis. In the present study we showed that caspase-6 is essential for the executive phase involving lamin and DNA fragmentation in both NO-induced and spontaneous eosinophil apoptosis. However, differences in the involvement of calpains suggest that the intracellular signalling in NO-induced apoptosis has specific features at the level of proteases. This study demonstrates new mechanisms for NO-induced and spontaneous apoptosis in human eosinophils., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The paper investigates the effect of thiol drugs (RSH) under oxidative stress condition using luminol-enhanced chemiluminescence technique. The examinations included N-acetylcysteine (NAC), N-acetylpenicillamine (NAP), penicillamine (PEN), mesna (MES), and tiopronin (TPR). The model systems contained isolated human erythrocytes (RBC), erythrocyte lysates (LYS) or erythrocyte membranes (MEM) exposed to tert-butyl hydroperoxide (t-BuOOH). Under the influence of RSH, a bimodal character of some experimental chemiluminescence curves was observed and the kinetic solution was considered as the sum of two logistic-exponential processes. These chemiluminescence changes probably reflected two connected processes--scavenging by RSH of the t-BuOOH-induced free radicals and simultaneous generation of thiol-derived secondary free radicals. Individual differences in thiols interaction showed a multivariate set of the kinetic curve descriptors. The Principal Component Analysis (PCA) well distinguished subsets of RSH influence in systems with RBC or LYS. Generally, the action of NAC was exclusively pro-oxidant in both systems, with RBC and LYS. The behaviour of MES or NAP in these systems was also pro-oxidant but many times less prominent than NAC. Under the influence of TPR a dramatic switch in the anti-oxidant effect was observed in system with RBC to very pro-oxidant effect in LYS. The influence of PEN was analogical to TPR but very weak. This experimental model together with kinetic solution of the unique bimodal chemiluminescence curves, and PCA, supply new insights to the dual (anti- and pro-oxidant) effects of thiol drugs under oxidative stress condition., ((c) 2010 Elsevier Ireland Ltd. All rights reserved.)

D-penicillamine is an aminothiol that is cytotoxic to cancer cells and generates dose dependent reactive oxygen species (ROS) via copper catalyzed oxidation. However, the delivery of D-pen to cancer cells remains a challenge due to its high hydrophilicity, highly reactive thiol group and impermeability to the cell membrane. To overcome this challenge, we investigated a novel poly-L-glutamic acid (PGA) conjugate of D-pen (PGA-D-pen) where D-pen was conjugated to PGA modified with 2-(2-pyridyldithio)-ethylamine (PDE) via disulfide bonds. Confocal microscopy and cell uptake studies showed that the fluorescently labeled PGA-D-pen was taken up by human leukemia cells (HL-60) in a time dependent manner. Treatment of HL-60, murine leukemia cells (P388) and human breast cancer cells (MDA-MB-468) with PGA-D-pen resulted in dose dependent cytotoxicity and elevation of intracellular ROS levels. PGA-D-pen induced apoptosis in HL-60 cells which was verified by Annexin V binding. The in vivo evaluation of the conjugate in the P388 murine leukemia model (intraperitoneal) resulted in significant enhancement in the survival of CD2F1 mice over vehicle control.

Young and mature Artemisia princeps var. orientalis (APO, Compositae) are used as a health food and a medicinal plant, respectively, in Korea. Here, we identified the in vitro potent peroxynitrite (ONOO(-))-scavenging effect (IC(50), 0.26 microg/mL) of the components from the EtOAc fraction. Octadecylsilane column chromatography on the EtOAc fraction yielded two caffeoylquinic acid compounds, 3,5-di-O-caffeoyl-muco-quinic acid (1) and methyl 4,5-di-O-caffeoylquinate (2) by NMR spectroscopic data, which have not been reported before from APO. The IC(50) values of compounds 1 and 2 were 0.18 +/- 0.01 microg/mL and 0.12 +/- 0.00 microg/mL, respectively, lower than that of the positive control (L-penicillamine). HPLC data indicated that young APO (1: 30.3 mg/g dried weight, 2: 27.7 mg/g) contained considerably higher quantities of the two caffeoylquinic acids than mature APO (1: 1.77 mg/g dried weight, 2: 4.10 mg/g).

