Futile Redox Cycling Research Articles

Contribution of Oxidative Metabolism to Cocaine-Induced Liver and Kidney Damage

Cocaine is a potent psychoactive illicit substance and its abuse represents a major health burden worldwide. The pharmacodynamics and toxicity of cocaine have been extensively documented, and are generally associated to its affinity towards neurotransmitters transporters and several receptors. However, drug-related formation of reactive compounds, as is the case of pro-oxidant reactive species, and interaction at molecular level is still an understudied matter. The involvement of oxidative stress (OS) in cocaine-induced toxicity has been reported in both human and animal models, in several organs and systems, including heart, liver, kidney, and central nervous system (CNS). Cytochrome P450 (CYP450)-mediated cocaine metabolism yields the reactive pro-oxidant compound norcocaine (NCOC) and further oxidative metabolites. Special emphasis should be given to the stable radical norcocaine nitroxide (NCOC-NO·), which plays a key role in cocaine-induced hepatotoxicity, either by entering a futile redox cycle with an N-oxidative metabolite, or by being further oxidized to a highly reactive ion. In fact, cocaine-induced generation of reactive oxygen species (ROS) and consequent OS has been postulated based on the reactivity of cocaine N-oxidative metabolites. Depletion of cellular antioxidant defenses and impairment of mitochondrial respiration have also been considered important causes of ROS production, and subsequent cell death mediated by cocaine. The present review provides a thorough description of the current knowledge on cocaine oxidative metabolism and its role on drug-induced liver and kidney damage.

The role of base excision repair genes OGG1, APN1 and APN2 in benzo[a]pyrene-7,8-dione induced p53 mutagenesis

Lung cancer is primarily caused by exposure to tobacco smoke. Tobacco smoke contains numerous carcinogens, including polycyclic aromatic hydrocarbons (PAH). The most common PAH studied is benzo[a]pyrene (B[a]P). B[a]P is metabolically activated through multiple routes, one of which is catalyzed by aldo-keto reductase (AKR) to B[a]P-7,8-dione (BPQ). BPQ undergoes a futile redox cycle in the presence of NADPH to generate reactive oxygen species (ROS). ROS, in turn, damages DNA. Studies with a yeast p53 mutagenesis system found that the generation of ROS by PAH o-quinones may contribute to lung carcinogenesis because of similarities between the patterns (types of mutations) and spectra (location of mutations) and those seen in lung cancer. The patterns were dominated by G to T transversions, and the spectra in the experimental system have mutations at lung cancer hotspots. To address repair mechanisms that are responsible for BPQ induced damage we observed the effect of mutating two DNA repair genes OGG1 and APE1 (APN1 in yeast) and tested them in a yeast reporter system for p53 mutagenesis. There was an increase in both the mutant frequency and the number of G:C/T:A transversions in p53 treated with BPQ in ogg1 yeast but not in apn1 yeast. Knocking out APN2 increased mutagenesis in the apn1 cells. In addition, we did not find a strand bias on p53 treated with BPQ in ogg1 yeast. These studies suggest that Ogg1 is involved in repairing the oxidative damage caused by BPQ, Apn1 and Apn2 have redundant functions and that the stand bias seen in lung cancer may not be due to impaired repair of oxidative lesions.

Metabolism and Toxicity of Thioacetamide and Thioacetamide S-Oxide in Rat Hepatocytes

The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrolobular necrosis accompanied by increases in plasma transaminases and bilirubin. To elicit these effects, TA requires oxidative bioactivation, leading first to its S-oxide (TASO) and then to its chemically reactive S,S-dioxide (TASO(2)), which ultimately modifies amine-lipids and proteins. To generate a suite of liver proteins adducted by TA metabolites for proteomic analysis and to reduce the need for both animals and labeled compounds, we treated isolated hepatocytes directly with TA. Surprisingly, TA was not toxic at concentrations up to 50 mM for 40 h. On the other hand, TASO was highly toxic to isolated hepatocytes as indicated by LDH release, cellular morphology, and vital staining with Hoechst 33342/propidium iodide. TASO toxicity was partially blocked by the CYP2E1 inhibitors diallyl sulfide and 4-methylpyrazole and was strongly inhibited by TA. Significantly, we found that hepatocytes produce TA from TASO relatively efficiently by back-reduction. The covalent binding of [(14)C]-TASO is inhibited by unlabeled TA, which acts as a "cold-trap" for [(14)C]-TA and prevents its reoxidation to [(14)C]-TASO. This in turn increases the net consumption of [(14)C]-TASO despite the fact that its oxidation to TASO(2) is inhibited. The potent inhibition of TASO oxidation by TA, coupled with the back-reduction of TASO and its futile redox cycling with TA, may help explain phenomena previously interpreted as "saturation toxicokinetics" in the in vivo metabolism and toxicity of TA and TASO. The improved understanding of the metabolism and covalent binding of TA and TASO facilitates the use of hepatocytes to prepare protein adducts for target protein identification.

