Assessment of the effects of amphiphilic poly (N­vinylpyrrolidone) nanoparticles loaded with bortezomib on glioblastoma cell lines and zebrafish embryos.

Proteasome inhibitor bortezomib is an anticancer agent approved for treatment of multiple myeloma and mantle cell lymphoma. However, its application in other types of cancer, primarily in solid tumors, is limited due to poor pharmacokinetics, inefficient tissue penetration, low stability and frequent adverse effects. In the present study, a novel micellar nano-scaled delivery system was manufactured, composed of amphiphilic poly(N-vinylpyrrolidone) nanoparticles loaded with bortezomib. Similar nanoparticles loaded with prothionamide, a drug without anticancer effect, were used as control. The size and zeta potential of the obtained polymeric micelles were measured by dynamic light scattering. Bortezomib-loaded micelles exhibited significant cytotoxic activity in vitro in monolayer tumor cell cultures (IC50 ~6.5 µg/ml) and in 3D multicellular tumor spheroids (IC50 ~8.5 µg/ml) of human glioblastoma cell lines U87 and T98G. Additionally, the toxic effects in vivo were studied in zebrafish Danio rerio embryos, with an estimated 50% lethal concentration of 0.1 mg/ml. Considering that bortezomib and other molecules from the class of proteasome inhibitors are potent antitumor agents, nanodelivery approach can help reduce adverse effects and expand the range of its applications for treatment of various oncological diseases.

New method for risk assessment in environmental health: The paradigm of heavy metals in honey.

The release of heavy metals into the natural environment creates problems due to their persistence. They can accumulate in the food chain presenting a dangerous sign for ecosystems and human health. The metals in honey could be of agrochemical or industrial origin. Regular consumption of honey and bee products contaminated with various pollutants in high concentrations can cause serious health problems due accumulation of toxic substances in the body. In the current study, we aimed to determine the concentrations of chromium, cadmium, zinc, copper, lead and nickel in four types of honey (linden, acacia, rapeseed and polyfloral honey) and soil collected from three regions with different degrees of pollution. For the risk characterization, we used a new methodology that calculated the corrected estimated daily intake and the source hazard quotient for each metal and the adversity-specific hazard index. There was a strong influence of the degree of environmental pollution on the level of contaminants in the honey samples. In the case of a single chemical assessment, an HQ above 10 was obtained for Cd in linden, rapeseed and polyfloral honey from area 1 and an HQ above 1 was obtained for Cd in the other honey samples from the 3 areas, for Cu in all honey samples from all the 3 areas, for Pb in linden, rapeseed and polyfloral honey from area 1 and for Cr in linden honey for area 2. HIA calculated as a sum of all HQS of heavy metals in food reveals an increase and moderate risk for nephrotoxicity, bone demineralisation, cardiotoxicity, developmental toxicity, small decrease in body weight or body weight gain after consumption of honey impurified with heavy metals. A strict monitorization of heavy metals in honey samples from farmers should be done in order to protect the consumers.

Probabilistic risk assessment of exposure to multiple metals and pesticides through consumption of fruit juice samples collected from Iranian market.

The current study assessed the risk posed to Iranian consumers by oral exposure to a mixture of 20 pesticides and six metals in 96 fruit juice (FJ) samples (3 batches × 4 brands × 8 types of FJs) collected from Iran market. Concentrations of metals and pesticides in FJs were quantified by inductively coupled plasma-optical emission spectrometry (ICP-OES) and chromatography-mass spectrometry (GC-MS), respectively. The mean concentration of all pesticides was below the maximum residue limits (MRLs) set by the European Union (EU). The calculated target hazard quotients (THQs) and total hazard index (HI) were <1.0 for all pesticides residue, indicating no risk. For the carcinogenic metals (As, Ni, and Pb), estimated incremental lifetime cancer risks (ILCRs) at the 50th and 95th centiles were respectively 4.25 × 10-5 and 5.30 × 10-5 (for As), 2.85 × 10-5 and 3.71 × 10-5 (for Ni), and 2.84 × 10-8, and 3.97 × 10-8 (for Pb), indicating no risk. At the 50th and 95th centiles, HI for non-carcinogenic metals (Cd, Hg, and Cr) was <1.0, indicating no risk. Based on sensitivity analyses of the input variables, the concentration of metals and pesticides, and the FJs ingestion rate had significant influential impacts on the calculated THQ and HI.

