Benzene Exposure in Workers From a Waste Oil Regeneration Plant During Ordinary Activities by Air and Biological Monitoring.

BACKGROUND: In the regeneration of waste oil, a strategical technological process for the European Union circular economy action plan, exhausted oils are regenerated to produce high performing oil bases. Aim of this work was to assess the exposure to benzene in plant workers during ordinary activities. METHODS: 59 workers, potentially exposed to benzene, and 9 administrative workers from an Italian plant were monitored for the whole work shift with personal air samplers; urinary benzene (BEN-U) and S-phenyl mercapturic acid (SPMA) were measured by mass spectrometry methods in end-shift urine samples. Different job tasks were identified among workers. RESULTS: Median (minimum-maximum) airborne exposures to benzene were <0.9 (<0.9-6.3) and <0.9 (<0.9-0.9) µg/m3, BEN-U and SPMA levels were 0.094 (<0.015-3.095) µg/L and 0.15 (<0.10-9.67) µg/g crt and 0.086 (0.034-0.712) µg/L and <0.10 (<0.10-3.19) µg/g creatinine in workers and administrative workers, respectively. No differences were found among job tasks and between workers and administrative workers, while higher levels were found in smokers than in non-smokers. For all job tasks, the exposure to benzene was always below occupational limit values. CONCLUSIONS: This study has investigated for the first time the exposure to benzene of workers employed in the re-refining of exhaust oil. The results showed that normal production activities in regenerating used oils do not pose a risk of exposure to benzene in workers.

Follicular steroidogenesis in random start protocols for oocyte cryopreservation.

PURPOSE: Random start protocols are commonly used for oocyte cryopreservation in women with cancer. However, albeit generally reassuring, available evidence is still insufficient to rule out a sub-optimal cycle outcome. This study aimed to compare follicular steroidogenesis between women initiating the random start protocol in the luteal phase and those initiating in the follicular phase. METHODS: Consecutive women with cancer scheduled for oocyte cryostorage were prospectively recruited. We excluded those requiring a concomitant letrozole assumption. All women received a standardized protocol with recombinant FSH and GnRH antagonists. At the time of oocyte retrieval, follicular fluids were pooled, and a sample was collected and frozen at -80 °C. All samples were assayed concomitantly after thawing by liquid chromatography-tandem mass spectrometry. The concentration of 15 different steroid hormones was determined. RESULTS: Seventy-one women were recruited. Thirty-three initiated the ovarian stimulation in the luteal phase, while the remaining 38 initiated in the follicular phase. Baseline characteristics were generally similar. Cycle outcome did also not differ; the median (interquartile range) number of frozen mature oocytes was 9 (5-14) and 10 (5-21), respectively (p = 0.42). None of the 15 tested steroid hormones differed. CONCLUSIONS: The endocrine microenvironment surrounding oocytes is not markedly influenced by the phase of the menstrual cycle at the initiation of ovarian stimulation. This result further supports the validity of random start protocols.

Development and validation of an LC-MS/MS method for the quantitation of 30 legacy and emerging per- and polyfluoroalkyl substances (PFASs) in human plasma, including HFPO-DA, DONA, and cC6O4.

Per- and polyfluoroalkyl substances (PFASs) include persistent organic pollutants whose spread is still ubiquitous. Efforts to substitute substances of high concern with fluorinated alternatives, such as HFPO-DA (GenX), DONA (ADONA), and cC6O4, have been made. The aim of this work was to develop and validate an isotopic dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method suitable to quantify 30 PFASs in human plasma. Analytes included legacy PFASs (PFOA, PFOS, and PFHxS), fluorinated alternatives (PFBA, PFBS, 6:2 FTSA, HFPO-DA, DONA, and cC6O4), and newly identified compounds (F-53B and PFECHS). The sample preparation was rapid and consisted of simple protein precipitation and centrifugation. Calibration standards and quality control solutions were prepared with a human pooled plasma containing relatively low background levels of the considered analytes. A complete validation was carried out: the lower limits of quantitation (LLOQs) ranged from 0.009 to 0.245 µg/L; suitable linearity (determination coefficients, R2 0.989-0.999), precision (2.0-19.5%, relative standard deviation), and accuracy (87.9-113.1% of theoretical) were obtained for considered concentration ranges. No significant variations of analyte responses were recorded under investigated storage conditions and during matrix effect tests. The external verification confirmed the accuracy of the method, although limited to 12 analytes. The method was also applied to 38 human plasma samples to confirm its applicability. The developed assay is suitable for large-scale analyses of a wide range of legacy and emerging PFASs in human plasma. To our knowledge, this is the first published method including cC6O4 for human biomonitoring.

