Mechanism of action of sprG1-encoded type I toxins in Staphylococcus aureus: from membrane alterations to mesosome-like structures formation and bacterial lysis.

sprG1/SprF1 is a type I toxin-antitoxin system located on Staphylococcus aureus prophage. It has previously been shown that the two toxins, SprG131 and SprG144, encoded by the sprG1 gene, are two membrane-associated peptides structured in a single α-helix. Overexpression of these two peptides leads to growth inhibition and even S. aureus death. In this study, we investigated the involvement of each peptide in this toxicity, the sequence requirements necessary for SprG131 toxicity, and the mechanism of action of these two peptides. Our findings show that both peptides, when expressed individually, are able to stop growth, with higher toxicity observed for SprG131. The combination of a hydrophobic domain and a charged domain located only at the C-terminus is necessary for this toxicity, likely to retain the orientation of the transmembrane domain. A net cationic charge for SprG131 is not essential to induce a growth defect in S. aureus. Furthermore, we established a chronology of toxic events following overexpression to gain insights into the mode of action of SprG144 and SprG131. We demonstrated that mesosome-like structures are already formed when membrane is depolarized, about 20 min after peptides induction. This membrane depolarization occurs concomitantly with a depletion of intracellular ATP, leading to S. aureus growth arrest. Moreover, we hypothesized that SprG144 and SprG131 do not form large pores in the S. aureus membrane, as ATP is not excreted into the extracellular medium, and membrane permeabilization is delayed relative to membrane depolarization. The next challenge is to identify the conditions under which SprG144 and SprG131 are naturally expressed, and to uncover their potential roles during staphylococcal growth, colonization, and infection.

Mitochondrial remodeling and energy metabolism adaptations in colonic crypts during spontaneous epithelial repair after colitis induction in mice.

Mucosal healing has emerged as a therapeutic goal to achieve lasting clinical remission in ulcerative colitis. Intestinal repair in response to inflammation presumably requires higher energy supplies for the restoration of intestinal barrier and physiological functions. However, epithelial energy metabolism during intestinal mucosal healing has been little studied, whereas inflammation-induced alterations have been reported in the main energy production site, the mitochondria. The aim of the present work was to assess the involvement of mitochondrial activity and the events influencing their function during spontaneous epithelial repair after colitis induction in mouse colonic crypts. The results obtained show adaptations of colonocyte metabolism during colitis to ensure maximal ATP production for supporting energetic demand by both oxidative phosphorylation and glycolysis in a context of decreased mitochondrial biogenesis and through mitochondrial function restoration during colon epithelial repair. In parallel, colitis-induced mitochondrial ROS production in colonic epithelial cells was rapidly associated with transient expression of GSH-related enzymes. Mitochondrial respiration in colonic crypts was markedly increased during both inflammatory and recovery phases despite decreased expression of several mitochondrial respiratory chain complex subunits after colitis induction. Rapid induction of mitochondrial fusion was associated with mitochondrial function restoration. Finally, in contrast with the kinetics expression of genes involved in mitochondrial oxidative metabolism and in glycolysis, the expression of glutaminase was markedly reduced in the colonic crypts both during colitis and repair phases. Overall, our data suggest that the epithelial repair after colitis induction is characterized by a rapid and transient increased capacity for mitochondrial ATP production in a context of apparent restoration of mitochondrial biogenesis and metabolic reorientation of energy production. The potential implication of energy production adaptations within colonic crypts to sustain mucosal healing in a context of altered fuel supply is discussed.

Roseburia intestinalis Modulates PYY Expression in a New a Multicellular Model including Enteroendocrine Cells.

The gut microbiota contributes to human health and disease; however, the mechanisms by which commensal bacteria interact with the host are still unclear. To date, a number of in vitro systems have been designed to investigate the host-microbe interactions. In most of the intestinal models, the enteroendocrine cells, considered as a potential link between gut bacteria and several human diseases, were missing. In the present study, we have generated a new model by adding enteroendocrine cells (ECC) of L-type (NCI-H716) to the one that we have previously described including enterocytes, mucus, and M cells. After 21 days of culture with the other cells, enteroendocrine-differentiated NCI-H716 cells showed neuropods at their basolateral side and expressed their specific genes encoding proglucagon (GCG) and chromogranin A (CHGA). We showed that this model could be stimulated by commensal bacteria playing a key role in health, Roseburia intestinalis and Bacteroides fragilis, but also by a pathogenic strain such as Salmonella Heidelberg. Moreover, using cell-free supernatants of B. fragilis and R. intestinalis, we have shown that R. intestinalis supernatant induced a significant increase in IL-8 and PYY but not in GCG gene expression, while B. fragilis had no impact. Our data indicated that R. intestinalis produced short chain fatty acids (SCFAs) such as butyrate whereas B. fragilis produced more propionate. However, these SCFAs were probably not the only metabolites implicated in PYY expression since butyrate alone had no effect. In conclusion, our new quadricellular model of gut epithelium could be an effective tool to highlight potential beneficial effects of bacteria or their metabolites, in order to develop new classes of probiotics.