In Vitro Assessment of Poly-N-Vinylpyrrolidone/Acrylic Acid Nanoparticles Biocompatibility in a Microvascular Endothelium Model.

An amphiphilic copolymer of N-vinyl-2-pyrrolidone and acrylic acid-namely, p(VP-AA)-OD6000 (p(VP-AA))-was synthesized to prepare p(VP-AA) nanoparticles (NPs). Furthermore, the copolymer was linked with CFSE, and the so-prepared nanoparticles were loaded with the DiI dye to form D nanoparticles (DNPs). In this study, as demonstrated by immunofluorescence microscopy, immunofluorescence, and confocal microscopy, DNPs were readily taken up by human microvascular endothelial cells (HMEC-1) cells in a concentration-dependent manner. Upon uptake, both the CFSE dye (green stain) and the DiI dye (red stain) were localized to the cytoplasm of treated cells. Treatment with p(VP-AA) did not affect the viability of normal and challenged with LPS, HMEC-1 cells at 0.010 mg/mL and induced a dose-dependent decrease of these cells' viability at the higher concentrations of 0.033 and 0.066 mg/mL (p ≤ 0.01; p ≤ 0.001, respectively). Furthermore, we focused on the potential immunological activation of HMEC-1 endothelial cells upon p(VP-AA) NPs treatment by assessing the expression of adhesion molecules (E-Selectin, ICAM-1, and V-CAM). NPs treatments at concentrations utilized (p = NS) did not affect individual adhesion molecules' expression. p(VP-AA) NPs do not activate the endothelium and do not affect its viability at pharmacologically relevant concentrations.

Antibiotics in Raw Meat Samples: Estimation of Dietary Exposure and Risk Assessment.

The extensive use of antibiotics in livestock farming poses increased concerns for human health as residues of these substances are present in edible tissues. The aim of this study was the determination of the levels of four groups of antibiotics (sulfonamides-SAs, tetracyclines-TCs, streptomycines-STr and quinolones-QNLs) in meat samples (muscles, livers and kidneys from beef, chicken and pork) and the estimation of the dietary exposure to antibiotics from meat consumption and the potential hazard for human health. Fifty-four samples of raw meat were randomly collected in 2018 from the Cretan market, Greece and analyzed both with an enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry (LC-MS). According to the results derived from the ELISA method, only 2% of the meat samples were free from antibiotics, 2% were detected with 4 antibiotics and the great majority of the samples (87%) were detected with 2 to 3 antibiotics. SAs presented the highest detection frequencies for all samples whereas TCs were not detected in any bovine sample. The highest median concentration was detected for STr in bovine muscles (182.10 µg/kg) followed by QNLs (93.36 µg/kg) in pork kidneys whereas the chicken samples had higher burdens of QNLs compared to the other meat samples. LC-MS analysis showed that oxytetracycline (OTC) was the most common antibiotic in all samples. The highest median concentration of all antibiotics was detected for doxycycline (DOX) (181.73 µg/kg in pork kidney) followed by OTC in bovine liver (74.46 µg/kg). Risk characterization was applied for each of the two methods; The hazard quotients (HQ) did not exceed 0.059 for the ELISA method and 0.113 for the LC-MS method for any group of antibiotics, whereas the total hazard indexes (HI) were 0.078 and 0.021, respectively. The results showed the presence of different groups of antibiotics in meat from the Cretan market and that the health risk to antibiotics is low. A risk assessment analysis conducted for meat consumption and corrected for the aggregated exposure revealed no risk for the consumers.

Assessment of the combined effects of chromium and benzene on the rat neuroendocrine and immune systems.

