Smoking habit in parents and exposure to environmental tobacco smoke in elementary school children of Milan.

INTRODUCTION: Children with smoking parents are potentially exposed to Environmental Tobacco Smoke (ETS). The aims of this study were: 1) to assess ETS exposure in Milan schoolchildren, by measuring urinary cotinine (COT-U), 2) to compare the parents' perception of children ETS exposure, with the actual ETS exposure measured by COT-U, 3) to explore the factors influencing COT-U, including smoking bans at home, the season, and children characteristics. METHODS: One-hundred school children (7-11 years) and their parents were recruited for the study in Spring 2018 (n = 81) and in Winter 2019 (n = 94), 75 children participated to both campaigns, for a sum of 175 observations. A questionnaire was submitted to parents to collect information about smoking habits in the house. COT-U was measured by LC-MS/MS in spot urine sample collected in the morning from children. RESULTS: Detectable COT-U levels were found in 42% and 57% of children, in spring and winter, in contrast with 17% and 13% of parents acknowledging ETS exposure. Children living with smokers or e-cigarette users (vapers) (30% of the participants) had higher COT-U levels than children not living with smokers or vapers (median 0.67, 0.46, and <0.1 µg/L in spring, and 0.98, 0.85, and 0.11 µg/L in winter, respectively). Increasingly higher COT-U levels were observed in children living in homes where smoking was completely banned, allowed in the external parts of the home, or allowed in some rooms. The multiple regression analysis confirmed the positive significant effect of living with smokers, a partial smoking ban and absence of smoking ban at home, the winter season, and BMI as determinants of COT-U. CONCLUSION: ETS exposure resulted in measurable urinary cotinine in children. Smoking parents underestimate exposure to ETS of their children. Living with smokers is a determinant of COT-U, only slightly mitigated by adopting partial smoking ban.

Minding the gap between cortisol levels measured with second-generation assays and current diagnostic thresholds for the diagnosis of adrenal insufficiency: a single-center experience.

PURPOSE: The current cut-offs for the diagnosis of adrenal insufficiency (AI) have been established using outdated immunoassays. We compared the cortisol concentrations measured with Roche Cortisol I (R1), the newly available Roche Cortisol II (R2), and liquid chromatography tandem mass spectrometry (LC-MS/MS), the gold standard procedure to measure steroids in patients undergoing the corticotropin (ACTH) test. METHODS: We enrolled 30 patients (age 47 ± 21 years) referred to undergo the ACTH test (1 or 250 µg). Cortisol was measured at 0, 30, and 60 min after stimulation with R1, R2, and LC-MS/MS. AI was diagnosed for R1-stimulated peak cortisol concentrations < 500 nmol/L. RESULTS: Mean cortisol concentrations measured with R2 and LC-MS/MS were comparable, while mean cortisol concentrations measured by R1 were higher than those of both R2 and LC-MS/MS (respectively, basal 411 ± 177, 287 ± 119, and 295 ± 119 nmol/L; at 30 min, 704 ± 204, 480 ± 132, and 500 ± 132 nmol/L; at 60 min, 737 ± 301, 502 ± 196, and 519 ± 201 nmol/L, p ≤ 0.01 for R1 vs. both R2 and LC-MS/MS at each point). Considering the 500 nmol/L cortisol peak cut-off, AI was diagnosed in 5/30 patients using R1 and in 12/30 using R2 (+ 140%). Based on the correlation between R1 and R2, the threshold of 500 nmol/L became 351 nmol/L (12.7 µg/dL) when cortisol was measured with R2, and 368 nmol/L (13.3 µg/dL) with LC-MS/MS. CONCLUSIONS: The use of more specific cortisol assays results in lower cortisol concentrations. This could lead to misdiagnosis and overtreatment when assessing AI with the ACTH test if a different cut-off for cortisol peak is not adopted.

Hair as a matrix to evaluate cumulative and aggregate exposure to pesticides in winegrowers.

