Monitoring of Polycyclic Aromatic Hydrocarbon Levels in Mussels (Mytilus galloprovincialis) from Aquaculture Farms in Central Macedonia Region, Greece, Using Gas Chromatography-Tandem Mass Spectrometry Method.

A new sensitive and selective gas chromatography tandem mass spectrometry (GC-MS/MS) method was developed for the analysis of 26 polycyclic aromatic hydrocarbons (PAHs), including 16 Environmental Protection Agency (EPA) and 15 + 1 European Union (EU) PAHs, in mussel samples from aquaculture farms in Thermaikos and Strymonian Gulf, Central Macedonia Region, in three sampling periods. Concentrations were found at moderate to low values at all sampling sites, without exceeding maximum levels set by EU. Low molecular weight PAHs were predominant in all samples. Seasonal variation of the concentrations was observed; values were slightly higher in the winter period. Use of diagnostic ratios for potential sources of PAHs showed both petrogenic and pyrolitic origin. In comparison to other related studies of mussels from the Mediterranean Sea, Greek mussels cultivated in the studied gulfs are low in contaminants due to minimal environmental pollution effects. Low concentrations of PAHs are in compliance with the low values of other POPs which were found in the mussels.

Preliminary assessment of general population exposure to perfluoroalkyl substances through diet in Greece.

Food and drinking water intake are the major routes of exposure to perfluoroalkyl substances (PFASs) for humans. In this study an initial oral exposure assessment of the non-occupationally exposed general Greek population to PFASs is presented. Levels of PFOA and PFOS determined in the present study in 128 food items from the Greek market and previously published analytical results of 43 water samples and 76 egg samples from Greece were combined with food availability data of the DAFNE-ANEMOS database for the calculation of oral intake of PFASs. Fish form the food group with the highest concentration in both PFOA and PFOS. For the general Greek population the estimated average daily intake in PFOA and PFOS when applying the lowerbound mean concentrations in food and drinking water samples is 0.49 and 0.91 ng kg-1 body weight (b.w.) respectively. The main contributor to oral PFAS intake is fish. A pharmacokinetic model is applied to estimate total intake from blood serum levels reported previously and the predicted values are in agreement with lowerbound estimated values. Weekly intake estimated values are below the tolerable weekly intake recently proposed by EFSA.

Prenatal exposure to PFOS and PFOA in a pregnant women cohort of Catalonia, Spain.

This study was aimed at assessing the prenatal exposure to perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) in a cohort of pregnant women living in Reus (Tarragona County, Catalonia, Spain). These chemicals were biomonitored in maternal plasma during the first trimester of pregnancy, at delivery, and in cord blood. The dietary exposure of PFOS and PFOA was estimated by using questionnaires of food frequency and water intake, as well as data on food levels previously reported in the same area. In addition, the exposure through air inhalation and indoor dust ingestion was also calculated. Finally, a physiologically-based pharmacokinetic (PBPK) model was applied in order to establish the prenatal exposure of the fetus/child and to adjust exposure assessment vs. biomonitoring results. Probabilistic calculations of fetal exposure were performed by forward internal dosimetry and Monte-Carlo simulation. Mean plasma levels of PFOA were 0.45, 0.13 and 0.12 ng/mL at the first trimester, at delivery and in cord plasma, while those of PFOS were 2.93, 2.21, and 1.17 ng/mL, respectively. Traces of PFOS were found in all samples in the trimester and at delivery, and almost in all cord blood samples. Transplacental transfers of PFOS and PFOA were estimated to be around 70% and 60%, respectively. A temporal decrease trend in plasma levels of PFOS and PFOA was noticed, when comparing current values with data obtained 10 years ago in the same area. In agreement with many other studies, dietary intake was the main route of exposure to PFOS and PFOA in our cohort of pregnant women. It is an important issue to establish the exposure in critical windows periods such as fetal development to perfluoroalkylated substances, but also to other endocrine disrupting chemicals.

Biomarkers of exposure in environment-wide association studies - Opportunities to decode the exposome using human biomonitoring data.

