Coupling Urinary Trihalomethanes and Metabolomic Profiles of Type II Diabetes: A Case-Control Study.

Abiding by the exposome paradigm, incorporation of external and internal exposure metrics using metabolomics tools is warranted to refine the etiology of type II diabetes (T2D). A small (n = 51) age- and sex-matched case-control study was conducted in Cyprus coupling urinary trihalomethanes (THMs) with T2D. The objectives were to (i) perform a comparative assessment of different deconvolution parameters in compound identification and (ii) evaluate the association between differentially expressed metabolites and either urinary THM or T2D status. Untargeted urinary metabolomics was performed with a GC-MS triple quadrupole mass spectrometry system. Results of three deconvolution searches each yielding >130 metabolites were used in subsequent analyses. The number of differentially expressed compounds by T2D status or the urinary THM levels (above or below median) differed among the three searches. The identity of these compounds was also confirmed using known standards (level 1 identification). In multivariate logistic regression, 3-aminoisobutyric acid was an important predictor of lower odds of T2D after adjusting for known risk factors. The widespread incorporation of metabolomics in population studies accounting for environmental exposures will eventually pave the way for the exposome characterization, also improving our understanding of the disease process.

Household cleaning activities as noningestion exposure determinants of urinary trihalomethanes.

Previous epidemiological studies linking drinking water total trihalomethanes (THM) with pregnancy disorders or bladder cancer have not accounted for specific household cleaning activities that could enhance THM exposures. We examined the relation between household cleaning activities (washing dishes/clothes, mopping, toilet cleaning, and washing windows/surfaces) and urinary THM concentrations accounting for water sources, uses, and demographics. A cross-sectional study (n = 326) was conducted during the summer in Nicosia, Cyprus, linking household addresses to the geocoded public water pipe network, individual household tap water, and urinary THM measurements. Household tap water THM concentrations ranged between 3-129 µg L(-1), while the median (Q1, Q3) creatinine-adjusted urinary THM concentration in females (669 ng g(-1) (353, 1377)) was significantly (p < 0.001) higher than that in males (399 ng g(-1), (256, 681)). Exposure assessment, based on THM exposure equivalency units, showed that hand dishwashing, mopping, and toilet cleaning significantly (p < 0.001) increased urinary THM levels. The effect of dishwashing by females ≥36 y of age remained significant, even after adjusting for potential confounders. No significant (p > 0.05) association was observed between ingestion-based THM exposure equivalency units and urinary THM. Noningestion routes of THM exposures during performance of routine household cleaning activities were shown for the first time to exert a major influence on urinary THM levels. It is warranted that future pregnancy-birth cohorts include monitoring of noningestion household THM exposures in their study design.

Spatial and seasonal variability of urinary trihalomethanes concentrations in urban settings.

A complex network of sources and routes of exposure to disinfection by-products (DBP), such as trihalomethanes (THM) has been driving the wide variability of daily THM intake estimates in environmental epidemiological studies. We hypothesized that the spatiotemporal variability of THM exposures could be differentially expressed with their urinary levels among residents whose households are geographically clustered in district-metered areas (DMA) receiving the same tap water. Each DMA holds unique drinking-water pipe network characteristics, such as pipe length, number of pipe leaking incidences, number of water meters by district, average minimum night flow and average daily demand. The present study assessed the spatial and seasonal variability in urinary THM levels among residents (n=310) of geocoded households belonging to two urban DMA of Nicosia, Cyprus, with contrasting water network properties. First morning urine voids were collected once in summer and then in winter. Results showed that the mean sum of the four urinary THM analytes (TTHM) was significantly higher during summer for residents of both areas. Linear mixed effects models adjusted for age, season and gender, illustrated spatially-resolved differences in creatinine-adjusted urinary chloroform and TTHM levels between the two studied areas, corroborated by differences observed in their pipe network characteristics. Additional research is warranted to shed light on the contribution of spatially-resolved and geographically-clustered environmental exposures coupled with internal biomarker of exposure measurements towards better understanding of health disparities within urban centers.