Detoxication of Benzo[a]pyrene-7,8-dione by Sulfotransferases (SULTs) in Human Lung Cells

Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Human aldo-keto reductases catalyze the metabolic activation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active o-quinones. Benzo[a]pyrene-7,8-dione a representative PAH o-quinone is reduced back to the corresponding catechol to generate a futile redox-cycle. We investigated whether sulfonation of PAH catechols by human sulfotransferases (SULT) could intercept the catechol in human lung cells. RT-PCR identified SULT1A1, -1A3, and -1E1 as the isozymes expressed in four human lung cell lines. The corresponding recombinant SULTs were examined for their substrate specificity. Benzo[a]pyrene-7,8-dione was reduced to benzo[a]pyrene-7,8-catechol by dithiothreitol under anaerobic conditions and then further sulfonated by the SULTs in the presence of 3'-[(35)S]phosphoadenosine 5'-phosphosulfate as the sulfonate group donor. The human SULTs catalyzed the sulfonation of benzo[a]pyrene-7,8-catechol and generated two isomeric benzo[a]pyrene-7,8-catechol O-monosulfate products that were identified by reversed phase HPLC and by LC-MS/MS. The various SULT isoforms produced the two isomers in different proportions. Two-dimensional (1)H and (13)C NMR assigned the two regioisomers of benzo[a]pyrene-7,8-catechol monosulfate as 8-hydroxy-benzo[a]pyrene-7-O-sulfate (M1) and 7-hydroxy-benzo[a]pyrene-8-O-sulfate (M2), respectively. The kinetic profiles of three SULTs were different. SULT1A1 gave the highest catalytic efficiency (k(cat)/K(m)) and yielded a single isomeric product corresponding to M1. By contrast, SULT1E1 showed distinct substrate inhibition and formed both M1 and M2. Based on expression levels, catalytic efficiency, and the fact that the lung cells only produce M1, it is concluded that the major isoform that can intercept benzo[a]pyrene-7,8-catechol is SULT1A1.

An NQO1 Substrate with Potent Antitumor Activity That Selectively Kills by PARP1-Induced Programmed Necrosis

Agents, such as β-lapachone, that target the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to induce programmed necrosis in solid tumors have shown great promise, but more potent tumor-selective compounds are needed. Here, we report that deoxynyboquinone kills a wide spectrum of cancer cells in an NQO1-dependent manner with greater potency than β-lapachone. Deoxynyboquinone lethality relies on NQO1-dependent futile redox cycling that consumes oxygen and generates extensive reactive oxygen species (ROS). Elevated ROS levels cause extensive DNA lesions, PARP1 hyperactivation, and severe NAD+ /ATP depletion that stimulate Ca2+ -dependent programmed necrosis, unique to this new class of NQO1 "bioactivated" drugs. Short-term exposure of NQO1+ cells to deoxynyboquinone was sufficient to trigger cell death, although genetically matched NQO1- cells were unaffected. Moreover, siRNA-mediated NQO1 or PARP1 knockdown spared NQO1+ cells from short-term lethality. Pretreatment of cells with BAPTA-AM (a cytosolic Ca2+ chelator) or catalase (enzymatic H2O2 scavenger) was sufficient to rescue deoxynyboquinone-induced lethality, as noted with β-lapachone. Investigations in vivo showed equivalent antitumor efficacy of deoxynyboquinone to β-lapachone, but at a 6-fold greater potency. PARP1 hyperactivation and dramatic ATP loss were noted in the tumor, but not in the associated normal lung tissue. Our findings offer preclinical proof-of-concept for deoxynyboquinone as a potent chemotherapeutic agent for treatment of a wide spectrum of therapeutically challenging solid tumors, such as pancreatic and lung cancers.