BACKGROUND: The European Union's 7th Framework Programme (EU's FP7) project HEALS - Health and Environment-wide Associations based on Large Population Surveys - aims a refinement of the methodology to elucidate the human exposome. Human biomonitoring (HBM) provides a valuable tool for understanding the magnitude of human exposure from all pathways and sources. However, availability of specific biomarkers of exposure (BoE) is limited. OBJECTIVES: The objective was to summarize the availability of BoEs for a broad range of environmental stressors and exposure determinants and corresponding reference and exposure limit values and biomonitoring equivalents useful for unraveling the exposome using the framework of environment-wide association studies (EWAS). METHODS: In a face-to-face group discussion, scope, content, and structure of the HEALS deliverable "Guidelines for appropriate BoE selection for EWAS studies" were determined. An expert-driven, distributed, narrative review process involving around 30 individuals of the HEALS consortium made it possible to include extensive information targeted towards the specific characteristics of various environmental stressors and exposure determinants. From the resulting 265 page report, targeted information about BoE, corresponding reference values (e.g., 95th percentile or measures of central tendency), exposure limit values (e.g., the German HBM I and II values) and biomonitoring equivalents (BEs) were summarized and updated. RESULTS: 64 individual biological, chemical, physical, psychological and social environmental stressors or exposure determinants were included to fulfil the requirements of EWAS. The list of available BoEs is extensive with a number of 135; however, 12 of the stressors and exposure determinants considered do not leave any measurable specific substance in accessible body specimens. Opportunities to estimate the internal exposure stressors not (yet) detectable in human specimens were discussed. CONCLUSIONS: Data about internal exposures are useful to decode the exposome. The paper provides extensive information for EWAS. Information included serves as a guideline - snapshot in time without any claim to comprehensiveness - to interpret HBM data and offers opportunities to collect information about the internal exposure of stressors if no specific BoE is available.

Systematic investigation of organochlorine pesticides and polychlorinated biphenyls blood levels in Greek children from the Rhea birth cohort suggests historical exposure to DDT and through diet to DDE.

The blood levels of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) have been thoroughly investigated in Greek children from the Rhea birth cohort study. This investigation aimed to assess exposure levels, explore their possible relationship with children's age and sex, and indicate potential sources of exposure. Exposure patterns and common sources of PCBs and OCPs were analyzed using bivariate and multivariate statistics. A total of 947 blood samples from study participants were analyzed for OCP and PCB exposure, with 375 samples collected at 4 years old, 239 at 6.5 years old, and 333 at 11 years old. Elevated levels of DDE were observed in 6.5-year-old children compared to corresponding levels in other European countries. Higher levels of DDE were found in 4-year-old children, with the lowest concentrations in the 11-year-old group. The DDT/DDE ratio was consistently less than 1 among all the examined subjects. These results indicate exposure to DDT and DDE both in utero and through breastfeeding and dietary intake. For the entire cohort population, the highest concentration was determined for PCB 28, followed by PCBs 138, 153, and 180. The sum of the six indicator PCBs implied low exposure levels for the majority of the cohort population. Spearman correlations revealed strong associations between PCBs and OCPs, while principal component analysis identified two different groupings of exposure. DDE exhibited a correlation with a series of PCBs (153, 156, 163, 180), indicating a combined OCP-PCB source, and an anticorrelation with others (52, 28, 101), implying a separate and competing source.

Placental leucine aminopeptidase efficiently generates mature antigenic peptides in vitro but in patterns distinct from endoplasmic reticulum aminopeptidase 1.

All three members of the oxytocinase subfamily of M1 aminopeptidases, endoplasmic reticulum aminopeptidase 1 (ERAP1), ERAP2, and placental leucine aminopeptidase (PLAP), also known as insulin-regulated aminopeptidase, have been implicated in the generation of MHC class I-presented peptides. ERAP1 and 2 trim peptides in the endoplasmic reticulum for direct presentation, whereas PLAP has been recently implicated in cross-presentation. The best characterized member of the family, ERAP1, has unique enzymatic properties that fit well with its role in Ag processing. ERAP1 can trim a large variety of long peptide sequences and efficiently accumulate mature antigenic epitopes of 8-9 aa long. In this study, we evaluate the ability of PLAP to process antigenic peptide precursors in vitro and compare it with ERAP1. We find that, similar to ERAP1, PLAP can trim a variety of long peptide sequences efficiently and, in most cases, accumulates appreciable amounts of correct length mature antigenic epitope. Again, similar to ERAP1, PLAP continued trimming some of the epitopes tested and accumulated smaller products effectively destroying the epitope. However, the intermediate accumulation properties of ERAP1 and PLAP are distinct and epitope dependent, suggesting that these two enzymes may impose different selective pressures on epitope generation. Overall, although PLAP has the necessary enzymatic properties to participate in generating or destroying MHC class I-presented peptides, its trimming behavior is distinct from that of ERAP1, something that supports a separate role for these two enzymes in Ag processing.

PFASs intake from fish, eggs and drinking water in Greece in relation to the safety limits for weekly intake proposed in the EFSA scientific opinion of 2020.

