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Nanoparticles are extensively used in industrial products or as food additives. However, despite their contribution to improving our quality of life, concerns have been raised regarding their potential impact on occupational and public health. To speed up research assessing nanoparticle-related hazards, this study was undertaken to identify early markers of harmful effects on the lungs. Female Sprague Dawley rats were either exposed to crystalline silica DQ-12 with instillation, or to titanium dioxide P25, carbon black Printex-90, or multi-walled carbon nanotube Mitsui-7 with nose-only inhalation. Tissues were collected at three post-exposure time points to assess short- and long-term effects. All particles induced lung inflammation. Histopathological and biochemical analyses revealed phospholipid accumulation, lipoproteinosis, and interstitial thickening with collagen deposition after exposure to DQ-12. Exposure to the highest dose of Printex-90 and Mitsui-7, but not P25, induced some phospholipid accumulation. Comparable histopathological changes were observed following exposure to P25, Printex-90, and Mitsui-7. Comparison of overall gene expression profiles identified 15 potential early markers of adverse lung outcomes induced by spherical particles. With Mitsui-7, a distinct gene expression signature was observed, suggesting that carbon nanotubes trigger different toxicity mechanisms to spherical particles.

International audience; Volatile organic solvents are frequently present in industrial atmospheres. Their lipophilic properties mean they quickly reach the brain following inhalation. Acute exposure to some solvents perturbs the middle ear reflex, which could jeopardize cochlear protection against loud noises. As the physiological mechanisms involved in this protective reflex are highly complex, in vivo rodent models are required to allow rapid and reliable identification of any adverse effects of solvents on the middle ear reflex (MER).In this study, MER amplitude was measured in anesthetized Brown-Norway rats by monitoring the decrease in distortion product otoacoustic emissions (DPOAEs) caused by a contralateral stimulation. Our screening test consisted in measuring the impact of inhalation of solvent vapors at 3000 ppm for 15 minutes on the MER amplitude. We had previously studied a selection of aromatic solvents with this model; here, we extended the analysis to volatile compounds from other chemical families. The results obtained shed light on the mechanisms involved in the interactions between solvents and their neuronal targets. Thus, benzene and chlorobenzene had the greatest effect on MER (≥ +1.8 dB), followed by a group composed of toluene, styrene, p-xylene, m-xylene, tetrachloroethylene and cyclohexane, which had a moderate effect on the MER (between +0.3 and +0.7 dB). Finally, trichloroethylene, nhexane, methyl-ethyl-ketone, acetone, o-xylene, and ethylbenzene had no effect on the MER.Thus, the effect of solvents on the MER is not simply linked to their lipophilicity, rather it depends on specific interactions with neuronal targets. These interactions appear to be governed by the compound's chemical structure, e.g. the presence of an aromatic ring and its steric hindrance. In addition, perturbation of the MER by a solvent is independent of its toxic effects on cochlear cells. As the MER plays a protective role against exposure to high-intensity noises, these findings could have a significant impact in terms of prevention for subjects exposed to both noise and solvents.

Despite their numerous possible applications, the potential impact of carbon engineered nanomaterials (CEN) on human health, especially after inhalation exposure, is still questioned. Quantification of CEN in the respiratory system is a recurring issue and deposition and pulmonary biopersistence data are essential for toxicological evaluation. In this context, a fully validated standard method for CEN quantification in lung tissue is therefore imperative. The present method, based on the National Institute for Occupational Safety and Health 5040 method for atmospheric elemental and organic carbon analysis as well as on previous developments on biological matrices, involves a simple thermogravimetric analysis (TGA) of lyophilized samples, possibly preceded by a step of chemical digestion of the tissues depending on the nature of CEN investigated. The analytical method was validated for 4 CEN (carbon black as well as 3 long and thick or short and thin carbon nanotubes) for selectivity, linearity, detection and quantification limits, bias, and within-batch and between-batch precision. Calibration curves show linearity in the range of 1-40 mg/g lyophilized lung. Limits of detection for the different CEN range from 6 to 18 µg in 20 mg dry test sample. On average, within-batch precision was kept below 20 and 10% for analysis with or without a prior digestion step, respectively, whereas the corresponding between-batch precision levels reached almost 20 and 15%, respectively. The method was successfully applied to toxicological investigations for the quantitative analysis of CEN contents in rat lung exposed by inhalation.

