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.

Prenatal and childhood exposure to per-/polyfluoroalkyl substances (PFASs) and its associations with childhood overweight and/or obesity: a systematic review with meta-analyses.

BACKGROUND: Per-/polyfluoroalkyl substances (PFASs) are persistent organic pollutants and suspected endocrine disruptors. OBJECTIVE: The aim of this work was to conduct a systematic review with meta-analysis to summarise the associations between prenatal or childhood exposure to PFASs and childhood overweight/obesity. METHODS: The search was performed on the bibliographic databases PubMed and Embase with text strings containing terms related to prenatal, breastfeeding, childhood, overweight, obesity, and PFASs. Only papers describing a biomonitoring study in pregnant women or in children up to 18 years that assessed body mass index (BMI), waist circumference (WC), or fat mass in children were included. When the estimates of the association between a PFAS and an outcome were reported from at least 3 studies, a meta-analysis was conducted; moreover, to correctly compare the studies, we developed a method to convert the different effect estimates and made them comparable each other. Meta-analyses were performed also stratifying by sex and age, and sensitivity analyses were also performed. RESULTS: In total, 484 and 779 articles were retrieved from PubMed and Embase, respectively, resulting in a total of 826 articles after merging duplicates. The papers included in this systematic review were 49: 26 evaluating prenatal exposure to PFASs, 17 childhood exposure, and 6 both. Considering a qualitative evaluation, results were conflicting, with positive, negative, and null associations. 30 papers were included in meta-analyses (19 prenatal, 7 children, and 4 both). Positive associations were evidenced between prenatal PFNA and BMI, between PFOA and BMI in children who were more than 3 years, and between prenatal PFNA and WC. Negative associations were found between prenatal PFOS and BMI in children who were 3 or less years, and between PFHxS and risk of overweight. Relatively more consistent negative associations were evidenced between childhood exposure to three PFASs (PFOA, PFOS, and PFNA) and BMI, in particular PFOS in boys. However, heterogeneity among studies was high. CONCLUSION: Even though heterogeneous across studies, the pooled evidence suggests possible associations, mostly positive, between prenatal exposure to some PFASs and childhood BMI/WC; and relatively stronger evidence for negative associations between childhood exposure to PFASs and childhood BMI.

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.

Cardiogenic Shock Integrated PHenotyping for Event Reduction: A Pilot Metabolomics Analysis.

Cardiogenic shock (CS) portends a dismal prognosis if hypoperfusion triggers uncontrolled inflammatory and metabolic derangements. We sought to investigate metabolomic profiles and temporal changes in IL6, Ang-2, and markers of glycocalyx perturbation from admission to discharge in eighteen patients with heart failure complicated by CS (HF-CS). Biological samples were collected from 18 consecutive HF-CS patients at admission (T0), 48 h after admission (T1), and at discharge (T2). ELISA analytical techniques and targeted metabolomics were performed Seven patients (44%) died at in-hospital follow-up. Among the survivors, IL-6 and kynurenine were significantly reduced at discharge compared to baseline. Conversely, the amino acids arginine, threonine, glycine, lysine, and asparagine; the biogenic amine putrescine; multiple sphingolipids; and glycerophospholipids were significantly increased. Patients with HF-CS have a metabolomic fingerprint that might allow for tailored treatment strategies for the patients' recovery or stabilization.

Ricerca scientifica ed educazione ambientale: l'approccio partecipativo dei progetti MAPS-MI nelle scuole di Milano.

The accumulated scientific evidence regarding the effects of air pollution on health calls for urgent and effective action to protect the most vulnerable part of the population, such as children and the elderly. In this context, the participatory approach represents a viable option to empower citizens and increase their level of awareness and participation. The involvement of civil society is a potential ally in tackling the still open research challenges. The MAPS-MI 2018-2019 Mapping air pollution in the catchment area of an elementary school of Milan and MAPS-MI 2022-2023 Mobility, environment and participation in the schools of Milan projects are here presented.The main targets of the projects are primary and middle school children, their parents and teachers, with some activities extended to the entire citizenship. Starting from the survey on parents' habits and opinions about mobility and air pollution, to the air quality and personal exposure assessment, and the field-testing and development of an environmental education module, the MAPS-MI approach aims to increase knowledge and awareness about air pollution and to involve stakeholders in experimenting practices towards change. The first results suggest that adopting a participatory approach in the fields of exposure science and environmental epidemiology is a winning choice in terms of quality research, participation and community impact.

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.

Development and Application of an LC-MS/MS Untargeted Exposomics Method with a Separated Pooled Quality Control Strategy.

