[Determination of 3-nitropropionic acid in poisoning samples by ultra-performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: An ultra-performance liquid chromatography-tandem mass spectrometric method was established for determination of 3-nitropropionic acid of sugarcane, sugarcane bagasse, vomit, serum and urine. METHODS: The 3-nitropropionic acid in poisoning samples was extracted by acetonitrile in super-sonic instrument. The supernatant was cleaned up with PSA column and eluted with 10% ammonia water-methanol (10: 90, V/V), then the purified solution was concentrated by nitrogen, dissolved with water (containing 0.4% formic acid) and cleaned by 0.22 µm millipore filter. The filtrate was detected by ultra-performance liquid chromatography-tandem mass spectrometry, identified by electrospray ionization (ESI) in negative mode using multiple reaction monitoring, and quantified with external standards of sample matrix matching. The sample extract was separated on an acquity BEH C18 column (2.1 mm x 150 mm x 1.7 µm) by gradient elution in 10 minutes with acetronitrile-water as mobile phase. RESULTS: The calibration curves of 3-nitropropionic acid residues showed good linearity in the range of 1.0 - 50 µg/kg with correlation coefficient of 0.9993 or 0.9998. The detection limits of the method were from 0.06 µg/kg to 0.30 µg/kg, and limits of quantitation ranged from 0.20 µg/kg to 1.0 µg/kg. The recoveries of three spiking levels (1.0, 10.0 and 100.0 µg/kg) ranged from 86.9% to 102.0%, and the relative standard deviations of 1.80%- 4.19% were obtained. CONCLUSION: The method for determination of 3-nitropropionic acid in poisoning samples by UPLC-MS/MS is of operation convenience, less interference from impurities and good accuracy, which could provide evidence and treatment for mouldy sugarcane poisoning.

N-Acetylcysteine reverses mitochondrial dysfunctions and behavioral abnormalities in 3-nitropropionic acid-induced Huntington's disease.

Mitochondrial dysfunction is a major event involved in the pathogenesis of Huntington's disease (HD). The present study evaluates the role of N-acetyl-L-cysteine (NAC) in preventing mitochondrial dysfunctions in a 3-nitropropionic acid (3-NP)-induced model of HD. Administration of 3-NP to rats (Wistar strain) resulted in significant inhibition of mitochondrial complexes II, IV and V in the striatum. However, no significant effect on complex I was observed. Increased generation of reactive oxygen species and lipid peroxidation was observed in mitochondria of 3-NP-treated animals. Endogenous antioxidants (thiols and manganese-superoxide dismutase) were lowered in mitochondria of 3-NP-treated animals. 3-NP-treated animals showed increased cytosolic cytochrome c levels and mitochondrial swelling. Increased expressions of caspase-3 and p53 were also observed in 3-NP-treated animals. Histopathological examination of the striata of 3-NP-treated animals revealed increased neural space, neurodegeneration and gliosis. This was accompanied by cognitive and motor deficits. NAC treatment, on the other hand, was found to be effective in reversing 3-NP-induced mitochondrial dysfunctions and neurobehavioral deficits. Our findings suggest a beneficial effect of NAC in HD.

Acute intentional self-poisoning with a herbicide product containing fenoxaprop-P-ethyl, ethoxysulfuron, and isoxadifen ethyl: a prospective observational study.

BACKGROUND: Herbicides are commonly ingested for self-harm, but relatively little has been published on poisoning with herbicides other than paraquat and glyphosate. We report here a case series of patients with acute exposure to a combination herbicide (brand name Tiller Gold or Whip Super) containing the selective phenoxy herbicide compounds fenoxaprop-P-ethyl and ethoxysulfuron and a safener isoxadifen ethyl. METHOD: Clinical data on all patients presenting with Tiller Gold or Whip Super poisoning to two General Hospitals in Sri Lanka from 2002-2008 were collected prospectively until discharge. RESULTS: Eighty-six patients with a history of Tiller Gold or Whip Super ingestion were included. The main clinical features were an epigastric burning sensation and vomiting; however, most of those who vomited had received gastric lavage or forced emesis. Eight patients had a reduced level of consciousness on admission (Glasgow coma scale 9-14) that resolved without intervention over several hours. Only symptomatic and supportive care was required. The median hospital stay was 1 day (IQR: 1-2) and the case fatality was zero (95% confidence interval: 0-4.2%). This low case fatality compared favorably with the case fatality of other common herbicides in our cohort: paraquat >40%, propanil >10%, 4-chloro-2-methylphenoxyacetic acid > 5%, and glyphosate >2%. CONCLUSION: This combination herbicide product appears to be safe in patients with acute self-poisoning, particularly in comparison with other herbicides, and causing few clinical features.

