Reference Values on Children's Hair for 28 Elements (Heavy Metals and Essential Elements) Based on a Pilot Study in a Representative Non-Contaminated Local Area.

Studies have been published, and laboratories offer services of measuring elements in hair as biomarkers of environmental exposure and/or control of essential elements (trace or macro). These reported values can have only sense if compared with adopted reference values. In this work, we propose provisional reference values based on a pilot child population. The concentrations of 28 elements were measured in children's hair samples. An observational, descriptive, cross-sectional study was conducted in a typical child population in the Mediterranean region void of excessive pollution problems to analyze 419 hair samples of children aged 3-12 years. Children were selected by a simple random method from eight primary education schools in different municipal districts, which included urban, rural and industrial areas. Samples of around 100 mg were washed and acid digested by an optimized procedure. All measures were performed using ICP-MS with Sc, Y and Re as internal standards. The statistical analysis was performed by two approaches: (a) considering all the data and (b) without outliers (second-order atypical data) to compare them with other published studies. The distribution curves in all the elements studied were asymmetric and did not fit the theoretical normality distributions. Therefore, the analysis based on percentiles was more appropriate. In most elements, only slight differences were observed with sex or age, which did not justify proposing separate reference ranges. From the results of this study, provisional reference values are proposed following two criteria: (a) simple application of the table of percentiles built by removing outlier values and (b) values after a detailed analysis case-by-case, considering other data as the distribution profile and other published data of each element. Although the pilot sample was from a limited area, it was carefully selected to be representative of a general non-contaminated population. With this limitation, the proposed reference values might be useful for researchers and physicians until a wider geographical study is available for a large number of elements.

Case study: risk associated to wearing silver or graphene nanoparticle-coated facemasks for protection against COVID-19.

The world is living a pandemic situation derived from the worldwide spreading of SARS-CoV-2 virus causing COVID-19. Facemasks have proven to be one of the most effective prophylactic measures to avoid the infection that has made that wearing of facemasks has become mandatory in most of the developed countries. Silver and graphene nanoparticles have proven to have antimicrobial properties and are used as coating of these facemasks to increase the effectivity of the textile fibres. In the case of silver nanoparticles, we have estimated that in a real scenario the systemic (internal) exposure derived from wearing these silver nanoparticle facemasks would be between 7.0 × 10-5 and 2.8 × 10-4 mg/kg bw/day. In addition, we estimated conservative systemic no effect levels between 0.075 and 0.01 mg/kg bw/day. Therefore, we estimate that the chronic exposure to silver nanoparticles derived form facemasks wearing is safe. In the case of graphene, we detected important gaps in the database, especially regarding toxicokinetics, which prevents the derivation of a systemic no effect level. Nevertheless, the qualitative approach suggests that the risk of dermal repeated exposure to graphene is very low, or even negligible. We estimated that for both nanomaterials, the risk of skin sensitisation and genotoxicity is also negligible.

Roles of NTE protein and encoding gene in development and neurodevelopmental toxicity.

Neuropathy Target Esterase (NTE) is a membrane protein codified by gene PNPLA6. NTE was initially discovered as a target of the so-called organophosphorus-induced delayed polyneuropathy triggered by the inhibition of the NTE-associated esterase center by neuropathic organophosphorus compounds (OPs). The physiological role of NTE might be related to membrane lipid homeostasis and seems to be involved in adult organisms in maintaining nervous system integrity. However, NTE is also involved in cell differentiation and embryonic development. NTE is expressed in embryonic and adult stem cells, and the silencing of Pnpla6 by interference RNA in D3 mouse cells causes significant alterations in several genetic pathways related to respiratory tube and nervous system formation, and in vasculogenesis and angiogenesis. The silencing of gene PNPLA6 in human NT2 cells at the beginning of neurodifferentiation causes severe phenotypic alterations in neuron-like differentiated cells; e.g. reduced electrical activity and the virtual disappearance of markers of neural tissue, synapsis and glia. These phenotypic effects were not reproduced when NTE esterase activity was inhibited by neuropathic OP mipafox instead of being silenced at the genetic level. Neuropathic OP chlorpyrifos seems able to induce neurodevelopmental alterations in animals. However, the effects of chlorpyrifos in the expression of biomarker genes of differentiation in D3 cells differ considerably from the effects induced by Pnpla6 silencing. In conclusion, available information suggests that PNPLA6 and/or the NTE protein play a role in early neurodifferentiation stages, although this role is not dependent upon the esterase NTE center. Therefore, impairments caused by OPs, such as chlorpyrifos, on neurodevelopment are not due to inhibition of NTE esterase enzymatic activity.

