Prenatal Exposure to Titanium Dioxide Nanoparticles Alters Maternal Gut Microbiota and Elevates Fasting Blood Glucose in Rats

Abstract

Titanium dioxide nanoparticles (TiO₂ NPs) are ubiquitous in cosmetics, food additives, and paints, exposing pregnant women to unavoidable contact. While their antibacterial activity is well documented, the impact on maternal gut microbiota during pregnancy remains unclear. We exposed pregnant Sprague‑Dawley rats to 5 mg/kg bw/day TiO₂ NPs (days 5–18 of gestation) and monitored fecal microbiota at gestation days (GD) 0, 10 and 17. Alpha‑diversity indices (Shannon, Simpson, Chao1) did not differ significantly between control and exposed groups, yet beta‑diversity analysis revealed distinct community shifts, particularly a decrease in Ellin6075 and Dehalobacteriaceae and an increase in Clostridiales after exposure. Functional inference (PICRUSt) indicated enrichment of type 2 diabetes mellitus‑related pathways and suppression of taurine metabolism during the second trimester. Correspondingly, fasting blood glucose rose significantly at GD 10 (P < 0.05) and GD 17 (P < 0.01). These findings suggest that TiO₂ NP exposure perturbs the maternal gut microbiome and elevates glucose levels, potentially increasing gestational diabetes risk in humans.

Introduction

Titanium dioxide nanoparticles (TiO₂ NPs) rank among the most widely used nanomaterials, present in sunscreens, food additives, inks, and paints [1, 2]. Their small size facilitates release into the environment and subsequent ingestion or inhalation, making exposure unavoidable for pregnant women. Prior animal studies have linked TiO₂ NP exposure to ovarian dysfunction, altered monoaminergic neurotransmission, and adverse pregnancy outcomes [3–5]. However, the mechanisms underpinning these effects remain poorly understood. Notably, TiO₂ NPs exhibit broad-spectrum antibacterial activity [6], raising concerns that they might disrupt the gut microbiota—an ecosystem increasingly recognized as pivotal for metabolic regulation, immune tolerance, and pregnancy maintenance [7–10]. While adult rodents exposed to TiO₂ NPs exhibit hyperglycemia [11], it is unknown whether pregnant females experience similar metabolic disturbances or whether gut microbiota alterations mediate such effects. This study addresses these gaps by examining gut microbial dynamics and fasting glucose in a rat model of prenatal TiO₂ NP exposure.

Materials and Methods

Study Design

Building on a human exposure model [12], female Sprague‑Dawley rats received daily oral gavage of 5 mg/kg bw TiO₂ NPs (days 5–18 of gestation). Controls received 0.5% methylcellulose vehicle. Body weight was recorded prior to each dose.

Animals

Adult SD rats (n = 16; 12 weeks old females, 14 weeks old males) were housed under controlled temperature (22 ± 2 °C) and humidity (40–60%) with a 12‑h light/dark cycle. After acclimation, females were paired with males (1:1) and vaginal plugs confirmed pregnancy (GD 0.5). Pregnant females were then randomly assigned to control (n = 4) or exposure (n = 4) groups.

TiO₂ NP Preparation and Administration

Commercial TiO₂ NPs (Sigma‑Aldrich 13463‑67‑7) were dispersed in 0.5% methylcellulose (5 mg/ml) and sonicated (100 W, 30 min). Hydrodynamic diameter in vehicle was ~199.5 nm (DLS).

Sample Collection and DNA Extraction

Fecal pellets were collected at GD 0, 10 and 17, snap‑frozen at −80 °C, and total DNA isolated with the PowerSoil kit (Mo Bio). DNA quantity was assessed via NanoDrop.

16S rRNA Gene Sequencing and Bioinformatics

The V3‑V4 region was amplified and sequenced on an Illumina MiSeq platform. Raw reads were processed in QIIME: merging, chimera removal, OTU clustering (97% similarity), and taxonomic assignment. Alpha and beta diversity metrics (Shannon, Simpson, Chao1, NMDS) were calculated. Differential taxa were identified by LefSe (LDA > 2). Functional potentials were inferred with PICRUSt, focusing on diabetes‑related pathways and taurine metabolism.

Blood Glucose Measurement

Fasting venous blood (12‑h fast) was collected via caudal vein at GD 0, 10 and 17. Glucose levels were measured immediately using the Roche ACCU‑CHEK® Performa glucometer.

Statistical Analysis

Data were analyzed with GraphPad Prism 6. One‑way ANOVA with Dunnett’s post‑hoc test assessed significance; P < 0.05 was considered significant.

Results and Discussion

TiO₂ NP Characterization

TEM imaging confirmed nearly spherical particles (~21 nm primary diameter). The reported purity was ≥99.5% with a surface area of 35–65 m²/g. These characteristics align with prior studies indicating that nanosized TiO₂ can elevate blood glucose in adult rodents [11, 16].

Microbiota Dynamics During Normal Pregnancy

Alpha diversity (Shannon, Simpson, Chao1) remained stable across GD 0, 10 and 17 in control rats (Fig. 2a). NMDS analysis showed no clear clustering by gestation day (Fig. 2b). Venn diagrams revealed 164 shared OTUs among the three time points, indicating progressive acquisition of specific taxa (Fig. 2c). These observations are consistent with literature reporting modest microbiome shifts during pregnancy [21, 22].

Impact of TiO₂ NP Exposure on Microbial Diversity

After exposure, alpha diversity displayed a slight increase in Shannon and a significant rise in Simpson index (P < 0.05) but remained unchanged in Chao1 (Fig. 3a). NMDS plots did not show a major separation, yet the number of unique OTUs decreased at GD 10 and 17 (Fig. 3c). The data suggest that TiO₂ NPs selectively suppress dominant taxa, allowing previously suppressed microbes to expand—a plausible mechanism given their broad antibacterial activity.

Second‑Trimester Microbiota Alterations

At GD 10, alpha diversity was unaffected, but NMDS revealed a clear separation between control and exposed groups (Fig. 4b). LefSe identified reduced abundance of Ellin6075 and increased Clostridiales (Fig. 4d). PICRUSt predicted enrichment of type 2 diabetes pathways and decreased taurine/hypotaurine metabolism (Fig. 4e). These functional shifts align with the known role of gut microbes in glucose homeostasis and taurine’s hypoglycemic effects [31, 32].

Late‑Trimester Microbiota Changes

At GD 17, alpha diversity remained unchanged, but NMDS indicated significant compositional divergence (Fig. 5b). OTU counts were lower in exposed rats (Fig. 5c). LefSe confirmed persistent depletion of Ellin6075 and reduction of Dehalobacteriaceae (Fig. 5d). No diabetes‑related functional changes were observed at this stage, suggesting the second trimester is the critical window for metabolic disruption.

Fasting Blood Glucose Outcomes

Consistent with functional predictions, fasting glucose increased in exposed rats at GD 10 (P < 0.05) and GD 17 (P < 0.01) (Fig. 5e). Although the rise at GD 17 (~0.5 mM) did not reach clinical thresholds for gestational diabetes [34], it indicates a measurable metabolic perturbation that could influence fetal development [35].

Conclusion

Our findings demonstrate that prenatal exposure to TiO₂ nanoparticles perturbs the maternal gut microbiota—particularly reducing Ellin6075 and Dehalobacteriaceae, increasing Clostridiales—and leads to elevated fasting glucose levels during pregnancy. These results highlight a potential mechanism linking TiO₂ NP exposure to gestational diabetes risk, warranting further investigation in human populations.