salicylates and copper-bis(3-5-diisopropylsalicylate)

Oxidative stress has been suggested as a potential contributor to the development of diabetic complications. In this study, we investigated the protective effect of a strong antioxidant copper complex against streptozotocin (STZ)-induced diabetes in animals. Out of four copper complexes used, copper(II) (3,5-diisopropyl salicylate)4 (Cu(II)DIPS) was found to be the most potent antioxidant-copper complex. Pretreatment with Cu(II)DIPS (5 mg/kg) twice a week prior to the injection of streptozotocin (50 mg/kg) has reduced the level of hyperglycemia by 34 % and the mortality rate by 29 %. Injection of the same dosage of the ligand 3,5-diisopropyl salicylate has no effect on streptozotocin-induced hyperglycemia. The same copper complex has neither hypoglycemic activity when injected in normal rats nor antidiabetic activity when injected in STZ-induced diabetic rats. The protective effect of Cu(II)DIPS could be related to its strong antioxidant activity compared to other copper complexes median effective concentration (MEC) = 23.84 μg/ml and to Trolox MEC = 29.30 μg/ml. In addition, it reduced serum 8-hydroxy-2'-deoxyguanosine, a biomarker of oxidative DNA damage, by 29 %. This effect may explain why it was not effective against diabetic rats, when β Langerhans cells were already destroyed. Similar protective activities were reported by other antioxidants like Trolox.

Topics: Animals; Antioxidants; Blood Glucose; Chromans; Coordination Complexes; Copper; Diabetes Mellitus, Experimental; Male; Rats; Rats, Sprague-Dawley; Salicylates

Renin-angiotensin system (RAS) plays a central role in the development and progression of diabetic nephropathy. There is a growing body of evidence that advanced glycation end products (AGE) and inflammation contribute to diabetic nephropathy as well. However, the pathophysiological crosstalk between the RAS and AGE in inflammatory reactions in glomerular endothelial cells (ECs) remains unknown. In this study, we examined whether and how irbesartan, an angiotensin II type 1 receptor blocker (ARB), inhibited the AGE-induced vascular cell adhesion molecule-1 (VCAM-1) gene expression in cultured human glomerular ECs. Irbesartan or an anti-oxidant N-acetylcysteine inhibited the AGE-induced increase in reactive oxygen species (ROS) generation and subsequently blocked up-regulation of VCAM-1 mRNA levels in glomerular ECs. AGE significantly stimulated angiotensin II production by glomerular ECs. Furthermore, irbesartan completely suppressed up-regulation of VCAM-1 mRNA levels in AGE plus angiotensin II-exposed glomerular ECs. Our present data suggest that there exists a crosstalk between the RAS and AGE in inflammatory reactions in glomerular ECs. Irbesartan may play a protective role against diabetic nephropathy by blocking the deleterious effects of AGE-elicited angiotensin II and ROS.

Topics: Acetylcysteine; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Antioxidants; Biphenyl Compounds; Cells, Cultured; Endothelial Cells; Gene Expression; Glycation End Products, Advanced; Humans; Hydrogen Peroxide; Irbesartan; Kidney Glomerulus; Peptidyl-Dipeptidase A; Renin; Salicylates; Serum Albumin, Bovine; Superoxides; Tetrazoles; Up-Regulation; Vascular Cell Adhesion Molecule-1

Opposing effects have been ascribed to nitric oxide (NO) on retinal microvascular survival. We investigated whether changes in the redox state may contribute to explain apparent conflicting actions of NO in a model of oxygen-induced retinal vasoobliteration. Retinal microvascular obliteration was induced by exposing 7-day-old rat pups (P7) for 2 or 5 days to 80% O(2). The redox state of the retina was assessed by measuring reduced glutathione and oxidative and nitrosative products malondialdehyde and nitrotyrosine. The role of NO on vasoobliteration was evaluated by treating animals with nitric oxide synthase (NOS) inhibitors (N-nitro-l-arginine; L-NA) and by determining NOS isoform expression and activity; the contribution of nitrosative stress was also determined in animals treated with the degradation catalyst of peroxynitrite FeTPPS or with the superoxide dismutase mimetic CuDIPS. eNOS, but not nNOS or iNOS, expression and activity were increased throughout the exposure to hyperoxia. These changes were associated with an early (2 days hyperoxia) decrease in reduced glutathione and increases in malondialdehyde and nitrotyrosine. CuDIPS, FeTPPS, and L-NA treatments for these 2 days of hyperoxia nearly abolished the vasoobliteration. In contrast, during 5 days exposure to hyperoxia when the redox state rebalanced, L-NA treatment aggravated the vasoobliteration. Interestingly, VEGFR-2 expression was respectively increased by NOS inhibition after short-term (2 days) exposure to hyperoxia and decreased during the longer hyperoxia exposure. Data disclose that the dual effects of NO on newborn retinal microvascular integrity in response to hyperoxia in vivo depend on the redox state and seem mediated at least in part by VEGFR-2.

Topics: Animals; Animals, Newborn; Antioxidants; Glutathione; Isoenzymes; Malondialdehyde; Metalloporphyrins; Microcirculation; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxidation-Reduction; Oxidative Stress; Oxygen; Rats; Rats, Sprague-Dawley; Retina; Retinal Diseases; Retinal Vessels; Salicylates; Tyrosine; Vascular Endothelial Growth Factor Receptor-2

This research was performed to determine whether or not treatment of burn-injured rats with Cu(II)2(3,5-diisopropylsalicylate)4(Cu(II)2(3,5-DIPS)4) facilitated recovery from burn-injury. Four groups of adult male rats received a standard skin burn 1 h before an initial subcutaneous treatment which was continued daily for three days with either 0, 5, 10 or 20micromol Cu(II)2(3,5-DIPS)4/kg body mass. A fifth group was given no treatment. A sixth group served as a non-burn-injured non-treated normal control group. At 3 h and on days 1, 2, 3, 7 and 14 post-burn-injury blood samples were obtained from rats in all groups for the determination of leukocyte, platelet and erythrocyte counts, clotting times, hemoglobin and hematocrit values. Total protein and middle mass peptides in plasma, as well as plasma lipid and erythrocyte membrane peroxidation products were determined on days 7 and 14. Burn wound healing and body mass were determined daily from day 0 to 6 with a notation of crust rejection by day 14. Treatment with Cu(II)2(3,5-DIPS)4 produced effects consistent with a facilitation of Cu-dependent immune-mediated physiological inflammatory responses to burn injury. It is concluded that treatment of burn injury with Cu(II)2(3,5-DIPS)4 supports Cu-dependent physiological responses involved in overcoming burn injury, which may have been further optimized by continued treatment beyond day 2, the last day of treatment.

Topics: Animals; Blood Proteins; Burns; Erythrocyte Count; Erythrocyte Membrane; Hematocrit; Hemoglobins; Leukocyte Count; Lipid Peroxidation; Male; Platelet Count; Rats; Rats, Inbred Strains; Salicylates; Whole Blood Coagulation Time

The ability of Cu(II)(2)(3,5-diisopropylsalicylate)(4), CuDIPS, which exhibits superoxide dismutase (SOD)-like activity, to prevent cisplatin-induced nephrotoxicity was examined in rats. Rats were divided into four groups and treated as follows: (i) vehicle control; (ii) cisplatin (16 mg/kg, intraperitoneally); (iii) CuDIPS (10 mg/kg, intraperitoneally); and (iv) cisplatin plus CuDIPS. Rats were sacrificed 3 days post-treatment. Cisplatin alone resulted in significantly increased plasma creatinine and urea. Administration of 10 mg/kg CuDIPS prevented the cisplatin-induced elevation of plasma creatinine and urea and protected against kidney damage. Relative to controls, rats that received cisplatin treatment displayed a decrease of reduced glutathione (GSH) and elevated platinum and thiobarbituric acid reactive substances (TBARS) levels in the kidney. In comparison with controls, activities of antioxidant enzymes (SOD, CAT, GSH-Px and GSH-Rd) were also reduced in the kidney of rats treated with cisplatin. Administration of 10 mg/kg CuDIPS prevented cisplatin-induced alterations in renal platinum, GSH, TBARS, and antioxidant enzyme activities. This study suggests that the protection offered by CuDIPS against cisplatin-induced nephrotoxicity is partly related to maintenance of renal antioxidant systems.

Topics: Animals; Antineoplastic Agents; Antioxidants; Body Weight; Catalase; Cisplatin; Creatinine; Glutathione; Glutathione Peroxidase; Glutathione Reductase; Kidney; Male; Platinum; Rats; Salicylates; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Urea

To evaluate the effects of ultraviolet-induced environmental trauma on human skin cells, primary normal human epidermal keratinocytes were exposed to ultraviolet-B radiation (290-320 nm). We found that relatively low doses of ultraviolet-B (62.5-500 mJ per cm2) caused dose-dependent increases in 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a biomarker of oxidative DNA damage. Unirradiated normal human epidermal keratinocytes contained 1.49 (+/- 0.11) 8-oxo-dG per 10(6) 2'-deoxyguanosine (dG) residues in cellular DNA, which increased linearly to as high as 6.24 (+/- 0.85) 8-oxo-dG per 10(6) dG after irradiation with 500 mJ per cm2. Further, this oxidative damage was reduced by 60.7% when the cells were pretreated with 1 mM mannitol. As hydrogen peroxide (H2O2) is known to be generated during oxidative stress, its accumulation in ultraviolet-B-irradiated normal human epidermal keratinocytes was also assessed and correlated to 8-oxo-dG formation. An ultraviolet-B-induced increase in H2O2 was observed in normal human epidermal keratinocytes and its production was inhibited by the addition of catalase. Based on the ability of a neutral molecule like H2O2 to permeate membranes, our data indicate that, after ultraviolet-B irradiation, H2O2 migrates from the cytosol to the nucleus where it participates in a Fenton-like reaction that results in the production of hydroxyl radicals (OH*), which may then cause 8-oxo-dG formation in cellular DNA. This conclusion is supported by our data showing that OH* scavengers, such as mannitol, are effective inhibitors of oxidative DNA base damage. Although increased levels of 8-oxo-dG were previously found in immortalized mouse keratinocytes exposed to ultraviolet-B radiation, we now report the induction of 8-oxo-dG in normal human skin keratinocytes at ultraviolet-B doses relevant to human skin exposure.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Antineoplastic Agents; Antioxidants; beta Carotene; Cells, Cultured; Deoxyguanosine; Diuretics, Osmotic; DNA Damage; Epidermal Cells; Epidermis; Free Radical Scavengers; Humans; Hydrogen Peroxide; Hydroxyl Radical; Keratinocytes; Mannitol; Oxidation-Reduction; Salicylates; Thiourea; Ultraviolet Rays

