Free Radical Biology and Medicine

The oxidized glutathione mimetic NOV-002 is a unique anti-tumor agent that not only has the ability to inhibit tumor cell proliferation, survival, and invasion, but in some settings can also ameliorate cytotoxic chemotherapy-induced hematopoietic and immune suppression. However, the mechanisms by which NOV-002 protects the hematopoietic and immune systems against the cytotoxic effects of chemotherapy are not known. Therefore, in this study we investigated the mechanisms of action of NOV-002 using a mouse model in which hematopoietic and immune suppression was induced by cyclophosphamide (CTX) treatment. We found that NOV-002 treatment in a clinically comparable dose regimen attenuated CTX-induced reduction in bone marrow hematopoietic stem and progenitor cells (HSPCs) and reversed the immunosuppressive activity of myeloid-derived suppressor cells (MDSCs), which led to a significant improvement in hematopoietic and immune functions. These effects of NOV-002 may be attributable to its ability to modulate cellular redox. This suggestion is supported by the finding that NOV-002 treatment upregulated the expression of superoxide dismutase 3 and glutathione peroxidase 2 in HSPCs, inhibited CTX-induced increases in reactive oxygen species production in HSPCs and MDSCs, and attenuated CTX-induced reduction of the ratio of reduced glutathione to oxidized glutathione in splenocytes. These findings provide a better understanding of the mechanisms whereby NOV-002 modulates chemotherapy-induced myelosuppression and immune dysfunction and a stronger rationale for clinical utilization of NOV-002 to reduce chemotherapy-induced hematopoietic and immune suppression.

L-012, a luminol-based chemiluminescent (CL) probe, is widely used in vitro and in vivo to detect NADPH oxidase (Nox)-derived superoxide (O2(•-)) and identify Nox inhibitors. Yet understanding of the free radical chemistry of L-012 probe is still lacking. We report that peroxidase and H2O2 induce superoxide dismutase (SOD)-sensitive, L-012-derived CL in the presence of oxygen. O2(•-) alone does not react with L-012 to emit luminescence. Self-generated O2(•-) during oxidation of L-012 and luminol-analogs artifactually induce CL inhibitable by SOD. These aspects make assays based on luminol analogs less than ideal for specific detection and identification of O2(•-) and NOX inhibitors.

In the present study we sought to determine the ability of the chemiluminescence dye 8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012) to detect superoxide in different biological systems. In human whole blood or isolated leukocytes, the sensitivity of the luminol analogue L-012 to detect superoxide was higher as compared with luminol, lucigenin, coelenterazine, and the fluorescence dye dihydroethidine. In isolated leukocytes as well as aortic rings from control (New Zealand White) and hyperlipidemic (Watanabe heritable hyperlipidemic) rabbits, L-012-enhanced chemiluminescence was successful in detecting differences in superoxide formation under basal conditions and on stimulation with the direct activator of protein kinase C, phorbol 12,13-dibutyrate (PDBu). The effects of PDBu were abrogated by gliotoxin and inhibitors of protein kinase C such as chelerythrine, identifying NAD(P)H oxidase as the significant superoxide source. Experiments using electron paramagnetic resonance and the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide revealed that in contrast to lucigenin, L-012 is not subject to redox cycling. These findings indicate that L-012-enhanced chemiluminescence represents a sensitive and reliable probe to detect superoxide in whole blood, inflammatory cells, and vascular tissue.

Production of reactive oxygen and nitrogen species (ROS/RNS) is an important part of the inflammatory response, but prolonged elevated levels of ROS/RNS as under chronic inflammation can contribute to the development of disease. Monitoring ROS/RNS in living animals is challenging due to the rapid turnover of ROS/RNS and the limited sensitivity and specificity of ROS/RNS probes. We have explored the use of the chemiluminescent probe L-012 for noninvasive imaging of ROS/RNS production during inflammation in living mice. Various inflammatory conditions were induced, and L-012-dependent luminescence was recorded with an ultrasensitive CCD camera. Strong luminescent signals were observed from different regions of the body corresponding to inflammation. The signal was reduced by administration of the SOD mimetic tempol, the NADPH oxidase inhibitor apocynin, and the inhibitor of nitric oxide synthesis L-NAME, signifying the requirement for the presence of ROS/RNS. Additionally, the L-012 signal was abolished in mice with a mutation in the Ncf1 gene, encoding a protein in the NADPH oxidase complex 2, which generates ROS/RNS during inflammation. In conclusion, L-012 is well distributed in the mouse body and mediates a strong ROS/RNS-dependent luminescent signal in vivo and is useful for monitoring the development and regulation of inflammation in living organisms.

