Frédéric Cosnier

Impact of noise or styrene exposure on the kinetics of presbycusis

Presbycusis, or age-related hearing loss is a growing problem as the general population ages. In this longitudinal study, the influence of noise or styrene exposure on presbycusis was investigated in Brown Norway rats. Animals were exposed at 6 months of age, either to a band noise centered at 8 kHz at a Lex,8h = 85 dB (86.2 dB SPL for 6 h), or to 300 ppm of styrene for 6 h per day, five days per week, for four weeks. Cubic distortion product otoacoustic emissions (2f1-f2 DPOAEs) were used to test the capacity of the auditory receptor over the lifespan of the animals. 2f1-f2DPOAE measurements are easy to implement and efficiently track the age-related deterioration of mid- and high-frequencies. They are good indicators of temporary auditory threshold shift, especially with a level of primaries close to 60 dB SPL. Post-exposure hearing defects are best identified using moderate, rather than high, levels of primaries. Like many aging humans, aging rats lose sensitivity to high-frequencies faster than to medium-frequencies. Although the results obtained with the styrene exposure were not entirely conclusive, histopathological data showed the presbycusis process to be enhanced. Noise-exposed rats exhibit a loss of spiral ganglion cells from 12 months and a 7 dB drop in 2f1-f2DPOAEs at 24 months, indicating that even moderate-intensity noise can accelerate the presbycusis process. Even though the results obtained with the styrene exposure are less conclusive, the histopathological data show an enhancement of the presbycusis process.

Methyl Mercapturate Synthesis: An Efficient, Convenient and Simple Method

A safe and simple method for methyl S-arylmercapturate synthesis is described. Thirteen such compounds, to be used afterwards in metabolism studies, have been obtained with yields ranging from 71 to 99.6%. These compounds were obtained using a sulfa-Michael addition and synthesized by adding the corresponding thiophenols to a mixture composed of methyl 2-acetamidoacrylate (MAA), potassium carbonate and a phase transfer catalyst, Aliquat 336. MAA, the initial synthon, was itself isolated in quasi quantitative yield following a fully described synthesis.

Biomarkers of toluene exposure in rats: mercapturic acids versus traditional indicators (urinary hippuric acid and o-cresol and blood toluene)

Abstract 1. Toluene (TOL) is a neurotoxic, ototoxic and reprotoxic solvent which is metabolized via the glutathione pathway, producing benzylmercapturic, o-, m- and p-toluylmercapturic acids (MAs). These metabolites could be useful as biomarkers of TOL exposure. 2. The aims of this study were (1) to provide data on MAs excretion in rat urine following TOL exposure by inhalation, (2) to compare them to data from traditional TOL biomarkers, i.e. TOL in blood (Tol-B), and urinary hippuric acid (HA) and o-cresol (oCre) and (3) to establish a relationship between these different indicators and the airborne TOL concentration (Tol-A). 3. Sprague–Dawley rats were exposed to a range of TOL concentrations. Blood and urine were collected and analyzed to determine biomarker levels. 4. Levels of the four MAs correlate strongly with Tol-A (comparable to the correlation with Tol-B). 5. MAs are thus clearly superior to oCre and HA as potential markers of exposure to TOL.

Neurobehavioral toxicity of a repeated exposure (14 days) to the airborne polycyclic aromatic hydrocarbon fluorene in adult Wistar male rats.

Fluorene is one of the most abundant polycyclic aromatic hydrocarbons in air and may contribute to the neurobehavioral alterations induced by the environmental exposure of humans to PAHs. Since no data are available on fluorene neurotoxicity, this study was conducted in adult rats to assess the behavioral toxicity of repeated fluorene inhalation exposure. Male rats (n = 18/group) were exposed nose-only to 1.5 or 150 ppb of fluorene 6 hours/day for 14 consecutive days, whereas the control animals were exposed to non-contaminated air. At the end of the exposure, animals were tested for activity and anxiety in an open-field and in an elevated-plus maze, for short-term memory in a Y-maze, and for spatial learning in an eight-arm maze. The results showed that the locomotor activity and the learning performances of the animals were unaffected by fluorene. In parallel, the fluorene-exposed rats showed a lower level of anxiety than controls in the open-field, but not in the elevated-plus maze, which is probably due to a possible difference in the aversive feature of the two mazes. In the same animals, increasing blood and brain levels of fluorene monohydroxylated metabolites (especially the 2-OH fluorene) were detected at both concentrations (1.5 and 150 ppb), demonstrating the exposure of the animals to the pollutant and showing the ability of this compound to be metabolized and to reach the cerebral compartment. The present study highlights the possibility for a 14-day fluorene exposure to induce some specific anxiety-related behavioral disturbances, and argues in favor of the susceptibility of the adult brain when exposed to volatile fluorene.