This study assessed the hypothalamic-pituitary-adrenocortical (HPA) axis and lymphoid organs (thymus, spleen, and bone marrow) of Wistar rats treated with a mixture of chromium and benzene. Animals were assessed at three time-points (45, 90 and 135 days) following oral mixture exposure. The hypothalamus-pituitary system was examined in light and electron microscopy. Lymphoid organs underwent a morphological assessment and the immunophenotype of splenocytes was characterized immunohistochemically using monoclonal antibodies. Splenocytes cytokine production of was determined by ELISA after Con-A stimulation. Combined exposure to chromium and benzene in average doses of 20 mg Cr (VI)/kg body weight/day and 0.6 ml benzene/kg body weight/day impaired the responsiveness of the central compartment of the HPA axis, as evidenced by functional activation of the secretory activity of the hypothalamus and pituitary gland, which was not followed by a sufficient extrusion of nonapeptides at the neurohypophysis and hypothalamic median eminence. Chromium and benzene exposure reduced the thymus mass, thymocytes count, and caused a number of structural and functional changes indicative of transient thymus involution. In the spleen, exposure to both chemicals resulted in lymphoreticular hyperplasia and plasma cell-macrophage transformation (also observed in lymph nodes). Apoptosis of thymocytes and lymphocytes was also observed in T-zones of the spleen. Notably, the effects were similar to those observed earlier for the single agents, under the same experimental conditions, without evidence of additivity.

An exposome connectivity paradigm for the mechanistic assessment of the effects of prenatal and early life exposure to metals on neurodevelopment.

The exposome paradigm through an integrated approach to investigating the impact of perinatal exposure to metals on child neurodevelopment in two cohorts carried out in Slovenia (PHIME cohort) and Greece (HERACLES cohort) respectively, is presented herein. Heavy metals are well-known neurotoxicants with well-established links to impaired neurodevelopment. The links between in utero and early-life exposure to metals, metabolic pathway dysregulation, and neurodevelopmental disorders were drawn through urinary and plasma untargeted metabolomics analysis, followed by the combined application of in silico and biostatistical methods. Heavy metal prenatal and postnatal exposure was evaluated, including parameters indirectly related to exposure and health adversities, such as sociodemographic and anthropometric parameters and dietary factors. The primary outcome of the study was that the identified perturbations related to the TCA cycle are mainly associated with impaired mitochondrial respiration, which is detrimental to cellular homeostasis and functionality; this is further potentiated by the capacity of heavy metals to induce oxidative stress. Insufficient production of energy from the mitochondria during the perinatal period is associated with developmental disorders in children. The HERACLES cohort included more detailed data regarding diet and sociodemographic status of the studied population, allowing the identification of a broader spectrum of effect modifiers, such as the beneficial role of a diet rich in antioxidants such as lycopene and ω-3 fatty acids, the negative effect the consumption of food items such as pork and chicken meat has or the multiple impacts of fish consumption. Beyond diet, several other factors have been proven influential for child neurodevelopment, such as the proximity to pollution sources (e.g., waste treatment site) and the broader living environment, including socioeconomic and demographic characteristics. Overall, our results demonstrate the utility of exposome-wide association studies (EWAS) toward understanding the relationships among the multiple factors that determine human exposure and the underlying biology, reflected as omics markers of effect on neurodevelopment during childhood.

[Comment] COVID­19 vaccine safety.

In response to the SARS­CoV­2 outbreak, and the resulting COVID­19 pandemic, a global competition to develop an anti­COVID­19 vaccine has ensued. The targeted time frame for initial vaccine deployment is late 2020. The present article examines whether short­term, mid­term, and long­term vaccine safety can be achieved under such an accelerated schedule, given the myriad vaccine­induced mechanisms that have demonstrated adverse effects based on previous clinical trials and laboratory research. It presents scientific evidence of potential pitfalls associated with eliminating critical phase II and III clinical trials, and concludes that there is no substitute currently available for long­term human clinical trials to ensure long­term human safety.

Assessment of Amphiphilic Poly-N-vinylpyrrolidone Nanoparticles' Biocompatibility with Endothelial Cells in Vitro and Delivery of an Anti-Inflammatory Drug.

