Mixtures of Chemical Pollutants at European Legislation Safety Concentrations: How Safe Are They?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentration

Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations

Multiple liver insults synergize to accelerate experimental hepatocellular carcinoma

Taqman array data from mouse models of chemically-induced hepatocellular carcinom

Fibroblast activation protein is dispensable in the anti-influenza immune response in mice

<div><p>Fibroblast activation protein alpha (FAP) is a unique dual peptidase of the S9B serine protease family, being capable of both dipeptidyl peptidase and endopeptidase activities. FAP is expressed at low level in healthy adult organs including the pancreas, cervix, uterus, submaxillary gland and the skin, and highly upregulated in embryogenesis, chronic inflammation and tissue remodelling. It is also expressed by cancer-associated stromal fibroblasts in more than 90% of epithelial tumours. FAP has enzymatic and non-enzymatic functions in the growth, immunosuppression, invasion and cell signalling of tumour cells. FAP deficient mice are fertile and viable with no gross abnormality, but little data exist on the role of FAP in the immune system. FAP is upregulated in association with microbial stimulation and chronic inflammation, but its function in infection remains unknown. We showed that major populations of immune cells including CD4⁺ and CD8⁺ T cells, B cells, dendritic cells and neutrophils are generated and maintained normally in FAP knockout mice. Upon intranasal challenge with influenza virus, FAP mRNA was increased in the lungs and lung-draining lymph nodes. Nonetheless, FAP deficient mice showed similar pathologic kinetics to wildtype controls, and were capable of supporting normal anti-influenza T and B cell responses. There was no evidence of compensatory upregulation of other DPP4 family members in influenza-infected FAP-deficient mice. FAP appears to be dispensable in anti-influenza adaptive immunity.</p></div

Effects of genetic deficiency of FAP on the numbers and phenotype of leukocytes at steady state.

<p>Organs were harvested from FAP knockout (gko; closed symbols) and wildtype (WT; open symbols) C57BL/6 mice. <b>A.</b> The numbers of thymic CD4^-CD8^- double negative (DN), CD4⁺CD8⁺ double positive (DP) CD4⁺CD8^- and CD8⁺CD4^- single positive (SP) populations. <b>B.</b> The numbers of splenic CD4⁺ and CD8⁺ T cells. <b>C.</b> The percentages of CD44^- naïve, CD25⁺ regulatory and CD44^hi activated/ memory CD4⁺ T cells in the thymus and the spleen. <b>D.</b> The percentages of CD8⁺ T cell subsets gated according to their expression of CD44 and CD62L in the thymus and the spleen. <b>E.</b> The numbers of dendritic cells (DC) in the spleen, peripheral LN (pLN), mesenteric LN (mLN) and thymus in FAP knockout and WT mice at steady state. <b>F.</b> The geometric mean fluorescence index (MFI) of surface expression of CD86 (top) and B7H1/PD-L1 (bottom) on DC in the spleen, pLN, mLN and thymus. <b>G.</b> The numbers of splenic CD11b⁺Ly6G⁺ neutrophils. <b>H.</b> The numbers of splenic CD19⁺ B cells. Each symbol represents one mouse and the bar represents the mean. Except for part <b>F</b>, results were pooled from two independent experiments of n = 4 mice each. For <b>F</b>, results from one of three experiments (n = 4 mice each) are shown. Statistical significance was tested using the Mann–Whitney test. * p<0.05, ** p<0.01.</p

T and B cell responses to influenza in FAP knockout (gko) and wildtype (WT) mice.

