Mechanistic aspects of paraquat toxicity in E. coli. A spin trapping study

Mechanistic aspects of paraquat monocation radical (PQ.+) and copper involvement in paraquat toxicity have been examined using E. coli B cells. Electron spin resonance (ESR) spectrometry combined with cell survival studies were used to explore the correlation between radical production and biological damage. The line broadening agent oxalato-chromiate (CrOx) was used to characterize the anoxic partition of PQ.+ inside and outside the cell. In the presence of CrOx the ESR signal was totally eliminated, indicating that intracellular species were undetectable and that, contrary to previous reports, PQ.+ exclusively accumulates outside the cell. The PQ.+ radical does not react with H2O2 but disappears in the presence of H2O2 when catalytic traces of Cu(II) are present. Spin-trapping studies using DMPO showed that in aerobic environment paraquat-induced O2 radicals are detectable exclusively in the extracellular compartment. The correlation between PQ.+ appearance and the biological damage is not simple. PQ.+ non-toxically accumulates, in the absence of oxygen and either Cu(II) or H2O2. By contrast, with both H2O2 and Cu(II) the cells are rapidly killed but PQ.+ was undetectable. These results reconfirm the key catalytic mediatory function of transition metals in paraquat toxicity.     

Nitric oxide and nitroxides can act as efficient scavengers of protein-derived free radicals

Nitric oxide ((*)NO) may act as either a pro-oxidant or an antioxidant in biological systems. Although (*)NO and nitroxide radicals react slowly with most molecules, they react at near diffusion-controlled rates with other radicals and may therefore be efficient protective agents. This study assessed the ability of (*)NO and nitroxides to intercept specific protein-derived radicals and compared the efficacy of these species. Three protein radical systems were investigated as follows: BSA-derived radicals generated via radical transfer from H(2)O(2)-activated horseradish peroxidase, radicals formed on myoglobin via reaction with H(2)O(2), and carbon-centered radicals formed from amino acid hydroperoxides on exposure to Fe(2+)-EDTA. In each case, radicals were generated in the absence or presence of (*)NO or nitroxides of different size and charge. Concentration-dependent loss of the protein radicals was detected by electron paramagnetic resonance with both (*)NO and nitroxides and time-dependent consumption of (*)NO using an (*)NO electrode. The protein oxidation product dityrosine was significantly reduced by (*)NO and nitroxides, and 3,4-dihydroxyphenylalanine levels were reduced by nitroxides but not (*)NO. Overall, these studies demonstrate that (*)NO and nitroxides are efficient near-stoichiometric scavengers of protein radicals and, hence, are potential protective agents against protein oxidation reactions and resulting damage. These reactions show little dependence on nitroxide structure or charge.     

The relationship between structure and antioxidative activity of piperidine nitroxides

We have investigated the relationship between structure and antioxidative activity of piperidine nitroxides which were substituted by different groups at the 4-position. All of the tested piperidine nitroxides inhibited malondialdehyde (MDA) generation caused either spontaneously or by a hydroxyl free radical generation system (Fe2+-ascorbic acid) in homogenates of liver, heart and kidney of rats, and antagonized H2O2-induced haemolysis from rat erythrocytes in a concentration-dependent manner. The same rank was followed: Bis-(4-amino-2,2,6,6-tetramethyl piperidinooxyl) (4-BIS-Tempo) and 4-azido-2,2,6,6-tetramethyl piperidinooxyl (4-N(3)-Tempo) > 4-isothiocyanate-2,2,6,6-tetramethyl piperidinooxyl (4-ISO-Tempo), 4-2', 4'-dinitrophenylhy-drazone-2,2,6,6-tetramethyl piperidinooxyl (4-D-Tempo), 4-sulfonate-2,2,6,6-tetramethyl piperidinooxyl (4-S-Tempo) and 4-amino-2,2,6,6-tetramethyl piperidinooxyl (4-NH(2)-Tempo) > 4-acetate ester-2,2,6,6-tetramethyl piperidinooxyl (4-A-Tempo) and 4-benzoate-2,2,6,6-tetramethyl piperidinooxyl (4-B-Tempo). With the exception of 4-A-Tempo and 4-D-Tempo, the tested piperidine nitroxides inhibited superoxide anion (O(2*-)) release from neutrophils stimulated by zymosan. The concentration required for inhibiting O(2*-) release was higher than that of inhibiting MDA formation and haemolysis. However, 4-amino-2,2,6,6-tetramethyl piperidine (4-NH2-TempH) and other 4-position substitutes, such as NaN3 and isothiocyanate, had no effects on MDA formation, haemolysis or O(2*-) release. The results indicated that nitroxides have a wide range of scavenging reactive oxygen species (ROS) actions. The nitroxide moiety was the essential group while the 4-position substitutes could influence the activity of nitroxides on scavenging ROS.     

