Pere Boadas-Vaello

Determination of cyanide and volatile alkylnitriles in whole blood by headspace solid-phase microextraction and gas chromatography with nitrogen phosphorus detection

Simultaneous determination of cyanide and volatile alkylnitriles such as acetonitrile, cis- and trans-crotononitrile, allylnitrile and butyronitrile at low ppb concentration on whole blood (rat and mice) by headspace solid-phase microextraction (HS-SPME) followed by gas chromatography (GC) with nitrogen phosphorus detection has been achieved for the first time. SPME extraction time and temperature were optimized using a star experimental design. Optimum conditions for cyanide extraction were chosen to analyze unspiked blood samples containing alkylnitriles as that analyte occurs at the lowest concentrations. For all analytes, the developed methodology yielded good quality parameters. In all cases, good reproducibility (relative standard deviation < or =12%), detection limits (<3ng mL(-1)) and quantification limits (<4 ng mL(-1)) were recorded.

The targets of acetone cyanohydrin neurotoxicity in the rat are not the ones expected in an animal model of konzo

Konzo is a neurotoxic motor disease caused by excess consumption of insufficiently processed cassava. Cassava contains the cyanogenic glucoside linamarin, but konzo does not present the known pathological effects of cyanide. We hypothesized that the aglycone of linamarin, acetone cyanohydrin, may be the cause of konzo. This nitrile rapidly decomposes into cyanide and acetone, but the particular exposure and nutrition conditions involved in the emergence of konzo may favor its stabilization and subsequent acute neurotoxicity. A number of preliminary observations were used to design an experiment to test this hypothesis. In the experiment, young female Long-Evans rats were given 10mM acetone cyanohydrin in drinking water for 2 weeks, and then 20mM for 6 weeks. Nutrition deficits associated with konzo were modeled by providing tapioca (cassava starch) as food for the last 3 of these weeks. After this period, rats were fasted for 24h in order to increase endogenous acetone synthesis, and then exposed to 0 (control group) or 50 micromol/kg-h of acetone cyanohydrin for 24h (treated group) through subcutaneous osmotic minipump infusion (n=6/group). Motor activity and gait were evaluated before exposure (pre-test), and 1 and 6 days after exposure. Brains (n=4) were stained for neuronal degeneration by fluoro-jade B. Rats exposed to 50 micromol/kg-h of acetone cyanohydrin showed acute signs of toxicity, but no persistent motor deficits. Two animals showed fluoro-jade staining in discrete thalamic nuclei, including the paraventricular and the ventral reuniens nuclei; one also exhibited labeling of the dorsal endopiriform nucleus. Similar effects were not elicited by equimolar KCN exposure. Therefore, acetone cyanohydrin may cause selective neuronal degeneration in the rat, but the affected areas are not those expected in an animal model of konzo.

Allylnitrile Metabolism by CYP2E1 and Other CYPs Leads to Distinct Lethal and Vestibulotoxic Effects in the Mouse

This study addressed the hypothesis that the vestibular or lethal toxicities of allylnitrile depend on CYP2E1-mediated bioactivation. Wild-type (129S1) and CYP2E1-null male mice were exposed to allylnitrile at doses of 0, 0.5, 0.75, or 1.0 mmol/kg (po), following exposure to drinking water with 0 or 1% acetone, which induces CYP2E1 expression. Induction of CYP2E1 activity by acetone in 129S1 mice and lack of activity in null mice was confirmed in liver microsomes. Vestibular toxicity was assessed using a behavioral test battery and illustrated by scanning electron microscopy observation of the sensory epithelia. In parallel groups, concentrations of allylnitrile and cyanide were assessed in blood after exposure to 0.75 mmol/kg of allylnitrile. Following allylnitrile exposure, mortality was lower in CYP2E1-null than in 129S1 mice, and increased after acetone pretreatment only in 129S1 mice. This increase was associated with higher blood concentrations of cyanide. In contrast, no consistent differences were recorded in vestibular toxicity between 129S1 and CYP2E1-null mice, and between animals pretreated with acetone or not. Additional experiments evaluated the effect on the toxicity of 1.0 mmol/kg allylnitrile of the nonselective P450 inhibitor, 1-aminobenzotriazole, the CYP2E1-inhibitor, diallylsulfide, and the CYP2A5 inhibitor, methoxsalen. In 129S1 mice, aminobenzotriazole decreased both mortality and vestibular toxicity, whereas diallylsulfide decreased mortality only. In CYP2E1-null mice, aminobenzotriazole and methoxsalen, but not diallylsulfide, blocked allylnitrile-induced vestibular toxicity. We conclude that CYP2E1-mediated metabolism of allylnitrile leads to cyanide release and acute mortality, probably through alpha-carbon hydroxylation, and hypothesize that epoxidation of the beta-gamma double bond by CYP2A5 mediates vestibular toxicity.