Associations of urinary and dietary cadmium with urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and blood biochemical parameters.

Cadmium is a heavy metal with established adverse effects on human health, namely on bone, liver and kidney function and the cardiovascular system. We assessed cadmium exposure and its correlation with biomarkers of toxicity. We recruited 137 non-smoking blood donors without a history of chronic disease or cancer who resided in the Northern Italy province of Reggio Emilia (mean age 47 years, range 30-60 years) in the 2017-2019 period. We used a semi-quantitative food frequency questionnaire to estimate dietary cadmium intake and urine samples to assess concentrations of urinary cadmium and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Median urinary cadmium and 8-oxodG concentrations were 0.21 µg/L (interquartile range (IQR): 0.11-0.34 µg/L) and 3.21 µg/g creatinine (IQR: 2.21-4.80 µg/g creatinine), respectively, while median dietary cadmium intake was 6.16 µg/day (IQR: 5.22-7.93 µg/day). We used multivariable linear and spline regression models to estimate mean differences exposure concentrations. Dietary and urinary cadmium were positively correlated, and both were positively and linearly correlated with 8-oxodG. We found a positive association of urinary cadmium with blood alanine aminotransferase (ALT), total cholesterol, low-density lipoprotein (LDL)-cholesterol and thyroid-stimulating hormone (TSH) concentrations. We also observed a positive association with triglycerides, in both linear (beta regression coefficient = 77.03, 95% confidence interval 32.27-121.78) and non-linear spline regression analyses. Despite the positive correlation between dietary and urinary cadmium estimates, dietary cadmium intake showed inconsistent results with the study endpoints and generally weaker associations, suggesting a decreased capacity to reflect actual cadmium exposure. Overall, these findings suggest that even low levels of cadmium exposure may adversely alter hematological and biochemical variables and induce oxidative stress.

Effect of letrozole on follicular fluid steroids concentrations in cancer patients undergoing oocyte cryopreservation.

PURPOSE: To investigate the impact of letrozole administration on follicular steroid hormones during controlled ovarian hyperstimulation for fertility preservation. METHODS: One hundred and nineteen women with cancer undergoing oocytes retrieval for fertility preservation were recruited. All women underwent ovarian hyperstimulation according to a random start protocol. Those with hormone-sensitive tumors also received letrozole, an aromatase inhibitor aimed at keeping peripheral estrogen levels low. At the time of oocytes retrieval, a sample of follicular fluid was collected and frozen. All samples were assayed concomitantly after thawing, by liquid chromatography tandem mass spectrometry. The concentration of 15 steroid hormones was determined and results were compared between women who did and did not receive letrozole. RESULTS: Fifty-two women were treated with letrozole, while 67 were not. Statistically significant differences emerged for 12 of the 15 tested steroids. They were the following: cortisol, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), estradiol, androstenedione, testosterone, dihydrotestosterone (DHT), 17-hydroxyprogesterone, progesterone and corticosterone. The most striking differences were observed for testosterone that showed a more than 200-time increase in women receiving letrozole. Estradiol was conversely reduced to a third. CONCLUSIONS: The endocrine microenvironment surrounding oocytes is markedly perturbed by the concomitant assumption of letrozole. Robust clinical evaluation is pressingly needed to rule out any detrimental effect on the chance of live birth with the use of these oocytes.

Oral Vitamin D supplementation impacts gene expression in granulosa cells in women undergoing IVF.

