Lipid peroxidation by bovine heart submitochondrial particles stimulated by 1,1′-dimethyl-4,4′-bipyridylium dichloride (paraquat)

Paraquat enhanced the NADH-dependent lipid peroxidation of bovine heart submitochondrial particles in the presence of ADE-Fe³⁺ chelate. The enhancement at physiological pH was about 3-fold. The pH optimum of the lipid peroxidation was shifted from pH 6.5 by paraquat. The submitochondrial particles catalyzed the reduction of paraquat when incubated anaerobically with NADH, whereas they did not reduce paraquat with succinate. The reduction was inhibited by phydroxymercuribenzoate or amytal, but it was not inhibited by rotenone, antimycin A or cyanide. The respiratory-chain inhibitors similarly affected the NADH-dependent O₂ consumption stimulated by paraquat, indicating that the NADH-dehydrogenase is involved in the reduction of paraquat at a region between the mercurial-sensitive site and the rotenone-sensitive site. The NADH-dependent reduction of ADP-Fe³⁺ chelate, a key step in lipid peroxidation, was stimulated by paraquat about 5-fold at physiological pH. The stimulation could mainly be ascribed to the direct electron transfer from a paraquat radical to the chelate and partially to the electron transfer from O₂^? produced by the reoxidation of the paraquat radical. ADP-Fe²⁺ produced lipid hydroperoxide in liposomes and decomposed cumene hydroperoxide. These reactions, the initiation reaction and the propagation reaction of peroxidation, were stimulated by paraquat. These results suggest that paraquat enhanced lipid peroxidation by stimulating (1) the reduction of ADP-Fe³⁺ chelate, and (2) the ADP-Fe²⁺-dependent initiation and propagation reactions of the peroxidation.     

Mechanism of nifurtimox toxicity in different forms of Trypanosoma cruzi

Addition of nifurtimox (a nitrofuran derivative) to NAD(P)H-containing homogenates of Trypanosoma cruzi (epi-, trypo- or amastigote forms) determined the appearance of an e.p.r. spectrum that could be identified as corresponding to the nitroaromatic anion radical. The anion radical signal was observed after an induction period that depended on the oxygen concentration and the pyridine nucleotide in the incubation medium. Incubation of intact T. cruzi forms with nifurtimox also led to the appearance of the anion radical signal. The nitroaromatic anion radical, which is assumed to be the first product of nitroreductase activity, reacted with oxygen under aerobic conditions, as shown by the increased rate of superoxide anion and hydrogen peroxide production after addition of nifurtimox to homogenates of T. cruzi in the presence of NAD(P)H. The nifurtimox-induced peroxide production was higher with T. cruzi amastigotes than with epi- or trypomastigotes.     

Comparison of the oxime-induced reactivation of erythrocyte and muscle acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity

The purpose of these experiments was to compare oxime-induced reactivation rate constants of acetylcholinesterase from different human tissue sources inhibited by organophosphorus compounds. To this end, preliminary testing was necessary to generate a stable system both for working with erythrocytes and musculature. We established a dynamically working in vitro model with a fixed enzyme source in a bioreactor that was perfused with acetylthiocholine, Ellman's reagent and any agent of interest (e.g. nerve agents, oximes) and analyzed in a common HPLC flow-through detector. The enzyme reactor was composed of a particle filter (Millex-GS, 0.22 microm) containing a thin layer of membrane-bound acetylcholinesterase and was kept at constant temperature in a water bath. At constant flow the height of absorbance was directly proportional to the enzyme activity. To start with, we applied this system to human red cell membranes and then adapted the system to acetylcholinesterase of muscle tissue. Homogenate (Ultra-Turrax and Potter-Elvehjem homogenizer) of human muscle tissue (intercostal musculature) was applied to the same particle filter and perfused in a slightly modified way, as done with human red cell membranes. We detected no decrease of acetylcholinesterase activity within 2.5h and we reproducibly determined reactivation rate constants for reactivation with obidoxime (10 microM) or HI 6 (30 microM) of sarin-inhibited human muscle acetylcholinesterase (0.142+/-0.004 min(-1) and 0.166+/-0.008 min(-1), respectively). The reactivation rate constants of erythrocyte and muscular acetylcholinesterase differed only slightly, highlighting erythrocyte acetylcholinesterase as a proper surrogate marker.     