To compare the potency of 2 cytokines, interleukin 17 (IL-17) and IL-1beta, on rat cartilage proteoglycan synthesis with special attention to nitric oxide (NO) and peroxynitrite formation.. Chondrocytes in alginate beads were stimulated with human recombinant (rh) IL-17 (0.03 to 300.0 ng/ml) and/or rhIL-1beta (0.25 to 25.0 ng/ml) in the presence or not of L-NMMA or CuDips. Alternatively, rats were injected with either IL-17 (10.0 micro g) or IL-1beta (1.0 micro g) into each knee joint. NO concentrations were determined by a spectrofluorimetric assay, proteoglycan synthesis by 35SO4-2 incorporation, peroxynitrite generation by immunostaining for 3-nitrotyrosine, and IL-1beta mRNA expression by reverse transcription-polymerase chain reaction.. IL-17 inhibited proteoglycan synthesis and increased NO production, both in vitro and in vivo, without inducing expression of IL-1beta mRNA in cartilage. Additive effects were observed when IL-17 was combined with low concentrations of IL-1. Surprisingly, a similar NO synthesis between IL-1 and IL-17 led to a less suppressive effect of IL-17 on cartilage anabolism than with IL-1. Both in vitro and in vivo, peroxynitrite formation was extensive with IL-1beta, but negligible or nonexistent with IL-17. L-NMMA and CuDips completely corrected the suppressive effect of IL-1beta on proteoglycan synthesis, unlike with IL-17.. These data showed that NO is weakly involved in the IL-17 mediated inhibition of proteoglycan synthesis in rat. NO overload may not be predictive of any inhibitory effect on cartilage anabolism, but instead superoxide is a key regulator of NO contribution to chondrocyte dysfunction. Since IL-17 is a NO-producing cytokine with additive effects when combined with IL-1, it may play a pivotal role in cartilage destruction during rheumatoid arthritis, for which infiltrating cells produce high levels of superoxide and proinflammatory cytokines.

Topics: Animals; Cartilage, Articular; Cells, Cultured; Chondrocytes; Dose-Response Relationship, Drug; Drug Combinations; Hindlimb; Injections, Intra-Articular; Interleukin-1; Interleukin-17; Joints; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Peroxynitrous Acid; Proteoglycans; Rats; Rats, Wistar; Recombinant Proteins; RNA, Messenger; Salicylates

Nitric oxide (NO) is thought to mediate most effects of interleukin-1 (IL-1) on cartilage. In vitro evidence includes the decreased synthesis of extracellular matrix components, the abnormal cell renewal, the decreased production of IL-1 receptor antagonist, the induction of apoptosis and the enhanced sensitivity of chondrocytes to oxidative stress. Studies in NOS2(-/-) mice or administration of NO synthase inhibitors in animal models of joint disorders have confirmed its potent pathophysiological role in cartilage. Using L-NMMA (1 mM), as a NO synthase inhibitor, and CuDips (10 microM), as a SOD mimetic, we provide evidence that the inhibitory potency of IL-1beta on proteoglycan synthesis and its stimulating effect on COX-2 activity depend both on NO and O2-* production. Peroxynitrite formation is further demonstrated by the occurrence of 3-nitrotyrosines in chondrocytes stimulated in vitro with 2.5 ng/ml IL-1 and in femoral condyles of rats injected locally with 1 microg IL-1. Preliminary data suggest that such contribution of reactive oxygen species is not shared in common by IL-17, another NO-producing cytokine. We conclude that superoxide is a key modulator of NO-mediated effects in chondrocyte stimulated with IL-1 and that a combined therapy with NO synthase inhibitors and antioxidants may be promising for a full cartilage protection.

Topics: Animals; Cartilage Diseases; Cartilage, Articular; Cells, Cultured; Chondrocytes; Depression, Chemical; Enzyme Inhibitors; Free Radical Scavengers; Humans; Interleukin-1; Interleukin-17; Male; Models, Animal; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Proteoglycans; Rats; Rats, Wistar; Salicylates

Previous work from our laboratory indicated that the bile salt sodium deoxycholate (NaDOC) induced apoptosis in cultured cells and in normal goblet cells of the colonic mucosa. We also reported that the normal-appearing flat mucosa of patients with colon cancer exhibited apoptosis resistance. Using immunofluorescence in conjunction with confocal microscopy, we now report that high physiological concentrations (0.5 mM) of NaDOC result in the formation of nitrotyrosine residues, a footprint for the formation of reactive nitrogen species, including peroxynitrite, in plasma membrane-associated proteins of HT-29 cells. Because peroxynitrite is formed from the reaction between nitric oxide and superoxide anion, we specifically looked at the role of nitric oxide and superoxide anion in NaDOC-induced apoptosis. Pretreatment of cells with the inhibitor/antioxidants, N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, copper (II) 3,5-diisopropyl salicylate hydrate, a superoxide dismutase mimetic compound, and Trolox, a water-soluble analog of alpha-tocopherol, alone or in combination, sensitized cells to apoptosis induced by 0.5 mM NaDOC. These results suggest that nitric oxide may be part of a signaling pathway that is responsible for apoptosis resistance. The results also indicate that nitric oxide does not appear to protect cells against NaDOC-induced apoptosis by scavenging superoxide anion.

Topics: Apoptosis; Bile Acids and Salts; Colonic Neoplasms; Deoxycholic Acid; Enzyme Inhibitors; Fluorescent Antibody Technique; Free Radical Scavengers; Humans; Microscopy, Confocal; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Salicylates; Superoxides; Tumor Cells, Cultured; Tyrosine

The copper(II) complex of 3,5-diisopropylsalicylate is a lipophilic water-insoluble binuclear complex, Cu(II)2(3,5-DIPS)4, that has attracted interest because of a wide range of pharmacological activities. This study was undertaken to examine bonding interactions between the complex and human serum albumin (HSA) to help elucidate the mode of transport of the complex in vivo. Electron paramagnetic resonance, numerical magnetic resonance and UV-visible absorption spectroscopic studies were performed using 200 microM aqueous solutions (pH 7.5) of HSA to which had been added up to three molar equivalents of CuCl2, CuSO4, or Cu(II)2(3,5-DIPS)4. Both EPR and UV-visible spectra demonstrated the presence of more than one copper bonding site on HSA, and proton NMR spectra showed that the 3,5-DIPS ligand is also bonded to HSA. These results indicate that there is no observable direct coordination of the ligand to copper in the presence of HSA, and that the majority of the copper and 3,5-DIPS bond to HSA at separate sites. Addition of solid Cu(II)2(3,5-DIPS)4 to HSA at pH 7.5 similarly resulted in spectra suggest that there are no ternary Cu(II)(3,5-DIPS), Cu(II)(3,5-DIPS)2, or Cu(II)2(3,5-DIPS)4 complexes formed with HSA. It is concluded that any ternary complexes formed in the presence of HSA are below the spectroscopic detection limits and represent less than 5% of the total copper.

Topics: Electron Spin Resonance Spectroscopy; Humans; Macromolecular Substances; Magnetic Resonance Spectroscopy; Protein Binding; Salicylates; Serum Albumin; Spectrophotometry, Ultraviolet

Constitutive production of hydroxyl radicals from four established cancer cell lines was detected as spin adducts of 5,5-dimethyl-l-pyroline-N-oxide (DMPO), using an electron spin resonance spectrometer. The generated hydroxyl radicals was decreased in three out of four cancer cell lines when incubated in vitro for 3 h with TNF-alpha No direct scavenging effect of TNF-alpha on hydroxyl radicals or superoxide anions was observed in the in vitro radical generation system. The modulation of intracellular reactive oxygen species of these cancer cells by adding menadione or CuDIPS to the culture medium changed the antiproliferative effect of TNF-alpha on the cells. The ultrastructural localization of the radical-generating sites in cancer cells was visualized using the diaminobenzidine/horseradish peroxide histochemical system at the electron microscopic level. The hydrogen peroxide-dependent formation of electron-dense materials localized at the mitochondrial membranes was decreased after the treatment of the cancer cells with TNF-alpha. These data indicate that the reduction of radical generation in cancer cells by TNF-alpha may be an early mechanism that contributes to the antiproliferative effect of this cytokine on some cancer cells.

Topics: Cell Division; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Humans; Hydroxyl Radical; Microscopy, Electron; Reactive Oxygen Species; Salicylates; Spin Labels; Superoxides; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vitamin K

Topics: Down-Regulation; Hypoglycemic Agents; NADPH Dehydrogenase; Nitric Oxide Synthase; Salicylates

Addition of H2O2 at 100 microM, or 1 mM, to the culture medium of BHK-21 fibroblasts results in increased intracellular levels of H2O2. Whilst exposure of BHK-21 cells to lower levels of H2O2 (1 microM) actually stimulates proliferation, these higher oxidant concentrations not only depress proliferation rates but also lead to an increase in the appearance of apoptotic-like cells in the cultures. Other agents such as inhibitors of glutathione peroxidase and catalase, or mimics of superoxide dismutase, which also bring about elevated cellular levels of H2O2 in BHK-21 cells, similarly lead to decreased proliferation and an apparent increase in cells with apoptopic features. Thus intracellular conditions which are considered more prooxidant than normal, appear to favour apoptosis over proliferation in BHK-21 fibroblasts. Additionally these abnormal cellular conditions also appear to favour excessive DNA replication, in remaining non-apoptotic cells.