Homologous recombination repair-dependent cytotoxicity of the benzotriazine di- N-oxide CEN-209: Comparison with other hypoxia-activated prodrugs

CEN-209 (SN30000) is a second-generation benzotriazine di- N-oxide currently in advanced preclinical development as a hypoxia-activated prodrug (HAP). Herein we describe the DNA repair-, hypoxia- and one-electron reductase-dependence of CEN-209 cytotoxicity. We deployed mutant CHO cell lines to generate DNA repair profiles for CEN-209, and compared the profiles with those for other HAPs. Hypoxic selectivity of CEN-209 was significantly greater than PR-104A and the nitro-chloromethylbenzindoline (nCBI/SN29428) and comparable to tirapazamine and TH-302. CEN-209 was selective for homologous recombination (HR) repair-deficient cells ( Rad51d −/−), but less so than nitrogen mustard prodrugs TH-302 and PR-104A. Further, DNA repair profiles for CEN-209 differed under oxic and hypoxic conditions, with oxic cytotoxicity more dependent on HR. This feature was conserved across all three members of the benzotriazine di- N-oxide class examined (tirapazamine, CEN-209 and CEN-309/SN29751). Enhancing one-electron reduction of CEN-209 by forced expression of a soluble form of NADPH:cytochrome P450 oxidoreductase (sPOR) increased CEN-209 cytotoxicity more markedly under oxic than hypoxic conditions. Comparison of oxygen consumption, H 2O 2 production and metabolism of CEN-209 to the corresponding 1-oxide and nor-oxide reduced metabolites suggested that enhanced oxic cytotoxicity in cells with high one-electron reductase activity is due to futile redox cycling. This study supports the hypothesis that both oxic and hypoxic cell killing by CEN-209 is mechanistically analogous to tirapazamine and is dependent on oxidative DNA damage repaired via multiple pathways. However, HAPs that generate DNA interstrand cross-links, such as TH-302 and PR-104, may be more suitable than benzotriazine di- N-oxides for exploiting reported HR repair defects in hypoxic tumour cells.

Aldo-Keto Reductases Protect Lung Adenocarcinoma Cells from the Acute Toxicity of B[a]P-7,8-trans-Dihydrodiol

Tobacco smoke exposure stimulates the expression of genes that are likely to be involved in the metabolism of its combustion products such as polycyclic aromatic hydrocarbons (PAH). Four of the smoke induced genes are aldo-keto reductases (AKR), enzymes that metabolically activate PAH to PAH o-quinones. Alternatively, PAHs are metabolized to (±)-anti-diol epoxides, such as (±)-anti-benzo[a]pyrene diol epoxide ((±)-anti-BPDE)), by the combined action of P4501A1/1B1 and epoxide hydrolase. (±)-anti-BPDE forms DNA adducts directly, while PAH o-quinones cause DNA damage by oxidative stress through a futile redox cycle. To address the role of AKRs in PAH cytotoxicity, we compared the cytotoxicity of PAH metabolites and the effects of overexpressing AKR1A1 in lung cells. (±)-anti-BPDE and B[a]P-7,8-trans-dihydrodiol, an intermediate in (±)-anti-BPDE metabolism, are toxic to A549 cells at concentrations with an IC(50) of ∼2 μM. In contrast, the PAH o-quinone B[a]P-7,8-dione was about 10-fold less toxic to A549 cells with an IC(50) > 20 μM. Similar differences in cytoxicity were observed with two other PAH o-quinones (benz[a]anthracene-3,4-dione and 7,12-dimethylbenz[a]anthracene-3,4-dione) compared with their respective diol-epoxide counterparts (BA-3,4-diol-1,2-epoxide and DMBA-3,4-diol-1,2-epoxide). In addition, both anti-BPDE and B[a]P-7,8-trans-dihydrodiol induced p53 expression ∼6 h post-treatment at concentrations as low as 1 μM consistent with extensive DNA damage. B[a]P-7,8-dione treatment did not induce p53 but generated reactive oxygen species (ROS) in A549 cells and induced the expression of oxidative response genes in H358 cells. We also observed that overexpression of AKR1A1 in H358 cells, which otherwise have low levels of AKR expression, protected cells 2-10-fold from the toxic effects of B[a]P-7,8-trans-dihydrodiol. These data suggest that overexpression of AKRs may protect lung cancer cells from the acute toxic effects of PAH.

