Maternal inhalation of carbon black nanoparticles induces neurodevelopmental changes in mouse offspring

Abstract Background Engineered nanoparticles are smaller than 100 nm and designed to improve or creating even new physico-chemical properties. Consequently, toxicological properties of materials may change as size reaches the nm size-range. We examined outcomes related to the central nervous system in the offspring following maternal inhalation exposure to nanosized carbon black particles (Printex 90). Methods Time-mated mice (NMRI) were exposed by inhalation, for 45 min/day to 0, 4.6 or 37 mg/m3 aerosolized carbon black on gestation days 4–18, i.e. for a total of 15 days. Outcomes included maternal lung inflammation (differential cell count in bronchoalveolar lavage fluid and Saa3 mRNA expression in lung tissue), offspring neurohistopathology and behaviour in the open field test. Results Carbon black exposure did not cause lung inflammation in the exposed females, measured 11 or 28–29 days post-exposure. Glial fibrillary acidic protein (GFAP) expression levels were dose-dependently increased in astrocytes around blood vessels in the cerebral cortex and hippocampus in six weeks old offspring, indicative of reactive astrogliosis. Also enlarged lysosomal granules were observed in brain perivascular macrophages (PVMs) in the prenatally exposed offspring. The number of parvalbumin-positive interneurons and the expression levels of parvalbumin were decreased in the motor and prefrontal cortices at weaning and 120 days of age in the prenatally exposed offspring. In the open field test, behaviour was dose-dependently altered following maternal exposure to Printex 90, at 90 days of age. Prenatally exposed female offspring moved a longer total distance, and especially males spent significantly longer time in the central zone of the maze. In the offspring, the described effects were long-lasting as they were present at all time points investigated. Conclusion The present study reports for the first time that maternal inhalation exposure to Printex 90 carbon black induced dose-dependent denaturation of PVM and reactive astrocytes, similarly to the findings observed following maternal exposure to Printex 90 by airway instillation. Of note, some of the observed effects have striking similarities with those observed in mouse models of neurodevelopmental disorders

Associations Between Metal Levels in Whole Blood and IgE Concentrations in Pregnant Women Based on Data From the Japan Environment and Children’s Study

Background: Metal exposures could possibly affect allergic responses in pregnant women, although no studies have yet shown a clear relationship between the two, and such exposures might also affect the development of allergic diseases in children. Methods: We investigated the relationship between metal concentrations in whole blood and immunoglobulin E (IgE; total and specific) in 14,408 pregnant women who participated in the Japan Environment and Children’s Study. The subjects submitted self-administered questionnaires, and blood samples were collected from them twice, specifically, during the first trimester and again during the second=third trimester. Concentrations of the metals Cd, Pb, Hg, Se, and Mn, as well as serum total and allergen-specific IgEs for egg white, house dust-mites (HDM), Japanese cedar pollen (JCP), animal dander, and moth, were measured. Allergen-specific IgE(s) were divided based on concentrations <0.35 or ≥0.35UA=mL, and the metal levels were divided into quartiles. Results: Multivariable logistic regression analysis showed that there was a significant negative correlation between HDM- and animal dander-specific IgEs and Hg and Mn concentrations. Conversely, there was a significant positive relationship between JCP-specific IgE and Hg and Se concentrations. Conclusions: Metal exposures may be related to both increases and decreases in allergen-specific IgEs in pregnant women.This work was supported by the Ministry of the Environment, Japan.Supplementary data related to this article can be found at https://doi.org/10.2188/jea.JE20180098

Pretreatment with N-acetyl cysteine suppresses chronic reactive astrogliosis following maternal nanoparticle exposure during gestational period

<p>Early pregnant employees are potentially and unintendedly exposed to industrial chemicals including nanoparticles. Developmental toxicity of nanoparticle exposure has been concerned because exposure to fine particle including carbon black nanoparticle (CB-NP) during the brain developmental stage enhances the risk of brain disorders. Maternal CB-NP exposure dose-dependently induces astrogliosis, which is an abnormal increase in the reactive astrocytes with glial fibrillary acidic protein (GFAP) and aquaporin-4 overexpression due to the destruction of nearby neurons and blood vessels. The present study aimed to investigate protective effects of antioxidants on the histopathological denaturation with astrogliosis following maternal CB-NP exposure in offspring mice, thereby to evaluate the role of oxidative stress on the developmental toxicity. Pregnant ICR mice were treated with CB-NP by intranasal instillation on gestational days 5 and 9. N-acetyl cysteine (NAC) or ascorbic acid was intraperitoneally administered to the pregnant mice 1 h prior to CB-NP instillation. The brains were collected from 6- to 12-week-old offspring mice and analyzed using western blotting and immunohistochemistry. NAC suppressed GFAP overexpression in 6- and 12-week-old offspring mice following maternal CB-NP exposure. However, NAC did not suppress aquaporin-4 overexpression following maternal CB-NP exposure. Ascorbic acid did not suppress, but rather slightly and significantly enhanced the expression of GFAP and aquaporin-4. These results indicate that astrogliosis by maternal CB-NP exposure is partially prevented by NAC pretreatment. Oxidative stress is a possible key factor of developmental neurotoxicity of maternal NP exposure. This study will contribute to elucidating the mechanisms underlying the effects of developmental neurotoxicity of NPs.</p