Background Carbon disulfide (CS2) exacerbates the effect of noise on hearing, and disrupts the vestibular system. The goal of this study was to determine whether these effects are also observed with intermittent CS2 exposure. Methods Rats were exposed for 4 weeks (5 days/week, 6 h/day) to a band noise at 106 dB SPL either alone or combined with continuous (63 ppm or 250 ppm) or intermittent (15 min/h or 2 × 15 min/h at 250 ppm) CS2. Hearing function was assessed by measuring distortion product otoacoustic emissions (DPOAEs); balance was monitored based on the vestibulo-ocular reflex (VOR). Functional measurements were performed before, at the end of exposure and 4 weeks later. Histological analyses of the inner ear were also performed following exposure and after the 4-week recovery period. Results The results obtained here confirmed that CS2 exposure exerts two differential temporary effects on hearing: (1) it attenuates the noise-induced DPOAE decrease below 6 kHz probably through action on the middle ear reflex when exposure lasts 15 min per hour, and (2) continuous exposure to 250 ppm for 6 h extends the frequency range affected by noise up to 9.6 kHz (instead of 6 kHz with noise alone). With regard to balance, the VOR was reversibly disrupted at the two highest doses of CS2 (2 × 15 min/h and continuous 250 ppm). No morphological alterations to the inner ear were observed. Conclusion These results reveal that short periods of CS2 exposure can alter the sensitivity of the cochlea to noise at a dose equivalent to only 10 times the short-term occupational limit value, and intermittent exposure to CS2 (2 × 15 min/h) can alter the function of the vestibular system.

Occupational noise can damage workers’ hearing, and the phenomenon is even more dangerous when noise is associated with an ototoxic solvent. Aromatic solvents are known to provoke chemical-induced hearing loss, but little is known about the effects on hearing of carbon disulfide (CS 2 ) when combined with noise. Co-exposure to CS 2 and noise may have a harmful effect on hearing, but the mechanisms involved are not well understood. For instance, CS 2 is not thought to have a cochleotoxic effect, but rather it is thought to cause retrocochlear hearing impairment. In other words, CS 2 could have a distal neuropathic effect on the auditory pathway. However, a possible pharmacological effect of CS 2 on the central nervous system (CNS) has never been mentioned in the literature. The aim of this study was to assess, in rats, the effects of a noise (continuous vs. impulse), associated with a low concentration of CS 2 [(short-term threshold limit value) x 10 as a safety factor] on the peripheral auditory receptor. The noise, whatever its nature, was an octave band noise centered at 8 kHz, and the 250-ppm CS 2 exposure lasted 15 min per hour, 6 h per day, for 5 consecutive days. The impact of the different experimental conditions on hearing loss was assessed using distortion product oto-acoustic emissions and histological analyses. Although the LEX,8 h (8-h time-weighted average exposure) for the impulse noise was lower (84 dB SPL) than that for the continuous noise (89 dB SPL), it appeared more damaging to the organ of Corti, in particular to the outer hair cells. CS 2 exposure alone did not have any effect on the organ of Corti, but co-exposure to continuous noise with CS 2 was less damaging than exposure to continuous noise alone. In contrast, the cochleo-traumatic effects of impulse noise were significantly enhanced by co-exposure to CS 2 . Therefore, CS 2 can clearly modulate the middle-ear reflex function. In fact, CS 2 may have two distinct effects: firstly, it has a pharmacological effect on the CNS, modifying the trigger of the acoustic reflex; and secondly, it can make the organ of Corti more susceptible to impulse noise. The pharmacological effects on the CNS and the effects of CS 2 on the organ of Corti are discussed to try to explain the overall effect of the solvent on hearing. Once again, the results reported in this article show that the temporal structure (continuous vs. impulse) of noise should be taken into consideration as a key parameter when establishing hearing conservation regulations.

Hexavalent chromium (Cr(VI)) compounds are classified as carcinogenic to humans. Whereas chromium measurements in urine and plasma attest to the last few hours of total chromium exposure (all oxidation states of chromium), chromium in red blood cells (RBC) is attributable specifically to Cr(VI) exposure over the last few days. Before recommending Cr in RBC (CrIE) as a biological indicator of Cr(VI) exposure, in vivo studies must be undertaken to assess its reliability. The present study examines the kinetics of Cr(VI) in rat after a single intravenous dose of ammonium dichromate. Chromium levels were measured in plasma, red blood cells and urine. The decay of the chromium concentration in plasma is one-phase-like (with half-life time of 0.55 day) but still measurable two days post injection. The excretion of urinary chromium peaks between five and six hours after injection and shows large variations. Intra-erythrocyte chromium (CrIE) was very constant up to a minimum of 2 days and half-life time was estimated to 13.3 days. Finally, Cr(III) does not interfere with Cr(VI) incorporation in RBC. On the basis of our results, we conclude that, unlike urinary chromium, chromium levels in RBC are indicative of the amount of dichromate (Cr(VI)) in blood.

