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.

Evaluation of 2-Thiothiazolidine-4-Carboxylic Acid, a Common Metabolite of Isothiocyanates, as a Potential Biomarker of Cruciferous Vegetable Intake.

SCOPE: Cruciferous vegetable consumption is associated with favorable health outcomes. Bioactive compounds arising in these, especially isothiocyanates, exert effects that contribute to prevention of disease, in large part through the attenuation of inflammation and oxidative stress. However, much about isothiocyanate metabolites and their role as biomarkers of crucifer intake remain unknown. METHODS AND RESULTS: The utility and limitations of 2-thiothiazolidine-4-carboxylic acid (TTCA) as a urinary biomarker of broccoli beverage intake are tested in a randomized crossover clinical trial where 50 participants consumed either a glucoraphanin-rich (GRR) or sulforaphane-rich (SFR) beverage. Compared to run-in and wash-out periods, significantly higher urinary TTCA is observed after broccoli beverage consumption. Measurements also show that TTCA is present in beverage powders and in all tested cruciferous vegetables. GRR results in excretion of ≈87% of the ingested TTCA while SFR results in excretion of ≈176%. Elevated urinary TTCA is observed in rats administered 100 µmol kg-1 SFN. Unlike SFN, TTCA does not activate Nrf2-mediated cytoprotective signaling. CONCLUSION: Collectively, TTCA appears to be a common isothiocyanate-derived metabolite that has the capacity to be utilized as a biomarker of cruciferous vegetables that would be beneficial for objective and quantitative tracking of intake in studies.

Stable Tb(III)-Based Metal-Organic Framework: Structure, Photoluminescence, and Chemical Sensing of 2-Thiazolidinethione-4-carboxylic Acid as a Biomarker of CS2.

A novel three-dimensional microporous framework, [Tb(pddb)phen(ox)0.5] n (Tb-MOF), was synthesized hydrothermally with V-shaped 4,4'-(pyridine-2,6-diyl)dibenzoic acid (H2pddb), oxalate (ox), and 1,10-phenanthroline (phen). The framework of Tb-MOF features one-dimensional channels functionalized with pyridine-N Lewis base groups and the absence of coordinated and lattice water molecules in the structure. The Tb-MOF exhibits high thermostability (up to 385 °C) and chemical stability in a wide pH range (4-11) and common organic solvents as well as boiling water. The luminescence investigations of the Tb-MOF in common solvents, water with different pH values, and inorganic ions were performed. Results show that the Tb-MOF has high luminescence stability and the ability to probe Fe3+ ions. Significantly, the Tb-MOF with particularly high water stability can be first developed as a highly selective and sensitive luminescent sensor for the biomarker 2-thiazolidinethione-4-carboxylic acid (TTCA) via fluorescence quenching. The low detection limit (1 ppm), reusability, and high antidisturbance together make the Tb-MOF become a promising sensor for the practical detection of TTCA in urine systems, and for the first time realize the detection of urinary TTCA through fluorescence spectrometry based on an Ln-MOF sensor.

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.

Sensitive analysis and pharmacokinetic study of epalrestat in C57BL/6J mice.

Epalrestat is clinically applied for the management of diabetic peripheral neuropathy, yet its pharmacokinetic properties are not well understood. In this study, a rapid and sensitive LC-MS/MS method was established for assaying epalrestat in bio-samples of mice. The method was validated and it showed a good linearity over the range of 2-5000ng/mL, a precision of less than 12.3%, and recovery and matrix effects of 112.5-123.6% and 87.9-89.5%, respectively. After administration of a single dose of epalrestat administered, the exposure level of AUC0-∞ was positively dose-dependent and the mean Cmax, AUC0-12h, T1/2, and MRT were 36.23±7.39µg/mL, 29,086.5µg/Lh, 1.2h and 1.8h, respectively. Epalrestat was highly exposed in stomach, intestine, liver and kidney, and only a small amount was detected in brain, urine and feces. Multi-dose of epalrestat significantly increased MRT and apparent volume of distribution (Vd) relative to those of a single-dose.

No evidence of cardiovascular toxicity in workers exposed below 5 ppm carbon disulfide.

