Urinary Thioproline and Thioprolinyl Glycine as Specific Biomarkers of Formaldehyde Exposure in Humans.

Assessment of personal formaldehyde (FA) exposure is most commonly carried out using formate as a biomarker, as it is the major product from FA metabolism. However, formate could also have originated from the metabolism of other endogenous and exogenous substances or from dietary intake, which may give rise to overestimated results with regard to FA exposure. We have developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with an isotope-dilution method for rigorous quantitation of two major urinary FA conjugation products: thioproline (SPro) and thioprolinyl glycine (SPro-Gly), formed in the reaction between FA and endogenous cysteine or cysteinyl glycine, respectively, as marker molecules to assess personal FA exposure. Using this newly developed method, we measured the FA exposure levels in cigarette smokers, occupants of a chemistry research laboratory and typical domestic household, and visitors to a Chinese temple with a Pearson correlation coefficient greater than 0.94, showing a strong linear correlation between urinary adduct levels and the airborne FA level. It is believed that quantitation of urinary SPro and SPro-Gly may represent a noninvasive, interference-free method for assessing personal FA exposure.

A novel quantification method for sulfur-containing biomarkers of formaldehyde and acetaldehyde exposure in human urine and plasma samples.

A novel method for the quantification of the sulfur-containing metabolites of formaldehyde (thiazolidine carboxylic acid (TCA) and thiazolidine carbonyl glycine (TCG)) and acetaldehyde (methyl thiazolidine carboxylic acid (MTCA) and methyl thiazolidine carbonyl glycine (MTCG)) was developed and validated for human urine and plasma samples. Targeting the sulfur-containing metabolites of formaldehyde and acetaldehyde in contrast to the commonly used biomarkers formate and acetate overcomes the high intra- and inter-individual variance. Due to their involvement in various endogenous processes, formate and acetate lack the required specificity for assessing the exposure to formaldehyde and acetaldehyde, respectively. Validation was successfully performed according to FDA's Guideline for Bioanalytical Method Validation (2018), showing excellent performance with regard to accuracy, precision, and limits of quantification (LLOQ). TCA, TCG, and MTCG proved to be stable under all investigated conditions, whereas MTCA showed a depletion after 21 months. The method was applied to a set of pilot samples derived from smokers who consumed unfiltered cigarettes spiked with 13C-labeled propylene glycol and 13C-labeled glycerol. These compounds were used as potential precursors for the formation of 13C-formaldehyde and 13C-acetaldehyde during combustion. Plasma concentrations were significantly lower as compared to urine, suggesting urine as suitable matrix for a biomonitoring. A smoking-related increase of unlabeled biomarker concentrations could not be shown due to the ubiquitous distribution in the environment. While the metabolites of 13C-acetaldehyde were not detected, the described method allowed for the quantification of 13C-formaldehyde uptake from cigarette smoking by targeting the biomarkers 13C-TCA and 13C-TCG in urine.Graphical abstract.

Continuous exposure to low-frequency noise and carbon disulfide: Combined effects on hearing.

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 régulations.

[Validity of urinary 2-thiothiazolidine-4-carboxylic acid (TTCA) as biomarker of exposure to very low concentrations of carbon disulphide: preliminary results]. / Validiti dell'acido 2-tiotiazolidin-4-carbossilico (TTCA) urinario quale indicatore biologico dell'esposizione a concentrazioni molto basse di solfuro di carbonio: risultati preliminori.

The possibility to use urinary 2-thiothiazolidine-4-carboxylic acid (TTCA) as biomarker of occupational exposure to very low doses of carbon disulphide (CS2) was evaluated preliminarily in 10 workers employed in a chemical plant where rubber vulcanization accelerators are produced, and in 10 workers, residents in the same geographical area and not occupationally exposed to CS2 and dithiocarbamates (DTC). Exposure to airborne CS2 was assessed, only for exposed workers, by both personal and area samplers. For the determination of TTCA, a spot urine sample was collected for each worker, exposed and non exposed, at the end of work-shift. A questionnaire probing lifestyle and dietary habits and non occupational exposure to CS2 and DTC was administered to all workers involved in the study. Environmental exposure to CS2 in 2007 ranged between 0.21 mg/m3 and 0.73 mg/m3 for personal sampling, and between 0.23 mg/m3 and 0.41 mg/m3 for area sampling. Urinary TTCA levels resulted very low and did not show any significant difference between exposed (Median: 10.8 microg/g creat; Range: 6.1-26.4 microg/g creat) and non exposed workers (Median: 9.3 microg/g creat; Range: 3.0-33.0 microg/g creat), while higher, but not significant concentrations of TTCA were observed in smokers than in non smokers (p = 0.09). No correlation was found between urinary TTCA levels and environmental exposure to CS2, age, body mass index, smoking and dietary habits. In conclusion, the low sensibility and specificity in the assessment of occupational exposure to low doses of CS2 in workers compared to general population subjects, makes urinary TTCA a biomarker with a low usefulness in biological monitoring. ACGIH, besides, should also introduce "B" (background) notation, at present not considered for the BEI indicated for urinary TTCA.