Food consumption has been recognized as the most significant contributor to human exposure to polyfluoroalkyl and perfluoroalkyl substances (PFASs) for the general population. In 2020, EFSA introduced for the first time safety limit of 4.4 ng/kg body weight (bw) for weekly intake for the sum of perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorononanoic acid (PFNA) and perfluorohexane sulfonic acid (PFHxS) which are all perfluoralkyl acids (PFAAs) that belong to PFASs group. Fish and eggs have been found to contribute significantly, almost 50 % to PFOS and PFOA dietary intake of the Greek population. In the present study, estimation of human intake of these four PFASs from fish, eggs and drinking water consumption is attempted. Data from EFSA food consumption database for fish and eggs are used for assessment. Mean weekly intake estimated is above the tolerable weekly intake (TWI) recently proposed, mainly due to fish consumption. Exceedance of the proposed TWI emphasizes the need for continuous monitoring of levels of PFASs in food in parallel with efforts to lower these levels.

Polycyclic aromatic hydrocarbons and trace elements dietary intake in inhabitants of Athens, Greece, based on a duplicate portion study.

Concentration levels of 26 polycyclic aromatic hydrocarbons and 20 trace elements were measured in dietary samples, in order to estimate the dietary intake and the potential exposure risk of the Greek population. Dietary samples were collected with the help of 30 volunteers inhabitants of Athens, Greece, according to the duplicate portion study (DPS) method where the determination of the compounds is conducted in the final consumed meal. DPS was selected as an alternative to a total diet study for its feasibility, low cost and realistic image that it provides. PAHs determination was performed by gas chromatography tandem mass spectrometry (GC-MS/MS) and trace elements determination by inductively coupled plasma mass spectrometry (ICP-MS). The estimated mean daily dietary exposure on 26 PAHs for an adult was estimated at 101.47 ng kg-1 body weight day-1, while moderate to low exposure was related to the type and amount of food consumed. Exposure levels to PAHs do not indicate a possible risk for the health of the study group. For toxic, potentially essential and/or essential trace elements the daily exposure calculated for Greek population was in accordance with the reference values of the health-based guidances and compared to previous diet studies reported for the population of other countries was in general similar to the lower bound exposure.

Detection of interactions of the beta-amyloid peptide with small molecules employing transferred NOEs.

The interaction of pineal hormone melatonin, the histological dye thioflavin T, and the olive tree polyphenol oleuropein, with the 28 amino acid residue N-terminal fragment of the beta-amyloid peptide (beta-AP) of Alzheimer's disease, [beta-AP(1-28)], was detected in solution through the observation of transferred NOEs (trNOEs) in 1D and 2D NOE spectroscopy (NOESY) experiments. The trNOE method is applied for the first time in the detection of interactions of soluble beta-AP(1-28) with small molecules and may provide a means of screening for the identification of possible inhibitors of the formation of neurotoxic beta-AP assemblies.

Folding in solution of the C-catalytic protein fragment of angiotensin-converting enzyme.

Angiotensin-converting enzyme (ACE) is a key molecule of the renin-angiotensin-aldosterone system which is responsible for the control of blood pressure. For over 30 years it has become the target for fighting off hypertension. Many inhibitors of the enzyme have been synthesized and used widely in medicine despite the lack of ACE structure. The last 5 years the crystal structure of ACE separate domains has been revealed, but in order to understand how the enzyme works it is necessary to study its structure in solution. We present here the cloning, overexpression in Escherichia coli, purification and structural study of the Ala(959) to Ser(1066) region (ACE_C) that corresponds to the C-catalytic domain of human somatic angiotensin-I-converting enzyme. ACE_C was purified under denatured conditions and the yield was 6 mg/l of culture. Circular dichroism (CD) spectroscopy indicated that 1,1,1-trifluoroethanol (TFE) is necessary for the correct folding of the protein fragment. The described procedure can be used for the production of an isotopically labelled ACE(959-1066) protein fragment in order to study its structure in solution by NMR spectroscopy.

Synthesis and characterization of novel natural product-Gd(III) MRI contrast agent conjugates.

Several novel gadolinium chelates conjugated with paclitaxel, colchicine and thyroxine have been prepared as MRI contrast agents targeted to tubulin and thyroxine-binding globulin, respectively.

An effective and low cost carbon based clean-up method for PCDD/Fs and PCBs analysis in food.

