Association between mediterranean diet and metal(loid) exposure in 4-5-year-old children living in Spain.

Even relatively low levels of metals exposure may impact health, particularly among vulnerable populations such as infants and young children. However, little is known about the interplay between simultaneous metal exposures, common in real-life scenarios, and their association with specific dietary patterns. In this study, we have evaluated the association between adherence to Mediterranean diet (MD) and urinary metal concentrations individually and as an exposure mixture in 713 children aged 4-5-years from the INMA cohort study. We used a validated food frequency questionnaire to calculate two MD indexes scores: aMED and rMED. These indexes gather information on various food groups within the MD and score differently. To measure urinary concentrations of cobalt, copper, zinc, molybdenum, selenium, lead, and cadmium as exposure biomarkers, we used inductively coupled plasma mass spectrometry (ICP-MS), coupled with an ion chromatography (IC) equipment for arsenic speciation analysis. We applied linear regression and quantile g-computation, adjusted for confounders, to analyse the association between MD adherence and exposure to the metal mixture. High adherence to MD such as the quintile (Q) 5 MD was associated with higher urinary arsenobetaine (AsB) levels than Q1, with ß values of 0.55 (confidence interval - CI 95% 0.01; 1.09) for aMED and 0.73 (CI 95% 0.13; 1.33) for rMED. Consumption of fish was associated with increased urinary AsB but reduced inorganic arsenic concentrations. In contrast, the aMED vegetables consumption increased urinary inorganic arsenic content. A moderate level of adherence to MD (Q2 and Q3) was associated with lower copper urinary concentrations than Q1, with ß values of -0.42 (CI 95% -0.72; -0.11) for Q2 and -0.33 (CI 95% -0.63; -0.02) for Q3, but only with aMED. Our study, conducted in Spain, revealed that adhering to the MD reduces exposure to certain metals while increasing exposure to others. Specifically, we observed increase in exposure to non-toxic AsB, highlighting the significance of consuming fish/seafood. However, it is crucial to emphasize the necessity for additional efforts in reducing early-life exposure to toxic metals, even when adhering to certain food components of the MD.

Full life cycle test with Eisenia fetida - copper oxide NM toxicity assessment.

Copper oxide nanomaterials (CuONM) are widely used, e.g. as antimicrobial coatings, wood preservatives,fertilizers, etc. Life cycle aspects of Copper Nanomaterials (CuONM) toxicity have been scarcely studied in earthworms, as the focus has been on standard survival and reproduction toxicity tests. Standard tests do not allow for an understanding of which life cycle stage is the most sensitive, and how this can be impacted by CuONMs toxicity to cause longer term population level effects. Since CuONM may release free Cu ions (Cu2+) it is relevant to compare CuONM toxicity with CuCl2 salt. Therefore, the aim of the present study was to assess the effects of CuONM and CuCl2 throughout the different stages of the full life cycle (FLC) of Eisenia fetida while comparing it with the OECD standard test. Additional endpoints included juvenile survival, growth, maturation, besides survival and reproduction. The FLC test showed that e.g. juvenile survival was especially susceptible during the first 28 days post-hatching, neither juvenile growth nor time to maturity were related to exposure concentration. Both CuONM and CuCl2 caused a concentration-dependent decrease in population growth, while a hormesis effect was observed at low concentrations of CuCl2. A reduction in instantaneous growth rate was observed at higher concentrations. The FLC test and the OECD test had different exposure history therefore the ECx values are not comparable across the test systems. Hence, the FLC test enabled the detection of the most vulnerable developmental stages and elucidate different life stage sensitivities between the two Cu exposures.

Building confidence in skin sensitisation potency assessment using new approach methodologies: report of the 3rd EPAA Partners Forum, Brussels, 28th October 2019.

Skin sensitising substances that induce contact allergy and consequently risk elicitation of allergic contact dermatitis (ACD) remain an important focus regarding the replacement of animal experimentation. Current in vivo methods, notably the local lymph node assay (LLNA) refined and reduced animal usage and led to a marked improvement in hazard identification, characterisation and risk assessment. Since validation, regulatory confidence in the LLNA approach has evolved until it became the first choice assay in most regulated sectors. Currently, hazard identification using the LLNA is being actively replaced by a toolbox of non-animal approaches. However, there remains a need to increase confidence in the use of new approach methodologies (NAMs) as replacements for LLNA sensitiser potency estimation. The EPAA Partners Forum exchanged the current state of knowledge on use of NAMs in various industry sectors and regulatory environments. They then debated current challenges in this area and noted several ongoing needs. These included a requirement for reference standards for potency, better characterisation of applicability domains/technical limitations of NAMs, development of a framework for weight of evidence assessments, and an increased confidence in the characterisation of non-sensitisers. Finally, exploration of an industry/regulator cross-sector user-forum on skin sensitisation was recommended.

