Age-related cognitive impairments in mice with a conditional ablation of the neural cell adhesion molecule

Most of the mechanisms involved in neural plasticity support cognition, and aging has a considerable effect on some of these processes. The neural cell adhesion molecule (NCAM) of the immunoglobulin superfamily plays a pivotal role in structural and functional plasticity and is required to modulate cognitive and emotional behaviors. However, whether aging is associated with NCAM alterations that might contribute to age-related cognitive decline is not currently known. In this study, we determined whether conditional NCAM-deficient mice display increased vulnerability to age-related cognitive and emotional alterations. We assessed the NCAM expression levels in the hippocampus and medial prefrontal cortex (mPFC) and characterized the performance of adult and aged conditional NCAM-deficient mice and their age-matched wild-type littermates in a delayed matching-to-place test in the Morris water maze and a delayed reinforced alternation test in the T-maze. Although aging in wild-type mice is associated with an isoform-specific reduction of NCAM expression levels in the hippocampus and mPFC, these mice exhibited only mild impairments in working/episodic-like memory performance. However, aged conditional NCAM-deficient mice displayed pronounced impairments in both the delayed matching-to-place and the delayed reinforced alternation tests. Importantly, the deficits of aged NCAM-deficient mice in these working/episodic-like memory tasks could not be attributed to increased anxiety-like behaviors or to differences in locomotor activity. Taken together, these data indicate that reduced NCAM expression in the forebrain might be a critical factor for the occurrence of cognitive impairments during aging

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

Aquest article conté una errata annexadaOtotoxicity 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

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 ageing

Calyx junction dismantlement and synaptic uncoupling precede hair cell extrusion in the vestibular sensory epithelium during sub-chronic 3,3'-iminodipropionitrile ototoxicity in the mouse

The cellular and molecular events that precede hair cell (HC) loss in the vestibular epithelium during chronic ototoxic exposure have not been widely studied. To select a study model, we compared the effects of sub-chronic exposure to different concentrations of 3,3′-iminodipropionitrile (IDPN) in the drinking water of two strains of mice and of both sexes. In subsequent experiments, male 129S1/SvImJ mice were exposed to 30 mM IDPN for 5 or 8 weeks; animals were euthanized at the end of the exposure or after a washout period of 13 weeks. In behavioral tests, IDPN mice showed progressive vestibular dysfunction followed by recovery during washout. In severely affected animals, light and electron microscopy observations of the vestibular epithelia revealed HC extrusion towards the endolymphatic cavity. Comparison of functional and ultrastructural data indicated that animals with fully reversible dysfunction did not have significant HC loss or stereociliary damage, but reversible dismantlement of the calyceal junctions that characterize the contact between type I HCs (HCI) and their calyx afferents. Immunofluorescent analysis revealed the loss of calyx junction proteins, Caspr1 and Tenascin-C, during exposure and their recovery during washout. Synaptic uncoupling was also recorded, with loss of pre-synaptic Ribeye and post-synaptic GluA2 puncta, and differential reversibility among the three different kinds of synaptic contacts existing in the epithelium. qRT-PCR analyses demonstrated that some of these changes are at least in part explained by gene expression modifications. We concluded that calyx junction dismantlement and synaptic uncoupling are early events in the mouse vestibular sensory epithelium during sub-chronic IDPN ototoxicity

Repeated Sigma-1 Receptor Antagonist MR309 Administration Modulates Central Neuropathic Pain Development After Spinal Cord Injury in Mice

Up to two-thirds of patients affected by spinal cord injury (SCI) develop central neuropathic pain (CNP), which has a high impact on their quality of life. Most of the patients are largely refractory to current treatments, and new pharmacological strategies are needed. Recently, it has been shown that the acute administration of the σ1R antagonist MR309 (previously developed as E-52862) at 28 days after spinal cord contusion results in a dose-dependent suppression of both mechanical allodynia and thermal hyperalgesia in wild-type CD-1 Swiss female mice. The present work was addressed to determine whether MR309 might exert preventive effects on CNP development by repeated administration during the first week after SCI in mice. To this end, the MR309 (16 or 32 mg/kg i.p.) modulation on both thermal hyperalgesia and mechanical allodynia development were evaluated weekly up to 28 days post-injury. In addition, changes in pro-inflammatory cytokine (TNF-α, IL-1β) expression and both the expression and activation (phosphorylation) of the N-methyl-D-aspartate receptor subunit 2B (NR2B-NMDA) and extracellular signal-regulated kinases (ERK1/2) were analyzed. The repeated treatment of SCI-mice with MR309 resulted in significant pain behavior attenuation beyond the end of the administration period, accompanied by reduced expression of central sensitization-related mechanistic correlates, including extracellular mediators (TNF-α and IL-1β), membrane receptors/channels (NR2B-NMDA) and intracellular signaling cascades (ERK/pERK). These findings suggest that repeated MR309 treatment after SCI may be a suitable pharmacologic strategy to modulate SCI-induced CNP development