Sample preparation is of critical importance in dioxin analysis of food and feed samples. It is a complex procedure that includes lipid extraction followed by the application of chromatographic separation techniques, aiming in removing undesirable interferences from the matrix. The separation of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) from polychlorinated biphenyls (PCBs) is achieved by carbon-based materials which should have high fat capacity in order to be suitable for lipid-containing matrices. Automated methods are available but due to high cost and use of high amounts of solvents, manual methods are also applied. An active carbon material (Carbosphere) with high fat capacity that has been used in the past for manual methods is no longer commercially available. The present study assesses an alternative active carbon material, FU 4652, that can be used for the separation of PCDD/Fs and non-ortho PCBs. Mono-ortho and 6 non-dioxin-like PCBs are also analyzed. The method was validated according to the analytical criteria set in EU regulations 589/2014 and 709/2014. Control samples analyzed for the evaluation of the above material were olive oil reference samples spiked with PCDD/Fs and dioxin-like PCBs at two concentration levels. The new method was tested successfully on food samples of interlaboratory trials organized in previous years. Farmed fish samples collected within national surveillance programs for the years 2016-2017 were analyzed with the method developed. The results obtained indicate that the FU 4652 carbon sorbent has high fat capacity and is capable of separating congeners with good recoveries.

A HER-2/neu peptide admixed with PLA microspheres induces a Th1-biased immune response in mice.

The elimination of cancer cells requires strong cellular immune responses, and these responses are induced by the activation of Th1 lymphocytes. In this work, the possibility of inducing a Th1 type of immune response in vivo by mixing a HER-2/neu synthetic CTL (cytotoxic T lymphocyte) peptide [HER-2/neu (789-797)], with poly-lactide (PLA) microspheres was investigated. Various formulations of the peptide were administered to HLA-A2.1 transgenic (HHD) mice. Cellular experiments, assessing proliferation and cytokine determination in splenocyte culture supernatants, were carried out in order to evaluate the type of immune response to the antigen. The in vivo administration of the peptide antigen admixed with the PLA microspheres induced a potent immune response which was comparable to that induced by the combination of the antigen in complete Freund's adjuvant (CFA). Furthermore, the cytokine profile produced by the T lymphocytes of the immunized animals indicated that the combination of the peptide antigen with the PLA microspheres induced a strong Th1 biased immune response to the antigen. The time of peptide incubation with the microspheres prior to administration did not affect the immune response, which further simplifies the preparation of this type of vaccine. The results justify further investigation of the possibility of inducing effective cellular immune responses against cancer cells overexpressing HER-2/neu molecules by simply mixing appropriate HER-2/neu peptide antigens with PLA microspheres.

Levels of dioxins, furans and PCBs in human serum and milk of people living in Greece.

In an attempt to evaluate the background levels of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), non-ortho, mono-ortho and indicator polychlorinated biphenyls (PCBs) in people living in an urban (Athens) and a rural (Kozani) area of Greece, blood serum and mother milk samples were analyzed. Analytical results are reported in this study. Statistically significantly differences were observed between regions, sexes and ages. Contamination levels in blood and human milk from Greece reported here are low compared to the previously reported dioxin data from other European countries and give no indication of particular health risk.

PCDDs, PCDFs and PCBs in farmed fish produced in Greece: Levels and human population exposure assessment.

Fish is among the essential components of Mediterranean diet and has beneficial effects on human health. Farmed fish is an affordable alternative to wild fish and a significant food export product for Greece. Published studies worldwide have reported significant levels of environmental pollutants in fish tissues. Especially for PCDDs/Fs and PCBs, the studies suggest that the most important contribution to human dietary intake is from fish and seafood. In the present study, we investigate the levels of PCDDs/Fs, dioxin-like and non dioxin-like PCBs in the most common farmed fish species produced in Greece i.e. sea bass, sea bream and rainbow trout. These species are widely consumed in Greece and are also exported to many countries worldwide. The mean levels found were WHO-PCDD/F-TEQ: 0.22 pg g(-1) wet weight (w.w.), WHO-PCDD/F-PCB-TEQ: 0.88 pg g(-1) w.w. for sea bream, WHO-PCDD/F-TEQ: 0.13 pg g(-1) w.w., WHO-PCDD/F-PCB-TEQ: 0.68 pg g(-1) w.w. for sea bass and WHO-PCDD/F-TEQ: 0.10 pg g(-1) w.w., WHO-PCDD/F-PCB-TEQ: 0.43 pg g(-1) w.w. for rainbow trout. For non dioxin-like PCBs, mean sum values found were 8.02 ng g(-1) w.w. for sea bream, 5.24 ng g(-1) w.w. for sea bass and 2.90 ng g(-1) w.w. for rainbow trout. All concentrations found were far below maximum levels set by the European Union and in the same range as wild-caught fish also presented for comparison. Daily intake from the consumption of farmed fish species examined is calculated at 1.3 pg WHO-TEQ kg(-1) b.w., which is at the lowest end of TDI values proposed by the WHO.