Topics: Amitrole; Animals; Apoptosis; Cell Adhesion; Cell Death; Cell Division; Cell Line; Cricetinae; Culture Media; DNA; Electrophoresis, Agar Gel; Fibroblasts; Hydrogen Peroxide; Microscopy, Electron; Nucleosomes; Salicylates; Succimer

Peroxynitrite activates the cyclooxygenase activities of constitutive and inducible prostaglandin endoperoxide synthases by serving as a substrate for the enzymes' peroxidase activities. Activation of purified enzyme is induced by direct addition of peroxynitrite or by in situ generation of peroxynitrite from NO coupling to superoxide anion. Cu,Zn-superoxide dismutase completely inhibits cyclooxygenase activation in systems where peroxynitrite is generated in situ from superoxide. In the murine macrophage cell line RAW264.7, the lipophilic superoxide dismutase-mimetic agents, Cu(II) (3,5-diisopropylsalicylic acid)2, and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin dose-dependently decrease the synthesis of prostaglandins without affecting the levels of NO synthase or prostaglandin endoperoxide synthase or by inhibiting the release of arachidonic acid. These findings support the hypothesis that peroxynitrite is an important modulator of cyclooxygenase activity in inflammatory cells and establish that superoxide anion serves as a biochemical link between NO and prostaglandin biosynthesis.

Topics: Animals; Arachidonic Acid; Cell Line; Enzyme Activation; Glutathione Peroxidase; Kinetics; Macrophages; Metalloporphyrins; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Salicylates; Superoxides

Intracellular levels of H2O2 in BHK-21 cells are not static but decline progressively with cell growth. Exposure of cells to inhibitors of catalase, or glutathione peroxidase, not only diminishes this decline but also depresses rates of cell proliferation, suggesting important growth regulatory roles for those antioxidant enzymes. Other agents which also diminish the growth-associated decline in intracellular levels of H2O2, such as the superoxide dismutase mimic, copper II-(3,5-diisopropylsalicylate)2, or docosahexaenoic acid, also reduced cell proliferation. In contrast, proliferation can be stimulated by the addition of 1 microM exogenous H2O2 to the culture medium. Under these conditions, however, intracellular levels of H2O2 are unaffected, whereas there is a reduction in intracellular levels of glutathione. It is argued that critical balances between intracellular levels of both H2O2 and glutathione are of significance in relation both to growth stimulation and inhibition. In addition growth stimulatory concentrations of H2O2, whilst initially leading to increased intracellular levels of lipid peroxidation breakdown products, appear to "trigger" their metabolism, possibly through aldehyde dehydrogenase, whose activity is also stimulated by H2O2.

Topics: Aldehyde Dehydrogenase; Amitrole; Animals; Cell Division; Cell Line; Cricetinae; Culture Media, Serum-Free; Docosahexaenoic Acids; Hydrogen Peroxide; Kidney; Kinetics; Lipid Peroxidation; Lipid Peroxides; Salicylates; Thiomalates; Time Factors

A mouse model of hypersensitivity pneumonitis was generated by challenge with a thermophilic actinomycete. Oxygen radical scavengers were administered to challenged mice: vitamin E at 1000 units daily, polyethylene glycol-superoxide dismutase (SOD) at 500 units daily, polyethylene glycol-catalase at 10,000 units daily, 1,3,dimethyl-2-thiourea (DMTU) at 2 mg daily, and the biomimetic SOD, copper(II) [diisopropyl salicylate]2 (CuDIPS) at 1 mg daily. At three weeks after actinomycete challenge, a 10-fold increase in bronchoalveolar (BAL) cell number was observed. Treatments with catalase or DMTU were without effect on the BAL cell number in challenged mice. However, infusion of vitamin E was associated with an increased BAL cell influx (15-fold increase at two and three weeks). Similarly, treatment with PEG-SOD and CuDIPS resulted in an increase in cell number at two and three weeks. PEG-SOD or CuDIPS treatment resulted in a strong neutrophilia, whereas control challenged mice had a cellular influx mostly of macrophages and lymphocytes. Vitamin E treatment of challenged mice led to an increased T lymphocyte recruitment at two and three weeks. In vitro studies showed that actinomycete challenge was associated with an enhancement of alveolar macrophage O2- release, which was blocked by PEG-SOD, vitamin E, or DSC treatment but was unaffected by catalase or DMTU treatment. In control challenged mice, there was a 25-fold increase in the BAL albumin concentration at two weeks. PEG-SOD, vitamin E, or CuDIPS treatment all decreased the albumin concentration; the three modulators also diminished lung fibrosis at two or three weeks, as seen by a decrease in lung hydroxyproline and collagen synthesis by lung fibroblasts. Examination of sections from lungs of challenged animals showed evidence of cellular infiltrates around the bronchi and the blood vessels. Challenged mice given continuous infusions of vitamin E, SOD, or CuDIPS had lung histological scores that were significantly lower than control challenged mice or challenged mice treated with catalase or DMTU. Thus, therapies based on O2- scavenging or treatment with a general antioxidant such as vitamin E may hold some promise in the treatment of hypersensitivity pneumonitis.

Topics: Animals; Antigens, Fungal; Antioxidants; Bronchoalveolar Lavage Fluid; Collagen; Farmer's Lung; Fibroblasts; Free Radical Scavengers; Hydroxyproline; Macrophages, Alveolar; Mice; Mice, Inbred C57BL; Micromonosporaceae; Neutrophils; Pulmonary Fibrosis; Reactive Oxygen Species; Salicylates; Specific Pathogen-Free Organisms; Superoxide Dismutase; T-Lymphocytes; Thiourea; Vitamin E

Purposes of this work were to develop an enzyme system as an in vitro model of the NADPH-dependent component of nitric oxide synthase (NOS) and examine the plausible down-regulation of this system and brain NOS by copper (II)2(3,5-diisopropylsalicylate)4[Cu(II)2(3,5-DIPS)4] as a mechanism accounting for its analgesic, anticonvulsant, and other pharmacological activities. Porcine heart diaphorase (PHD) was found to oxidize 114 microM NADPH with the corresponding reduction of an equivalent amount of 2,6-dichlorophenolindophenol (DCPIP). Addition of Cu(II)2(3,5-DIPS)4 to the reaction mixture decreased the reduction of DCPIP without substantially affecting the oxidation of NADPH. The IC50 for Cu(II)2(3,5-DIPS)4 in inhibiting the reduction of DCPIP was 1.5 microM. Mechanistically, this inhibition of DCPIP reduction was found to be due to the ability of Cu(II)2(3,5-DIPS)4 to serve as a catalytic electron acceptor for reduced PHD, which was enhanced by the presence of a large concentration of DCPIP and inhibited by a large concentration of NADPH. Oxidation of NADPH by PHD in the absence of DCPIP was linearly related to the concentration of Cu(II)2(3,5-DIPS)4 through the concentration range of 5-25 microM Cu(II)2(3,5-DIPS)4 with 50% recovery of NADPH oxidation by PHD at a concentration of 16 microM Cu(II)2(3,5-DIPS)4. Whole rat brain tissue sections incubated in medium containing an NADPH-generating system and nitroblue tetrazolium chloride (NBT) were less intensely stained when Cu(II)2(3,5-DIPS)4 was added to the medium. It is concluded that Cu(II)2(3,5-DIPS)4 serves as an electron acceptor in down-regulating PHD reduction of DCPIP and in down-regulating NOS in brain tissue sections. A decrease in NO synthesis in animal models of seizure, pain, and other disease states with Cu(II)2(3,5-DIPS)4 may account for the anticonvulsant, analgesic, and other pharmacological activities of this complex.

Topics: Animals; Azo Compounds; Brain; Down-Regulation; Electron Transport; Hypoglycemic Agents; Molecular Structure; Myocardium; NADP; NADPH Dehydrogenase; Nitric Oxide Synthase; Oxidation-Reduction; Salicylates; Spectrophotometry; Swine

In HeLa cells evidence is provided that active oxygen species such as hydrogen peroxide and superoxide at low levels are important growth regulatory signals. They may constitute a novel regulatory redox system of control superimposed upon the established cell growth signal transduction pathways. Whilst for example hydrogen peroxide can be added exogenously to elicit growth responses in these cells, it is clear that cellularly generated superoxide and hydrogen peroxide are important. Experiments with superoxide dismutase, superoxide dismutase mimics and inhibitors of both superoxide dismutase and xanthine oxidase suggest that superoxide generated intracellularly and superoxide released extracellularly are both relevant to growth control in HeLa cells.

Topics: Allopurinol; Catalase; Cell Division; Ditiocarb; HeLa Cells; Humans; Hydrogen Peroxide; Oxidation-Reduction; Oxypurinol; Reactive Oxygen Species; Salicylates; Signal Transduction; Superoxide Dismutase; Superoxides; Tetrazolium Salts; Thiazoles; Xanthine Oxidase

The ability of a biomimetic superoxide dismutase agent, copper(II)(3,5-diisopropylsalicylate)2 (CuDIPS), to modulate benzoyl peroxide (BzPo)-induced tumor promotion and progression in mouse skin multistage carcinogenesis was evaluated. The results showed a significant inhibition of tumor incidence by CuDIPS pretreatment during promotion-progression. Different types of tumors were developed: papillomas, keratoacanthomas and squamous cell carcinomas. There was a significant increase in the keratoacanthoma-papilloma ratio when the period of treatment with BzPo was prolonged, which was inhibited by CuDIPS pretreatment. CuDIPS induced a significant inhibition of malignant conversion. Our results suggest that reactive oxygen species could be important in BzPo-induced promotion and progression.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antineoplastic Agents; Benzoyl Peroxide; Carcinoma, Squamous Cell; Cocarcinogenesis; Female; Incidence; Keratoacanthoma; Mice; Papilloma; Salicylates; Skin Neoplasms

Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, hydroxyl radical, and hypochlorous acid have been implicated in the pathogenesis of inflammation and tissue injury in colitis. To determine whether or not anti-ROS agents can decrease the severity of colitis, we evaluated the effects of three known anti-ROS agents: catalase, WR-2721, and Cu(II)2(3,5-DIPS)4 on acetic acid-induced colonic inflammation in rats. Histologically, all three compounds significantly decreased the severity of colonic inflammation. The anti-ROS activity of these compounds was also tested using the luminol-enhanced chemiluminescence assay. Catalase, WR-2721, or Cu(II)2(3,5-DIPS)4 significantly inhibited luminol-enhanced chemiluminescence produced by inflamed colonic mucosa. These findings suggest that ROS, and in particular superoxide, hydrogen peroxide, and/or one of its secondarily derived species, may play an important role in acetic acid-induced colitis. Further studies are needed to determine the potential effectiveness of these compounds in human colitis.