Distinct mechanisms of cell-kill by triapine and its terminally dimethylated derivative Dp44mT due to a loss or gain of activity of their copper(II) complexes

Triapine, currently being evaluated as an antitumor agent in phase II clinical trials, and its terminally dimethylated derivative Dp44mT share the α-pyridyl thiosemicarbazone backbone that functions as ligands for transition metal ions. Yet, Dp44mT is approximately 100-fold more potent than triapine in cytotoxicity assays. The aims of this study were to elucidate the mechanisms underlying their potency disparity and to determine their kinetics of cell-kill in culture to aid in the formulation of their clinical dosing schedules. The addition of Cu²⁺ inactivated triapine in a 1:1 stoichiometric fashion, while it potentiated the cytotoxicity of Dp44mT. Clonogenic assays after finite-time drug-exposure revealed that triapine produced cell-kill in two phases, one completed within 20 min that caused limited cell-kill, and the other occurring after 16 h of exposure that produced extensive cell-kill. The ribonucleotide reductase inhibitor triapine at 0.4 μM caused immediate complete arrest of DNA synthesis, whereas Dp44mT at this concentration did not appreciably inhibit DNA synthesis. The inhibition of DNA synthesis by triapine was reversible upon its removal from the medium. Cell death after 16 h exposure to triapine paralleled the appearance of phospho-(γ)H2AX, a marker of DNA double-strand breaks induced by collapse of DNA replication forks after prolonged replication arrest. In contrast to triapine, Dp44mT produced robust cell-kill within 1 h in a concentration-dependent manner. The short-term action of both agents was prevented by thiols, indicative of the involvement of reactive oxygen species. The time dependency in the production of cell-kill by triapine should be considered in treatment regimens.     

Cr(VI) induces mitochondrial-mediated and caspase-dependent apoptosis through reactive oxygen species-mediated p53 activation in JB6 Cl41 cells

Cr(VI) compounds are known to cause serious toxic and carcinogenic effects. Cr(VI) exposure can lead to a severe damage to the skin, but the mechanisms involved in the Cr(VI)-mediated toxicity in the skin are unclear. The present study examined whether Cr(VI) induces cell death by apoptosis or necrosis using mouse skin epidermal cell line, JB6 Cl41 cells. We also investigated the cellular mechanisms of Cr(VI)-induced cell death. This study showed that Cr(VI) induced apoptotic cell death in a dose-dependent manner, as demonstrated by the appearance of cell shrinkage, the migration of cells into the sub-G1 phase, the increase of Annexin V positively stained cells, and the formation of nuclear DNA ladders. Cr(VI) treatment resulted in the increases of mitochondrial membrane depolarization and caspases activation. Electron spin resonance (ESR) and fluorescence analysis revealed that Cr(VI) increased intracellular levels of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion radical in dose-dependent manner. Blockage of p53 by si-RNA transfection suppressed mitochondrial changes of Bcl-2 family composition, mitochondrial membrane depolarization, caspase activation and PARP cleavage, leading to the inhibition of Cr(VI)-induced apoptosis. Further, catalase treatment prevented p53 phosphorylation stimulated by Cr(VI) with the concomitant inhibition of caspase activation. These results suggest that Cr(VI) induced a mitochondrial-mediated and caspase-dependent apoptosis in skin epidermal cells through activation of p53, which are mainly mediated by reactive oxidants generated by the chemical.     

Emodin induces apoptosis in human promyeloleukemic HL-60 cells accompanied by activation of caspase 3 cascade but independent of reactive oxygen species production

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active constituent of Rheum palmatum, and showed inhibitory activity on lipopolysaccharide-induced NO production in our previous study. However, the apoptosis-inducing activity of emodin has remained undefined. Among three structurally related anthraquinones, including emodin, physcion, and chrysophanol, emodin showed the most potent cytotoxic effects on HL-60 cells, accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including an increase in DNA ladder intensity, morphological changes, appearance of apoptotic bodies, and an increase in hypodiploid cells. Emodin at apoptosis-inducing concentrations causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity, according to cleavage of caspase 3 substrates poly(ADP-ribose) polymerase and D4-GDI proteins, the appearance of cleaved caspase 3 fragments being detected in emodin- but not physcion- or chrysophanol-treated HL-60 cells. A decrease in the anti-apoptotic protein, Mcl-1, was detected in emodin-treated HL-60 cells, whereas other Bcl-2 family proteins including Bax, Bcl-2, Bcl-XL, and Bad remained unchanged. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated emodin-induced DNA ladders, associated with the blockage of PARP and D4-GDI cleavage. Free radical scavenging agents including NAC, catalase, SOD, ALL, DPI, L-NAME and PDTC showed no preventive effect on emodin-induced apoptotic responses, whereas NAC, CAT and PDTC prevented HL-60 cells from ROS (H(2)O(2))-induced apoptosis through inhibition of caspase 3 cascades. Induction of catalase, but not SOD, activity was detected in emodin-treated HL-60 cells by in gel activity assays, and H(2)O(2)-induced intracellular peroxide level was significantly reduced by prior treatment of emodin in HL-60 cells. Our experiments provide evidence that emodin is an effective apoptosis inducer in HL-60 cells through activation of the caspase 3 cascade, but that it is independent of ROS production.     