Biopersistence and translocation to extrapulmonary organs of titanium dioxide nanoparticles after subacute inhalation exposure to aerosol in adult and elderly rats.

The increasing industrial use of nanoparticles (NPs) has raised concerns about their impact on human health. Since aging and exposure to environmental factors are linked to the risk for developing pathologies, we address the question of TiO2 NPs toxicokinetics in the context of a realistic occupational exposure. We report the biodistribution of titanium in healthy young adults (12-13-week-old) and in elderly rats (19-month-old) exposed to 10mg/m3 of a TiO2 nanostructured aerosol 6h/day, 5days/week for 4 weeks. We measured Ti content in major organs using inductively coupled plasma mass spectrometry immediately and up to 180days after the end of exposure. Large amounts of titanium were initially found in lung which were slowly cleared during the post-exposure period. From day 28, a small increase of Ti was found in the spleen and liver of exposed young adult rats. Such an increase was however never found in their blood, kidneys or brain. In the elderly group, translocation to extra-pulmonary organs was significant at day 90. Ti recovered from the spleen and liver of exposed elderly rats was higher than in exposed young adults. These data suggest that TiO2 NPs may translocate from the lung to extra-pulmonary organs where they could possibly promote systemic health effects.

Inhaled toluene can modulate the effects of anesthetics on the middle-ear acoustic reflex

Toluene (Tol) is an organic solvent widely used in the industry. It is also abused as an inhaled solvent, and can have deleterious effects on hearing. Recently, it was demonstrated that Tol has both anticholinergic and antiglutamatergic effects, and that it also inhibits voltage-dependent Ca(2+) channels. This paper describes a study of the effects of inhaled Tol on rats anesthetized with isoflurane, pentobarbital, or a mixture of ketamine/xylazine. Hearing was tested using distortion product oto-acoustic emissions (DPOAEs) associated with a contralateral noise to evaluate contraction of the middle-ear muscles. This allowed us to assess the interactions between the effects of Tol and anesthesia on the central nervous system (CNS). Although both anesthetics and Tol are known to inhibit the middle-ear acoustic reflex, our data indicated that inhaled Tol counterbalances the effects of anesthetic in a dose-dependent manner. In other terms, Tol can increase the amplitude of the middle-ear reflex in anesthetized rats, whatever the nature of the anesthetic used. This indicates that inhaling Tol (a Ca(2+)-channel-blocking drug) modifies the potency of anesthesia, and thereby the amplitude of the middle-ear reflex.

The tonotopicity of styrene-induced hearing loss depends on the associated noise spectrum

The neuropharmacological and cochleotoxic effects of styrene can exacerbate the impact of noise on the peripheral auditory receptor. The mechanisms through which co-exposure to noise and styrene impairs hearing are complex as the slowly developing cochleotoxic process can be masked in the short-term by the rapid pharmacological effect on the central nervous system. The current investigation was therefore designed to delineate the auditory frequency range sensitive to noise, to styrene, and to noise and styrene combined. In case of different frequency ranges targeted by noise and styrene, it would be possible to point out the main factor responsible for cases of deafness by looking at the location of the audiometric deficits. Male Brown-Norway rats were exposed to 600-ppm styrene, to an octave band noise centered at 8 kHz, or to both noise and styrene. The noise exposure was of two different types: impulse noise with a LEX,8h (equivalent continuous noise level averaged over 8 h) of 80 dB and continuous noise with a LEX,8 h of 85 dB SPL. Hearing was tested using a non-invasive technique based on distortion product otoacoustic emissions. Hearing data were completed with histological analysis of cochleae. The results showed that exposure to styrene alone caused outer hair cell losses in the apical cochlear region, which discriminates low frequencies. In contrast, noise-induced hearing loss was located at half an octave above the central frequency of the spectrum, around 10-12 kHz. Damage due to impulse noise was significantly exacerbated by styrene, and the noise spectrum defined the location of the cochlear trauma. Combined exposure caused greater cell losses than the sum of losses measured with the impulse noise and styrene alone. The fact that the tonotopicity of the styrene-induced damage depends on the associated noise spectrum complicates the diagnosis of styrene-related hearing loss with a tone-frequency audiometric approach. In conclusion, there is not really a frequency specificity of impairments due to styrene.