Topics: Acetates; Acetic Acid; Amifostine; Animals; Catalase; Colitis; Female; Luminescent Measurements; Rats; Rats, Inbred F344; Reactive Oxygen Species; Salicylates

Copper(II)2(3,5-diisopropylsalicylate)4 [Cu(II)2(3,5-DIPS)4] has been found to have antiinflammatory, antiulcer, anticancer, anticonvulsant, antimutagenic, antidiabetic, analgesic, and radiation protection and recovery activities. It has also been found to reduce ischemia-reperfusion injury. Because of these activities it was of interest to understand how this compound is transported in the body to affected tissues. Evidence supporting the suggested formation of ternary human serum albumin (HSA)-Cu(II)(3,5-DIPS)2 or Cu(II)2(3,5-DIPS)4 complexes was obtained using ultraviolet spectrophotometry, dialysis, and atomic absorption spectrophotometry or atomic emission spectroscopy. Superoxide dismutase (SOD)-mimetic activity was also determined using the xanthine/xanthine oxidase/cytochrome c system. Ultraviolet spectra of aqueous solution mixtures of Cu(II)2(3,5-DIPS)4 in equilibrium with 2Cu(II)(3,5-DIPS)2 and HSA as well as aqueous solutions of solid Cu(II)2(3,5-DIPS)4 obtained by stirring the solid with an aqueous solution of HSA showed no obvious change in absorbance to indicate ternary complex formation. However, comparison of ultraviolet spectra taken before and after dialysis supports the suggested bonding of Cu(II)(3,5-DIPS)2 or Cu(II)2(3,5-DIPS)4 to HSA. Comparison of copper concentrations before and after dialysis also supports the suggested bonding of Cu(II)(3,5-DIPS)2 or Cu(II)2(3,5-DIPS)4 to HSA. Based upon these data it is plausible that Cu(II)(3,5-DIPS)2 or Cu(II)2(3,5-DIPS)4 form stable ternary complexes with HSA. These stable ternary complexes were also found to have SOD-mimetic activity.

Topics: Copper; Dialysis; Humans; Salicylates; Serum Albumin; Superoxide Dismutase

Mice are relatively resistant to the lethal effects of endotoxin. Sensitivity to lipopolysaccharide (LPS) and monophosphoryl lipid A (MPL) can be enhanced by concurrently loading animals with D-galactosamine (D-gal). Significant diurnal variation in susceptibility to lethal toxicity was observed in D-gal loaded mice upon LPS or MPL immunostimulant challenge. In mice treated with either MPL or MPL plus D-gal, at the time of greatest toxic sensitivity, serum TNF levels were significantly higher than was seen in mice treated at a time of low sensitivity. Peritoneal exudate cells (PECs) harvested from mice treated with either D-gal or MPL displayed enhanced in vitro superoxide (SO) production. Simultaneous treatment with D-gal and MPL led to a synergistic enhancement of SO production above that induced by either xenobiotic alone. Pretreatment with the SO dismutase mimetic Cu(II) (diisopropyl salicylate)2 significantly protected mice from the lethal toxicity of D-gal-MPL challenge. PECs harvested from these same mice failed to display the elevated in vitro SO production reported above. SO elaboration in vivo, presumably by hepatocytes, PECs, and possibly other cells, subsequent to D-gal loading and LPS or MPL challenge, appears to play an important role in the lethal toxicity observed. The diurnal variation in toxicity reported in this animal model may result from TNF modulation of SO production in vivo.

Topics: Animals; Ascitic Fluid; Circadian Rhythm; Female; Galactosamine; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred ICR; Salicylates; Superoxides; Tumor Necrosis Factor-alpha

The oxidative stress induced in vivo by benzoyl peroxide (BzPo) or 12-O-tetradecanoylphorbol-13-acetate (TPA) was evaluated in terms of chemiluminescence (CL) emitted by SENCAR mouse skin, a non-invasive method that allows an estimation of overall oxidative stress. The ability of a biomimetic superoxide dismutase, copper(II)(3,5-diisopropylsalicylate)2 (CuDIPS), to inhibit that response was also evaluated. A single application of BzPo to mouse skin resulted in a dose-dependent increase in CL up to 0.083 mumol. Sequential treatment with BzPo in a dose used for tumor promotion resulted in a fall in CL induced by the second topical application. There were no differences between initiated and non-initiated mice in their responses to BzPo-induced CL. CuDIPS, an inhibitor of tumor promotion, was an effective inhibitor of CL in all the protocols evaluated. Conversely, ZnDIPS and DIPS did not inhibit CL. Phenolic antioxidants induced partial inhibition of CL. Unlike BzPo treatment, a single application of TPA up to 105 nmol did not induce an increase in CL, but the second topical application with TPA in a dose used for tumor promotion resulted in a small but significant increase in CL. However, these values of CL were much smaller than the CL induced by BzPo. Our results show a differential response of the skin in terms of the oxidative stress induced by BzPo or TPA.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antineoplastic Agents; Benzoyl Peroxide; Butylated Hydroxyanisole; Butylated Hydroxytoluene; Luminescent Measurements; Mice; Mice, Inbred Strains; Oxidation-Reduction; Salicylates; Skin; Tetradecanoylphorbol Acetate

L-cysteine, D-penicillamine, and L-glutathione were oxidized to symmetrical disulfides in the presence of Cu(II)(3,5-DIPS)2 and air-oxygen at physiologic pH, 7.3. Air-oxygen caused the oxidation of thiol reduced copper, Cu(I), to Cu(II), as evidenced by expected spectrophotometric changes in these reaction mixtures. L-cysteine, D-penicillamine, and L-glutathione formed mixed disulfides and TNB with the addition of DTNB to solutions of these thiols. The observed order of reactivity for these thiols with DTNB was: L-cysteine greater than D-penicillamine greater than L-glutathione. Surprisingly, Cu(II)(3,5-DIPS)2 converted these mixed disulfides to their symmetrical disulfides and DTNB, and although the initial conversion rate was rapid, complete conversion required more than two hours. These observations suggest caution with regard to the spectrophotometric determination of thiols immediately after the addition of Ellman's reagent. These results also clarify an earlier report concerning the oxidation of thiols by Cu(II)(o-phenanthroline)2 and offer caution with regard to the determination of thiols using DTNB in the presence of copper complexes. Spectrophotometric data are provided in support of the suggestion that analysis of plasma or cellular samples for thiols be done in the absence of copper(II) complexes to avoid false negative results.

Topics: Cysteine; Disulfides; Dithionitrobenzoic Acid; Drug Interactions; Glutathione; Nitrobenzoates; Oxidation-Reduction; Penicillamine; Salicylates; Sulfhydryl Compounds

Topics: Animals; Bone Marrow; Copper; Female; Free Radicals; Hematopoietic Stem Cells; Immunity; Iron; Male; Manganese; Mice; Mice, Inbred C57BL; Radiation-Protective Agents; Salicylates; Spleen; Thymus Gland; Zinc

These studies were intended to compare the effects of aspirin, 3,5-diisopropysalicylic acid (3,5-DIPS), and indomethacin with those of their copper complexes: Cu(II)2(aspirinate)4, Cu(II)2(3,5-DIPS)4, and Cu(II)2(indomethacinate)4 as well as Cu(II)2(acetate)4 on polymorphonuclear leukocyte (PMNL) random and directional migration, in addition to their anti-inflammatory activities. Experiments were performed both in vivo and in vitro. In vitro modifications of PMNL migration were measured with the Boyden chamber using N-formyl-methionyl-leucyl-phenylalanine (fMLP) as the chemoattractant and in the agarose assay using fMLP and serum chemotactic derivatives of complement as chemoattractants. In vivo anti-inflammatory activities of these compounds were determined after induction of a serum-induced pleurisy in the rat, and measurement of exudate volume and number of exudative cells 4 hr later. Copper complexes of non-steroidal anti-inflammatory drugs (NSAIDs) were found to be more effective in decreasing random migration and chemotaxis of PMNLs than their parent drugs or Cu(II)2(acetate)4 in in vitro studies. Only chemotaxis was found to be reduced significantly for PMNLs obtained from pleuritic rats after in vivo treatment and the order of copper complex effectiveness was: Cu(II)2(indomethacinate)4 greater than Cu(II)2(3,5-DIPS)4 greater than Cu(II)2(aspirinate)4. All doses of Cu(II)2(acetate)4 administered in vivo failed to affect chemotactic activity. Copper complexes of NSAIDs were also more effective than their parent drugs as anti-inflammatory agents, and Cu(II)2(acetate)4 had no anti-inflammatory activity in this model of inflammation. The order of anti-inflammatory activity was: Cu(II)2(indomethacinate)4 greater than Cu(II)2(3,5-DIPS)4 greater than Cu(II)2(aspirinate)4.

Topics: Acetates; Acetic Acid; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Cell Movement; Chemotaxis, Leukocyte; Indomethacin; Molecular Structure; Neutrophils; Organometallic Compounds; Pleurisy; Rats; Salicylates

We have previously observed that the interaction of an oxidant generated by polymorphonuclear leukocytes (PMNs) with (+-)-trans benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) resulted in covalent binding to DNA and elicited bacterial mutagenesis (PNAS 82:5194, 1985). We now report that this interaction also induces sister chromatid exchanges (SCEs) in Chinese hamster V-79 cells. This genotoxic response required stimulation of the PMNs by phorbol ester as no effect was observed with unstimulated cells. Likewise, no intrinsic activity of BP-7,8-diol alone was noted. The addition of azide, CuDIPS, or taurine markedly inhibited the induction of SCEs by the combination of BP-7,8-diol and stimulated PMNs, further suggesting the involvement of myeloperoxidase in the activation of the polycyclic aromatic hydrocarbon. The (-) isomer of BP-7,8-diol as well as 7,8-dihydro-BP were more active than (+)-BP-7,8-diol in inducing SCEs. By contrast, benzo[a]pyrene or derivatives lacking a double bond at the 9,10 position were not effective in inducing SCEs above the level seen with phorbol ester-stimulated PMNs. These observations serve to underscore the potential for myeloperoxidase-dependent activation of xenobiotics by PMNs to result in a localized genotoxic environment.