Comparative study of oxime-induced reactivation of erythrocyte and muscle AChE from different animal species following inhibition by sarin or paraoxon

Standard treatment of acute poisoning by organophosphorus compounds (OP) includes administration of an antimuscarinic (e.g. atropine) and of an oxime-based reactivator of OP-inhibited acetylcholinesterase (AChE). A recently introduced dynamically working in vitro model with real-time determination of membrane-bound AChE activity was shown to be a very versatile and promising model to investigate oxime-induced reactivation kinetics of OP-inhibited enzyme. In this assay, human AChE from erythrocytes or muscle tissue was immobilized on a particle filter. This bioreactor was continuously perfused with substrate and chromogen and AChE activity was analyzed on-line in a flow-through detector. The model has been successfully adopted to Rhesus monkey, swine and guinea pig erythrocytes and intercostal muscle AChE. In addition, the basic kinetic constants of inhibition, aging, spontaneous- and oxime-induced-reactivation of erythrocyte AChE from these species were determined with a standard static model. The major findings were, in part substantial species differences in the inhibition (sarin, paraoxon) and reactivation kinetics (obidoxime, HI 6) of erythrocyte AChE, but comparable kinetics of inhibition and reactivation between erythrocyte and muscle AChE. Hence, these data provide further support of the assumption that erythrocyte AChE is an adequate surrogate of muscle (synaptic) AChE and admonish that major species differences have to be considered for the design and evaluation of therapeutic animal models.     

Kinetic analysis of the protection afforded by reversible inhibitors against irreversible inhibition of acetylcholinesterase by highly toxic organophosphorus compounds

In organophosphate poisoning, the underlying mechanism of the therapeutic efficacy of carbamate prophylaxis, which was successfully tested in animal experiments, still awaits complete understanding. In particular, it is unclear whether survival is improved by increased acetylcholinesterase activity during the acute phase, when both carbamate and organophosphate are present. This question should be solved experimentally by means of a dynamically working in vitro model. Immobilized human erythrocytes were continuously perfused while acetylcholinesterase activity was monitored in real-time by a modified Ellman method. The concentrations of reversible inhibitors and of paraoxon were varied to assess the influence of both components on the enzyme activity under steady-state conditions. Physostigmine, pyridostigmine and huperzine A were tested for their prophylactic potential. Upon pretreatment with these reversible inhibitors the enzyme was inhibited by 20-90%. Additional perfusion with 1 microM paraoxon for 30 min resulted in a residual activity of 1-4%, at low and high pre-inhibition, respectively. The residual activity was significantly higher than in the absence of reversibly blocking agents (0.3%). After discontinuing paraoxon, the activity increased even in the presence of the reversible blockers. Stopping the reversibly blocking agents resulted in 10-35% recovery of the enzyme activity, depending on the degree of pre-inhibition. The experimental results agreed with computer simulations upon feeding with the essential reaction rate constants, showing that physostigmine was somewhat superior to pyridostigmine in enhancing residual activity in the presence of 1 microM paraoxon for 30 min. The model predicts that inhibitors with a faster dissociation rate, e.g. huperzine A, may be superior in case of a 'hit-and-run' poison such as soman.     

Reactivation of tabun-hAChE investigated by structurally analogous oximes and mutagenesis

The nerve agent tabun inhibits the essential enzyme acetylcholinesterase (AChE) by a rapid phosphoramidation of the catalytic serine residue. Oximes, such as K027 and HLö-7, can reactivate tabun-inhibited human AChE (tabun-hAChE) whereas the activity of their close structural analogue HI-6 is notably low. To investigate HI-6, K027 and HLö-7, residues lining the active-site gorge of hAChE were substituted and the effects on kinetic parameters for reactivation were determined. None of the mutants (Asp74Asn, Asp74Glu, Tyr124Phe, Tyr337Ala, Tyr337Phe, Phe338Val and Tyr341Ala) were able to facilitate HI-6-mediated reactivation of tabun-hAChE. In contrast, Tyr124Phe and Tyr337Phe induce a 2-2.5-fold enhancement of the bimolecular rate constant for K027 and HLö-7. The largest effects on the dissociation constant (3.5-fold increase) and rate constant (20-fold decrease) were observed for Tyr341Ala and Asp74Asn, respectively. These findings demonstrate the importance of residues located distant from the conjugate during the reactivation of tabun-hAChE.     