Measurement of ketamine and xylazine in rat brain by liquid-liquid extraction and gas chromatography-mass spectrometry.

INTRODUCTION In human and veterinary medicine, the injectable drugs ketamine and xylazine are mainly used in combination to induce, and then maintain general anaesthesia; they also provide pain and stress relief. Some side-effects have been reported on the auditory brainstem response, a method is therefore required to determine their concentrations in the brain. METHODS This paper presents a method to determine nanogramme quantities of ketamine and xylazine in rat brain using liquid-liquid extraction and gas chromatography-mass spectrometry in selective ion monitoring mode. The technique requires only 0.5 g of sample, and uses xylazine d6 as an internal standard. RESULTS The method was linear between 0.86 and 34.4 μg/g of brain. Limits of quantification were 378 and 87 ng (approximately 0.76 and 0.17 μg/g of brain) for ketamine and xylazine, respectively. The reliability of the method in terms of accuracy, within-day and between-day precision was also demonstrated. For xylazine, bias and intra-day precision were good (<3.0%), as was between-day precision (<10.5%); the equivalent values for ketamine were 7%, 11.1% and 20.9%, respectively. Stability of the analytes in the matrix at -80 °C was assessed over five months; both compounds were found to be stable for at least 1 month, even at very low concentrations. The procedure was successfully applied to determine (for the first time) the in vivo brain levels of both drugs in animals following systemic administration. DISCUSSION The procedure will be useful in future studies of the side-effects of these drugs, and their interactions with other compounds.

Glutathione pathway in ethylbenzene metabolism: novel biomarkers of exposure in the rat.

Glutathione pathway was specifically studied in rats exposed by inhalation to a range of ethylbenzene vapours (5-2000 ppm). Urines were collected during exposure (6h) and over the 18 h following the exposure. The potential metabolites coming from either side-chain or ring oxidation were synthesized: 1-, 2-phenylethylmercapturic acids (1-, and 2-PEMA) and 2-, 3- and 4-ethylphenylmercapturic acids (2-, 3-, and 4-EPMA). Their synthesis was fully described and the molecules characterized. Urine samples were analysed using a selective HPLC-fluorescence method. Among the five metabolites, 2-PEMA was never observed in any urine sample. By contrast, 1-PEMA was discovered in its two diastereomeric forms, and it was shown that one of them was mainly present. 2-EPMA, 3-EPMA and 4-EPMA (in the ratio 1:2:6) were also found, and their combined excretion levels were similar to that of 1-PEMA. The atmospheric concentrations and urinary excretions yielded very close correlations which allow us to consider these mercapturic acids as novel ethylbenzene exposure biomarkers.

Metabolism of inhaled methylethylketone in rats

Abstract Methylethylketone (MEK) is widely used in industry, often in combination with other compounds. Although nontoxic, it can make other chemicals harmful. This study investigates the fate of MEK in rat blood, brain and urine as well as its hepatic metabolism following inhalation over 1 month (at 20, 200 or 1400 ppm). MEK did not significantly accumulate in the organism: blood concentrations were similar after six-hour or 1-month inhalation periods, and brain concentrations only increased slightly after 1 month’s exposure. Urinary excretion, based on the major metabolites, 2,3-butanediols (± and meso forms), accounted for less than 2.4% of the amount inhaled. 2-Butanol, 3-hydroxy-2-butanone and MEK itself were only detectable in urine in the highest concentration conditions investigated, when metabolic saturation occurred. Although MEK exposure did not alter the total cytochrome P450 concentration, it induced activation of both CYP1A2 and CYP2E1 enzymes. In addition, the liver glutathione concentration (reduced and oxidized forms) decreased, as did glutathione S-transferase (GST) activity (at exposure levels over 200 ppm). These metabolic data could be useful for pharmacokinetic model development and/or verification and suggest the ability of MEK to influence the metabolism (and potentiate the toxicity) of other substances.