Topics: Animals; Azides; Benzo(a)pyrene; Biotransformation; Cells, Cultured; Humans; Neutrophils; Salicylates; Sister Chromatid Exchange; Superoxide Dismutase; Taurine; Tetradecanoylphorbol Acetate

We have previously reported that copper(II)2(3,5-diisopropylsalicylate)4 (Cu-DIPS), administered 3 h before exposure to lethal irradiation, significantly increased the survival rate of mice. Agents that can improve recovery from irradiation are of particular importance for accidental radiation exposure if they are effective when given after exposure. In the present study, we showed that Cu-DIPS had radiation recovery activity when administered subsequent to radiation exposure. Mice were exposed to 800 cGy irradiation and 3 h later injected with vehicle or 20, 40, or 60 mumol/kg Cu-DIPS. The 30-day survival rate was significantly increased at all doses of Cu-DIPS tested. Survival increased from 47% for vehicle-treated mice to 78% (p less than 0.001) for mice treated with 40 mumol/kg. The recovery of hemopoietic activity was assessed in similarly treated mice 14 and 24 days after irradiation. The postirradiation Cu-DIPS treatment significantly increased spleen weights, bone marrow cellularity, and hemopoietic activity in the spleen and bone marrow compared to vehicle-treated controls. Enhanced recovery of hemopoietic activity included both committed progenitor granulocyte-macrophage colony-forming units (GM-CFU) and more primitive stem cells (endogenous spleen colony-forming units, CFU-Se). The number of CFU-Se at 14 days, the number of bone marrow GM-CFU at 24 days, and bone marrow cellularity at 24 days appear to be better predictors of survival rates than other parameters.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Hematopoiesis; Mice; Mice, Inbred C57BL; Organ Size; Radiation Injuries, Experimental; Radiation-Protective Agents; Salicylates; Spleen; Time Factors

The hypoxanthine - xanthine oxidase system generates an extracellular flux of superoxide anion radical (O2.-) and hydrogen peroxide (H2O2). Catalase but not superoxide dismutase (SOD) protects V79 cells exposed to the hypoxanthine - xanthine oxidase system, showing that H2O2 is the major reactive oxygen species involved in the cytotoxicity of such a system. In contrast to SOD, the lipophilic SOD like compound CuII (diisopropylsalicylate)2 (CuDIPS) exhibits some protection at non cytotoxic concentration. It is also found that methanol partially protects cells exposed to the hypoxanthine-xanthine oxidase system. It appears that in our experimental conditions (temperature, ionic strength and pH) the protective effect afforded by methanol and CuDIPS is due to the inhibition of the xanthine oxidase activity.

Topics: Animals; Catalase; Cell Line; Cell Survival; Hydrogen Peroxide; Hypoxanthine; Hypoxanthines; Methanol; Oxygen Consumption; Salicylates; Superoxide Dismutase; Xanthine Oxidase

Copper(II)2(3,5-diisopropylsalicylate)4 [Cu(II)2(3,5-DIPS)4] is a synthetic copper complex with a variety of effects, including radiation recovery, anti-inflammatory, and accelerated wound healing activities. When C57BL/6 mice were injected subcutaneously with 80 mumol/kg Cu(II)2(3,5-DIPS)4 their spleens significantly enlarged. This splenomegally lasted for at least 3 weeks and was accompanied by increased myelopoiesis in the spleen. Bone marrow had no significant change in cellularity or myelopoiesis. The treatment with Cu(II)2(3,5-DIPS)4 had only minor effects on the ability of spleen cells to respond to mitogenic or antigenic stimulation. Cu(II)2(3,5-DIPS)4 did stimulate lymphopoiesis, since it accelerated the recovery of immune reactivity following exposure to 8 Gy whole body irradiation. Copper levels in the spleens and bone marrow of unirradiated mice treated with Cu(II)2(3,5-DIPS)4 were not significantly elevated 24 hr after treatment and copper levels were transiently reduced 7 days after treatment.

Topics: Animals; Bone Marrow; Colony-Forming Units Assay; Copper; Hematopoiesis; Liver; Mice; Salicylates; Spleen

Copper (II)2(3,5-Diisopropylsalicylate)4(H2O)2 has been found to have antiinflammatory, antiulcer, anticonvulsant, anticancer, anticarcinogenic, antimutagenic, and radiation recovery activities and it prevents reperfusion injury. To study pharmacokinetic parameters accounting for these pharmacological effects the double labeled 67Cu(II)2(carboxy-14C-3,5-diisopropylsalicylate)4 complex was synthesized and used to obtain these parameters. Treatment of mice with 1 mumol of this complex revealed that 67Cu was distributed to blood, liver, kidney, intestine, lung, thymus, femur, muscle, spleen, brain, urine, and feces within 0.5 hr and patterned changes in 67Cu content of these tissues and excreta were found throughout the 96 hr term of this study.

Topics: Animals; Biological Availability; Chemical Phenomena; Chemistry; Chronic Disease; Copper; Mice; Organ Specificity; Salicylates

The superoxide dismutase mimetic compound, Cu(II) (3,5-diisopropylsalicylate)2 (CuDIPS) inhibited soluble Ca2+/phospholipid dependent protein, kinase (protein kinase C) in rat liver, competing with ATP. The Ca2+/phospholipid- and TPA-stimulated phosphorylation of endogenous proteins were also inhibited by CuDIPS. In vitro and in vivo CuDIPS as well as CuSO4 reduced the activity of TPA-stimulated protein kinase C, while 3,5-diisopropylsalicylate lacked this effect. Our results indicate that CuDIPS interacts with the catalytic domain of the enzyme and the inhibition of protein kinase C may be due to copper(II) ions.

Topics: Adenosine Triphosphate; Animals; Calcium; Cytosol; Dose-Response Relationship, Drug; Liver; Male; Phosphatidylserines; Phosphorylation; Protein Kinase C; Proteins; Rats; Rats, Inbred Strains; Salicylates; Tetradecanoylphorbol Acetate

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits growth and induces terminal squamous differentiation of normal human bronchial cells when added to the culture media [J. C. Willey, A. J. Saladino, C. Ozanne, J. F. Lechner, and C. C. Harris, Carcinogenesis (lond.), 5: 209-215, 1984]. We have investigated the possibility of oxygen free radicals being involved as intermediates in this process. Electron paramagnetic resonance measurements using the spin-trapping agent 5,5-dimethyl-1-pyrroline-1-oxide failed to detect oxygen free radicals in bronchial epithelial cells exposed to TPA, although oxy radicals were detected in bronchial epithelial cells after a nontoxic exposure to menadione, and in human neutrophils after exposure to TPA. Addition to the culture media of free radical scavenger, i.e., reduced glutathione, N-acetylcysteine, D-alpha-tocopherol, copper (II) (3,5-diisopropylsalicyclic acid)2, or the combination of superoxide dismutase and catalase did not affect the dose-dependent growth inhibition of TPA on the bronchial epithelial cells. Moreover, exposure of the bronchial epithelial cells to TPA did not result in increased DNA single strand breaks measured by alkaline elution, as would be expected with a free radical mediated mechanism. Thus, our results argue against the importance of oxygen free radicals in the inhibition of growth and the induction of squamous differentiation by TPA in normal human bronchial epithelial cells.

Topics: Acetylcysteine; Antineoplastic Agents; Bronchi; Catalase; Cell Survival; DNA Damage; Electron Spin Resonance Spectroscopy; Epithelial Cells; Epithelium; Free Radicals; Glutathione; Humans; Kinetics; Salicylates; Superoxide Dismutase; Tetradecanoylphorbol Acetate; Vitamin E

A keratinocyte-mediated mutagenesis assay, and the murine skin multistage carcinogenesis tumor model were used to survey the chemopreventive properties of Cu(II)(3,5-diisopropylsalicylate)2 [CuDIPS] and its analogs. Supplementation of cocultures of newborn SENCAR keratinocytes and Chinese hamster lung fibroblasts (V79 cells) with CuDIPS, 3,5-diisopropylsalicylate (DIPS), and CuSO4 resulted in dose-dependent killings of V79 cells (LD50 of 34, 75, 960 microM, respectively), and inhibitions of benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) mutagenesis (ED50 of 13, 95, 80 microM, and 40, 125, 110 microM, respectively). Analyses of dose-response curves suggest (i) CuDIPS preferentially inhibits BP mutagenesis; (ii) the antimutagenic activity of CuDIPS towards DMBA and the cytotoxicity of the copper complex are derived from the DIPS component of the chelate; (iii) the antimutagenic activity of CuDIPS towards BP requires both copper and DIPS; and (iv) DIPS and CuDIPS induced cytotoxicity is required for inhibition of mutagenesis. Inhibition of mutagenesis by CuDIPS was not mediated by modulation of promutagen metabolism because antimutagenic concentrations of the chelate had no significant effects on DMBA- and BP-dependent cytotoxicities. Topical pretreatment of SENCAR mice with CuDIPS (100-4000 nmol) 15 min prior to initiation with DMBA or BP resulted in small (38% maximum) non-dose-responsive reductions of papillomas/mouse following 20 weeks of promotion.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antineoplastic Agents; Benzo(a)pyrene; Cell Line; Cell Survival; Cells, Cultured; Copper; Copper Sulfate; Epidermal Cells; Epidermis; Mice; Mice, Inbred Strains; Mutation; Papilloma; Salicylates; Skin Neoplasms

We have previously reported that copper(II)2(3,5-diisopropylsalicylate)4 (Cu-DIPS) significantly increased the survival rate of mice exposed to lethal irradiation. To examine whether Cu-DIPS affected hemopoietic activity, groups of mice were treated with Cu-DIPS or vehicle and assayed for in vitro interleukin 3 (IL-3)-dependent colony-forming units (CFU-C) and for committed progenitor granulocyte-macrophage CFU (GM-CFU). Cu-DIPS increased the number of splenic IL-3 CFU-C by five- to sixfold 7 days after treatment and splenic GM-CFU by 12-fold on day 24. These increases were accompanied by a 50% increase in spleen weight. Bone marrow IL-3 CFU-C and GM-CFU were not affected at 7 or 14 days after treatment, but were somewhat depressed at 24 days. In irradiated (8.0 Gy) mice treated with Cu-DIPS or vehicle, splenic IL-3 CFU-C and GM-CFU were undetectable 7 days after irradiation, but recovered more rapidly in Cu-DIPS-treated mice. By 24 days splenic IL-3 CFU-C in Cu-DIPS-treated mice recovered to 150% of normal (unirradiated) values and GM-CFU recovered to 270% of normal, whereas irradiated control values remained at 25% and 7%, respectively. The recovery of bone marrow hemopoiesis was slower than spleen, but 42 days after irradiation Cu-DIPS-treated mice had higher levels of bone marrow IL-3 CFU-C (eightfold) and GM-CFU (4.6-fold) than vehicle-treated mice. Cu-DIPS stimulated sixfold increases in renewable, pluripotent stem cells as measured by the in vivo assay of endogenous colony-forming units (CFU-Se).

