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Background An important aspect of nanomaterial (NM) risk assessment is establishing relationships between physicochemical properties and key events governing the toxicological pathway leading to adverse outcomes. The difficulty of NM grouping can be simplified if the most toxicologically relevant dose metric is used to assess the toxicological dose-response. Here, we thoroughly investigated the relationship between acute and chronic inflammation (based on polymorphonuclear neutrophil influx (% PMN) in lung bronchoalveolar lavage) and the retained surface area in the lung. Inhalation studies were performed in rats with three classes of NMs: titanium dioxides (TiO2) and carbon blacks (CB) as poorly soluble particles of low toxicity (PSLT), and multiwall carbon nanotubes (MWCNTs). We compared our results to published data from nearly 30 rigorously selected articles. Results This analysis combined data specially generated for this work on three benchmark materials - TiO2 P25, the CB Printex-90 and the MWCNT MWNT-7 - following subacute (4-week) inhalation with published data relating to acute (1-week) to subchronic (13-week) inhalation exposure to the classes of NMs considered. Short and long post-exposure recovery times (immediately after exposure up to more than 6 months) allowed us to examine both acute and chronic inflammation. A dose-response relationship across short-term and long-term studies was revealed linking pulmonary retained surface area dose (measured or estimated) and % PMN. This relationship takes the form of sigmoid curves, and is independent of the post-exposure time. Curve fitting equations depended on the class of NM considered, and sometimes on the duration of exposure. Based on retained surface area, long and thick MWCNTs (few hundred nm long with an aspect ratio greater than 25) had a higher inflammatory potency with 5 cm2/g lung sufficient to trigger an inflammatory response (at 6% PMN), whereas retained surfaces greater than 150 cm2/g lung were required for PSLT. Conclusions Retained surface area is a useful metric for hazard grouping purposes. This metric would apply to both micrometric and nanometric materials, and could obviate the need for direct measurement in the lung. Indeed, it could alternatively be estimated from dosimetry models using the aerosol parameters (rigorously determined following a well-defined aerosol characterization strategy).

The difficulty of nanomaterial (NM) grouping can be simplified if the most toxicologically relevant dose metric is used to assess the toxicological dose-response. Retained surface area could be useful for this purpose. Here, we thoroughly investigated the relationship between acute and chronic inflammation (based on polymorphonuclear neutrophil influx (% PMN) in lung bronchoalveolar lavage) and the retained surface area in the lung. 4-week inhalation studies were performed in rats with three benchmark materials: the TiO2 P25 and the carbon black Printex-90 as poorly soluble particles of low toxicity (PSLT), and the multiwall carbon nanotube (MWCNT) MWNT-7. We compared our results to published data from nearly 30 rigorously selected articles relating to acute (1-week) to subchronic (13-week) inhalation exposure to the classes of NMs considered. Short and long post-exposure recovery times (immediately after exposure up to more than 6 months) allowed us to examine both acute and chronic inflammation. A dose-response relationship across short-term and long-term studies was revealed linking pulmonary retained surface area dose (measured or estimated) and % PMN. This relationship, taking the form of sigmoid curves, is dependent on the class of NM considered and sometimes on the duration of exposure but is independent of the post-exposure time. Based on retained surface area, long and thick MWCNTs (few hundred nm long with an aspect ratio greater than 25) had a higher inflammatory potency with 4 cm²/g lung sufficient to trigger an inflammatory response (at 6% PMN), whereas retained surfaces greater than 150 cm²/g lung were required for PSLT.

Although aging is associated with a higher risk of developing respiratory pathologies, very few studies have assessed the impact of age on the adverse effects of inhaled nanoparticles. Using conventional and transcriptomic approaches, this study aimed to compare in young (12–13-week-old) and elderly (19-month-old) fisher F344 rats the pulmonary toxicity of an inhaled nanostructured aerosol of titanium dioxide (TiO2). Animals were nose-only exposed to this aerosol at a concentration of 10 mg/m3 for 6 h per day, 5 days per week for 4 weeks. Tissues were collected immediately (D0), and 28 days after exposure (D28). A pulmonary influx of neutrophilic granulocytes was observed in exposed rats at D0, but diminished with time while remaining significant until D28. Similarly, an increased expression of several genes involved in inflammation at the two post-exposure time-points was seen. Apart from an age-specific pulmonary influx of lymphocyte, only slight differences in physio-pathological responses following TiO2 exposure between young and elderly animals were noticed. Conversely, marked age-related differences in gene expression profiles were observed making possible to establish lists of genes specific to each age group and post-exposure times. These results highlight different signaling pathways that were disrupted in rats according to their age.