To evaluate the contribution of reactive nitrogen species to inflammation by asbestos, Fischer 344 rats were exposed to crocidolite or chrysotile asbestos by inhalation to determine whether increases occurred in nitric oxide (NO.) metabolites from alveolar macrophages (AMs). AMs from animals inhaling asbestos showed significant elevations (p < .05) in nitrite/nitrate levels which were ameliorated by NG-monomethyl-L-arginine (NMMA), an inhibitor of inducible nitric oxide synthase (iNOS) activity. Temporal patterns of NO. generation from AMs correlated with neutrophil influx in bronchoalveolar lavage samples after asbestos inhalation or bleomycin instillation, another model of pulmonary fibrosis. To determine the molecular mechanisms and specificity of iNOS promoter activation by asbestos, RAW 264.7 cells, a murine macrophage-like line, and AMs isolated from control rats were exposed to crocidolite asbestos in vitro. These cells showed increases in steady-state levels of iNOS mRNA in response to asbestos and more dramatic increases in both iNOS mRNA and immunoreactive protein after addition of lipopolysaccharide (LPS). After transfection of an iNOS promoter/luciferase reporter construct, RAW 264.7 cells exposed to LPS, crocidolite asbestos and its nonfibrous analog, riebeckite, revealed increases in luciferase activity whereas cristobalite silica had no effects. Studies suggest that NO. generation may be important in cell injury and inflammation by asbestos.

The effects of IRFI-048 (2,3-dihydro-5-methoxy-4,6,7-trimethyl-2-benzofuranyl acetic acid), a selective analogue of Vitamin E, on myocardial tissue injury were examined in anaesthetized rats subjected to 60-min occlusion of the left coronary artery followed by 60-min reperfusion. Infarct size (Evan's blue and tetrazolium stain), serum creatinphosphokinase (CPK), plasma malonaldehyde (MAL), cardiac myeloperoxidase (MPO) activity, and ST-segment of electrocardiogram (ECG) and survival rate were evaluated. Postischaemic reperfusion produced severe cardiac necrosis, caused neutrophil (PMNs) infiltration (evaluated by MPO activity) in the jeopardized tissue, increased serum CPK and plasma MAL, raised ST-segment of ECG, and decreased survival rate. IRFI-048, (200 and 400 mg/kg o.s.) given to the rats 6 h before occlusion, caused a reduction of necrotic area expressed as a percentage of either the area at risk or the total left ventricle, decreased MPO activity both in the area at risk (from 3.2 +/- 0.3 U x 10(-3)/g tissue to 1.1 +/- 0.4 U x 10(-3)/g tissue; p < .005) and in the necrotic area (from 5.7 +/- 0.9 U x 10(-3)/g tissue to 1.8 +/- 0.5 U x 10(-3)/g tissue; p < .001), attenuated the rise of ST-segment of ECG (from 0.51 +/- 0.14 mV in the vehicle group to 0.28 +/- 0.11 mV in the treated group; p < .005), reduced the increase of plasma MAL and serum CPK during reperfusion (from 42 +/- 5.3 nmol/ml to 15 +/- 3.1 nmol/ml and 139 +/- 13 IU/100 ml to 58 +/- 7.5 IU/100 ml, respectively; p < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

The scavenging effects of tea catechins and their epimerized, acylated, and glucostylated derivatives on 1,1-diphenyl-2-picrythydrazyl (DPPH) radical were evaluated by electron spin resonance spectrometry. Tea catechins and their epimers were shown to have 50% radical scavenging ability in the concentration range of 1 to 3 microM. No significant differences were observed between the scavenging activities of tea catechins and their epimers, and, hence, the scavenging effects of catechins are not dependent on their sterical structure. The relationship between scavenging ability and the structure of tea catechins was also examined with acylated and glucosylated catechin derivatives. It is suggested that the galloyl moiety attached to flavan-3-ol at 3 position has a strong scavenging ability on the DPPH radical as well as the ortho-trihydroxyl group in the B ring, which elevates the radical scavenging efficiency above that of the ortho-dihydroxyl group; as has been recognized in other flavonoids such as flavones. The results obtained from the reactivity of tea catechins with the DPPH radical at different pHs suggest not only that the ortho-trihydroxyl group and the galloyl moiety contribute to maintaining the DPPH radical scavenging ability more effectively in a wide range of conditions from acidic to alkaline, but also that the radical scavenging efficiency of the ortho-dihydroxyls in the B ring is limited in neutral to alkaline regions. The difference between the scavenging abilities of the trihydroxyls (probably in the galloyl moiety) and the dihydroxyls can be explained in terms of redox potentials. It is concluded that the ortho-trihydroxyl group in the B ring and the galloyl moiety at 3 position of flavan-3-ol skeleton are the most important structural features for displaying an excellent scavenging ability on the DPPH radical.

