Sandra Saldaña-Ruíz

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.

Loss of neurofilaments in the neuromuscular junction in a rat model of proximal axonopathy

C. Soler‐Martín, Ú. Vilardosa, S. Saldaña‐Ruíz, N. Garcia and J. Llorens (2012) Neuropathology and Applied Neurobiology38, 61–71

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.

Cocoa Diet Prevents Antibody Synthesis and Modifies Lymph Node Composition and Functionality in a Rat Oral Sensitization Model

Cocoa powder, a rich source of polyphenols, has shown immunomodulatory properties in both the intestinal and systemic immune compartments of rats. The aim of the current study was to establish the effect of a cocoa diet in a rat oral sensitization model and also to gain insight into the mesenteric lymph nodes (MLN) activities induced by this diet. To achieve this, three-week-old Lewis rats were fed either a standard diet or a diet with 10% cocoa and were orally sensitized with ovalbumin (OVA) and with cholera toxin as a mucosal adjuvant. Specific antibodies were quantified, and lymphocyte composition, gene expression, and cytokine release were established in MLN. The development of anti-OVA antibodies was almost totally prevented in cocoa-fed rats. In addition, this diet increased the proportion of TCRγδ+ and CD103+CD8+ cells and decreased the proportion of CD62L+CD4+ and CD62L+CD8+ cells in MLN, whereas it upregulated the gene expression of OX40L, CD11c, and IL-1β and downregulated the gene expression of IL-17α. In conclusion, the cocoa diet induced tolerance in an oral sensitization model accompanied by changes in MLN that could contribute to this effect, suggesting its potential implication in the prevention of food allergies.

Vestibulotoxic Properties of Potential Metabolites of Allylnitrile

This study addressed the hypothesis that epoxidation of the double bond in allylnitrile mediates its vestibular toxicity, directly or after subsequent metabolism by epoxide hydrolases. The potential metabolites 3,4-epoxybutyronitrile and 3,4-dihydroxybutyronitrile were synthesized and characterized. In aqueous solutions containing sodium or potassium ions, 3,4-epoxybutyronitrile rearranged to 4-hydroxybut-2-enenitrile, and this compound was also isolated for study. Male adult Long-Evans rats were exposed to allylnitrile or 3,4-epoxybutyronitrile by bilateral transtympanic injection, and vestibular toxicity was assessed using a behavioral test battery and scanning electron microscopy (SEM) observation of the sensory epithelia. Overt vestibular toxicity was caused by 3,4-epoxybutyronitrile at 0.125 mmol/ear and by allylnitrile in some animals at 0.25 mmol/ear. Additional rats were exposed by unilateral transtympanic injection. In these studies, behavioral evidences and SEM observations demonstrated unilateral vestibular toxicity after 0.125 mmol of 3,4-epoxybutyronitrile and bilateral vestibular toxicity after 0.50 mmol of allylnitrile. However, 0.25 mmol of allylnitrile did not cause vestibular toxicity. Unilateral administration of 0.50 mmol of 3,4-dihydroxybutyronitrile or 4-hydroxybut-2-enenitrile caused no vestibular toxicity. The four compounds were also evaluated in the mouse utricle explant culture model. In 8-h exposure experiments, hair cells completely disappeared after 3,4-epoxybutyronitrile at concentrations of 325 or 450μM but not at concentrations of 150μM or lower. In contrast, no difference from controls was recorded in utricles exposed to 450μM or 1.5mM of allylnitrile, 3,4-dihydroxybutyronitrile, or 4-hydroxybut-2-enenitrile. Taken together, the present data support the hypothesis that 3,4-epoxybutyronitrile is the active metabolite of allylnitrile for vestibular toxicity.

