Occupational exposure to environmental tobacco smoke in hospitality venues: are genetic- or proteomics-based biomarkers predictive of respiratory diseases?

Environmental tobacco smoke (ETS) is recognized as an occupational hazard in the hospitality industry. Although Portuguese legislation banned smoking in most indoor public spaces, it is still allowed in some restaurants/bars, representing a potential risk to the workers’ health, particularly for chronic respiratory diseases. The aims of this work were to characterize biomarkers of early genetic effects and to disclose proteomic signatures associated to occupational exposure to ETS and with potential to predict respiratory diseases development. A detailed lifestyle survey and clinical evaluation (including spirometry) were performed in 81 workers from Lisbon restaurants. ETS exposure was assessed through the level of PM 2.5 in indoor air and the urinary level of cotinine. The plasma samples were immunodepleted and analysed by 2D-SDSPAGE followed by in-gel digestion and LC-MS/MS. DNA lesions and chromosome damage were analysed innlymphocytes and in exfoliated buccal cells from 19 cigarette smokers, 29 involuntary smokers, and 33 non-smokers not exposed to tobacco smoke. Also, the DNA repair capacity was evaluated using an ex vivo challenge comet assay with an alkylating agent (EMS). All workers were considered healthy and recorded normal lung function. Interestingly, following 2D-DIGE-MS (MALDI-TOF/TOF), 61 plasma proteins were found differentially expressed in ETS-exposed subjects, including 38 involved in metabolism, acute-phase respiratory inflammation, and immune or vascular functions. On the other hand, the involuntary smokers showed neither an increased level of DNA/chromosome damage on lymphocytes nor an increased number of micronuclei in buccal cells, when compared to non-exposed non-smokers. Noteworthy, lymphocytes challenge with EMS resulted in a significantly lower level of DNA breaks in ETS-exposed as compared to non-exposed workers (P<0.0001) suggestive of an adaptive response elicited by the previous exposure to low levels of ETS. Overall, changes in proteome may be promising early biomarkers of exposure to ETS. Likewise, alterations of the DNA repair competence observed upon ETS exposure deserves to be further understood. Work supported by Fundação Calouste Gulbenkian, ACSS and FCT/Polyannual Funding Program.

Application of mathematical models to mycotoxins children risk assessment: a case study of Portuguese children exposure to co-occurring mycotoxins in processed cereal-based foods

People, animals and the environment can be exposed to multiple chemicals at once from a variety of sources, but current risk assessment is usually carried out based on one chemical substance at a time. In human health risk assessment, ingestion of food is considered a major route of exposure to many contaminants, namely mycotoxins, a wide group of fungal secondary metabolites that are known to potentially cause toxicity and carcinogenic outcomes. Mycotoxins are commonly found in a variety of foods including those intended for consumption by infants and young children and have been found in processed cereal-based foods available in the Portuguese market. The use of mathematical models, including probabilistic approaches using Monte Carlo simulations, constitutes a prominent issue in human health risk assessment in general and in mycotoxins exposure assessment in particular. The present study aims to characterize, for the first time, the risk associated with the exposure of Portuguese children to single and multiple mycotoxins present in processed cereal-based foods (CBF). Portuguese children (0-3 years old) food consumption data (n=103) were collected using a 3 days food diary. Contamination data concerned the quantification of 12 mycotoxins (aflatoxins, ochratoxin A, fumonisins and trichothecenes) were evaluated in 20 CBF samples marketed in 2014 and 2015 in Lisbon; samples were analyzed by HPLC-FLD, LC-MS/MS and GC-MS. Daily exposure of children to mycotoxins was performed using deterministic and probabilistic approaches. Different strategies were used to treat the left censored data. For aflatoxins, as carcinogenic compounds, the margin of exposure (MoE) was calculated as a ratio of BMDL (benchmark dose lower confidence limit) to the aflatoxin exposure. The magnitude of the MoE gives an indication of the risk level. For the remaining mycotoxins, the output of exposure was compared to the dose reference values (TDI) in order to calculate the hazard quotients (ratio between exposure and a reference dose, HQ). For the cumulative risk assessment of multiple mycotoxins, the concentration addition (CA) concept was used. The combined margin of exposure (MoET) and the hazard index (HI) were calculated for aflatoxins and the remaining mycotoxins, respectively. 71% of CBF analyzed samples were contaminated with mycotoxins (with values below the legal limits) and approximately 56% of the studied children consumed CBF at least once in these 3 days. Preliminary results showed that children exposure to single mycotoxins present in CBF were below the TDI. Aflatoxins MoE and MoET revealed a reduced potential risk by exposure through consumption of CBF (with values around 10000 or more). HQ and HI values for the remaining mycotoxins were below 1. Children are a particularly vulnerable population group to food contaminants and the present results point out an urgent need to establish legal limits and control strategies regarding the presence of multiple mycotoxins in children foods in order to protect their health. The development of packaging materials with antifungal properties is a possible solution to control the growth of moulds and consequently to reduce mycotoxin production, contributing to guarantee the quality and safety of foods intended for children consumption.