Blood trihalomethane and urinary haloacetic acid concentrations in relation to hypertension: An observational study among 1162 healthy men.

Disinfection byproducts (DBPs) have demonstrated cardiovascular and reproductive toxicity. However, the associations and mechanisms of DBP exposure in relation to hypertension among healthy young men, which are critical for gaining new insights into the prevention and treatment of male subfertility, remain unclear. In 2017-2018, we recruited 1162 healthy Chinese men. A single blood sample was collected and measured for trihalomethane (THM) concentrations (n = 956). Up to 2930 repeated urinary samples were collected at baseline and during follow-up periods and determined for haloacetic acid concentrations. Oxidative stress (OS) biomarkers were measured in within-subject pooled urinary samples (n = 1003). In total, 403 (34.68 %) participants were diagnosed with stage 1-2 hypertension (≥130/80 mmHg) and 108 (9.29 %) stage 2 hypertension (≥140/90 mmHg). In adjusted models, blood bromodichloromethane (BDCM) concentrations were positively associated with the risk of stage 1-2 and stage 2 hypertension [ORs= 1.48 (95 % CI: 1.15, 1. 91) and 1.65 (95 % CI: 1.08, 2.51), respectively, per 2.7-fold increase in BDCM concentrations]. Additionally, we found positive associations between DBP exposure biomarkers and urinary concentrations of 4-hydroxy-2-nonenal-mercapturic acid and 8-hydroxy-2-deoxyguanosine. However, these OS biomarkers were unrelated to hypertension. Our results suggest that BDCM exposure may be associated with a greater risk of hypertension among healthy young men.

Optimization of a novel procedure for determination of VOCs in water and human urine samples based on SBSE coupled with TD-GC-HRMS.

In this study, stir-bar sorptive extraction and thermal desorption followed by gas chromatography coupled with high resolution mass spectrometry was applied for determination of halo-organic compounds (bromodichloromethane, dibromochloromethane, bromoform, and tetrachloroethylene) in water and human urine samples. Time of extraction and stirring speed were optimized. The results show that the optimum extraction time is 30 min with 600 rpm of stirring speed with Twister of 20 mm in length and 1.0-mm film thickness of PDMS (126 microL). The calibration curves, limits of detection and quantification for all compounds were calculated. This procedure is characterized by very low limits of detection and quantitation: lower than 0.0017 microg/L and good repeatability for all four volatile compounds. This new analytical procedure was identified to be easy, reliable, sensitive, and requires only small amounts of sample. It can constitute a good alternative to well-known procedures based on application of head space and gas chromatography coupled with electron capture detection.

Cohort-friendly protocol for a sensitive and fast method for trihalomethanes in urine using gas chromatography-Triple quadrupole mass spectrometry.

A cohort-friendly biomonitoring protocol has been developed for measuring biomarkers of exposure to disinfection by-products (trihalomethanes, THM) in urine using small initial volume (3mL) and short analysis time (∼10min) that facilitates the throughput of a large number of samples. The objective of this study was to optimise a cohort-friendly biomonitoring protocol for the determination of four THM analytes in human urine using gas chromatography coupled with triple quadrupole mass spectrometry (GC-QqQ-MS/MS). The proposed methodology will facilitate the inclusion of such urinary THM measurements into large population health studies.

Time of the day dictates the variability of biomarkers of exposure to disinfection byproducts.

