Simultaneous determination of aromatic and chlorinated compounds in urine of exposed workers by dynamic headspace and gas chromatography coupled to mass spectrometry (dHS-GC-MS).

Mixed exposure to chemical products is a topical issue for occupational health and often includes exposure to volatile organic compounds (VOCs). As very few methods are available for evaluating these mixed exposures, the aim of this work was to develop a simple biomonitoring method to assess simultaneous occupational exposures to chlorinated and aromatic VOCs by analyzing the unmetabolized fraction of the VOCs in the urine of workers. Volatile organic compounds were analyzed using dynamic headspace gas chromatography coupled to mass spectrometry (dHS-GC-MS), and 11 unmetabolized urinary VOCs were measured into headspace phase, without any time-consuming pretreatment. Simultaneously, a standardized collection protocol was designed to avoid VOC losses or the contamination of urinary samples. The calibration samples were real urines, spiked with known amounts of the VOC mixtures studied. Test investigations were performed on potentially exposed workers in three factories in order to assess the effectiveness of both the collection protocol and analytical method. A satisfactory level of sensitivity was achieved, with limits of quantification (LOQ) between 10 and 15 ng/L obtained for all VOCs (except for styrene at 50 ng/L). Calibration curves were linear in the 0-20 µg/L range tested, with R2 correlation coefficients of 0.991 to 0.998. At the lowest concentration tested (0.08 µg/L), within-day precision varied from 2.1 to 5.5% and between-day precision ranged from 2.7 to 8.5%. Sample stability at -20 °C required that urinary samples be analyzed within 3 months. Even though the urinary concentrations of VOCs used in the plants were mostly quite low, significant differences between post-shift and pre-shift were observed. In conclusion, a fast, sensitive, specific and easy-to-use method has been developed for extracting VOCs from human urine using dHS-GC-MS. The method described has proven to be reliable for assessing current occupational exposure to chlorinated and aromatic VOCs in France.

Metabolism of inhaled methylethylketone in rats.

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.

Impact of coexposure on toluene biomarkers in rats.

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.

Adverse effects of diisooctyl phthalate on the male rat reproductive development following prenatal exposure.

In a first study, rats were given diisooctyl phthalate (DIOP, CAS 27554-26-3) at 0, 0.1, 0.5, and 1g/kg/day, by gavage, on gestation days 6-20 (GD). There was a significant increase in resorptions at 1g/kg/day and a reduction in fetal weights at 0.5 and 1g/kg/day. Malpositioned testes were observed in fetuses at 1g/kg/day, and supernumerary lumbar ribs and ossification delay at 0.5 and 1g/kg/day. In a follow-up study, DIOP administered on GD 12-19 reduced fetal testicular testosterone at 0.1g/kg/day and above. Finally, postnatal reproductive assessment was conducted in adult male offspring prenatally exposed to DIOP on GD 12-21. Abnormalities of reproductive system (e.g. hypospadias, non scrotal testes, and hypospermatogenesis) were observed in a few adult males at 0.5g/kg/day, and with a high incidence at 1g/kg/day. Thus, DIOP displayed an antiandrogenic activity and disrupted the male reproductive development.

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

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.

Mercapturic acids derived from toluene in rat urine samples: identification and measurement by gas chromatography-tandem mass spectrometry.

Toluene is one of the most widely used CMR chemicals in industry. Worker exposure to this compound is regulated in France, but new, more sensitive methods are required to effectively monitor this exposure. A gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed and fully validated for the simultaneous determination of urinary toluene mercapturic acids derived from side chain and ring oxidation, i.e., benzylmercapturic acid and the three isomers o-, m- and p-toluylmercapturic acids, respectively. The method involves a simple and efficient two-step preparation procedure consisting of liquid-liquid extraction of the urinary acids followed by a microwave-assisted esterification of the isolated compounds using 2-propanol. The method meets all the required validation criteria: high selectivity, intra-day and inter-day precision ranges between 1.0 % and 12.4 %, with close to 100 % recovery. Linearity has been shown over the reduced concentration range 0.03-0.5 mg/L whereas a multiplicative model (ln-ln transformation) had to be used to describe the full range of concentrations 0.03-20 mg/L. The limits of detection for the four analytes, ranging from 2.8 to 5.5 µg/L, made the method suitable for their identification and quantification in urine from rats inhaling toluene in the 2 to 200 ppm concentration range. All urine samples from exposed rats contained measurable amounts of all metabolites. This is the first time that o- and m-toluylmercapturic acids have been shown to occur. Our results confirm the hypothesis that toluene mercapturic acids derived from ring oxidation exist in three forms.

