Pulmonary exposure to metallic nanomaterials during pregnancy irreversibly impairs lung development of the offspring

Abstract

Due to the growing commercial applications of manufactured nanoparticles (NPs), toxicological studies on NPs, especially during the critical window of development, are of major importance. The aim of the study was to assess the impact of respiratory exposure to metallic and metal oxide NPs during pregnancy on lung development of the offspring and to determine the key parameters involved in lung alterations. Pregnant mice were exposed to weekly doses of 100 μg (total dose 300 μg) of titanium dioxide (TiO₂), cerium oxide (CeO₂), silver (Ag) NPs or saline solution by nonsurgical intratracheal instillation. The offspring lungs were analyzed at different stages of lung development: fetal stage (gestational day 17.5), pulmonary alveolarization (post-delivery day 14.5) and lung maturity (post-delivery day 49.5). Regardless of the type of NP, maternal exposure during gestation induced long-lasting impairment of lung development of the offspring. This effect was accompanied by: i) decreased placental efficiency together with the presence of NPs in placenta, ii) no increase of inflammatory mediators present in amniotic fluid, placenta or offspring lungs and iii) decreased pulmonary expression of vascular endothelial growth factor-α (VEGF-α) and matrix metalloproteinase 9 (MMP-9) at the fetal stage, and fibroblast growth factor-18 (FGF-18) at the alveolarization stage. Respiratory exposure to metallic NPs during pregnancy induces stereotyped impairment of lung development with a lasting effect in adult mice, independently of the chemical nature of the NP.

Keywords:

• Nanoparticles
• toxicology
• lung development
• pregnancy

Acknowledgments

We thank Dr. V. Cohignac for her help in the animal experiments. The expert technical assistance of M. Surenaud is also gratefully acknowledged. Special thanks to Dr. P. Andujar, Dr. P. Launois, S. Rouziere, Dr. T. Fournier, Dr. PH. Jarreau, S. Valentino, Dr. P. Chavatte Palmer, Dr. A. Tarrade and Dr. J. Tran Van Nhieu for their helpful comments.

Ethics approval and consent to participate

Ethical approval for all animal experimentation was obtained from the local ethics committee (N° 12-104, ComEth Afssa/ENVA/UPEC, Maisons-Alfort, France).

Disclosure statement

The authors declare that they have no competing interests.

Additional material

Nanoparticle preparation and characterization

For determination of NPs size by transmission electron microscopy (TEM; JEOL JEM 1400, Peabody, MA), NPs were suspended in distilled water at a concentration of 100 μg/mL and sonicated in an ice bath at 37 kHz for 10 min (Elmasonic S 30 H, Mägenwil, Switzerland). The suspension (1 μL) was immediately deposited on a TEM copper grid, and pictures of the NPs were taken systematically at predefined widely separated positions at a magnification of 250,000 at 120 kV. The primary size of the NPs was measured using imageJ software (National Institute of Health, Rockville Pike, MD) based on examination of more than 1500 particles (Rice et al., 2013).

Dynamic light scattering (DLS; NanoZS, Malvern, UK) was carried out to determine the zeta potential and hydrodynamic diameter of the various NPs. For this purpose, NPs were suspended in sterile NaCl at a concentration of 10 mg/mL and were dispersed by sonication in an ice bath for 10 min just before DLS analysis. NPs suspensions were then injected in a capillary zeta cell for measurement of the zeta potential and hydrodynamic diameter (Brar & Verma, 2011).

The crystallinity of TiO₂ NPs and CeO₂ NPs was determined by X-ray diffractometer (XRD, PANalytical X’Pert PRO, Limeil-Brévannes, France). NPs powders were embedded in a silicon wafer with 500 μL of ethanol and were analyzed at room temperature for 5 h (Labille et al., 2010). The specific surface area of TiO₂ NPs was measured by a Micromeritics Flowsorb 2300 (Norcross, GA) using Brunauer–Emmett–Teller (BET) analysis. The specific surface area of CeO₂ NPs and Ag NPs was estimated by TEM by the following equation: where ρ is the particle density and r is the radius of the spherical NPs determined by TEM (considering that CeO₂ NPs and Ag NPs had a spherical shape).

To investigate possible Ag ion release from the Ag NPs suspension, an additional experiment was performed. Ag NPs suspension (10 mg/mL) in NaCl was sonicated in an ice bath for 10 min and was placed in ultrafiltration tubes (Millipore, Saint-Quentin en Yvelines, France) and, after ultracentrifugation at 4000 g for 40 min, the liquid phase containing the Ag ionic form was analyzed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Perkin Elmer NexIon 300X, Waltham, MA) (Ma et al., 2013).

Additional information

Funding

This work was carried out in the framework of the Labex SERENADE (ANR-11-LABX-0064) and A*MIDEX project (ANR-11-IDEX-0001-02), funded by the “Investissements d’Avenir” French Government program managed by the French National Research Agency (ANR), and, DHU A-TVB.