Effects of Maternal Exposure to Ultrafine Carbon Black on Brain Perivascular Macrophages and Surrounding Astrocytes in Offspring Mice

<div><p>Perivascular macrophages (PVMs) constitute a subpopulation of resident macrophages in the central nervous system (CNS). They are located at the blood-brain barrier and can contribute to maintenance of brain functions in both health and disease conditions. PVMs have been shown to respond to particle substances administered during the prenatal period, which may alter their phenotype over a long period. We aimed to investigate the effects of maternal exposure to ultrafine carbon black (UfCB) on PVMs and astrocytes close to the blood vessels in offspring mice. Pregnant mice were exposed to UfCB suspension by intranasal instillation on gestational days 5 and 9. Brains were collected from their offspring at 6 and 12 weeks after birth. PVM and astrocyte phenotypes were examined by Periodic Acid Schiff (PAS) staining, transmission electron microscopy and PAS-glial fibrillary acidic protein (GFAP) double staining. PVM granules were found to be enlarged and the number of PAS-positive PVMs was decreased in UfCB-exposed offspring. These results suggested that in offspring, “normal” PVMs decreased in a wide area of the CNS through maternal UfCB exposure. The increase in astrocytic GFAP expression level was closely related to the enlargement of granules in the attached PVMs in offspring. Honeycomb-like structures in some PVM granules and swelling of astrocytic end-foot were observed under electron microscopy in the UfCB group. The phenotypic changes in PVMs and astrocytes indicate that maternal UfCB exposure may result in changes to brain blood vessels and be associated with increased risk of dysfunction and disorder in the offspring brain.</p></div

Quantitative observation of PAS-positive PVMs.

<p>A: The number of PAS-positive PVMs in each brain region (n = 5/group). Numbers in the following regions were significantly decreased in UfCB-exposed offspring; cerebral cortex (Cx), hippocampus (HIP), hypothalamus (Hy), midbrain (MBr), cerebellum (Cb), and medulla oblongata (MO). Asterisks indicate statistical significance (* P<0.05, **P<0.01, ***P<0.001). Data are shown as mean ±SD. B: The regions of the brain. Abbreviations: Olf, olfactory bulb; Cx, cerebral cortex; cc, corpus callosum; Str, striatum; HIP, hippocampus; Th, thalamus; Hy, hypothalamus; MBr, midbrain; Po, pons; Cb, cerebellum; MO, medulla oblongata.</p

Light micrographs of GFAP-positive astrocytes and PAS-positive PVMs of 6-week-old male mice.

<p>All scale bars represent 10 μm. (A–C) 6-μm paraffin sections and (D–I) 10-μm frozen sections. A: PVM surrounding the cerebral blood vessels of a 6-week-old control mouse. GFAP-positive astrocytes were very few in number at sites attached to blood vessels with PVMs with small (approximately 1 μm) PAS-positive granules (arrow). B, C: PVM surrounding cerebral blood vessels of a mouse in the UfCB group. The PVM possessed enlarged granules (arrows). Many GFAP-positive astrocytes were observed at sites attached to PVMs with enlarged granules. D: GFAP-positive astrocytes and PAS-positive PVM (arrows) surrounding cerebral blood vessels of mouse in the UfCB group. E–I: Enlarged views of (D). Around one cerebral vessel, GFAP-positive astrocytic end-feet were detected (E, G, H) at a site attached to PVM with enlarged PAS-positive granules (G, 2.2 μm; H, 1.6 μm), but (F, I) not surrounding PVMs with small PAS-positive granules (smaller than 1.1 μm).</p

Light micrographs of GFAP-positive astrocytes of the wide-field.

<p>Scale bars represent (A, C) 200 m or (B, D) 100 m. A, C: The frontal cortex of 6-week-old male mice of (A) the control group (B) UfCB-exposed offspring. B, D: Enlarged views of A and C. (B) Few GFAP-positive astrocytes were observed in the grey matter in the control group, while (D) many GFAP-positive astrocytes were detected in the grey matter in UfCB-exposed offspring. GFAP-positive astrocytes were not observed at any sites attached to blood vessels with PVMs with small (approximately 1 m) PAS-positive granules (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094336#pone-0094336-g007" target="_blank">Figure 7A</a>), but were found at blood vessels with PVMs that had enlarged (approximately 2-3 m) PAS-positive granules in the UfCB-exposed offspring (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094336#pone-0094336-g007" target="_blank">Figure 7B, C</a>). Moreover, GFAP-positive astrocytic end-feet were detected at sites attached to PVMs with enlarged PAS-positive granules (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094336#pone-0094336-g007" target="_blank">Figure 7E, G, H</a>), and not surrounding PVMs with small PAS-positive granules (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094336#pone-0094336-g007" target="_blank">Figure 7F, I</a>) around one blood vessel in the UfCB-exposed offspring (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094336#pone-0094336-g007" target="_blank">Figure 7D-I</a>). These results suggested that the increase in the expression level of GFAP in astrocytic end-feet around blood vessels in the grey matter was correlated with enlargement of PAS-positive granules of PVM .</p