Role of lipid peroxidation and DNA damage in paraquat toxicity and the interaction of paraquat with ionizing radiation.

Since the introduction of paraquat (PQ) as a herbicide in 1963, there have been many speculations concerning the critical lesion in PQ toxicity. Damage to membrane lipids might be an initial event leading to PQ-induced cell killing. The ability of PQ to induce lipid peroxidation was tested in liver homogenates of the mouse. Lipid peroxidation was indeed induced by PQ and shown to be dose dependent, starting to be significant at 2.5 mM. Subsequently, a possible correlation between lipid peroxidation and PQ-induced cell death was investigated in mouse fibroblasts (LM) and Ehrlich ascites tumour (EAT) cells using a clonogenic assay. It was found that in order to be cytotoxic PQ needs enzymatic activation (incubation at 37 degrees). In both cell lines, PQ-induced cell killing appeared to be dose dependent, starting at a dose of 0.5 mM. Supplementation of LM cells with the antioxidant vitamin E had no effect on PQ-induced cell killing and modification of the membranes of LM cells by incorporation of the polyunsaturated fatty acid 20:4 (arachidonic acid) did not sensitize the cells to PQ toxicity. PQ had no effect on the glutathione (GSH) level in EAT cells and complete GSH depletion by DL-buthionine-(SR)-sulfoximine could not sensitize the cells to PQ toxicity. In LM cells PQ-induced cell killing was enhanced after complete GSH depletion by DEM. This sensitization might, however, be attributed to the binding of DEM to proteins. From these results it seems unlikely that lipid peroxidation is the primary cause for PQ-induced cell killing. Another critical target in PQ toxicity is DNA. This possibility was investigated in EAT cells. PQ was found to induce DNA damage (detected by the alkaline unwinding assay) in the same dose range that caused cell death. A good correlation was obtained for cell killing after PQ treatment and DNA damage measured 2 hr after 37 degrees post-incubation. A proposed possible interaction between PQ and X-rays was also investigated. In EAT cells, X-ray-induced cell death was significantly enhanced by pre-incubation with PQ at doses of 0.5 mM and above. At the level of 10% survival an enhancement factor of 1.6 could be observed by treatment with 1 mM PQ when cell killing by PQ is not taken into account. Induction as well as processing of radiation-induced DNA damage seems to be unaffected by pre-incubation with PQ. The mechanism of radiosensitization by PQ is yet unclear.     

吡咯啉氮氧自由基及衍生物抗大鼠不同组织脂质过氧化损伤

目的　在体外研究新合成吡咯啉氮氧自由基及衍生物对大鼠肝细胞膜、线粒体、红细胞及卵黄磷脂脂质过氧化的保护作用 ,并探讨其作用机制。方法　采用Fenton反应法诱导大鼠肝细胞膜、肝线粒体、卵黄磷脂脂质过氧化 ,通过硫代巴比妥酸 (TBA)法测定丙二醛 (MDA)含量 ;415nm处测定H2 O2 诱导的红细胞溶血作用 ;邻苯三酚自氧化法检测其对超氧阴离子 (O·2 )清除作用。结果　带一个活性基团 (NO·)的化合物A、B可明显地抑制MDA的产生 (P <0 0 1) ,抗H2 O2 诱导的红细胞溶血作用 ,但并不影响O·2 的产生 ;具有两个活性基团 (NO·)的化合物C作用略为明显 ,IC50 <31 2 5mg·L-1;而不带活性基团 (NO·)的化合物D无抗氧化能力。结论　稳定性氮氧自由基化合物通过清除生物体系中羟基自由基 (·OH)发挥其抗脂质过氧化作用 ,而对·OH的捕捉可能是通过活性基团NO·实现的     