Manganese levels in newborns' hair by maternal sociodemographic, dietary and environmental factors.

Exposure to manganese (Mn) has been associated with neurodevelopmental problems in children. Mn exposure begins in utero, and maternal sociodemographic, dietary and environmental factors may affect Mn levels in newborns' hair. This study aimed to characterize in utero Mn exposure using Mn levels in newborns' hair as a biomarker of prenatal exposure, and to analyze its relationship with sociodemographic, dietary and environmental factors in the Spanish INMA-Gipuzkoa cohort. Overall, 638 pregnant women were recruited in the first trimester of pregnancy and a hair sample was obtained from 473 newborns. Sociodemographic, dietary and environmental data were recorded through two questionnaires administered in the first and third trimesters of pregnancy. The median Mn concentration in newborns´ hair was 0.31 µg/g (P5 = 0.02; P95 = 1.23). Mn levels in newborns´ hair were associated with smoking during pregnancy (ß = 0.222; 95% CI = 0.065-0.379) and with maternal PM2.5 exposure (ß = 0.025; 95% CI = 0.004; 0.047), as well as with cereal and pasta intake in the first trimester of pregnancy (ß = 0.002; 95% CI = 0.0004; 0.003). In conclusion, fetal exposure to Mn was associated with smoking and exposure to atmospheric pollution during pregnancy.

In vitro RHE skin sensitisation assays: Applicability to challenging substances.

In the last 20 years, alternative approaches to the identification of skin sensitisation hazards have been at the forefront of the 3Rs and have helped refine the validation and acceptance processes. However, experience with the local lymph node assay showed that, post-validation, challenges still occurred, particularly when a wider diversity of chemical substances was addressed, a situation which will arise with validated in vitro alternatives. In the present work, a range of substances potentially challenging to assess in current nonanimal OECD test guidelines were evaluated in several of the emerging in vitro alternatives. Twelve such substances (of which just over half were known skin sensitisers) were assessed in 4 assays, all based on reconstructed human epidermis (RHE) models. For hazard identification, the overall predictive accuracy ranged around 70% for three assays, although for one (SensCeeTox), it fell below 50% when human data was used as the benchmark. In most cases, sensitivity was high, such that sensitisation was overpredicted. As the substances were challenging to assess in other nonanimal methods, the results indicate that the 3D RHE models may be a useful tool for assessing skin sensitisation potentials without needing to revert to animal use.

Selection of an optimal culture medium and the most responsive viability assay to assess AgNPs toxicity with primary cultures of Eisenia fetida coelomocytes.

Earthworm immune cells (coelomocytes) have become a target system in ecotoxicology due to their sensitivity against a wide range of pollutants, including silver nanoparticles (AgNPs). Presently, in vitro approaches (viability assays in microplate, flow cytometry, cell sorting) with primary cultures of Eisenia fetida coelomocytes have been successfully used to test the toxicity and the dissimilar response of cell subpopulations (amoebocytes and eleocytes) after PVP-PEI coated AgNPs and AgNO3 exposures. In order to obtain reliable data and to accurately assess toxicity with coelomocytes, first an optimal culture medium and the most responsive assay were determined. AgNPs posed a gradual decrease in coelomocytes viability, establishing the LC50 value in RPMI-1640 medium at 6 mg/l and discarding that the observed cytotoxicity was attributable to its coating agent PVP-PEI. Exposure to AgNPs caused selective cytotoxicity in amoebocytes, which correlated with the Ag concentrations measured in sorted amoebocytes and reinforced the idea of dissimilar sensitivities among amoebocytes and eleocytes. Silver nano and ionic forms exerted similar toxicity in coelomocytes. The in vitro approaches with coelomocytes of E. fetida performed in this study have the capacity to predict impairments caused by pollutants at longer exposure levels and thus, provide rapid and valuable information for eco(nano)toxicology.

Interspecies assessment factors and skin sensitization risk assessment.

For many endpoints in toxicology, an interspecies safety factor remains a standard requirement. However, for skin sensitization, the hazard and potency predictions, notably from the local lymph node assay (LLNA) have been shown to correlate well with human data. Despite this, there are always exceptions, both over and under predictions. For this reason it has been suggested that an interspecies factor of 15 would accommodate potential "errors". An alternative approach is suggested in which an evidence-based strategy is taken: the large majority of the information indicates a human:LLNA ratio of 1, therefore a corrective factor would best be applied where our knowledge of the underlying chemistry of sensitization indicates that it is necessary.