Chronic effects of two rutile TiO2 nanomaterials in human intestinal and hepatic cell lines.

BACKGROUND: TiO2 nanomaterials (NMs) are present in a variety of food and personal hygiene products, and consumers are exposed daily to these NMs through oral exposition. While the bulk of ingested TiO2 NMs are eliminated rapidly in stool, a fraction is able to cross the intestinal epithelial barrier and enter systemic circulation from where NMs can be distributed to tissues, primarily liver and spleen. Daily exposure to TiO2 NMs, in combination with a slow rate of elimination from tissues, results in their accumulation within different tissues. Considerable evidence suggests that following oral exposure to TiO2 NMs, the presence of NMs in tissues is associated with a number of adverse effects, both in intestine and liver. Although numerous studies have been performed in vitro investigating the acute effects of TiO2 NMs in intestinal and hepatic cell models, considerably less is known about the effect of repeated exposure on these models. In this study, we investigated the cytotoxic effects of repeated exposure of relevant models of intestine and liver to two TiO2 NMs differing in hydrophobicity for 24 h, 1 week and 2 weeks at concentrations ranging from 0.3 to 80 µg/cm2. To study the persistence of these two NMs in cells, we included a 1-week recovery period following 24 h and 1-week treatments. Cellular uptake by TEM and ToF-SIMS analyses, as well as the viability and pro-inflammatory response were evaluated. Changes in the membrane composition in Caco-2 and HepaRG cells treated with TiO2 NMs for up to 2 weeks were also studied. RESULTS: Despite the uptake of NM-103 and NM-104 in cells, no significant cytotoxic effects were observed in either Caco-2 or HepaRG cells treated for up to 2 weeks at NM concentrations up to 80 µg/cm2. In addition, no significant effects on IL-8 secretion were observed. However, significant changes in membrane composition were observed in both cell lines. Interestingly, while most of these phospholipid modifications were reversed following a 1-week recovery, others were not affected by the recovery period. CONCLUSION: These findings indicate that although no clear effects on cytotoxicity were observed following repeated exposure of differentiated Caco-2 and HepaRG cells to TiO2 NMs, subtle effects on membrane composition could induce potential adverse effects in the long-term.

Genotoxic impact of aluminum-containing nanomaterials in human intestinal and hepatic cells.

Exposure of consumers to aluminum-containing nanomaterials (Al NMs) is an area of concern for public health agencies. As the available data on the genotoxicity of Al2O3 and Al0 NMs are inconclusive or rare, the present study investigated their in vitro genotoxic potential in intestinal and liver cell models, and compared with the ionic form AlCl3. Intestinal Caco-2 and hepatic HepaRG cells were exposed to Al0 and Al2O3 NMs (0.03 to 80 µg/cm2). Cytotoxicity, oxidative stress and apoptosis were measured using High Content Analysis. Genotoxicity was investigated through γH2AX labelling, the alkaline comet and micronucleus assays. Moreover, oxidative DNA damage and carcinogenic properties were assessed using the Fpg-modified comet assay and the cell transforming assay in Bhas 42 cells respectively. The three forms of Al did not induce chromosomal damage. However, although no production of oxidative stress was detected, Al2O3 NMs induced oxidative DNA damage in Caco-2 cells but not likely related to ion release in the cell media. Considerable DNA damage was observed with Al0 NMs in both cell lines in the comet assay, likely due to interference with these NMs. No genotoxic effects were observed with AlCl3. None of the Al compounds induced cytotoxicity, apoptosis, γH2AX or cell transformation.

Myelinosome Organelles in the Retina of R6/1 Huntington Disease (HD) Mice: Ubiquitous Distribution and Possible Role in Disease Spreading.