Reactions of nitric oxide, peroxynitrite, and carbonate radicals with nitroxides and their corresponding oxoammonium cations

Cyclic nitroxides effectively protect biological systems against radical-induced damage. However, the mechanism of the reactions of nitroxides with nitrogen-derived reactive species and carbonate radicals is far from being elucidated. In the present study, the reactions of several representative piperidine- and pyrrolidine-based nitroxides with *NO, peroxynitrite, and CO3*- were investigated, and the results are as follows: (i) There is no evidence for any direct reaction between the nitroxides and the *NO. In the presence of oxygen, the nitroxides are readily oxidized by *NO2, which is formed as an intermediate during autoxidation of *NO. (ii) *NO reacts with the oxoammonium cations to form nitrite and the corresponding nitroxides with k1 = (9.8 +/- 0.2) x 10(3) and (3.7 +/- 0.1) x 10(5) M(-1) s(-1) for the oxoammonium cations derived from 2,2,6,6-tetramethylpiperidine-1-oxyl (TPO) and 3-carbamoyl-proxyl (3-CP), respectively. (iii) CO3*- oxidizes all nitroxides tested to their oxoammonium cations with similar rate constants of (4.0 +/- 0.5) x 10(8) M(-1) s(-1), which are about 3-4 times higher than those determined for H-abstraction from the corresponding hydroxylamines TPO-H and 4-OH-TPO-H. (iv) Peroxynitrite ion does not react directly with the nitroxides but rather with their oxoammonium cations with k(10) = (6.0 +/- 0.9) x 10(6) and (2.7 +/- 0.9) x 10(6) M(-1) s(-1) for TPO+ and 3-CP+, respectively. These results provide a better insight into the complex mechanism of the reaction of peroxynitrite with nitroxides, which has been a controversial subject. The small effect of relatively low concentrations of nitroxides on the decomposition rate of peroxynitrite is attributed to their ability to scavenge efficiently *NO2 radicals, which are formed during the decomposition of peroxynitrite in the absence and in the presence of CO2. The oxoammonium cations, thus formed, are readily reduced back to the nitroxides by ONOO-, while forming *NO and O2. Hence, nitroxides act as true catalysts in diverting peroxynitrite decomposition from forming nitrating species to producing nitrosating ones.     

Does the anaerobic formation of hydroxyl radicals by paraquat monocation radicals and hydrogen peroxide require the presence of transition metals?

This in vitro study investigated the formation of hydroxyl radicals (*OH) under anaerobic conditions through the direct reaction between paraquat radicals (PQ(+)*) and hydrogen peroxide (H(2)O(2)) by quantitative UV-VIS and electron spin resonance (ESR) spectroscopy. PQ(+)* was formed by paraquat reduction using either sodium dithionite or the xanthine/xanthine oxidase reaction as electron donors. The anaerobic formation of PQ(+)* was quantified both by measuring light absorption at 605 nm or by ESR techniques respectively, using either the absorption coefficient or ultramarine as a stable spin standard. Detection of *OH took place with aid of the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline- N-oxide (DEPMPO). Generation or addition of H(2)O(2) to PQ(+)* eliminates the 35-line ESR signal of PQ(+)* and subsequently generates the 8-line ESR signal of the DEPMPO-OH adduct. The elimination of PQ(+)* as well as the formation of OH-DEPMPO adduct was not influenced by 1.0 mM deferoxamine, indicating that iron or other transition metals are, at least under anoxic conditions, not necessarily involved in the generation of the most aggressive reactive oxygen species *OH.     