Abstract 3468: Aldo-keto reductases protect lung epithelial cells from polycyclic aromatic hydrocarbons at the expense of increased oxidative stress

Abstract Tobacco exposure stimulates genes that are likely to be involved in the metabolism of tobacco and its combustion products. Four induced genes are aldo-keto reductases (AKR), a family of enzymes that reduce aldehydes and ketones to alcohols. AKRs act on a range of substrates including polycyclic aromatic hydrocarbons (PAH). AKRs convert PAH to form o-quinones. Alternatively, PAHs are metabolized to (+)-anti-benzo[α]pyrene diol epoxide ((+)-anti-BPDE)) by the combined action of P4501A1/1B1 and epoxide hydrolase. (+)-anti-BPDE forms DNA adducts directly, while PAH o-quinones cause oxidative stress through a futile redox cycle. To address the role of AKRs in PAH toxicity, we compared the toxicities of PAH metabolites and the effects of overexpressing AKR 1A1 in lung cells. We find that anti-BPDE and BP,-7,8-diol, an intermediate product, are toxic to A549 cells at concentrations with an IC50 of ∼2 µM. In addition, both anti-BPDE and BP 7,8-diol induce p53 expression in 6 hrs post-treatment at concentrations as low as 1 µM consistent with extensive DNA damage. In contrast, we found that the PAH o-quinone B[a]P-7,8-dione was about 10-fold less toxic to A549 cells with an IC 50 > 20 µM. B[a]P-7,8-dione treatment also did not induce p53 expression in 24 hrs at 20 µM. However, B[a]P-7,8-dione treatment generated reactive oxygen species (ROS) in A549 cells as previously reported. We also observed that overexpression of AKR 1A1 in H358 cells, which otherwise have low levels of AKR expression, protected cells 2-10 fold from the toxic effects of BP, 7,8-diol. These data suggest that overexpression of AKRs may protect lung cancer cells from the acute toxic effects of PAH at the expense of increasing the burden oxidative stress Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3468.

Semiquinone Radicals from Oxygenated Polychlorinated Biphenyls: Electron Paramagnetic Resonance Studies

Polychlorinated biphenyls (PCBs) can be oxygenated to form very reactive hydroquinone and quinone products. A guiding hypothesis in the PCB research community is that some of the detrimental health effects of some PCBs are a consequence of these oxygenated forms undergoing one-electron oxidation or reduction, generating semiquinone radicals (SQ•−). These radicals can enter into a futile redox cycle resulting in the formation of reactive oxygen species, that is, superoxide and hydrogen peroxide. Here, we examine some of the properties and chemistry of these semiquinone free radicals. Using electron paramagnetic resonance (EPR) to detect SQ•− formation, we observed that (i) xanthine oxidase can reduce quinone PCBs to the corresponding SQ•−; (ii) the heme-containing peroxidases (horseradish and lactoperoxidase) can oxidize hydroquinone PCBs to the corresponding SQ•−; (iii) tyrosinase acting on PCB ortho-hydroquinones leads to the formation of SQ•−; (iv) mixtures of PCB quinone and hydroquinone form SQ•− via a comproportionation reaction; (v) SQ•− are formed when hydroquinone-PCBs undergo autoxidation in high pH buffer (≈>pH 8); and, surprisingly, (vi) quinone-PCBs in high pH buffer can also form SQ•−; (vii) these observations along with EPR suggest that hydroxide anion can add to the quinone ring; (viii) H2O2 in basic solution reacts rapidly with PCB-quinones; and (ix) at near-neutral pH SOD can catalyze the oxidization of PCB-hydroquinone to quinone, yielding H2O2. However, using 5,5-dimethylpyrroline-1-oxide (DMPO) as a spin-trapping agent, we did not trap superoxide, indicating that generation of superoxide from SQ•− is not kinetically favorable. These observations demonstrate multiple routes for the formation of SQ•− from PCB-quinones and hydroquinones. Our data also point to futile redox cycling as being one mechanism by which oxygenated PCBs can lead to the formation of reactive oxygen species, but this is most efficient in the presence of SOD.

Motexafin Gadolinium-Induced Cell Death Correlates with Heme oxygenase-1 Expression and Inhibition of P450 Reductase-Dependent Activities

