Basal Ti level in the human placenta and meconium and evidence of a materno-foetal transfer of food-grade TiO2 nanoparticles in an ex vivo placental perfusion model

Abstract

Background: Titanium dioxide (TiO₂) is broadly used in common consumer goods, including as a food additive (E171 in Europe) for colouring and opacifying properties. The E171 additive contains TiO₂ nanoparticles (NPs), part of them being absorbed in the intestine and accumulated in several systemic organs. Exposure to TiO₂-NPs in rodents during pregnancy resulted in alteration of placental functions and a materno-foetal transfer of NPs, both with toxic effects on the foetus. However, no human data are available for pregnant women exposed to food-grade TiO₂-NPs and their potential transfer to the foetus. In this study, human placentae collected at term from normal pregnancies and meconium (the first stool of newborns) from unpaired mothers/children were analysed using inductively coupled plasma mass spectrometry (ICP-MS) and scanning transmission electron microscopy (STEM) coupled to energy-dispersive X-ray (EDX) spectroscopy for their titanium (Ti) contents and for analysis of TiO₂ particle deposition, respectively. Using an ex vivo placenta perfusion model, we also assessed the transplacental passage of food-grade TiO₂ particles.

Results: By ICP-MS analysis, we evidenced the presence of Ti in all placentae (basal level ranging from 0.01 to 0.48 mg/kg of tissue) and in 50% of the meconium samples (0.02-1.50 mg/kg), suggesting a materno-foetal passage of Ti. STEM-EDX observation of the placental tissues confirmed the presence of TiO₂-NPs in addition to iron (Fe), tin (Sn), aluminium (Al) and silicon (Si) as mixed or isolated particle deposits. TiO₂ particles, as well as Si, Al, Fe and zinc (Zn) particles were also recovered in the meconium. In placenta perfusion experiments, confocal imaging and SEM-EDX analysis of foetal exudate confirmed a low transfer of food-grade TiO₂ particles to the foetal side, which was barely quantifiable by ICP-MS. Diameter measurements showed that 70 to 100% of the TiO₂ particles recovered in the foetal exudate were nanosized.

Conclusions: Altogether, these results show a materno-foetal transfer of TiO₂ particles during pregnancy, with food-grade TiO₂ as a potential source for foetal exposure to NPs. These data emphasize the need for risk assessment of chronic exposure to TiO₂-NPs during pregnancy.

Keywords: E171 food additive; Foetus; Human placenta; Nanoparticles; Titanium dioxide.

Fig. 1

SEM analysis of size distribution of food-grade TiO₂ particles. a Scanning electron microscopy image of TiO₂ particles in E171 water suspension. Scale bar = 300 nm. b Primary size distribution by number of TiO₂ particles, based on the diameters of 600 particles measured on SEM images

Fig. 2

STEM-EDX analysis of TiO₂ particles basal distribution in human placenta (a) and meconium (b). (A1) (S) TEM -micrographs showing cluster of four particles. Left hand corner micrograph is acquired in STEM-HAADF mode where particles appear brighter than biological matrix, due to the detection of elastic electron scattered by high Z number particles (chemical contrast). (A2) STEM-EDX elemental maps and sum spectrum identifying Ti, O, Al, and Si as main elements, and (A3) corresponding EDX analysis. All scale bars = 100 nm. (B1) TEM image illustrating particle content in meconium and coupled to EDX analysis (B2) with Ti, O, Al and Si identified as main elements over 3 particles

Fig. 3

Mean transfer profile of particles during 1 h of placental perfusion with E171 using confocal microscopy. Placenta were perfused with food-grade TiO₂ (E171, 15 μg/mL in PM) and counted as laser-diffracting particle spots over 1 h of experiment. Each data is the mean ± SEM of 4 to 6 independent experiments

Fig. 4

Analysis of TiO₂ particles by SEM-EDX in foetal exudate. a SEM images of particles agglomerated in the dried PM after sample preparation of foetal exudate taken up from the 20–30 min period of perfusion. b Corresponding EDX analyses of particles into agglomerates showing the presence of C, Cl, S, Ti and O (Si signal from SEM wafer). c SEM-EDX cartography of particles showing Ti (red) and O (green) elements, and corresponding merge image. White arrows indicate examples of particles negative for Ti and O in this preparation. Scale bar = 200 nm

Fig. 5

Size distribution of TiO₂ particles in the maternal perfusion medium and the foetal exudate. Primary size distribution of TiO₂ particles by number based on diameters measured on SEM-EDX images corresponding to samples of foetal exudate collected between 20 and 30 mn of E171 perfusion in 2 placentae, and compared to E171 suspension (solid line) in the perfusion medium (PM). P1 = 144 particles, P2 = 156 particles, and E171 in PM = 300 particles

Fig. 6

Tissue distribution of TiO₂ particles in the E171 perfused placenta. a TEM-Bright-Field micrographs coupled to EDX analysis showing food-grade TiO₂ particles into the syncytiotrophoblast microvilli. b Chemical characterization of TiO₂ particles by STEM-EDX mapping based on STEM-HAADF micrographs for particle detection, and elemental maps and sum spectrum. Scale bars in (B) = 1 μm