Inducible nitric oxide synthase catalyzes ethanol oxidation to alpha-hydroxyethyl radical and acetaldehyde

The physiologic function of nitric oxide synthases, independent of the isozyme, is well established, metabolizing L-arginine to L-citrulline and nitric oxide (NO). This enzyme can also transfer electrons to O2, affording superoxide (O2*-) and hydrogen peroxide (H2O2). We have demonstrated that NOS1, in the presence of L-arginine, can biotransform ethanol (EtOH) to alpha-hydroxyethyl radical (CH3*CHOH). We now report that a competent NOS2 with l-arginine can, like NOS1, oxidize EtOH to CH3*CHOH. Once this free radical is formed, it is metabolized to acetaldehyde as shown by LC-ESI-MS/MS and HPLC analysis. These observations suggest that NOS2 can behave similarly to cytochrome P-450 in the catalysis of acetaldehyde formation from ethanol via the generation of alpha-hydroxyethyl radical when L-arginine is present.     

Cadmium induces autophagy through ROS-dependent activation of the LKB1-AMPK signaling in skin epidermal cells

Cadmium is a toxic heavy metal which is environmentally and occupationally relevant. The mechanisms underlying cadmium-induced autophagy are not yet completely understood. The present study shows that cadmium induces autophagy, as demonstrated by the increase of LC3-II formation and the GFP-LC3 puncta cells. The induction of autophagosomes was directly visualized by electron microscopy in cadmium-exposed skin epidermal cells. Blockage of LKB1 or AMPK by siRNA transfection suppressed cadmium-induced autophagy. Cadmium-induced autophagy was inhibited in dominant-negative AMPK-transfected cells, whereas it was accelerated in cells transfected with the constitutively active form of AMPK. mTOR signaling, a negative regulator of autophagy, was downregulated in cadmium-exposed cells. In addition, cadmium generated reactive oxygen species (ROS) at relatively low levels, and caused poly(ADP-ribose) polymerase-1 (PARP) activation and ATP depletion. Inhibition of PARP by pharmacological inhibitors or its siRNA transfection suppressed ATP reduction and autophagy in cadmium-exposed cells. Furthermore, cadmium-induced autophagy signaling was attenuated by either exogenous addition of catalase and superoxide dismutase, or by overexpression of these enzymes. Consequently, these results suggest that cadmium-mediated ROS generation causes PARP activation and energy depletion, and eventually induces autophagy through the activation of LKB1-AMPK signaling and the down-regulation of mTOR in skin epidermal cells.     

Cutaneous irritation in the topical application of 30 antineoplastic agents to New Zealand white rabbits

Of 30 antineoplastic agents studied for their primary irritation potential in rabbits, 9 showed some potential for irritation. Five of these 9 agents produced a significant dermal irritation. None of the irritation observed was considered to be irreversible skin damage. The study further showed a strong correlation between irritation observed by the Draize method and acute inflammation evaluated histopathologically. There was a tendency toward increased epidermal thickness of irritated skin sites. None of the agents produced gross or microscopically visible lesions in the internal organs observed.     

Very stable superoxide radical adducts of 5-ethoxycarbonyl- 3,5-dimethyl-pyrroline N-oxide (3,5-EDPO) and its derivatives

Oxygen radicals are involved in the onset of many diseases. Adequate spin traps are required for identification and localisation of free radical formation in biological systems. Superoxide spin adducts with half-lives up to 20 min at physiological pH have recently been reported to be formed from derivatives of the spin trap 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO). This is a major improvement over DMPO (t(1/2) ca. 45 s), and even DEPMPO (t(1/2) ca. 14 min). In this study, an additional methyl group was introduced into position 3 or 4 of the pyrroline ring which greatly increases the stability of the respective superoxide spin adducts. In addition, the ethoxy group of EMPO was exchanged by either a propoxy- or an iso-propoxy group in order to test the influence of increasing lipophilic properties of the investigated spin traps. The structure of all compounds was confirmed by (1)H and (13)C-NMR with full signal assignment. In comparison with EMPO (t(1/2) ca. 8 min) or DEPMPO (t(1/2) ca. 14 min), the superoxide adducts of all novel spin traps were considerably higher (t(1/2) ca. 12-55 min). In addition, various other spin adducts obtained from oxygen-centered as well as carbon-centered radicals (e.g. derived from methanol or linoleic acid hydroperoxide) were also detected.     