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.

Toluene-induced hearing loss in the guinea pig.

Toluene is a high-production industrial solvent, which can disrupt the auditory system in rats. However, toluene-induced hearing loss is species dependent. For instance, despite long-lasting exposures to high concentrations of aromatic solvent, no study has yet succeeded in causing convincing hearing loss in the guinea pig. This latter species can be characterized by two metabolic particularities: a high amount of hepatic cytochrome P-450s (P-450s) and a high concentration of glutathione in the cochlea. It is therefore likely that the efficiency of both the hepatic and cochlear metabolisms plays a key role in the innocuousness of the hearing of guinea pigs to exposure to solvent. The present study was carried out to test the auditory resistance to toluene in glutathione-depleted guinea pigs whose the P-450 activity was partly inhibited. To this end, animals on a low-protein diet received a general P-450 inhibitor, namely SKF525-A. Meanwhile, they were exposed to 1750 ppm toluene for 4 weeks, 5 days/week, 6 h/day. Auditory function was tested by electrocochleography and completed by histological analyses. For the first time, a significant toluene-induced hearing loss was provoked in the P-450-inhibited guinea pigs. However, the ototoxic process caused by the solvent exposure was different from that observed in the rat. Only the stria vascularis and the spiral fibers were disrupted in the apical coil of the cochlea. The protective mechanisms developed by guinea pigs are discussed in the present publication.

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.

Toluene-induced hearing loss in phenobarbital treated rats.

Exposure to aromatic organic solvents may induce hearing loss in rats, the cochlea being the primary target. The aim of this study which was carried out in rat, was to evaluate the impact of the hepatic metabolism of toluene on its ototoxic potency. To this end, the solvent hepatic metabolism was shifted by treating the rats with 50 mg/kg/d of phenobarbital (PhB), a potent inducer of the microsomal cytochromes P450 system, alcohol and aldehyde dehydrogenases, and glutathione-S-transferases. The two main urinary metabolites of the oxidative and conjugate pathways [hippuric (HA) and benzyl mercapturic acids (BMA) respectively] confirmed the efficacy of the PhB treatment. For the PhB-induced rats, the amount of excreted HA increased by 43% and the amount of BMA by 35%. Auditory function impairments were assessed using auditory-evoked potentials. On completion of the auditory tests, the organs of Corti were dissected to evaluate hair cell losses. The permanent auditory threshold shifts were approximately 15 dB greater in the toluene-exposed rats than in the PhB-induced rats. Both the functional and morphological data confirmed that PhB treatment can decrease the ototoxic potency of toluene.

Ototoxicity of the three xylene isomers in the rat.

Numerous experiments have shown that the aromatic solvents can affect the auditory system in the rat, the cochlea being targeted first. Solvents differ in cochleotoxic potency: for example, styrene is more ototoxic than toluene or xylenes. The goal of this study was to determine the relative ototoxicity of the three isomers of xylene (o-, m- or p-xylene). Moreover, by dosing with the two urinary metabolites of xylene, methylhippuric (MHAs) and mercapturic acids (MBAs), this study points toward a causal relationship between the cochleotoxic effects and potential reactive intermediates arising from the biotransformation of the parent molecules. Separate groups of rats were exposed by inhalation to one isomer following this schedule: 1800 ppm, 6 h/d, 5 d/wk for 3 wk. Auditory thresholds were determined with brainstem-auditory evoked potentials. Morphological analysis of the organ of Corti was performed by counting both sensory and spiral ganglion cells. Among the three isomers, only p-xylene was cochleotoxic. A 39-dB permanent threshold shift was obtained over the tested frequencies range from 8 to 20 kHz. Whereas outer hair cells were largely injured, no significant morphological change was observed within spiral ganglia. The concentrations of urinary p-, o- or m-MHA were greater (p-MHA: 33.2 g/g; o-MHA: 7.8 g/g; m-MHA: 20.4 g/g) than those obtained for MBAs (p-MBA: 0.04 g/g; o-MBA: 6.2 g/g; m-MBA: 0.03 g/g). Besides, there is a large difference between o-MBA (6.2 g/g) and p-MBA (0.04 g/g). As a result, since the cysteine conjugates are not determinant in the ototoxic process of xylenes, the location of the methyl groups around the benzene nucleus could play a key role.