Visual deficit is one of the complications of Huntington disease (HD), a fatal neurological disorder caused by CAG trinucleotide expansions in the Huntingtin gene, leading to the production of mutant Huntingtin (mHTT) protein. Transgenic HD R6/1 mice expressing human HTT exon1 with 115 CAG repeats recapitulate major features of the human pathology and exhibit a degeneration of the retina. Our aim was to gain insight into the ultrastructure of the pathological HD R6/1 retina by electron microscopy (EM). We show that the HD R6/1 retina is enriched with unusual organelles myelinosomes, produced by retinal neurons and glia. Myelinosomes are present in all nuclear and plexiform layers, in the synaptic terminals of photoreceptors, in the processes of retinal neurons and glial cells, and in the subretinal space. In vitro study shows that myelinosomes secreted by human retinal glial Müller MIO-M1 cells transfected with EGFP-mHTT-exon1 carry EGFP-mHTT-exon1 protein, as revealed by immuno-EM and Western-blotting. Myelinosomes loaded with mHTT-exon1 are incorporated by naive neuronal/neuroblastoma SH-SY5Y cells. This results in the emergence of mHTT-exon1 in recipient cells. This process is blocked by membrane fusion inhibitor MDL 28170. Conclusion: Incorporation of myelinosomes carrying mHTT-exon1 in recipient cells may contribute to HD spreading in the retina. Exploring ocular fluids for myelinosome presence could bring an additional biomarker for HD diagnostics.

ING2 tumor suppressive protein translocates into mitochondria and is involved in cellular metabolism homeostasis.

ING2 (Inhibitor of Growth 2) is a tumor suppressor gene that has been implicated in critical biological functions (cell-cycle regulation, replicative senescence, DNA repair and DNA replication), most of which are recognized hallmarks of tumorigenesis occurring in the cell nucleus. As its close homolog ING1 has been recently observed in the mitochondrial compartment, we hypothesized that ING2 could also translocate into the mitochondria and be involved in new biological functions. In the present study, we demonstrate that ING2 is imported in the inner mitochondrial fraction in a redox-sensitive manner in human cells and that this mechanism is modulated by 14-3-3η protein expression. Remarkably, ING2 is necessary to maintain mitochondrial ultrastructure integrity without interfering with mitochondrial networks or polarization. We observed an interaction between ING2 and mtDNA under basal conditions. This interaction appears to be mediated by TFAM, a critical regulator of mtDNA integrity. The loss of mitochondrial ING2 does not impair mtDNA repair, replication or transcription but leads to a decrease in mitochondrial ROS production, suggesting a detrimental impact on OXPHOS activity. We finally show using multiple models that ING2 is involved in mitochondrial respiration and that its loss confers a protection against mitochondrial respiratory chain inhibition in vitro. Consequently, we propose a new tumor suppressor role for ING2 protein in the mitochondria as a metabolic shift gatekeeper during tumorigenesis.

Mitochondrial Aurora kinase A induces mitophagy by interacting with MAP1LC3 and Prohibitin 2.

Epithelial and haematologic tumours often show the overexpression of the serine/threonine kinase AURKA. Recently, AURKA was shown to localise at mitochondria, where it regulates mitochondrial dynamics and ATP production. Here we define the molecular mechanisms of AURKA in regulating mitochondrial turnover by mitophagy. AURKA triggers the degradation of Inner Mitochondrial Membrane/matrix proteins by interacting with core components of the autophagy pathway. On the inner mitochondrial membrane, the kinase forms a tripartite complex with MAP1LC3 and the mitophagy receptor PHB2, which triggers mitophagy in a PARK2/Parkin-independent manner. The formation of the tripartite complex is induced by the phosphorylation of PHB2 on Ser39, which is required for MAP1LC3 to interact with PHB2. Last, treatment with the PHB2 ligand xanthohumol blocks AURKA-induced mitophagy by destabilising the tripartite complex and restores normal ATP production levels. Altogether, these data provide evidence for a role of AURKA in promoting mitophagy through the interaction with PHB2 and MAP1LC3. This work paves the way to the use of function-specific pharmacological inhibitors to counteract the effects of the overexpression of AURKA in cancer.

Avrilella dinanensis gen. nov., sp. nov., a novel bacterium of the family Flavobacteriaceae isolated from human blood.