Mechanistic details of the interaction of 1,10-phenanthroline and its copper complex with Ehrlich ascites tumor cells were examined, using inhibition of cell proliferation, DNA breakage, and increased membrane permeability as indices of cellular damage. The metal chelating agent, 1,10-phenanthroline (OP), the 1:0.5 complex of 1,10-phenanthroline and CuCl2 [(OP)2Cu], and CuCl2 inhibited growth of Ehrlich ascites tumor cell monolayers during 48-h treatments by 50% at about 3.5, 2, and 70 nmol/10(5) cells/mL, respectively. (OP)2Cu at 10 nmol/10(5) cells also enhanced uptake of trypan blue dye during 6 h of treatment, while dye uptake in OP- and CuCl2-treated cells remained similar to controls. DNA breakage, measured by DNA alkaline elution, was produced during 1-h treatments with (OP)2Cu at drug/cell ratios similar to those producing growth inhibition. Copper uptake was similar for both (OP)2Cu and CuCl2. Electron spin resonance (ESR) spectroscopy suggested that cellular ligands bind copper added as (OP)2Cu or CuCl2 and then undergo time-dependent reductions of Cu(II) to Cu(I) for both forms. Inhibition of (OP)2Cu-induced single-strand scission and trypan blue uptake by scavengers of activated oxygen is consistent with participation of superoxide and H2O2 in both processes. In contrast, superoxide dismutase (SOD) did not reduce the magnitude of the fraction of cellular DNA appearing in lysis fractions prior to alkaline elution of (OP)2Cu-treated cells. Dimethyl sulfoxide (DMSO) inhibited uptake of trypan blue dye but did not inhibit DNA strand scission produced by (OP)2Cu. Thus, multiple mechanisms for generation of oxidative damage occur in (OP)2Cu-treated cells. Growth inhibition produced by OP or (OP)2Cu, as well as the low levels of strand scission produced by OP, was not reversed by scavengers.

DNA-targeting copper(II) reagents have emerged as suitable drug candidates owing to the clinical success of the copper-activated, natural chemotherapeutic drug bleomycin. This agent and the synthetic chemical nuclease copper(II) bis-1,10-phenanthroline represent important templates for inorganic drug design owing to their ability to initiate free radical DNA scission. Herein, we report the synthesis and biological properties of 1:1:1 square-planar copper(II) complexes incorporating the dicarboxylate o-phthalate and 1,10-phenanthroline (1) or 2,2'-dipyridyl (2) ligands. Their broad-spectrum chemotherapeutic potential has been assessed at 24- and 96-h intervals, along with that of the clinical agent cisplatin, using breast (MCF-7), prostate (DU145), colon (HT29), and intrinsically cisplatin-resistant ovarian (SK-OV-3) human cancer cells. 1 represents a potent cytotoxic agent with IC(50) values ranging from 5.6 to 3.4μM across all cell lines, including SK-OV-3. The production of endogenous reactive oxygen species within SK-OV-3 cancer cells was monitored using the fluorophore 2',7'-dichlorodihydrofluorescin diacetate, and results indicate a concentration-dependent propensity toward ROS generation by 1 and 2 that mirrors their antitumoral behavior. DNA interaction studies, using fluorescence and viscosity measurements, were conducted in tandem with the DNA-targeting drugs actinomycin D and pentamidine, using calf thymus DNA, poly[d(A-T)(2)], and poly[d(G-C)(2)], with intercalation of 1 and 2 at the minor groove appearing to be the likely interaction mode. DNA cleavage reactions using superhelical plasmid DNA, in the presence of exogenous reductant, l-ascorbic acid, revealed excellent agreement between double-stranded DNA scission capability and antitumoral IC(50) concentration. The presence of double-strand DNA breaks (DSBs) was confirmed within SK-OV-3 cancer cells using immunodetection of γ-H2AX foci by confocal microscopy and flow cytometry, with complex 1 quantitatively producing superior numbers of DSBs compared with complex 2. Superoxide dismutase and catalase mimetic activity assays were conducted, and these activities are related to the ability of both complexes to cleave DNA through free radical generation.