Heme oxygenase-1 (HO1), which oxidizes heme to biliverdin, CO, and free iron, conveys protection against oxidative stress and is antiapoptotic. Under stress conditions, some porphyrin derivatives can inhibit HO1 and trigger cell death. Motexafin gadolinium (MGd) is an expanded porphyrin that selectively targets cancer cells through a process of futile redox cycling that decreases intracellular reducing metabolites and protein thiols. Here, we report that hematopoietic-derived cell lines that constitutively express HO1 are more susceptible to MGd-induced apoptosis than those that do not. MGd used in combination with tin protoporphyrin IX, an inhibitor of HO1, resulted in synergistic cell killing. Consistent with these cell culture observations, we found that MGd is an inhibitor of heme oxygenase-1 activity in vitro. We demonstrate that inhibition of HO1 reflects an interaction of MGd with NADPH-cytochrome P450 reductase, the electron donor for HO1, that results in diversion of reducing equivalents from heme oxidation to oxygen reduction. In accord with this mechanism, MGd is also an in vitro inhibitor of CYP2C9, CYP3A4, and CYP4A1. Inhibition of HO1 by MGd may contribute to its anticancer activity, whereas its in vitro inhibition of a broad spectrum of P450 enzymes indicates that a potential exists for drug-drug interactions.

Motexafin gadolinium induces oxidative stress and apoptosis in hematologic malignancies

Redox mechanisms have been shown to be important in malignant cell survival and are a system that may be modified for the treatment of hematologic malignancies. Motexafin gadolinium (MGd) is a synthetic expanded porphyrin that selectively accumulates in tumor cells and oxidizes various intracellular metabolites, including ascorbate, nicotinamide adenine dinucleotide phosphate, glutathione, and protein thiols, to generate reactive oxygen species in a process known as futile redox cycling. The rationale for its use in hematologic malignancies is that, like naturally occurring porphyrins, it tends to concentrate selectively in cancer cells, and it has a novel mechanism of action of inducing redox stress and triggering apoptosis in a broad range of malignancies. MGd induces apoptosis in B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and highly resistant myeloma cell lines. Furthermore, MGd is additive or synergistic with ionizing radiation, several chemotherapy agents, and rituximab in vitro and in vivo tumor models. Through gene expression profiling, various stress-related genes are upregulated in response to MGd, including genes encoding metallothioneins, heat shock proteins, and heme oxygenase. Preliminary results from clinical trials with MGd in hematopoietic malignancies have shown that it is well tolerated, with minimal hematologic side effects in both; it has single agent activity in very heavily pretreated chronic lymphocytic leukemia /small lymphocytic lymphoma patients, and it has induced prompt complete remissions in combination with 90Yttrium-ibritumomab (Y-90 Zevalin; Biogen Idec Inc., Cambridge, MA) for relapsed non-Hodgkin's lymphoma in the first two cohorts of patients enrolled. Various clinical trials studying MGd as a single agent and in combination with radiation and/or chemotherapy for the treatment of hematologic malignancies are ongoing.

Development of β-Lapachone Prodrugs for Therapy Against Human Cancer Cells with Elevated NAD(P)H:Quinone Oxidoreductase 1 Levels

beta-Lapachone, an o-naphthoquinone, induces a novel caspase- and p53-independent apoptotic pathway dependent on NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 reduces beta-lapachone to an unstable hydroquinone that rapidly undergoes a two-step oxidation back to the parent compound, perpetuating a futile redox cycle. A deficiency or inhibition of NQO1 rendered cells resistant to beta-lapachone. Thus, beta-lapachone has great potential for the treatment of specific cancers with elevated NQO1 levels (e.g., breast, non-small cell lung, pancreatic, colon, and prostate cancers). We report the development of mono(arylimino) derivatives of beta-lapachone as potential prodrugs. These derivatives are relatively nontoxic and not substrates for NQO1 when initially diluted in water. In solution, however, they undergo hydrolytic conversion to beta-lapachone at rates dependent on the electron-withdrawing strength of their substituent groups and pH of the diluent. NQO1 enzyme assays, UV-visible spectrophotometry, high-performance liquid chromatography-electrospray ionization-mass spectrometry, and nuclear magnetic resonance analyses confirmed and monitored conversion of each derivative to beta-lapachone. Once converted, beta-lapachone derivatives caused NQO1-dependent, mu-calpain-mediated cell death in human cancer cells identical to that caused by beta-lapachone. Interestingly, coadministration of N-acetyl-l-cysteine, prevented derivative-induced cytotoxicity but did not affect beta-lapachone lethality. Nuclear magnetic resonance analyses indicated that prevention of beta-lapachone derivative cytotoxicity was the result of direct modification of these derivatives by N-acetyl-l-cysteine, preventing their conversion to beta-lapachone. The use of beta-lapachone mono(arylimino) prodrug derivatives, or more specifically a derivative converted in a tumor-specific manner (i.e., in the acidic local environment of the tumor tissue), should reduce normal tissue toxicity while eliciting tumor-selective cell killing by NQO1 bioactivation.