Some volatile aromatic solvents have similar or opposite effects to anesthetics in the central nervous system. Like for anesthetics, the mechanisms of action involved are currently the subject of debate. This paper presents an in vivo study to determine whether direct binding or effects on membrane fluidity best explain how solvents counterbalance anesthesia's depression of the middle-ear reflex (MER). Rats were anesthetized with a mixture of ketamine and xylazine while also exposed to solvent vapors (toluene, ethylbenzene, or one of the three xylene isomers) and the amplitude of their MER was monitored. The depth of anesthesia was standardized based on the magnitude of the contraction of the muscles involved in the MER, determined by measuring cubic distortion product oto-acoustic emissions (DPOAEs) while triggering the bilateral reflex with contralateral acoustic stimulation. The effects of the aromatic solvents were quantified based on variations in the amplitude of the DPOAEs. The amplitude of the alteration to the MER measured in anesthetized rats did not correlate with solvent lipophilocity (as indicated by logKow values). Results obtained with the three xylene isomers indicated that the positions of two methyl groups around the benzene ring played a determinant role in solvent/neuronal cell interaction. Additionally, Solid-state Nuclear Magnetic Resonance (NMR) spectra for brain microsomes confirmed that brain lipid fluidity was unaffected by solvent exposure, even after three days (6h/day) at an extremely high concentration (3000ppm). Therefore, aromatic solvents appear to act directly on the neuroreceptors involved in the acoustic reflex circuit, rather than on membrane fluidity. The affinity of this interaction is determined by stereospecific parameters rather than lipophilocity.

The number of workers potentially exposed to nanoparticles (NPs) in industrial processes is constantly increasing, even though the toxicological effects of these compounds have not yet been fully characterized. The hazards associated with this exposure can be assessed most relevantly by toxicology studies involving inhalation of nanoaerosols by animals. In this paper, we describe and characterize an aerosol generated in a nose-only exposure system used to study the respiratory effects of NPs in rat; this system was designed to meet the most stringent requirements for animal testing in terms of protection of operators against risks associated with NPs and biohazards and to comply with the OECD guidelines for chemical testing. The inhalation facility was fully validated by exposing Fisher 344 rats to TiO2 P25 aerosols at 10 mg m–3. Aerosol monitoring and in-depth characterization were ensured by real-time devices (condensation particle counter, optical particle sizer, scanning mobility particle sizer, aerodynamic particle sizer and electrical low pressure impactor) and samples taken for off-line analyses (gravimetric analysis, mass size distribution from cascade impactor, TEM observations). The test atmosphere was stable in terms of concentrations and distributions (mass or number) between different inhalation towers on a given day and between days (intra-experiment), as well as between inhalation campaigns (between experiments). In terms of the respiratory deposition profile, preliminary results after exposure for one month indicate that this system is relevant, and should therefore be appropriate for in vivo inhalation toxicity studies.

International audience; The number of workers potentially exposed to nanoparticles (NPs) during industrial processes is increasing, although the toxicological properties of these compounds still need to be fully characterized. As NPs may be aerosolized during industrial processes, inhalation represents their main route of occupational exposure. Here, the short- and long-term pulmonary toxicological properties of titanium dioxide were studied, using conventional and molecular toxicological approaches. Fischer 344 rats were exposed to 10 mg/m3 of a TiO2 nanostructured aerosol (NSA) by nose-only inhalation for 6 h/day, 5 days/week for 4 weeks. Lung samples were collected up to 180 post-exposure days. Biochemical and cytological analyses of bronchoalveolar lavage (BAL) showed a strong inflammatory response up to 3 post-exposure days, which decreased overtime. In addition, gene expression profiling revealed overexpression of genes involved in inflammation that was maintained 6 months after the end of exposure (long-term response). Genes involved in oxidative stress and vascular changes were also up-regulated. Long-term response was characterized by persistent altered expression of a number of genes up to 180 post-exposure days, despite the absence of significant histopathological changes. The physiopathological consequences of these changes are not fully understood, but they should raise concerns about the long-term pulmonary effects of inhaled biopersistent NPs such as TiO2.

The number of workers potentially exposed to nanoparticles (NPs) during industrial processes is increasing, although the toxicological properties of these compounds still need to be fully characterized. As NPs may be aerosolized during industrial processes, inhalation represents their main route of occupational exposure. Here, the short- and long-term pulmonary toxicological properties of titanium dioxide were studied, using conventional and molecular toxicological approaches. Fischer 344 rats were exposed to 10 mg/m