Nervous and Vestibular Toxicities of Acrylonitrile and Iminodipropionitrile

A recent article by Khan et al. (2009) in Toxicological Sciences deals with the putative mechanisms and target sites of acrylonitrile (ACN) and iminodipropionitrile (IDPN) in rats, and concludes that ‘‘the brain and vestibule appear to be major target sites of ACN and IDPN respectively.’’ We think that the article raises several points that deserve comment. The data reported by Khan et al. (2009) for ACN include transient acute behavioral effects. Surprisingly, the authors do not explain how the effects recorded in Table 1 were assessed, and fail to cite the detailed evaluation of these effects reported by Ghanayem et al. (1991) and Farooqui et al. (1995) among others. These previous studies reasonably identified the effects as being cholinomimetic, and therefore mediated, to a significant extent, by the peripheral nervous system. In the case of IDPN, Khan et al. cite our work revealing the ‘‘correlation’’ between vestibular hair cell degeneration and the behavioral effects of IDPN and allylnitrile, and similarly cite other studies suggesting that a number of brain neurotransmitter systems may be involved in the behavioral deficits. We stress that these behavioral deficits are identical to those of a bilateral labyrinthectomy (Llorens and Rodriguez-Farre, 1997; Llorens et al., 1993), and that the association of hair cell degeneration with the behavioral syndrome is found in dose-response studies in acute, repeated, and chronic dosing in rats (Balbuena and Llorens, 2001; Llorens and Rodriguez-Farre, 1997; Llorens et al., 1993; Seoane et al., 2001), in several other animal species, including mice, guinea pigs and Perezi frogs (Soler-Martin et al., 2007)—not only for IDPN and allylnitrile, but also for crotononitrile (Balbuena and Llorens, 2003; Boadas-Vaello et al., 2005, 2007; Llorens et al., 1998). Crotononitrile (CH3– CH1⁄4CH–CN) shows a great structural similarity with ACN and, whereas both the IDPN-like behavioral changes and the vestibular pathology are induced by the cis-isomer, neither effect is induced by the transisomer, which has a different set of behavioral and pathological effects (Balbuena and Llorens, 2003; Boadas-Vaello et al., 2005; Seoane et al., 2005). The available evidence thus indicates that there is no need to turn to other pathological effects to explain the major effects of vestibulotoxic nitriles on spontaneous motor behavior. Of course, this does not exclude the possibility that other effects may exist, and in fact IDPN also causes neurofilamentous axonopathy (Chou and Hartmann, 1964) which is the main effect in chronic low dose exposure (Clark et al., 1980; Llorens and Dememes, 1996; Llorens and Rodriguez-Farre, 1997), and also damages other sensory systems (Barone et al., 1995; Crofton et al., 1994; Genter et al., 1992; Selye, 1957; Seoane et al., 1999). However, any statement on nitrile effects should be based on reliable data. This is not the case of the claims by Khan et al. on tyrosine hydroxylase (TH) expression in the striatum: only one rat per group was examined, using a technique which ends with a chromogenic reaction subsequently rated by naked eye observation alone. Regarding the striatum, in previous studies we found no change in dopamine concentrations following IDPN exposure in rats (Seoane et al., 1999). Another point is the fact that Khan et al used a single rat per group (in fact the same animals used for TH staining) to examine the vestibular sensory epithelia. Again, this number invalidates the statements included in the article (and in the abstract as well), regarding differential integrity of the sensory epithelium in the different treatment groups—this shortcoming being aggravated by the substandard quality of the vestibular histology. The article by Khan et al. includes more standard data on the effects of the nitriles on reduced glutathione in the central nervous system. The results indicate a greater effect for ACN than for IDPN, but we wonder whether the IDPN data are meaningful. The doubt arises from the fact that a technical grade IDPN (90%) was used in the study and so unidentified compounds in this ‘‘IDPN’’ may have been responsible for this effect. Although the evaluation of technical grade chemicals is often of toxicological interest, they may not be a good choice if they are being used as reference compounds. 1 To whom correspondence should be addressed at Departament de Ciencies Fisiologiques II, Universitat de Barcelona, Feixa Llarga s/n, 08907 Hospitalet de Llobregat, Spain. Fax: þ34-93-402-4268. E-mail: jllorens@ub.edu.

Second International Congress on Chocolate and Cocoa in Medicine Held in Barcelona, Spain, 25-26th September 2015

Cocoa powder is a product derived from the beans of the Theobroma cacao tree, which is considered a good source of fiber (26%–40%), proteins (15%–20%), carbohydrates (about 15%) and lipids (10%–24%; generally, 10%–12%).[...]