Nanoparticles (NPs) produced from amphiphilic derivatives of poly-N-vinylpyrrolidone (Amph-PVP), composed of various molecular weight polymeric hydrophilic fragments linked into hydrophobic n-alkyl chains of varying lengths, were previously shown to exert excellent biocompatibility. Although routes of administration can be different, finally, most nanosystems enter the blood circulation or lymphatic vessels, and by this, they establish direct contact with endothelial cells. In this study, Amph-PVP NPs and fluorescently labeled Amph-PVP-based NPs, namely "PVP" NPs (Amph-PVP-NPs (6000 Da) unloaded) and "F"-NPs (Amph-PVP-NPs (6000 Da) loaded with fluorescent FITC), were synthesized to study Amph-PVP NPs interactions with HMEC-1 endothelial cells. PVP NPs were readily uptaken by HMEC-1 cells in a concentration-dependent manner, as demonstrated by immunofluorescence imaging. Upon uptake, the FITC dye was localized to the perinuclear region and cytoplasm of treated cells. The generation of lipopolysaccharide (LPS)-induced activated endothelium model revealed an increased uptake of PVPNPs, as shown by confocal microscopy. Both unloaded PVP NPs and F-NPs did not affect EC viability in the 0.01 to 0.066 mg/mL range. Furthermore, we focused on the potential immunological activation of HMEC-1 endothelial cells upon PVPNPs treatment by assessing the expression of their E-Selectin, ICAM-1, and VCAM-1 adhesion receptors. None of the adhesion molecules were affected by NP treatments of both activated by LPS and nonactivated HMEC-1 cells, at the utilized concentrations (p = NS). In this study, PVP (6000 Da) NPs were used to encapsulate indomethacin, a widely used anti-inflammatory drug. The synthesized drug carrier complex did not affect HMEC-1 cell growth and expression of E-selectin, ICAM-1, and VCAM-1 adhesion receptors. In summary, PVP-based NPs are safe for use on both basal and activated endothelium, which more accurately mimics pathological conditions. Amph-PVP NPs are a promising drug delivery system.

Phthalate metabolites concentrations in amniotic fluid and maternal urine: Cumulative exposure and risk assessment.

Phthalates are used in industry as plasticizers or additives in everyday products and they have been considered as endocrine disrupting chemicals. Maternal exposure during pregnancy has been associated with neonatal exposure, preterm birth and impacts in the reproductive and respiratory systems. The aim of this study is to determine six phthalate metabolites (mono isobutyl phthalate, miBP, mono n-butyl phthalate, mnBP, mono benzyl phthalate, mBzP, mono ethylhexyl phthalate, mEHP, mono 2-ethyl-5-hydroxyhexyl phthalate, mEHHP, mono 2-ethyl-5-oxohexyl-phthalate, mEOHP) in amniotic fluid and urine from 100 pregnant women. Participants answered questionnaires for the use of plastics and cosmetics, dietary habits, health effects, pregnancy problems, health and infant development. Positive amniotic fluid samples ranged from 1% to 21% and urine from 27% to 54%. The median levels for amniotic fluid were 2.3 µg/L - 10.7 µg/L and for urine 4.9 µg/L - 46.7 µg/L. The major results include significant correlations between urinary phthalates indicating their common sources of exposure, the frequent use of deodorant was significantly associated with higher urinary miBP (p = 0.050) and mnBP (p = 0.028) and a weak inverse association was found for the use of make-up products with mBzP (p = 0.053). The frequent use of plastic food containers was significantly associated with urinary mEHP (p = 0.026), and a positive trend was noticed for mEHP in amniotic fluid (p = 0.093). An association although weak was found between urinary mEHP and lower birth length (rs = 0.396, p = 0.062). No other associations were found for infant health problems or development. The daily intake of the total phthalates was calculated 5.4 µg/kg body weight/day which corresponds to hazard index 0.10 and exposure follows the declining trend that has been observed the last decades.

Multiresidue analysis of insecticides and fungicides in apples from the Greek market. Applying an alternative approach for risk assessment.