At neutral pH values 1,10-phenanthroline forms a colored complex with Fe(II), but it does not form such a complex with Fe(III). On the contrary, only Fe(III) forms with desferal a yellow complex with a g = 4.3 electron paramagnetic resonance (EPR) signal, but Fe(II) is rapidly oxidized by desferal to Fe(III), which gives then a yellow complex. On the basis of these facts, a method for simultaneous determination of both Fe(II) and Fe(III) ions was elaborated using a desferal-phenanthroline mixture. Two ways of detecting Fe(II) and Fe(III) have been suggested: (1) the spectrophotometric method for transparent water solutions, and (2) the EPR-spectrometric method for turbid solutions and tissue homogenates. The latter method was applied for determination of free and weakly bound iron in rat liver. The Fe(II) amount in intact liver was 22.2 +/- 7.6 nmol/g of wet tissue; free Fe(III) was not found.

The kinetics of the reaction between the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and methylated urates was studied. Urates that had methyl groups on the 1,3,9, or on the 1 and 3 or 1 and 9 nitrogens reacted with DPPH 15 to 77% faster than uric acid. Urates substituted with methyl groups on the 7 nitrogen or on both the 3 and 9 nitrogens reacted with DPPH at rates that were less than 0.1 that of uric acid. 3,7,9-Trimethyluric acid and 1,3,7,9-tetramethyluric acid reacted with DPPH at barely detectable rates. DPPH reacted with uric acid, the monomethylated urates, and some of the dimethylated urates in a ratio of 2:1. DPPH reacted with other dimethylated and trimethylated urates in a ratio of 1:1. Semiempirical MNDO calculations indicate that the most stable radical of uric acid is formed by hydrogen abstraction from the 3, 7 or 9 position. The most stable species resulting from loss of a second hydrogen lack hydrogens at the 3 and 7 positions or the 7 and 9 positions. For maximum reactivity with DPPH, methylated uric acid derivatives must have a hydrogen at nitrogen 7 and one of the hydrogens at either the 3 or 9 position.

Experimental acute intoxication by prooxidant haloalkanes produces marked stimulation of hepatic lipid peroxidation and cytolysis, which is followed by tissue regeneration. Our aim was to clarify the role of oxidative imbalance in the activation of the redox-sensitive transcription factor, activator protein-1 (AP-1), which is involved in tissue repair. Rats were poisoned with a very low concentration of carbon tetrachloride, given alone or in combination with another hepatotoxin, 1,2-dibromoethane, to provide varying extents of oxidative damage. The level of AP-1-DNA binding was analyzed by electrophoretic mobility shift assay on liver extracts, obtained from rats killed 6 h after poisoning. Stimulation of lipid peroxidation and AP-1 upregulation were already established when the hepatic damage due to carbon tetrachloride +/-1,2-dibromoethane was beginning to appear. Rat supplementation with the antioxidant vitamin E completely inhibited AP-1 upregulation, thus supporting a causative role of membrane lipid oxidation in the observed modulation of the transcription factor. Moreover, activation of Kupffer cells appears to be a crucial step in the increased AP-1 binding to DNA, the latter being largely prevented by gadolinium chloride, a macrophage-specific inhibitor.

Tight linkage between aging and oxidative stress is indicated by the observations that reactive oxygen species generated under various conditions of oxidative stress are able to oxidize nucleic acids, proteins, and lipids and that aging is associated with the accumulation of oxidized forms of cellular constituents, and also by the fact that there is an inverse relationship between the maximum life span of organisms and the age-related accumulation of oxidative damage. Nevertheless, validity of the oxidative stress hypothesis of aging is questioned by (i) the failure to establish a causal relationship between aging and oxidative damage and (ii) lack of a consistent correlation between the accumulation of oxidative damage and aging. The present discussion is focused on the complexity of the aging process and suggests that discrepancies between various studies in this area are likely due to the fact that aging is not a single process and that the lack of consistent experimental results is partly explained by individual variations. Even so, there is overwhelming support for a dominant role of oxidative stress in the aging of some individuals.