[The research of the pathogen responsible for Pleurocybella porrigens related encephalopathy: investigation of 3-nitropropionic acid hypothesis].

Pleurocybella porrigens related encephalopathy exhibits consciousness disturbance and convulsion in the patients after taking and patients show bilateral basal ganglia lesion resulted in high mortality rate. This encephalopathy is a very similar to the moldy sugarcane encephalopathy epidemic in China in the past. We investigated the relationship between Pleurocybella porrigens related encephalopathy and 3-nitropropionic acid which had caused the moldy sugarcane encephalopathy. We have tried to detect 3-NPA in the various specimens from patients and Pleurocybella porrigens, but failed. Further examinations for elucidating the causation of Pleurocybella porrigens related encephalopathy are needed.

Celastrol protects against MPTP- and 3-nitropropionic acid-induced neurotoxicity.

Oxidative stress and inflammation are implicated in neurodegenerative diseases including Parkinson's disease (PD) and Huntington's disease (HD). Celastrol is a potent anti-inflammatory and antioxidant compound extracted from a perennial creeping plant belonging to the Celastraceae family. Celastrol is known to prevent the production of proinflammatory cytokines, inducible nitric oxide synthase and lipid peroxidation. Mice were treated with celastrol before and after injections of MPTP, a dopaminergic neurotoxin, which produces a model of PD. A 48% loss of dopaminergic neurons induced by MPTP in the substantia nigra pars compacta was significantly attenuated by celastrol treatment. Moreover, celastrol treatment significantly reduced the depletion in dopamine concentration induced by MPTP. Similarly, celastrol significantly decreased the striatal lesion volume induced by 3-nitropropionic acid, a neurotoxin used to model HD in rats. Celastrol induced heat shock protein 70 within dopaminergic neurons and decreased tumor necrosis factor-alpha and nuclear factor kappa B immunostainings as well as astrogliosis. Celastrol is therefore a promising neuroprotective agent for the treatment of PD and HD.

A novel systemically active caspase inhibitor attenuates the toxicities of MPTP, malonate, and 3NP in vivo.

Molecular machinery involved in apoptosis plays a role in neuronal death in neurodegenerative disorders such as Parkinson's disease (PD) and Huntington's disease (HD). Several caspase inhibitors, such as the well-known peptidyl inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone (zVADfmk), can protect neurons from apoptotic death caused by mitochondrial toxins. However, the poor penetrability of zVADfmk into brain and toxicity limits its use therapeutically. In the present study, a novel peptidyl broad-spectrum caspase inhibitor, Q-VD-OPH, which offers improvements in potency, stability, and toxicity over zVADfmk, showed significant protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 3-nitropropionic acid (3NP), and malonate toxicities. Q-VD-OPH significantly reduced dopamine depletion in striatum produced by MPTP administration and prevented MPTP-induced loss of dopaminergic neurons in the substantia nigra. It significantly reduced the size of striatal lesions produced by intrastriatal malonate injections and systemic administration of 3NP. Western blots performed on tissues from the midbrain following administration of MPTP or the striatum in 3NP-treated animals showed increases of the active forms of caspase-9 and caspase-8, as well as the caspase-8-mediated proapoptotic protein Bid, which were inhibited Q-VD-OPH treatment. These findings suggest that systematically active broad-spectrum caspase inhibitors maybe useful in the treatment of neurodegenerative diseases such as PD and HD.

Subacute systemic 3-nitropropionic acid intoxication induces a distinct motor disorder in adult C57Bl/6 mice: behavioural and histopathological characterisation.

