Copper (α)/cis-platinum-loaded nanogels as an adjuvant potentiate disulfiram's antitumor efficacy.

Disulfiram (DSF) is nontoxic and exerts anticancer activity by forming highly toxic chelates via its metabolite diethyldithiocarbamate with transition metal ions. However, there are not enough such ions in the human body to maintain the therapeutic effect. Herein, we report nanogels that complex copper ions or cis-platinum (CDDP) for tumor metal delivery to potentiate DSF's antitumor efficacy. We synthesized zwitterionic poly[N-(3-(methacryloyloxy-2-hydroxy)propyl)]-N-methyl glycine (PGMA-SAR) capable of chelating copper ions or CDDP and formed nanogels with suitable size and zeta potential. The intravenously injected nanogels circulated long in the blood compartment and delivered a high concentration of metal ions to the tumor. Separately administered DSF could sequester the metal ions from the nanogels and form highly cytotoxic complexes with potent in vitro and in vivo anticancer activity. This study provides a new strategy to potentiate DSF in anticancer treatment.

Diethyldithiocarbamate-copper nanocomplex reinforces disulfiram chemotherapeutic efficacy through light-triggered nuclear targeting.

To circumvent the huge cost, long R&D time and the difficulty to identify the targets of new drugs, repurposing the ones that have been clinically approved has been considered as a viable strategy to treat different diseases. In the current study, we outlined the rationale for repurposing disulfiram (DSF, an old alcohol-aversion drug) to treat primary breast cancer and its metastases. Methods: To overcome a few shortcomings of the individual administration of DSF, such as the dependence on copper ions (Cu2+) and limited capability in selective targeting, we here artificially synthesized the active form of DSF, diethyldithiocarbamate (DTC)-Cu complex (CuET) for cancer therapeutics. To achieve a greater efficacy in vivo, smart nanomedicines were devised through a one-step self-assembly of three functional components including a chemically stable and biocompatible phase-change material (PCM), the robust anticancer drug (CuET) and a near-infrared (NIR) dye (DIR), namely CuET/DIR NPs. A number of in vitro assays were performed including the photothermal efficacy, light-triggered drug release behavior, nuclear localization, DNA damage and induction of apoptosis of CuET/DIR NPs and molecular mechanisms underlying CuET-induced repression on cancer metastatic behaviors. Meanwhile, the mice bearing 4T1-LG12-drived orthotopic tumors were employed to evaluate in vivo biodistribution and anti-tumor effect of CuET/DIR NPs. The intravenous injection model was employed to reflect the changes of the intrinsic metastatic propensity of 4T1-LG12 cells responding to CuET/DIR NPs. Results: The rationally designed nanomedicines have self-traceability for bioimaging, long blood circulation time for enhanced drug accumulation in the tumor site and photo-responsive release of the anticancer drugs. Moreover, our data unearthed that CuET/DIR nanomedicines behave like "Trojan horse" to transport CuET into the cytoplasm, realizing substantial intracellular accumulation. Upon NIR laser irradiation, massive CuET would be triggered to release from the nanomedicines and reach a high local concentration towards the nucleus, where the pro-apoptotic effects were conducted. Importantly, our CuET/DIR nanomedicines revealed a pronounced capability to leash breast cancer metastases through inhibition on EMT. Additionally, these nanomedicines showed great biocompatibility in animals. Conclusion: These combined data unearthed a remarkably enhanced tumor-killing efficacy of our CuET nanomedicines through nuclear targeting. This work may open a new research area of repurposing DSF as innovative therapeutic agents to treat breast cancer and its metastases.

Diethyldithiocarbamate encapsulation reduces toxicity and promotes leishmanicidal effect through apoptosis-like mechanism in promastigote and ROS production by macrophage.