Topics: Animals; Bone Marrow; Colony-Forming Units Assay; Female; Hematopoiesis; Hematopoietic Stem Cells; Mice; Mice, Inbred C57BL; Radiation Injuries, Experimental; Radiation-Protective Agents; Salicylates; Spleen; Whole-Body Irradiation

Paraquat exerts a cytotoxic effect on Chinese hamster ovary cells in culture via the superoxide radical (O2-). We have described a superoxide dismutase (SOD) mimic based on manganese (DF-Mn) which consists of a one-to-one complex between desferrioxamine B (Desferal) and MnO2. It is a small molecular weight molecule, easy to prepare and possesses considerable stability. It is now shown to protect mammalian cells from paraquat toxicity. Thus, 20 microM DF-Mn affords up to complete protection against the cytotoxicity of 200 microM paraquat in Chinese hamster ovary cells. Desferrioxamine B or MnO2 alone gave no protection. MnCl2 or catalase provided little or no protection against the paraquat, respectively. Equivalent amounts of human Cu-Zn SOD in terms of activity, also provided no protection. Copper diisopropylsalicylate (CuDIPS) provided limited, yet significant, protection, but this is explained in terms other than SOD activity. Finally, at higher concentrations, purified human SOD, exerts a limited toxicity as well as a protective ability against paraquat (similar to DF-Mn) both of which are eliminated upon heat denaturation of the enzyme. It appears that the SOD mimic, DF-Mn, can enter mammalian cells and can protect against the cytotoxic effects of O2-.

Topics: Animals; Cell Line; Cell Survival; Cricetinae; Cricetulus; Deferoxamine; Dose-Response Relationship, Drug; Drug Combinations; Female; Manganese; Manganese Compounds; Molecular Weight; Ovary; Oxides; Paraquat; Salicylates; Superoxide Dismutase

The present study was designed to compare the skin tumor promoting and epidermal ornithine decarboxylase (ODC) inducing activities of various structural analogs of anthralin (1,8-dihydroxy-9-anthrone) and chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone). Groups of 30 SENCAR mice each were initiated with 7,12-dimethylbenz[a]anthracene and 2 weeks later promoted with once- or twice-weekly applications of various doses of these anthrone derivatives. Carbon-10 (C10)-acyl derivatives of anthralin were active skin tumor promoters in the range of 25-440 nmol per mouse. 10-Acetylanthralin was significantly more active than 10-myristoyl-anthralin at low doses (e.g. 25 and 50 nmol per mouse) and nearly as potent as the unsubstituted compound. Higher doses (greater than or equal to 100 nmol per mouse) of this derivative were toxic, hence, reducing the final papilloma response. On a relative activity scale where anthralin is 1.0, these derivatives had activities that were approximately 0.7 and 0.2, respectively. 10,10-Dipropylanthralin was totally inactive at the doses tested. C6-Substituted derivatives of chrysarobin demonstrated diverse tumor promoting activities when tested in the range of 25-440 nmol per mouse. On a relative activity scale where chrysarobin is 1.0, 6-methoxychrysarobin (physcion anthrone) was approximately 0.9, whereas 6-hydroxychrysarobin (emodin anthrone) had no activity. Chrysophanic acid (1,8-dihydroxy-3-methyl-9,10-anthraquinone) was also inactive as a tumor promoter at the doses tested. In general, the tumor promoting activities of these anthrone derivatives correlated very well with their ability to induce epidermal ODC after a single topical application indicating an important role for this enzyme in skin tumor promotion by anthones. The ability of C10-substituted derivatives of anthralin to undergo base catalyzed oxidation in vitro correlated with both ODC inducing and tumor promoting activities. In addition, copper(II)bis(diisopropylsalicylate) was found to inhibit both ODC induction and skin tumor promotion by chrysarobin. These latter data, when taken together, suggest a role for oxidation at C10 in skin tumor promotion by anthrone derivatives.

Topics: Animals; Anthracenes; Anthralin; Enzyme Induction; Female; Free Radicals; Mice; Ornithine Decarboxylase; Salicylates; Skin; Skin Neoplasms; Structure-Activity Relationship

ESR spectra have been obtained after addition of either a cupric phenylhydantoin or a cupric diisopropylsalicylate complex to Ehrlich ascites tumor cells. It is shown that some of the complex remains in the cupric state. Because the ESR parameters of these complexes in the presence of cells differ from the ESR parameters for these complexes in the absence of cells, in the presence of cells either adducts or new cupric complexes are formed. The fast motion of these complexes, as determined from room temperature ESR spectra, is characteristic of complexes with molecular weights less than 1500.

Topics: Animals; Carcinoma, Ehrlich Tumor; Chemical Phenomena; Chemistry; Electron Spin Resonance Spectroscopy; Hydantoins; In Vitro Techniques; Mice; Organometallic Compounds; Salicylates

When Chinese hamster fibroblasts were exposed to hydrogen peroxide or to a system consisting of xanthine oxidase and hypoxanthine, which generates superoxide anion plus hydrogen peroxide, sister-chromatid exchanges (SCEs) were formed in a dose-dependent manner. When the iron-complexing agent o-phenanthroline was present in the medium, however, the production of these SCEs was completely inhibited. This fact indicates that the Fenton reaction: Fe2+ + H2O2----OH0 + OH- + Fe3+ is responsible for the production of SCEs. When O2- and H2O2 were generated inside the cell by incubation with menadione, the production of SCE was prevented by co-incubation with copper diisopropylsalicylate, a superoxide dismutase mimetic agent. The most likely role of O2- is as a reducing agent of Fe3+: O2- + Fe3+----Fe2+ + O2, so that the sum of this and the Fenton reaction, i.e., the iron-catalyzed Haber-Weiss reaction, provides an explanation for the active oxygen species-induced SCE: H2O2 + O2(-)----OH- + OH0 + O2. According to this view, the OH radical thus produced is the agent which ultimately causes SCE. These results are discussed in comparison with other mechanisms previously proposed for induction of SCE by active oxygen species.

Topics: Animals; Cell Cycle; Cell Line; Cricetinae; Drug Synergism; Free Radicals; Hydrogen Peroxide; Hydroxides; Iron; Phenanthrolines; Salicylates; Sister Chromatid Exchange; Superoxides; Vitamin K

Copper(II)(3,5-diisopropylsalicylate)2 (Cu-DIPS), administered subcutaneously to mice at 80 mg/kg body weight, had marked radioprotective activity. Given 3 h before exposure to 8.0 Gy (800 rad) irradiation, Cu-DIPS increased the 42-day survival from 40% to 86%. Seven days after exposure to 8.0 Gy, there were severe reductions in spleen weight (73%) and cellularity (98%) in both Cu-DIPS- and vehicle-treated mice. Viable spleen cells collected 7 days after irradiation were totally unresponsive to mitogenic or antigenic stimulation regardless of Cu-DIPS or vehicle treatment, suggesting that Cu-DIPS did not prevent radiation-induced damage to mature lymphocytes. At 14 days, when Cu-DIPS-treated mice started to show improved survival over vehicle-treated mice, spleen weights and cellularity were 2.5- and 3.5-fold higher, respectively, in Cu-DIPS-treated mice. Treatment with Cu-DIPS not only enhanced splenic repopulation, but also accelerated the reappearance of both B and T cell reactivities. Spleen cell responsiveness to the B cell mitogen, lipopolysaccharide (LPS), and the T cell mitogen, concanavalin A (Con A), regenerated significantly faster in Cu-DIPS-treated mice. Cu-DIPS also significantly accelerated the regeneration of T-dependent antibody induction. Based on these assays of immunocompetence, Cu-DIPS-treated mice had, on average, a seven-fold greater capacity to respond to immune stimulation than vehicle-treated mice 24 days after irradiation.

Topics: Animals; Antibody Formation; Antineoplastic Agents; B-Lymphocytes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Organ Size; Salicylates; Spleen; T-Lymphocytes

Our previous studies had suggested a link between bile salt stimulation of colonic epithelial proliferation and the release and oxygenation of arachidonate via the lipoxygenase pathway. In the present study, we examined the role of reactive oxygen versus end products of arachidonate metabolism via the cyclooxygenase and lipoxygenase pathways in bile salt stimulation of rat colonic epithelial proliferation. Intracolonic instillation of 5 mM deoxycholate increased mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA. Responses to deoxycholate were abolished by the superoxide dismutase mimetic CuII (3,5 diisopropylsalicylic acid)2 (CuDIPS), and by phenidone or esculetin, which inhibit both lipoxygenase and cyclooxygenase activities. By contrast, indomethacin potentiated the response. Phenidone and esculetin suppressed deoxycholate-induced increases in prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and 5, 12, and 15-hydroxyeicosatetraenoic acid (HETE), whereas CuDIPS had no effect. Indomethacin suppressed only PGE2. Deoxycholate (0.5-5 mM) increased superoxide dismutase sensitive chemiluminescence 2-10-fold and stimulated superoxide production as measured by cytochrome c reduction in colonic mucosal scrapings or crypt epithelium. Bile salt-induced increases in chemiluminescence were abolished by CuDIPS, phenidone, and esculetin, but not by indomethacin. Intracolonic generation of reactive oxygen by xanthine-xanthine oxidase increased colonic mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA approximately twofold. These effects were abolished by superoxide dismutase. The findings support a key role for reactive oxygen, rather than more distal products of either the lipoxygenase or cyclooxygenase pathways, in the stimulation of colonic mucosal proliferation by bile salts.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Bile Acids and Salts; Cell Cycle; Deoxycholic Acid; DNA; Female; Free Radicals; Indomethacin; Intestinal Mucosa; Lipoxygenase; Luminescent Measurements; Ornithine Decarboxylase; Oxygen; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Rats; Salicylates; Superoxide Dismutase; Superoxides; Umbelliferones; Xanthine Oxidase