D-cycloserine in the basolateral amygdala prevents extinction and enhances reconsolidation of odor-reward associative learning in rats.

It is well established that D-cycloserine (DCS), a partial agonist of the NMDA receptor glycine site, enhances learning and memory processes. Although the effects of DCS have been especially elucidated in the extinction and reconsolidation of aversive behavioral paradigms or drug-related behaviors, they have not been clearly determined in appetitive tasks using natural reinforcers. The current study examined the effects of pre-retrieval intra-basolateral amygdala (BLA) infusions of DCS on the extinction and reconsolidation of an appetitive odor discrimination task. Rats were trained to discriminate between three odors, one of which was associated with a palatable food reward, and, 20 min prior to extinction learning (experiment 1) or reactivation (experiment 2), they received bilateral intra-BLA infusions of DCS or vehicle. In experiment 1, DCS infusion reduced the rate of extinction learning, weakened extinction retention in a post-extinction test and enhanced reacquisition of the ODT task. In experiment 2, DCS improved subsequent memory expression in the reconsolidation test performed one day after the reactivation session. Such results indicate the involvement of BLA NMDA receptors in odor-food reward associative memory and suggest that DCS may potentiate the persistence or strength of the original memory trace.

Vestibular damage in chronic ototoxicity: A mini-review

Ototoxicity is a major cause of the loss of hearing and balance in humans. Ototoxic compounds include pharmaceuticals such as aminoglycoside antibiotics, anti-malarial drugs, loop diuretics and chemotherapeutic platinum agents, and industrial chemicals including several solvents and nitriles. Human and rodent data indicate that the main target of toxicity is hair cells (HCs), which are the mechanosensory cells responsible for sensory transduction in both the auditory and the vestibular system. Nevertheless, the compounds may also affect the auditory and vestibular ganglion neurons. Exposure to ototoxic compounds has been found to cause HC apoptosis, HC necrosis, and damage to the afferent terminals, of differing severity depending on the ototoxicity model. One major pathway frequently involved in HC apoptosis is the c-jun N-terminal kinase (JNK) signaling pathway activated by reactive oxygen species, but other apoptotic pathways can also play a role in ototoxicity. Moreover, little is known about the effects of chronic low-dose exposure. In rodent vestibular epithelia, extrusion of live HCs from the sensory epithelium may be the predominant form of cell demise during chronic ototoxicity. In addition, greater involvement of the afferent terminals may occur, particularly the calyx units contacting type I vestibular HCs. As glutamate is the neurotransmitter in this synapse, excitotoxic phenomena may participate in afferent and ganglion neuron damage. Better knowledge of the events that take place in chronic ototoxicity is of great interest, as it will increase understanding of the sensory loss associated with chronic exposure and aging.

Transient alteration of the vestibular calyceal junction and synapse in response to chronic ototoxic insult in rats.