PURPOSE: Carbon disulfide (CS2), used in the viscose process, is well known for having multiple health effects, including on the cardiovascular system, in workers with long-term exposure higher than 10 ppm. The mechanisms of those effects are, however, not precisely defined, and it remains uncertain whether cardiovascular toxicity may occur at exposure levels lower than 10 ppm. Therefore, the purpose of this study is to explore the health impact of low CS2 exposure levels using an array of preclinical biomarkers of cardiovascular risk. METHODS: Exposure intensity was determined by measuring urinary 2-thiothiazolidine-4-carboxylic acid (TTCA) in 117 workers from two plants using the viscose process, sampled in multiples phases (2003, 2006 and 2013). A cumulative exposure index (CEI) and a recent exposure index (REI) were calculated for each worker, and shiftwork was documented to account for potential confounding. Cardiovascular parameters included blood pressure, total, LDL and HDL cholesterol, triglycerides, C-reactive protein dosed in serum with high sensitivity (HsCRP), N-terminal pro-brain natriuretic peptide, and albuminuria/creatininuria ratio (UACR). Potential biological confounders were fasting blood glucose and serum creatinine. Bivariate and multivariate regression analyses were used to trace relationships between cardiovascular risk biomarkers and other variables, including CEI, REI and shiftwork duration. RESULTS: Median REI and CEI were 0.05 mg TTCA/g creat and 21.5 mg TTCA/g creat*months, respectively. While expected associations, such as between HsCRP and LDL Cholesterol, were found, significant associations between cardiovascular risk markers and CS2 exposure indexes (CEI or REI) were not detected. Shiftwork duration was positively associated with UACR in workers with elevated fasting blood glucose. CONCLUSION: In practice, when CS2 exposure levels are kept below 5 ppm (TTCA < 2.2 mg/g creat), it does not appear useful to perform a systematic monitoring of total serum cholesterol or its subfractions, or of the new biomarkers of cardiovascular risk (NTproBNP, HsCRP, UACR) investigated in the present study. It appears important to carefully monitor the existence of diabetes that may justify avoiding shiftwork.

Current and historical individual data about exposure of workers in the rayon industry to carbon disulfide and their validity in calculating the cumulative dose.

INTRODUCTION: The objective of the study was to investigate how exposure to carbon disulfide (CS2) in a rayon-manufacturing plant has changed within two decades and whether it is possible to calculate valid data for the individual cumulative exposure. METHODS: The data for CS2 concentration in air and biological exposure monitoring (2-thio-1,3-thiaxolidine-4-carboxylic acid (TTCA) in urine) from two cross-sectional studies, performed in 1992 (n = 362) and 2009 (n = 212) in a German rayon-manufacturing plant, were compared to data obtained from company-internal measurements between the studies. RESULTS: Using the data from the cross-sectional studies and company-internal data, cumulative external exposure and the cumulative internal exposure were calculated for each worker. External and internal CS2 exposure of the employees decreased from 1992 (medians 4.0 ppm and 1.63 mgTTCA/g creatinine) to 2009 (medians 2.5 ppm and 0.86 mg/g). However, company-internal CS2 data do not show a straight trend for this period. The annual medians of the company-internal measurement of external exposure to CS2 have varied between 2.7 and 8.4 ppm, in which median values exceeded 5 ppm generally since 2000. The annual medians for the company-internal biomonitoring assessment ranged between 1.2 and 2.8 mg/g creatinine. The cumulative CS2 exposure ranged from 8.5 to 869.5 ppm years for external exposure and between 1.30 and 176.2 mg/g creatinine years for the internal exposure. Significant correlations were found between the current air pollution and the internal exposure in 2009 but also between the cumulative external and internal CS2 exposure. CONCLUSIONS: Current exposure data, usually collected in cross-sectional studies, rarely allow a reliable statement on the cumulative dose, because of higher exposure in the past and of fluctuating courses of exposure. On the other hand, company-internal exposure data may be affected by non-representative measurement strategies. Some verification of the reliability of cumulative exposure data may be possible by testing the correlation between cumulative exposure data of external assessment and biological monitoring.

[The optimization of 2-thiothiazolidine-4-carboxylic acid back extraction in urine and determination by HPLC].

OBJECTIVE: Establishment of determination method of 2-thiothiazolidine-4-carboxylic acid (TTCA) in urine with HPLC. METHODS: A volume of 0.5 ml hydrochloric acid (2 mol/L) and 0.5 ml pure water was added into 1 ml urine, and then extracted by 4 ml of diethyl ether by shaking for 2 min. Remove the water phase in a tube with plug and extract again, mix the two extraction diethyl ether together, take 4 ml by adding 2 ml borax-monopotassium phosphate buffer and shaking for 2 min to extract, then take the water phase to detect. A C(18) column and UV detector were used for separating and detecting. The wavelength was 273 nm, the flow rate was 1.0 ml/min, and the injection volume was 20 µl. RESULTS: TTCA has a good linearity (r = 0.9995) over the concentration of1 1 ∼ 10 µg and the minimum detectable concentration of TTCA in urine was 0.1 µg/ml. The within-day precision (RSD) were 8.4%, 3.0% and 1.7%, the between-day precision (RSD) were 11%, 3.8%, 1.9%, respectively. The extraction recovery were between 80% ∼ 102%. CONCLUSION: The method was accurate and sensitive to detect TTCA in urine.