STUDY QUESTION: Does oral Vitamin D supplementation alter the hormonal milieu of follicular fluid (FF) and the transcriptomic profile of luteinised granulosa cells (GCs) in women with Vitamin D deficiency undergoing IVF? SUMMARY ANSWER: A transcriptomic signature relevant to oral Vitamin D supplementation in luteinised GCs was demonstrated, although Vitamin D supplementation did not alter hormone levels in FF. WHAT IS KNOWN ALREADY: Vitamin D deficiency is linked to lower live birth rates among women undergoing IVF. It is unclear whether Vitamin D elicits a targeted action in reproductive physiology or is a surrogate marker of overall well-being. Several in-vitro studies, but none in vivo, have examined the impact of Vitamin D on the periovulatory follicle, focusing on GCs as a proxy marker of oocyte competence. STUDY DESIGN, SIZE, DURATION: We present a report of secondary outcomes from the SUNDRO clinical trial, which was launched in 2016 to determine whether Vitamin D supplementation can improve the IVF outcomes of women who are deficient in Vitamin D (<30 ng/ml). FF samples of 145 women who were randomised to receive Vitamin D or placebo from March 2017 to January 2019 were collected. All follicles that were aspirated in our study measured ≥11 mm on the day of hCG trigger. The first cohort of samples was collected from the dominant follicle of each participant and utilised for hormone profiling (n = 50 Vitamin D, n = 45 Placebo). For the second cohort, the follicle aspirates of each participant were pooled to create a single FF sample, which was used for the isolation of GCs for gene expression studies (n = 20 Vitamin D, n = 30 placebo). Six of the samples from the second cohort were used for RNA-sequencing analysis (n = 3 Vitamin D, n = 3 placebo). PARTICIPANTS/MATERIALS, SETTING, METHODS: Two academic infertility units were involved in the recruitment of the participants, who received a single dose of oral 25-hydroxyvitamin D (600 000 IU) or placebo, 2-12 weeks before oocyte retrieval. Women in both groups were deficient in Vitamin D, aged 18-39 years with a normal BMI (18-25 kg/m2) and <3 previous IVF cycles. The FF was aspirated at the time of oocyte retrieval and stored. Liquid chromatography tandem mass spectrometry was used to measure FF abundance of 25-hydroxyvitamin D, aldosterone, androstenedione, cortisol, cortisone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone, oestradiol (E2), 17-OH-hydroxyprogesterone, progesterone (P4) and testosterone. GCs were isolated from pooled FFs and the transcriptome was evaluated by RNA-sequencing and RT-PCR. Ingenuity pathway analysis (IPA) was used to assess the top canonical pathways and upstream regulators mediating the action of Vitamin D. MAIN RESULTS AND THE ROLE OF CHANCE: At oocyte retrieval, FF concentration of 25-hydroxyvitamin D was 2.8-fold higher (P < 0.001) in the Vitamin D group (39.5 ng/ml; n = 50) compared to placebo (13.8 ng/ml; n = 45) but no other hormonal differences were detected. In the placebo group, but not the Vitamin D group, weak correlations of 25-hydroxyvitamin D concentration with P4 (r = 0.31, P = 0.03) and E2 (r = 0.45, P = 0.002) were observed. RNA-sequencing identified 44 differentially expressed genes in the GCs of patients who received Vitamin D (n = 3) compared to placebo (n = 3). RT-PCR demonstrated upregulation of VDR (vitamin D receptor), GSTA3 (glutathione S-transferase A3) and IL21R (interleukin 21 receptor), and downregulation of P T GS2 (prostaglandin-endoperoxide synthase 2), KLF4 (kruppel-like factor 4), T RP C4 (transient receptor potential cation channel subfamily C member 4), VEGF (vascular endothelial growth factor), RXRB (retinoid X receptor beta) and AGER (advanced glycosylation end-product specific receptor) genes in the Vitamin D (n = 17) versus placebo (n = 27) group. IPA suggested roles of Vitamin D in antioxidant defence. LIMITATIONS, REASONS FOR CAUTION: Interpretation of the data is influenced by our intervention strategy (2-12 weeks prior to retrieval). As folliculogenesis may last 5-6 months, our protocol can only examine with confidence the impact of Vitamin D on the final stages of follicular growth. Furthermore, we examined the hormonal profile of the dominant follicle only, while the GC data reflect the transcriptome of all (pooled) follicles large enough to be used for IVF. Luteinised GCs from controlled ovarian stimulation were used in this study, which may be functionally distinct from the GCs of developing follicles. Moreover, the sample size for RNA-sequencing analysis was low (n = 3 per group), regardless of validation by RT-PCR that was performed on a larger cohort, introducing complexity to the IPA analysis, which required an input of data with P-adjusted <0.08 instead of <0.05 to be informative. WIDER IMPLICATIONS OF THE FINDINGS: This is the first in-vivo study to show that Vitamin D supplementation alters gene expression in luteinised GCs. In contrast to some in-vitro evidence, no effect of the intervention on expression of genes encoding steroidogenic enzymes was observed. Unlike other studies, our results suggest that supplementation with Vitamin D is unlikely to directly influence hormone availability in FF. Our findings instead reinforce the hypothesis that Vitamin D could be considered one of the gatekeepers in protecting against an exaggerated response to ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S): The study has been funded by the Italian Ministry of Health (RF-2013-02358757) following peer review in the competitive 'Bando di Ricerca Finalizzata e Giovani Ricercatori 2013' for the clinical trial SUNDRO (EudraCT registration number 2015-004233-27). There are no competing interests. TRIAL REGISTRATION NUMBER: EudraCT registration number 2015-004233-27.

Simultaneous Quantification of Bisphenol A, Its Glucuronide Metabolite, and Commercial Alternatives by LC-MS/MS for In Vitro Skin Absorption Evaluation.

