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FEBS 2023 - the 47th FEBS Congress, 08-12 juillet, Tours, France
Allouche, R., Hafeez, Z., Dary, A., Genay, M., Miclo, L.
Streptococcus thermophilus is a dairy starter granted “Generally Recognized as Safe” by the FDA and “Qualified Presumption of Safety” by EFSA. A significant part of the world's population ingests this bacterium when consuming fermented products. Some strains of S. thermophilus, either in the live or heat-inactivated state, and peptides released after shaving and hydrolysis of the surface proteins of some strains of this bacterium displayed anti-inflammatory activity in vitro (Allouche et al.,2022). S. thermophilus cells could undergo lysis during their passage through the digestive tract. Consequently, its intracellular proteins could be hydrolysed by endogenous proteases leading to the release of peptides. We hypothesized that peptides generated from digestion of intracellular protein of S. thermophilus might also contribute to its overall anti-inflammatory effect. Therefore, intracellular proteins from S. thermophilus CNRZ-21N strain were recovered after sonication. After fractionation by size exclusion chromatography, the resulting 3-10 kDa protein fraction was hydrolysed by Corolase PP, a mixture of pancreatic proteases. MS-MS analysis showed that most of the identified peptides belonged to the ribosomal proteins. The hydrolysed fraction showed anti-inflammatory activity on macrophagelike THP-1 cells inflamed by LPS since their secretion of IL-8 and IL-1β cytokines and expression level of Pro-IL-1β were reduced. The results suggest that the peptides released from a fraction of intracellular proteins of S. thermophilus after digestion by Corolase PP may contribute to the anti-inflammatory activity of this bacterium and could be used as a functional ingredient to prevent lowgrade inflammation.
5th Edition of Innovations in Food Science and Human Nutrition (IFHN-2022), 20-21 septembre, Barcelone, Espagne
Allouche, R., Hafeez, Z., Dary-Mourot, A., Genay, M., Miclo, L.
23ème Colloque du Club des Bactéries Lactiques, 08-10 juin, Rennes, France
Allouche, R., Hafeez, Z., Papier, F., Dary-Mourot, A., Genay, M., Miclo, L.
61st Society Of Toxicology annual meeting & ToxExpo, 21-27 mars, San Diego, États-Unis
Morel, C., Emond, C., Duca, R., Debaugnies, F., Borde, P., Paoli, J., Hardy, E., Van Nieuwenhuyse, A., Schroeder, H., Grova, N.
Valproic acid-exposed rat is widely recognized as a validated model to assess Autism Spectrum disorders (ASD-VPA model). However, this model had never been studied for a co-exposure to pollutants like brominated flame retardants (e.g. α-hexabromocyclododecane, α-HBCDD). Both compounds interact on the same xenobiotic metabolism pathway resulting for VPA in the formation of delta-4 VPA, a reactive metabolite known to induce hepatic toxicity. Before assessing the role of α-HBCDD as a factor of susceptibility in the ASD-VPA model, it was essential to characterize VPA pharmacokinetic in α-HBCDD-co-exposure context. The aim of this work was thus to assess the pharmaco- and toxicokinetic involving the VPA/α-HBCDD co-exposure in rat. A sub-acute oral dose of α-HBCDD in oil (100 ng/kg/day) was administered in Wistar female rats for 12 days followed by a single intraperitoneal VPA dose (IP, 500 mg/kg bw) at D12 or a daily oral VPA dose for 3 days (PO, 500 mg/kg bw/day, D10-12). Sequential blood samplings were carried out from 20 min to 18 h after the last VPA administration. The results showed that α-HBCDD did not influence the VPA kinetic independently of the administration mode. The consecutive PO administration of VPA for 3 days did not allow to reach the IP Cmax and was 3.75 times lower than the IP one. In parallel, the toxicokinetic of α-HBCDD was carried out using the same sequential time points. Comparable Cmax values were observed for α-HBCDD only or α-HBCDD-VPA exposed rats by IP whereas a 3-fold increase in α-HBCDD Cmax was highlighted in rats administered with VPA PO. Such discrepancy could suggest an inhibition of CYP3A4 by the delta-4 VPA when VPA is administered for 3 days. A lag time seems therefore to be required to inhibit the metabolic pathway of α-HBCDD. A similar protocol was then carried out to demonstrate the efficiency of VPA/α-HBCDD co-exposure in pregnant rats. This preliminary study enabled to valid the usefulness of the VPA model in a context of α-HBCDD co-exposure, and pointed out, for the first time, the ability of VPA to enhance the internal effective dose of α-HBCDD.