Organophosphorus pesticide chlorpyrifos and its metabolites alter the expression of biomarker genes of differentiation in D3 mouse embryonic stem cells in a comparable way to other model neurodevelopmental toxicants.

There are discrepancies about whether chlorpyrifos is able to induce neurodevelopmental toxicity or not. We previously reported alterations in the pattern of expression of biomarker genes of differentiation in D3 mouse embryonic stem cells caused by chlorpyrifos and its metabolites chlorpyrifos-oxon and 3,5,6-trichloro-2-pyridinol. Now, we reanalyze these data comparing the effects on these genes with those caused in the same genes by retinoic acid, valproic acid, and penicillin-G (model compounds considered as strong, weak, and non-neurodevelopmental toxicants, respectively). We also compare the effects of chlorpyrifos and its metabolites on the cell viability of D3 cells and 3T3 mouse fibroblasts with the effects caused in the same cells by the three model compounds. We conclude that chlorpyrifos and its metabolites act, regarding these end-points, as the weak neurodevelopmental toxicant valproic acid, and consequently, a principle of caution should be applied avoiding occupational exposures in pregnant women. A second independent experiment run with different cellular batches coming from the same clone obtained the same result as the first one.

An integrated approach for detecting embryotoxicity and developmental toxicity of environmental contaminants using in vitro alternative methods.

The main available alternatives for testing embryotoxicity are cellular tests with stem cells and in vitro-ex vivo tests with embryos. In cellular tests, the most developed alternative is the embryonic stem cell test, while the most developed tests involving embryos are the zebrafish and whole embryo culture test. They are technically more complex than cellular tests, but offer the advantage of determining the expectable phenotypic alteration caused by the exposure. Many efforts are currently being made, basically through proteomic and genomic approaches, in order to obtain improvements in predictivity of these tests. Development is a very complex process, and it is highly unlikely that a single alternative test can yield satisfactory performance with all types of chemicals. We propose a step-wise approach where model complexity, and consequently technical skills and economical costs, gradually increase if needed. The first level would be run short cellular assays to detect effects in early differentiation stages. The second level would involve longer cellular embryotoxicity tests to search embryotoxicants that have an effect on late differentiation stages. The third stage would consider tests with embryos because they allow the determination of hazards based on molecular and morphological alterations, and not only on differentiating cells.

Cytotoxic effect against 3T3 fibroblasts cells of saffron floral bio-residues extracts.

For every kilogram of saffron spice produced, about 63 kg of floral bio-residues (FB) (tepals, stamens and styles) are thrown away. Extracts of these bio-residues in water (W1), water:HCl (100:1, v/v) (W2), ethanol (E3), ethanol:HCl (100:1, v/v) (E4), dichloromethane (D5) and hexane (H6) were prepared. Their composition in flavonols and anthocyanins, and their effect on cell viability were determined. W1 was the richest in kaempferol 3-sophoroside (30.34 mg/g dry FB) and delphinidin 3,5-diglucoside (15.98 mg/g dry FB). The highest tested concentration (900 µg/ml) of W1, W2, E4, D5 and H6 did not significantly decrease the cell viability. Only E3 at that concentration caused a significant decrease of 38% in the cell viability. Therefore, all extracts studied are not cytotoxic at concentrations lower than 900 µg/ml, and W1 is proposed as the optimal for food applications due to its greater contribution of phenolic compounds.

Chlorpyrifos and its metabolites alter gene expression at non-cytotoxic concentrations in D3 mouse embryonic stem cells under in vitro differentiation: considerations for embryotoxic risk assessment.