INTRODUCTION: Vineyard is a crop where a large number of pesticides are applied; exposure to pesticides may occur in farmers and the general population living close to the treated area. This work aimed to investigate hair as a matrix for the assessment of cumulative and aggregate exposure to pesticides in potentially exposed individuals. METHODS: Twenty agricultural workers (AW), 4 agricultural worker relatives (AR), and 5 research staff members (RS) were involved in the study. Hair samples were collected before and after the application season (PRE- and POST-EXP samples) to obtain 18 paired samples. Records with the name and the quantity of applied pesticides were obtained; twenty-seven pesticides were measured in hair by solvent extraction and LC-MS/MS. RESULTS: During the study season, AW applied 14 different pesticides with median amount ranging from 12 to 7200 g. The most popular pesticides were dimethomorph, penconazole, cyazofamid, fenamidone and quinoxyfen, applied from 94 to 69% of AW. In AW, in PRE-EXP samples the majority of used pesticides was detectable (with detection rates from 6 to 88%), with median concentrations of few pg/mg hair; in the POST-EXP samples the frequency of detected values increased (from 25 to 100%), with median concentrations up to two orders of magnitude higher. In AR, most pesticides were quantifiable only in POST-EXP samples and with lower concentration in comparison with AW; in RS, in both PRE- and POST-EXP samples only a few pesticides were quantifiable with very low levels. In AW, a linear correlation (r = 0.682 on log-transformed data, p < 0.01) was found between the total amounts of applied pesticides during the season and their concentration in hair. CONCLUSION: The study shows that the majority of assessed pesticides was incorporated into hair of AW and AR. The increased frequency of detection and level at the end of the season and the correlation between pesticide in hair and the amount of applied pesticides, reinforce the use of hair for quantitative biomonitoring of cumulative exposure to pesticides.

Identification of Metabolites of the Fungicide Penconazole in Human Urine.

Penconazole (PEN) is a fungicide used in agriculture that has been classified as hazardous to humans and the environment. The objective of this work was to identify PEN urinary metabolites in humans and propose a biomarker for PEN exposure. Five urine samples were collected from agricultural workers who worked with and were exposed to PEN. Samples were analyzed by liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry, with the source operating in the electrospray ionization mode. Metabolites previously identified in animal studies were searched as possible metabolites in humans. Candidate metabolites were first identified by multiple reaction monitoring following the protonated molecular ions that generated the protonated triazole moiety, which is expected to be present in all PEN metabolites; second, the isotopic patterns of the molecular ions were checked for consistency with the presence of two chlorine atoms; third, the full mass spectra were evaluated for consistency with the molecular structure. Seven different oxidized metabolites were found, both in the free and glucuronide conjugate forms. The major metabolite was the monohydroxyl-derivative PEN-OH (median molar fraction approximately 0.92 as a sum of free and glucuronide conjugated form). The product of further oxidation was the carboxyl-derivate PEN-COOH (median molar fraction approximately 0.03). After hydrolysis with ß-glucuronidase, the free compounds were quantified in the presence of deuterated PEN as an internal standard; PEN-OH levels ranged from 230 to 460 µg/L, and PEN-COOH levels ranged from 5.2 to 16.7 µg/L. We propose a pathway for PEN metabolism in humans and suggest PEN-OH, after hydrolysis of glucuronide conjugates, as a biomarker for monitoring human exposure to PEN.

Identification and quantification of metabolites of the fungicide tebuconazole in human urine.

Tebuconazole (TEB) is a fungicide used in agriculture; the objective of this work was to identify and quantify TEB metabolites in human urine. Samples from seven vineyard workers exposed to TEB were submitted to liquid chromatography interfaced with a triple quadrupole mass spectrometer, equipped with an electron spray source, and a linear ion trap to gain a profile of candidate metabolites. Based on the presence of the ion m/z 70 in the MS/MS spectra, which corresponds to protonated triazole (a specific moiety of TEB), and the isotopic pattern of the molecular ions, typical of molecules with one chlorine atom, hydroxyl and carboxyl derivatives of TEB, that is, TEB-OH and TEB-COOH, were identified as major metabolites, both as free molecules and as glucuronide (Glc) conjugates. The mean molar fractions were 0.67, 0.13, 0.13, and 0.07 for TEB-O-Glc, TEB-OH, TEB-COO-Glc, and TEB-COOH. Urine samples were submitted to hydrolysis with ß-glucuronidase, and the free compounds were quantified in the presence of deuterated TEB (TEB-d6) as the internal standard (IS), by multiple reaction monitoring (MRM) mode. The assay was linear in the ranges of 0.2-600 µg/L and 0.1-240 µg/L for TEB-OH and TEB-COOH, respectively; precision, accuracy, and the limit of quantification (LOQ) were <3.1%, 98-103%, and 0.3 µg/L for both analytes. An evaluation of matrix effects showed that the use of TEB-d6 controlled these sources of bias. The urinary levels of TEB-OH and TEB-COOH in specimens collected from farmers exposed to TEB ranged from 10 to 473 and from 3 to 159 µg/L, respectively.

