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.

Pregnancy outcome in women infected with HIV-1 receiving combination antiretroviral therapy before versus after conception.

OBJECTIVE: The potential adverse effects of antiretroviral drugs during pregnancy are discrepant and few studies, mostly from Europe, have provided information about pregnancy outcomes of those already on treatment at conception. The aim of this study was to investigate the impact of antiretrovirals (ARVs) on pregnancy outcome according to the timing of treatment initiation in a cohort of pregnant women from Brazil infected with HIV. METHODS: A prospective cohort of 696 pregnant women followed up in one single centre between 1996 and 2006 was studied. Patients who had ARV treatment before pregnancy were compared with those treated after the first trimester. The outcomes evaluated were preterm delivery (PTD) (<37 weeks), severe PTD (<34 weeks), low birth weight (LBW) (<2500 g) and very LBW (<1500 g). RESULTS: Patients who were using ARVs pre-conception had higher rates of LBW (33.3% vs 16.5%; p<0.001) and a similar trend for PTD (26.3% vs 17.7%; p = 0.09). Stratification by type of therapy (dual vs highly active antiretroviral therapy (HAART)) according to timing of initiation of ARVs showed that patients who use HAART pre-conception have a higher rate of PTD (20.2% vs 10.2%; p = 0.03) and LBW (24.2% vs 10.2%; p = 0.002). After adjusting for several factors, HAART used pre-conception was associated with an increased risk for PTD (AOR 5.0; 95% CI 1.5 to 17.0; p = 0.009) and LBW (OR 3.6; 95% CI 1.7 to 7.7; p = 0.001). CONCLUSIONS: We identified an increased risk for LBW and PTD in patients who had HAART prior to pregnancy.

Successful prevention of hiv transmission from mother to infant in Brazil using a multidisciplinary team approach.

OBJECTIVES: To determine the HIV vertical transmission rate (VTR) and associated risk factors by use of zidovudine and infant care education in Brazil. METHODS: Since 1995, a prospective cohort of HIV infected pregnant women has been followed at the Federal University of Rio de Janeiro. A multidisciplinary team was established to implement the best available strategy to prevent maternal-infant HIV transmission. Patients with AIDS or low CD(4) and high viral load received anti-retroviral drugs in addition to zidovudine. Children were considered infected if they had 2 positive PCR-RNA tests between 1 and 4 months of age, or were HIV antibody positive after 18 months. Education regarding infant treatment and use of formula instead of breast feeding was provided. RESULTS: Between 1995 and August, 2000, HIV status was determined for 145 infants. Compliance with intra-partum treatment, infant treatment and use of formula was 88.2%. Intra-partum zidovudine treatment was completed in 134/145 (92.6%) of patients; 88.1% had rupture of membranes < 4 hours; 85.4% of mothers were asymptomatic. The mean CD(4) count was 428.4 cells and mean viral load 39,050 copies. HIV vertical transmission rate was 4/145 (2.75%; CI: 0.1%-5.4%). The only risk factor significantly associated with transmission was a failure to use zidovudine intra-partum in 2 of the 4 mothers (50% versus 6.4% in non-transmitting mothers). A trend toward low CD(4) and high viral load at entry, and rupture of membranes > 4 hours were associated with increased HIV transmission. CONCLUSION: HIV vertical transmission in Brazil was reduced to a level similar to other countries with the most effective prevention programs using a multidisciplinary team approach. A high level of compliance for use of anti-retroviral drugs, the provision of health education to mothers, and use of formula for all exposed infants.