Monoacylglycerol lipase inhibition by organophosphorus compounds leads to elevation of brain 2-arachidonoylglycerol and the associated hypomotility in mice

Three components of the cannabinoid system are sensitive to selected organophosphorus (OP) compounds: monoacylglycerol (MAG) lipase that hydrolyzes the major endogenous agonist 2-arachidonoylglycerol (2-AG); fatty acid amide hydrolase (FAAH) that cleaves the agonist anandamide present in smaller amounts; the CB1 receptor itself. This investigation considers which component of the cannabinoid system is the most likely contributor to OP-induced hypomotility in mice. Structure-activity studies by our laboratory and others rule against major involvement of a direct toxicant-CB1 receptor interaction for selected OPs. Attention was therefore focused on the OP sensitivities of MAG lipase and FAAH, assaying 19 structurally diverse OP chemicals (pesticides, their metabolites and designer compounds) for in vitro inhibition of both enzymes. Remarkably high potency and low selectivity is observed with three O-alkyl (C1, C2, C3) alkylphosphonofluoridates (C8, C12) (IC50 0.60-3.0 nM), five S-alkyl (C5, C7, C9) and alkyl (C10, C12) benzodioxaphosphorin oxides (IC50 0.15-5.7 nM) and one OP insecticide metabolite (chlorpyrifos oxon, IC50 34-40 nM). In ip-treated mice, the OPs at 1-30 mg/kg more potently inhibit brain FAAH than MAG lipase, but FAAH inhibition is not correlated with hypomotility. However, the alkylphosphonofluoridate-treated mice show dose-dependent increases in severity of hypomotility, inhibition of MAG lipase activity and elevation of 2-AG. Moderate to severe hypomotility is accompanied by 64 to 86% MAG lipase inhibition and about 6-fold elevation of brain 2-AG level. It therefore appears that OP-induced MAG lipase inhibition leads to elevated 2-AG and the associated hypomotility.     

Paramagnetic species in the plasma of dogs with lymphoma prior to and after treatment with doxorubicin. An ESR study

Doxorubicin is a potent cytostatic drug which is applied for the treatment of various kinds of malignant diseases. In spite of the routine use of this drug its major adverse effect, the dose-dependent cardiotoxicity, cannot be prevented yet. However, several clinical trials indicated that iron chelators are able to moderate the noxious effect more efficiently than radical scavenging antioxidants. This in turn supports the idea that doxorubicin-iron complexes are involved in triggering the cardiotoxicity of this drug by catalyzing the formation of oxygen radicals. However, both the mode of generation of doxorubicin-iron complexes and the consequences in vivo are not understood so far. In order to figure out whether or not doxorubicin can utilize iron from the transport protein transferrin for complex formation and prooxidative activities we studied the redox state of iron and its regulatory control by ceruloplasmin and ascorbate in the plasma of dogs suffering from malignant lymphoma by electron spin resonance spectroscopy. The respective electron spin resonance intensities prior to and after treatment with doxorubicin were compared with those from healthy controls. Our results revealed that dogs with lymphoma exhibit lower levels of paramagnetic copper in ceruloplasmin (-22%) and iron in transferrin (-33%) than healthy animals. Likewise the concentration of ascorbate radicals was lower in patients with lymphoma than in healthy subjects. The decreased cupric state of ceruloplasmin is equivalent to a diminished ferroxidase activity in plasma and therefore indicates indirectly an impaired antioxidant activity in these patients. Administration of doxorubicin in vivo further reduced the concentration of paramagnetic copper (-18%) and iron (-13%) while the concentration of ascorbate radicals remained unchanged. This decrease was also seen during the in vitro incubation of plasma with doxorubicin suggesting a direct interaction of the drug with the paramagnetic metal species. Model experiments revealed that the effect is based on a doxorubicin-induced release of iron from transferrin which is enhanced by ascorbate and the subsequent formation of doxorubicin-iron complexes. This mechanism was shown to trigger the formation of hydroxyl radicals from H(2)O(2) and to cause an oxidation of the antioxidant ceruloplasmin. Our data demonstrate that cardiotoxic doxorubicin-iron complexes are not only formed in cardiomyocytes itself as generally assumed, but are also present in the circulation. Therefore, these findings provide an additional rationale for potential benefit of iron chelators during doxorubicin chemotherapy.