Perfluoroalkylated substances in edible livers of farm animals, including depuration behaviour in young sheep fed with contaminated grass.

Perfluoroalkylated substances (PFASs) present a potential health risk for consumers. In animals these compounds are known to accumulate in livers. In order to determine potential PFASs contamination in commercially available livers, samples from farmed sheep, horses, cows, pigs and chicken were collected from the Dutch market. PFOS was the only detectable PFAS and its concentration was higher in free ranging animals like cows and sheep. The detected levels of PFOS in the liver samples were very low (up to 4.5 ng g(-1) ww). To further study the kinetic behaviour in foraging animals, samples from a study in which sheep were fed with grass obtained from a river floodplain, were examined. PFOS was the only detectable PFAS in the contaminated grass pellets, showing a level of about 0.5 µg kg(-1). Young blackhead sheep were fed with either clean or contaminated grass for a period up to 112 days. A time-dependent increase in liver PFOS concentrations was observed from 2.4 to 10.9 ng g(-1) ww after 8 and 112 days respectively. A time-dependent depuration was observed in livers of animals switched to clean grass after 56 days of exposure, from 9.2 to 4.7 ng g(-1) ww after 64 and 112 days respectively. The percentage of PFOS ingested from the grass and retained in the liver was estimated to be 12% at day 56, and decreased gradually to 6% after 56 days on clean grass, showing that the decrease in levels is not only caused by an increase in liver weight. Levels detected in commercial livers but also those in the sheep study would not lead to exceedance of the current TDI for PFOS set by EFSA. Therefore, it can be assumed that they do not present a risk for human health.

Perfluoroalkylated substances (PFASs) in home and commercially produced chicken eggs from the Netherlands and Greece.

Dietary intake is a major route of human exposure to perfluoroalkylated substances (PFASs). However, the available information on PFAS levels in food, including chicken eggs, is limited. In the present study, home produced and commercially produced eggs (organic, battery and free range eggs) were collected from the Netherlands (n = 95) and Greece (n = 76). The egg yolks were analysed for 11 PFASs by liquid chromatography-tandem mass spectrometry using isotope dilution. PFAS levels in yolk were higher in home produced eggs from the Netherlands (median 3.1, range < LOQ - 31.2 ng g(-1)) and Greece (median 1.1, range < LOQ - 15.0 ng g(-1)) compared to the eggs collected from supermarkets. In these eggs, all PFAS levels were below the LOQ of 0.5 ng g(-1), except for a small amount of perfluorooctane sulfonate (PFOS) in 1 sample in each country (1.1 ng g(-1) and 0.9 ng g(-1) for the Netherlands and Greece respectively). PFOS was the predominant PFAS, making up on average 85% of ∑PFASs. The highest PFOS concentration was detected in a Dutch home produced egg sample (24.8 ng g(-1)). The contamination pattern was similar in both countries with the long-chain PFASs (C ≥ 8) being most frequently detected, while short-chain PFASs were rarely found. The most likely cause of the contamination of home produced eggs is ingestion of soil through pecking. Although regular consumption of home produced eggs will lead to an increased PFOS exposure, it is not expected that it will lead to exceedance of the tolerable daily intake established by EFSA.

A novel and efficient method for cleavage of phenacylesters by magnesium reduction with acetic acid.

[reaction: see text] In the present study, we use magnesium turnings as a new deprotection reagent for the phenacyl group during orthogonal organic synthesis in the presence of other esters and sensitive protecting groups. By applying the new magnesium turnings/acetic acid deprotection method, phenacyl group can be more easily combined with other protecting groups that are not compatible with the zinc/acetic acid method.

Levels of perfluorinated compounds in raw and cooked Mediterranean finfish and shellfish.

Perfluorinated compounds (PFCs) were analyzed in several species of small Mediterranean fish and shellfish, all of which are popular in Greek diet. Analysis was conducted in raw samples and in samples cooked by the two ways preferred in Greek cuisine, i.e. fried in olive oil and grilled. PFCs above the detection limit were found in all raw samples except sardine, mussel and squid. The predominant PFC was PFOS (perfluorooctane sulfonate), the highest concentration of which was measured in picarel (20.4 ng g(-1) fresh weight). The PFOS values for the rest of the samples were between