Free radicals and other reactive species are generated in vivo and many of them can cause oxidative damage to DNA. Although there are methodological uncertainties about accurate quantitation of oxidative DNA damage, the levels of such damage that escape immediate repair and persist in DNA appear to be in the range that could contribute significantly to mutation rates in vivo. The observation that diets rich in fruits and vegetables can decrease both oxidative DNA damage and cancer incidence is consistent with this. By contrast, agents increasing oxidative DNA damage usually increase risk of cancer development. Such agents include cigarette smoke, several other carcinogens, and chronic inflammation. Rheumatoid arthritis and diabetes are accompanied by increased oxidative DNA damage but the pattern of increased cancer risk seems unusual. Other uncertainties are the location of oxidative DNA damage within the genome and the variation in rate and level of oxidative damage between different body tissues. In well-nourished human volunteers, fruits and vegetables have been shown to decrease oxidative DNA damage in several studies, but data from short-term human intervention studies suggest that the protective agents are not vitamin C, vitamin E, beta-carotene, or flavonoids.

1,2-Naphthoquinone (1,2-NQ) is electrophilic, and forms covalent bonds with protein thiols, but its two-electron reduction product 1,2-dihydroxynaphthalene (1,2-NQH(2)) is not, so enzymes catalyzing the reduction with reduced pyridine nucleotides as cofactors could protect cells from electrophile-based chemical insults. To assess this possibility, we examined proteins isolated from the 9000g supernatant from mouse liver for 1,2-NQ reductase activity using an HPLC assay procedure for the hydroquinone of 1,2-NQ and Cibacron Blue 3GA column chromatography and Western blot analysis with specific antibody to determine 1,2-NQ-bound proteins. Among the proteins with high affinities for pyridine nucleotides that also inhibited 1,2-NQ-protein adduct formation in the presence of NADH, a 37-kDa protein was found and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using recombinant human GAPDH, we found that this glycolytic enzyme indeed catalyzes the two-electron reduction of 1,2-NQ accompanied by extensive NADH consumption under 20% oxygen conditions. When either 1,2-NQH(2) or 1,2-NQ was incubated with GAPDH in the presence of NADH, minimal covalent bonding to the enzyme occurred compared to that in its absence. These results indicate that GAPDH can inhibit 1,2-NQ-based electrophilic protein modification by conversion to the nonelectrophilic 1,2-NQH(2) via an NADH-dependent process.

Several 1,2-dithiole-3-thiones are potent inhibitors of chemical-induced tumors in multiple tissues. Chemoprotection by 1, 2-dithiole-3-thiones has been associated with induction of detoxication enzymes, although several studies suggest that additional mechanisms may be involved. In this study, we examined the induction of hepatic antioxidant genes in rats treated with 3H-1, 2-dithiole-3-thione (D3T). After a 24 h D3T treatment, a 2.4-fold increase in catalase mRNA was observed, which was accompanied by a 1. 5-fold increase in catalase protein expression and a 2.3-fold increase in catalase activity. D3T also mediated 2.9-, 5.9-, and 3. 7-fold increases in the 1.0, 3.0, and 4.0 kb mRNA species of manganese superoxide dismutase (MnSOD), respectively. The induction of MnSOD mRNA by D3T was coincident with 1.7-fold and 4.6-fold increases in MnSOD protein and enzyme activity, respectively. Induction of gamma-glutamylcysteine synthetase mRNA by D3T was accompanied by an increase in glutathione levels. Nuclear run-on assays provided evidence that D3T enhances the transcription rate from MnSOD, catalase, and gamma-glutamylcysteine synthetase. In support of this view, D3T also activated an MnSOD promoter-reporter construct in transiently transfected HepG2 cells. In light of observations that antioxidant enzyme regulation may be altered during carcinogenesis, induction of these genes could provide a potentially important mechanism of action of chemoprotective 1, 2-dithiole-3-thiones.