Pesticides are widely used in agriculture and their proven high toxicity makes the need of monitoring their presence in food imperative. A multi residue method is applied in apples samples (81) collected from the Greek market for the monitoring of 40 analytes. Pesticides levels were evaluated by gas and liquid chromatography-mass spectrometry using a QuEChERs protocol. Risk for consumers was assessed by a newly developed methodology, employing the source related Hazard Quotient (HQs) and the adversity specific Hazard Index (HIA). The 84% of the apple's samples were positive for at least one pesticide, 21% for one pesticide, 55.6% for two to four pesticide residues and 7.4% for more than 4 pesticide residues. The most frequently detected compound was carbendazim (45.7%) followed by chlorpyrifos (44.4.0%). The mean detected concentration levels varied from 0.169 ppm (fluopyram) to 0.005 ppm (triazophos). 19 of the 40 investigated pesticides were not detected in any apple sample. For all individual pesticides, the source related Hazard Quotient (HQs) was <1 indicating no risk. HIA, resulting from the sum of all HQs was found HIA < 1 in nine out of ten toxicity groups, except to the neurotoxicity group, which presented HIA 2.258, indicating moderate risk.

Toxicity assessment of particulate matter emitted from different types of vehicles on marine microalgae.

Air pollution caused by vehicle emissions remains a serious environmental threat in urban areas. Sedimentation of atmospheric aerosols, surface wash, drainage water, and urbane wastewater can bring vehicle particle emissions into the aquatic environment. However, the level of toxicity and mode of toxic action for this kind of particles are not fully understood. Here we explored the aquatic toxic effects of particulate matter emitted from different types of vehicles on marine microalgae Porphyridium purpureum and Heterosigma akashiwo. We used flow cytometry to evaluate growth rate inhibition, changes in the level of esterase activity, changes in membrane potential and size changes of microalgae cells under the influence of particulate matter emitted by motorcycles, cars and specialized vehicles with different types of engines and powered by different types of fuel. Both microalgae species were highly influenced by the particles emitted by diesel-powered vehicles. These particle samples had the highest impact on survival, esterase activity, and membrane potential of microalgae and caused the most significant increase in microalgae cell size compared to the particles produced by gasoline-powered vehicles. The results of the algae-bioassay strongly correlate with the data of laser granulometry analyses, which indicate that the most toxic samples had a significantly higher percentage of particles in the size range less than 1 µm. Visual observation with an optical microscope showed intensive agglomeration of the particles emitted by diesel-powered vehicles with microalgae cells. Moreover, within the scope of this research, we did not observe the direct influence of metal content in the particles to the level of their aquatic toxicity, and we can conclude that physical damage is the most probable mechanism of toxicity for vehicle emitted particles.

Critical assessment and integration of separate lines of evidence for risk assessment of chemical mixtures.

Humans are exposed to multiple chemicals on a daily basis instead of to just a single chemical, yet the majority of existing toxicity data comes from single-chemical exposure. Multiple factors must be considered such as the route, concentration, duration, and the timing of exposure when determining toxicity to the organism. The need for adequate model systems (in vivo, in vitro, in silico and mathematical) is paramount for better understanding of chemical mixture toxicity. Currently, shortcomings plague each model system as investigators struggle to find the appropriate balance of rigor, reproducibility and appropriateness in mixture toxicity studies. Significant questions exist when comparing single-to mixture-chemical toxicity concerning additivity, synergism, potentiation, or antagonism. Dose/concentration relevance is a major consideration and should be subthreshold for better accuracy in toxicity assessment. Previous work was limited by the technology and methodology of the time, but recent advances have resulted in significant progress in the study of mixture toxicology. Novel technologies have added insight to data obtained from in vivo studies for predictive toxicity testing. These include new in vitro models: omics-related tools, organs-on-a-chip and 3D cell culture, and in silico methods. Taken together, all these modern methodologies improve the understanding of the multiple toxicity pathways associated with adverse outcomes (e.g., adverse outcome pathways), thus allowing investigators to better predict risks linked to exposure to chemical mixtures. As technology and knowledge advance, our ability to harness and integrate separate streams of evidence regarding outcomes associated with chemical mixture exposure improves. As many national and international organizations are currently stressing, studies on chemical mixture toxicity are of primary importance.