Novel Epigallocatechin-3-Gallate (EGCG) Derivative as a New Therapeutic Strategy for Reducing Neuropathic Pain after Chronic Constriction Nerve Injury in Mice

Abstract Neuropathic pain is common in peripheral nerve injury and often fails to respond to ordinary medication. Here, we investigated whether the two novel epigallocatechin-3-gallate (EGCG) polyphenolic derivatives, compound 23 and 30, reduce the neuropathic pain in mice chronic constriction nerve injury (CCI). First, we performed a dose-response study to evaluate nociceptive sensation after administration of EGCG and its derivatives 23 and 30, using the Hargreaves test at 7 and 21 days after injury (dpi). We daily administered EGCG, 23 and 30 (10 to 100 mg/Kg; i.p.) during the first week post-CCI. None of the doses of compound 23 caused significant pain diminution, whereas 50mg/kg was optimal for both EGCG and 30 to delay the latency of paw withdrawal. With 50 mg/Kg, we showed that EGCC prevented the thermal hyperalgesia from 7 to 21 dpi and compound 30 from 14 to 56 dpi. To evaluate the molecular mechanisms underpinning why EGCG and compound 30 differentially prevented the thermal hyperalgesia, we studied several biochemical parameters in the dorsal horn of the spinal cord at 14 and 56 dpi. We showed that the effect observed with EGCG and compound 30 was related to the inhibition of fatty acid synthase (FASN), a known target of these polyphenolic compounds. Additionally, we observed that EGCG and compound 30 reduced the expression of CCI-mediated inflammatory proteins and the nuclear localization of nuclear factor-kappa B at 14 dpi, but not at 56 dpi. We also strongly detected a decrease of synaptic plasma membrane levels of N-methyl-D-asparte receptor 2B in CCI-mice treated with compound 30 at 56 dpi. Altogether, compound 30 reduced the chronic thermal hyperalgesia induced by CCI better than the natural compound EGCG. Thus, our findings provide a rationale for the preclinical development of compound 30 as an agent to treat neuropathic pain

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

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 α-carbon hydroxylation, and hypothesize that epoxidation of the β-γ double bond by CYP2A5 mediates vestibular toxicity

Novel epigallocatechin-3-gallate (EGCG) derivative as a new therapeutic strategy for reducing neuropathic pain after chronic constriction nerve injury in mice

Neuropathic pain is common in peripheral nerve injury and often fails to respond to ordinary medication. Here, we investigated whether the two novel epigallocatechin-3-gallate (EGCG) polyphenolic derivatives, compound 23 and 30, reduce the neuropathic pain in mice chronic constriction nerve injury (CCI). First, we performed a dose-response study to evaluate nociceptive sensation after administration of EGCG and its derivatives 23 and 30, using the Hargreaves test at 7 and 21 days after injury (dpi). We daily administered EGCG, 23 and 30 (10 to 100 mg/Kg; i.p.) during the first week post-CCI. None of the doses of compound 23 caused significant pain diminution, whereas 50mg/kg was optimal for both EGCG and 30 to delay the latency of paw withdrawal. With 50 mg/Kg, we showed that EGCC prevented the thermal hyperalgesia from 7 to 21 dpi and compound 30 from 14 to 56 dpi. To evaluate the molecular mechanisms underpinning why EGCG and compound 30 differentially prevented the thermal hyperalgesia, we studied several biochemical parameters in the dorsal horn of the spinal cord at 14 and 56 dpi. We showed that the effect observed with EGCG and compound 30 was related to the inhibition of fatty acid synthase (FASN), a known target of these polyphenolic compounds. Additionally, we observed that EGCG and compound 30 reduced the expression of CCI-mediated inflammatory proteins and the nuclear localization of nuclear factor-kappa B at 14 dpi, but not at 56 dpi. We also strongly detected a decrease of synaptic plasma membrane levels of N-methyl-D-asparte receptor 2B in CCI-mice treated with compound 30 at 56 dpi. Altogether, compound 30 reduced the chronic thermal hyperalgesia induced by CCI better than the natural compound EGCG. Thus, our findings provide a rationale for the preclinical development of compound 30 as an agent to treat neuropathic pain