Polyphasic taxonomic analysis was performed on a novel bacterium, designated UR159T, isolated in 2016 from human blood of a septic patient hospitalized in France. Preliminary 16S rRNA gene sequence-based phylogenetic analysis indicated that strain UR159T belonged to the family Flavobacteriaceae, forming a distinct phyletic line distantly related (<94% sequence similarity) to known species of the family. Further phenotypic, chemotaxonomic and genomic analyses were performed. Cells were non-motile, oxidase-negative, catalase-positive Gram-negative rods. It was strictly aerobic yielding yellow-pigmented colonies, and was metabolically rather inert. Major fatty acids were iso-branched fatty acids, predominantly iso-C15:0 (55.5%) and iso-C17:1ω9c (8.8%). Whole genome sequencing revealed a 2.3-Mbp genome encoding a total of 2262 putative genes with a genomic DNA G+C content at 37.6mol%. The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values between strain UR159T and the most closely related members of the Flavobacteriaceae family were <75% and <39%, respectively, much below the established cut-offs for ANI (<95-96%) and isDDH (<70%) for species and genus delineation. Average Amino Acid Identity (AAI) percentages were also estimated and were lower than 65% (cut-off proposed for genus delineation for uncultivated prokaryotes) in all cases, except for F. marinum that was just at the limit (65.1%). Based on these findings, we propose it as a new genus and species, Avrilella dinanensis gen. nov., sp. nov. (type strain UR159T=CIP 111616T=DSM 105483T).

Extracellular vesicles released by polycyclic aromatic hydrocarbons-treated hepatocytes trigger oxidative stress in recipient hepatocytes by delivering iron.

A growing body of evidences indicate the major role of extracellular vesicles (EVs) as players of cell communication in the pathogenesis of liver diseases. EVs are membrane-enclosed vesicles released by cells into the extracellular environment. Oxidative stress is also a key component of liver disease pathogenesis, but no role for hepatocyte-derived EVs has yet been described in the development of this process. Recently, some polycyclic aromatic hydrocarbons (PAHs), widespread environmental contaminants, were demonstrated to induce EV release from hepatocytes. They are also well-known to trigger oxidative stress leading to cell death. Therefore, the aim of this work was to investigate the involvement of EVs derived from PAHs-treated hepatocytes (PAH-EVs) in possible oxidative damages of healthy recipient hepatocytes, using both WIF-B9 and primary rat hepatocytes. We first showed that the release of EVs from PAHs -treated hepatocytes depended on oxidative stress. PAH-EVs were enriched in proteins related to oxidative stress such as NADPH oxidase and ferritin. They were also demonstrated to contain more iron. PAH-EVs could then induce oxidative stress in recipient hepatocytes, thereby leading to apoptosis. Mitochondria and lysosomes of recipient hepatocytes exhibited significant structural alterations. All those damages were dependent on internalization of EVs that reached lysosomes with their cargoes. Lysosomes thus appeared as critical organelles for EVs to induce apoptosis. In addition, pro-oxidant components of PAH-EVs, e.g. NADPH oxidase and iron, were revealed to be necessary for this cell death.

Adding the oxygen carrier M101 to a cold-storage solution could be an alternative to HOPE for liver graft preservation.

BACKGROUND & AIMS: Hypothermic oxygenated machine perfusion (HOPE) is a promising technique for providing oxygen to the liver during graft preservation; however, because of associated logistical constraints, addition of an oxygen transporter to static cold-storage solutions (SCS) might be easier. M101 is marine worm haemoglobin that has been shown to improve kidney preservation in the clinic when added to SCS. This study evaluated the effects of the addition of M101 to SCS on the quality of pig liver graft preservation. METHODS: Pig liver grafts were preserved using SCS, HOPE, or SCS+M101, and the liver functions were compared during cold preservation and after orthotopic allotransplantation (OLT) in pigs. RESULTS: During preservation of the liver grafts, mitochondrial function, ATP synthesis, antioxidant capacities, and hepatocyte architecture were better preserved, and free radical production, antioxidant activities, and inflammatory mediators were lower, with HOPE or SCS+M101 than with SCS alone. However, after 1 h of preservation, liver functions with HOPE were superior to those with SCS+M101. After 6 h of preservation and OLT, blood levels of aspartate and alanine aminotransferases and lactate dehydrogenase increased with a peak effect at Day 1 post-transplant; values were similar with HOPE and SCS+M101, and were significantly lower than those in the SCS group. At Days 1 and 3, tumor necrosis factor α levels remained lower with HOPE and SCS+M101 vs. SCS. At Day 7, liver cell necrosis and inflammation were less marked in both oxygenated groups. CONCLUSIONS: When added to SCS, M101 effectively oxygenates liver grafts during preservation, preventing post-transplant injury; although graft performances are below those achieved with HOPE. LAY SUMMARY: When transported between donors and recipients, even cold-stored liver grafts need oxygen to maintain their viability. To provide them with oxygen, we added a marine worm super haemoglobin (M101) to the cold-storage solution UWCS. Using a pig liver transplant model, we revealed that livers cold stored with UWCS+M101 showed improved oxygenation compared with simple cold-storage solutions, but did not reach the oxygenation level achieved with machine perfusion.