A novel deep learning architecture outperforming 'off­the­shelf' transfer learning and feature­based methods in the automated assessment of mammographic breast density.

Potentially suspicious breast neoplasms could be masked by high tissue density, thus increasing the probability of a false­negative diagnosis. Furthermore, differentiating breast tissue type enables patient pre­screening stratification and risk assessment. In this study, we propose and evaluate advanced machine learning methodologies aiming at an objective and reliable method for breast density scoring from routine mammographic images. The proposed image analysis pipeline incorporates texture [Gabor filters and local binary pattern (LBP)] and gradient­based features [histogram of oriented gradients (HOG) as well as speeded­up robust features (SURF)]. Additionally, transfer learning approaches with ImageNet trained weights were also used for comparison, as well as a convolutional neural network (CNN). The proposed CNN model was fully trained on two open mammography datasets and was found to be the optimal performing methodology (AUC up to 87.3%). Thus, the findings of this study indicate that automated density scoring in mammograms can aid clinical diagnosis by introducing artificial intelligence­powered decision­support systems and contribute to the 'democratization' of healthcare by overcoming limitations, such as the geographic location of patients or the lack of expert radiologists.

Predictive power of the Triticum root elongation test for the assessment of novel anti­proliferative therapies.

The use of alternative techniques to reduce the number of animals used in anticancer research is an issue of current interest. The aim of this study was to validate the use of a simple and efficient alternative tool for the assessment of the potential of novel anti­proliferative agents. A set of 20 compounds with various mechanisms were tested in the Triticum aestivum root elongation assay, using aminophylline as negative control. Hierarchical cluster analyses were performed using the furthest neighbor method based on Euclidean distance measure, and the compounds were statistically analyzed in reference to their anti­proliferative pattern registered in the NCI60 human tumor cell line anticancer drug screen. A correlation between the Triticum test results and the NCI60 anti­proliferative profile was made for a number of human cells that we defined as the Triticum cell panel. Linear equations were computed that can be used to transform the inhibitory effect measured in any future Triticum assay in order to predict the effect on particular human cells. Of the tested anti­proliferative agents, methotrexate, colchicine, cantharidin, cisplatin and verapamil produced a growth inhibition over 50%. On the whole, the findings of this study suggest that the Triticum test can be used to detect several types of anti­proliferative mechanisms, particularly those targeting tubulin, rendering it a useful tool with which to identify novel mitotic spindle inhibitors.

Indicator PCBs in farmed and wild fish in Greece - Risk assessment for the Greek population.

Health benefits of fish consumption could be counterbalanced by the intake of contaminants after long term fish consumption, burdened even in trace levels. The presence of the indicator PCBs (NDL-PCBs and PCB 118) in farmed and wild seabream and seabass was evaluated. For the determination of PCB, a GC-MS method was developed and evaluated. The association of PCB accumulation in fish with seasonality, locality, production mode and species was also investigated. A new approach for the risk characterisation after exposure to NDL-PCB through fish consumption in Greece was developed, based on the real exposure and the permitted maximum levels of both aggregated dietary exposure and exposure through fish consumption. PCB levels determined in fish were below established permitted limits (6.24 ng/g 95th percentile), while PCB levels and congener distribution varied significantly between farmed and wild fish (p = 0.001). Seasonality was highlighted as an important factor affecting NDL-PCBs accumulation, with high levels coinciding with the reproduction period of each species. Differences were also depicted for sampling sites, with PCB 118 presenting significantly higher values in open seas while NDL-PCB congeners in closed seas. Risk assessment of NDL-PCB intake through fish consumption corrected for the aggregated exposure revealed no risk for the consumers.

Quantification of 4-Methylimidazole in soft drinks, sauces and vinegars of Greek market using two liquid chromatography techniques.