Colloque annuel de la Société Cerveau et Maladies Cérébrovasculaires 2021, 28 mai, Dématérialisé
Morel, C., Maguin Gaté, K., Christophe, A., Jubreaux, J., Paoli, J., Degiorgio, C., Bouillaud-Kremarik, P., Grova, N., Schroeder, H.
Introduction: Evidence is now growing that exposure to environmental pollutants during the critical period of early-life brain development may be a strong risk factor, contributing to the emergence of neurobehavioral disorders later in life. The present study aims at evaluating the developmental neurotoxicity of an early exposure to an environmental chemical of high concern for human health, hexabromocyclododecane (HBCDD), a persistent organobromine flame retardant, in comparison with VPA, a common anti-epileptic drug known to induce developmental disorders including teratogenicity and neurobehavioral disturbances. HBCDD is a brominated flame retardant added to foam materials as a technical mixture of 3 isomers with the α-one to be of most concern for human health. Nowadays, little is known about the neurotoxicity of this isomer. A previous study (Maurice et al., Toxicol. Teratol., 2015) highlighted that a daily exposure of rat pups to α-HBCDD during gestation and lactation (66 ng/kg/day) induced early disturbances in locomotor maturation, exploratory activity and level of anxiety later in life.
Material et Methods: In order to assess neurobehavioral impairments induced by a perinatal exposure to HBCDD or VPA in the F1 generation at different postnatal ages from PND1 to PND122, pregnant Wistar rats were divided into three groups: Control, VPA, and HBCDD. HBCDD-exposed rats were administered daily p.o. during gestation and lactation (GD0 to PND21) with 100 ng/kg/day of α-HBCDD isomer in oil, whereas the two other groups received the vehicle only. At GD12, VPA-treated rats received a single i.p. injection of VPA at a dose of 600 mg/kg whereas the two other groups were injected with the vehicle only. Pups were tested for their early behavioral development from PND3 to PND21 using a standardized test battery. Anxiety and social behavior were assessed at two time points (juvenile PND 41 and adult age PND 122) by using the elevated-plus-maze and the social recognition test.
Results: The present results point out the ability of both compounds to induce subtle behavioral disturbances that may be quite different between the two chemicals.
Conclusion: The results of the social recognition test associated with the measurements of the cytochrome oxydase activity and epigenetic changes in various brain regions, currently under progress, should provide us additional information on the impact of an early exposure to HBCDD or VPA on behavioral impairments later in life.
Séminaire de l'école doctorale SIReNa, 26 mars, Dématérialisé
Morel, C., Christophe, A., Maguin-Gaté, K., Jubreaux, J., Degiorgio, C., Bouillaud-Kremarik, P., Grova, N., Schroeder, H.
Evidence is now growing that early-life environmental pollutant exposure during the critical period of brain development may be an important risk factor, contributing to the emergence of neurobehavioral disorders later in life (Grova et al., 2019). In this context, our team previously highlighted that a daily exposure of rat pups to the α isomer of HBCDD, a brominated flame retardant largely added to polystyrene building materials, during gestation and lactation (66 ng/kg/day) induced disturbances in locomotor maturation, exploratory activity and level of anxiety over the first 6 weeks of postnatal life (Maurice et al., 2015). The present study therefore aims at evaluating the developmental neurotoxicity of an early exposure to this chemical that is considered as a compound of high concern for human health, in comparison with valproic acid (VPA), a common anti-epileptic drug known to induce developmental disorders and contribute to the emergence of autism spectrum disorders. HBCDD-exposed dams were administered daily p.o. from GD0 to PND21 with 100 ng/kg/day of α-HBCDD isomer in oil, whereas the two other groups received the vehicle only. At GD12, VPA-treated rats received a single i.p. injection of VPA at a dose of 600 mg/kg whereas the two other groups were injected with the vehicle only. Pups were tested for their early behavioral development from PND3 to PND21 using a standardized test battery. At PND21, brains were collected and cortex removed for further analysis. The results pointed out the ability of both compounds to induce subtle significant behavioral changes during the neurodevelopment with the reduction in the time spent to grasp a rotating grid in VPA-exposed pups at PND9-11 and the increase in the time to move back in the neogative geotaxis task in the HBCDD-treated rats at PND8-10. No significant modification in the olfactory discriminative test (PN9-PND11) has been observed among groups. Cortical protein expression was analyzed for the neuroinflammation and synaptic plasticity, demonstrating a significant increase in the level of glial fibrillary acidic protein (GFAP) associated with a diminution of synaptophysin in the VPA-treated pups whereas HBCDD-exposed rats showed only an increasing level of expression of GFAP. In conclusion, both results suggest the ability of both compounds to impair slightly the brain and behavior development of rat pups in a different way according to the chemicals. The measurements of the cytochrome oxydase activity in various brain regions, currently under progress, should provide us additional information on the impact of an early exposure to HBCDD or VPA on behavioral impairments later in life.