Identification of gene markers for formaldehyde exposure in humans.

BACKGROUND: Formaldehyde (FA) is classified as a human carcinogen and has been linked to increased leukemia rates in some epidemiologic studies. Inhalation of FA induces sensory irritation at relatively low concentrations. However, little is known concerning the cellular alterations observed after FA exposure in humans. OBJECTIVES: Our aim was to profile global gene expression in Hs 680.Tr human tracheal fibroblasts exposed to FA and to develop biomarkers for the evaluation of FA exposure in humans. METHODS AND RESULTS: We used gene expression analysis, and identified 54 genes designated as FA responsive. On the basis of these data, we conducted an exploratory analysis of the expression of these genes in human subjects exposed to high or low levels of FA. We monitored FA exposure by measuring the urinary concentration of thiazolidine-4-carboxylate (TZCA), a stable and quantitative cysteinyl adduct of FA. Nine genes were selected for real-time PCR analysis; of these, BHLHB2, CCNL1, SE20-4, C8FW, PLK2, and SGK showed elevated expression in subjects with high concentrations of TZCA. CONCLUSION: The identification of gene marker candidates in vitro using microarray analysis and their validation using human samples obtained from exposed subjects is a good tool for discovering genes of potential mechanistic interest and biomarkers of exposure. Thus, these genes are differentially expressed in response to FA and are potential effect biomarkers of FA exposure.

Determination of thiazolidine-4-carboxylates in urine by chloroformate derivatization and gas chromatography-electron impact mass spectrometry.

The derivatization method of thiazolidine-4-carboxylic acid (TZCA) and methyl-thiazolidine-4-carboxylic acid (Me-TZCA) in urine with alcohol/chloroformate was achieved. TZCA and Me-TZCA were derivatized in one step in urine with ethyl chloroformate in 1 min at room temperature. The derivatives of TZCA and Me-TZCA had very good chromatographic properties and offered very sensitive response for gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS). On the basis of derivatization, the method for simultaneous determination of TZCA and Me-TZCA in human urine was developed. Deuterated Me-TZCA (Me-TZCA-d(4)) was synthesized as the internal standard (IS) for the analysis of urine samples. TZCA and Me-TZCA were derivatized and extracted from urine at pH 9.5 with toluene, and then the dried extract was dissolved with 100 microl ethyl acetate and injected in GC/MS system. The recoveries of TZCA and Me-TZCA were about 102 and 103%, respectively, at the concentration of 0.05 mg/l. The method detection limits (MDL) were 1.0 and 0.5 microg/l, respectively, for TZCA and Me-TZCA in 1 ml human urine. The coefficients of variation of TZCA and Me-TZCA were less than 6% at the concentrations of 0.05 and 0.2 mg/l, respectively. To assess the formation of TZCA during inhalation with formaldehyde (FA) (about 3.1 and 38.1 ppm FA in air), urine samples from rats were taken during 3 days after initiation of treatment. The mean amount of TZCA determined was 0.07 mg/l in control group and 0.18 mg/l during treatment with 3.1 ppm. The TZCA levels increased up to about 1.01 mg/l during treatment with 38.1 ppm. It is planned to study whether urinary TZCA can be used as an indicator in the biological monitoring of exposure to FA.

Levels of 2-thiothiazolidine-4-carboxylic acid (TTCA) and effect modification of polymorphisms of glutathione-related genes in vulcanization workers in the southern Sweden rubber industries.

OBJECTIVES: Workers in the rubber industry are exposed to a complex mixture of hazardous substances and have increased risk of developing several diseases. However, there is no up to date survey examining the exposure in the Swedish rubber industry. One of the toxic compounds in the industry is carbon disulfide (CS(2)), which is biotransformed to 2-thiothiazolidine-4-carboxylic acid (TTCA). TTCA is used as a biomarker of CS(2) exposure, but there seem to exist inter- and intraindividual variability; which could partly be due to genetic variation. The aim of the study was to determine TTCA levels and the modifying effects of glutathione-related genes in a group of Swedish rubber workers. METHODS: Urine was collected from both exposed workers and controls during the last 4 h of the work shift. The level of TTCA in urine was analyzed by liquid chromatograpy tandem mass spectrometry. Genotyping of the single nucleotide polymorphisms GCLC-129, GCLM-588, GSTA1-52, GSTP1-105 and GSTP1-114 and deletions of GSTM1 and GSTT1 were performed with real-time PCR or ordinary PCR and subsequent agarose electrophoresis. RESULTS: The highest levels of TTCA were found among workers curing with salt bath, hot air, microwaves or fluid-bed, and lower levels were found among workers curing with injection and compression molding. Furthermore, with respect to GSTM1 and GSTT1 there were statistically significant differences in TTCA-levels between genotypes among exposed workers but not among controls. The other five polymorphisms had no impact on the TTCA levels. CONCLUSIONS: The present study demonstrates relatively high levels of TTCA in urine from Swedish rubber workers. Polymorphisms in GSTM1 and GSTT1 modify the levels.