Data on motor behavioural disorders induced by systemic 3-nitropropionic acid, an irreversible inhibitor of mitochondrial succinate dehydrogenase and their histopathological correlates in mice, are sparse. We thus further characterised the subacute 3-nitropropionic-acid-induced motor disorder and its time course in C57Bl/6 mice using standard behavioural tests, histopathological correlates and in vivo magnetic resonance imaging. Firstly, we studied two intoxication paradigms (340 and 560 mg 3-nitropropionic acid/kg, 7 days) compared to controls. The low-dose regimen induced only slight motor changes (reduced hindlimb stride length and rearing). The high-dose regimen induced significant (P<0.05) behavioural and sensorimotor integration deficits (pole test, rotarod, stride length, open-field spontaneous activity) but with 37.5% lethality at week one. The clinical motor disorder consisted of hindlimb clasping and dystonia, truncal dystonia, bradykinesia and impaired postural control. Histopathologically, there were discrete lesions of the dorsolateral striatum in 62.5% of mice together with a 32% reduction (P<0.0001) of the striatal volume, reduced caldbindin-D28K immunoreactivity in the lateral striatum, and met-enkephalin and substance P in the striatal output pathways. There was also a significant (P<0.05) 30-40% dopaminergic cell loss within the substantia nigra pars compacta. Secondly, we validated a semi-quantitative behavioural scale to describe the time course of the motor deficits and to predict the occurrence of striatal damage. We sought to determine whether it could also be disclosed in vivo by magnetic resonance imaging. The scale correlated with the striatal volume reduction (r(2)=0.57) and striatal cell loss (r(2)=0.87) but not with the loss of striatal dopaminergic terminals (dopamine transporter binding). Increased T2-signal intensity within the striatal lesion correlated with the cell loss (r(2)=0.66). We conclude that systemic administration of 3-nitropropionic acid in C57Bl/6 mice induces a distinct motor disorder and dose-dependent striatonigral damage, which are potentially useful to model human diseases of the basal ganglia.

Mitochondrial toxin 3-nitropropionic acid induces cardiac and neurotoxicity differentially in mice.

We investigated the effects of 3-nitropropionic acid (3NPA), a previously characterized neurotoxin, in four strains of mice to better understand the molecular basis of variable host responses to this agent. Unexpectedly, we found significant cardiac toxicity that always accompanied the neurotoxicity in all strains of mice in acute and subacute/chronic toxicity testing. Caudate putamen infarction never occurred without cardiac toxicity. All mouse strains tested are sensitive to 3NPA although the C57BL/6 and BALB/c mice require more exposure than 129SVEMS and FVB/n mice. Cardiac toxicity alone was found in 50% of symptomatic mice tested and morphologically, the cardiac toxicity is characterized by diffuse swelling of cardiomyocytes and multifocal coagulative contraction band necrosis. In subacute to chronic exposure, atrial thrombosis, cardiac mineralization, cell loss, and fibrosis are combined with cardiomyocyte swelling and necrosis. Ultrastructurally, mitochondrial swelling occurs initially, followed by disruption of myofilaments. Biochemically, isolated heart mitochondria from the highly sensitive 129SVEMS mice have a significant reduction of succinate dehydrogenase activity, succinate oxygen consumption rates, and heart adenosine triphosphate after 3NPA treatment. The severity of morphological changes parallels the biochemical alterations caused by 3NPA, consistent with cardiac toxicity being a consequence of the effects of 3NPA on succinate dehydrogenase. These experiments show, for the first time, that 3NPA has important cardiotoxic effects as well as neurotoxic effects, and that cardiac toxicity possibly resulting from inhibition of the succinate dehydrogenase in heart mitochondria, contributes to the cause of death in 3NPA poisoning in acute and subacute/chronic studies in mice.

Determination of glufosinate ammonium and its metabolite, 3-methylphosphinicopropionic acid, in human serum by gas chromatography-mass spectrometry following mixed-mode solid-phase extraction and t-BDMS derivatization.

A method for the analysis of glufosinate ammonium (GLUF) and its metabolite 3-methylphosphinicopropionic acid (MPPA) in human serum by gas chromatography-mass spectrometry (GC-MS) was developed. Employing a mixed-mode cartridge with both anion exchange action and weak nonpolar interaction, we extracted GLUF and MPPA from the serum and carried out GC-MS analysis of their tert-butyldimethylsilyl derivatives. The detection limits of GLUF and MPPA were 10 pg and 1 pg, respectively. Full mass spectra of 100 pg GLUF and of 10 pg MPPA were easily obtainable. The recovery rate of 90.0+/-11.9% (or better) when the serum concentrations of GLUF and MPPA were 10-0.1 microg/mL. Results of 23 serum samples, from patients with GLUF poisoning, measured by this method correlate well with those derived from the conventional high-performance liquid chromatography method (r = 0.996). The developed GC-MS method is likely to become a useful analytical technique in clinical settings.

Early N-acetylaspartate depletion is a marker of neuronal dysfunction in rats and primates chronically treated with the mitochondrial toxin 3-nitropropionic acid.

