Identification of a Gene Signature Predicting (Nano)Particle-Induced Adverse Lung Outcome in Rats.

Nanoparticles are extensively used in industrial products or as food additives. However, despite their contribution to improving our quality of life, concerns have been raised regarding their potential impact on occupational and public health. To speed up research assessing nanoparticle-related hazards, this study was undertaken to identify early markers of harmful effects on the lungs. Female Sprague Dawley rats were either exposed to crystalline silica DQ-12 with instillation, or to titanium dioxide P25, carbon black Printex-90, or multi-walled carbon nanotube Mitsui-7 with nose-only inhalation. Tissues were collected at three post-exposure time points to assess short- and long-term effects. All particles induced lung inflammation. Histopathological and biochemical analyses revealed phospholipid accumulation, lipoproteinosis, and interstitial thickening with collagen deposition after exposure to DQ-12. Exposure to the highest dose of Printex-90 and Mitsui-7, but not P25, induced some phospholipid accumulation. Comparable histopathological changes were observed following exposure to P25, Printex-90, and Mitsui-7. Comparison of overall gene expression profiles identified 15 potential early markers of adverse lung outcomes induced by spherical particles. With Mitsui-7, a distinct gene expression signature was observed, suggesting that carbon nanotubes trigger different toxicity mechanisms to spherical particles.

Retained particle surface area dose drives inflammation in rat lungs following acute, subacute, and subchronic inhalation of nanomaterials.

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).

Prenatal air pollution exposure to diesel exhaust induces cardiometabolic disorders in adulthood in a sex-specific manner.

BACKGROUND: Results from observational and experimental studies indicate that exposure to air pollutants during gestation reduces birth weight, whereas little is known on potential cardiometabolic consequences for the offspring at adulthood. OBJECTIVES: Our aim was to evaluate the long-term effects of gestational exposure to diesel engine exhaust (DE) on adult offspring phenotype in a rabbit model. METHODS: The protocol was designed to mimic human exposure in large European cities. Females rabbits were exposed to diluted (1 mg/m3) DE (exposed, n = 9) or clean air (controls, n = 7), from 3 days after mating, 2 h/d and 5 d/wk in a nose-only inhalation system throughout gestation (gestation days 3-27). After birth and weaning, 72 offspring (47 exposed and 25 controls) were raised until adulthood (7.5 months) to evaluate their cardio-metabolic status, including the monitoring of body weight and food intake, fasting biochemistry, body composition (iDXA), cardiovascular parameters and glucose tolerance. After a metabolic challenge (high fat diet in males and gestation in females), animals were euthanized for postmortem phenotyping. RESULTS: Sex-specific responses to maternal exposure were observed in adult offspring. Age-related increases in blood pressure (p = 0.058), glycaemia (p = 0.029), and perirenal fat mass (p = 0.026) as well as reductions in HDL-cholesterol (p = 0.025) and fat-to-body weight ratio (p = 0.011) were observed in exposed males, suggesting a metabolic syndrome. Almost only trends were observed in exposed females with higher triglycerides and decreased bone density compared to control females. Metabolic challenges triggered or amplified some biological responses, especially in females. CONCLUSIONS: In utero exposure to air pollution predisposed rabbit offspring to cardiometabolic disorders in a sex-specific manner.

Exposure to TiO2 Nanostructured Aerosol Induces Specific Gene Expression Profile Modifications in the Lungs of Young and Elderly Rats.

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.

Adverse outcome pathways as a tool for the design of testing strategies to support the safety assessment of emerging advanced materials at the nanoscale.

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.

Deciphering the Impact of Early-Life Exposures to Highly Variable Environmental Factors on Foetal and Child Health: Design of SEPAGES Couple-Child Cohort.

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.

Effects of first-generation in utero exposure to diesel engine exhaust on second-generation placental function, fatty acid profiles and foetal metabolism in rabbits: preliminary results.

Atmospheric pollution has major health effects on directly exposed subjects but intergenerational consequences are poorly characterized. We previously reported that diesel engine exhaust (DE) could lead to structural changes in the placenta of in utero exposed rabbits (first generation, F1). The effects of maternal exposure to DE were further studied on second-generation (F2) rabbits. Pregnant F0 females were exposed to filtered, diluted DE (1 mg/m3, median particle diameter: 69 nm) or clean filtered air (controls) for 2 h/day, 5 days/week by nose-only exposure during days 3-27 post-conception (dpc). Adult female offspring (F1) were mated to control males: F1 tissues and F2 foeto-placental units were collected at 28 dpc and placental structure and gene expression (microarray) analysed. Fatty acid profiles were determined in foetal and maternal plasma, maternal liver and placenta. In F1, compared to controls, hepatic neutral lipid contents were increased in exposed animals without change in the blood biochemistry. In F2, the placental lipid contents were higher, with higher monounsaturated fatty acids and reduced pro-inflammatory arachidonic acid (AA), without placental structural changes. Conversely, the proportion of anti-inflammatory n-3 polyunsaturated fatty acids in F2 plasma was increased while that of AA was decreased. Gene set enrichment analyses (GSEA) of F2 placenta transcriptomic data identified that the proteasome complex and ubiquitin pathways genes were over-represented and ion channel function and inflammation pathways genes were under-represented in exposed animals. These preliminary results demonstrate that diesel engine exhaust exposure and in utero indirect exposure should be considered as a programming factor within the context of the DOHaD (Developmental Origins of Health and Disease) with a probable intergenerational transmission.