Exposure to fine-particulate air pollution is a major global health concern because it is associated with reduced birth weight and an increased risk of cardiovascular disease. Here we have investigated the potential for exposure to diesel exhaust during pregnancy to influence mammary gland development and milk composition. Female rabbits were therefore exposed by nose-only inhalation to either diluted diesel exhaust fumes (1 mg/m3) or clean air for 2h/day, 5 days/week, from the 3rd to the 27th days of pregnancy. On Day 28 of pregnancy, mammary glands were collected from twelve females (six controls and six diesel-exposed) and assessed for morphological and functional alterations. Milk samples were collected from eighteen dams (nine controls and nine diesel-exposed) during early (days 2 to 4) and established (days 13 to 16) lactation to verify the composition of fatty acids and major proteins and leptin levels. The mammary alveolar lumina contained numerous fat globules, and stearoyl CoA reductase expression was higher in mammary epithelia from diesel exhaust-exposed rabbits, which together suggested increased mammary lipid biosynthesis. Gas chromatography analysis of the composition of milk fatty acids revealed a sharp rise in the total fatty acid content, mainly due to monounsaturated fatty acids. Liquid chromatography-mass spectrometry analysis of milk samples enabled identification and quantification of the main rabbit milk proteins and their main phosphorylated isoforms, and revealed important changes to individual casein and whey protein contents and to their most phosphorylated isoforms during early lactation. Taken together, these findings suggest that repeated daily exposure to diesel exhaust fumes during pregnancy at urban pollution levels can influence lipid metabolism in the mammary gland and the lipid and protein composition of milk. As milk may contribute to metabolic programming, such alterations affecting milk composition should be taken into account from a public health perspective.

In humans, studies based on Developmental Origins of Health and Disease (DOHaD) concept and targeting short half-lived chemicals, including many endocrine disruptors, generally assessed exposures from spot biospecimens. Effects of early-life exposure to atmospheric pollutants were reported, based on outdoor air pollution levels. For both exposure families, exposure misclassification is expected from these designs: for non-persistent chemicals, because a spot biospecimen is unlikely to capture exposure over windows longer than a few days; for air pollutants, because indoor levels are ignored. We developed a couple-child cohort relying on deep phenotyping and extended personal exposure assessment aiming to better characterize the effects of components of the exposome, including air pollutants and non-persistent endocrine disruptors, on child health and development. Pregnant women were included in SEPAGES couple-child cohort (Grenoble area) from 2014 to 2017. Maternal and children exposure to air pollutants was repeatedly assessed by personal monitors. DNA, RNA, serum, plasma, placenta, cord blood, meconium, child and mother stools, living cells, milk, hair and repeated urine samples were collected. A total of 484 pregnant women were recruited, with excellent compliance to the repeated urine sampling protocol (median, 43 urine samples per woman during pregnancy). The main health outcomes are child respiratory health using early objective measures, growth and neurodevelopment. Compared to former studies, the accuracy of assessment of non-persistent exposures is expected to be strongly improved in this new type of birth cohort tailored for the exposome concept, with deep phenotyping and extended exposure characterization. By targeting weaknesses in exposure assessment of the current approaches of cohorts on effects of early life environmental exposures with strong temporal variations, and relying on a rich biobank to provide insight on the underlying biological pathways whereby exposures affect health, this design is expected to provide deeper understanding of the interplay between the Exposome and child development and health.