Environmental fate and effect of biodegradable electro-spun scaffolds (biomaterial)-a case study.

Poly-ε-caprolactone (PCL) based medical devices are increasingly produced and thus, their presence in the environment is likely to increase. The present study analysed the biodegradation of PCL electro-spun scaffolds (alone) and PCL electro-spun scaffolds coated with human recombinant (hR) collagen and Bovine Achilles tendon (BAT) collagen in sewage sludge and in soil. Additionally, an eco-toxicological test with the model organism Enchytraeus crypticus was performed to assess environmental hazard of the produced materials in soils. The electro-spun scaffolds were exposed to activated sludge and three different soils for various time periods (0-7-14-21-28-56-180 days); subsequently the degradation was determined by weight loss and microscopical analysis. Although no toxicity occurred in terms of Enchytraeus crypticus reproduction, our data indicate that biodegradation was dependent on the coating of the material and exposure condition. Further, only partial PCL decomposition was possible in sewage treatment plants. Collectively, these data indicate that electro-spun PCL scaffolds are transferred to amended soils.

Effects of soil organic matter content on cadmium toxicity in Eisenia fetida: implications for the use of biomarkers and standard toxicity tests.

Bioavailability is affected by soil physicochemical characteristics such as pH and organic matter (OM) content. In addition, OM constitutes the energy source of Eisenia fetida, a well established model species for soil toxicity assessment. The present work aimed at assessing the effects of changes in OM content on the toxicity of Cd in E. fetida through the measurement of neutral red uptake (NRU) and mortality, growth, and reproduction (Organisation for Economic Co-operation and Development [OECD] Nos. 207 and 222). Complementarily, metallothionein (MT) and catalase transcription levels were measured. To decrease variability inherent to natural soils, artificial soils (Organization for Economic Cooperation and Development 1984) with different OM content (6, 10, and 14%) and spiked with Cd solutions at increasing concentrations were used. Low OM in soil decreased soil ingestion and Cd bioaccumulation but also increased Cd toxicity causing lower NRU of coelomocytes, 100 % mortality, and stronger reproduction impairment, probably due to the lack of energy to maintain protection mechanisms (production of MT).Cd bioaccumulation did not reflect toxicity, and OM played a pivotal role in Cd toxicity. Thus, OM content should be taken into account when using E. fetida in in vivo exposures for soil health assessment.

PFAS levels and exposure determinants in sensitive population groups.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants. The first exposure to PFAS occurs in utero, after birth it continues via breast milk, food intake, environment, and consumer products that contain these chemicals. Our aim was to identify determinants of PFAS concentrations in sensitive population subgroups- pregnant women and newborns. METHODS: Nine European birth cohorts provided exposure data on PFAS in pregnant women (INMA-Gipuzkoa, Sabadell, Valencia, ELFE and MoBa; total N = 5897) or newborns (3xG study, FLEHS 2, FLEHS 3 and PRENATAL; total N = 940). PFOS, PFOA, PFHxS and PFNA concentrations were measured in maternal or cord blood, depending on the cohort (FLEHS 2 measured only PFOS and PFOA). PFAS concentrations were analysed according to maternal characteristics (age, BMI, parity, previous breastfeeding, smoking, and food consumption during pregnancy) and parental educational level. The association between potential determinants and PFAS concentrations was evaluated using multiple linear regression models. RESULTS: We observed significant variations in PFAS concentrations among cohorts. Higher PFAS concentrations were associated with higher maternal age, primipara birth, and educational level, both for maternal blood and cord blood. Higher PFAS concentrations in maternal blood were associated with higher consumption of fish and seafood, meat, offal and eggs. In cord blood, higher PFHxS concentrations were associated with daily meat consumption and higher PFNA with offal consumption. Daily milk and dairy consumption were associated with lower concentrations of PFAS in both, pregnant women and newborns. CONCLUSION: High detection rates of the four most abundant PFAS demonstrate ubiquitous exposure of sensitive populations, which is of concern. This study identified several determinants of PFAS exposure in pregnant women and newborns, including dietary factors, and these findings can be used for proposing measures to reduce PFAS exposure, particularly from dietary sources.

Defective expression of cytochrome P450 proteins in the liver of the genetically obese Zucker rat.