The acknowledged risk of deliberate release of radionuclides into local environments by terrorist activities has prompted a drive to improve novel materials and methods for removing internally deposited radionuclides. These decorporation treatments will also benefit workers in the nuclear industry, should an exposure occur. Cuprimine and Syprine are oral therapeutics based on the active ingredients D-penicillamine and N,N'-bis-(2-aminoethyl)-1,2-ethanediamine dihydrochloride, respectively. These therapeutic drugs have been used for several decades to treat Wilson's disease, a genetic defect leading to copper overload, by chelation and accelerated excretion of internally deposited copper. Studies were undertaken to evaluate these FDA-approved drugs for the in vivo decorporation of radioactive cobalt (Co) and polonium (Po) using male Wistar-Han rats. In these studies, Co or Po was administered to animals by IV injection, followed by oral gavage doses of either Cuprimine or Syprine. Control animals received the radionuclide alone. For Co studies, animals received a single dose of Cuprimine or Syprine, while for Po studies animals were repeatedly dosed at 24-h intervals for a total of 5 doses. Results show that Syprine significantly increased urinary elimination and skeletal concentrations of Co compared to controls. While Cuprimine had little effect on total excretion of Co, the skeletal, kidney, liver, muscle, and stomach tissues had significantly lower radioactivity compared to control animals. The low overall excretion of Po made it difficult to reliably measure urinary or fecal radioactivity and draw a definitive conclusion on the effect of Cuprimine or Syprine treatment on excretion. However, Cuprimine treatment was effective at reducing spleen levels of Po compared to controls. Similarly, Syprine treatment produced statistically significant reductions of Po in the spleen and skeletal tissues compared to control animals. Based on these promising findings, further studies to evaluate the dose-response pharmacokinetic profiles for decorporation are warranted.

Objective: This study presents the results of antioxidative and pro-oxidative efficacy of cysteamine and D-penicillamine (D-pen) in comparison to L-glutathione (L-GSH) on high-molar-mass hyaluronan (HA) degradation by cupric ions plus ascorbic acid., Methods: The substance tested was applied in the degradative system cupric ions plus ascorbate: (i) before the reaction onset or also (ii) 1 h after the reaction started. The results obtained were compared with that one recorded by using the degradative system in the absence of the substance tested. To monitor HA degradation kinetics, rotational viscometry was applied. Moreover, the standard ABTS and DPPH assays were used., Results: By using the method of rotational viscometry, D-pen showed dual effect: initial inhibitory effect on •OH radicals was changed to a pro-oxidative one in the dose and time dependent manner. Both L-GSH and cysteamine were recorded to be more effective scavengers of •OH radicals than D-pen. Cysteamine demonstrated to be an excellent scavenger also of alkoxyl- and peroxyl- type radicals. Based on IC50 values, gained by ABTS assay, it is evident that D-pen showed higher radical scavenging capacity compared to cysteamine. Similar results were observed also in DPPH assay, although in this assay less effective radical scavenging capacities of both substances tested were recorded., Conclusions: On the basis of the results obtained, it can be stated that D-pen can produce hydrogen peroxide or •OH radicals and can inhibit the production of these oxidants. Our results showed that both L-GSH and cysteamine are similarly effective in inhibiting of HA degradation. Moreover, cysteamine demonstrated to be a significant inhibitor of alkoxyl- and peroxyl- type radicals generated from C-type macroradical of HA.