Protective action of Omega-3 on paraquat intoxication in Drosophila melanogaster

ABSTRACT

Paraquat (PQ) (1,1?-dimethyl-4-4?-bipyridinium dichloride) is the second most widely used herbicide worldwide; however, in countries different sales and distribution remain restricted. Chronic exposure to PQ leads to several diseases related to oxidative stress and mitochondrial dysfunctions including myocardial failure, cancer, and neurodegeneration and subsequently death depending upon the dose level. The aim of this study was to examine if diet supplementation with eicosapentaenoic and docosahexaenoic acids (EPA and DHA, omega-3 long-chain fatty acids) serves a protective mechanism against neuromuscular dysfunctions mediated by PQ using Drosophila melanogaster as a model with focus on mitochondrial metabolism. PQ ingestion (170 mg/kg b.w. for 3 d) resulted in a decreased life span and climbing ability in D. melanogaster. In the brain, PQ increased thioflavin fluorescence and reduced either 4?,6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclei staining and neuronal nuclei protein (NeuN) positive neurons, indicating amyloid formation and neurodegenetation, respectively. In the thorax, PQ ingestion lowered citrate synthase activity and respiratory functions indicating a reduction in mitochondrial content. PQ elevated Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) mRNA expression levels, indicative of high calcium influx from cytosol to mitochondrial matrix. In brain and thorax, PQ also increased hydrogen peroxide (H2O2) production and impaired acetylcholinesterase (AChE) activity. Concomitant EPA/DHA ingestion (0.31/0.19 mg/kg b.w.) protected D. melanogaster against PQ-induced toxicity preserving neuromuscular function and slowing down the rate of aging. In brain and thorax, these omega-3 fatty acids inhibited excess H2O2 production and restored AChE activity. EPA/DHA delayed amyloid deposition in the brain, and restored low citrate synthase activity and respiratory functions in the thorax. The effects in the thorax were attributed to stimulated mRNA expression level of genes involved either in mitochondrial dynamics or biogenesis promoted by EPA/DHA: dynamin-related protein (DRP1), mitochondrial assembly regulatory factor (MARF), mitochondrial dynamin like GTPase (OPA1), and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α). In conclusion, diet supplementation with EPA/DHA appears to protect D. melanogaster muscular and neuronal tissues against PQ intoxication.     

Effect of preinduction of metallothionein on paraquat toxicity in mice

The effect of pretreatment with metallothionein (MT)-inducing metals (Zn, Cu, Bi, Co, Cd or Hg) on paraquat (PQ) toxicity was investigated in mice. PQ lethality was remarkably reduced by pretreatment with the above MT-inducing metals. The protective effect of pretreatment with these metals on PQ lethality was significantly correlated with MT levels in the lung, a target tissue of PQ toxicity, in mice administered MT-inducing metals, but not with MT in the liver or kidney. The increase in pulmonary lipid peroxidation in mice treated with PQ was significantly inhibited by Zn pretreatment. Zn was the most effective of the MT-inducing metals used in this experiment in protecting mice against PQ lethality. Of those monitored, the only pulmonary free radical scavenging factor increased by Zn pretreatment was MT. Other free radical scavenging factors (activities of Superoxide dismutase, glutathione peroxidase and catalase, and concentration of non-protein thiols level) were not influenced by Zn treatment. These results indicate that the induction of pulmonary MT protects against the lethality and lung toxicity of PQ. Pulmonary MT may scavenge free radicals produced by PQ, thereby protecting against lethal pulmonary toxicity.     

Effect of hypoxia on tert-butylhydroperoxide-induced oxidative injury in hepatocytes

Toxicity of t-butylhydroperoxide (t-BuOOH) was studied at different steady state O2 concentrations under conditions at which O2 deficiency alone did not cause cell death. t-BuOOH-induced cell death was more rapid in hypoxic than normoxic cells; the maximal rate of cell death occurred in anoxic cells. t-BuOOH elimination was independent of O2 concentration and was complete within 15 min; t-Butanol was produced at the same rate and was the only product detected by gas chromatography. Measurement of radical production by formation of adducts of the spin-trapping agent N-tert-butylphenylnitrone showed that the amount of radicals trapped was 0.02% of the amount of peroxide added and was the same under anoxic and oxygenated (214 microM O2) conditions. These results show that the O2 dependence of t-BuOOH-induced toxicity is not related to quantitative alterations in its metabolism. Lipid peroxidation was lowest in anoxic cells and increased as the O2 concentration was increased to 1.07 mM O2, showing that enhanced toxicity during hypoxia and anoxia was not due to enhanced lipid peroxidation. In contrast, O2 deficiency impaired the ability of cells to maintain and recovery GSH and NADPH pools after addition of t-BuOOH. GSH was decreased to a greater extent in anoxic cells than in normoxic cells, and the GSH content remained lower in these cells for up to 30 min. This decrease was due both to a decrease in the rate of synthesis and to decreased supply of the NADPH needed for the reduction of GSSG. Taken together, these results show that O2 deficiency has little effect on metabolism of t-BuOOH but impairs the ability of cells to maintain cellular GSH and renders them more susceptible to injury from oxidizing agents. This suggests that oxidative injury under hypoxia or following ischemia may not require a marked stimulation in generation of oxidative species but may occur as a consequence of the impaired ability to tolerate or repair oxidative injury.     