The hepatic expression of xenobiotic-metabolising cytochrome P450 isoforms in the genetically obese Zucker rat, a model of obesity, was compared to that of its lean littermate. Cytochrome P450 (CYP) levels were determined using diagnostic substrates and/or immunologically in Western blot analyses. When compared with the lean Zucker rat, the obese animal exhibited hyperglycaemia, hypercholesterolaemia, marked hyperinsulinaemia and hypertriglyceridaemia but was normoketonaemic. CYP3A and CYP1A2 levels were higher in the obese Zucker rat when compared with the lean littermate but, in contrast, a protein recognised by human CYP2D6 and, to a lesser extent, CYP2C11 levels were lower. Pretreatment with acetone, dexamethasone and clofibrate resulted in enhanced p-nitrophenol hydroxylase (CYP2E), erythromycin N-demethylase (CYP3A) and lauric acid hydroxylase (CYP4A) activities respectively in the liver of the lean Zucker rat but, in contrast, the obese Zucker rat was refractive to such treatment; similarly, hepatic apoprotein levels of the CYP2E and CYP4A subfamilies were increased markedly only in the lean Zucker rat. It is concluded that CYP2E, CYP3A and CYP4A subfamilies are poorly expressed in the obese Zucker rat, and this rat strain may serve as a good model for elucidating the molecular mechanisms of induction of these cytochrome P450 proteins.

Marked inhibition of hepatic cytochrome P450 activity in cholesterol-induced atherosclerosis in rabbits.

The objective of the present study was to investigate the expression of major xenobiotic-metabolising cytochrome P450 proteins, and of other enzyme systems, in hepatic and extrahepatic tissues of rabbits rendered atherosclerotic by the dietary administration of 1% cholesterol diets for 8 weeks. Individual cytochrome P450 proteins were monitored using diagnostic substrates and immunologically in Western blot analysis. The activity of all hepatic isoforms studied was depressed in the atherosclerotic animals; when, however, apoprotein levels were determined immunologically, no major differences were evident between the control and the atherosclerotic rabbits. In vitro studies indicated that neither cholesterol nor palm oil inhibited cytochrome P450 activity. The effects of cholesterol treatment leading to atherosclerosis on kidney, heart and lung cytochrome P450 activities were isoform- and tissue-specific; no change was evident in the heart activities, but in the lung and kidney cytochrome P450 activities were clearly modulated by the treatment with cholesterol. Apoprotein levels did not always parallel the changes in activities. Western blot analysis of aortic cytochromes P450 revealed that administration of cholesterol-rich diets enhanced CYP2B and CYP3A apoprotein levels. Cholesterol feeding to rabbits gave rise to a marked decrease in hepatic glutathione S-transferase activity but did not influence glutathione reductase or total glutathione levels. The same treatment had no effect on catalase, glutathione peroxidase and superoxide dismutase. It is concluded that treatment of rabbits with cholesterol-rich diets leading to atherosclerosis gives rise to profound changes in the expression of cytochrome P450 proteins in the liver and other tissues; possible mechanisms are discussed.

Zonation in the digestive tract of Eisenia fetida: implications in biomarker measurements for toxicity assessment.

Eisenia fetida is a model species for soil health assessment and different biomarkers that detect either the presence of bioavailable contaminants or their biological effect have been developed. These parameters are performed in a target tissue or whole earthworm, without considering the marked zonation in histological organisation, enzyme activities and gene expression pattern existing along the body. Thus, the present work was aimed at (a) characterising the morphofunctional heterogeneity along the digestive tract of E. fetida in tissue morphology and turnover, lysosomal enzyme markers (ß-glucuronidase, ß-GUS; hexosaminidase, HEX), lipofuscin contents (LPF) and metallothionein (MT) and catalase (CAT) gene expression; and (b) determining whether the responsiveness to Cd exposure varies among tissues and along the digestive tract. HEX and ß-GUS exhibited a heterogeneous distribution pattern along and across the digestive tract and Cd exposure caused a marked decrease of HEX and an increase of ß-GUS activity. Likewise, the significant decrease of cell turnover and the induction of MT transcription were was zone-dependent. Therefore, in was concluded that the consideration of the zonation when applying biomarker for toxicity assessment would reduce the intrinsic variability that results from overlooking the marked morphofunctional heterogeneity that exists in annelids along their body axis.

Extrahepatic expression of P450 proteins in insulin-dependent diabetes mellitus.