The substance 4-methylimidazole (4-MEI) has raised several concerns regarding its toxicity to humans, although no harmonized classification has yet been decided. The regulatory limits for food products set by various authorities in Europe and the USA differ considerably. The purpose of the present study is to compare two liquid chromatography techniques in order to determine the levels of 4-MEI in food products from the Greek market and roughly estimate the possible exposure and relevant health risk for the consumers. A total of thirty-four samples (soft drinks, beers, balsamic vinegars, energy drinks and sauces) were collected and analyzed. The quality parameters for both analytical methodologies (linearity, accuracy, inter day precision, recovery) are presented. No detectable levels of 4-MEI are found in beers and soft drink samples, other than cola type. On the other hand, 4-MEI was detected in all cola type soft drinks (15.8-477.0 ng/ml), energy drinks (57.1%, 6.6-22.5 ng/ml) and vinegar samples (66.7%, 9.7-3034.7 ng/ml), while only one of the sauce samples was found to have a detectable level of 17.5 ng/ml 4-MEI.

Human exposure to chemical mixtures: Challenges for the integration of toxicology with epidemiology data in risk assessment.

Little is known about the potential adverse effects from longterm exposure to complex mixtures at low doses, close to health-based reference values. Traditional chemical-specific risk assessment based on animal testing may be insufficient and the lack of toxicological studies on chemical mixtures remains a major regulatory challenge. Hence, new methodologies on cumulative risk assessment are being developed but still present major limitations. Evaluation of chemical mixture effects requires an integrated and systematic approach and close collaboration across different scientific fields, particularly toxicology, epidemiology, exposure science, risk assessment and statistics for a proper integration of data from all these disciplines. Well designed and conducted epidemiological studies can take advantage of this new paradigm and can provide insight to support the correlation between humans low-dose exposures and diseases, thus avoiding the uncertainty associated with extrapolation across species. In this regard, human epidemiology studies may play a significant role in the new vision of toxicity testing. However, this type of information has not been fully considered in risk assessment, mainly due to the inherent limitations of epidemiologic studies. An integrated approach of in vivo, in vitro and in silico data, together with systematic reviews or meta-analysis of high quality epidemiological studies will improve the robustness of risk assessment of chemical mixtures and will provide a stronger basis for regulatory decisions. The ultimate goal is that experimental and mechanistic data can lend support and biological plausibility to the human epidemiological observations.

Environmental metal contamination and health impact assessment in two industrial regions of Romania.

We investigated two Romanian industrial regions- Copsa Mica and Zlatna, to assess the current situation of soil pollution and bioaccumulation of Pb, Cd, Cu and Zn in different vegetable species and possible risks to consumers. Both total and mobile forms of the metals were determined in soil samples, and metal content in the edible parts of root vegetable samples was also assessed. The concentrations of Pb and Zn in soil were higher in Copsa Mica than in Zlatna (566mg/kg vs 271mg/kg for Pb and 1143mg/kg vs 368mg/kg for Zn)·The metal mobility in soil from Copsa Mica decreases in the order Zn>Cu>Cd>Pb (1.88mg/kg, 0.40mg/kg, 0.22mg/kg, 0.16mg/kg, respectively), while in Zlatna, the order was Cu>Zn>Pb>Cd (0.88mg/kg, 0.29mg/kg, 0.04mg/kg, 0.01mg/kg, respectively), apparently depending on metal and soil conditions. In Copsa Mica, the amount of Pb and Cd in vegetable samples exceeded the maximum permissible limits in carrots (median concentration 0.32mg/kg for Pb and Cd) and in yellow onions (median concentration 0.24mg/kg for Cd). In Zlatna region, the content of Cd exceeded the maximum limits in yellow onions (median concentration 0.11mg/kg). The amount of Pb was higher than the maximum acceptable level in carrots from the Zlatna region (median concentration 0.12mg/kg). Cu and Zn levels were within the normal range in all vegetable samples. In the Zlatna region, the transfer factors for Pb and Cd were higher in carrots (median values of 9.9 for Pb and 21.0 for Cd) compared to carrots harvested in Copsa Mica (median values of 4.0 for Pb and 2.0 for Cd). Daily intake rates of metals through local vegetable consumption exceeded the limit values established by the European Food Safety Authority for Pb (1.2 to 2.4 times) and Cd (5.5 to 9.1 times) in both regions, with potential adverse health effects for the local population. The results highlight the need for total soil remediation action before fruit and vegetables produced in these polluted areas can be safely consumed.