Bisphenol A (BPA) is the most used color developer in thermal paper products such as cashiers' receipts, followed by Bisphenol S (BPS), Wincon 8 (D-8), and Pergafast 201 (PF201). These chemicals can migrate from the paper onto the skin and possibly be absorbed and metabolized. Until now, D-8 and PF201 have not been analyzed in biological matrices, nor has a method been developed to simultaneously quantify them, even though they are often found as mixtures. Our aim was to develop and validate a method to quantify BPA, its glucuronide metabolite (BPA-G), BPS, D-8, and PF201 in in vitro skin absorption samples. After solid-phase extraction and reversed-phase chromatography, we quantified the substances in saline that had been in contact with human dermis for 24 h using a triple-quadrupole mass detector equipped with an electrospray ionization source. We assessed the method in three in vitro skin absorption assays using ex vivo human skin from one skin donor per test substance. The quantification ranges of our method were 0.2-200 µg/L for BPA and 0.2-20 µg/L for BPA-G, BPS, D-8, and PF201. Accuracies were within ±8% of nominal concentrations. Intra-day and total precisions (%RSD) were <10% for all analytes, except for BPA in low-concentration quality control solutions (low QCs) (12.2% and 15.5%, respectively). Overall, the process efficiency was 100-113% for all analytes, except BPS low and high QCs (80% and 71%, respectively) and BPA low QCs (134%). The absorbed dose ranged from 0.02% to 49% depending on the test substance, and was not determinable for PF201. This is the first analytical method to quantify simultaneously BPA, BPA-G, and BPA alternatives in saline from in vitro skin absorption samples.

Development and validation of a liquid chromatography/tandem mass spectrometry method to quantify metabolites of phthalates, including di-2-ethylhexyl terephthalate (DEHTP) and bisphenol A, in human urine.

RATIONALE: Several phthalates and bisphenol A are endocrine-disrupting chemicals (EDCs). Recently, their use has been partially restricted and less toxic compounds, such as di-2-ethylhexyl terephthalate (DEHTP), have been placed on the market. The aim of this work was to develop and validate a method for the simultaneous quantitation of bisphenol A and urinary metabolites of phthalates, including DEHTP. METHODS: An isotopic dilution high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method for the determination of bisphenol A (BPA), monobenzyl phthalate (MBzP), mono-2-ethyl-5-carboxypentyl phthalate (MECPP), mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP), mono-2-ethyl-5-hydroxyhexyl terephthalate (MEHHTP), monoethyl phthalate (MEP), and mono-n/i-butyl phthalates (MnBP/MiBP) in human urine was developed. A complete validation was carried out and the method was applied to 36 non-occupationally exposed adults. RESULTS: Limits of quantitation ranged from 0.02 (MECPP) to 1 µg/L (MnBP and MiBP). Relative standard deviations below 10% indicated a suitable precision; accuracy, evaluated using a standard reference material, ranged from 74.3% to 117.5%; isotopically labelled internal standards were suitable for correcting the matrix effect. The accuracy was confirmed by the successful participation in an external verification exercise. However, for terephthalates, the validation was incomplete due to the lack of reference materials and external verification. Levels of the investigated chemicals in subjects were in line with those previously reported. CONCLUSIONS: An LC/MS/MS assay for the simultaneous measurement of BPA and phthalate metabolites in human urine was developed and validated; it is useful to investigate exposure in epidemiological studies involving the general population.

Assessment of penconazole exposure in winegrowers using urinary biomarkers.

Penconazole (PEN) is a fungicide used in agriculture. The aim of this work was to evaluate the exposure to PEN in vineyard workers focusing on urinary biomarkers. Twenty-two agricultural workers were involved in the study; they were investigated during PEN applications and re-entry work, performed for 1-4 consecutive working days, for a total of 42 mixing and applications and 12 re-entries. Potential and actual dermal exposure, including hand exposure, were measured using pads and hand washes. Urine samples were collected starting before the first application, continuing during the work shift, and ending 48 h after the last shift. The determination of PEN in dermal samples and PEN metabolites in urine was performed by liquid chromatography tandem mass spectrometry. Dermal potential body exposure and actual total exposure showed median levels ranging from 18 to 3356µg and from 21 to 111 µg, respectively. Urinary monohydroxyl-derivative PEN-OH was the most abundant metabolite; its excretion rate peaked within 24 h after the work shift. In this period, median concentrations of PEN-OH and the carboxyl-derivative PEN-COOH ranged from 15.6 to 27.6 µg/L and from 2.5 to 10.2 µg/L, respectively. The concentration of PEN-OH during the work shift, in the 24 h after and in the 25-48 h after the work shift were correlated with actual body and total dermal exposure (Pearson's r from 0.279 to 0.562). Our results suggest that PEN-OH in the 24 h post-exposure urine is a promising candidate for biomonitoring PEN exposure in agricultural workers.