Production of superoxide radical in reductive metabolism of a synthetic food-coloring agent, indigocarmine, and related compounds

Indigocarmine, which is widely used as a synthetic colouring agent for foods and cosmetics in many countries, was reduced to its leuco form and decolorized by rat liver microsomes with NADPH under anaerobic conditions. The reductase activity was enhanced in liver microsomes of phenobarbital-treated rats, and inhibited by diphenyliodonium chloride, a NADPH-cytochrome P450 reductase (P450 reductase) inhibitor, but was not inhibited by SKF 525-A or carbon monoxide. Indigocarmine reductase activity was exhibited by purified rat P450 reductase. In contrast, when indigocarmine was incubated with rat liver microsomes and NADPH under aerobic conditions, superoxide radical was produced and its production was inhibited by superoxide dismutase and diphenyliodonium chloride. When indigocarmine was incubated with purified rat P450 reductase in the presence of NADPH, superoxide radical production was enhanced 17.7-fold (similar to the enhancement of indigocarmine-reducing ability) as compared with that of rat liver microsomes. A decrease of one molecule of NADPH was accompanied with formation of about two molecules of superoxide radical. P450 reductase exhibited little reductase activity towards indigo and tetrabromoindigo, which also afforded little superoxide radical under aerobic conditions. These results indicate that indigocarmine is reduced by P450 reductase to its leuco form, and superoxide radical is produced by autoxidation of the leuco form, through a mechanism known as futile redox cycling.

Properties and utility of the peculiar mixed disulfide in the bacterial glutathione transferase B1-1.

Bacterial glutathione transferases appear to represent an evolutionary link between the thiol:disulfide oxidoreductase and glutathione transferase superfamilies. In particular, the observation of a mixed disulfide in the active site of Proteus mirabilis glutathione transferase B1-1 is a feature that links the two families. This peculiar mixed disulfide between Cys10 and one GSH molecule has been studied by means of ESR spectroscopy, stopped-flow kinetic analysis, radiochemistry, and site-directed mutagenesis. This disulfide can be reduced by dithiothreitol but even a thousand molar excess of GSH is poorly effective due to an unfavorable equilibrium constant of the redox reaction (K(eq) = 2 x 10(-4)). Although Cys10 is partially buried in the crystal structure, in solution it reacts with several thiol reagents at a higher or comparable rate than that shown by the free cysteine. Kinetics of the reaction of Cys10 with 4,4'-dithiodipyridine at variable pH values is consistent with a pK(a) of 8.0 +/- 0.1 for this residue, a value about 1 unit lower than that of the free cysteine. The 4,4'-dithiodipyridine-modified enzyme reacts with GSH in a two-step mechanism involving a fast precomplex formation, followed by a slower chemical step. The natural Cys10-GSH mixed disulfide exchanges rapidly with free [3H]GSH in a futile redox cycle in which the bound GSH is continuously replaced by the external GSH. Our data suggest that the active site of the bacterial enzyme has intermediate properties between those of the recently evolved glutathione transferases and those of the thiol:disulfide oxidoreductase superfamily.

Redox cycling by motexafin gadolinium enhances cellular response to ionizing radiation by forming reactive oxygen species

Purpose: To examine the mechanism of radiation enhancement by motexafin gadolinium (Gd-Tex) in vitro.Methods and Materials: Oxidation of ascorbate and NADPH by Gd-Tex was evaluated in a neutral buffer. Growth inhibition of human uterine cancer cell line MES-SA was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. Clonogenic assays were used to measure radiation response in MES-SA, A549 human lung carcinoma, E89, a CHO cell line variant deficient in glucose-6-phosphate dehydrogenase activity, and murine lymphoma cell lines LYAR and LYAS.Results: Gd-Tex catalyzed the oxidation of NADPH and ascorbate under aerobic conditions, forming hydrogen peroxide. Decreased viability was observed in MES-SA cells incubated with Gd-Tex in media containing NADPH or ascorbate. Gd-Tex and ascorbate increased fluorescence in dichlorofluorescin acetate-treated cultures. Synergistic effects on the aerobic radiation response in MES-SA and A549 were seen using Gd-Tex in combination with l-buthionine-(S,R)-sulfoximine (BSO). Incubation with Gd-Tex in the presence of ascorbate increased the aerobic radiation response of E89 and the apoptosis-sensitive B-cell line (LYAS).Conclusions: Gd-Tex sensitizes cells to ionizing radiation by increasing oxidative stress as a consequence of futile redox cycling. Optimization of the concentration of ascorbate (or other reducing species) may be required when evaluating Gd-Tex activity in vitro.