Non-persistent environmental chemicals (NOPEC) are xenobiotics with short half-lives of elimination (<7h). Similar to chronopharmacokinetics, NOPEC metabolism may follow diurnal patterns of cytochrome P450 activity. The role of circadian liver clock in shaping NOPEC metabolism and their concomitant measurements of biomarkers of exposure and effect remains poorly understood in real-life human settings. Metabolic activation (toxication) by CYP2E1 converts trihalomethanes (THM) to harmful metabolites. We investigated the diurnal variation of urinary THM exposures and their metabolism patterns as catalyzed by CYP2E1 redox activity, using the surrogate marker of 4-hydroxynonenal (4HNE). We implemented three time-series trials with adult volunteers conducting specific household cleaning activities at predefined times of a day. Circadia variation of 4HNE was assessed with a cosinor model and its mesor levels increased with THM exposure. The time of exposure within the day dictated the magnitude of urinary THM levels and their toxication effect; in all three trials and relative to urinary THM levels before the activity, lower and higher median THM were measured right after the activity in morning and afternoon/night, respectively. This is consistent with higher reported CYP2E1 redox activity in light/active phase. Population health studies should incorporate time-stamped biomarker data to improve the understanding of chronic disease processes.

Coupling external with internal exposure metrics of trihalomethanes in young females from Kuwait and Cyprus.

The Eastern Mediterranean and the Middle Eastern regions are both understudied in terms of possible environmental health risks for their populations. Water scarcity and desalination treatment provide the general population of countries from these regions (e.g., Kuwait and Cyprus) with unique tap water characteristics. This study investigated the association between external (tap water and 24 h personal air samples) and internal (urine) THM exposure metrics that reflected information about THM-related habits and activities collected using questionnaires and time activity diaries. The study population comprised of young females residing in either Kuwait (n=13) or Cyprus (n=22). First morning urine voids were collected on 2 consecutive days. Urinary creatinine-adjusted total THM (TTHM) levels were higher in Kuwait (median (interquartile range): 1044 (814, 1270) ng/g) than in Cyprus (691 (510, 919) ng/g, P<0.05). Median personal air TTHM levels in Kuwait (1.4 (0.7, 1.7) µg/m3) were higher than those in Cyprus (0.9 (0.5, 1.4) µg/m3), but did not reach statistical significance (P=0.17). Median tap water TTHM in Kuwait (6.7 (5.4, 11.6) µg/l) did not correlate with urinary or air THM and they were lower than those in Cyprus (29.5 (20.1, 48.0) µg/l; P<0.01). Despite that tap water did not contain chloroform (TCM), TCM was detected in both air and urine samples in Kuwait, suggesting other TCM exposure sources, such as household cleaning activities. Total duration of activities and mopping were significantly correlated with air and urine THM in Kuwait, as reported in the time activity diary. Personal air and urine exposure metrics were correlated in Kuwait (TTHM ρ=0.62, P<0.05), but not in Cyprus (TTHM ρ=-0.32, P>0.05). Time-activity diaries and urinary THM seemed to be useful measures of THM exposures in Kuwait. Coupling both external with internal exposure metrics could find use in population health studies towards further refining the association between environmental exposures and health outcomes.

Sensitive headspace gas chromatography-mass spectrometry determination of trihalomethanes in urine.

A sensitive and straightforward method for the determination of trihalomethanes (THMs) in urine by using headspace extraction technique has been developed. Chemical and instrumental variables were studied in order to optimize the method for sensitivity: an excess of KCl (4 g per 12 ml of urine), an oven temperature of 85 degrees C and an equilibration time of 30 min were selected. The use of the mass spectrometer in selected ion monitoring mode allows achieving linear ranges between 10 and 5000 ng/l and detection limits from 3 to 10 ng/l, for 12 ml of urine. The stability of the urine sample during storage at 4 and -20 degrees C was also evaluated: THMs remained stable for up to 2 days and 2 months, respectively. Finally, the method was successfully applied to study the THM uptake from swimmers of an indoor swimming pool, as well as non-swimmers. This study revealed that the concentrations of THMs in urine increased approximately three times for chloroform and bromodichloromethane after swimming activity. In addition, THMs in unchanged form were mainly excreted within 2-3h after the end of exposure.

A comparison of three solvent-free techniques coupled with gas chromatography for determining trihalomethanes in urine samples.