Background: Cigarette smoke (CS) is the major risk factor for aerodigestive tract cancers such as lung and oral cancers., Methods: In in vitro models of lung and oral cancers, we found D-penicillamine (PenA) to be a most potent protector against CS, both in the absence and presence of saliva (a highly pro-oxidative condition)., Results: The survival rate of lung cancer cells and oral cancer cells was reduced by CS in the absence of saliva by 39-45% (p < 0.01) and by 55-60% (p < 0.01) in the presence of saliva. The addition of 5 mM PenA to cell medium prior to CS exposure limited cell loss to 22-25% only (p < 0.01). Similarly, the iron chelator desferal protected the cells only in the presence of saliva. PenA also protected against a CS-induced increase in carbonyls (oxidized proteins) and decrease in p53 levels (in the presence of saliva) and mitochondrial membrane potential (a hallmark of CS-induced apoptotic cell death). Malfunctioning p53 often characterizes carcinogenesis of CS-induced cancers., Conclusions: Redox-active iron and copper in pleural fluid and saliva, upon encounter with CS, may be responsible for this carcinogenesis, mediated via alteration of p53 function. Chelation of redox-active metals may be an efficient tool for prevention of CS-induced lung and oral cancers. The superiority of PenA results from its copper-chelating action as well as its antialdehyde and anti-inflammatory capabilities., (Copyright 2010 S. Karger AG, Basel.)

N-acetyl cysteine (NAC) and penicillamine (PEN) have been shown to induce apoptosis in multiple types of human cancer cells; however, the molecular mechanism underlying this activity is unclear. This study was designed to identify the genes responsible for apoptosis induction by NAC and PEN. We found that glucose-regulated protein 78 (GRP78) was upregulated in HeLa cells following treatment with NAC or PEN. GRP78 is a central regulator of endoplasmic reticulum (ER) stress and has been used as a marker of ER stress. Additionally, both the activating transcription factor 6 (ATF6) protein and X box-binding protein 1 (XBP1) mRNA were processed, which facilitates the expression of C/EBP homologous protein (CHOP), a key-signaling component of ER stress-induced apoptosis. Furthermore, the PERK-ATF4 pathway, which also induces the expression of CHOP, was activated in NAC-treated cells. The role of the ER stress pathway was further confirmed through the small interfering RNA (siRNA)-mediated knockdown of CHOP, which attenuated NAC and PEN-induced apoptosis. These results demonstrate that NAC- and PEN-induced apoptosis in HeLa cells is mediated by the ER stress pathway., (2009 Wiley-Liss, Inc.)

Introduction: Brain magnetic resonance imaging (MRI) studies on Wilson's disease (WD) show lack of correlations between neurological and neuroimaging features. Long-term follow-up reports with sequential brain MRI in patients with neurological WD comparing different modalities of treatment are scarce., Methods: Eighteen patients with neurological WD underwent pretreatment and posttreatment brain MRI scans to evaluate the range of abnormalities and the evolution along these different periods. All patients underwent at least two MRI scans at different intervals, up to 11 years after the beginning of treatment. MRI findings were correlated with clinical picture, clinical severity, duration of neurological symptoms, and treatment with two different drugs. Patients were divided into two groups according to treatment: D: -penicillamine (D-P), zinc (Zn), and Zn after the onset of severe intolerance to D-P., Results: MRI scans before treatment showed, in all patients, hypersignal intensity lesions on T2- and proton-density-weighted images bilaterally and symmetrically at basal nuclei, thalamus, brain stem, cerebellum, brain cortex, and brain white matter. The most common neurological symptoms were: dysarthria, parkinsonism, dystonia, tremor, psychiatric disturbances, dysphagia, risus sardonicus, ataxia, chorea, and athetosis., Conclusions: From the neurological point of view, there was no difference on the evolution between the group treated exclusively with D-P and the one treated with Zn. Analysis of MRI scans with longer intervals after the beginning of treatment depicted a trend for neuroimaging worsening, without neurological correspondence, among patients treated with Zn. Neuroimaging pattern of evolution was more favorable for the group that received exclusively D-P.