Gene expression analysis of the lung following paraquat administration in rats using DNA microarray

Gene expression changes in the lungs induced by paraquat (PQ) administration were studied in rats using DNA microarrays that were detectable for 1,090 genes per DNA microarray. The rats were subjected to subacute PQ exposure (7 mg/kg, s.c., daily for eight administrations). Two days after the final administration, the rats were divided into two groups. Group 1 experienced significant body weight loss and displayed signs of subacute PQ toxicity, but Group 2 showed no significant effects due to the PQ treatment. A control group, Group 3, was also included. In the comparison of the gene expression levels in the animals from Group 1 or Group 2 to the control animals treated by vehicle, 48 genes in Group 1 and 29 genes from Group 2 were differentially expressed. The twenty-eight genes were common to these two groups. These differentially expressed genes following paraquat treatment were classified as follows: 5 neurotransmitter receptor genes; 4 transporter genes; 4 voltage-gated ion channel genes; 2 lipid metabolism enzyme genes; 2 G-proteins involved in endocytosis and exocytosis genes; 7 cytokine genes; 4 ADP ribosylation genes involved in cell death and regeneration; CFTR gene, which is the causal gene for cystic fibrosis; neurofibromatosis type 1 gene, which is the causal gene for the neurofibromatosis type 1 that is known to accompany pulmonary fibrosis; and the causal gene for spinocerebellar ataxia. These genes may prove to be the keys for the elucidation of the mechanism of PQ toxicity, e.g. PQ-induced pulmonary fibrosis.     

Alternative electron acceptors: Proposed mechanism of paraquat mitochondrial toxicity

Paraquat (PQ) is a relatively safe and effective herbicide used all over the world. PQ is very toxic to all living organisms; and many cases of acute poisoning and death have been reported over the past decade.

The main suggested potential mechanism for PQ toxicity is the production of superoxide radicals from the metabolism of the PQ by microsomal enzyme systems, and by inducing mitochondrial toxicity.

Mitochondria are considered to be a major source of reactive oxygen species in cells and according to this hypothesis, PQ, through suitable oxidation and reduction processes, is able to participate in the redox system in mitochondria. The potential ability of PQ to accept electrons from complex (I, II, III, IV) leads to rapid reaction with molecular oxygen to yield superoxide anion which can lead to the formation of more toxic reactive oxygen species, e.g., hydroxyl radical, often taken as the main toxicant.

Lipid peroxidation due to PQ has been implicated in a number of deleterious effects such as increased membrane rigidity, osmotic fragility, decreased mitochondrial components, reduced mitochondrial survival and lipid fluidity.

The biological effect of reactive oxygen species (ROS) is controlled by a wide spectrum of enzymatic and non-enzymatic defense mechanisms such as superoxide dismutas (SOD), catalase (CAT) and glutathione.

According to this hypothesis, the chemical cascades lead to the reduction of PQ, which reacts quite rapidly with molecular oxygen to yield superoxide anion. The generation of free radicals and lipid peroxidation are the main factors that lead to mitochondrial damage.     

Glutathione status of isolated rabbit lungs. Effects of nitrofurantoin and paraquat perfusion with normoxic and hyperoxic ventilation

Thirty-minute perfusion of isolated rabbit lungs with a Krebs-Ringer bicarbonate buffer containing 420 microM paraquat (PQ) or nitrofurantoin (NF) resulted in increases in lung oxidized glutathione (GSSG) content of 589 and 2656%, respectively, over control levels. The degree of glutathione efflux was also increased with both agents, i.e. 77 and 238% above control leakage for PQ and NF respectively. The pulmonary toxicity of both compounds is known to be heightened by conditions of hyperoxia(O2). Ventilation of lungs with 95% O2-5% CO2 did not, in itself, significantly alter glutathione efflux, GSH or GSSG levels. However, ventilation with 95% O2-5% CO2 increased lung GSSG levels in PQ-perfused lungs 225% over PQ-air-perfused lungs, a combined effect not observed with NF-O2, wherein mean GSSG levels were only 72% of that observed with NF-air. Glutathione efflux in PQ-O2-treated lungs declined somewhat (20%) compared to that observed with PQ-air, but a significant increase in the amount of glutathione efflux was seen with NF-O2-treated lungs, i.e. 120 and 310%, respectively, over that attributable to NF or O2 alone. Although the biochemical mechanisms of toxicity of these compounds are thought to be very similar, the disparate degree of GSH oxidation observed with equimolar levels of PQ and NF may indicate differences in reactivity towards glutathione and other lung sulfhydryl pools. The stimulation of the oxidative effects of PQ and NF on lung GSH due to hyperoxic ventilation may be related to the reported O2 enhancement of their toxicity.     