[The rervising of biological exposure index for carbon disulfide exposuring].

OBJECTIVE: To study the biological exposure index of carbon disulfide in China. METHODS: High-performance liquid chromatography (HPLC) was used to detect the levels of 2-thiothiazolidine-4-carboxylic acid (TTCA) in the urine of the workers after working shift end, Gas chromatography was used to detect the concentrations of the carbon disulfide in the workplace air. The relationship between the urine TTCA levels and the concentrations of the carbon disulfide was analyzed, the biological exposure index and judgement result from PC-TWA were compared. RESULTS: The levels of TTCA in urine of workers occupationally exposed to carbon disulfide were closely and positively related with the concentrations of the carbon disulfide in the workplace air. The regression equation was Y = 0.265X - 0.165, The biological exposure index of carbon disulfide were calculated by regression equation according to occupational exposure limits of carbon disulfide in China. CONCLUSION: The biological exposure index of CS(2) in China might be revised for 1.2 mg/g Cr.

Determination of the active metabolite of moguisteine in human plasma and urine by LC-ESI-MS method and its application in pharmacokinetic study.

In this study, a sensitive and reproducible electro-spray ionization liquid chromatography-mass spectrometry (LC-ESI-MS) method was established to determine the concentration of M1, the main active metabolite of moguisteine in human plasma and urine. The analysis was performed on a Diamonsil® C18(2) column (150 mm × 4.6 mm, 5 µm) with the mobile phase consisting of 0.1% formic acid-acetonitrile (57:43, v/v, pH=3.0) at a flow rate of 0.8 mL min⁻¹. The pseudo-molecular ions [M+H]+ (m/z 312.2 for M1 and 446.3 for glipizide) were selected as the target ions for quantification in the selected ion monitoring (SIM) mode. Plasma samples were analyzed after being processed by acidification with formic acid and protein precipitation with acetonitrile. Urine samples were appropriately diluted with blank urine for analysis. Calibration curve was ranged from 0.02 to 8 µg mL⁻¹. The extraction recovery in plasma was over 90%. Both the inter- and intra-day precision values were less than 7.5%, and the accuracy was in the range from -6.0% to 6.0%. This is the first reported LC-ESI-MS method for analyzing M1 in human plasma and urine. The method was successfully applied to the pharmacokinetic study after oral administration of single-dose and multiple-dose of moguisteine tablets in healthy Chinese subjects.

[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.

Symptoms and immunologic markers among vulcanization workers in rubber industries in southern Sweden.

OBJECTIVES: The aim of this study was to estimate the risk of symptoms and the possible derangement of levels of immunologic markers for contemporary Swedish rubber workers. Furthermore, the relation between exposure and these biomarkers of response was examined using urinary levels of 2-thiothiazolidine-4-carboxylic acid (TTCA), which reflect the exposure. METHODS: Included in the study were 166 exposed workers and 117 controls. Medical and occupational histories were obtained in structured interviews. Symptoms were recorded, and immunologic markers were analyzed in blood. Urinary levels of TTCA were determined by liquid chromatography tandem mass spectrometry. RESULTS: Compared with the controls, the exposed workers had increased risks of eye symptoms [odds ratio (OR) 3.0], nose bleeds (OR 4.0), burning and dry throat (OR 3.0), hoarseness (OR 2.4), severe dry cough (OR 3.8), nausea (OR 4.3), and headache (OR 2.5). When the exposed workers were divided into three groups according to the TTCA levels, the highest risks were observed among the exposed workers with intermediate TTCA levels. Furthermore, the exposed workers in all of the TTCA subgroups had elevated concentrations of total immunoglobulin G when compared with the controls. Elevated concentrations of leukocytes, neutrophils, and eosinophils were observed in the group with high TTCA levels. CONCLUSIONS: This work shows an increased risk of several symptoms and elevated levels of some immunologic markers among exposed workers in Swedish rubber industries. In addition, relationships between urinary levels of TTCA and some biomarkers of response were reported.

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.