Oxidative Stress and 1-Methyl-2-nitroimidazole Cytotoxicity

The 2-nitroimidazoles have been used clinically to radiosensitize resistant hypoxic cells, but a dose-limiting peripheral neuropathy has restricted their therapeutic effectiveness. A model compound, 1-methyl-2-nitroimidazole (INO 2), was used to investigate the possible role of oxidative stress in this normal tissue toxicity. Chinese hamster ovary (CHO) cells were 10–15 times more resistant to 20 mM INO 2 under aerobic than hypoxic conditions. In comparison, a pair of transformed rat embryo fibroblasts (ER17-1 wtp53 and ER12L5 mtp53), differing in their p53 genotype, were approximately 3- to 4-fold more sensitive than Chinese hamster ovary cells to INO 2 under aerobic conditions, but had the same sensitivity as Chinese hamster ovary cells under hypoxic conditions. These results are consistent with an earlier hypothesis that the mechanism of aerobic toxicity is different from that of hypoxic toxicity (nitroreduction) and show that neither toxicity is dependent on cellular p53 status. There was an increase in the production of reactive oxygen intermediates and a decrease in the antioxidant glutathione following aerobic exposure to INO 2, which correlated with cell survival in all three cell lines. No evidence of reductive adducts of the 2-nitroimidazole 2-(2-nitro-1 H-imidazol-1-yl)- N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5) was found by immunofluorescent techniques in aerobic cells. Differing activities of the antioxidant enzymes superoxide dismutase and catalase could be correlated with INO 2 aerobic cytotoxicity. DNA strand breaks, as measured by the comet assay, paralleled the appearance of INO 2 aerobic cytotoxicity in all three cell lines. Taken together, these results strongly support the conclusion that the aerobic toxicity of INO 2 is due to active oxygen species created by futile redox cycling of the parent compound.

Efficient redox cycling of nitroquinoline bioreductive drugs due to aerobic nitroreduction in Chinese hamster cells

Nitroquinoline bioreductive drugs with 4-alkylamino substituents undergo one-electron reduction in mammalian cells, resulting in futile redox cycling due to oxidation of the nitro radical anion in aerobic cultures, and eventual reduction to the corresponding amines in the absence of oxygen. Rates of drug-induced oxygen consumption (R) due to redox cycling in cyanide-treated AA8 cell cultures were determined for 17 nitroquinolines. There was a linear dependence of log R on the one-electron reduction potential at pH 7 (E 7 1) with a slope of 7.1 V −1, excluding compounds with substituents ortho to the nitro group. The latter had anomalously low rates of oxygen consumption relative to E 7 1, suggesting that interaction with the active site of nitroreductases is impeded sterically for such compounds. Absolute values of R (and the observed E 7 1 dependence) were well predicted by a simple kinetic model that used rates of net nitroreduction to the amines under anoxia as a measure of the rates of one-electron reduction in aerobic cells. This indicates that redox cycling of 4-alkylaminonitroquinolines occurs at high efficiency in aerobic cells, suggesting that there are no quantitatively significant fates of nitro radical anions in cells other than their reaction with oxygen (or their spontaneous disproportionation under hypoxia).

Genotoxic effects of 3-amino-1,2,4-benzotriazine-1, 4-dioxide (SR 4233) and nitrogen mustard- N-oxide (nitromin) in Walker carcinoma cells under aerobic and hypoxic conditions

As judged by alkaline elution, exposure of Walker cells to either 3-amino-1,2,4-benzotriazine-1, 4-dioxide (SR 4233) or nitromin results in a dose-dependent increase in DNA damage due to single-strand breaks. With nitromin or SR 4233 there was little difference in the extent of DNA single-strand breaks between Walker cells incubated either hypoxically or aerobically. In contrast, there was a 24-fold enhancement in the differential hypoxic/aerobic response to SR 4233 in clonogenic studies. Following incubation of cells with nitrogen mustard, DNA cross-linking is observed. Bioreduction of nitromin would be expected to yield nitrogen mustard as the putative reactive metabolite. However, only DNA strand-breaks could be detected in Walker cells incubated with nitromin, suggesting that reduction of this pro-drug to nitrogen mustard was not a major activation pathway. In cells incubated under aerobic conditions, SR 4233 induces oxidative DNA damage, as indicated by the formation of 8-hydroxydeoxyguanosine, suggesting the involvement of futile redox cycling. In rats dosed with SR 4233 in vivo, significantly higher levels of 8-hydroxydeoxyguanosine could be detected in liver, compared to vehicle-dosed controls.