Neurotoxicitat i metabolisme de nitrils a la rata i el ratolí

[cat] Els nitrils són compostos químics que contenen el grup ciano: C N. Són molt abundants en la naturalesa, però la seva síntesi és també molt important pel gran ventall d'aplicacions que aquests aporten a la indústria. A la naturalesa els trobem, entre d'altres, en forma de glucòsids cianogènics com és el cas de l'amigdalina a les ametlles amargants i la linamarina a la mandioca, i en forma d'aminonitrils, forma que es troba a les lleguminoses del gènere Lathyrus. A la indústria s'utilitzen com a dissolvents i productes intermediaris en la síntesi de plàstics, fibres artificials, resines, productes farmacèutics i altres substàncies químiques. El consum humà o animal de determinats nitrils provoca símptomes similars a la intoxicació per cianur. Aquest fet suggereix que en la metabolització d'aquests nitrils es produeix l'alliberament d'aquest compost i que seria el responsable de la intoxicació aguda. Alguns nitrils però, en aquest procés de metabolització alliberen menys cianur o n'alliberen més lentament i aquests són els que provoquen neurotoxicitat i síndromes neurològiques. L'estudi d'aquest tipus de nitrils adquireix importància com a models de neurotoxicitat i de síndromes de toxicitat humanes causades per nitrils d'origen natural. A la primera part d'aquest treball de tesi s'ha pogut caracteritzar exhaustivament les dianes del 3,3'-iminodipropionitril (IDPN), l'al·lilnitril, el cis-crotononitril, el trans-crotononitril i el 2,4-hexadienenitril al sistema nerviós de la rata i establir la relació entre les lesions estructurals i els efectes comportamentals que aquests provoquen. Per a aquesta caracterització global, s'ha treballat amb un disseny experimental que combina experiments histopatològics amb tests d'avaluació comportamental animal. Els resultats han permès dividir els nitrils neurotòxics en dos grups, els que afecten al sistema nerviós central (trans-crotononitril i hexadienenitril) i els que afecten els sistemes sensorials (IDPN, al·lilnitril i cis-crotononitril). A la segona part de la tesi es demostra que la toxicitat vestibular de nitrils no és un fenomen exclusiu de la rata, sinó que ocorre també en altres espècies incloent-hi mamífers i no mamífers. Aquest resultats obren la porta a l'ús de soques de ratolins transgènics en l'estudi d'aquesta toxicitat. A la tercera part de la tesi s'estudia el paper de la metabolització d'aquests nitrils per a provocar els efectes neurotòxics. Concretament s'ha avaluat la bioactivació de la toxicitat vestibular del cis-crotononitril i l'al·lilnitril mitjançant el citocrom P450 2E1 utilitzant ratolins 129S1, ratolins transgènics pel citocrom P4502E1 i inhibidors selectius (Dial·lilsulfur) i no selectius (Aminobenzatriazol) d'aquest citocrom. A més, en aquesta segona part s'explica el procés d'optimització d'una metodologia que permet detectar simultàniament cianur i nitrils volàtils en sang, que combina la tècnica SPME (microextracció en fase sòlida) i la cromatografia de gasos amb detector NPD (detector nitrogen-fòsfor). Amb aquests treballs es demostra que tant l'al·lilnitril com el cis-crotononitril són substrats del citocrom P450 2E1 però que mitjançant aquest citocrom s'activa una via cianogènica no necessàriament vestibular. Els resultats obtinguts ens han permès concloure que la toxicitat vestibular d'aquets nitrils depèn d'un citocrom P450 diferent al 2E1 i que la via metabòlica amb efectes vestibulars més probable per a l'al·lilnitril és la de l'epoxidació del doble enllaç en posició beta-gamma.