Firefighters' exposure biomonitoring: Impact of firefighting activities on levels of urinary monohydroxyl metabolites

The concentrations of six urinary monohydroxyl metabolites (OH-PAHs) of polycyclic aromatic hydrocarbons, namely 1-hydroxynaphthalene, 1-hydroxyacenaphthene, 2-hydroxyfluorene, 1-hydroxyphenanthrene, 1-hydroxypyrene (1OHPy), and 3-hydroxybenzo[a]pyrene, were assessed in the post-shift urine of wildland firefighters involved in fire combat activities at six Portuguese fire corporations, and compared with those of non-exposed subjects. Overall, median levels of urinary individual and total OH-PAHs (ΣOH-PAHs) suggest an increased exposure to polycyclic aromatic hydrocarbons during firefighting activities with ΣOH-PAH levels in exposed firefighters 1.7-35 times higher than in non-exposed ones. Urinary 1-hydroxynaphthalene and/or 1-hydroxyacenapthene were the predominant compounds, representing 63-98% of ΣOH-PAHs, followed by 2-hydroxyfluorene (1-17%), 1-hydroxyphenanthrene (1-13%), and 1OHPy (0.3-10%). A similar profile was observed when gender discrimination was considered. Participation in fire combat activities promoted an increase of the distribution percentage of 1-hydroxynaphthalene and 1-hydroxyacenaphthene, while contributions of 1-hydroxyphenanthrene and 1OHPy decreased. The detected urinary 1OHPy concentrations (1.73×10(-2) to 0.152μmol/mol creatinine in exposed subjects versus 1.21×10(-2) to 5.44×10(-2)μmol/mol creatinine in non-exposed individuals) were lower than the benchmark level (0.5μmol/mol creatinine) proposed by the American Conference of Governmental Industrial Hygienists. This compound, considered the biomarker of exposure to PAHs, was the less abundant one from the six analyzed biomarkers. Thus the inclusion of other metabolites, in addition to 1OHPy, in future studies is suggested to better estimate firefighters' occupational exposure to PAHs. Moreover, strong to moderate Spearman correlations were observed between individual compounds and ΣOH-PAHs corroborating the prevalence of an emission source.

Polycyclic aromatic hydrocarbons at fire stations: firefighters’ exposure monitoring and biomonitoring, and assessment of the contribution to total internal dose

This work characterizes levels of eighteen polycyclic aromatic hydrocarbons (PAHs) in the breathing air zone of firefighters during their regular work shift at eight Portuguese fire stations, and the firefighters' total internal dose by six urinary monohydroxyl metabolites (OH-PAHs). Total PAHs (ΣPAHs) concentrations varied widely (46.4-428ng/m(3)), mainly due to site specificity (urban/rural) and characteristics (age and layout) of buildings. Airborne PAHs with 2-3 rings were the most abundant (63.9-95.7% ΣPAHs). Similarly, urinary 1-hydroxynaphthalene and 1-hydroxyacenaphthene were the predominant metabolites (66-96% ΣOH-PAHs). Naphthalene contributed the most to carcinogenic ΣPAHs (39.4-78.1%) in majority of firehouses; benzo[a]pyrene, the marker of carcinogenic PAHs, accounted with 1.5-10%. Statistically positive significant correlations (r≥0.733, p≤0.025) were observed between ΣPAHs and urinary ΣOH-PAHs for firefighters of four fire stations suggesting that, at these sites, indoor air was their major exposure source of PAHs. Firefighter's personal exposure to PAHs at Portuguese fire stations were well below the existent occupational exposure limits. Also, the quantified concentrations of post-shift urinary 1-hydroxypyrene in all firefighters were clearly lower than the benchmark level (0.5μmol/mol) recommended by the American Conference of Governmental Industrial Hygienists.