Determination of tebuconazole and penconazole fungicides in rat and human hair by liquid chromatography/tandem mass spectrometry.

RATIONALE: Tebuconazole (TEB) and penconazole (PEN) are widely applied fungicides and environmental contaminants; their toxicological properties include possible effects to the unborn child, therefore, the evaluation of human exposure is relevant to risk assessment. Hair is a non-invasive specimen that incorporates pollutants allowing an extended exposure window to be surveyed. The aim of this work was to develop and validate an assay for the determination of TEB and PEN in human hair. METHODS: Under optimised conditions, analytes were extracted by soaking hair in acetonitrile, in the presence of deuterated analogues, under heating and agitation. Chemical separation was achieved using a C18 reversed-phase chromatographic column and detection and quantification were performed, after positive electrospray ionization, by triple quadrupole mass spectrometry operating in selected reaction monitoring mode. RESULTS: The assay validation showed a linear dynamic range up to 5 µg/L or 200 pg/mg hair, inter- and intra-run precisions <6%, and accuracies within 5% of spiked concentrations. Limits of quantification were 0.001 µg/L or 1 pg/mg hair for both TEB and PEN. Matrix effect experiments showed that the isotope dilution approach allowed for the control of bias sources. TEB and PEN were determined in hair of rats exposed to a low dose of TEB and in hair of agricultural workers exposed to TEB and/or PEN during the application season, indicating that both chemicals are incorporated into the hair upon exposure. CONCLUSIONS: The results of this study indicate that the developed assay is useful to evaluate the exposure to TEB and PEN in humans.

Immunosuppressive drugs in whole blood: validation of a commercially available liquid chromatography/tandem mass spectrometry kit and comparison with immunochemical assays.

RATIONALE: In the determination of immunosuppressive drugs cyclosporine A (CSA), tacrolimus (TARO), sirolimus (SIRO), and everolimus (EVE) in whole blood there is an open debate about which is the best assay between immunochemistry and liquid chromatography/tandem mass spectrometry (LC/MS/MS). This work is aimed to explore this topic, focusing on the use of updated assays and the analysis of a large number of samples. METHODS: A certified in vitro diagnostic kit coupled with a medical device LC/MS/MS was validated and applied to the analysis of 1192 blood samples of patients treated with immunosuppressive drugs. The results were compared with those obtained by immunoassays. RESULTS: The LC/MS/MS approach was found to provide linear, stable, precise, and accurate results, with lower limits of quantification of 12.5, 1.1, 1.2, and 1.2 µg/L for CSA, TACRO, SIRO, and EVE, respectively. With this method 80 samples were analysed and reported within a single work shift. A correlation was observed between the LC/MS/MS and immunoassay data, with Spearman correlation coefficients of 0.980 (n = 260) for CSA, 0.836 for TACRO (n = 562), 0.898 for SIRO (n = 113), and 0.904 for EVE (n = 257). Passing-Bablock regression showed the presence of constant and proportional biases for most of the drugs. A Blond-Altman graph showed differences between the assays, with immunoassays generally overestimating the drugs. CONCLUSIONS: The LC/MS/MS certified kit was validated for the detection of immunosuppressant drugs in whole blood and it provided a high-throughput method that is consistent with the requirements of clinical laboratories. The comparison of patient data between LC/MS/MS and up-dated immunoassays shows that a significant discrepancy still exists, especially for CSA and SIRO, confirming the greater specificity associated with use of the LC/MS/MS assay Copyright © 2017 John Wiley & Sons, Ltd.

Determinants of active and environmental exposure to tobacco smoke and upper reference value of urinary cotinine in not exposed individuals.