Chronic occupational exposure to carbon disulfide (CS2) has debilitating motor and sensory effects in humans, which can increase the risk of falls. Although no mention of vestibulotoxic effects is contained in the literature, epidemiological and experimental data suggest that CS2 could cause low-frequency hearing loss when associated with noise exposure. Low-frequency noise might also perturb the peripheral balance receptor through an as-yet unclear mechanism. Here, we studied how exposure to a low-frequency noise combined with 250-ppm CS2 affected balance in rats. Vestibular function was tested based on post-rotary nystagmus recorded by a video-oculography system. These measurements were completed by behavioral tests and analysis of the cerebellum to measure expression levels for gene expression associated with neurotoxicity. Assays were performed prior to and following a 4-week exposure, and again after a 4-week recovery period. Functional measurements were completed by histological analyses of the peripheral organs.Nystagmus was unaltered by exposure to noise alone, while CS2 alone caused a moderate 19% decrease of the saccade number. In contrast, coexposure to 250-ppm CS2 and low-frequency noise decreased both saccade number and duration by 33% and 34%, respectively. After four weeks, recovery was only partial but measures were not significantly different from pre-exposure values. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of cerebellar tissue revealed a slight but significant modification in expression levels for two genes linked to neurotoxicity in CS2-exposed animals. However, neither histopathological changes to the peripheral receptor nor behavioral differences were observed. Based on all these results, we propose that the effects of CS2 were due to reversible neurochemical disturbance of the efferent pathways managing post-rotatory nystagmus. Because the nervous structures involving the vestibular function appear particularly sensitive to CS2, post-rotary nystagmus could be used as an early, non-invasive measurement to diagnose CS2 intoxication as part of an occupational conservation program.

International audience; 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.; La méthyléthylcétone (MEK) est largement utilisée dans l'industrie, souvent en combinaison avec d'autres composés. Bien que non toxique, elle peut rendre d'autres produits chimiques nocifs. Cette étude s'est intéressée au devenir de la MEK dans le sang, le cerveau et l'urine ainsi qu'à son métabolisme hépatique chez le rat exposé par inhalation pendant 1 mois à 20, 200 ou 1400 ppm. La MEK ne s'est pas accumulée de manière significative dans l'organisme: les concentrations sanguines mesurées après 6 heures ou 1 mois d'exposition par inhalation étaient similaires, tandis que les concentrations cérébrales n'ont que légèrement augmenté après 1 mois d'exposition. L'excrétion urinaire, par l'intermédiaire du 2,3-butanediol (métabolite principal présent sous les formes ± et méso), représentait moins de 1,3% de la quantité inhalée. Les autres métabolites, le 2-butanol, la 3-hydroxy-2-butanone ainsi que la MEK (sous sa forme inchangée) n'étaient détectable dans l'urine que pour des concentrations atmosphériques élevées au delà desquelles un phénomène de saturation métabolique se met en place. Bien que l'exposition à la MEK n'ait pas modifié la concentration totale en cytochromes P450, les activités enzymatiques des deux isoformes CYP1A2 et CYP2E1 ont été induites. En outre, la concentration en glutathion dans le foie (formes réduite et oxydée) a diminué, tout comme l'activité des glutathion-S-transférases (GST). Ces données métaboliques fournissent une preuve supplémentaire sur la capacité de la MEK à influencer le métabolisme (et potentialiser la toxicité) d'autres substances.

Carbon disulfide (CS2) is used in industry; it has been shown to have neurotoxic effects, causing central and distal axonopathies.However, it is not considered cochleotoxic as it does not affect hair cells in the organ of Corti, and the only auditory effects reported in the literature were confined to the low-frequency region. No reports on the effects of combined exposure to low-frequency noise and CS2 have been published to date. This article focuses on the effects on rat hearing of combined exposure to noise with increasing concentrations of CS2 (0, 63,250, and 500ppm, 6h per day, 5 days per week, for 4 weeks). The noise used was a low-frequency noise ranging from 0.5 to 2kHz at an intensity of 106dB SPL. Auditory function was tested using distortion product oto-acoustic emissions, which mainly reflects the cochlear performances. Exposure to noise alone caused an auditory deficit in a frequency area ranging from 3.6 to 6 kHz. The damaged area was approximately one octave (6kHz) above the highest frequency of the exposure noise (2.8kHz); it was a little wider than expected based on the noise spectrum.Consequently, since maximum hearing sensitivity is located around 8kHz in rats, low-frequency noise exposure can affect the cochlear regions detecting mid-range frequencies. Co-exposure to CS2 (250-ppm and over) and noise increased the extent of the damaged frequency window since a significant auditory deficit was measured at 9.6kHz in these conditions.Moreover, the significance at 9.6kHz increased with the solvent concentrations. Histological data showed that neither hair cells nor ganglion cells were damaged by CS2. This discrepancy between functional and histological data is discussed. Like most aromatic solvents, carbon disulfide should be considered as a key parameter in hearing conservation regulations.