N-acetylaspartate (NAA) quantification by 1H-magnetic resonance spectroscopy has been commonly used to assess in vivo neuronal loss in neurodegenerative disorders. Here. the authors used ex vivo and in vivo 1H-magnetic resonance spectroscopy in rat and primate models of progressive striatal degeneration induced by the mitochondrial toxin 3-nitropropionate (3NP) to determine whether early NAA depletions could also be associated with neuronal dysfunction. In rats that were treated for 3 days with 3NP and had motor symptoms, the authors found a significant decrease in NAA concentrations, specifically restricted to the striatum. No cell loss or dying cells were found at this stage in these animals. After 5 days of 3NP treatment, a further decrease in striatal NAA concentrations was observed in association with the occurrence of dying neurons in the dorsolateral striatum. In 3NP-treated primates, a similar striatal-selective and early decrease in NAA concentrations was observed after only a few weeks of neurotoxic treatment, without any sign of ongoing cell death. This early decrease in striatal NAA was partially reversed after 4 weeks of 3NP withdrawal. These results demonstrate that early NAA depletions reflect a reversible state of neuronal dysfunction preceding cell degeneration and suggest that in vivo quantification of NAA 1H-magnetic resonance spectroscopy may become a valuable tool for assessing early neuronal dysfunction and the effects of potential neuroprotective therapies in neurodegenerative disorders.

Excitotoxicity is required for induction of oxidative stress and apoptosis in mouse striatum by the mitochondrial toxin, 3-nitropropionic acid.

Excitotoxicity is implicated in the pathogenesis of several neurologic diseases, such as chronic neurodegenerative diseases and stroke. Recently, it was reported that excitotoxicity has a relationship to apoptotic neuronal death, and that the mitochondrial toxin, 3-nitropropionic acid (3-NP), could induce apoptosis in the striatum. Although striatal lesions produced by 3-NP could develop through an excitotoxic mechanism, the exact relationship between apoptosis induction and excitotoxicity after 3-NP treatment is still not clear. The authors investigated the role of excitotoxicity and oxidative stress on apoptosis induction within the striatum after intraperitoneal injection of 3-NP. The authors demonstrated that removal of the corticostriatal glutamate pathway reduced superoxide production and apoptosis induction in the denervated striatum of decorticated mice after 3-NP treatment. Also, the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, prevented apoptosis in the striatum after 3-NP treatment for 5 days, whereas the non-NMDA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, was ineffective. The authors also evaluated the initial type of neuronal death by 3-NP treatment for different durations from 1 to 5 days. In early striatal damage, apoptotic neuronal death initially occurred after 3-NP treatment. Our data show that excitotoxicity related to oxidative stress initially induces apoptotic neuronal death in mouse striatum after treatment with 3-NP.

Anti-death properties of TNF against metabolic poisoning: mitochondrial stabilization by MnSOD.

The cytokine tumor necrosis factor (TNF) is toxic to some mitotic cells, but protects cultured neurons from a variety of insults by mechanisms that are unclear. Pretreatment of neurons or astrocytes with TNF caused significant increases in MnSOD activity, and also significantly attenuated 3-nitropropionic acid (3-NP) induced superoxide accumulation and loss of mitochondrial transmembrane potential. In oligodendrocytes, however, MnSOD activity was not increased, and 3-NP toxicity was unaffected by TNF. Genetically engineered PC6 cells that overexpress MnSOD also were resistant to 3-NP-induced damage. TNF pretreatment and MnSOD overexpression prevented 3-NP induced apoptosis, and shifted the mode of death from necrosis to apoptosis in response to high levels of 3-NP. Mitochondria isolated from either MnSOD overexpressing PC6 cells or TNF-treated neurons maintained resistance to 3-NP-induced loss of transmembrane potential and calcium homeostasis, and showed attenuated release of caspase activators. Overall, these results indicate that MnSOD activity directly stabilizes mitochondrial transmembrane potential and calcium buffering ability, thereby increasing the threshold for lethal injury. Additional studies showed that levels of oxidative stress and striatal lesion size following 3-NP administration in vivo are increased in mice lacking TNF receptors.

[Demonstration by x-ray computed tomography of renal involvement caused by NSAID poisoning]. / Mise en évidence par tomodensitométrie de l'atteinte rénale des AINS dans le cadre d'intoxication.

We describe the contribution of computed tomography in the determination of the renal damage after using tiaprofenic acid. Computed tomography realized six hours after injection of contrast medium revealed corticomedullary enhancement secondary to vascular and tubular stasis. This stasis is compatible with interstitial edema in relation with usually reversible lesions of tubulo-interstitial nephritis induced by of nonsteroidal anti-inflammatory drugs.

Consistent striatal damage in rats induced by 3-nitropropionic acid and cultures of arthrinium fungus.