The use of compounds from natural or synthetic sources and nanotechnology may represent an alternative to develop new drugs for the leishmaniasis treatment. DETC is an inhibitor of the SOD1 enzyme, which leads to increased ROS production, important for the elimination of Leishmania. Thus, our objective was to assess the leishmanicidal in vitro effect of free Diethydithiocarbamate (DETC) and DETC loaded in beeswax-copaiba oil nanoparticles (DETC-Beeswax-CO Nps) on L. amazonensis forms and elucidate the possible mechanisms involved in the parasite death. DETC-Beeswax-CO Nps presented size below 200 nm, spherical morphology, negative zeta potential, and high encapsulation efficiency. Free DETC reduced the viability of promastigotes and increase ROS production, lower the mitochondrial membrane potential, cause phosphatidylserine exposure, and enhance plasma membrane permeability, in addition to promoting morphological changes in the parasite. Free DETC proved toxic in the assessment of toxicity to murine macrophages, however, the encapsulation of this compound was able to reduce these toxic effects on macrophages. DETC-Beeswax-CO Nps exerted anti-amastigote effect by enhancing the production of ROS, superoxide anion, TNF-α, IL-6, and reduced IL-10 in macrophages. Therefore, free DETC induces antipromastigote effect by apoptosis-like; and DETC-Beeswax-CO Nps exerted anti-leishmanial effect due to pro-oxidant and pro-inflammatory response.

Tumor Homing Reactive Oxygen Species Nanoparticle for Enhanced Cancer Therapy.

Multifunctional nanoparticles that carry chemotherapeutic agents can be innovative anticancer therapeutic options owing to their tumor-targeting ability and high drug-loading capacity. However, the nonspecific release of toxic DNA-intercalating anticancer drugs from the nanoparticles has significant side effects on healthy cells surrounding the tumors. Herein, we report a tumor homing reactive oxygen species nanoparticle (THoR-NP) platform that is highly effective and selective for ablating malignant tumors. Sodium nitroprusside (SNP) and diethyldithiocarbamate (DDC) were selected as an exogenous reactive oxygen species (ROS) generator and a superoxide dismutase 1 inhibitor, respectively. DDC-loaded THoR-NP, in combination with SNP treatment, eliminated multiple cancer cell lines effectively by the generation of peroxynitrite in the cells (>95% cell death), as compared to control drug treatments of the same concentration of DDC or SNP alone (0% cell death). Moreover, the magnetic core (ZnFe2O4) of the THoR-NP can specifically ablate tumor cells (breast cancer cells) via magnetic hyperthermia, in conjunction with DDC, even in the absence of any exogenous RS supplements. Finally, by incorporating iRGD peptide moieties in the THoR-NP, integrin-enriched cancer cells (malignant tumors, MDA-MB-231) were effectively and selectively killed, as opposed to nonmetastatic tumors (MCF-7), as confirmed in a mouse xenograft model. Hence, our strategy of using nanoparticles embedded with ROS-scavenger-inhibitor with an exogenous ROS supplement is highly selective and effective cancer therapy.

Diethyldithiocarbamate loaded in beeswax-copaiba oil nanoparticles obtained by solventless double emulsion technique promote promastigote death in vitro.

Leishmaniasis is considered a neglected tropical disease that represents a Public Health problem due to its high incidence. In the search of new alternatives for Leishmaniasis treatment diethyldithiocarbamate (DETC) has shown an excellent leishmanicidal activity and the incorporation into drug carrier systems, such as solid lipid nanoparticles (SLNs), is very promising. In the present work DETC loaded in beeswax nanoparticles containing copaiba oil were obtained by the double emulsion/melt technique. The nanoparticles were characterized and leishmanicidal activity against L. amazonensis promastigotes forms and cytotoxicity in murine macrophages were evaluated. SLNs presented size below 200 nm, spherical morphology, negative charge surface, high encapsulation efficiency, above 80%, and excellent stability. Moreover, Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) analyses were performed to evaluate the chemical structure and possible interactions between DETC and SLNs. SLNs provided a protection for DETC, decreasing its cytotoxic effects in macrophages, which led to an improvement in the selectivity against the parasites, which almost doubled from free DETC (11.4) to DETC incorporated in SLNs (18.2). These results demonstrated that SLNs had a direct effect on L. amazonensis promastigotes without affect the viability of macrophage cell, can be a promising alternative therapy for the cutaneous treatment of L. amazonensis.

Antibacterial activity of disulfiram and its metabolites.