Specific inhibitors of the production of reactive oxygen species (ROS) by the NADPH oxidases (Nox's) are potentially important therapeutic agents in the wide range of human diseases that are characterized by excessive ROS production. It has been proposed that VAS2870 (3-benzyl-7-(2-benzoxazolyl)thio-1,2,3- triazolo[4,5-d]pyrimidine), identified as an inhibitor of Nox2 by small-molecule screening, may serve as an example of such an agent. Here we show that VAS2870 inhibits ROS production in the sarcoplasmic reticulum (SR) of mammalian skeletal muscle, previously identified with Nox4, and thereby abrogates O(2)-coupled redox regulation of the ryanodine receptor-Ca(2+) channel (RyR1). However, we also find that VAS2870 modifies directly identified cysteine thiols within RyR1. Mass spectrometric analysis of RyR1 exposed in situ to VAS2870 and of VAS2870-treated glutathione indicated that thiol modification is through alkylation by the benzyltriazolopyrimidine moiety of VAS2870. Thus, VAS2870 exerts significant off-target effects, and thiol alkylation by VAS2870 (and closely related Nox inhibitors) may in fact replicate some of the effects of ROS on cellular thiol redox status. In addition, we show that SR-localized Nox4 is inhibited by other thiol-alkylating agents, consistent with a causal role for cysteine modification in the inhibition of ROS production by VAS2870.

Iron chelates of inositol 1,2,3-trisphosphate and inositol 1,2,3,6-tetrakisphosphate lacked free coordination sites and prevented the iron-catalyzed oxidation of ascorbic acid and peroxidation of arachidonic acid. In contrast, iron chelates of inositol 1,2,6-trisphosphate and inositol 1,2,5,6-tetrakisphosphate contained available coordination sites, permitted iron-catalyzed ascorbic acid oxidation, and enhanced arachidonic acid peroxidation. It was concluded that the 1,2,3-trisphosphate grouping of inositol hexakisphosphate was responsible for the inhibition of iron-catalyzed hydroxyl radical formation. The structure of the chelate with the phosphates in an axial-equatorial-axial configuration appeared to be the only possible inositol trisphosphate that could form bonds between six oxygen atoms and the six coordination sites on iron. Km values for cleavage by Escherichia coli alkaline phosphatase were as follows: inositol 1,2,3-trisphosphate, 56 microM; inositol 1,2,6-trisphosphate, 35 microM; inositol 1,2,3,6-tetrakisphosphate, 139 microM; and inositol 1,2,5,6-tetrakisphosphate, 100 microM. The initial hydrolysis rates of 200 microM solutions of the latter three isomers by E. coli alkaline phosphatase were not affected by an equimolar concentration of iron, whereas the rate for inositol 1,2,3-trisphosphate decreased in the presence of iron to 50% of the control. Therefore, the antioxidant potential of inositol 1,2,3-trisphosphate and inositol 1,2,3,6-tetrakisphosphate in cells and other biological systems may be fortified by the resistance of their iron chelates to enzymatic hydrolysis of the functional 1,2,3-trisphosphate array.

Currently, obesity is considered a systemic inflammation; however, the effects of obesity on the vulnerability of dopaminergic neurons to oxidative stress are not fully defined. We evaluated the effects of high-fat diet-induced obesity (HF DIO) on neurotoxicity in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Eight weeks after a HF or matched normal diet, a severe decrease in the levels of striatal dopamine and of nigral microtubule-associated protein 2, manganese superoxide dismutase, and tyrosine hydroxylase was observed in obese mice treated with subtoxic doses of MPTP (20 mg/kg) compared with the matched lean group. In addition, the levels of nitrate/nitrite and thiobarbituric acid-malondialdehyde adducts in the substantia nigra of obese mice were reciprocally elevated or suppressed by MPTP. Interestingly, striatal nNOS phosphorylation and dopamine turnover were elevated in obese mice after MPTP treatment, but were not observed in lean mice. The nitrotyrosine immunoreactivity for evaluation of nigral nitrogenous stress in obese mice with MPTP was higher than that in matched lean mice. At higher doses of MPTP (60 mg/kg), the mortality was higher in obese mice than in lean mice. These results suggest that DIO may increase the vulnerability of dopaminergic neurons to MPTP via increased levels of reactive oxygen and nitrogen species, and the role of nNOS phosphorylation in the MPTP toxicities and dopamine homeostasis should be further evaluated.