Pooled quality controls (QCs) are usually implemented within untargeted methods to improve the quality of datasets by removing features either not detected or not reproducible. However, this approach can be limiting in exposomics studies conducted on groups of exposed and nonexposed subjects, as compounds present at low levels only in exposed subjects can be diluted and thus not detected in the pooled QC. The aim of this work is to develop and apply an untargeted workflow for human biomonitoring in urine samples, implementing a novel separated approach for preparing pooled quality controls. An LC-MS/MS workflow was developed and applied to a case study of smoking and non-smoking subjects. Three different pooled quality controls were prepared: mixing an aliquot from every sample (QC-T), only from non-smokers (QC-NS), and only from smokers (QC-S). The feature tables were filtered using QC-T (T-feature list), QC-S, and QC-NS, separately. The last two feature lists were merged (SNS-feature list). A higher number of features was obtained with the SNS-feature list than the T-feature list, resulting in identification of a higher number of biologically significant compounds. The separated pooled QC strategy implemented can improve the nontargeted human biomonitoring for groups of exposed and nonexposed subjects.

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.

A systematic review on biomonitoring of individuals living near or working at solid waste incinerator plants.

Background: Solid waste incinerators (SWI) emit several pollutants among which polychlorodibenzodioxins and furans (PCDD/Fs), polychlorobiphenyls, metals, monocyclic and polycyclic aromatic hydrocarbons (PAHs).Aim of the study: To present a systematic review of peer-reviewed literature on human biological monitoring of exposure and effect following potential exposure to SWI pollutants to bring together evidences and to highlight strengths and deficiencies of the studies conducted so far.Methods: Relevant studies on biomonitoring of individuals living near or working at SWIs were selected through three steps: (1) a literature search in the Medline, CAplus, and Embase database; (2) the retrieved abstracts were screened by four independent reviewers; (3) the full text of the relevant papers was read, papers were pooled in studies, and then analyzed to highlight strengths and weaknesses. Studies with the strongest epidemiological design and/or the largest sample size were identified as reference studies.Results: One hundred and thirty-two papers, pooled in 82 studies, were included in the review: 67 on general population, 52 on SWI workers, and 14 on both groups. The most frequently investigated biomarkers were PCDD/Fs in plasma (87). Several studies presented limitations, such as a small samples size, scarce information on confounders, and a poor statistical analysis. Some earlier studies showed an increase of PCDD/Fs, lead, and PAHs in individuals (mainly workers) exposed to emissions from old SWIs; studies from the year 2000 showed no increase of biomarkers or biomarkers within the range of the general population; decreasing trends were observed in prospective studies.Conclusions: Most studies presented methodological pitfalls; reference studies showed no or a limited evidence of the impact of SWI on exposure and effect biomarkers.

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.

The first steps of industrial hygiene and occupational toxicology at the Clinica del Lavoro di Milano under the guidance of Luigi Devoto.

INTRODUCTION: The Clinica del Lavoro, the first clinic for occupational diseases of the world, was inaugurated in Milan on 20 March 1910; its first director was Luigi Devoto, who was in charge until 1935. The purpose of this work is to review the activities of industrial hygiene and toxicology carried out at the Clinica del Lavoro under the guidance of Devoto. METHODS: Documents published by the Istituti Clinici di Perfezionamento, a group of clinics of which the Clinica del Lavoro was part, record the birth and organization of this structure and the presence of a laboratory of chemistry; documents by Devoto and other authors were also retrieved to extrapolate specific information on activities of industrial hygiene and toxicology. RESULTS: The Clinica del Lavoro, at the time of its inauguration, included four laboratories: of chemistry, clinical physics, histopathology and bacteriology. The chemistry lab was located on the first floor and was composed of 6 well-lit rooms, modernly equipped with work benches that could accommodate 12 people. In Devoto's view, the chemistry laboratory, supported by that of clinical physics, had to assess the toxicological properties of chemicals commonly found in the workplace and to reveal the mechanisms of induction of damage to humans. In the first 30 years of activity, the Clinica del Lavoro investigated various diseases deriving from exposure to chemical agents, including saturnism, or lead intoxication, mercurialism, phosphorism, benzolism, sulfocarbonism, dust diseases. Several assays were developed and applied to measure toxicants in different biological and environmental mean as evidenced by scientific publications starting from 1920. CONCLUSION: In Devoto's view, industrial hygiene and toxicology were essential tools for the research and prevention of occupational diseases since the first years of activity of the Clinica del Lavoro.