The three gaseous neuromodulators nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) are endogenously produced in vertebrate retinas. The NO/cyclic guanosine monophosphate (cGMP) and CO/cGMP pathways have been previously shown to interact synergistically in the turtle retina to increase cGMP levels. In this study, we examined H2S as a modulator of cGMP-like immunoreactivity (-LI) and its interactions with the NO/CO/cGMP signaling pathways in the tiger salamander retina. Stimulation with NO donor or CO significantly increased cGMP-LI from basal levels in bipolar and amacrine cells and in stratified arborizations in the inner plexiform layer. Stimulation with a combination of NO donor and CO significantly increased cGMP-LI above that seen with either stimulation alone. Nitric oxide synthase inhibitors reduced CO-induced cGMP-LI, suggesting that CO-induced cGMP-LI is not produced from direct activation of soluble guanylate cyclase. Exogenous H2S alone, from the donor NaHS, did not significantly modify cGMP-LI in dosages ranging from 2 to 1,200 microM NaHS, but there was a significant decrease in NO-induced cGMP-LI in the presence of 200 muM NaHS. This reduction of NO-induced cGMP-LI was not significantly affected by the addition of CuCl2, suggesting that the decrease was not a result of H2S and NO sequestering to form a novel nitrosothiol. NaHS did not have any significant effect on CO-induced cGMP-LI levels. Our results concur with previous studies showing synergistic interactions between NO and CO/cGMP retinal signaling pathways. We now show that H2S inhibits NO-induced cGMP-LI but not CO-induced cGMP-LI. In conclusion, all three gaseous neuromodulators have interactive roles in modulating retinal cGMP signaling., (Copyright 2009 Wiley-Liss, Inc.)

Cigarette smoke is a mixture of chemicals that cause direct or indirect oxidative stress in different cell lines. We investigated the effect of nonfractionated cigarette smoke extract (CSE) on protein carbonylation in human THP-1 cells. Cells were exposed to various concentrations (2.5-20%) of CSE for 30 min, and protein carbonylation was assessed by use of the sensitive 2,4-dinitrophenylhydrazine immuno-dot blot assay. CSE-induced protein carbonylation exhibited a dose-response relation with CSE concentrations. However, with prolonged exposure to CSE, significant decrements were observed when compared with the 30 min exposure. Cotreatment of THP-1 cells with antioxidants (N-acetyl-cysteine, S-allyl-cysteine, and alpha-tocopherol) and copper(II) ion chelators (d-penicillamine) during CSE exposure significantly reduced protein carbonylation, whereas cotreatment with antioxidants (vitamin C and trolox) and a metal chelator (EDTA), iron chelator (1,10-phenanthroline), or copper(I) chelator (neocuprin) did not decrease CSE-induced protein carbonylation in THP-1 cells. These results suggest that protein carbonylation is induced by CSE in THP-1 cells via a copper(II)-catalyzed reaction and not an iron-catalyzed reaction. Furthermore, the copper(II) ions involved in this CSE-induced protein carbonylation are derived from the intracellular pool, not via uptake from the extracellular medium. We speculate that natural copper(II) chelators may prevent some of the health problems caused by cigarette smoking, including lung disease, renal failure, and diabetes.

Oviductal fluid molecules, such as sulphated glycosaminoglycans and disulphide-reductants, may represent periovulatory signals for the release of spermatozoa from the oviductal reservoir in the bovine species. Disulphide-reductants release spermatozoa through the reduction of sperm-surface disulphides to sulphhydryls (SH). Herein, we studied sperm-surface protein SH through labelling with maleimidylpropionyl biocytin in the initial sperm suspension, in the subpopulations able and unable to adhere to the in vitro cultured oviductal epithelium, and in spermatozoa released either through the disulphide-reductant penicillamine (PEN) or the sulphated glycosaminoglycan heparin (HEP). Adhesion assays were performed to study the ability of released spermatozoa to readhere to the oviductal epithelium. Results showed that the level of SH in sperm-surface proteins was: 1) low in adhering spermatozoa; 2) high in spermatozoa unable to adhere; and 3) markedly increased in released spermatozoa. Adhesion assays showed that: 1) PEN-released spermatozoa promptly recovered adhesion after removal of the disulphide-reductant and could be released again in response to PEN; 2) conversely, a limited number of HEP-released spermatozoa was able to readhere to the oviductal epithelium and this ability was not affected by HEP removal. Recovery of adhesion was associated to reoxidation of sperm-surface protein SH and to the reversal of capacitation. In conclusion, redox modulation of sperm-surface protein SH is involved in the release of spermatozoa adhering to the oviduct in vitro; the reversible action of disulphide-reductants might be responsible for intermittent phases of adhesions and releases; and the irreversible action of HEP indicates that it may represent a terminal releasing signal.