Journée doctorale virtuelle franco-allemande et transfrontalière : Biotechnologies et Sciences de la Vie, 10 novembre, dématérialisée
Allouche, R., Hafeez, Z., Dary-Mourot, A., Genay, M., Miclo, L.
Séminaire de l'École Doctorale SIReNa, 13 février, Nancy, France
Allouche, R., Hafeez, Z., Dary, A., Genay, M., Miclo, L.
Streptococcus thermophilus is widely used as a starter culture in the dairy industry and has been awarded generally recognized as safe status (GRAS) by the American Food and Drug Administration. Some strains of S. thermophilus display an anti-inflammatory activity in vitro (Junjua et al., 2016). Inflammation is a part of the regular host reaction to injury or infection caused by pathogens, damaged cells, irritants and allergens. However, the mechanism of action by which this bacterium modulates inflammatory response remains unclear. It has been shown that the hydrolysis of food proteins or endogenous proteins by some digestive proteases releases peptides with various biological activities. Such peptides can also be generated by the surface proteolytic system of Lactic Acid Bacteria (Hafeez et al., 2014) as S. thermophilus, which produces bioactive peptides from bovine caseins (Miclo et al., 2012). These peptides are inactive within the sequence of the parent protein and display their activity after a hydrolysis step. Thus, the assumption that peptides generated in the gastro-intestinal tract from hydrolysis of S. thermophilus surface or intracellular proteins could display an anti?inflammatory activity and contribute to the overall anti-inflammatory effect of the bacterium can be made. Therefore, it is interesting to explore the role of such peptides in the modulation of inflammation. In a first approach, this study aims to investigate the anti-inflammatory properties of hydrolysates genrated after hydrolysis by gastrointestinal enzymes of surface proteins of S. thermophilus. The method involves the recovery of bacterial surface polypeptides by shaving with pepsin. Supernatant obtained after shaving was analysed by RP-HPLC and showed the release of peptides. The next challenge constitutes evaluation in in vitro cell model of anti-inflammatory activity of the peptides obtained and the characterisation of these peptides by mass spectrometry. This study will lead to novel insights into the modulation of host inflammatory response through probable action of peptides obtained from S. thermophilus.
Colloque Annuel de la Société Cerveau et Maladies Cérébrovasculaires - Maturation et vieillissement du système cérébrovasculaire, 28-29 mars, Lille, France
Olivier, J.-L.
Nos travaux antérieurs ont montré qu’un régime riche en acide arachidonique augmente la sensibilité des souris à la neurotoxicité des oligomères de peptide Aβ, principaux agents de la maladie d’Alzheimer, en termes d’atteintes des capacités cognitives et d’expression de protéines impliqués dans la transmission synaptique. Dans ce travail, nous avons étudié l’action positive de souches de Streptococcus thermophilus dont certaines présentent des propriétés anti-inflammatoires, sur l’association négative régime alimentaire riche en acide arachidonique – neurotoxicité des oligomères de peptide Aβ. Nous avons cherché à déterminer les mécanismes sous-jacents, notamment le rôle de la barrière hémato-encéphalique dans la transmission du signal intestin - cerveau.