This study examined the characteristics of the active oxygen species involved in generation of the reactive intermediate of methoxychlor which covalently binds to liver microsomal proteins. The possibility that the active oxygen participating in the above reaction is the superoxide anion (O2-) or a species generated from O2- was examined with the help of superoxide dismutase (SOD) and with an SOD-mimetic agent, CuDIPS [Cu2+(3,5-diisopropylsalicylic acid)2]. It was observed that, whereas CuDIPS inhibited covalent binding of methoxychlor metabolite(s), SOD did not. However, ZnDIPS [Zn2+(3,5-diisopropylsalicylic acid)2], which exhibits no SOD-mimetic activity, did not inhibit covalent binding. Furthermore, both CuDIPS and ZnDIPS had little or no effect on the formation of demethylated (polar) metabolites of methoxychlor, demonstrating that the inhibition of covalent binding by CuDIPS was not merely due to a general inhibition of the hepatic monooxygenase system. These findings suggested that O2- was involved in covalent binding, but was not accessible to SOD. Additional support for O2- involvement stems from the observation that alpha-tocopheryl acid succinate markedly inhibited covalent binding of methoxychlor. The possibility that hydrogen peroxide (H2O2) was involved in covalent binding of methoxychlor appears unlikely. Catalase had no effect on covalent binding when NADPH was the cofactor, and the use of H2O2 in place of NADPH did not yield covalent binding. Certain scavengers of hydroxyl radical (ethanol, t-butanol and benzoate) inhibited, and other known scavengers (DMSO and mannitol) did not inhibit, covalent binding. EDTA stimulated binding, desferal (desferrioxamine) exhibited no effect on binding, and diethylenetriaminepentaacetic acid (DETAPAC) inhibited binding. A possible explanation for this observation is that the Fe2+ needed for generation of X OH is much more easily obtained from Fe3+-EDTA than from Fe3+-desferal, which resists reduction. The inhibitory effect by DETAPAC may be due to chelation of another metal which is needed for the reaction. Lastly, certain scavengers of singlet oxygen inhibited covalent binding with little effect on the formation of polar metabolites of methoxychlor. In conclusion, these studies support the involvement of X OH and singlet oxygen, possibly derived from O2-, in the formation of the reactive methoxychlor intermediate.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Benzoates; Benzoic Acid; beta Carotene; Butanols; Carotenoids; Catalase; Deferoxamine; Edetic Acid; Ethanol; Histidine; Hydrogen Peroxide; Mannitol; Methoxychlor; Microsomes, Liver; NADP; Oxygen; Pentetic Acid; Proteins; Salicylates; Superoxide Dismutase; Superoxides; tert-Butyl Alcohol; Tryptophan; Zinc

Superoxide dismutase mimetic copper(II) complexes, such as copper(II)(3,5-diisopropylsalicylate)2 (CuDIPS), inhibit phorbol ester stimulated tumor promotion in mouse skin. Therefore, CuDIPS was tested as a potential inhibitor of another effect of phorbol esters, induction of interleukin 2 (IL2) synthesis, in the mouse thymoma cell line EL4. CuDIPS inhibited phorbol ester induced IL2 production in a concentration dependent manner with a 50% inhibitory concentration of about 10 microM. However, the ligand 3,5-diisopropylsalicylic acid also inhibited the induction of IL2 by phorbol esters (50% inhibitory concentration, 15 microM). Since the superoxide dismutase mimetic activity of CuDIPS is not stable in the presence of ethylenediaminetetraacetic acid, the effects of CuDIPS could be due to the free ligand and not to the intact metallocomplex. Consequently, a series of extremely stable copper(II) macrocyclic compounds was synthesized, and the reduction potential, superoxide dismutase mimetic activity, and ability to inhibit phorbol ester induced IL2 production were determined for each. Of the copper(II) macrocyclic complexes studied, only the most potent superoxide dismutase mimetic compound was found to inhibit phorbol ester induced IL2 production. Copper(II) complexes had to be added no later than 4 following phorbol ester administration to be effective inhibitors of the IL response, suggesting that these compounds act subsequent to the binding of phorbol esters but prior to the transcription of IL2 messenger RNA. Adherence of EL4 cells to substrate in response to phorbol esters was unaffected by copper(II) compounds. In summary, copper(II) compounds with appropriate reduction potentials can act within a defined time period to inhibit some, but not all, of the effects of phorbol esters on EL4 cells.

Topics: Animals; Cations, Divalent; Cell Adhesion; Copper; Interleukin-2; Mice; Phorbol Esters; Protein Biosynthesis; Salicylates; Solubility; Superoxide Dismutase; Superoxides; Time Factors

The superoxide dismutase (SOD) biomimetic copper(II) (3,5-diisopropylsalicylate)2 (CuDIPS) has been previously reported to inhibit the tumorigenicity of a polycyclic aromatic hydrocarbon (PAH) requiring metabolic activation. We have used the Ames Salmonella typhimurium revertant assay to survey the effects of CuDIPS and its analogs on the metabolic activation of the PAH benzo[a]pyrene (BP) by liver homogenates. Supplementation of homogenates from normal and Aroclor 1254-treated SENCAR mice with varied concentrations of CuDIPS resulted in a dose-dependent noncompetitive inhibition of BP mutagenesis. Cytochrome P-450 reductase activity in liver homogenates and microsomal preparations was also inhibited by concentrations of CuDIPS possessing antimutagenic activity. Neither DIPS nor ZnDIPS, analogs of CuDIPS lacking SOD activity, inhibited mutagenesis or P-450 reductase activity. CuSO4, which has SOD activity, was almost as effective as CuDIPS on a molar basis in inhibiting mutagenesis and P-450 reductase activity. The inhibitory effects of CuDIPS and CuSO4 could not be attributed to their SOD activity since bovine liver superoxide dismutase, at a 100-fold excess of CuDIPS-SOD activity, had no effect on their activity. Collectively these findings suggest that the in vitro antimutagenic activity of CuDIPS is independent of its salicylate structure and is mediated through a copper-dependent but non SOD-associated inhibition of P-450 reductase activity.

Topics: Animals; Antineoplastic Agents; Benzo(a)pyrene; Biotransformation; Hydrogen-Ion Concentration; Mice; Mutation; NADPH-Ferrihemoprotein Reductase; Salicylates; Superoxide Dismutase

Bile acids increase the proliferative activity of rat colonic epithelium. However, the mechanisms responsible are unknown. The present study examined the relationships between deoxycholate (DOC) induced surface cell sloughing, as measured by loss of DNA into the lumen and by light microscopy, and the subsequent increases in mucosal ornithine decarboxylase activity and [3H]thymidine (dThd) incorporation into mucosal DNA induced by deoxycholate. Intracolonic instillation of DOC (10 mumol; 5 mM) resulted in a progressive increase in luminal DNA content which was significant by 1 min and maximal by 1 h. No further increase in luminal DNA occurred between 1 and 4 h after DOC. Similarly, light microscopy demonstrated a progressive loss of surface epithelium between 10 min and 1 h after DOC instillation. By 4 h after DOC, the colonic mucosal surface was normal histologically. The rapid repair of the epithelial surface occurred without a detectable increase in [3H]dThd incorporation into DNA within 4 h. The latter finding thus suggested that upward migration of nondividing crypt epithelial cells rather than the rapid initiation of new DNA synthesis and new mitotic activity was responsible for surface repair. Enhanced proliferative activity of colonic mucosa, as measured by increased [3H]dThd incorporation into DNA, did occur subsequently (12 to 24 h) after instillation of DOC. The dose response of early surface cell loss and the subsequent proliferative response to DOC were identical, consistent with a link between these two DOC mediated events. However, two observations suggested that surface epithelial loss alone was not sufficient to trigger the proliferative response to DOC: intracolonic instillation of DOC followed by removal of the DOC solution at 1 h, at which time surface epithelial loss was maximal, did not result in an increase in ornithine decarboxylase activity or [3H]dThd incorporation into DNA when these parameters were assessed at 4 h or 12 to 48 h, respectively; phenidone, an antioxidant and radical scavenger, and bis[(3,5-diisopropyl-salicylato) (O,O) copper(II), a lipophilic agent with superoxide dismutase activity, abolished the DOC mediated proliferative response but did not prevent the early loss of surface cells. The results imply that events other than or in addition to surface cell loss are necessary for the expression of the action of DOC to stimulate the proliferative activity of colonic epithelium.

Topics: Animals; Cell Cycle; Colon; Deoxycholic Acid; DNA; Dose-Response Relationship, Drug; Epithelial Cells; Epithelium; Intestinal Mucosa; Ornithine Decarboxylase; Pyrazoles; Rats; Salicylates

Oxidants, such as those generated by metabolically activated phagocytes in inflammation, have been implicated in the metabolic activation of carcinogens, and in this study we demonstrate that the interaction of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP 7,8-dihydrodiol) with phorbol ester-stimulated polymorphonuclear leukocytes (PMNs) results in the generation of both a chemiluminescent intermediate and one that covalently binds to DNA. Cu(II)(3,5-diisopropylsalicylic acid)2 (CuDIPS), a biomimetic superoxide dismutase, and azide, a myeloperoxidase inhibitor, inhibited both of these reactions, indicating a dependency on oxygen-derived oxidants in these hydrocarbon-activation processes. Concordant with the formation of a carcinogen-DNA adduct, the admixture of BP 7,8-dihydrodiol and phorbol ester-stimulated PMNs elicited mutagenesis in Salmonella typhimurium strain TA100. 7,8-Dihydro-BP and BP cis-7,8-dihydrodiol were also mutagenic, whereas derivatives lacking a double bond at the 9,10 position were not. These results demonstrate that oxidants generated by metabolically stimulated PMNs can activate penultimate polycyclic aromatic hydrocarbons to a genotoxic metabolite and further defines a role for inflammation in carcinogenesis.