||SUMMARY: Nitriles are increasingly used in the chemical industry, and they are also common in crop plants. They cause acute lethality, osteolathyrism and neurotoxicity. In the first part of this thesis, we compared the behavioural effects of IDPN, allylnitrile, cis-crotononitrile, trans-crotononitrile, and hexadienenitrile in the rat using test methods sensitive to the ECC (Excitation with Choreiform and Circling movements) syndrome or to the trans-crotononitrile syndrome. In addition, we assessed the CNS effects of these nitriles with the Fluoro-Jade B stain, which selectively labels degenerating neurons. In conclusion, the present and previous data indicate that neurotoxic nitriles induce one of two syndromes of abnormal motor behaviour: (1) the ECC syndrome caused by IDPN, allylnitrile, and cis-crotononitrile, and resulting from degeneration of the vestibular sensory hair cells with no CNS toxicity role, and (2) the syndrome of faltering movements, caused by trans-crotononitrile and hexadienenitrile, and resulting from degeneration of the inferior olive neurons. In the second part of this thesis, we demonstrate that nitriles that are toxic to the vestibular and auditory systems of the rat are also ototoxic to other animal species, both mammals and no mammals. The findings open up the possibility of using species other than the rat to study this toxic effect of nitriles and the possibility of using nitriles for inner ear research performed in diverse animal species. In the third part of this thesis, we assessed the hypotheses that the vestibular or lethal toxicities of cis-crotononitrile and allylnitrile depend on CYP2E1-mediated bioactivation. Wild-type (129S1) and CYP2E1-null mice were exposed to cis-crotononitrile and allylnitrile in either a baseline condition or following exposure to 1% acetone in drinking water to induce CYP2E1 expression. In parallel groups, concentrations of cis-crotononitrile, allylnitrile and cyanide were assessed in whole blood using an optimized SPME GC-NPD methodology. Also further groups of animals were used to test the effect the non selective P450 inhibitor 1-aminobenzotriazole and the CYP2E1-selective inhibitor diallylsulfide on the toxicity of allylnitrile. We conclude that allylnitrile and cis-crotononitrile are CYP2E1 substrates and that CYP2E1-mediated metabolism of these nitriles leads to cyanide release and acute mortality, while a different P450 system bioactivates these nitriles for vestibular toxicity. We propose that CYP2E1 acts on allylnitrile by alfa-carbon hydroxylation and that an action on the beta-gamma-double bound is more likely responsible for the vestibular effect.SUMMARY: Nitriles are increasingly used in the chemical industry, and they are also common in crop plants. They cause acute lethality, osteolathyrism and neurotoxicity. In the first part of this thesis, we compared the behavioural effects of IDPN, allylnitrile, cis-crotononitrile, trans-crotononitrile, and hexadienenitrile in the rat using test methods sensitive to the ECC (Excitation with Choreiform and Circling movements) syndrome or to the trans-crotononitrile syndrome. In addition, we assessed the CNS effects of these nitriles with the Fluoro-Jade B stain, which selectively labels degenerating neurons. In conclusion, the present and previous data indicate that neurotoxic nitriles induce one of two syndromes of abnormal motor behaviour: (1) the ECC syndrome caused by IDPN, allylnitrile, and cis-crotononitrile, and resulting from degeneration of the vestibular sensory hair cells with no CNS toxicity role, and (2) the syndrome of faltering movements, caused by trans-crotononitrile and hexadienenitrile, and resulting from degeneration of the inferior olive neurons. In the second part of this thesis, we demonstrate that nitriles that are toxic to the vestibular and auditory systems of the rat are also ototoxic to other animal species, both mammals and no mammals. The findings open up the possibility of using species other than the rat to study this toxic effect of nitriles and the possibility of using nitriles for inner ear research performed in diverse animal species. In the third part of this thesis, we assessed the hypotheses that the vestibular or lethal toxicities of cis-crotononitrile and allylnitrile depend on CYP2E1-mediated bioactivation. Wild-type (129S1) and CYP2E1-null mice were exposed to cis-crotononitrile and allylnitrile in either a baseline condition or following exposure to 1% acetone in drinking water to induce CYP2E1 expression. In parallel groups, concentrations of cis-crotononitrile, allylnitrile and cyanide were assessed in whole blood using an optimized SPME GC-NPD methodology. Also further groups of animals were used to test the effect the non selective P450 inhibitor 1-aminobenzotriazole and the CYP2E1-selective inhibitor diallylsulfide on the toxicity of allylnitrile. We conclude that allylnitrile and cis-crotononitrile are CYP2E1 substrates and that CYP2E1-mediated metabolism of these nitriles leads to cyanide release and acute mortality, while a different P450 system bioactivates these nitriles for vestibular toxicity. We propose that CYP2E1 acts on allylnitrile by alfa-carbon hydroxylation and that an action on the beta-gamma-double bound is more likely responsible for the vestibular effect.