Idiosyncratic drug reactions (IDRs) represent a major clinical problem, and at present, the mechanisms involved are still poorly understood. One animal model that we have used for mechanistic studies of IDRs is penicillamine-induced autoimmunity in Brown Norway (BN) rats. Previous work in our lab found that macrophage activation preceded the clinical autoimmune syndrome. It is thought that one of the interactions between T cells and macrophages involves reversible Schiff base formation between an amine on T cells and an aldehyde on macrophages, but the identity of the molecules involved is unknown. It is also known that penicillamine reacts with aldehyde groups to form a thiazolidine ring, which unlike a Schiff base, is essentially irreversible. Such binding could lead to macrophage activation. Generalized macrophage activation could lead to the observed autoimmune reaction. Hydralazine and isoniazid also react with aldehydes to form stable hydrazones, and they also cause an autoimmune lupus-like syndrome. In this study, isolated spleen cells from male BN rats were incubated with biotin-aldehyde-reactive probe (ARP, a hydroxylamine), biotin-hydrazide, or D-penicillamine. At all concentrations, ARP, hydrazide, and penicillamine preferentially "stained" macrophages relative to other spleen cells. In addition, preincubation of cells with penicillamine or hydralazine decreased ARP staining of macrophages, which further indicates that most of the ARP binding to macrophages involves binding to aldehyde groups. This provides support for the hypothesis that the interaction between aldehyde-containing signaling molecules on macrophages and penicillamine could be the initial event of penicillamine-induced autoimmunity. Several of the proteins to which ARP binds were identified, and some such as myosin are attractive candidates to mediate macrophage activation.

Nitric oxide (NO) plays important roles in CNS and smooth muscle function. Here we reveal an additional function in peripheral sensory transmission. We hypothesized that endogenous NO modulates the function of gastrointestinal vagal afferent endings. The nonselective NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester hydrochloride increased responses to tactile mechanical stimuli of mucosal afferent endings in two species, in some cases severalfold. This was mimicked by a neuronal NOS inhibitor but not an endothelial NOS inhibitor. NOS inhibitors did not affect the responsiveness of smooth muscle afferent endings, suggesting that the endogenous source of NO is exclusively accessible to mucosal receptors. The role of the NO-soluble guanylyl cyclase (sGC)-cGMP pathway was confirmed using the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one and the cGMP phosphodiesterase 5' inhibitor sildenafil. The first enhanced and the second inhibited mechanosensory function. Exogenous NO, from the donor S-nitroso-N-acetylpenicillamine, significantly reduced mechanosensitivity of both types of ending. Up to one-third of stomach-projecting afferent neurons in the nodose ganglia expressed neuronal NOS (nNOS). However, anterograde-traced vagal endings were nNOS negative, indicating NOS is not transported peripherally and there are alternative sources of NO for afferent modulation. A subpopulation of enteroendocrine cells in the gut mucosa were nNOS positive, which were found anatomically in close apposition with mucosal vagal afferent endings. These results indicate an inhibitory neuromodulatory role of epithelial NO, which targets a select population of vagal afferents. This interaction is likely to play a role in generation of symptoms and behaviors from the upper gastrointestinal system.