Consequences of exposure to diesel exhaust during gestation on feto-placental development. Insight from animal models. Air pollution is a major threat for human health, causing 3.7 million premature deaths in 2012 worldwide. Elderly people, children and people suffering from asthma in outbursts of air pollution peaks are the most concerned whereas pregnant women are most often neglected. Epidemiological surveys and studies using animal models indicate that the components of air pollution can disturb placental function, reduce foetal growth and induce oxidative stress and inflammation in the mother, placenta and foetus. On the long term, these effects can contribute to the current worldwide increase in adult metabolic diseases in the context of the Developmental Origins of Health and Disease (DOHaD). It is therefore important to include pregnant women as recipients of preventative messages for air pollution and to carry on research on long term effects using relevant animal models and procedures., La pollution atmosphérique est un problème majeur pour la santé humaine, causant 3,7 millions de décès prématurés en 2012 dans le monde. Les personnes âgées, les enfants ou les asthmatiques sont les cibles des messages de prévention lors de pics de pollution alors que les femmes enceintes sont le plus souvent négligées. Pourtant, les études épidémiologiques et les travaux sur les modèles animaux indiquent que la pollution atmosphérique et ses composants peuvent perturber la fonction placentaire, réduire la croissance foetale et induire un stress oxydant et une inflammation. A long terme, ces effets peuvent participer à l’augmentation des risques de maladies métaboliques chez l’adulte. Il est donc important d’inclure les femmes enceintes dans les messages de prévention lors de pics de pollution atmosphérique et de continuer la recherche sur les effets à long terme grâce au recours aux modèles animaux., Valentino Sarah, Couturier-Tarrade Anne, Chavatte-Palmer Pascale. Exposition aux gaz d’échappement diesel durant la gestation : quelles conséquences sur le développement foeto-placentaire ? Apport des modèles animaux. In: Bulletin de l'Académie Vétérinaire de France tome 169 n°3, 2016. pp. 208-217.

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BACKGROUND Following embryo transfer (ET), the size and breed of the recipient mare can affect fetal development and subsequent post natal growth rate and insulin sensitivity in foals. OBJECTIVES To investigate placental adaptation in pregnancies where increased or restricted fetal growth was induced through ET between Pony, Saddlebred and Draught horses. STUDY DESIGN In vivo experiment. METHODS Control Pony (P, n = 21) and Saddlebred (S, n = 28) pregnancies were obtained by artificial insemination. Increased pregnancies were obtained by transferring Pony (P-D, n = 6) and Saddlebred (S-D, n = 8) embryos into Draught mares. Restricted pregnancies were obtained by transferring Saddlebred embryos into Pony mares (S-P, n = 6). Placental weight and surface were recorded and samples collected for stereology and analysis of expression of genes involved in placental growth, vascularisation and nutrient transport. Data were analysed by linear model. RESULTS S-P foals were growth retarded when compared with controls despite increased gestational length. Placental weight was reduced but placental surface density and volume fraction were increased. Placental expression of genes involved in growth and development and nutrient transfer was strongly reduced. In contrast, placental size and weight were increased in enhanced growth P-D and S-D foals. The trophoblastic surface density and the allantoic vessels surface density were decreased in P-D and S-D, respectively, both with very few modifications in gene expression. MAIN LIMITATIONS Control embryos were produced by artificial insemination whereas experimental embryos were produced by ET. CONCLUSIONS Placental structure and gene expression are modified after ET into a smaller or larger breed than that of the embryo. These adaptations contribute to the observed phenotype of foal growth restriction or enhanced growth at birth.

Maternal exposure to diluted diesel engine exhaust alters placental function of the first and second generation in rabbit model. 8. International Symposium on Nanotechnology, Occupational and Environmental Health

International audience; Ponies and sometimes draft horses are often used as experimental models for horses although size and metabolic parameters are known to vary between horse breeds. So far, there is little information about differences of placental structure and no information about differences of placental function between breeds. The aim of this study was to investigate differences in placental size, structure and function at birth in relation to foal size and weight in ponies, Saddlebred and draft horses. Pony, Saddlebred and draft horse pregnancies were obtained by artificial insemination over 2 successive breeding seasons. Foals and total fetal membranes (TFM) were weighed and placentas measured for surface area at term. Placentas were sampled above the umbilical cord insertion. Surface density and volume fraction of the different cellular components of the placenta were measured on histological sections using stereology. The expression of genes involved in growth and development, nutrient transfer and vascularization was compared between groups. Foals and TFM were lighter at birth in ponies than Saddlebred horses, and both were lighter compared to draft horses. The surface density and volume fraction of microcotyledonary vessels was increased in pony compared to Saddlebred placentas. The relative expression of genes involved in growth and development was different between breeds and increased with maternal, fetal and placental weight. Primiparous dams produced lighter foals and smaller placentas, associated with a decreased volume fraction of microcotyledonary vessels and genes involved in growth and development and vascularization. Foal sex had little effect on placental structure and function as the expression of only one gene differed according to sex, with EGFR expression being decreased in placentas of females compared to males. In conclusion, foal and placental weight, as well as placental expression of genes involved in growth and development were correlated with maternal size. Placental structure also differed between breeds, with a stronger difference between ponies and both breeds of horses.