AIMS: Disulfiram (Antabuse™) and its metabolites formed in vivo were evaluated as antibacterial agents against thirty species of Gram-positive and Gram-negative bacteria. The synergistic potential of disulfiram (DSF) and metabolite diethyldithiocarbamate (DDTC) with approved antibiotics were also compared by isobologram (checkerboard) analysis. METHODS AND RESULTS: Standard microdilution susceptibility testing showed that most DSF metabolites did not possess appreciable antibacterial activity except for DDTC in Bacillus anthracis. Checkerboard studies revealed similarities between the combination drug effects of DSF and DDTC with standard antibiotics. CONCLUSIONS: It was concluded from the susceptibility data that the metabolites would not extend the antibacterial spectrum of DSF in vivo. The data also suggest that the DDTC by-product of DSF metabolism potentiates the antibacterial activity of DSF as both a standalone and combination agent. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a greater understanding of the antibacterial effects of Antabuse and its metabolites. This research also demonstrates the potential application of DSF as an antibiotic adjuvant for the treatment of resistant staph infections.

Evaluation of the selective toxic effect of the charge switchable diethyldithiocarbamate-loaded nanoparticles between hepatic normal and cancerous cells.

Liver cancer is mainly originated by cancer stem cells (CSCs). Due to difference in pH between normal and tumor cell microenvironments, targeting hepatic CSCs exploiting pH-dependent charge switchable nanoparticles (NPs) is extremely required to limit nonselective toxicity to normal hepatocytes (NHCs) and to completely eliminate the root of cancer origin. In this study, NPs were prepared from cationic chitosan and then coated with anionic albumin namely uncoated and coated NPs, respectively. Both NPs were loaded with diethyldithiocarbamate (DDC) which is an inhibitor of the critical enzyme, aldehyde dehydrogenase (ALDH) 1A1, for CSCs survival. The charge switchable of coated DDC-loaded NPs in neutral and acidic pH (-19 and +28.5 mv, respectively) was illustrated. This special privilege of coated NPs mediated DDC releasing in a slightly acidic pH (tumor microenvironment) rather than a neutral pH (microenvironment of normal cells). Thence, these coated NPs showed the highest selective apoptosis-mediated toxicity only in murine hepatoma cells (Hepa) that may attribute to suppression of NF-κB expression and ALDH1A1 activity, subsequently collapsing 89.7% CD133+CSCs. These new findings declare that coated NPs could be promising safe selective anticancer drug for targeting hepatic CSCs and that requires additional future investigations using animal models of liver cancer.

Rhus verniciflua Stokes Extract and Its Flavonoids Protect PC-12 Cells against H2O2-Induced Cytotoxicity.

Rhus verniciflua Stokes (RVS), an herbal medicine found in East Asia, was extracted and further fractionated to investigate its antioxidant capacity and neuroprotective effects. The RVS ethyl acetate (EtOAc) fraction had the highest level of total phenolics and antioxidant capacity among all solvent fractions tested. Pretreatment of PC-12 cells with the EtOAc fraction effectively attenuated H2O2-induced oxidative damage. Furthermore, the EtOAc fraction significantly attenuated caspase-3 activity, resulting in inhibition of H2O2-induced apoptosis. We identified and quantified fustin, sulfuretin, and butein in the EtOAc fraction using accurate mass quadrupole time-of-flight mass spectrometry and reversed-phase high-performance liquid chromatography. The intracellular antioxidant capacity and superoxide dismutase (SOD) activity were significantly increased in PC-12 cells treated with the EtOAc fraction and with individual flavonoids. When cells were pretreated with the EtOAc fraction or individual flavonoids and then co-incubated with diethyldithiocarbamic acid (an inhibitor of SOD activity), cell viability against H2O2-induced oxidative stress was attenuated. These results suggest that the RVS EtOAc fraction and its flavonoid constituents protect PC-12 cells against H2O2-induced neurotoxicity through their antioxidant properties.

Investigations on GSK-3ß/NF-kB signaling in stress and stress adaptive behavior in electric foot shock subjected mice.