1. Multiple exposures are ubiquitous in industrial environments. In this article, we highlight the risks faced by workers and complete the data available on the metabolic impact of a common mixture: toluene (TOL) and methylethylketone (MEK). 2. Rats were exposed by inhalation under controlled conditions either to each solvent individually, or to mixtures of the two. How the interaction between the two solvents affected their fate in the blood and brain, their main relevant urinary metabolites (o-cresol, benzylmercapturic acid for TOL and 2,3-butanediols for MEK) and their hepatic metabolism were investigated. 3. Although the cytochrome P450 concentration was unchanged, and the activities of CYP1A2 and CYP2E1 isoforms were not additively or synergistically induced by co-exposure, TOL metabolism was inhibited by the presence of MEK (and vice versa). Depending on the relative proportions of each compound in the mixture, this sometimes resulted in a large increase in blood and brain concentrations. Apart from extreme cases (unbalanced mixtures), the amount of o-cresol and benzylmercapturic acid (and to a lesser extent 2,3-butanediols) excreted were proportional to the blood solvent concentrations. 4. In a co-exposure context, ortho-cresol and benzylmercapturic acid can be used as urinary biomarkers in biomonitoring for employees to relatively accurately assess TOL exposure.

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 cytogenetic alterations in leukocytes and the increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer observed in workers occupationally exposed to styrene have been associated with its hepatic metabolisation into styrene-7,8-oxide, an epoxide which can induce DNA damages. However, it has been observed that styrene-7,8-oxide was also found in the atmosphere of reinforced plastic industries where large amounts of styrene are used. Since the main route of exposure to these compounds is inhalation, in order to gain new insights regarding their systemic genotoxicity, Fisher 344 male rats were exposed in full-body inhalation chambers, 6 h/day, 5 days/week for 4 weeks to styrene-7,8-oxide (25, 50, and 75 ppm) or styrene (75, 300, and 1000 ppm). Then, the induction of micronuclei in circulating reticulocytes and DNA strand breaks in leukocytes using the comet assay was studied at the end of the 3rd and 20th days of exposure. Our results showed that neither styrene nor styrene-7,8-oxide induced a significant increase of the micronucleus frequency in reticulocytes or DNA strand breaks in white blood cells. However, in the presence of the formamidopyridine DNA glycosylase, an enzyme able to recognize and excise DNA at the level of some oxidized DNA bases, a significant increase of DNA damages was observed at the end of the 3rd day of treatment in leukocytes from rats exposed to styrene but not to styrene-7,8-oxide. This experimental design helped to gather new information regarding the systemic genotoxicity of these two chemicals and may be valuable for the risk assessment associated with an occupational exposure to these molecules.

A convenient and reliable gas chromatographic method was developed for the simultaneous determination of six aromatic acid metabolites of styrene and styrene-oxide in rat urine; i.e., benzoic (BA), phenylacetic (PAA), mandelic (MA), phenylglyoxylic (PGA), hippuric (HA) and phenylaceturic (PAUA) acids. The method involves a one-pot esterification-extraction procedure, performed directly on urine without prior treatment. Analyses were performed on a RTX-1701 capillary column and the recovered isopropyl esters derivatives were detected by flame ionization detection. The analytical method was validated for selectivity, linearity, detection and quantification limits, recovery and intra-day and inter-day precisions. Calibration curves showed linearity in the range of 8-800 mg/L, except for HA and PAUA (40-800 mg/L). Limits of detection were between 0.2 (PPA) and 7.0 (PAUA) mg/L. The intra-day precisions determined at three concentrations levels were less than 5% for BA, PAA, MA and PGA and 9% for HA and PAUA, respectively. The corresponding mean inter-day precisions for these two groups were 8 and 16%, respectively. The method was successfully applied to quantitatively analyze styrene, styrene-oxide, ethylbenzene and toluene metabolites in urine samples from rats exposed by inhalation to these compounds at levels close to the occupational threshold limit values. Provided that this method can be transposed to human urine, it could have applications as part of biological monitoring for workers exposed to styrene or related compounds.

Glutathione pathway was specifically studied in rats exposed by inhalation to a range of ethylbenzene vapours (5–2000 ppm). Urines were collected during exposure (6 h) 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.

Exposure to bitumen fumes during paving and roofing activities may represent an occupational health risk. To date, most of the studies performed on the biological effect of asphalt fumes have been done with regard to their content in carcinogenic polycyclic aromatic hydrocarbons (PAH). In order to gain an additional insight into the mechanisms of action of bitumen fumes, we studied their pulmonary effects in rodents following inhalation using the microarray technology. Fisher 344 rats were exposed for 5 days, 6 h/day to bitumen fumes generated at road paving temperature (170 degrees C) using a nose-only exposition device. With the intention of studying the early transcriptional events induced by asphalt fumes, lung tissues were collected immediately following exposure and gene expression profiles in control and exposed rats were determined by using oligonucleotide microarrays. Data analysis revealed that genes involved in lung inflammatory response as well as genes associated with PAH metabolization and detoxification were highly expressed in bitumen-exposed animals. In addition, the expression of genes related to elastase activity and its inhibition which are associated with emphysema was also modulated. More interestingly genes coding for monoamine oxidases A and B involved in the metabolism of neurotransmitters and xenobiotics were downregulated in exposed rats. Altogether, these data give additional information concerning the bitumen fumes biological effects and would allow to better review the health effects of occupational asphalt fumes exposure.