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Reproductive effects of gestational exposure to diesel exhaust in a rabbit model. XIV. International Congress of Toxicology

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Le placenta est un organe transitoire, apparu avec la viviparité. Il se forme après l’implantation de l’embryon sur la muqueuse utérine et dérive d’interactions cellulaires et moléculaires complexes entre les tissus utérins et embryonnaires. Chez les mammifères, diverses formes de placentation existent mais cet organe assure le même rôle chez toutes les espèces : des fonctions d'échanges nutritionnels, endocrines et immunologiques. Le placenta est l’organe clé de la régulation du métabolisme fœtal. Cette régulation implique divers échanges entre mère et foetus par des mécanismes de transport à travers le trophoblaste et les cellules endothéliales fœtales, le sang maternel et le sang fœtal n’étant jamais en contact. Le bon fonctionnement placentaire assure une crois-sance fœtale harmonieuse. Une forte corrélation existe entre un faible poids de naissance et le risque de développer une pathologie métabolique à l’âge adulte telle que l’obésité, le diabète de type II ou l’hypertension. Ces observations ont conduit au concept de " programmation foetale " ou DOHaD (Developemental Origin of Health and Disease) (Barker 1990). Il a été montré que les perturbateurs de l’environnement maternel durant la gestation (malnutrition, stress, polluants…) peuvent affecter l’organogenèse du fœtus ou sa croissance, entraînant une adaptation métabolique du fœtus à ce nouvel environnement en agissant sur l’expression de gènes placentaires, la vascularisation, l’hémodynamique ou les processus inflammatoires. Par conséquent le placenta joue un rôle critique dans la programmation de la croissance fœtale et du devenir de l’adulte. L’équipe étudie le rôle du placenta dans l’établissement du phénotype fœtal, post-natal et intergénérationnel en lien avec des perturbations périconceptionnelles et/ou gestationnelles de l’environnement maternel. Le lapin constitue un modèle de prédilection car la structure du placenta est proche de celle du placenta humain. Nous décrivons ici l’ultrastructure du placenta de lapin dans des conditions physiologiques normales ou après inhalation par la mère gestante de gaz d’échappement diesel filtrés.

Short-term effects of daily exposure to Diesel exhaust during gestation on the olfactory system development in rabbits pups. XIV International Congress of Toxycology

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National audience; Plusieurs études épidémiologiques chez l’Homme ont montré qu’une exposition maternelle à la pollution atmosphérique induisait un Retard de Croissance Intra-Utérin (RCIU) associée à une diminution du poids placentaire. Cependant, l’exploration plus approfondie des mécanismes reste limitée chez l’Homme. Dans ce projet, nous avons donc développé un modèle lapin afin d’étudier les effets potentiels de la pollution atmosphérique au cours de la gestation. Les lapines gestantes ont été exposées à un mélange représentatif de la pollution atmosphérique, c’est-à-dire des gaz d’échappement de moteur diesel dilués (groupe P) ou de l’air filtré (groupe C), 1h le matin et 1h l’après-midi, 5 jours par semaine, de 3 à 27 jpc (terme = 31 jpc). A 28 jpc, les lapines ont été euthanasiées (N=7 lapines, groupe P et N=6 lapines, groupe C), les fœtus et les placentas ont été collectés, pesés et mesurés. Les données ont été analysées avec des ANOVA en prenant comme co-facteurs la taille de la portée, le sexe fœtal et la mère. Nous avons établi que l’exposition des lapines aux gaz d’échappement n’affecte pas le taux d’implantation. A 28 jpc, 68P et 50C unités fœto-placentaires ont été collectées. Les fœtus P sont plus légers que les fœtus C (p