The present study was designed to explore the role of GSK-3ß and NF-kB signaling in electric foot shock-induced stress and stress adaptation. Mice were subjected to foot shocks of 0.5mA intensity and 1s duration of 1h to produce acute stress. Animals were exposed to the same stressor for 5 days to induce stress adaptation. The behavioral alterations were assessed using the actophotometer, hole board, open field and social interaction tests. The serum corticosterone levels were assessed as a marker of the HPA axis. The levels of total GSK-3ß, p-GSK-3ß-S9 and p-NF-kB were determined in the hippocampus, frontal cortex and amygdala. Acute electric foot shock stress produced behavioral and biochemical changes; decreased the levels of p-GSK-3ß-S9, produced no change in total GSK-3ß levels and increased p-NF-kB levels in the brain. However, repeated exposure of foot shock stress restored the behavioral and biochemical changes along with normalization of p-GSK-3ß-S9 and p-NF-kB levels. Administration of AR-A01, a selective GSK-3ß inhibitor, or diethyldithiocarbamic acid (DDTC), a selective NF-kB inhibitor, diminished acute stress-induced behavioral and biochemical changes. Furthermore, AR-A014418 normalized acute stress-induced alterations in p-GSK-3ß-S9 and p-NF-kB levels, however, DDTC selectively restored NF-kB levels without any change in p-GSK-3ß-S9 levels. It probably suggests that NF-kB is a downstream mediator of the GSK-3 signaling cascade. It may conclude that acute stress associated decrease in p-GSK-3ß-S9 and increase in p-NF-kB levels in the brain contribute in the development of behavioral and biochemical alterations and normalization of GSK-3ß/NF-kB signaling may contribute in stress adaptive behavior in response to repeated electric foot shock-subjected mice.

A comparative study of stress-mediated immunological functions with the adjuvanticity of alum.

The efficacy of a vaccine is generally dependent on an adjuvant, which enhances the immune functions and alum has been widely used in human immunization. Alum activates the intracellular stress sensors inflammasomes, but whether these are responsible for the adjuvanticity is controversial. The objectives of this investigation were to examine the hypothesis that alum-mediated adjuvanticity is a function of stress and conversely that stress agents will elicit adjuvanticity. The investigation was carried out in BALB/c mice by SC immunization with ovalbumin (OVA) mixed with alum. This elicited inflammasomes, with significant activation of caspase 1, production of IL-1ß, and adjuvanticity, demonstrated by enhancing OVA-specific serum IgG antibodies, CD4(+) T cells, and proliferation. The novel finding that alum induced HSP70 suggests that stress is involved in the mechanism of adjuvanticity. This was confirmed by inhibition studies with PES (phenylethynesulfonamide), which disrupts inducible HSP70 function, and inhibited both inflammasomes and the adjuvant function. Parallel studies were pursued with an oxidative agent (sodium arsenite), K-releasing agent (Gramicidin) and a metal ionophore (dithiocarbamate). All 3 stress agents induced HSP70, inflammasomes, and the adjuvant functions. Furthermore, up-regulation of membrane associated IL-15 on DC and CD40L on T cells in the animals treated with alum or the stress agents mediate the interactions between splenic CD11c DC and CD4(+) or CD8(+) T cells. The results suggest that the three stress agents elicit HSP70, a hallmark of stress, as well as inflammasomes and adjuvanticity, commensurate with those of alum, which may provide an alternative strategy in developing novel adjuvants.

Recovery of axonal myelination sheath and axonal caliber in the mouse corpus callosum following damage induced by N,N-diethyldithiocarbamate.