Superoxide dismutases (SODs) have been found to decrease tumor formation and angiogenesis. SOD gene therapy, as with many other gene transfer strategies, may not completely inhibit tumor growth on its own. Thus, concomitant therapies are necessary to completely control the spread of this disease. We hypothesized that intratumoral injection of AdSOD in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) chemotherapy would synergistically inhibit breast cancer growth. Our data indicate that BCNU when combined with SOD overexpression increased oxidative stress as suggested by elevated glutathione disulfide (GSSG) production in one of three breast cancer cell lines tested, at least in part due to glutathione reductase (GR) inactivation. The increased oxidative stress caused by BCNU combined with adenovirally expressed SODs, manganese or copper zinc SOD, decreased growth and survival in the three cell lines tested in vitro, but had the largest effect in the MDA-MB231 cell line, which showed the largest amount of oxidative stress. Delivery of MnSOD and BCNU intratumorally completely inhibited MDA-MB231 xenograft growth and increased nude mouse survival in vivo. Intravenous (iv) BCNU, recapitulating clinical usage, and intratumoral AdMnSOD delivery, to provide tumor specificity, provided similar decreased growth and survival in our nude mouse model. This cancer therapy produced impressive results, suggesting the potential use of oxidative stress-induced growth inhibitory treatments for breast cancer patients.

The nucleophilic addition of GSH to quinonoid compounds, characterized as a 1,4-reductive addition of the Michael type, was studied with p-benzoquinone- and 1,4-naphthoquinone epoxides with different degree of methyl substitution. Identification and evaluation of molecular products from the above reaction were assessed by h.p.l.c. with either reductive or oxidative electrochemical detection, based on the redox properties retained in the molecular products formed. It was found that the degree of methyl substitution of the quinone epoxide, from either the 1,4-naphthoquinone- or p-benzoquinone epoxide series, determined their rate of reaction with GSH. The reductive addition implied the rearrangement of the quinone structure with opening of the epoxide ring yielding as the primary product a hydroxy-glutathionyl substituted adduct of either p-benzohydroquinone or 1,4-naphthohydroquinone. The primary product undergoes elimination reactions and redox transitions which bring about a number of secondary molecular products. The distribution pattern of the latter depends on the degree of methyl substitution of the quinone epoxide studied and on the concentration of O2 in the solution. The occurrence of the hydroxy-substituent in position alpha, adjacent to the carbonyl group, enhances the autoxidation properties of the compound resulting in an augmented O2 consumption and H2O2 production. Therefore, it could be expected that the chemical reactivity of the products originating from the thiol-mediated nucleophilic addition to quinone epoxides would be of toxicological interest.

The α-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB's cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37°C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other α-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml-1) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(•), and those with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(•) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 μM) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity.

The short-term toxicities of 2-methyl-1,4-naphthoquinone and a series of 2,3-dialkyl-1,4-naphthoquinones have been determined in rats and compared with their ability to cause oxidative damage to erythrocytes in vitro. In accord with previous results, 2-methyl-1,4-naphthoquinone caused marked oxidative damage to erythrocytes in vitro and haemolytic anaemia in rats. The dialkylnaphthoquinones were also haemolytic agents in vivo, with 2,3-dimethyl-1,4-naphthoquinone being particularly active. Unlike the monoalkyl derivative, however, these substances caused little or no damage to red cells in vitro. The in vivo toxicity of dialkylnaphthoquinones cannot, therefore, be predicted on the basis of in vitro cytotoxicity tests.

In this work we investigated the toxicity of a polyphenolic p-benzoquinone derivative, the tetrahydroxy-1,4-quinone (THQ) toward V79 Chinese hamster fibroblasts and analyzed the role of H2O2 and Ca2+ in that mechanism. The exposure of exponentially growing cultures to THQ, in the presence of 1.0 mM Ca2+, caused a dose-dependent inhibition of cell growth and DNA synthesis. Complete prevention of those effects by catalase indicated that H2O2-induced damages should underlie both toxic processes. Further detection of a rise in the intracellular free Ca2+ concentration ([Ca2+]i) in cells exposed to THQ plus Ca2+, together with the partial protection conferred by the intracellular Ca(2+)-chelator fura-2 against cell growth inhibition, indicated that a disruption of Ca2+ homeostasis is a determinant event in THQ cytotoxicity. Furthermore, the intracellular accumulation of rhodizonic acid (RDZ), the primary oxidative product of THQ, indicated that THQ, or its corresponding semiquinone form, was entering the cells and undergoing further autoxidation to RDZ. It was also evidenced that mitochondria represent an important target in the development of THQ toxicity, as shown by the disruption of the transmembrane electrical potential (delta psi) of isolated rat liver mitochondria, as well as by the Ca(2+)-release by mitochondria of permeabilized V79 cells. We concluded that disruption of Ca2+ homeostasis and generation of H2O2 are critically involved in THQ-induced impairment of DNA replication and mitochondrial functions, leading ultimately to cell growth inhibition.