The birth of modern industrial hygiene and occupational toxicology. A historical reconstruction through the analysis of the contributions of three protagonists.

BACKGROUND: The Clinica del Lavoro of Milan provided several contributions to industrial hygiene and occupational toxicology during the twentieth century. OBJECTIVES: Describe the first years of the laboratory of industrial hygiene of Milan through three figures who played a leading role: Enrico Carlo Vigliani, Nicola Zurlo and Gianmario Cavagna. METHODS: Scientific literature of the period 1948-1970 was investigated, also interviewing first-hand witnesses of that period. RESULTS: Enrico Vigliani was the first European scholar to understand the importance of a laboratory of industrial hygiene within his institution. Thanks to the support of private (Montecatini) and public (INAIL) institutions he succeeded in creating a laboratory in 1948. Nicola Zurlo, who directed this structure in the first thirty years, conducted innovative studies on chronic mercury intoxication, lead intoxication and silicosis, designing and creating instruments for capturing and analyzing atmospheric dust and protection devices. He conducted analysis of the health effects of organophosphorus insecticides and started to study the air pollution. Zurlo also provided an epistemological and methodological content to the discipline. Gianmario Cavagna, one of the first Italian toxicologists, contributed to the discovery of the origin of fevers caused by the inhalation of metal fumes and to the studies on the pathogenesis of byssinosis, hypothesizing a role of bacterial endotoxins in the genesis of this disease. CONCLUSIONS: The contributions provided by these three protagonists to industrial hygiene and occupational toxicology were relevant and made in those years the Clinica del Lavoro of Milan as a landmark, not only in Italy but also abroad.

Benzene and leukemia: from scientific evidence to regulations. A historical example.

BACKGROUND: Benzene is a highly flammable, highly volatile liquid aromatic hydrocarbon. It has been used in many industrial processes as a solvent or a starting material. At the beginning of the twentieth century, it was very widely used in the workplace, especially in printing and in the shoe manufacturing and rubber industries. Although benzene was first recognized to cause aplastic anemia, its association with leukemia has been investigated only since the 1930s. In 1963, Italy was one of the first countries in the world to adopt a law to ban benzene as a solvent in work activities. OBJECTIVES: This study analyzed the contribution of the Clinica del Lavoro in Milan, Italy, to studies of the relationship between exposure to benzene and leukemia. METHODS: Scientific literature and historical sources on benzene and leukemia in the twentieth century were reviewed, and interviews with a first-hand witness of that period were conducted. RESULTS: By 1928, several scholars had reported anecdotal cases of leukemia among workers exposed to benzene. Enrico Vigliani was the first to collect all of these cases and to try to conduct statistical analysis on these data, in order to support the association between benzene and leukemia. In the 1960s, Vigliani and Alessandra Forni showed that benzene could cause chromosome aberrations in the bone marrow that could produce leukemic clones. CONCLUSIONS: As a result of these studies and the subsequent regulations which banned benzene, exposure conditions changed in the workplace in the last few decades. The resulting low concentrations have prompted researchers to investigate new exposure biomarkers and to study any related health problems.

Digital PCR (dPCR) analysis reveals that the homozygous c.315-48T>C variant in the FECH gene might cause erythropoietic protoporphyria (EPP).

Alterations in the ferrochelatase gene (FECH) are the basis of the phenotypic expressions in erythropoietic protoporphyria. The phenotype is due to the presence of a mutation in the FECH gene associated in trans to the c.315-48 T > C variant in the intron 3. The latter is able to increase the physiological quota of alternative splicing events in the intron 3. Other two variants in the FECH gene (c.1-252A > G and c.68-23C > T) have been found to be associated to the intron 3 variant in some populations and together, they constitute a haplotype (ACT/GTC), but eventually, their role in the alternative splicing event has never been elucidated. The absolute number of the aberrantly spliced FECH mRNA molecules and the absolute expression of the FECH gene were evaluated by digital PCR technique in a comprehensive cohort. The number of splicing events that rose in the presence of the c.315-48 T > C variant, both in the heterozygous and homozygous condition was reported for the first time. Also, the percentage of the inserted FECH mRNA increased, even doubled in the T/C cases, compared to T/T cases. The constant presence of variants in the promoter and intron 2 did not influence or modulate the aberrant splicing. The results of FECH gene expression suggested that the homozygosity for the c.315-48 T > C variant could be considered pathological. Thus, this study identified the homozygotes for the c.315-48 T > C variant as pathological. By extension, when the samples were categorised according to the haplotypes, the GTC haplotype in homozygosis was pathological.

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.