The aim of this study was to determine the effect of exogenous nitric oxide (NO) on the induction of murine splenic immune response to Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) in vitro. BALB/c mice were immunized with A. actinomycetemcomitans LPS and a control group was sham-immunized. Spleen cells were obtained, cultured and stimulated with A. actinomycetemcomitans LPS with or without the presence of S-nitroso acetyl-penicillamine (SNAP), a NO donor, and carboxy-PTIO, an NO scavenger. Culture supernatants were assessed for inducible nitric oxide synthase (iNOS) activity, specific IgG subclass levels, and both IFN-gamma and IL-4 levels. The results showed that in A. actinomycetemcomitans LPS-stimulated cells, SNAP enhances iNOS activity but inhibits the levels of specific IgG2a and IFN-gamma suggesting a Th1 response. The effect of SNAP on these immune parameters was ablated by carboxy-PTIO. These results suggest that exogenous NO may suppress the Th1-like immune response of A. actinomycetemcomitans LPS-stimulated murine spleen cells.

Objective: To investigate the effect of "Wenyang Huazhuo Tongluo recipe" (WYHZTLR) on proliferation and cell cycle of systemic sclerosis (SSc) skin fibroblasts in vitro by serum pharmacologic technique., Methods: Prednisone, D-penicillamine and WYHZTLR contained serum from patients with SSc were respectively added into cultured normal dermal fibroblast cell-line and SSc lesional dermal fibroblast cell-line. Then the cells were incubated for 48 hours. Cell proliferation and cell cycle were examined by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively., Results: Western medicine Chinese medicine and integrated Chinese and western medicine could markedly inhibit the proliferation of normal dermal fibroblasts and SSc lesional dermal fibroblasts in vitro compared with control group (P < 0.05 or P < 0.01). But 20% serum in integrated Chinese and western medicine group could markedly inhibit the proliferation of fibroblasts compared with western medicine group (P < 0.01). Different drug-contained serum of WYHZTLR could markedly increase the percentage of G0-G1 stage cells (P < 0.01) and depress the percentage of sphase cells and G2-M stage cells compared with control group (P < 0.05 or P < 0.01)., Conclusion: WYHZTLR can block the progression of SSc skin fibroblasts into sphase and G2-M stage and inhibit the proliferation of SSc skin fibroblasts, and WYHZTLR was unselectively interfered with normal and SSc fibroblasts in vitro.

The present study investigates the direct action and the underlying mechanism(s) of epigallocatechin-3-gallate (EGCG) vasomotor effects on the bovine isolated ophthalmic artery. Adjacent rings were cut from each artery and mounted in a wire miograph system for isometric recording. Concentration-response curves for EGCG were constructed by adding cumulative concentrations of the drug to arterial rings pre-contracted with 5-HT (1 microM). Effects of mechanical endothelial cell removal and of selective blockers of the nitric oxide (NO)/cGMP pathways were investigated on the EGCG relaxant responses. EGCG relaxed ophthalmic arteries and maximum relaxation was 78.4+/-2.64%. Mechanical removal of endothelium, blockade of soluble guanylyl cyclase by 1H-1,2,4-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ, 1 and 5 microM) or inhibition of nitric oxide (NO) synthase by N(G)-nitro-L-arginine (L-NAME, 50 and 100 microM) reduced significantly the relaxant response to catechin; moreover, the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 100 microM) significantly increased the vasorelaxant responses to EGCG. Relaxation to EGCG was inhibited by iberiotoxin (200 nM), a blocker of big-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, whereas the blockade of K(ATP) channel by glibenclamide (5 microM) and of small-conductance Ca(2+)-activated K(+) (SK(Ca)) channel by apamin (100 nM) elicited no effect. Interestingly, also inhibition of phosphoinositide-3-kinase (PI3K) by wortmannin (100 nM) and of Akt by SH6 (1 microM) markedly decreased the EGCG-evoked vasorelaxation. These data suggest that EGCG induced vasorelaxation in ophthalmic arteries with endothelium-intact via the activation of the NO/cGMP signalling pathway and defined an intriguing role for PI3K and Akt as upstream mediators for activation of NO-mediated relaxant responses.