The aims of this study were (1) to explore the behavioral and sociodemographic factors influencing urinary cotinine (COT-U) levels in active smokers and in environmental tobacco smoke (ETS)-exposed individuals, (2) to assess the specificity and sensitivity of the questionnaire for identifying active smokers and nonsmokers, and (3) to derive the upper reference value of COT-U in non-ETS exposed individuals. The COT-U levels of 495 adults (age range 18-69 years) who classified themselves as active smokers (29%) or as nonsmokers with (17%) or without (83%) ETS exposure were quantified by LC-MS-MS (quantification limit: 0.1µg/L, range of linearity: 0.1-4000µg/L). Median COT-U levels in these groups were 883, 1.38, and 0.39µg/L, respectively. Significant determinants of COT-U levels in active smokers were the number of cigarettes per day, type of smoking product, smoking environment, as well as time between the last cigarette and urine collection. Among ETS-exposed nonsmokers, significant determinants were living with smokers, being exposed to smoke at home, ETS exposure duration, as well as time between the last exposure and urine collection. When a 30-µg/L COT-U cut-off value was used to identify active daily smoking, the sensitivity and specificity of the questionnaire were 94% and 98%, respectively. For ETS exposure, the COT-U value of 1.78 (0.90 confidence interval 1.75-1.78) µg/L, corresponding to the 95th percentiles of the COT-U distribution in non-ETS-exposed participants, is proposed as upper reference value to identify environmental exposure.

Biological Monitoring of Occupational Exposure to Polycyclic Aromatic Hydrocarbons at an Electric Steel Foundry in Tunisia.

Occupational exposures during iron and steel founding have been classified as carcinogenic to humans, and the exposure to polycyclic aromatic hydrocarbons (PAHs) in this industrial setting may contribute to cancer risk. The occupational exposure to PAHs was assessed in 93 male workers at an electric steel foundry in Tunisia by biomonitoring, with the aims of characterizing the excretion profile and investigating the influence of job title and personal characteristics on the biomarkers. Sixteen 2-6 ring unmetabolized PAHs (U-PAHs) and eight hydroxylated PAH metabolites (OHPAHs) were analyzed by gas chromatography-triple quadrupole tandem mass spectrometry and liquid chromatography triple quadrupole tandem mass spectrometry, respectively. Among U-PAHs, urinary naphthalene (U-NAP) was the most abundant compound (median level: 643ng l(-1)), followed by phenanthrene (U-PHE, 18.5ng l(-1)). Urinary benzo[a]pyrene (U-BaP) level was <0.30ng l(-1) Among OHPAHs, 2-hydroxynaphthalene (2-OHNAP) was the most abundant metabolite (2.27 µg l(-1)). Median 1-hydroxypyrene (1-OHPYR) was 0.52 µg l(-1) Significant correlations among urinary biomarkers were observed, with Pearson's r ranging from 0.177 to 0.626. 1-OHPYR was correlated to benzo[a]pyrene, but not to five- and six-rings PAHs. A multiple linear regression model showed that job title was a significant determinant for almost all U-PAHs. In particular, employees in the steel smelter workshop had higher levels of high-boiling U-PAHs and lower levels of low-boiling U-PAHs than those of workers with other job titles. Among OHPAHs, this model was significant only for naphthols and 1-hydroxyphenanthrene (1-OHPHE). Smoking status was a significant predictor for almost all biomarkers. Among all analytes, U-PHE and 1-OHPHE were the less affected by tobacco smoke, and they were significantly correlated with both low- and high-molecular-weight compounds, and their levels were related to job titles, so they could be proposed as suitable biomarkers of PAH exposure at steel foundries. Based on 1-OHPYR levels, our findings show that occupational exposure of these workers was similar to that reported in recent studies of electric steel foundry workers. The multianalytic approach is useful in revealing different exposure levels among job titles.

Dermal exposure and risk assessment of tebuconazole applicators in vineyards.

INTRODUCTION: Models used in the pre-marketing evaluation do not cover all work scenarios and may over- or underestimate exposure. OBJECTIVES: Uncertainties present in the extrapolation from pre-marketing to the post-marketing warrant exposure and risk assessment in real-life working conditions. METHODS: Seven vineyard pesticide applicators were followed for a total of 12 work-days. A data collection sheet was developed specifically for this study. Workers' body exposure, hands, and head exposure were measured. Tebuconazole was analyzed using LC-MS/MS. RESULTS: Median potential and actual body exposures were 22.41 mg/kg and 0.49 mg/kg of active substance applied, respectively. The median protection factor provided by the coverall was 98% (range: 90-99%). Hand exposure was responsible for 61% of total actual exposure, and was reduced by more than 50% in workers using gloves. The German Model underestimated the exposure in one work-day, and grossly overestimated it in 3 work-days. CONCLUSIONS: High levels of potential body exposure were efficiently controlled by the cotton coverall. Use of personal protective devices, especially chemically-resistant gloves and head cover is the main determinant of skin protection. Field studies on pesticide exposure in real-life conditions and development of methods and tools for easier risk assessment are necessary to complement and confirm the risk assessment done in the authorization process.

Metabolomic profiling of Prader-Willi syndrome compared with essential obesity.