Road paving workers are exposed to bitumen fumes (CAS No. 8052-42-4), a complex mixture of volatile compounds and particles containing carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons. However, epidemiological and experimental animal studies failed to draw unambiguous conclusions concerning their toxicity. In order to gain better insights on their genotoxic potential, we used an experimental design able to generate bitumen fumes at road paving temperature (temperature: 170 °C, total particulate matter: 100 mg/m 3 ) and perform a nose-only exposure of Big Blue ® transgenic rodents 6 h/day for five consecutive days. The mutagenic properties of bitumen fumes were determined by analyzing the mutation frequency and spectrum of the neutral reporter gene cII inserted into the rodent genome. We previously observed in mouse lung, that bitumen fumes did not induce an increase of cII mutants, a modification of the mutation spectrum, nor the formation of DNA adducts. Since DNA adducts were found in the lungs of rats exposed to asphalt fumes in similar conditions, we decided to carry out an analogous experiment with Big Blue ® rats. A DNA adduct was detected 3 and 30 days after the end of treatment suggesting that these genetic alterations were quite steady. Thirty days after exposure, the cII mutant frequency was similar in control and exposed rats. In addition, a slight but not significant modification of the mutation spectrum associated with an increase of G:C to T:A and A:T to C:G transversions was noticeable in the treated animals. Then, these data failed to demonstrate a pulmonary mutagenic potential for bitumen fumes generated at road paving temperature in our experimental conditions despite the presence of a DNA adduct. These results may provide information concerning the pulmonary mechanism of action of this aerosol and may contribute to the occupational health hazard assessment.

During the hot application of bitumen containing materials, e.g., in hot paving or roofing, fumes are emitted that contain traces of polycyclic aromatic compounds (PACs) including heterocyclic and/or substituted PACs. Previous studies of DNA adduct formation by bitumen and coal-tar fume condensates (BFCs and CTFCs, respectively) indicated that the genotoxic compounds responsible for DNA adduct formation in BFCs and CTFCs were of different nature. Moreover, it was suggested that the major adduct found in the lungs and also in skin and lymphocytes of BFC-treated rats might be usable as a marker of exposure to bitumen fumes.

During the hot application of bitumen-containing materials, e.g. in road paving or roofing, fumes are emitted that contain traces of polycyclic aromatic compounds (PACs). Although worker's exposure to these fumes is low, it might lead to health problems. For studying DNA adduct formation as a consequence of inhalation of bitumen fumes we developed and validated an inhalation system (a dynamic fume generator plus a nose only inhalation chamber). This paper presents and discusses the analytical results from the different laboratories involved in this study on the fumes sampled in the inhalation chamber during three series of experiments where the animals were exposed to fumes at the 5 mg/m 3 and 50 mg/m 3 level, coming from bitumen heated at 200° C and, as a positive control, fumes from coal tar, heated to 110°C at the 5 mg/m 3 level. The following parameters were controlled: temperatures at different key places in the generator; humidity of the chamber; the bitumen or coal tar flow rate; and Total Particulate Matter (TPM). Analyses were performed for Benzene Soluble Matter (BSM), the EPA polycyclic aromatic hydrocarbon (PAH) mixture and for a number of heteroatom-containing PACs. The data show that the coal tar fumes produced at 110 C were very volatile and that most of the differences in particulate matter found between the laboratories can be attributed to evaporative losses. The bitumen fumes boil 25-50 C higher and contain higher boiling compounds. A comparison is made between the PAC exposure profiles for bitumen experiments aimed at 5 and 50 mg/m 3 . Although the same molecules are found in both fumes their proportion is dramatically different. This effect is largest with the 2- and 3-ring PACs, the ratio of the concentrations found in the 50 mg/m 3 TPM concentration to that in the 5 mg/m 3 experiment gradually declines from 5500 for acenaphthene to 500 for pyrene, for the 5-ring PACs this ratio is 20-30. As function of their vapour pressure, the ratios of the concentrations of the hetero PACs follow the same trend as that of the 16 EPA PAHs and are of the same order of magnitude. In conclusion, for the compounds investigated, the equipment delivers a fume atmosphere in a reproducible manner. The 50 mg/m 3 bitumen fumes are not representatives of field fumes. The reason for these quantitative differences is unclear and further work would be needed to clarify this. Nevertheless it was felt that these fumes at 50 mg/m 3 might be a useful tool for qualitative detection of DNA adducts in an animal exposure study. © 2000 British Occupational Hygiene Society. Published by Elsevier Science Ltd. All rights reserved.