5th International Systems Biomedicine Symposium - Systems neuroscience: bridging the scales of the brain, 5 novembre, Esch-sur-Alzette, Luxembourg
Roth, S., Lamartinière, Y., Fernandes, S.B., Mériaux, S., Maguin Gaté, K., Guebels, P., Godderis, L., Duca, R.C., Bouillaud-Kremarik, P., Turner, J.D., Schroeder, H., Grova, N.
Short- and long-term behavioral impairments related to anxiety, sexual and social behavior have recently been demonstrated in rats daily exposed during gestation and lactation (GD0 to PND21) to α-hexabromocyclododecane (α-HBCDD, 66 ng/kg/day of body weight), a brominated flame retardant of very high concern. The present study is aimed at examining the effects of such exposure on the potent mechanism leading to the phenotypes observed in the cerebellum of male pups at PND14. This brain region is known for its high sensitivity to environmental disturbances occurring essentially during the early phase of brain development. In the cerebellum, we initially assumed that perinatal exposure to α-HBCDD may i) lead to epigenetic changes in the 6-methyl Adenine (6-mA), which has been identified as genuine epigenetic mark through different techniques (LC-MS/MS, DotBlot and immunochemistry) and ii) induce neuroinflammation which could also exert key influence in neuronal dysfunction. The results revealed that α-HBCDD is able to alter the expression levels of proteins associated with neuroinflammation such as GFAP (+10% compared with controls for males and -18% compared to controls for females) and S100β (+ 19% compared with controls in males, p<0.05) which could later interfere in brain development and functioning. Concomitantly, a decrease in males (-30%) in 6mA has been noticed in this part of the brain, suggesting the ability of this contaminant to induce early reduction in DNA methylation. Corresponding IlluminaR sequencing proved that many differential methylated regions (DMR) can be identified on the different chromosomes at PND14 with a particular attention to be paid to changes on the Y and mitochondrial chromosome. This early decrease in 6mA signal intensity was also observed in the cerebellum at the adult stage (PND270) for both female and male exposed animals compared with controls, with a significant interaction between HBCDD and sex (p<0.01). Detailed gene analysis, currently under evaluation, should enable us to understand how the impact of early life α-HBCDD exposure on gene expression induces chronic neuroinflammation and changes in neurotransmission pathways with the occurrence of behavioural impairments later in life as a consequence.
26th International Symposium on Polycyclic Aromatic Compounts (ISPAC), 08-12 septembre, Örebro, Suède
Morel, C., Schroeder, H., Paoli, J., Charalambous, E., Thiebault, C., Guebels, P., Dosen, A., Turner, J.D., Genay, M., Grova, N.
There is growing evidence that supplementation with probiotics improves intestinal transit,
induces systemic protective immune responses and presents beneficial effect on stress
and anxiety. Concomitantly, exposure to Polycyclic Aromatic Hydrocarbons (PAH),
especially in juvenile, proves to induce cognitive developmental delay and behavioral
impairments related to anxiety.
This study provides a proof of concept on the use of probiotic beneficial effect to
counteract the neurotoxic effects induced by PAH. It was carried out by using six groups
of 12 Swiss female mice each . Three groups were daily fed with a mixture of probiotics
for 8 weeks whereas the others received the vehicule only. After 1 month of probiotic
supplementation, the 3 groups of each conditions were exposed by oral gavage to a
mixture of 16-PAHs (3 times per week, 0, 20 and 200 µg/kg, 4 weeks). Neurobehavioural
status related to exploration, anxiety and immediate learning were studied during the last
week of PAH exposure. Faeces were collected, -before, -after 4 weeks of probiotic
supplementation and -at the end of PAH exposure to assess the microbiota balance and
the probiotic viability along the gastro-intestinal tract. Preliminary data showed that
probiotics enable a faster growth of mice compared to controls (p<0.05) and reduce the
loss of weight observed in PAH-treated groups. Enumeration of probiotic strains at several
time point, pointed out that the probiotics survive along the gastro-intestinal tract, but PAH
seems to affect their viability as well as this of microbiota at 200 µg/kg of bw. Analysis of
microbiota by 16S ribosomal RNA gene sequencing should confirm this result. In the lightdark
apparatus, supplementation with probiotics partially restore the decrease of the level
of activity observed in mice exposed to PAH 200 µg/kg (p<0.05). Behavioral analyses
currently under evaluation should enable us to understand how PAH-induced
neurotoxicity and if probiotics may prevent their detrimental effects.