Disulfiram is an aldehyde dehydrogenase inhibitor used for the treatment of alcohol dependence and of cocaine addiction. It has been demonstrated that subchronic administration of disulfiram or N,N-diethyldithiocarbamate (DEDTC), the main derivative of disulfiram, to rats can produce central-peripheral distal axonopathy. However, few data regarding the axonal effects of these compounds in the central nervous system exist. Our previous studies have revealed DEDTC-induced axonal damage in the mouse brain during the course of postnatal development, together with alterations in axonal pathfinding and in the myelination process, with partial recovery during the post-treatment period. In order to gather new data about how this axonal damage and recovery occurs in the central nervous system, we performed an ultrastructural analysis of the axons located in the corpus callosum from mice treated with DEDTC during postnatal development. The axonal caliber throughout the axonal area, the maximum axonal diameter, the maximum fiber diameter, and the axonal circularity, at different postnatal stages [from postnatal day (P)9 to P30], were analyzed. In addition, parameters related to the myelinization process (number of myelinated axons, sheath thickness, and the ratio of myelinated axons to total axons) were evaluated. A reduction in the average value of axonal caliber during treatment and a delay in the axonal myelination process were detected. Whereas early recovery of individual axons occurred after treatment (P22), complete recovery of myelinated axons occurred at late postnatal stages (P42). Therefore, chronic treatment with dithiocarbamates requires periods of rest to encourage the recovery of myelinated axons.

Survival role of superoxide dismutase 1 on human granulosa luteinized cells in vitro.

OBJECTIVE: Deleterious effects of free radicals do not only result from the amount of free radicals produced but also are related to the efficiency and to the activities of enzymatic antioxidant systems. We investigated the effect of exogenous superoxide dismutase (SOD1) or Cu-chelating agent diethyldithiocarbamate (DDC) on the apoptosis (caspase-3 activity) of human granulosa luteinized cells (hGLC) in vitro. METHODS: The effects of SOD1 and DDC were studied using in vitro culture system, caspase-3 and the total SOD activity in hGLCs were measured using AcDEVD-PNA substrate and Beaushap and Fridovich methods, respectively, after 48 h of the culture period. RESULTS: The activity of SOD1 was the lowest in GLCs treated with 100 µM DDC as compared to control cells and to the cells supplemented with Cu, Zn-SOD or DDC (10 µM). The effect of DDC was associated with elevated caspase-3 activity as compared to control cells. CONCLUSIONS: It was demonstrated for the first time that the supplementation of cultured hGLCs with Cu2+, Zn2+-SOD (200 U/ml) maintains the viability of hGLCs via caspase-3 suppression.

Preparation of the core-shell structure adriamycin lipiodol microemulsions and their synergistic anti-tumor effects with diethyldithiocarbamate in vivo.

We prepared the core-shell structure adriamycin lipiodol microemulsions (ADM-CSLMs) and evaluated their in vivo antitumor effects in combination with Diethyldithiocarbamate (DDC). Two types of ADM-CSLMs, adriamycin liposome-lipiodol microemulsion(ADM-LLM) and adriamycin microsphere lipiodol microemulsion (ADM-MLM), were prepared through the emulsification method. The drug loading and encapsulation efficiency of ADM-CSLMs were measured by the high-performance liquid chromatograph (HPLC). The size and shape of the ADM-CSLMs were determined by an atom force microscopy (AFM), a transmission electron microscopy (TEM), and a particle size analyzer, respectively. The synergistic effects of DDC and ADM-CSLMs for cancer treatment of carcinoma drug-resistance cell was evaluated by the MTT method, the activation of superoxide dismutase (SOD) was detected by chemiluminescence, and the ADM accumulation in cells was measured by flow cytometry. Walker-256 carcinoma was transplanted to the livers of the male SD rats, ADM-CSLMs were administrated to the livers of the rats by intervention hepatic artery embolization through microsurgery. The tumor growth and animal survival were evaluated. The results show that the average diameter of ADM-LLM and ADM-MLM were 4.23 ± 1.2 µm and 4.67 ± 1.4 µm, respectively, and their ADM encapsulation efficiency were 83.7% and 87.2% with respect to loading efficiency of 82 µg/ml and 91 µg/ml. The tumor growth and animal survival in two of the ADM-CSLMs combined with DDC groups were significantly higher than that of ADM only treatment, ADM liposome combined with DDC (P < 0.01), as well as the ADM microsphere combined with DDC (P < 0.01). Therefore, ADM-CSLMs are useful carriers for the treatment of carcinoma and their anti-tumor effect can be enhanced by DDC in a suitable concentration.

Bioavailability and catalytic properties of copper and iron for Fenton chemistry in human cerebrospinal fluid.