ABSTRACT Ototoxicity is known to cause permanent loss of vestibule function through degeneration of sensory hair cells (HCs). However, functional recovery has been reported during washout after chronic ototoxicity, although the mechanisms underlying this reversible dysfunction are unknown. Here, we study this question in rats chronically exposed to the ototoxic compound 3,3′-iminodipropionitrile (IDPN). Pronounced alterations in vestibular function appeared before significant loss of HCs or stereociliary coalescence became evident by ultrastructural analyses. This early dysfunction was fully reversible if the exposure was terminated promptly. In cristae and utricles, the distinct junctions formed between type I HCs (HCI) and calyx endings were completely dismantled at these early stages of reversible dysfunction, and completely rebuilt during washout. Immunohistochemical observations revealed loss and recovery of the junction proteins CASPR1 and tenascin-C and RT-PCR indicated that their loss was not due to decreased gene expression. KCNQ4 was mislocalized during intoxication and recovered control-like localization after washout. At early stages of the intoxication, the calyces could be classified as showing intact or lost junctions, indicating that calyceal junction dismantlement is triggered on a calyx-by-calyx basis. Chronic toxicity also altered the presence of ribeye, PSD-95 and GluA2 puncta in the calyces. These synaptic alterations varied between the two types of calyx endings (formed by calyx-only or dimorphic afferents) and some persisted at the end of the washout period. The present data reveal new forms of plasticity of the calyx endings in adult mammals, including a robust capacity for rebuilding the calyceal junction. These findings contribute to a better understanding of the phenomena involved in progressive vestibular dysfunction and its potential recovery during and after ototoxic exposure. Summary: New forms of damage and repair have been identified in the vestibular sensory epithelium using a rat model of chronic ototoxicity and recovery that causes reversible vestibular dysfunction.

Reduced Systemic Toxicity and Preserved Vestibular Toxicity Following Co-treatment with Nitriles and CYP2E1 Inhibitors: a Mouse Model for Hair Cell Loss

Several nitriles, including allylnitrile and cis-crotononitrile, have been shown to be ototoxic and cause hair cell degeneration in the auditory and vestibular sensory epithelia of mice. However, these nitriles can also be lethal due in large part to the microsomal metabolic release of cyanide, which is mostly dependent on the activity of the 2E1 isoform of the cytochrome P450 (CYP2E1). In this study, we co-administered mice with a nitrile and, to reduce their lethal effects, a selective CYP2E1 inhibitor: diallylsulfide (DAS) or trans-1,2-dichloroethylene (TDCE). Both in female 129S1/SvImJ (129S1) mice co-treated with DAS and cis-crotononitrile and in male RjOrl:Swiss/CD-1 (Swiss) mice co-treated with TDCE and allylnitrile, the nitrile caused a dose-dependent loss of vestibular function, as assessed by a specific behavioral test battery, and of hair cells, as assessed by hair bundle counts using scanning electron microscopy. In the experiments, the CYP2E1 inhibitors provided significant protection against the lethal effects of the nitriles and did not diminish the vestibular toxicity as assessed by behavioral effects in comparison to animals receiving no inhibitor. Additional experiments using a single dose of allylnitrile demonstrated that TDCE does not cause hair cell loss on its own and does not modify the vestibular toxicity of the nitrile in either male or female 129S1 mice. In all the experiments, high vestibular dysfunction scores in the behavioral test battery predicted extensive to complete loss of hair cells in the utricles. This provides a means of selecting animals for subsequent studies of vestibular hair cell regeneration or replacement.

Learning deficits in an odor reward-task induced by parafascicular thalamic lesions are ameliorated by pretraining D-cycloserine in the prelimbic cortex.

We investigated whether the N-methyl-D-aspartate (NMDA) receptor partial agonist D-cycloserine (DCS) infused into the prelimbic cortex (PLC) would reverse the learning deficits caused by bilateral excitotoxic lesions of the parafascicular nucleus (PFn) in an odor discrimination task (ODT). Rats with PFn lesions received a bilateral infusion of DCS (10 μg/side) into the PLC 20 min before ODT acquisition. The task retention was evaluated in a drug-free test carried out 24 h later. DCS significantly attenuated the PFn lesion-induced deficits as measured by both latency to nose-poke the rewarded odor and number of errors committed during ODT acquisition and retention. Therefore, DCS may be an enhancing memory treatment in animal models of cognitive impairment, such as PFn-lesioned rats. The PFn contribution to learning and memory may possibly be linked to its role in the modulation of glutamatergic PLC activity.