Occupational and environmental exposures to bitumen fumes during road paving or roofing activities represent a safety issue since this complex mixture of volatile compounds and particles contains carcinogenic polycyclic aromatic hydrocarbons (PAH). However, epidemiological and experimental animal studies failed to draw unambiguous conclusions concerning bitumen fumes toxicity. In order to gain better insights on their genotoxic potential, we used an experimental device able to generate bitumen fumes at 170°C (upper range used during road paving operations) with a total particulate matter of 112 ± 13 mg/m3 and a mass median aerodynamic diameter of 4.6 μ m. The nose-only exposure of Big Blue® transgenic rats was performed 6 h/day for 5 consecutive days. Biological exposure to this aerosol was monitored by measuring the rat urinary levels of 1-hydroxypyrene and lungs were collected at various time-points after the end of treatment for further analysis. As determined by the 32P post-labeling method, no DNA bu...

1. Toluene (TOL) is widely used in industry. Occupational exposure to TOL is commonly assessed using TOL in blood, hippuric acid and ortho-cresol. Levels of these biomarkers may depend on factors potentially interfering with TOL biotransformation, such as the presence of other solvents in the workplace. Mercapturic acids (MAs) could be an alternative to the "traditional" TOL biomarkers. 2. This study aims (1) to investigate in rat the effects of an exposure to vapours mixtures on the TOL metabolism, and (2) to assess how well MAs performed in these contexts compared to the traditional TOL biomarkers. 3. Rats were exposed by inhalation to binary mixtures of TOL with n-butanol (BuOH), ethyl acetate (EtAc), methyl ethyl ketone (MEK) or xylenes (XYLs); biological exposure indicators were then measured. 4. Depending on the compounds in the mixture and their concentrations, TOL metabolism was accelerated (with BuOH), unchanged (with EtAc) or inhibited (with XYLs and MEK). Inhibition leads to an increase in blood TOL concentrations, even at authorized atmospheric concentrations, which may potentiate the effect of TOL. 5. MAs excretions are little affected by coexposure scenarios, their levels correlating well with atmospheric TOL levels. They could thus be suitable bioindicators of atmospheric TOL exposure.

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.

The mechanisms of action involved in the neurotoxicity of solvents are poorly understood. In vitro studies have suggested that the effects of some solvents might be due to the formation of reactive oxygen species (ROS). This study assesses hydroxyl radical (OH) generation and measures malondialdehyde (MDA) levels in the cerebral tissue of rats exposed to six solvents (n-hexane, n-octane, toluene, n-butylbenzene, cyclohexane and 1,2,4-trimethylcyclohexane). Three of these solvents have been shown to generate ROS in studies carried out in vitro on granular cell cultures from rat cerebellum. We assessed OH production by quantifying the rate of formation of 3,4-dihydroxybenzoic acid using a trapping agent, 4-hydroxybenzoic acid, infused via the microdialysis probe, into the prefrontal cortex of rats exposed intraperitoneally to the solvents. Extracellular MDA was quantified in microdialysates collected from the prefrontal cortex of rats exposed, 6h/day for ten days, to 1000ppm of the solvents (except for n-butylbenzene, generated at 830ppm) in inhalation chambers. Tissue levels of free and total MDA were measured in different brain structures for rats acutely (intraperitoneal route) and sub-acutely (inhalation) exposed to solvents. None of the six solvents studied increased the production of hydroxyl radicals in the prefrontal cortex after acute administration. Nor did they increase extracellular or tissue levels of MDA after 10 days' inhalation exposure. On the other hand, a decrease in the concentrations of free MDA in brain structures was observed after acute administration of n-hexane, 1,2,4-trimethylcyclohexane, toluene and n-butylbenzene. Therefore, data of this study carried out in vivo did not confirm observations made in vitro on cell cultures.

An experimental protocol has been developed for the use of rat tracheal explant to evaluate the effects of inhaled chemicals on the mucociliary function. Rats were exposed for 4 h or 7 days (24 h per day) to different concentrations of toluene diisocyanate (TDI). Each rat trachea was subsequently removed rapidly and placed in a humid chamber maintained at 37 degrees C. Mucociliary function was evaluated by video measurements of the two following parameters: mucociliary beating frequency (MCBF) and number of active (A+), partially active (A +/-) and inactive areas (A-). In control tracheas; all areas were active and the MCBF showed hardly any variation around 15.3 Hz. In rats exposed to ca. 0.27 or 0.54 ppm of TDI for 4 h, tracheas showed a significant decrease in the number of active areas and a significant decrease in the MCBF. The same changes were observed in tracheas from rats exposed to approximately 0.05 and 0.10 ppm of TDI for 7 days. After a 7 days recovery period the number of active areas and the MCBF were similar in exposed and control rats except in the group exposed for 7 days to the highest concentrations; these showed only partial recovery. The results of this study showed that this ex vivo method is useful for detecting mucociliary dysfunction.