Protection against sr 4233 (tirapazamine) aerobic cytotoxicity by the metal chelators desferrioxamine and tiron

Purpose : Metal chelating agents and antioxidants were evaluated as potential protectors against aerobic SR 4233 cytotoxicity in Chinese hamster V79 cells. The differential protection of aerobic and hypoxic cells by two metal chelators, desferrrioxamine and Tiron, is discussed in the context of their potential use in the on-going clinical trials with SR 4233. Methods and Materials : Cytotoxicity was evaluated using clonogenic assay. SR 4233 exposure was done in glass flasks as a function of time either alone or in the presence of the following agents: superoxide dismutase, catalase, 5,5-dimethyl-l-pyrioline, Trolox, ICRF-187, desferrioxamine, Tiron (1,2-dihydroxybenzene-3,5-disulfonate), and ascorbic acid. Experiments done under hypoxic conditions were carried out in specially designed glass flasks that were gassed with humidified nitrogen/carbon dioxide mixture and with a side-arm reservoir from which SR 4233 was added to cell media after hypoxia was obtained. Electron paramagnetic resonance studies were also performed. Results : Electron paramagnetic resonance and spectrophotometry experiments suggest that under aerobic conditions SR 4233 undergoes futile redox cycling to produce superoxide. Treatment of cells during aerobic exposure to SR 4233 with the enzymes superoxide dismutase and catalase, the spin trapping agent DMPO, the water-soluble vitamin E analog Trolox, and the metal chelator ICRF-187 provided little or no protection against aerobic SR 4233 cytotoxicity. However, two other metal chelators, desferrioxamine and Tiron, afforded significant protection against aerobic SR 4233 cytotoxicity (protection factors at 50% survival were 3.8 and 3.1, respectively), while exhibiting minimal protection to hypoxic cells treated with SR 4233. Conclusions : One potential mechanism of aerobic cytotoxicity is redox cycling of SR 4233 with molecular oxygen resulting in several. potentially toxic oxidative species that overburden the intrinsic intracellular detoxification systems such as superoxide dismutase, catalase, and glutathione peroxidase. This study identifies two metal chelating agents, desferrioxarnine and Tiron, that were able to protect against aerobic but not hypoxic SR 4233 cytotoxicity.

Bioreductive metabolism of SR-4233 (win 59075) by whole cell suspensions under aerobic and hypoxic conditions: Role of the pentose cycle and implications for the mechanism of cytotoxicity observed in air

Purpose : Measurement of pentose cycle (PC) activity is shown to be a noninvasive means for monitoring the reduction of SR-4233 in whole cells. Comparing these measurements to the actual measurements of drug loss under aerobic and hypoxic conditions helps to define the mechanism for the associated aerobic toxicity. Methods and Materials : SR-4233 is activated to a toxic species by bioreductive metabolism. NADPH is required for the activation of the drug by purified enzymes, cell homogenates and whole cells. In vivo the NADPH:NADP+ ratio is maintained by the oxidation of glucose via the oxidative limb of the pentose cycle. By measuring radiolabeled 14CO 2 released as a product of this oxidation one can get an accurate measurement of the rate of drug metabolism in whole cells. These results are compared to measurements of drug consumption under aerobic and hypoxic conditions using an HPLC assay. Results : SR-4233 stimulates pentose cycle activity to a greater extent in air then under hypoxia, however, in the presence of added catalase, pentose cycle activity is stimulated to a similar extent under both conditions. The higher levels of PC activity observed in air are due to the production of hydrogen peroxide by the nitroxide free radical undergoing futile redox cycling. The contribution of H 2O 2 to the observed aerobic cytotoxicity of SR-4233 is minimal however, since toxicity is only slightly reduced in the presence of exogenous catalase and antioxidants such as vitamin E. The level of PC stimulation by SR-4233 suggests that the rate of electron addition to the drug is independent of O 2 concentration. The loss of drug from the incubation medium, i.e., conversion to a stable intermediate species, occurs approximately five times faster under nitrogen than in air for A549 cells. It is the rate of drug loss from the cell and not the rate of reduction which best correlates with the observed aerobic and hypoxic toxicity. Conclusion : Toxicity in air and in nitrogen is directly related to the rate of drug reduction, i.e., at equivalent levels of drug loss we observe equal levels of cytotoxicity.