Investigations into the mechanism of paraquat toxicity utilizing a cell culture system

Paraquat (PQ) and diquat (DQ) produced a dose-dependent inhibition of cellular proliferation in the P388D₁, “macrophage-like” cells in culture. DQ (IC₅₀ = 1.92 × 10^?5m) was not significantly more potent than PQ (IC₅₀ = 1.05 × 10^?4m). The dose-response lines did not deviate significantly from parallelism. Both herbicides had no effect on lipid peroxidation. Ferric pyrophosphate, a known stimulator of lipid peroxidation, produced a dose- and time-dependent stimulation. The effects produced by ferric pyrophosphate indicated that the absence of an effect due to PQ or DQ was not due to an insensitivity of the system. In addition, increasing the oxygen tension did not enhance the toxicity (as measured by cell growth) of PQ. There was no indication of PQ-induced membrane damage as indicated by the leakage of the cytoplasmic enzyme, lactate dehydrogenase. The effect of PQ and DQ on macromolecular synthesis was evaluated as a possible mechanism by which cell growth may have been inhibited. Both herbicides produced a dose- and time-dependent inhibition of DNA synthesis. DQ, but not PQ, inhibited RNA synthesis. Neither PQ nor DQ altered protein synthesis. The inhibition of DNA synthesis did not appear to be due to an alteration of the availability of the precursors. Mitochondrial function, as measured by oxygen consumption, was not affected, suggesting that the inhibition of DNA synthesis was not due to an alteration of energy metabolism. Cyclic nucleotide levels were also not affected, suggesting that the inhibition of cell growth and DNA synthesis was not due to an alteration of cellular control processes. These results suggest that inhibition of macromolecular synthesis may be involved in the molecular mechanism of toxicity of PQ.     

Induction of single strand scission in bacteriophage phi X174 replicative form I DNA by mitomycin C

The action of mitomycin C on double-stranded replicative form I DNA (RF I DNA; supercoiled, covalently closed, circular duplex DNA) of bacteriophage phi X174 was investigated using the technique of agarose gel electrophoresis. Mitomycin C reduced with sodium hydrosulfite (sodium dithionite, Na2S2O4) caused single strand scission in phi X174 RF I DNA in the presence of Cu2+. Cu2+ was essential for this DNA cleave action, and other transition metal ions such as Fe2+, Fe3+, Mn2+, Co2+ and Zn2+ were of no effect. This DNA strand scission was inhibited by catalase (EC 1.11.1.6) and various radical scavengers. This DNA strand scission was caused by free oxygen radicals generated during autoxidation of reduced mitomycin C in the presence of Cu2+.     

Reactivity of paraquat with sodium salicylate: formation of stable complexes

Sodium salicylate (NaSAL) has been shown to be a promising antidote for the treatment of paraquat (PQ) poisonings. The modulation of the pro-oxidant and pro-inflammatory pathways, as well as the anti-thrombogenic properties of NaSAL are probably essential features for the healing effects provided by this drug. Nevertheless, a possible direct chemical reactivity between PQ and NaSAL is also a putative pathway to be considered, this hypothesis being the ground of the present study. In accordance, it is shown, for the first time that PQ and NaSAL react immediately in aqueous medium and within 2-3 min in the solid state. Photographs and scanning electron photomicrographs indicated that a new chemical entity is formed when both compounds are mixed. This assumption was corroborated by the evaluation of the melting point, and through several analytical techniques, namely ultraviolet/visible spectroscopy, nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry/mass spectrometry (GC/MS/MS), liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry (LC/ESI/MS/MS) and infrared spectroscopy, which revealed that stable charge-transfer complexes are formed when PQ is mixed with NaSAL. LC/ESI/MS/MS allowed obtaining the stoichiometry of the charge-transfer complexes. In order to increase resolution, single value decomposition, acting as a filter, showed that the charge-transfer complexes with m/z 483, 643 and 803 correspond to the pseudo-molecular ions, respectively 1:2, 1:3 and 1:4 (PQ:NaSAL). In conclusion, these results provided a new and important mechanism of action of NaSAL against the toxicity mediated by PQ.     