17th biannual meeting of the International Neurotoxicology Association (INA-17) , 28 septembre - 3 octobre, Düsseldorf, Allemagne
Roth, S., Lamartinière, Y., Fernandes, S.B., Mériaux, S., Maguin Gaté, K., Guebels, P., Godderis, L., Duca, R.C., Bouillaud-Kremarik, P., Turner, J.D., Schroeder, H., Grova, N.
Short- and long-term behavioral impairments related to anxiety, sexual and social behavior have been recently demonstrated in rats daily exposed during gestation and lactation (GD0 to PND21) to α-hexabromocyclododecane (α-HBCDD, 66 ng/kg/day of body weight), a brominated flame retardant of very high concern. The present study aimed at examining the effects of such exposure on potent mechanism leading to the observed phenotypes in the cerebellum of male pups at PND14. This part of the brain is known as a structure of high sensitivity to environmental disturbances occurring during the early phase of brain development and of later maturation compared to other brain regions. In cerebellum, we assume that a perinatal exposure to α-HBCDD may i) lead to epigenetic changes of the 6-methyl Adenine (6-mA) that has been identified as genuine epigenetic mark through different techniques (LC-MS/MS, DotBlot and immunochemistry) and ii) induce neuroinflammation which could also play a key role in neuronal dysfunction. The results showed that α-HBCDD is able to change the expression levels of proteins associated to neuroinflammation such as GFAP (+10% compared to controls in male and -18% compared to control in females) and S100β (+ 19% compared to controls in male, p<0.05) which later on could interfere with brain development and functioning. Concomitantly, a decrease in male (-30%) in 6mA has been noticed in this part of the brain, suggesting the ability of this contaminant to induce an early reduction of DNA methylation. Corresponding IlluminaR sequencing proved that many differential methylated regions (DMR) can be identified on the different chromosomes at PND14 with a particular attention to be put on changes on the Y and mitochondrial chromosome. Results under progress showed that this early decrease in 6mA signal intensity was also stated in the cerebellum at the adult stage (PND270) for both female and male-exposed animals compared to controls with a significant interaction between HBCDD and sex (p<0.01). Detailed gene analysis, currently under evaluation, should enable us to understand how the impact of early life α-HBCDD exposure on gene expression may induce chronic neuroinflammation and changes in neurotransmission pathways with the occurrence of behavioural impairments later in life as a consequence.
Microbes – 15ème Congrès National de la Société Française de Microbiologie, 30 septembre - 2 octobre, Paris, France
Morel, C., Schroeder, H., Paoli, J., Charalambous, E., Thiebault, C., Guebels, P., Dosen, A., Turner, J.D., Genay, M., Grova, N.
Introduction and objectives:
There is growing evidence that supplementation with probiotics improves intestinal transit, induces systemic protective immune responses and presents beneficial effect on stress and anxiety. Concomitantly, exposure to Polycyclic Aromatic Hydrocarbons (PAH), especially in juvenile, proves to induce cognitive developmental delay and behavioral impairments related to anxiety. This study provides a proof of concept on the use of probiotic beneficial effect to counteract the neurotoxic effects induced by PAH.
Material and methods:
We studied six groups of 12 Swiss female mice each. Three groups were daily fed with a mixture of probiotics for 8 weeks whereas the others received the vehicule only. After 1 month of probiotic supplementation, the 3 groups of each conditions were exposed by oral gavage to a mixture of 16-PAHs (3 times per week, 0, 20 and 200 µg/kg, 4 weeks). Neurobehavioural status related to exploration, anxiety and immediate learning were studied during the last week of PAH exposure. Faeces were collected, -before, -after 4 weeks of probiotic supplementation and -at the end of PAH exposure to assess the microbiota balance and the probiotic viability along the gastro-intestinal tract.