The effects of organophosphate insecticide chlorpyrifos (CPF) on development are currently under discussion. CPF and its metabolites, chlorpyrifos-oxon (CPO) and 3,5,6-trichloro-2-pyridinol (TClP), were more cytotoxic for D3 mouse embryonic stem cells than for differentiated fibroblasts 3T3 cells. Exposure to 10 µM CPF and TClP and 100 µM CPO for 12 h significantly altered the in vitro expression of biomarkers of differentiation in D3 cells. Similarly, exposure to 20 µM CPF and 25 µM CPO and TClP for 3 days also altered the expression of the biomarkers in the same model. These exposures caused no significant reduction in D3 viability with mild inhibition of acetylcholinesterase and neuropathy target esterase by CPF and severe inhibition by CPO. We conclude that certain in vivo exposure scenarios are possible, which cause inhibition of acetylcholinesterase but without clinical symptoms that reach high enough systemic CPF concentrations able to alter the expression of genes involved in cellular differentiation with potentially hazard effects on development. Conversely, the risk for embryotoxicity by CPO and TClP was very low because the required exposure would induce severe cholinergic syndrome.

Characterization and evolution of exposure to volatile organic compounds in the Spanish shoemaking industry over a 5-year period.

This study measured inhalation exposure to 13 volatile organic compounds (VOCs) among workers in the leatherwear industry in Spain, examined the changes in those exposures over a 5-year period, and documented local exhaust ventilation practices that affected exposure. In collaboration with an occupational risk prevention company, air samples were collected from 849 workers' personal breathing zones using personal air pumps with activated charcoal tubes. VOCs were analyzed using a GC/MS-optimized method modified in our laboratory from that proposed by Spanish authorities (INSHT). Airborne concentrations were compared with occupational exposure limit (OEL) values from the European authorities. The most frequently detected VOCs were acetone (98.1%), toluene (94.8%), n-hexane (71.2%) and other C6-C7 branched alkyl hydrocarbons (97.5%). Other frequently detected VOCs were MEK (64.9%), ethylacetate (60.7%), and cyclohexane (29.3%). Benzene was detected in 24.6% of samples. Although all the samples were taken while workers performed tasks judged to have the highest VOC exposure potential, only 14% of samples showed excessive aggregate exposure, and chemical-specific OELs were exceeded in a relatively small number of cases: 7.2% for n-hexane, 2.8% for toluene, 0.6% for acetone, and 0.4% for hexane isomers. Over the study period, a diminished use of n-hexane in solvent formulations and an increased use of branched hexane and heptane isomers were observed. Six factors relating to work location conditions and types were evaluated. Most high-exposure cases were associated with three task types. The presence of local exhaust ventilation was an important exposure control, but significant exposures despite the use of local exhaust were observed. Although n-hexane exposures significantly decreased over the study period, the overall level of VOC exposure did not decrease. More effective exposure prevention measures need to be implemented.

Cell viability effects and antioxidant and antimicrobial activities of Tunisian date syrup (Rub El Tamer) polyphenolic extracts.

The aqueous-acetone polyphenolic extract of the traditionally derived date syrup, known as "Rub El Tamer", was analyzed using RP-HPLC-DAD and ESI-MS. The phenolic content of the extract was 394.53 ± 1.13 mg per 100 g of syrup with caffeoylsinapylquinic acid as the most abundant compound (72.23%). The extract exhibited strong antioxidant activities as evaluated using the ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)), DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric reducing antioxidant power) methods. The extract antimicrobial potential against a range of microorganism strains showed that Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus cereus were the most sensitive bacteria with MBC in the range of 0.5-0.05 mg/mL. Furthermore, in the presence of the syrup extract (8.18-131 µg/mL), the Human SH-SY5Y neuroblastoma and the 3T3 fibroblast cell lines showed dissimilar reduction of viability suggesting a higher cytotoxic effect against tumorigenic cells. Our results provide new insights into date syrup characterization which should stimulate further studies of this hot desert resource.