Introduction: Prader-Willi syndrome (PWS) is a rare disease, which shows a peculiar clinical phenotype, including obesity, which is different from essential obesity (EOB). Metabolomics might represent a valuable tool to reveal the biochemical mechanisms/pathways underlying clinical differences between PWS and EOB. The aim of the present (case-control, retrospective) study was to determine the metabolomic profile that characterizes PWS compared to EOB. Methods: A validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) targeted metabolomic approach was used to measure a total of 188 endogenous metabolites in plasma samples of 32 patients with PWS (F/M = 23/9; age: 31.6 ± 9.2 years; body mass index [BMI]: 42.1 ± 7.0 kg/m2), compared to a sex-, age- and BMI-matched group of patients with EOB (F/M = 23/9; age: 31.4 ± 6.9 years; BMI: 43.5 ± 3.5 kg/m2). Results: Body composition in PWS was different when compared to EOB, with increased fat mass and decreased fat-free mass. Glycemia and HDL cholesterol were higher in patients with PWS than in those with EOB, while insulinemia was lower, as well as heart rate. Resting energy expenditure was lower in the group with PWS than in the one with EOB, a difference that was missed after fat-free mass correction. Carrying out a series of Tobit multivariable linear regressions, adjusted for sex, diastolic blood pressure, and C reactive protein, a total of 28 metabolites was found to be associated with PWS (vs. non-PWS, i.e., EOB), including 9 phosphatidylcholines (PCs) ae, 5 PCs aa, all PCs aa, 7 lysoPCs a, all lysoPCs, 4 acetylcarnitines, and 1 sphingomyelin, all of which were higher in PWS than EOB. Conclusions: PWS exhibits a specific metabolomic profile when compared to EOB, suggesting a different regulation of some biochemical pathways, fundamentally related to lipid metabolism.

High-throughput determination of cortisol, cortisone, and melatonin in oral fluid by on-line turbulent flow liquid chromatography interfaced with liquid chromatography/tandem mass spectrometry.

RATIONALE: Cortisol, cortisone, and melatonin (CORTol, CORTone, and MELA, respectively) are hormones related to stress and sleep disorders. Their detection is relevant to epidemiological studies aimed at investigating the effects of circadian cycle disruption. The aim of this study was to develop and evaluate a high-throughput assay for the detection of CORTol, CORTone, and MELA concentrations in non-invasively collected oral fluid samples. METHODS: A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to measure levels of CORTol, CORTone, and MELA in oral fluid samples in the presence of deuterated analogs was optimized and validated. A 50 µL aliquot of oral fluid sample, obtained by centrifugation of a chewed swab, was purified using on-line turbulent flow liquid chromatography. Analytes were then separated using C18 reversed-phase chromatography, subjected to positive ionization using an electrospray source, then quantitated using a triple quadrupole mass detector in the selected reaction monitoring mode. RESULTS: Limits of quantification and linear dynamic ranges were found to be 0.55 nmol/L, 5.5 nmol/L, and 0.004 nmol/L, and up to 28 nmol/L, 277 nmol/L, and 0.43 nmol/L for CORTol, CORTone, and MELA, respectively. Inter- and intra-run precisions as relative standard deviation values were <5%, and accuracies were within 95-106% of theoretical concentrations. An evaluation of matrix effects showed that the use of deuterated analogs controlled sources of bias. Furthermore, the total analysis time per sample was 13 min, resulting in a throughput of approximately 100 samples/day. CONCLUSIONS: To our knowledge, this is the first automated, high-throughput assay for the simultaneous quantification of CORTol, CORTone, and MELA in oral fluid specimens.

Effects of the therapy shift from cortisone acetate to modified-release hydrocortisone in a group of patients with adrenal insufficiency.