International audience

ln equids, placentationis diffus'e and the nutrient supply to the fetus is deterrnined byuterine size, which is correlated with materna!size. The size of the mare affects fetai development as shawn by embryo transfer (ET) between Panics and Thoroughbreds. In tum, insulin sensitivity in the newbom tüal and subsequent postnatal growth rate are affected. We enhanced or restricted fetal growth through ETusing Pony (P), Saddlebred (S), and Draft (D) horses and investigated placenta! morpho!ogy, structure, and fonction at tenn. Control pregnancies of P-P (n = 21), S-S (n = 28), and D-D (n = 8) were obtained by Al. Enhanced and restricted pregnancies were obtained by transferring P (P-D, n = 6) and S embryos (S-D, n = 8) into D mares orS embryos into P mares (S-P, n = 6), respective]y. Placenta!weight and surface were recorded at delivery. Samples were collected for stereology and RT-qPCR analysis of expression of genes involved in placenta!growth, vascularization, and nutrient transport. Housekeeping genes were RPL32, SCAMP3, and B2M. Data were analysed by Kruskal-Wallis followed by Dunn's post hoc test. Placenta!weight and surface were increased in S-S and in D-D compared with P-P, whereas S-S and D-D were not different. No histological changes were observed among contrais, but most genes had their expression decreased in P-P compared with S-S andD-D. The P-D foals hada 57% increased birthweight with heavier and largerplacentas than P-P foals. The S-D foals were similar to both S-S and D-D in te1ms ofbirthweight and placenta!weight and surface. No major modifications in placenta!histology or transcript levels were observed in both enhanced groups. In contrast, S-P foals had a 37% decreased birthweight with lighter and smaller placentas compared with S-S and S-D foals. Therewas no gross histological difference betweenS-P and S-S but the microcotyledonary surfacedensity was higher in S-P compared with S-D. Moreover, the expression of IGF2, !GF2R, SLC2Al, and eNOS was decreased in S-P compared with S-S. There was no difference in gene expression between S-P and P-P. In conclusion, intrauterine growth restriction led to marked changes in placenta!morphology, histology, and gene expression. The increased microcotyledonary surface density suggests a lengthening ofvillî, which could increase feto-maternal contact surface as a compensatory mechanism for the restricted uterine capacity. Surprisingly, placenta! adaptation to the restricted intrauterine enviromnent in S-P induced gene profiles resembling that of control P, whereas no difference was observed in enhanced pregnancies.

International audience; Epidemiological studies in humans and animal experiments indicate that gestational exposure to airborne pollution, in particular diesel engine exhaust (DE), reduces birth weight, with effects depending on exposure duration, gestational window and nanoparticle (NP) concentration. Furthermore, whether the inhaled NP can reach the fetuses via a transplacental transfer remains unknown. In this study we used a rabbit model to evaluate the transport of maternally inhaled DE NP from the lungs to the placenta and to fetal organs using transmission electron microscopy (TEM). Pregnant females were exposed to diluted (1mg/m3), filtered DE (mean NP diameter ≈ 69 nm) or clean air for 2 hours/day, 5 days/week by nose-only exposure during 20 days in a 31-day gestation. At 28th day post-conception, the females were euthanized. Maternal lung samples as well as placental labyrinthine area, fetal liver and spleen were collected randomly from 4 fetuses in each litter and subsequently treated according to a classical TEM preparation protocol. The presence of NP was evaluated on ultrathin sections (75 nm). NP were identified in lungs of exposed does, in the alveoli, in type 1 pneumocytes and within blood vessels. When observed in the lung of exposed females, NP were also observed in the placenta, i.e. in the maternal blood space, close to the microvillous membrane, in trophoblastic cells, and both free and in fetal red blood cells with the fetal compartment. No NP were observed in control placentas. Some NP were also identified in the liver and the spleen of fetuses whose placenta contained NP, demonstrating transplacental transfer and accumulation in fetal organs. These results demonstrate that maternal exposure to DE during gestation at levels close to urban pollution leads to the transfer of the inhaled NP from maternal lung to the placenta and fetal organs.