Topics: Azides; Benzopyrenes; Biotransformation; Dihydroxydihydrobenzopyrenes; DNA; Humans; Inflammation; Luminescent Measurements; Mutation; Neutrophils; Oxidation-Reduction; Peroxidase; Salicylates; Superoxides; Tetradecanoylphorbol Acetate

The effects of copper (II) (3,5-diisopropylsalicylate)2 (CuDIPS), which is a synthetic superoxide dismutase, on the hepatotoxicity of carbon tetrachloride and acetaminophen in fed and fasted animals were investigated. CuDIPS did not alter the covalent binding of metabolites of either of these chemicals to the hepatic endoplasmic reticulum. However, CuDIPS did inhibit the hepatotoxicity of carbon tetrachloride by inhibiting the induction of lipid peroxidation by carbon tetrachloride. CuDIPS had only a slight, and histologically insignificant, ability to decrease acetaminophen hepatotoxicity which is related to the inability of CuDIPS to prevent depletion of reduced glutathione by acetaminophen. The observation that fasting potentiates the hepatotoxicity of acetaminophen is emphasized, and the mechanism of this potentiation is suggested to be related to the depletion of reduced glutathione.

Topics: Acetaminophen; Animals; Antineoplastic Agents; Carbon Tetrachloride; Lipid Peroxides; Liver; Male; Mice; Rats; Rats, Inbred Strains; Salicylates; Superoxides

Administration of the antioxidants 2(3)-tert-butyl-4-hydroxyanisole (BHA) or 5-(P-methoxyphenyl)-3H-1,2-dithiol-3-thione (ADT) to female CD-1 mice starting 4 weeks after infection with 70 cercariae of Schistosoma mansoni resulted in a decrease in the size of the inner fibrotic region of the hepatic granuloma. The cellular composition of the granuloma was not altered by treatment with these two compounds. The administration of the specific superoxide scavenger copper diisopropylsalicylate (CuDIPS) resulted in a similar decrease in granuloma size, suggesting a role of superoxide radicals in the granulomatous response.

Topics: Anethole Trithione; Animals; Anisoles; Antioxidants; Butylated Hydroxytoluene; Chemotaxis, Leukocyte; Female; Free Radicals; Granuloma; Inflammation; Liver Diseases, Parasitic; Mice; Ovum; Oxygen; Salicylates; Schistosoma mansoni; Schistosomiasis mansoni; Superoxides; Triglycerides

The effect of phorbol ester tumour promoters on the release of superoxide anion radicals .O2- by human peripheral leukocytes and the role of such radicals in tumour promotion of mouse skin was studied. No significant difference was found between complete [12-O-tetradecanoylphorbol-13-acetate (TPA)] as compared with incomplete [12-O-retinoylphorbol-13-acetate (RPA), 12-O-(2Z,4E,6,8)tetradecatetraenoylphorbol-13-acetate (Ti8), mezerein] tumour promoters upon induction of .O2- when measured by the reduction of ferricytochrome c. The semisynthetic phorbol esters 12-O-ethacrynylphorbol-13-acetate (EPA) and 4-O-methyl-TPA were less active, and phorbol diacetate, phorbol and ionophore A 23187 were found to be inactive in stimulating superoxide anion radicals. TPA-induced .O2- release from leukocytes was strongly inhibited by Cu(II)-(diisopropylsalicylate)2 (CuDIPS), and, to a lesser extent, by ethacrynic acid, nordihydroguaiaretic acid and quercetin. Retinoic acid exhibited only a moderate inhibitory effect. No .O2- release was observed in epidermal cell cultures upon TPA treatment. When analysed by the alkaline elution technique, TPA-induced .O2- release from leukocytes did not lead to measurable DNA damage in co-cultivated keratinocytes even in the presence of DNA repair inhibitors. In multi-stage-tumourigenesis experiments including two-stage promotion, retinoic acid, ethacrynic acid and CuDIPS were unable to inhibit tumour promotion in mouse skin when applied in combination with TPA in first stage promotion. gamma-Irradiation at a dose level shown to cause DNA damage in vitro could not replace TPA as a stage I-promoting agent. It is concluded that superoxide anion radicals--if related to promotion at all--may play a role in stage II rather than in stage I of mouse skin tumour promotion.

Topics: Animals; Cells, Cultured; Chromosome Aberrations; DNA; Female; Free Radicals; Leukocytes; Mice; Mice, Inbred Strains; Salicylates; Skin Neoplasms; Superoxides; Tetradecanoylphorbol Acetate

Cu(II) (3,5-diisopropyl salicylate)2 (CuDIPS) which is an anti-inflammatory copper coordination compound (mol. wt. 503) possessing superoxide dismutase (SOD) activity was tested to determine its effect on 7,12-dimethylbenz[a]anthracene (DMBA)-induced initiation of tumors in mouse skin and on mutagenicity to 6-thioguanine resistance in a mouse keratinocyte mediated Chinese hamster V-79 cell system. A single application of CuDIPS (0.4 mg/mouse) administered at a short interval before DMBA application when followed by 20 weeks of promotion by TPA reduced the mouse skin tumor yield by 55%. When DMBA-induced cell-mediated mutagenesis was tested in the presence of CuDIPS a significant reduction in the number of V-79 6-thioguanine resistant mutants was observed.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Benz(a)Anthracenes; Biotransformation; Cocarcinogenesis; Cricetinae; Female; Mice; Salicylates; Skin Neoplasms

We have studied the effects of superoxide dismutase (SOD), catalase, Cu(II) (3,5-diisopropylsalicylate)2 (CuDIPS) and other copper compounds on radiation transformation in vitro using C3H 10T1/2 cells. When present only during irradiation, high concentrations of SOD in the medium enhanced transformation, while catalase, inactivated SOD (autoclaved), CuDIPS, cupric chloride and cuprous chloride inhibited the initiation phase of radiation transformation. SOD, catalase and CuDIPS did not affect the expression phase of radiation transformation. Suppression of the TPA enhancement of transformation by catalase was a highly significant effect, while the suppression by SOD was not of statistical significance. Our results suggest that hydrogen peroxide (H2O2) may be important in the cellular damage leading to malignant transformation.

Topics: Animals; Catalase; Cell Transformation, Neoplastic; Free Radicals; Hydrogen Peroxide; Mice; Salicylates; Superoxide Dismutase; Tetradecanoylphorbol Acetate

Superoxide disproportionation may partially account for the noteworthy radioprotectant activity of bis(3,5-diisopropylsalicylato)copper(II) [CuII(3,5-dips)2]. Groups of mice treated with CuII(3,5-dips)2 3 or 24 h before exposure to a lethal dose of gamma-radiation had survival rates of 33% and 58%, respectively. These results suggest that copper complexes might be developed for protection of normal tissues in association with cancer radiotherapy and protection against occupational exposures to hazardous radiation.

Topics: Animals; Cobalt Radioisotopes; Female; Indicators and Reagents; Mice; Mice, Inbred Strains; Radiation-Protective Agents; Salicylates; Structure-Activity Relationship; Superoxide Dismutase

Experimental diabetes can be produced by agents with specific toxicity for pancreatic islet B cells. This effect has been reported to be modified both in vitro and in vivo by various radical scavengers including the enzyme superoxide dismutase. Copper(II)(3,5-diisopropylsalicylate)2 is lipophilic and possesses superoxide dismutase bioactivity. Prior administration of this compound to male rats appeared to attenuate the severity of streptozotocin-induced diabetes as assessed by glycosuria and glucose tolerance. Diisopropylsalicylate, which has no superoxide dismutase activity, did not alter the severity of streptozotocin-induced diabetes. Rats treated with the copper complex, with streptozotocin or with a combination of the two agents gained 50% less weight than untreated controls, or rats treated with diisopropylsalicylate. The attenuation of diabetes by the copper-complex may represent partial protection of the B cells against streptozotocin damage, although an extrapancreatic, toxic effect cannot be ruled out.

Topics: Animals; Diabetes Mellitus, Experimental; Glucose Tolerance Test; Glycosuria; Male; Rats; Rats, Inbred Strains; Salicylates; Streptozocin; Superoxide Dismutase

Topics: Free Radicals; Humans; Neutrophils; Salicylates; Superoxides; Tetradecanoylphorbol Acetate

The effect of glutathione and a glutathione reductase inhibitor on the antitumor effect of Cu(II)(3,5-diisopropylsalicylate)2 (CuDIPS) was studied. CuDIPS is a low-molecular-weight copper coordination compound that exhibits superoxide dismutase-like activity. CuDIPS had antitumor activity against intraperitoneal Ehrlich ascites carcinoma in Swiss mice. A single ip injection of glutathione partially eliminated the antitumor effect of CuDIPS, whereas a single ip injection of 1,3-bis(2-chloroethyl)-1-nitrosourea enhanced the antitumor effect of CuDIPS. These results are consistent with the hypothesis that CuDIPS exerts part of its antitumor effect by producing H2O2.

Topics: Animals; Antineoplastic Agents; Body Weight; Carcinoma, Ehrlich Tumor; Chemical Phenomena; Chemistry; Glutathione; Glutathione Reductase; Male; Mice; Neoplasm Transplantation; Salicylates; Superoxide Dismutase; Time Factors

A low molecular weight, lipophilic, copper coordination complex with superoxide dismutase-mimetic activity inhibited biochemical and biological actions of a tumor promoter in mouse epidermis. Such inhibitory effects implicate reactive oxygen species in the tumor promotion process.

Topics: Animals; Antineoplastic Agents; Carcinogens; Female; Mice; Ornithine Decarboxylase; Papilloma; Salicylates; Skin Neoplasms; Superoxide Dismutase; Tetradecanoylphorbol Acetate

Growth of Ehrlich carcinomas in inbred CBA mice was retarded by im administration of Cu(II)(3,5-diisopropylsalicylate)2 (CuDIPS). CuDIPS is a low molecular weight (mol wt = 503) copper coordination compound that exhibits superoxide dismutase (SOD)-like activity. It has been used as an anti-inflammatory agent and is lipid-soluble. This property enables the compound to penetrate membranes, thus becoming an intracellular O2- scavenger. In the tumor system studied, the amounts of both copper- and zinc-containing SOD (CuZnSOD) and manganese-containing SOD are reduced. Injection of Orgotein (CuZnSOD from bovine liver) had no significant effect on tumor growth and host survival. When CuDIPS was administered at various doses, reduction in tumor size, delay of metastasis, and a significant increase in survival of the hosts were observed.

Topics: Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Injections, Intramuscular; Mice; Mice, Inbred CBA; Prognosis; Salicylates; Superoxide Dismutase