Pulmonary toxicity of two different multi-walled carbon nanotubes in rat: Comparison between intratracheal instillation and inhalation exposure.

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.

Impact of exposure to diesel exhaust during pregnancy on mammary gland development and milk composition in the rabbit.

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.

Repeated gestational exposure to diesel engine exhaust affects the fetal olfactory system and alters olfactory-based behavior in rabbit offspring.

BACKGROUND: Airborne pollution, especially from diesel exhaust (DE), is known to have a negative effect on the central nervous system in exposed human populations. However, the consequences of gestational exposure to DE on the fetal brain remain poorly explored, with various effects depending on the conditions of exposure, as well as little information on early developmental stages. We investigated the short-term effects of indirect DE exposure throughout gestation on the developing brain using a rabbit model. We analyzed fetal olfactory tissues at the end of gestation and tested behaviors relevant to pups' survival at birth. Pregnant dams were exposed by nose-only inhalation to either clean air or DE with a content of particles (DEP) adjusted to 1 mg/m3 by diluting engine exhaust, for 2 h/day, 5 days/week, from gestational day 3 (GD3) to day 27 (GD27). At GD28, fetal olfactory mucosa, olfactory bulbs and whole brains were collected for anatomical and neurochemical measurements. At postnatal day 2 (PND2), pups born from another group of exposed or control female were examined for their odor-guided behavior in response to the presentation of the rabbit mammary pheromone 2-methyl-3-butyn-2-ol (2MB2). RESULTS: At GD28, nano-sized particles were observed in cilia and cytoplasm of the olfactory sensory neurons in the olfactory mucosa and in the cytoplasm of periglomerular cells in the olfactory bulbs of exposed fetuses. Moreover, cellular and axonal hypertrophies were observed throughout olfactory tissues. Concomitantly, fetal serotoninergic and dopaminergic systems were affected in the olfactory bulbs. Moreover, the neuromodulatory homeostasis was disturbed in a sex-dependent manner in olfactory tissues. At birth, the olfactory sensitivity to 2MB2 was reduced in exposed PND2 pups. CONCLUSION: Gestational exposure to DE alters olfactory tissues and affects monoaminergic neurotransmission in fetuses' olfactory bulbs, resulting in an alteration of olfactory-based behaviors at birth. Considering the anatomical and functional continuum between the olfactory system and other brain structures, and due to the importance of monoamine neurotransmission in the plasticity of neural circuits, such alterations could participate to disturbances in higher integrative structures, with possible long-term neurobehavioral consequences.

Maternal exposure to diluted diesel engine exhaust alters placental function and induces intergenerational effects in rabbits.

BACKGROUND: Airborne pollution is a rising concern in urban areas. Epidemiological studies in humans and animal experiments using rodent models indicate that gestational exposure to airborne pollution, in particular diesel engine exhaust (DE), reduces birth weight, but effects depend on exposure duration, gestational window and nanoparticle (NP) concentration. Our aim was to evaluate the effects of gestational exposure to diluted DE on feto-placental development in a rabbit model. Pregnant females were exposed to diluted (1 mg/m(3)), filtered DE (NP diameter ≈ 69 nm) or clean air (controls) for 2 h/day, 5 days/week by nose-only exposure (total exposure: 20 days in a 31-day gestation). RESULTS: DE exposure induced early signs of growth retardation at mid gestation with decreased head length (p = 0.04) and umbilical pulse (p = 0.018). Near term, fetal head length (p = 0.029) and plasma insulin and IGF1 concentrations (p = 0.05 and p = 0.019) were reduced. Placental function was also affected, with reduced placental efficiency (fetal/placental weight) (p = 0.049), decreased placental blood flow (p = 0.009) and fetal vessel volume (p = 0.002). Non-aggregated and "fingerprint" NP were observed at various locations, in maternal blood space, in trophoblastic cells and in the fetal blood, demonstrating transplacental transfer. Adult female offspring were bred with control males. Although fetoplacental biometry was not affected near term, second generation fetal metabolism was modified by grand-dam exposure with decreased plasma cholesterol (p = 0.008) and increased triglyceride concentrations (p = 0.015). CONCLUSIONS: Repeated daily gestational exposure to DE at levels close to urban pollution can affect feto-placental development in the first and second generation.

A perspective on the developmental toxicity of inhaled nanoparticles.

This paper aimed to clarify whether maternal inhalation of engineered nanoparticles (NP) may constitute a hazard to pregnancy and fetal development, primarily based on experimental animal studies of NP and air pollution particles. Overall, it is plausible that NP may translocate from the respiratory tract to the placenta and fetus, but also that adverse effects may occur secondarily to maternal inflammatory responses. The limited database describes several organ systems in the offspring to be potentially sensitive to maternal inhalation of particles, but large uncertainties exist about the implications for embryo-fetal development and health later in life. Clearly, the potential for hazard remains to be characterized. Considering the increased production and application of nanomaterials and related consumer products a testing strategy for NP should be established. Due to large gaps in data, significant amounts of groundwork are warranted for a testing strategy to be established on a sound scientific basis.