Carla Soler-Martín

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.

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.

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

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.