Mesenchymal stem cells (MSCs) have received special attention for cardiomyoplasty because several studies have shown that they differentiate into cardiomyocytes both in vitro and in vivo. Nitric oxide (NO) is a free radical signaling molecule that regulates several differentiation processes including cardiomyogenesis. Here, we report an investigation of the effects of two NO agents (SNAP and DEA/NO), able to activate both cGMP-dependent and -independent pathways, on the cardiomyogenic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs). The cells were isolated, cultured and treated with NO agents. Cardiac- and muscle-specific gene expression was analyzed by indirect immunofluorescence, flow cytometry, RT-PCR and real-time PCR. We found that untreated (control) ADSCs and BM-MSCs expressed some muscle markers and NO-derived intermediates induce an increased expression of some cardiac function genes in BM-MSCs and ADSCs. Moreover, NO agents considerably increased the pro-angiogenic potential mostly of BM-MSCs as determined by VEGF mRNA levels.

Glucocorticoids exert an opposing rapid regulation of glutamate and GABA synaptic inputs to hypothalamic magnocellular neurons via the activation of postsynaptic membrane-associated receptors and the release of retrograde messengers. Glucocorticoids suppress synaptic glutamate release via the retrograde release of endocannabinoids and facilitate synaptic GABA release via an unknown retrograde messenger. Here, we show that the glucocorticoid facilitation of GABA inputs is due to the retrograde release of neuronal nitric oxide and that glucocorticoid-induced endocannabinoid synthesis and nitric oxide synthesis are mediated by divergent G-protein signaling mechanisms. While the glucocorticoid-induced, endocannabinoid-mediated suppression of glutamate release is dependent on activation of the G(alpha)s G-protein subunit and cAMP-cAMP-dependent protein kinase activation, the nitric oxide facilitation of GABA release is mediated by G(beta)gamma signaling that leads to activation of neuronal nitric oxide synthase. Our findings indicate, therefore, that glucocorticoids exert opposing rapid actions on glutamate and GABA release by activating divergent G-protein signaling pathways that trigger the synthesis of, and glutamate and GABA synapse-specific retrograde actions of, endocannabinoids and nitric oxide, respectively. The simultaneous rapid stimulation of nitric oxide and endocannabinoid synthesis by glucocorticoids has important implications for the impact of stress on the brain as well as on neural-immune interactions in the hypothalamus.

Astrocytes, the most abundant glial cell type in the brain, provide metabolic and trophic support to neurons and modulate synaptic activity. In response to a brain injury, astrocytes proliferate and become hypertrophic with an increased expression of intermediate filament proteins. This process is collectively referred to as reactive astrocytosis. Lipocalin 2 (lcn2) is a member of the lipocalin family that binds to small hydrophobic molecules. We propose that lcn2 is an autocrine mediator of reactive astrocytosis based on the multiple roles of lcn2 in the regulation of cell death, morphology, and migration of astrocytes. lcn2 expression and secretion increased after inflammatory stimulation in cultured astrocytes. Forced expression of lcn2 or treatment with LCN2 protein increased the sensitivity of astrocytes to cytotoxic stimuli. Iron and BIM (Bcl-2-interacting mediator of cell death) proteins were involved in the cytotoxic sensitization process. LCN2 protein induced upregulation of glial fibrillary acidic protein (GFAP), cell migration, and morphological changes similar to characteristic phenotypic changes termed reactive astrocytosis. The lcn2-induced phenotypic changes of astrocytes occurred through a Rho-ROCK (Rho kinase)-GFAP pathway, which was positively regulated by nitric oxide and cGMP. In zebrafishes, forced expression of rat lcn2 gene increased the number and thickness of cellular processes in GFAP-expressing radial glia cells, suggesting that lcn2 expression in glia cells plays an important role in vivo. Our results suggest that lcn2 acts in an autocrine manner to induce cell death sensitization and morphological changes in astrocytes under inflammatory conditions and that these phenotypic changes may be the basis of reactive astrocytosis in vivo.