The analysis of volatile organic compounds in samples of biological fluids characterized by complex matrices is highly challenging. This paper presents a comparison of the results obtained in this field using three solvent-free techniques: thin-layer headspace with autogenous generation of liquid sorbent (TLHS) and membrane separation of the trace substances (pervaporation, PV), both of which are coupled to direct aqueous injection gas chromatography-electron capture detection (TLHS-DAI-GC-ECD and PV-DAI-GC-ECD), as well as conventional static headspace analysis followed by GC analysis with ECD detection (HS-GC-ECD). Basic validation parameters of the HS-GC-ECD, TLHS-DAI-GC-ECD and PV-DAI-GC-ECD procedures were calculated for water and urine samples. The calibration curves for all procedures were linear within the concentration range examined. The intermediate precisions of the procedures were good and reached about 10% (for all analytes) for HS-GC-ECD and TLHS-DAI-GC-ECD. The poorest results were obtained for PV-DAI-GC-ECD: about 20% for all analytes. The lowest method detection limits were obtained for the TLHS-DAI-GC-ECD procedure: below 0.0022 microg/L for all analytes. The enrichment factors did not differ significantly between water and urine samples, indicating little or no matrix effect in all procedures.

Association between exposures to brominated trihalomethanes, hepatic injury and type II diabetes mellitus.

Non-alcoholic fatty liver disease (NAFLD) is considered the most common liver disorder in the Western world, commonly diagnosed in the majority of obese patients with type 2 diabetes mellitus (T2DM). Metabolic disrupting chemicals with short half-lives, such as those of halogenated structure (trihalomethanes, THM) have been linked with hepatic insulin resistance phenomena in animal studies. However, human studies evaluating the role of THM exposure on liver pathogenesis and T2DM disease process are scarce. The objectives of this study were to: i) determine the association of urinary brominated THM (BrTHM) levels and T2DM disease status, and ii) investigate the association between urinary BrTHM levels and serum alanine aminotransferase (ALT) concentrations, often used as surrogate markers of NAFLD. A pilot case-control study was conducted in Nicosia, Cyprus (n=95). Cases were physician-diagnosed T2DM patients and controls were healthy individuals. Liver enzymes, leptin and TNF-α were measured in sera, while urinary THM levels were measured using tandem mass spectrometry. Diabetics had higher levels of serum leptin, body mass index and ALT than the controls. Among all study participants those with serum ALT levels above the median (17IU/L) had higher mean tribromomethane (TBM) concentrations compared to those with serum ALT below 17IU/L. A significant increase in the odds of having above the median serum ALT levels [OR 6.38, 95% CI: 1.11, 42.84 (p=0.044)] was observed for each unit increase in creatinine-unadjusted urinary TBM levels, along with BMI and past smoking, after adjusting for possible confounders, such as urinary creatinine, age, sex, and leptin; no other THM compound showed a significant association with serum ALT. Logistic regression models for T2DM using the urinary BrTHM as exposure variables did not reach the predetermined level of significance. The interplay between exposures to BrTHM and the initiation of key pathophysiological events relating to hepatic injury (ALT) and inflammation (leptin) was recognized via the use of selected biomarkers of effect. Our evidence that THM could act as hepatic toxins with a further initiation of diabetogenic effects call for additional studies to help us better understand the disease process of the two co-morbidities (NAFLD and T2DM).

Chlorination by-products in drinking water and menstrual cycle function.

We analyzed data from a prospective study of menstrual cycle function and early pregnancy loss to explore further the effects of trihalomethanes (THM) on reproductive end points. Premenopausal women ((italic)n(/italic) = 403) collected urine samples daily during an average of 5.6 cycles for measurement of steroid metabolites that were used to define menstrual parameters such as cycle and phase length. Women were asked about consumption of various types of water as well as other habits and demographics. A THM level was estimated for each cycle based on residence and quarterly measurements made by water utilities during a 90-day period beginning 60 days before the cycle start date. We found a monotonic decrease in mean cycle length with increasing total THM (TTHM) level; at > 60 microg/L, the adjusted decrement was 1.1 days [95% confidence interval (CI), -1.8 to -0.40], compared with less than or equal to 40 microg/L. This finding was also reflected as a reduced follicular phase length (difference -0.94 day; 95% CI, -1.6 to -0.24). A decrement in cycle and follicular phase length of 0.18 days (95% CI, -0.29 to -0.07) per 10 microg/L unit increase in TTHM concentration was found. There was little association with luteal phase length, menses length, or cycle variability. Examining the individual THMs by quartile, we found the greatest association with chlorodibromomethane or the sum of the brominated compounds. Incorporating tap water consumption showed a similar pattern of reduced cycle length with increasing TTHM exposure. These findings suggest that THM exposure may affect ovarian function and should be confirmed in other studies.

