Maternal titanium dioxide nanomaterial inhalation exposure compromises placental hemodynamics

Abstract

The fetal consequences of gestational engineered nanomaterial (ENM) exposure are unclear. The placenta is a barrier protecting the fetus and allowing transfer of substances from the maternal circulation. The purpose of this study was to determine the effects of maternal pulmonary titanium dioxide nanoparticle (nano-TiO₂) exposure on the placenta and umbilical vascular reactivity. We hypothesized that pulmonary nano-TiO₂ inhalation exposure increases placental vascular resistance and impairs umbilical vascular responsiveness. Pregnant Sprague-Dawley rats were exposed via whole-body inhalation to nano-TiO₂ with an aerodynamic diameter of 188 ± 0.36 nm. On gestational day (GD) 11, rats began inhalation exposures (6 h/exposure). Daily lung deposition was 87.5 ± 2.7 μg. Animals were exposed for 6 days for a cumulative lung burden of 525 ± 16 μg. On GD 20, placentas, umbilical artery and vein were isolated, cannulated, and treated with acetylcholine (ACh), angiotensin II (ANGII), S-nitroso-N-acetyl-DL-penicillamine (SNAP), or calcium-free superfusate (Ca²⁺-free). Mean outflow pressure was measured in placental units. ACh increased outflow pressure to 53 ± 5 mmHg in sham-controls but only to 35 ± 4 mmHg in exposed subjects. ANGII decreased outflow pressure in placentas from exposed animals (17 ± 7 mmHg) compared to sham-controls (31 ± 6 mmHg). Ca²⁺-free superfusate yielded maximal outflow pressures in sham-control (63 ± 5 mmHg) and exposed (30 ± 10 mmHg) rats. Umbilical artery endothelium-dependent dilation was decreased in nano-TiO₂ exposed fetuses (30 ± 9%) compared to sham-controls (58 ± 6%), but ANGII sensitivity was increased (-79 ± 20% vs -36 ± 10%). These results indicate that maternal gestational pulmonary nano-TiO₂ exposure increases placental vascular resistance and impairs umbilical vascular reactivity.

Keywords: Engineered nanomaterials; Microcirculation; Placenta; Titanium dioxide nanoparticles.

Figure 1.. Nano-TiO₂ aerosol characterization.

(A) Transmission electron microscope image of a typical nano-TiO₂ agglomerate generated with the high-pressure acoustical generator, (B) Size distribution of the nano-TiO₂ aerosol (mobility diameter) sampled from the exposure chamber using a scanning mobility particle sizer (SMPS-light grey) and an aerodynamic particle sizer (APS-dark grey, negligible values). The red line represents a log-normal fit of the histogram (Count Median Diameter = 190 nm), (C) Size distribution of the nano-TiO₂ aerosol (aerodynamic diameter) using a high resolution electrical low-pressure impactor (ELPI+). The red line represents a log-normal fit of the histogram (Count Median Diameter = 188 ± 0.36 nm). (D) Real-time mass concentration measurements of the nano-TiO₂ aerosol during a typical inhalation exposure. The red line represents the target concentration, 12 mg/m³.

Figure 2.. Ex vivo placental perfusion preparation.

(A) Umbilical artery (1); vein (2); placenta (3). (B) Input (arterial) perfusion pressure (1) is held constant while vasoactive agonists are added. Output pressure (2) and flow (3) (μl/min) are variables responsive to placental resistance.

Figure 3.. Maternal nano-TiO₂ inhalation exposure impairs endothelium-dependent placental hemodynamics.

Mean outflow pressures were measured in sham-control and nano-TiO₂ exposed animals after the inflow pressure was increased in a stepwise manner and set at 0 mm Hg, 20 mm Hg, 40 mm Hg, 60 mm Hg and 80 mm Hg. Nano-TiO₂ inhalation exposure altered placental hemodynamics and decreased outflow (venous) pressure in placentas in normal superfusate (A) and placentas treated with ACh (B) (n=8)., (P ≤ 0.05) * vs. sham-control group.

Figure 4.. Maternal nano-TiO₂ inhalation exposure does not affect endothelium-independent placental hemodynamics but increases ANGII sensitivity.

Mean outflow pressures were measured in sham-control and nano-TiO₂ exposed animals after the inflow pressure was increased in a stepwise manner and set at 0 mm Hg, 20 mm Hg, 40 mm Hg, 60 mm Hg and 80 mm Hg. Maternal nano-TiO₂ inhalation exposure did not affect endothelial-independent placental hemodynamics (A) but increased ANGII sensitivity (B) (n=8). , (P ≤ 0.05) * vs. sham-control group

Figure 5.. Maternal nano-TiO₂ inhalation impairs calcium-free placental hemodynamics.

Mean outflow pressures were measured in sham-control and nano-TiO₂ exposed animals after the inflow pressure was increased in a stepwise manner and set at 0 mm Hg, 20 mm Hg, 40 mm Hg, 60 mm Hg and 80 mm Hg. Maternal nano-TiO₂ inhalation exposure altered placental hemodynamics and decreased the outflow pressure in placentas placed in calcium-free superfusate (n=8)., (P ≤ 0.05) * vs. sham-control group

Figure 6.. Maternal nano-TiO₂ inhalation exposure and placental mean outflow pressure.

Mean outflow pressure at 80 mm Hg inflow pressure in both sham-control and nano-TiO₂ exposed groups and all treatment conditions is shown (n=8)., (P ≤ 0.05) * vs. sham-control group

Figure 7.. Maternal nano-TiO₂ inhalation exposure impairs endothelium-dependent dilation of the umbilical artery and vein.

(A) Endothelium-dependent dilation of the umbilical artery, (B) endothelium-dependent dilation of the umbilical vein from sham-control and nano-TiO₂ exposed animals was determined using pressure myography (n=12–14). Statistics were analyzed with two-way ANOVA., (P ≤ 0.05) * vs. sham-control group

Figure 8.. Maternal nano-TiO₂ inhalation exposure impairs endothelium-independent dilation of the umbilical artery and vein.

(A) Endothelium-independent dilation of the umbilical artery, (B) endothelium-independent dilation of the umbilical vein from sham-control and nano-TiO₂ exposed animals was determined using pressure myography (n=12–14)., (P ≤ 0.05) * vs. sham-control group

Figure 9.. Maternal nano-TiO₂ inhalation exposure increases ANGII sensitivity of the umbilical artery.

(A) ANGII dose-response curve of the umbilical artery (A) and vein (B) from sham-control and nano-TiO₂ exposed animals was determined using pressure myography (n=12–14)., (P ≤ 0.05) * vs. sham-control group

Figure 10.. Placental immunohistochemistry.

Smooth muscle actin is green, Von-Willebrand Factor is red, and DAPI nuclear staining is blue. (A) Representative image showing immunohistochemistry of a placental section from maternal nano-TiO₂ exposed animal. The purpose was to identify the microvascular architecture and the structural components of the placenta. (B) An enlarged (40×) image of a placental microvessel displaying an intact endothelium (green).