A breakdown in homeostasis of redox-active metals represents an important factor for neurodegeneration. We have used EPR spectroscopy and BMPO spin-trap to investigate the catalytic properties and ligand modulation of redox activity of copper and iron in human cerebrospinal fluid (CSF). In contrast to iron, copper supplementation provoked a statistically significant increase in hydroxyl free radical generation in CSF treated with H(2)O(2). However, in a binary copper/iron containing Fenton system, iron catalytically activated copper. The chelator EDTA, which represents a model of physiological metal ligands, completely prevented copper's redox activity in CSF, while iron chelation led to a significant increase in hydroxyl radical generation, indicating that copper and iron do not only have diverse catalytic properties in the CSF but also that their redox activities are differently modulated by ligands. The application of DDC reduced hydroxyl radical generation in the CSF containing catalytically active metals (free Cu(2+) or Fe(3+)-EDTA complex). We conclude that chelators, such as DDC, are capable of preventing the prooxidative activity of both metals and may be suitable for reducing hydroxyl radical formation in certain pathophysiological settings.

DETC induces Leishmania parasite killing in human in vitro and murine in vivo models: a promising therapeutic alternative in Leishmaniasis.

BACKGROUND: Chemotherapy remains the primary tool for treatment and control of human leishmaniasis. However, currently available drugs present serious problems regarding side-effects, variable efficacy, and cost. Affordable and less toxic drugs are urgently needed for leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate, by microscopy and viability assays, that superoxide dismutase inhibitor diethyldithiocarbamate (DETC) dose-dependently induces parasite killing (p<0.001) and is able to "sterilize" Leishmania amazonensis infection at 2 mM in human macrophages in vitro. We also show that DETC-induced superoxide production (p<0.001) and parasite destruction (p<0.05) were reverted by the addition of the antioxidant N-acetylcysteine, indicating that DETC-induced killing occurs through oxidative damage. Furthermore, ultrastructural analysis by electron microscopy demonstrates a rapid and highly selective destruction of amastigotes in the phagosome upon DETC treatment, without any apparent damage to the host cell, including its mitochondria. In addition, DETC significantly induced parasite killing in Leishmania promastigotes in axenic culture. In murine macrophages infected with Leishmania braziliensis, DETC significantly induced in vitro superoxide production (p = 0.0049) and parasite killing (p = 0.0043). In vivo treatment with DETC in BALB/C mice infected with Leishmania braziliensis caused a significant decrease in lesion size (p<0.0001), paralleled by a 100-fold decrease (p = 0.0087) in parasite burden. CONCLUSIONS/SIGNIFICANCE: Due to its strong leishmanicidal effect in human macrophages in vitro, its in vivo effectiveness in a murine model, and its previously demonstrated in vivo safety profile in HIV treatment, DETC treatment might be considered as a valuable therapeutic option in human leishmaniasis, including HIV/Leishmania co-infection.

Effect of diethyldithiocarbamate (DDC) and ticlopidine on CYP1A2 activity and caffeine metabolism: an in vitro comparative study with human cDNA-expressed CYP1A2 and liver microsomes.

The aim of the present study was to test the effect of diethyldithiocarbamate (DDC), which is regarded as a cytochrome P450 (CYP) CYP2A6 and CYP2E1 inhibitor, and ticlopidine, an efficient CYP2B6, CYP2C19 and CYP2D6 inhibitor, on the activity of human CYP1A2 and the metabolism of caffeine (1-N-, 3-N- and 7-N-demethylation, and C-8-hydroxylation). The experiment was carried out in vitro using human cDNA-expressed CYP1A2 (Supersomes) and human pooled liver microsomes. The effects of DDC and ticlopidine were compared to those of furafylline (a strong CYP1A2 inhibitor). A comparative in vitro study provides clear evidence that ticlopidine and DDC, applied at concentrations that inhibit the above-mentioned CYP isoforms, potently (as compared to furafylline) inhibit human CYP1A2 and caffeine metabolism, in particular 1-N- and 3-N-demethylation.

Detection of phenoloxidase activity in early stages of the Pacific oyster Crassostrea gigas (Thunberg).