Results, discussion, conclusion:
Preliminary data showed that probiotics enable a faster growth of mice compared to controls (p<0.05) and reduce the loss of weight observed in PAH-treated groups. Enumeration of probiotic strains at several time point, pointed out that the probiotics survive along the gastro-intestinal tract, but PAH seems to affect their viability as well as this of microbiota at 200 µg/kg of bw. Analysis of microbiota by 16S ribosomal RNA gene sequencing should confirm this result. In the light-dark apparatus, supplementation with probiotics partially restore the decrease of the level of activity observed in mice exposed to PAH 200 µg/kg (p<0.05). Behavioral analyses currently under evaluation should enable us to understand how PAH-induced neurotoxicity and if probiotics may prevent their detrimental effects.
43ème Colloque de la Société de Neuroendocrinologie, 2-4 octobre, Tours, France
Pinchaud, K., Hafeez, Z., Chatel, J.-M., Chadi, S., Auger, S., Dary, A., Maguin Gaté, K., Olivier, J.-L.
L’acide arachidonique (ARA) est le second acide gras polyinsaturé dans le cerveau. L’apport d’ARA est associé à la consommation d’aliments d’origine animale qui semble sous-estimée dans les régimes occidentaux. Une précédente étude de notre laboratoire a montré qu’un régime enrichi en ARA à hauteur de 1% augmente la sensibilité des souris BalB/C males à la neurotoxicité des oligomères de peptides β-amyloïdes, considérés comme étant un des principaux acteurs de la maladie d’Alzheimer. L’objectif de cette étude est d’évaluer l’impact d’un régime enrichi en ARA sur les fonctions cérébrales au travers de modifications du microbiote intestinal et d’altération des communications intestin-cerveau. Pour cela, deux groupes de souris BalB/C ont été nourries avec un régime moyennement hyperlipidique HL-ARA (15% de lipides non enrichi en ARA) ou HL+ARA (15% de lipides et enrichi en ARA à hauteur de 1%) ou encore Std-ARA 5% de lipides non enrichi en ARA) durant 8 semaines complètes. Notre étude n’a montré aucune différence significative concernant le poids des animaux durant l’expérimentation dans chacun des groupes, excepté une augmentation du poids du tissu adipeux mésentérique pour le groupe HL-ARA. Une augmentation du groupe de Bifidobacteriaceae (anti-inflammatoires) dans le microbiote intestinal des souris nourries avec le régime HL-ARA a été mise en évidence comparé au groupe Std-ARA. Cette augmentation de Bifidobacteriaceae est corrélée avec une plus forte présence de lipides dans le régime mais cet effet est renversé par l’ajout d’ARA dans le régime. Aucune modification des marqueurs de l’inflammation n’a été détecté dans le plasma et les fèces de chacun des groupes de souris, mais une augmentation du marqueur des astrocytes GFAP a été observée dans l’hippocampe des souris nourries avec le régime HL+ARA. Il serait intéressant d’étudier les altérations de communication entre l’intestin et cerveau incluant les signaux neuroendocriniens afin de comprendre la cascade impliquée dans la modulation des fonctions cérébrales par l’intestin.
Arachidonic acid (ARA) is the second polyunsaturated fatty acid in the brain. ARA intake is associated with consumption of animal origin products and seems to be underestimated in western diet. A previous study in our laboratory showed that a diet containing 1% ARA increased the sensitivity of male Balb/C mice to the neurotoxicity of the amyloid-β peptide oligomers, considered as the main Alzheimer’s disease agents. The objective of this study was to evaluate the impact of dietary ARA intake on brain functions through gut microbiota modifications and alteration of gut-brain communications. For this, two groups of male BalB/C mice were orally fed with moderately high fat diet, i.e., HL-ARA (15% lipid without ARA) and HL+ARA (15% lipid with 1% ARA) and the third group was fed on standard diet (Std-ARA, 5% lipids without ARA) during 9 weeks. No significant difference was observed in weight gain among the 3 groups except an increase in mesenteric fat tissue in HL-ARA diet group. An increase in Bifidobacteriaceae group (potentially anti-inflammatory) in gut microbiota of HL-ARA diet group was noted compared to standard diet group. This increase in Bifidobacteriaceae was correlated to high lipid contents in diet but this effect was reversed in HL+ARA diet group. No modifications in inflammatory markers were highlighted in plasma and feces samples of the three groups. Contrariwise, higher expression levels of the Glial fibrillary acidic protein were observed in hippocampus of HL+ARA group. It could be interesting to further investigate alterations of the gut-brain communications including neuroendocrine signals.