<p><b>A & B.</b> FAP knockout (gko; closed symbols) and wildtype (WT; open symbols) mice received intravenous transfer of donor OT-1 cells and on the next day were intranasally inoculated with 100 pfu PR/8-OVA influenza virus. Organs were obtained on day 7 post-infection. <b>A.</b> The numbers of donor cells recovered from recipient lungs and mediastinal lymph nodes (mdLN) on day 7 post-infection. <b>B.</b> Cells harvested from the lungs and mdLN were restimulated <i>in vitro</i> in the presence of SIINFEKL peptide and the production of IL-2 (top) and IFN-γ (bottom) by donor OT-I cells was quantified by flow cytometry. The percentage of positive events in stained samples and fluorescence minus one (FMO) negative controls are shown. Each data point represents an individual mouse and the bar represents the mean. Results from one of three experiments are shown. Statistical significance was tested using the Mann–Whitney test. <b>C.</b> FAP knockout and WT mice were intranasally infected with 50 pfu PR/8 virus and sera were harvested on day 21 post-infection. Neutralising anti-influenza antibody titres in the sera were measured using haemagglutination inhibition (HI) assay. Each data point represents an individual mouse and the bar represents the mean. Statistical significance was tested using Kruskal-Wallis test with Dunn’s multiple comparison test. * p<0.05, *** p<0.001, ns not significant.</p

The pathologic response of FAP knockout (gko) versus wildtype (WT) mice to intranasal influenza infection and the associated changes in expression of DPP4 family members.

<p>FAP knockout (gko; closed symbols) and WT (open symbols) mice were intranasally infected with 50 pfu PR/8 influenza virus. <b>A.</b> Real-time quantitative PCR results showing relative expression of FAP normalised to the housekeeping gene <i>hprt</i> in the mediastinal lymph nodes (mdLN), and normalised to the <i>18S RNA</i> gene in the lungs (bottom) of uninfected (-) and infected (+) WT mice on day 7 post-infection. Statistical significance was tested using Mann-Whitney test. <b>B.</b> Weight loss response in influenza-infected 10-week old female FAP knockout (closed symbols) and WT (open symbols) mice (n = 7). Results from one of three replicate experiments are shown. Statistical significance was tested using Student’s t-test. * p< 0.05. <b>C.</b> Real-time quantitative PCR results showing relative expression of influenza virus (Inf A), normalised to the 18S RNA gene in the lungs of infected FAP knockout (closed symbols) and WT (open symbols) mice on day 7 post-infection (n = 5 per group). Inf A expression was not detected in uninfected lungs. Each symbol represents data from one mouse and the bar represents the mean±SEM. Statistical significance was tested using the Mann-Whitney test. <b>D.</b> Expression of DPP4 family members DPP4, DPP8 and DPP9 in uninfected and infected FAP knockout (closed symbols) and WT (open symbols) mice. Each symbol represents data from one mouse and the bar represents the mean±SEM. Results from one of two experiments are shown. Statistical significance was tested using the Mann-Whitney test.</p

Metalloproteins and phytochelatin synthase may confer protection against zinc oxide nanoparticle induced toxicity in Caenorhabditis elegans

Zinc oxide nanoparticles (ZnONPs) are used in large quantities by the cosmetic, food and textile industries. Here we exposed Caenorhabditis elegans wild-type and a metal sensitive triple knockout mutant (mtl-1;mtl-2;pcs-1) to ZnONPs (0-50mg/L) to study strain and exposure specific effects on transcription, reactive oxygen species generation, the biomolecular phenotype (measured by Raman microspectroscopy) and key endpoints of the nematode life cycle (growth, reproduction and lifespan). A significant dissolution effect was observed, where dissolved ZnO constituted over 50% of total Zn within a two day exposure to the test medium, suggesting that the nominal exposure to pure ZnONPs represents in vivo, at best, a mixture exposure of ionic zinc and nanoparticles. Nevertheless, the analyses provided evidence that the metallothioneins (mtl-1 and mtl-2), the phytochelatin synthase (pcs-1) and an apoptotic marker (cep-1) were transcriptionally activated. In addition, the DCFH-DA assay provided in vitro evidence of the oxidative potential of ZnONPs in the metal exposure sensitive triple mutant. Raman spectroscopy highlighted that the biomolecular phenotype changes significantly in the mtl-1;mtl-2;pcs-1 triple knockout worm upon ZnONP exposure, suggesting that these metalloproteins are instrumental in the protection against cytotoxic damage. Finally, ZnONP exposure was shown to decrease growth and development, reproductive capacity and lifespan, effects which were amplified in the triple knockout. By combining diverse toxicological strategies, we identified that individuals (genotypes) housing mutations in key metalloproteins and phytochelatin synthase are more susceptible to ZnONP exposure, which underlines their importance to minimize ZnONP induced toxicity