Airborne pollution has been associated with various adverse effects on human reproductive health, especially intrauterine growth retardation and early pregnancy loss. However, few studies have analysed its effect on early development. Diesel exhaust particles (DEP) have been shown to alter blastocyst formation when diluted in embryo culture medium (2010 Toxicol Sci. 117, 200–208), but no data are available concerning the effect of maternal inhalation of diesel exhaust on early embryo development. Our study has been designed to answer this question using rabbit as a model and DEP doses mimicking daily exposure to traffic in large European cities. New Zealand female rabbits were superovulated by means of 5 subcutaneous administration of pFSH for 3 days before mating, followed 10 to 12 h later by an intravenous administration of 30 IU of hCG at the time of mating (natural mating). Dams were exposed to a representative air pollution mixture; that is, diluted diesel engine exhaust (1 mg m–3; N = 14) or clean air (N = 12), for 1 h every morning and afternoon, from Day 3 to Day 6 post-coitum (dpc). At 6 dpc, in vivo-developed embryos were collected from uteri perfused with PBS and counted; their diameter was measured on pictures using ImageJ software (NIH, Bethesda, MD, USA). Another group of female rabbits was exposed to the same inhalation conditions from 3 to 27 dpc without superovulation treatment. Measures by ultrasound were performed on these dams at 7 dpc. Data were analysed by Mann-Whitney test and ANOVA, including dams as cofactor. At 6 dpc, number of embryos per dams was higher in exposed group compared with control (P

In equids, placentation is diffuse and the nutrient supply to the fetus is determined by uterine size, which is correlated with maternal size. The size of the mare affects fetal development as shown by embryo transfer (ET) between Ponies and Thoroughbreds. In turn, insulin sensitivity in the newborn foal and subsequent postnatal growth rate are affected. We enhanced or restricted fetal growth through ET using Pony (P), Saddlebred (S), and Draft (D) horses and investigated placental morphology, structure, and function at term. Control pregnancies of P-P (n = 21), S-S (n = 28), and D-D (n = 8) were obtained by AI. Enhanced and restricted pregnancies were obtained by transferring P (P-D, n = 6) and S embryos (S-D, n = 8) into D mares or S embryos into P mares (S-P, n = 6), respectively. Placental weight and surface were recorded at delivery. Samples were collected for stereology and RT-qPCR analysis of expression of genes involved in placental growth, vascularization, and nutrient transport. Housekeeping genes were RPL32, SCAMP3, and B2M. Data were analysed by Kruskal-Wallis followed by Dunn's post hoc test. Placental weight and surface were increased in S-S and in D-D compared with P-P, whereas S-S and D-D were not different. No histological changes were observed among controls, but most genes had their expression decreased in P-P compared with S-S and D-D. The P-D foals had a 57% increased birthweight with heavier and larger placentas than P-P foals. The S-D foals were similar to both S-S and D-D in terms of birthweight and placental weight and surface. No major modifications in placental histology or transcript levels were observed in both enhanced groups. In contrast, S-P foals had a 37% decreased birthweight with lighter and smaller placentas compared with S-S and S-D foals. There was no gross histological difference between S-P and S-S but the microcotyledonary surface density was higher in S-P compared with S-D. Moreover, the expression of IGF2, IGF2R, SLC2A1, and eNOS was decreased in S-P compared with S-S. There was no difference in gene expression between S-P and P-P. In conclusion, intrauterine growth restriction led to marked changes in placental morphology, histology, and gene expression. The increased microcotyledonary surface density suggests a lengthening of villi, which could increase feto-maternal contact surface as a compensatory mechanism for the restricted uterine capacity. Surprisingly, placental adaptation to the restricted intrauterine environment in S-P induced gene profiles resembling that of control P, whereas no difference was observed in enhanced pregnancies.