The presence of phenoloxidase (PO) activity was detected in different developmental stages of the Pacific oyster, Crassostrea gigas. A significant reduction in PO activity was observed from the 6h embryo stage to the day 11 larvae by spectrophotometry. A progressive increase was also observed from the day 13 larvae right through to the juvenile stage. The microscopy studies with '6h embryo' and adult samples confirmed the presence of PO activity. Various modulators of PO activity were used to study the triggering of pro-phenoloxidase (proPO) activating system of C. gigas but also to confirm the exact nature of the monitored activity. The enzyme activation mechanisms appear to differ with the developmental stage: bacterial lipopolysaccharides constitute an early elicitor of the proPO-PO system, whereas a purified trypsin triggers proPO-PO system in C. gigas spat. Phenoloxidase activity was totally suppressed by PO-specific inhibitors such as beta-2-mercaptoethanol, sodium diethyldithiocarbonate and tropolone. This study demonstrated the selective response of PO-like activity by different elicitors and suggested that proPO-PO activating system, which is supposed to play an important function in non-self recognition and host immune reactions in oyster, is expressed early in the Pacific oyster, C. gigas.

Inhibition of superoxide dismutase induces collagen production in cardiac fibroblasts.

BACKGROUND: The aim of this study was to determine whether inhibition of superoxide dismutase (SOD) with diethyldithiocarbamic acid (DETC) could affect the collagen production, the mRNA and protein expression of collagen types I and III, and fibronectin in control and angiotensin II (ANG II)-treated cardiac fibroblasts. Its effect was compared with the SOD mimetics tempol and EUK-8 and with polyethyleneglycol (PEG)-SOD. METHODS: Cardiac fibroblasts were cultured to confluence, incubated in serum-free Dulbecco's modified Eagle's medium for 24 h, preincubated with(out) the tested inhibitors for 1 h and further incubated with(out) ANG II (1 micromol/l) for 24 h. RESULTS: DETC dose-dependently inhibited the activity of CuZn-SOD in cardiac fibroblasts. Superoxide anion production was increased by DETC and decreased by tempol in control and ANG II-treated fibroblasts. DETC also reduced the intracellular generation of reactive oxygen species (ROS) (such as H2O2, hydroxyl radicals, hydroperoxides) in control and ANG II-treated fibroblasts, whereas tempol reduced the ROS production only in ANG II-treated fibroblasts. ANG II and DETC stimulated the collagen production and the collagen I and fibronectin content in fibroblasts. The SOD mimetics tempol and EUK-8 as well as PEG-SOD reduced the collagen production. ANG II and DETC stimulated the tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 levels, whereas tempol decreased the TIMP-2 content in control and ANG II-treated fibroblasts. Matrix metalloproteinase (MMP)-1 level was reduced by ANG II and DETC and increased by tempol. CONCLUSION: These data suggest a vital role of SOD and the formed ROS in the accumulation of collagen in cardiac fibroblasts.

Mechanisms of oxidative stress in porcine oocytes and the role of anti-oxidants.

The mechanisms of oxidative stress in in vitro maturing porcine oocytes and the effects of anti-oxidant supplementation of the medium in ameliorating these effects were investigated in the present study. In addition to intracellular reduced glutathione (GSH) concentrations and DNA fragmentation, the present study focused on superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase activity. The anti-oxidants used were N-acetylcysteine (NAC) and its derivative NAC-amide (NACA). The results indicate that when SOD is inhibited, supplementation of the maturarion medium with 1.5 mm NAC or NACA compensates for the decrease in SOD activity by reducing the degree of DNA fragmentation (P < 0.05). When GPx is inhibited, supplementation of the maturarion medium with 1.5 mm NAC alleviates the effects of no GPx activity, as indicated by a decrease in the degree of DNA fragmentation (P < 0.05). When the maturarion medium was supplemented with 1.5 mm NACA, intracellular GSH concentrations decreased (P < 0.05) and SOD and catalase activities increased (P < 0.05) along with the degree of DNA fragmentation. These results indicate that the mechanisms of alleviating oxidative stress in porcine oocytes are very complex and supplementing maturing oocytes with anti-oxidants may enhance enzyme activities and eliminate free radicals.