1. Male Wistar rats were rendered diabetic by the administration of a single intraperitoneal dose of streptozotocin; the levels of the xenobiotic-inducible P450 proteins were determined in the lung and kidney using diagnostic substrates and immunoblotting employing polyclonal antibodies. The glutathione conjugation system in the cytosol of these tissues was also investigated. 2. The onset of insulin-dependent diabetes did not influence the O-dealkylations of methoxyresorufin, ethoxyresorufin and pentoxyresorufin in either kidney or lung. 3. Lauric acid hydroxylase activity, however, was induced in the kidney but no activity was detectable in the lung. Immunoblot analysis of kidney microsomes using antibodies to P4504A1 revealed the presence of two bands, both of which were clearly inducible in diabetes. In pulmonary microsomes a single faint band was detected which also appeared to be higher in the diabetic rats. 4. Aniline p-hydroxylase activity was not detectable in the kidney, but activity was measurable in the lung and was suppressed in diabetes. Immunoblot analysis of pulmonary microsomes using antibodies to P4502E1 immunodetected a single band which was suppressed in diabetes. In the kidney microsomes a single band was also detected which was, however, markedly elevated in diabetes. 5. Glutathione S-transferase activity was modestly higher in the kidney, but not lung, of the diabetic animals. Glutathione reductase and total glutathione levels were not influenced by the presence of diabetes. 6. It is concluded that streptozotocin-induced insulin-dependent diabetes modulates extrahepatic P450 proteins, the effect being both tissue- and isoform-specific.

Expression and inducibility of cytochrome P450 proteins belonging to families 2,3 and 4 in the rabbit aorta.

The expression and inducibility of cytochrome P450 proteins belonging to families CYP2, CYP3 and CYP4 in the aorta of rabbits were investigated immunologically using polyclonal antibodies to the hepatic proteins. Antibodies to CYP2B recognised two proteins, only one of which was inducible by phenobarbitone. One protein was detected by anti-CYP2E1 which was modestly inducible by imidazole. A highly expressed protein was recognised by antibodies to CYP3A1, the levels of which diminished following treatment with rifampicin. Finally, antibodies to CYP4A1 immunoreacted with a single protein in the aorta which was refractive to treatment with ciprofibrate. The data demonstrate that the rabbit aorta expresses a number of cytochrome P450 families which, although inducible in the liver, are not always inducible in the aorta.

gfp-Tagged cells as a useful tool to study the survival of Escherichia coli in the presence of the river microbial community.

We have used an Escherichia coli strain DH5a containing pGreenTIR to study the survival of this bacterium in river water. As green fluorescence was maintained throughout survival both in dark and illuminated conditions, gfp-tagged E. coli cells were clearly distinguished from the microbial community of the river Butrón. gfp-tagged E. coli cells were monitored to estimate total density as well as the density of the culturable and viable (active electron transport system, CTC+) cells. Our results indicate that autochthonous bacteria and introduced E. coli are predated by flagellates. The autochthonous bacterial community behaves as predation-escaping prey, showing a tendency to cellular miniaturization and so maintaining the density of the population. In contrast, introduced E. coli behaves as predation-non-escaping prey, so E. coli was eliminated from the system. When comparing the elimination by predation of heat-treated and non-heated gfp-tagged E. coli cells we deduce that the flagellates do not discriminate between live and heat-treated cells. Finally, in the presence of the river microbial community, the E. coli cells appeared to be ingested before cellular deterioration could occur. Thus predation reduces the quantitative importance of the viable but nonculturable (VBNC) population of E. coli in the aquatic systems.

Expression and inducibility of cytochrome P450 proteins in the liver of chick embryo.

The expression and inducibility of cytochrome P450 proteins in the liver of chick embryo were investigated using substrate probes and/or immunologically using polyclonal antibodies to the mammalian isoforms. Antibodies to CYP1A1 recognised a single protein which was inducible by structurally diverse chemicals, including aminocompounds, and was parallelled by increases in the O-dealkylations of ethoxy- and methoxyresorufin and in the bioactivation of Glu-P-1. When probed with antibodies to CYP2E1 an immunoreacting protein was revealed which was induced by phenobarbital but not acetone; a second protein became apparent following the treatment with phenobarbital. The increase in apoprotein levels was accompanied by similar increases in p-nitrophenol hydroxylase. Antibodies to CYP2C11 recognised two immunorelated proteins, of which one was inducible by phenobarbital. The same inducer, but not dexamethasone, enhanced the N-demethylation of erythromycin but antibodies to rat CYP3A1 failed to detect any proteins. Finally, lauric acid hydroxylation was not detectable in chick embryo and, moreover, no immunoreacting band was visible following probing of microsomes with anti-CYP4A1. It is concluded that proteins immunorelated to the mammalian CYP1 and CYP2 families are expressed in chick embryo but the regulation of the latter family in the embryo by exogenous chemicals differs markedly from that established for mammals.