Arthrinium fungi were cultivated from the samples of mildewed sugarcane which caused acute encephalopathy and delayed dystonia in children. The Arthrinium cultures (AC) contained 5 mg/ml 3-nitropropionic acid (3-NPA) after being inactivated and concentrated. A neuropathological study was carried out in rats intoxicated with 3-NPA and AC, respectively. Consistent bilateral striatal necrosis was found in rats with both poisonings, and the severity was well correlated with persistent recumbency which was a clinical indicator of the development of morphological brain lesions. A reproducible animal model of striatal damage has been produced in rats by IP injections of 10 mg/kg 3-NPA, 6 times a day, with an interval of 1.5 h for 3-5 days. The clinical and neuropathological manifestations in rats dosed with AC were nearly the same as those dosed with 3-NPA. The striatal lesions induced by 3-NPA and AC in poisoned rats were in accordance with the bilateral lenticular hypodensity found by CT scanning in patients of mildewed sugarcane poisoning with delayed dystonia. This neuropathological evidence supports previous epidemiological and mycological findings which indicate that 3-NPA is the possible pathogen of acute mildewed sugarcane poisoning.

Hypothalamic neurons are resistant to the intoxication with 3-nitropropionic acid that induces lesions in the striatum and hippocampus via the damage in the blood-brain barrier.

Modulation of the function of the blood-brain barrier (BBB) in the hypothalamus was investigated after the intoxication with 3-nitropropionic acid (3-NPA) that inhibits the succinate dehydrogenase. 3-NPA was administered to rats for three days. Following transcardial perfusion, brain sections were studied by immunohistochemistry. On the 2nd or 3rd day after 3-NPA, strong immunoreactions for blood-borne macromolecules, IgG, appeared in the striatum and hippocampus. Glial fibrillary acidic protein (GFAP) positive astroglias distributed heterogeneously, and induced nitric oxide synthase (iNOS) positive cells appeared around the vessels. A week later, bilateral lesions were detected in these areas. In the hypothalamus, there appeared a moderate immunoreaction for IgG, but no expression of iNOS. GFAP positive astroglias were rich especially around the vessels, and no loss in microtubule-associated protein 2 (MAP2) immunoreaction was detected, suggesting an intact BBB structure and no neuronal loss following 3-NPA intoxication. Data indicate that hypothalamic neurons are resistant to 3-NPA that induces specific lesions in the striatum and hippocampus via the damage in the BBB.

Toxic effects of nonsteroidal anti-inflammatory drugs in overdose. An overview of recent evidence on clinical effects and dose-response relationships.

Nonsalicylate, nonsteroidal anti-inflammatory drugs (NSAIDs) can be divided into 4 chemical classes: acetic acids, fenamic acids, oxicams and propionic acids. Most NSAID overdoses result in a benign outcome. Of 50,614 exposures reported to poison centres in the United States in a 2-year period, 131 (0.26%) had a major outcome, with 10 deaths. Despite the generally mild effects reported in large patient series, isolated case reports have documented serious toxicity, such as seizures, hypotension, apnoea, coma and renal failure. The majority of these consequences occur after ingestion of substantial quantities by adults attempting suicide. Rarely, with ibuprofen and piroxicam, children who ingest small amounts in accidental exposure develop serious toxicity. Typical signs and symptoms of NSAID overdose include nausea, vomiting, headache, drowsiness, blurred vision and dizziness. Seizures are rarely documented across all NSAID classes, with the exception of mefenamic acid (where seizures occur in over one-third of cases), or following massive ingestion of other agents. Drugs in the propionic acid group have produced metabolic acidosis, respiratory depression and coma in severe cases. Ibuprofen is the agent with the most published data on overdose, probably because it is available without a prescription in many countries. Symptoms are unlikely after ingestion of 100 mg/kg or less, and are usually not life-threatening unless more than 400 mg/kg is ingested. There is some relationship between plasma concentrations and the potential for development of symptoms, but plasma concentrations have no impact on treatment decisions. Treatment of NSAID overdose is entirely supportive. Recent trends in emergency department procedures regarding gastric decontamination are evolving towards the recommended administration of activated charcoal without gastric emptying in patients presenting more than 1 hour after ingestion, although gastric lavage, followed by administration of activated charcoal, may be advisable in patients who present earlier. Home administration of syrup of ipecac is still recommended if treatment is given shortly after ingestion, with a few exceptions: for example, ipecac is contraindicated after ingestion of mefenamic acid or ibuprofen in amounts greater than 400 mg/kg. Urine alkalinisation and diuresis have been recommended to enhance the elimination of NSAIDs, based on a pKa in the range of 3 to 5. However, because the drugs are universally highly protein bound, with little unchanged renal excretion, this technique is not likely to be beneficial. Haemodialysis is also unlikely to enhance elimination, but may be required if oliguric renal failure develops. Multiple dose activated charcoal may be useful in enhancing elimination of NSAIDs with long half-lives, such as piroxicam and sulindac.