Partition coefficients for the trihalomethanes among blood, urine, water, milk and air.

Chloroform, bromodichloromethane, chlorodibromomethane, and bromoform comprise the trihalomethanes, a group of widespread and mildly lipophilic compounds that result from water chlorination and other sources. Many animal studies show the chronic toxicity and carcinogenicity of these compounds, and recent work has demonstrated the importance of both ingestion and inhalation exposure pathways. This study presents partition coefficients describing the equilibrium among biological compartments (air, water, blood, milk, urine) for the four THMs based on results of headspace gas chromatographic analyses performed under equilibrium conditions and at 37 degrees C. The calculated partition coefficients ranged from 2.92 to 4.14 for blood/water, 1.54-2.85 for milk/blood, and 3.41-4.93 for blood/urine, with the lowest being chloroform and the highest being bromoform. Both human and cow milk were tested, with similar results. The available samples of human milk may not fully account for differences in lipid content and possibly other factors that affect estimates of partition coefficients. Simultaneous measurements of milk and blood in exposed individuals are suggested to confirm laboratory results. Partition coefficients are predicted using the octanol-air partition coefficient, also measured in this study, and the octanol-water partition coefficient. Results are similar to literature estimates for liquid/air partitioning of chloroform and chlorodibromomethane, but they differ from predictions based on hydrophobicity and lipid content. High correlations between the derived partitioned coefficients and the molecular structure (number of Br atoms) and physical properties (molecular weight and boiling point) are found for these analogous chemicals. In humans, THMs are both stored and metabolized with relatively rapid clearance rates. The derived partition coefficients can help to interpret results of biological monitoring and predict the potential for the accumulation and transfer of chemicals, specifically by the application of physiologically-based pharmacokinetic models. THM exposures to potentially susceptible populations, e.g. nursing infants, can be predicted using either such models.

A sensitive and fast method for trihalomethanes in urine using gas chromatography-triple quadrupole mass spectrometry.

Because of the plethora of exposure sources and routes through which humans are exposed to trihalomethanes (THM), the limitation of their short half-lives could be overcome, if a highly sensitive method was available to quantify urinary THM concentrations at sub-ppb levels. The objective of this study was to develop a fast and reliable method for the determination of the four THM analytes in human urine. A sensitive methodology was developed for THM in urine samples using gas chromatography coupled with triple quadrupole mass spectrometry (GC-QqQ-MS/MS) promoting its use in epidemiological and biomonitoring studies. The proposed methodology enjoys limits of detection similar to those reported in the literature (11-80 ng L(-1)) and the advantages of small initial urine volumes (15 mL) and fast analysis per sample (12 min) when compared with other methods. This is the first report using GC-QqQ-MS/MS for the determination of THM in urine samples. Because of its simplicity and less time-consuming nature, the proposed method could be incorporated into detailed (hundreds of participants' urine samples) exposure assessment protocols providing valuable insight into the dose-response relationship of THM and cancer or pregnancy anomalies.

Obesity-mediated association between exposure to brominated trihalomethanes and type II diabetes mellitus: an exploratory analysis.