[spa] Los nitrilos son compuestos químicos que contienen el grupo ciano: -CN. Son muy abundantes en la naturaleza, pero su síntesis es también muy importante por el amplio rango de aplicaciones que estos aportan a la industria. En la naturaleza los encontramos en forma de glucósidos cianogénicos, como la amigdalina de las almendras amargas y la linamarina de la yuca, y en forma de aminonitrilos, forma que se encuentra en las leguminosas del género Lathyrus. En la industria se utilizan como disolventes i productos intermediarios en la síntesis de plásticos, fibras artificiales, resinas, productos farmacéuticos i otras sustancias químicas. El consumo humano o animal de determinados nitrilos provoca síntomas similares a la intoxicación por cianuro. Este hecho sugiere que en la metabolización de estos nitrilos se produce la liberación de este compuesto y que sería el responsable de la intoxicación aguda. Algunos nitrilos, en este proceso de metabolización, liberan menos cianuro o lo hacen de forma más lenta y estos son los que provocan neurotoxicidad y síndromes neurológicos. El estudio de estos nitrilos adquiere importancia como modelos de neurotoxicidad y síndromes neurológicos de toxicidad humana causados por nitrilos de origen natural. En la primera parte de este trabajo de tesis se ha podido caracterizar de forma exhaustiva las dianas del 3,3'-iminodipropionitrilo (IDPN), el alilnitrilo, el cis-crotononitrilo, el trans-crotononitrilo i el 2,4-hexadienenitrilo en el sistema nervioso central y establecer la relación entre las lesiones estructurales y los efectos comportamentales que estos provocan. Para esta caracterización global, se ha trabajado con un diseño experimental que combina experimentos hitopatológicos con tests de evaluación comportamental animal. Los resultados han permitido dividir los nitrilos neurotóxicos en dos grupos, los que afectan al sistema nervioso central (trans-crotononitrilo y hexadienenitrilo) y los que afectan a los sistemas sensoriales (IDPN, alilnitrilo y cis-crotononitrilo). En la segunda parte de la tesis se demuestra que la toxicidad vestibular de nitrilos no es un fenómeno exclusivo de la rata, sino que ocurre también en otras especies incluyendo mamíferos y no mamíferos. Estos resultados abren las puertas a la utilización de ratones transgénicos en el estudios de dicha toxicidad. En la tercera parte de la tesis se estudia el papel de la metabolización de estos nitrilos para provocar los efectos neurotóxicos. Concretamente se ha evaluado la bioactivación de la toxicidad vestibular del cis-crotononitrilo y el alilnitrilo mediante el citocromo P450 2E1 utilizando ratones 129S1, ratones transgénicos para el citocromo P450 2E1 y inhibidores selectivos (dialilsulfuro) y no selectivos (aminobenzatriazol) de este citocromo. Además, en esta tercera parte se explica el proceso de optimización de una metodología que permite detectar simultáneamente cianuro y nitrilos volátiles en sangre, que combina la técnica SPME (microextracción en fase sólida) y la cromatografía de gases con detector NPD (detector nitrógeno-fósforo). Con estos trabajos se demuestra que tanto el alilnitrilo como el cis-crotononitrilo son sustratos del citocromo P450 2E1 pero que mediante este citocromo se activa una vía cianogénica no necesariamente vestibular. Los resultados obtenidos nos han permitido concluir que la toxicidad vestibular de estos nitrilos depende de un citocromo P450 diferente al 2E1 y que la vía metabólica con efectos vestibulares más probable para el alilnitrilo es la epoxidación del doble enlace en posición beta-gamma