Multi-walled carbon nanotubes (MWCNTs), which vary in length, diameter, functionalization and specific surface area, are used in diverse industrial processes. Since these nanomaterials have a high aspect ratio and are biopersistant in the lung, there is a need for a rapid identification of their potential health hazard. We assessed in Sprague-Dawley rats the pulmonary toxicity of two pristine MWCNTs (the “long and thick” NM-401 and the “short and thin” NM-403) following either intratracheal instillation or 4-week inhalation in order to gain insights into the predictability and intercomparability of the two methods. The deposited doses following inhalation were lower than the instilled doses. Both types of carbon nanotube induced pulmonary neutrophil influx using both exposure methods. This influx correlated with deposited surface area across MWCNT types and means of exposure at two different time points, 1–3 days and 28–30 days post-exposure. Increased levels of DNA damage were observed across doses and time points for both exposure methods, but no dose-response relationship was observed. Intratracheal instillation of NM-401 induced fibrosis at the highest dose while lower lung deposited doses obtained by inhalation did not induce such lung pathology. No fibrosis was observed following NM-403 exposure. When the deposited dose was taken into account, sub-acute inhalation and a single instillation of NM-401 and NM-403 produced very similar inflammation and DNA damage responses. Our data suggest that the dose-dependent inflammatory responses observed after intratracheal instillation and inhalation of MWCNTs are similar and were predicted by the deposited surface area.

In equids, placentation is diffuse and the nutrient supply to the fetus is determined by uterine size, which is correlated to maternal size and parity. That the size of the mare affects fetal development has previously been shown by embryo transfer (ET) between Ponies and Thoroughbreds [Allen WR, et al; Reproduction 2002;123:445-53.]. We enhanced or restricted fetal growth through ET using Pony (P), Saddlebred (S) and Draft (D) horses and investigated placental morphology, histology and function at term. Control pregnancies of P-P (n1⁄421), S-S (n1⁄428) and D-D (n1⁄48) were obtained by artificial insemination. Enhanced and restricted pregnancies were obtained by transferring P (P-D, n1⁄46) and S embryos (S-D, n1⁄48) into D mares or S embryos into P mares (S-P, n1⁄46), respectively. Placental weight and gross surface area were recorded at delivery. Samples were collected for stereology (quantitative histology) and RT-qPCR analysis of expression of genes involved in placental growth, vascularization and nutrient transport. Statistical analysis of data was performed by Kruskal-Wallis with Dunn’s posthoc test. Data are expressed as means SEM. Mean placental weight and gross surface area were higher in S-S and in D-D compared to P-P (p

International audience; Characterization of the Exposome and its effects on children health is warranted. Consistency between human observations and results of toxicological experiments strengths the evidence in environmental health research, but is difficult to identify when epidemiological and toxicological studies are conducted independently. We present an original approach com-bining a cohort with intense exposure monitoring conducted in parallel with an animal experi-ment focused on air pollution effects. Pregnant women are being included in a monocentric couple­child cohort before 18 gestational weeks. Exposure to air pollutants (including NO2, benzene, PM2.5, soot) is assessed every 3 to 6 months by personal monitors in mothers and children. A large biobank is constituted including DNA, RNA for transcriptome assessment, serum, plasma, placenta, repeated pooled urine samples, meconium, child stools, milk and hair. The main health out-comes are respiratory health, fetal and child growth and neurodevelopment. In parallel, pre-gnant rabbits were exposed to diluted filtered diesel exhaust, feto­placental units were col-lected and offspring growth was monitored. To date the cohort includes 260 trios. Maternal median personal exposure is 19.1 µg/m3 (25th­75th centiles, 14.1­24.5) for NO2 and 1.2 µg/m3 (0.7­2.2) for benzene. The rabbit experiment showed transplacental transfer of diesel exhaust nanoparticles and demonstrated effects on placental vascularization and blood flow. Replication will be sought using placenta collected in the cohort. Our cohort constitutes an approach allowing accurate characterization of ear-ly­life exposures to air pollutants. Close collaboration between toxicological and epidemiolo-gical studies is a promising approach that enables cross­talk between both disciplines and ultimately could provide deeper understanding of the interplay between the Exposome, ‘omics markers and child health.