Extracellular vesicles produced by human and animal Staphylococcus aureus strains share a highly conserved core proteome.

Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extracellular vesicles (EVs) that are involved in cellular communication and enable inter-kingdom crosstalk, the delivery of virulence factors and modulation of the host immune response. The protein content of EVs determines their biological functions. Clarifying which proteins are selected, and how, is of crucial value to understanding the role of EVs in pathogenesis and the development of molecular delivery systems. Here, we postulated that S. aureus EVs share a common proteome containing components involved in cargo sorting. The EV proteomes of five S. aureus strains originating from human, bovine, and ovine hosts were characterised. The clustering of EV proteomes reflected the diversity of the producing strains. A total of 253 proteins were identified, 119 of which composed a core EV proteome with functions in bacterial survival, pathogenesis, and putatively in EV biology. We also identified features in the sequences of EV proteins and the corresponding genes that could account for their packaging into EVs. Our findings corroborate the hypothesis of a selective sorting of proteins into EVs and offer new perspectives concerning the roles of EVs in S. aureus pathogenesis in specific host niches.

The Ouzo effect: A tool to elaborate high-payload nanocapsules.

We investigate the encapsulation in hybridosomes®, a type of capsules unique regarding their structure and method of elaboration. Hybridosomes® are made of a single shell of inorganic nanoparticles (~5 nm) crosslinked with a polymer and are easily obtained via spontaneous emulsification in a ternary mixture THF/water/butylated hydroxytoluene (BHT). Our main finding is that an exceptionally high concentration of a hydrophobic model dye can be loaded in the hybridosomes®, up to 0.35 mol.L-1 or equivalently 170 g.L-1 or 450,000 molecules/capsule. The detailed investigation of the encapsulation mechanism shows that the dye concentrates in the droplets during the emulsification step simultaneously with capsule formation. Then it precipitates inside the capsules during the course of solvent evaporation. In vitro fluorescence measurements show that the nano-precipitated cargo can be transferred from the core of the hybridosomes® to the membrane of liposomes. In vivo studies suggest that the dye diffuses through the body during several days. The released dye tends to accumulate in body-fat, while the inorganic nanoparticles remain trapped into the liver and the spleen macrophages.

Hybrid gold nanoparticle-quantum dot self-assembled nanostructures driven by complementary artificial proteins.

Hybrid nanostructures are constructed by the direct coupling of fluorescent quantum dots and plasmonic gold nanoparticles. Self-assembly is directed by the strong affinity between two artificial α-repeat proteins that are introduced in the capping layers of the nanoparticles at a controlled surface density. The proteins have been engineered to exhibit a high mutual affinity, corresponding to a dissociation constant in the nanomolar range, towards the protein-functionalized quantum dots and gold nanoparticles. Protein-mediated self-assembly is evidenced by surface plasmon resonance and gel electrophoresis. The size and the structure of colloidal superstructures of complementary nanoparticles are analyzed by transmission electron microscopy and small angle X-ray scattering. The size of the superstructures is determined by the number of proteins per nanoparticle. The well-defined geometry of the rigid protein complex sets a highly uniform interparticle distance of 8 nm that affects the emission properties of the quantum dots in the hybrid ensembles. Our results open the route to the design of hybrid emitter-plasmon colloidal assemblies with controlled near-field coupling and better optical response.

[Autophagy and spermatozoa]. / Autophagie et spermatozoïde.

Spermiogenesis, the ultimate stage of spermatogenesis, is a process involving autophagy. At this stage, the acrosome is generated by vesicular fusion and most of the cytoplasm disappears. Autophagy, literally "eating oneself", allowing the elimination and replacement of proteins and nonfunctional organelles, ensures the recycling of cellular constituents and is a highly conserved cellular mechanism within eukaryotic cells. The machinery of autophagy is present in the spermatozoon, regulating the vitality and mobility of the cells. The environmental and behavioral impact on autophagy and the consequences on spermatogenesis are beginning to be studied. The purpose of this review is to synthesize current knowledge about autophagy in the mature male gamete.