(‐)‐Epigallocatechin‐3‐Gallate Antihyperalgesic Effect Associates With Reduced CX3CL1 Chemokine Expression in Spinal Cord

(‐)‐Epigallocatechin‐3‐gallate (EGCG) is a major polyphenol in green tea with beneficial effects on the neuropathic pain alleviation in animal models. Because chemokine fractalkine (CX3CL1) has been suggested as an important signal during neuropathic pain development, this study aimed to investigate whether CX3CL1 expression may be modulated by EGCG treatment reducing hyperalgesia in chronic constriction injured mice. To this end, Balb/c mice were subjected to a chronic constriction injury of sciatic nerve (CCI) and treated with EGCG or vehicle once a day during the first week following surgery. Thermal hyperalgesia was tested at 7 and 14 days post‐surgery, and the expression of CX3CL1 and its mRNA were analyzed in spinal cord at the end of the experimental period. Results revealed that EGCG treatment significantly reduced thermal hyperalgesia in CCI‐injured mice at short time, and this antihyperalgesic effect was associated with a down‐regulation of CX3CL1 protein expression in the spinal cord. On the other hand, EGCG treatment did not affect the CX3CL1 transcription. Overall, our results suggest a new role of EGCG‐treatment in an experimental model of neuropathic pain as a mediator of nociceptive signaling cross talk between neurons and glial cells in the dorsal horn of the spinal cord. Copyright

Epigallocatechin-3-gallate treatment to promote neuroprotection and functional recovery after nervous system injury

Traumatic spinal cord injury (SCI) causes motor paralysis, sensory anesthesia and autonomic dysfunction below the lesion site and additionally some SCI patients refer neuropathic pain together with these signs and symptoms. Clinical and experimental studies have revealed the main pathological changes of injured spinal cord implicated in all these signs and symptoms, including neuropathic pain. After few hours of traumatic SCI, it is usual to observe broken blood brain barrier with plasma and blood cells extravasation, cell necrosis, disruption of ascending and descending spinal cord pathways and increased potassium and glutamate. Glutamate contributes to excitotoxicity of neurons whereas potassium facilitates ectopic depolarization of survival neurons and activation of resident microglia. Reactive microglia cells are able to secrete several pro-inflammatory cytokines (e.g., tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), IL-6) and chemokines (C-C motif) ligand 2 (CCL2) or monocyte chemoattractant protein 1 (MCP1) that contribute to the reactivation and migration of more microglial cells located far to lesion site, and also astrocytes that contribute to the secretion of more pro-inflammatory agents. Chemokine attracts blood cells, including neutrophils, lymphocytes and monocytes that infiltrate on injured spinal cord parenchyma, and contribute to eliminate the cellular debris, but also secrete more pro-inflammatory agents. All these cellular and biochemical changes were observed during the first weeks post-injury. Finally, reactive astrocytes and microglial cells form the glial scar around the lesion site, and astrocytes secrete several proteoglycan that inhibit the re-growth of regenerated central axons across the lesion site. Apoptosis of oligodendrocytes, and wallerian degeneration of nude axons also were seen. The associated myelin proteins (e.g., NOGO, OMpG, MAG, LINGO) that appeared in the injured spinal cord parenchyma also contribute to inhibit the regeneration of central axons. In summary, disruption of spinal cord pathways, persistent pro-inflammatory environment, necrosis and apoptosis of neurons, glia and endothelial cells, and inhibitory environment to axonal regeneration are the main changes observed in injured spinal cord (Silva et al., 2014) (Figure ​1A1A and ​andBB).