National audience; Recent events related to pollution peaks have shown that we are continuously and insidiously submitted to air pollution. Epidemiological and experimental studies in rodents describe direct consequences on health with increasing respiratory and cardiovascular diseases and high susceptibility for metabolic diseases. Indirect consequences due to in utero exposure induce also intrauterine growth retardation in human. The goal of this study was to study the affects of in utero exposure to diesel exhausts, in rabbit , focusing on the characterization of F1 offspring's phenotype at adulthood. The phenotype of offspring animals has been studied from puberty to adulthood (7 .5 months) to evaluate the occurrence of cardio-metabolic disorders, by animal tracking (measurement of food intake, weight change) as well as cardio-metabolic functional tests (lasting blood analyses of lipid metabolism, analyses of body composition by iDXA, assessment of insulinaemic and glycemia response to a bolus of glucose during glucose tolerance test, blood pressure and heart rate measurements). Alter this in vivo experiment, the animals were euthanized and the insulin sensitive tissues were collected for biometry records. ln utero exposure to polluted air modified the adult offspring's phenotype, alter puberty, in a sex-specific manner. Polluted males exhibited a metabolic syndrome with hyperglycemia, more abdominal fat and rise of blood pressure with age compared to control males. Polluted males presented symptoms of hepatic steatosis (higher transaminase ALAT, heavier liver) and atherosclerosis (lower HDL-cholesterol). Polluted females presented hypertriglyceridemia and a decrease of bone mass and density compared to control tamale, indicating a higher susceptibility to bone pathologies. ln conclusion an indirect in utero exposure to air pollution was sufficient to affect durably the phenotype of animals that developed signs of insulin resistance with higher risk of cardiovascular morbidity, involving sax­specific pathways.

National audience; Mammary development and fonction was abnormal in exposed clams. The a!veolar lumina were characterized by a marked increase in fat globules number and volume (p< 0.05) . Total fatty acid (FA) content was increased (p< 0.05) in DE-exposed rabbit milk on days 2-4 of lactation, essentially due to higher medium-chain FA concentrations. Sex-specific responses to maternai exposure were observed in offspring after weaning. In-utero exposed males hacl metabolic disorders with increased blood pressure (p=0.058) , higher glycemia (p=0.019) , lower HDL-cholesterol (p=0.025) , reduced fat-to-bocly weight ratio (p=0.011) but in­ creased peri-renal fat mass (p=0.026). Females had only higher triglycerides (TG) compared to contrai offspring. Testicular and ovarian structure and fonction were analyzed by histology and immunohistochemistry, demonstrating no difference between groups. In contrast , there was more DN A fragmentation (p=0.0012) in epididymal sperm in the exposed group. Adult female offspring were bred with contrai males. Although fetoplacental biometry was not affected near term, 2nd generation fetal metabolism was modifiecl by grand-dam exposure with decreased plasma cholesterol (p=0.008) and increased TG concentrations (p=0.015). In conclusion, repeated gestational exposure to DE at levels close to urban pollution can affect feto-placental clevelopment and health over 2 generations.

Toxicity testing and regulation of advanced materials at the nanoscale, i.e. nanosafety, is challenged by the growing number of nanomaterials and their property variants requiring assessment for potential human health impacts. The existing animal-reliant toxicity testing tools are onerous in terms of time and resources and are less and less in line with the international effort to reduce animal experiments. Thus, there is a need for faster, cheaper, sensitive and effective animal alternatives that are supported by mechanistic evidence. More importantly, there is an urgency for developing alternative testing strategies that help justify the strategic prioritization of testing or targeting the most apparent adverse outcomes, selection of specific endpoints and assays and identifying nanomaterials of high concern. The Adverse Outcome Pathway (AOP) framework is a systematic process that uses the available mechanistic information concerning a toxicological response and describes causal or mechanistic linkages between a molecular initiating event, a series of intermediate key events and the adverse outcome. The AOP framework provides pragmatic insights to promote the development of alternative testing strategies. This review will detail a brief overview of the AOP framework and its application to nanotoxicology, tools for developing AOPs and the role of toxicogenomics, and summarize various AOPs of relevance to inhalation toxicity of nanomaterials that are currently under various stages of development. The review also presents a network of AOPs derived from connecting all AOPs, which shows that several adverse outcomes induced by nanomaterials originate from a molecular initiating event that describes the interaction of nanomaterials with lung cells and involve similar intermediate key events. Finally, using the example of an established AOP for lung fibrosis, the review will discuss various in vitro tests available for assessing lung fibrosis and how the information can be used to support a tiered testing strategy for lung fibrosis. The AOPs and AOP network enable deeper understanding of mechanisms involved in inhalation toxicity of nanomaterials and provide a strategy for the development of alternative test methods for hazard and risk assessment of nanomaterials.

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