The role of salivary cortisol measured by liquid chromatography-tandem mass spectrometry in the diagnosis of subclinical hypercortisolism.

OBJECTIVE: The use of late-night salivary cortisol (LNSalC) for diagnosing subclinical hypercortisolism (SH) is debated. No data are available regarding the role of LNSalC as measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in SH diagnosis. The aim of this study was to evaluate the diagnostic accuracy of LNSalC measured by LC-MS/MS in SH. DESIGN: Cross-sectional prospective study of outpatients. METHODS: In 70 consecutive patients with adrenal incidentalomas (AI), without signs and symptoms of hypercortisolism, we diagnosed SH in the presence of at least two of the following: cortisol after 1 mg overnight dexamethasone suppression test (1  mg DST) >83  nmol/l, 24-h urinary free cortisol (UFC) >193  nmol/24  h, and morning ACTH <2.2  pmol/l. The LNSalC levels by LC-MS/MS at 2300  h (normal values <2.8  nmol/l) and the presence of hypertension, type 2 diabetes mellitus (T2DM), and osteoporosis (OP) were assessed. RESULTS: The increased LNSalC levels (>2.8  nmol/l) had an 83.3% specificity (SP) and a 31.3% sensitivity (SN) for predicting the biochemical diagnosis of SH. The increased LNSalC had an 85.2% SP and a 55.6% SN for predicting the presence of hypertension, T2DM, and OP, while the combination of LNSalC >1.4  nmol/l (cutoff with 100% SN) plus 1 mg DST >50  nmol/l had an 88.9% SN and an 85.2% SP (similar to SH criterion at enrollment). CONCLUSIONS: In AI patients, LNSalC measured by LC-MS/MS appears to be useful in combination with 1 mg DST for diagnosing SH, while it is not useful as a single criterion.

[Identification of biomarkers of short- and medium-term exposure in urine and hair of subjects exposed to terbuthylazine]. / Identificazione di indici biologici di esposizione a breve e medio termine nelle urine e nei capelli di soggetti esposti a terbutilazina.

Terbuthylazine (TBA) is a herbicide widely used for weed control in corn crop. In mammals it is completely metabolized and excreted in urine, mostly as desetilterbutilazina (DET). This work aims to evaluate the use of urine and hair as matrices for monitoring short- and medium-term exposure to TBA. Levels of TBA and DET have been evaluated in hair and urine samples of 12 exposed farmers, 14 rural residents, 17 urban residents. In hair TBA was quantified in all samples of farmers and rural residents, but not of urban residents. In urine DET was detected in post-application samples of farmers but not in rural and urban residents. These results suggest that TBA in hair can be used as an index of cumulative exposure to TBA, while DET in the urine can be used as an index of short-term exposure in farmers.

[Time course of excretion of tebuconazole and its metabolites in vineyard workers]. / Cinetica di escrezione del tebuconazolo e dei suoi metaboliti in viticoltori.

Tebuconazole (TEB) is a fungicide widely used in vineyards. This work aimed at the identification of urinary metabolites of TEB for the biological monitoring of exposure, and to study their kinetics of excretion. Major urinary metabolites of TEB in rats are t-butyl-hydroxy-and-carboxy-tebuconazole (TEB-OH and TEB-COOH). TEB and these metabolites were determined in urine samples of 5 wine growers who collected each void before (24 hours), during and after (48 hours) TEB application. These chemicals were found in 95%, 100% and 100% of the samples with levels of < 1.5-13.4 microg/L for TEB, 5.2-749 microg/L for TEB-OH e 2.8-234 microg/l for TEB-COOH. TEB-OH is the major metabolite of TEB, its concentration increases at the end of exposure and peaks after 16-24 hours. TEB-COOH has similar pattern. TEB-OH and TEB -COOH are promising candidates for biological monitoring of TEB exposure; preliminary results suggest the day after the application as the best sampling time.