Role of CYP2E1-mediated metabolism in the acute and vestibular toxicities of nineteen nitriles in the mouse

Abstract

Allylnitrile, cis-crotononitrile, and 3,3′-iminodipropionitrile are known to cause vestibular toxicity in rodents, and evidence is available indicating that cis-2-pentenenitrile shares this effect. We evaluated nineteen nitriles for vestibular toxicity in wild type (129S1) and CYP2E1-null mice, including all the above, several neurotoxic nitriles, and structurally similar nitriles. A new acute toxicity test protocol was developed to facilitate evaluation of the vestibular toxicity by a specific behavioral test battery at doses up to sub-lethal levels while using a limited number of animals. A mean number of 8.5 ± 0.3 animals per nitrile, strain and sex was necessary to obtain evidence of vestibular toxicity and optionally an estimation of the lethal dose. For several but not all nitriles, lethal doses significantly increased in CYP2E1-null mice. The protocol revealed the vestibular toxicity of five nitriles, including previously identified ototoxic compounds and one nitrile (trans-crotononitrile) known to have a different profile of neurotoxic effects in the rat. In all five cases, both sexes were affected and no decrease in susceptibility was apparent in CYP2E1-null mice respect to 129S1 mice. Fourteen nitriles caused no vestibular toxicity, including six nitriles tested in CYP2E1-null mice at doses significantly larger than the maximal doses that can be tested in wild type animals. We conclude that only a subset of low molecular weight nitriles is toxic to the vestibular system, that species-dependent differences exist in this vestibular toxicity, and that CYP2E1-mediated metabolism is not involved in this effect of nitriles although it has a role in the acute lethality of some of these compounds.

Introduction

Nitriles are compounds containing cyano (R-CN) groups; they are common in crop plants and their use in the chemical and pharmaceutical industries is becoming increasingly frequent. Nitriles with large production volumes are used as solvents and chemical intermediates in a variety of processes, including the synthesis of plastics, nylons, and elastomers (DeVito, 2007). Among their major toxic effects are acute lethality, osteolathyrism and neurotoxicity, including sensory toxicity (DeVito, 1996, Llorens et al., 2011). One significant target for several nitriles is the inner ear: degeneration of the vestibular and/or auditory hair cells has been reported in rodents exposed to 3,3′-iminodipropionitrile (IDPN) (Crofton and Knight, 1991, Llorens et al., 1993, Crofton et al., 1994, Llorens and Rodríguez-Farré, 1997, Seoane et al., 2001), allylnitrile (Balbuena and Llorens, 2001, Gagnaire et al., 2001), cis-crotononitrile (Balbuena and Llorens, 2003), racemic crotononitrile (Llorens et al., 1998, Gagnaire et al., 2001) and cis-2-pentenenitrile (Gagnaire et al., 2001).

Cyanide release by nitrile metabolism was identified as a major cause of the acute lethality effect that characterizes many nitriles (Ohkawa et al., 1972, Willhite and Smith, 1981, Tanii and Hashimoto, 1984), and a major role was hypothesized for the alcohol/acetone-inducible isoform of the P450 cytochrome (CYP2E1) in this metabolism (Lewis et al., 1994). Ensuing studies demonstrated that the CYP2E1 enzyme metabolizes a number of low molecular weight alkyl nitriles (Ghanayem et al., 1999) and is responsible for the initial metabolism of acrylonitrile leading to cyanide release (Wang et al., 2002, Chanas et al., 2003, El Hadri et al., 2005). However, the role of this or other metabolic routes in sensory effects remains poorly understood. The data reported by Genter et al. (1994) suggested that CYP2E1-mediated metabolism is involved in the olfactory toxicity of IDPN. Recent work from our laboratory has demonstrated that this P450 isoform is associated with cyanide release and acute lethality in mice exposed to cis-crotononitrile and allylnitrile but, unlike for other CYPs, not with the vestibular toxicity of these nitriles (Boadas-Vaello et al., 2007, Boadas-Vaello et al., 2009). Therefore, the lethal and vestibulotoxic effects of some nitriles appear to result from competing pathways of xenobiotic metabolism. Thus, we hypothesized that other nitriles might generate vestibulotoxic metabolites but have not been revealed to be vestibulotoxic due to the lethality associated with their simultaneous metabolism to cyanide. Identification of such nitriles may be facilitated by the absence of CYP2E1 and will help future modeling of structure–activity relationships.

In the present study we evaluated nineteen nitriles for vestibular toxicity in wild-type and CYP2E1 knock-out mice (Lee et al., 1996). Because sex differences have been found in nitrile metabolism and toxicity in mice (Chanas et al., 2003, Boadas-Vaello et al., 2007), males and females were studied separately. The study used a new acute toxicity test protocol designed to facilitate the identification of the vestibular toxicity at sub-lethal doses by means of a systematic approach, while using a low number of animals per compound, sex, and strain, and simplifying the management of the animal colonies.