The expiratory bradypnoea indicative of upper airway irritation in mice was evaluated during a period of 60 min of nasal exposure to methyl-2-cyanoacrylate, ethyl-2-cyanoacrylate, isopropyl-2-cyanoacrylate and 2-methoxyethyl-2-cyanoacrylate vapors using nose only exposure. Irritation of the upper respiratory tract caused a concentration-dependent decrease in the respiratory rate. The maximum effect occurred within the first 10 min of exposure and was followed by a drop-off in the response during the remainder of the exposure period. The airborne concentration resulting in a 50% decrease in the respiratory rate of mice (RD(50)) was calculated for each chemical. The results show that the four chemicals had similar irritant potencies. The RD(50) values of methyl-2-cyanoacrylate, ethyl-2-cyanoacrylate, isopropyl-2-cyanoacrylate and 2-methoxyethyl-2-cyanoacrylate were 1.4, 0.7, 0.6 and 1.0 p.p.m. Tentative estimates of threshold limit values showed that 0.1 RD(50) was closer to the values recommended by the American Conference of Governmental Industrial Hygienists for methyl- and ethyl-2-cyanoacrylate than 0.03 RD(50). On the basis of a threshold limit value for short-term exposure limit (TLV STEL) equal to 0.1 RD(50), the TLV STELs for the four cyanoacrylates should not exceed 0.1 or 0.2 p.p.m.

Bitumen fumes emitted during road paving or roofing contain polycyclic aromatic hydrocarbons (PAHs). Experimental studies have been previously performed to test the carcinogenic potency of bitumen fumes. Some of them have been criticised either on the grounds that the fume condensates were not representative of fumes to which humans are exposed or because the fumes were never characterised in terms of particle size and poorly in terms of composition and concentration in the chambers. For a nose-only inhalation study, we have evaluated the ability of a new fume generation system to deliver stable and reproducible atmospheres of bitumen fumes to an inhalation chamber and investigated the representativity of the fumes generated at a concentration level of 5 mg/m 3 . The fume generator comprises: (1) an insulated 20 1 heated kettle (200 C for bitumen); (2) an insulated inlet pipe with a needle valve to adjust the flow of the test compound from the kettle; (3) a fume generation chamber equipped with a series of interchangeable channels of different width. The fume concentration in the exposure chamber can be controlled by changing the channel width or by restricting the evaporation surface with aluminium foil, and/or by changing the flow rate. Samples of the atmosphere in the chamber were collected and analysed for quantitative determination of total particulate matter (TPM), soluble matter, benzo[a]pyrene (B|a]P) content of the fumes and other PAHs, and evaluation of the particle size distribution. The representativeness of the fumes has been tested by comparison with fumes generated in the Shell small-scale fume rig, which was previously validated against field fumes collected during paving operations. Evaporative losses from the filters during sampling, transport and storage have been also assessed. At 5 mg/m 3 TPM, the agreement between laboratories was quite good for the TPM analyses and was good for the soluble matter and B|a]P. Evaporative losses may lead to underestimation of the true exposure level in the inhalation chambers but the use of an XAD-2 cartridge backup is one approach to partially recover losses which occur on the filter. The particle size distributions are somewhat different from those reported for fumes associated with roofing and indoor mastic laying works, in that we found more than 85% of particles to be smaller than 1 um, compared with 40% particles in the previous analyses. In conclusion, this equipment allows reproducible generation of fumes at the 5 mg/m 3 TPM that are fairly representative of those produced in the field with the same bitumen. © 2000 British Occupational Hygiene Society. Published by Elsevier Science Ltd. All rights reserved.

Multi-walled carbon nanotubes (MWCNTs), which vary in length, diameter, functionalization and specific surface area, are used in diverse industrial processes. Since these nanomaterials have a high aspect ratio and are biopersistant in the lung, there is a need for a rapid identification of their potential health hazard. We assessed in Sprague-Dawley rats the pulmonary toxicity of two pristine MWCNTs (the “long and thick” NM-401 and the “short and thin” NM-403) following either intratracheal instillation or 4-week inhalation in order to gain insights into the predictability and intercomparability of the two methods. The deposited doses following inhalation were lower than the instilled doses. Both types of carbon nanotube induced pulmonary neutrophil influx using both exposure methods. This influx correlated with deposited surface area across MWCNT types and means of exposure at two different time points, 1–3 days and 28–30 days post-exposure. Increased levels of DNA damage were observed across doses and time points for both exposure methods, but no dose-response relationship was observed. Intratracheal instillation of NM-401 induced fibrosis at the highest dose while lower lung deposited doses obtained by inhalation did not induce such lung pathology. No fibrosis was observed following NM-403 exposure. When the deposited dose was taken into account, sub-acute inhalation and a single instillation of NM-401 and NM-403 produced very similar inflammation and DNA damage responses. Our data suggest that the dose-dependent inflammatory responses observed after intratracheal instillation and inhalation of MWCNTs are similar and were predicted by the deposited surface area.