TITLE: Autophagie et spermatozoïde. ABSTRACT: La spermiogenèse, étape ultime de la spermatogenèse, est un processus qui fait intervenir des acteurs qui participe à l'autophagie. C'est en effet lors de cette étape que se forme l'acrosome par fusion vésiculaire et que disparaît la majeure partie du cytoplasme du spermatozoïde. L'autophagie (littéralement « se manger soi-même ¼), en permettant l'élimination et le remplacement continuel des protéines et des organites non fonctionnels, assure le recyclage des constituants de la cellule. C'est un mécanisme cellulaire très conservé au sein des cellules eucaryotes. La machinerie de l'autophagie est également présente dans les spermatozoïdes. Elle régule la vitalité de ces cellules et leur mobilité. Les conséquences environnementales et comportementales sur l'autophagie et sur la spermatogenèse commencent à être étudiées. Le but de cette revue est de synthétiser les connaissances actuelles concernant les processus d'autophagie dans le gamète mâle mature.

Protective Action of Ostreococcus tauri and Phaeodactylum tricornutum Extracts towards Benzo[a]Pyrene-Induced Cytotoxicity in Endothelial Cells.

Marine microalgae are known to be a source of bioactive molecules of interest to human health, such as n-3 polyunsaturated fatty acids (n-3 PUFAs) and carotenoids. The fact that some of these natural compounds are known to exhibit anti-inflammatory, antioxidant, anti-proliferative, and apoptosis-inducing effects, demonstrates their potential use in preventing cancers and cardiovascular diseases (CVDs). Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), is an ubiquitous environmental pollutant known to contribute to the development or aggravation of human diseases, such as cancer, CVDs, and immune dysfunction. Most of these deleterious effects are related to the activation of the polycyclic aromatic hydrocarbon receptor (AhR). In this context, two ethanolic microalgal extracts with concentrations of 0.1 to 5 µg/mL are tested, Ostreoccoccus tauri (OT) and Phaeodactylum tricornutum (PT), in order to evaluate and compare their potential effects towards B[a]P-induced toxicity in endothelial HMEC-1 cells. Our results indicate that the OT extract can influence the toxicity of B[a]P. Indeed, apoptosis and the production of extracellular vesicles were decreased, likely through the reduction of the expression of CYP1A1, a B[a]P bioactivation enzyme. Furthermore, the B[a]P-induced expression of the inflammatory cytokines IL-8 and IL1-ß was reduced. The PT extract only inhibited the expression of the B[a]P-induced cytokine IL-8 expression. The OT extract therefore seems to be a good candidate for counteracting the B[a]P toxicity.

PAHs increase the production of extracellular vesicles both in vitro in endothelial cells and in vivo in urines from rats.

Environmental contaminants, to which humans are widely exposed, cause or worsen several diseases, like cardiovascular diseases and cancers. Among these molecules, polycyclic aromatic hydrocarbons (PAHs) stand out since they are ubiquitous pollutants found in ambient air and diet. Because of their toxic effects, public Health agencies promote development of research studies aiming at increasing the knowledge about PAHs and the discovery of biomarkers of exposure and/or effects. Extracellular vesicles (EVs), including small extracellular vesicles (S-EVs or exosomes) and large extracellular vesicles (L-EVs or microvesicles), are delivery systems for multimolecular messages related to the nature and status of the originating cells. Because they are produced by all cells and detected within body fluids, EV releases could act as cell responses and thereby serve as biomarkers. To test whether EVs can serve as biomarkers of PAHs exposure, we evaluate the effects of these pollutants on EV production using an in vitro approach (human endothelial cell line, HMEC-1) and an in vivo approach (urine samples from PAHs-exposed rats). Our study indicates that, i) PAH exposure increases in vitro the EV production by endothelial cells and in vivo the release of EVs in urine, and that the stimulating effects of PAHs concern both S-EVs and L-EVs; ii) PAH exposure and more particularly exposure to B[a]P, can influence the composition of exosomes produced by endothelial cells; iii) the aryl hydrocarbon receptor, a cytosolic receptor associated to most deleterious effects of PAHs, would be involved in the PAH effects on the release of S-EVs, but not L-EVs. These results suggest that EVs may have utility for monitoring exposure to PAHs, and more particularly to B[a]P, considered as reference PAH, and to detect the related early cellular response prior to end-organ damages.