With the exception of chloroform, the rest of trihalomethanes (THM), the so-called brominated THM (Br-THM) are composed of bromine-substituted molecules with increased lipophilicity and potency to biological tissues. The THM are formed within disinfected tap water and their health effects, under research, range from cancer to adverse reproductive outcomes. However, the association between human exposures to Br-THM and the risk of developing type II diabetes mellitus (T2DM) and metabolic co-morbidities, such as obesity and non-alcoholic steatohepatitis has never been previously explored. The objective of this exploratory analysis was to address obesity-mediated associations between urinary concentrations of brominated THM and incidences of T2DM in a Cypriot adult population (n=326). First morning urine voids were collected once during summer and another time during winter while a detailed questionnaire was administered to participants. Creatinine-adjusted urinary Br-THM analyte concentrations were significantly (p<0.05) higher in T2DM cases when compared with those in healthy individuals. Multivariate logistic regression models adjusted for potential confounders showed that participants with ≥30 kg m(-2) BMI were at a higher T2DM risk (OR=8.42, 95% CI: 1.97, 45.5; p<0.01) when compared with that of normal weight participants (<25 kg m(-2)). About 4 times higher risk for developing T2DM was observed for individuals in the upper tertile of urinary Br-THM levels (OR=3.99, 95% CI: 1.07, 19.7; p<0.05) when compared with the lower tertile participants. Among the participants with BMI≥25 kg m(-2), urinary Br-THM levels were significantly (p<0.001) higher in diabetics than in healthy individuals. Ingestion and non-ingestion exposures to Br-THM deserve careful consideration in relevant epidemiological studies, as a possible environmental risk factor of T2DM.

Alveolar air and urine analyses as biomarkers of exposure to trihalomethanes in an indoor swimming pool.

The exposure of workers and swimmers at an indoor swimming pool to trihalomethanes (THMs) as a consequence of water chlorination was evaluated by analyzing alveolar air and urine samples. Environmental monitoring of THMs in water and ambient air was also performed in order to assess the possible correlation between environmental and biological samples. The sampling was done concurrently, taking the urine and alveolar air samples before and after the work shift for 15 workers and the swimming activity for 12 swimmers. A high THM uptake was observed in alveolar air and urine of subjects exposed, with chloroform being the most abundant THM. Mean chloroform levels in alveolar air and urine before exposure were 4 microg/ m3 and 475 ng/L, respectively. After 2 h of exposure, concentration increases of ca. 8 times in alveolar air and 2 times in urine were observed in workers. After 1 h swimming, the increases found in swimmers were ca. 20 and 3 times in alveolar air and urine, respectively. High increases have also been observed in bromodichloromethane levels. We have obtained excellent correlations between the chloroform concentrations found in the swimming pool ambient air/alveolar air, and between the urine/ alveolar air of the participants after exposure (r > 0.9). In conclusion, alveolar air provides better response sensitivity and shorter reaction time to external exposure than urine, being therefore the most sensitive biomarker.

Assessment of exposure of workers and swimmers to trihalomethanes in an indoor swimming pool.

A simultaneous study on workers' and swimmers' exposure to trihalomethanes (THMs) in an indoor swimming pool has been carried out by analyzing urine samples using the headspace and gas chromatography-mass spectrometry technique. The subjects of this study were male and female workers of an indoor swimming pool as well as swimmers regularly attending the pool. The results reported show that only chloroform and bromodichloromethane were detected in the urine of those people exposed, which can be used as a specific index of exposure to these compounds. THM uptake of swimmers after 1 h of swimming was higher than that of workers after a 4 h work shift since THM levels in the workers' urine were associated only with inhalation, while levels in swimmers' urine were mainly associated with dermal absorption, apart from inhalation and occasional ingestion, as well as increased uptake due to the physical stress (swimming). The kinetics of THM excretion in the urine of the participants exposed has been calculated after termination of the exposure to select the sampling time and determine the elimination process. An interval of 15 min after exposure was selected as the sampling time, and the absorbed dosage was eliminated by 2 h after exposure. A good correlation between THM concentrations found in the swimming pool water and the urinary THM concentrations of the people affected after exposure has also been obtained.