Objective: Patients with adrenal insufficiency (AI) may be exposed to supraphysiological glucocorticoids levels during standard treatment with cortisone acetate (CA) or immediate-release hydrocortisone (IR-HC). Recent studies, predominantly including patients in IR-HC treatment, suggested that modified-release hydrocortisone (MRH) provide a more physiological cortisol rhythm, improving metabolic control and quality of life. Our primary aim was to assess clinical and biochemical modifications in patients shifted from CA to MRH. Design/Methods: We designed a retrospective longitudinal study, enrolling 45 AI patients (22 primary and 23 secondary AI) treated exclusively with CA thrice daily, shifted to MRH once daily; 29/45 patients concluded at least 18-months follow-up (MRH-group). We recruited 35 AI patients continuing CA as a control group (CA-group). Biochemical and clinical data, including metabolic parameters, bone quality, and symptoms of under- or overtreatment were collected. In 24 patients, a daily salivary cortisol curve (SCC) performed before and one month after shifting to MRH was compared to healthy subjects (HS). Results: No significant changes in glycometabolic and bone parameters were observed both in MRH and CA-groups during a median follow-up of 35 months. A more frequent decrease in blood pressure values (23.1% vs 2.8%, p=0.04) and improvement of under- or overtreatment symptoms were observed in MRH vs CA-group. The SCC showed a significant steroid overexposure in both CA and MRH-groups compared to HS [AUC (area under the curve) = 74.4 ± 38.1 nmol×hr/L and 94.6 ± 62.5 nmol×hr/L respectively, vs 44.1 ± 8.4 nmol×hr/L, p<0.01 for both comparisons], although SCC profile was more similar to HS in MRH-group. Conclusions: In our experience, patients shifted from CA to equivalent doses of MRH do not show significant glycometabolic modifications but blood pressure control and symptoms of over-or undertreatment may improve. The lack of amelioration in glucose metabolism and total cortisol daily exposure could suggest the need for a dose reduction when shifting from CA to MRH, due to their different pharmacokinetics.

Metabolomic profiles in night shift workers: A cross-sectional study on hospital female nurses.

Background and aim: Shift work, especially including night shifts, has been found associated with several diseases, including obesity, diabetes, cancers, and cardiovascular, mental, gastrointestinal and sleep disorders. Metabolomics (an omics-based methodology) may shed light on early biological alterations underlying these associations. We thus aimed to evaluate the effect of night shift work (NSW) on serum metabolites in a sample of hospital female nurses. Methods: We recruited 46 nurses currently working in NSW in Milan (Italy), matched to 51 colleagues not employed in night shifts. Participants filled in a questionnaire on demographics, lifestyle habits, personal and family health history and work, and donated a blood sample. The metabolome was evaluated through a validated targeted approach measuring 188 metabolites. Only metabolites with at least 50% observations above the detection limit were considered, after standardization and log-transformation. Associations between each metabolite and NSW were assessed applying Tobit regression models and Random Forest, a machine-learning algorithm. Results: When comparing current vs. never night shifters, we observed lower levels of 21 glycerophospholipids and 6 sphingolipids, and higher levels of serotonin (+171.0%, 95%CI: 49.1-392.7), aspartic acid (+155.8%, 95%CI: 40.8-364.7), and taurine (+182.1%, 95%CI: 67.6-374.9). The latter was higher in former vs. never night shifters too (+208.8%, 95%CI: 69.2-463.3). Tobit regression comparing ever (i.e., current + former) and never night shifters returned similar results. Years worked in night shifts did not seem to affect metabolite levels. The Random-Forest algorithm confirmed taurine and aspartic acid among the most important variables in discriminating current vs. never night shifters. Conclusions: This study, although based on a small sample size, shows altered levels of some metabolites in night shift workers. If confirmed, our results may shed light on early biological alterations that might be related to adverse health effects of NSW.

Progestin-primed ovarian stimulation for fertility preservation in women with cancer: A comparative study.

BACKGROUND: In women scheduled for cancer treatment, oocytes cryopreservation is a well-established procedure. Random start protocols have been a substantial improvement in this setting, allowing to prevent delay in the initiation of cancer treatments. However, there is still the need to optimize the regimen of ovarian stimulation, to make treatments more patient-friendly and to reduce costs. METHODS: This retrospective study compares two periods (2019 and 2020), corresponding to two different ovarian stimulation regimens. In 2019, women were treated with corifollitropin, recombinant FSH and GnRH antagonists. Ovulation was triggered with GnRH agonists. In 2020, the policy changed, and women were treated with a progestin-primed ovarian stimulation (PPOS) protocol with human menopausal gonadotropin (hMG) and dual trigger (GnRH agonist and low dose hCG) Continuous data are reported as median [Interquartile Range]. To overcome expected changes in baseline characteristics of the women, the primary outcome was the ratio between the number of mature oocytes retrieved and serum anti-mullerian hormone (AMH) in ng/ml. RESULTS: Overall, 124 women were selected, 46 in 2019 and 78 in 2020. The ratio between the number of mature oocytes retrieved and serum AMH in the first and second period was 4.0 [2.3-7.1] and 4.0 [2.7-6.8], respectively (p = 0.80). The number of scans was 3 [3-4] and 3 [2-3], respectively (p<0.001). The total costs of the drugs used for ovarian stimulation were 940 € [774-1,096 €] and 520 € [434-564 €], respectively (p<0.001). CONCLUSIONS: Random start PPOS with hMG and dual trigger represents an easy and affordable ovarian stimulation protocol for fertility preservation in women with cancer, showing similar efficacy and being more friendly and economical.