Polycyclic Aromatic Hydrocarbons Can Trigger Hepatocyte Release of Extracellular Vesicles by Various Mechanisms of Action Depending on Their Affinity for the Aryl Hydrocarbon Receptor.

Extracellular vesicles (EVs) are membrane-enclosed nanostructures released by cells into the extracellular environment. As major actors of physiological intercellular communication, they have been shown to be pathogenic mediators of several liver diseases. Extracellular vesicles also appear to be potential actors of drug-induced liver injury but nothing is known concerning environmental pollutants. We aimed to study the impact of polycyclic aromatic hydrocarbons (PAHs), major contaminants, on hepatocyte-derived EV production, with a special focus on hepatocyte death. Three PAHs were selected, based on their presence in food and their affinity for the aryl hydrocarbon receptor (AhR): benzo[a]pyrene (BP), dibenzo[a,h]anthracene (DBA), and pyrene (PYR). Treatment of primary rat and WIF-B9 hepatocytes by all 3 PAHs increased the release of EVs, mainly comprised of exosomes, in parallel with modifying exosome protein marker expression and inducing apoptosis. Moreover, PAH treatment of rodents for 3 months also led to increased EV levels in plasma. The EV release involved CYP metabolism and the activation of the transcription factor, the AhR, for BP and DBA and another transcription factor, the constitutive androstane receptor, for PYR. Furthermore, all PAHs increased cholesterol levels in EVs but only BP and DBA were able to reduce the cholesterol content of total cell membranes. All cholesterol changes very likely participated in the increase in EV release and cell death. Finally, we studied changes in cell membrane fluidity caused by BP and DBA due to cholesterol depletion. Our data showed increased cell membrane fluidity, which contributed to hepatocyte EV release and cell death.

Lichen butyrolactone derivatives disrupt oral bacterial membrane.

We have previously demonstrated that out of the butyrolactones series synthesized based on the natural lichen metabolite lichesterinic acid, compound (B-13) was the most effective against oral bacteria. However, its antibacterial mechanism is still unknown. In this study, we have investigated its bacterial localization by synthesizing a fluorescently labeled B-13 with NBD while maintaining its antibacterial activity. We showed that this compound binds to Streptococcus gordonii cell surface, as demonstrated by HPLC analysis. By adhering to cell surface, B-13 induced cell wall disruption leading to the release of bacterial constituents and consequently, the death of S. gordonii, a Gram-positive bacterium. A Gram-negative counterpart, Porphyromanas gingivalis, showed also cracked and ruptured cells in the presence of B-13. Besides, we also demonstrated that the analog of B-13, B-12, has also induced disruption of P. gingivalis and S. gordonii. This study revealed that butyrolactones can be considered as potent antibacterial compounds against oral pathogens causing medical complications.

Aluminum in liver cells - the element species matters.

Aluminum (Al) can be ingested from food and released from packaging and can reach key organs involved in human metabolism, including the liver via systemic distribution. Recent studies discuss the occurrence of chemically distinct Al-species and their interconversion by contact with biological fluids. These Al species can vary with regard to their intestinal uptake, systemic transport, and therefore could have species-specific effects on different organs and tissues. This work aims to assess the in vitro hepatotoxic hazard potential of three different relevant Al species: soluble AlCl3 and two nanoparticulate Al species were applied, representing for the first time an investigation of metallic nanoparticles besides to mineral bound γ-Al2O3 on hepatic cell lines. To investigate the uptake and toxicological properties of the Al species, we used two different human hepatic cell lines: HepG2 and differentiated HepaRG cells. Cellular uptake was determined by different methods including light microscopy, transmission electron microscopy, side-scatter analysis, and elemental analysis. Oxidative stress, mitochondrial dysfunction, cell death mechanisms, and DNA damage were monitored as cellular parameters. While cellular uptake into hepatic cell lines occurred predominantly in the particle form, only ionic AlCl3 caused cellular effects. Since it is known, that Al species can convert one into another, and mechanisms including 'trojan-horse'-like uptake can lead to an Al accumulation in the cells. This could result in the slow release of Al ions, for which reason further hazard cannot be excluded. Therefore, individual investigation of the different Al species is necessary to assess the toxicological potential of Al particles.