Captopril inhibits the pulmonary toxicity of paraquat in rats

Paraquat (PQ) is a herbicide that is very toxic to all living organisms. It generates free radicals and leads to acute or chronic lung injury. Free radicals are often associated with fibrogenesis, which occurs in various disease states. The purpose of this study was to determine whether captopril prevents paraquat toxicity in lung tissue. Paraquat alone increased the level of lipid peroxidation (LPO) and the activity of superoxide dismutase (SOD) after 4, 12, 24 and 72 h of administration. Also, the level of hydroxyproline showed an increase after 24 h of paraquat administration. However, paraquat also decreased the level of glutathione (GSH) and the activity of glutathione peroxidase (GSH-Px). Captopril (50 mg/kg i.p.) and paraquat were simultaneously injected (40 mg/kg i.p.), and the captopril injection 1 h after paraquat ameliorated the biochemical toxicity induced by paraquat. This was evidenced by a significant reduction in LPO and balancing the endogenous antioxidant capacity by normalizing the activities of SOD and GSH-Px and the GSH content in the lung tissue. Moreover, captopril injection prevented the increase of hydroxyproline content as an index of lung fibrosis. From these results, the beneficial effects of captopril on paraquat toxicity appear to be through enhancement of the endogenous antioxidant system preventing the lung fibrosis.     

Changes in plasma antiproteases in paraquat poisoned rats using micro two-dimensional electrophoretic analysis.

Rats were subjected to subacute paraquat (PQ) exposure (7 mg/kg sc daily for 6-9 administrations). They were divided into a group that died from subacute toxicity, a group that recovered from the subacute toxicity and progressed to pulmonary fibrosis, and a group that showed no effects from the PQ. The rats with subacute fatal toxicity had a remarkable increase in a2M-1. Those which progressed to pulmonary fibrosis had remarkable decreases in a2M-2, a2M-3, and a2M-4 and a1AT. The rats that showed no effects from the PQ had relative increases in all molecular species of a2M. Increases in major a2M molecular species, a2M-1 and a2M-4, and a minor molecular species, a2M-3, were characteristically observed.     

Site-specific DNA damage induced by sulfite in the presence of cobalt(II) ion. Role of sulfate radical

The reactivities of sulfite (SO23-) with DNA in the presence of metal ions were investigated by a DNA sequencing technique using 32P-labeled DNA fragments obtained from human c-Ha-ras-1 protooncogene. Sulfite caused DNA damage in the presence of Co2+, Cu2+ and Mn2+, although sulfite alone or metal ion alone did not. The order of inducing effect on sulfite-dependent DNA damage (Co2+ much greater than Cu2+ greater than Mn2+ Fe3+) was consistent with that of accelerating effect on the initial oxygen consumption rate of sulfite autoxidation. The DNA damage induced by sulfite plus Co2+ was inhibited by 3,5-dibromo-4-nitrobenzenesulfonate, primary and secondary alchols, whereas it was not inhibited by SOD, catalase and tert-butyl alcohol. Incubation of DNA with sulfite plus Co2+ followed by the piperidine treatment led to the predominant cleavage at the positions of guanine especially located 5' to guanine. Sulfite plus Cu2+ gave a DNA cleavage pattern different from that induced by sulfite plus Co2+. The photolysis of peroxydisulfate (S2O28-), which is known to produce SO-4 radicals, gave a DNA cleavage pattern similar to that induced by sulfite plus Co2+. ESR studies using spin-trapping reagent revealed the production of spin adduct possibly of SO-3 radical in a solution of sulfite plus Cu2+, whereas much less spin adduct was produced by sulfite plus Co2+. The results suggest that sulfite is rapidly autoxidized in the presence of Co2+ to produce SO4- radical causing site-specific DNA damage.