Maternal, placental, and fetal distribution of titanium after repeated titanium dioxide nanoparticle inhalation through pregnancy
Introduction
Ambient particulate matter (PM) with diameters of 0.1 μm (100 nm) or less are considered to be part of the ultrafine fraction of particulate matter (PM0.1). Enabled by their small size, PM0.1, otherwise recognized as nanoparticles (NPs), can cross biological barriers with considerable ease [[1], [2], [3]]. Over recent years, the global increases in aerosolized particulate matter [4], as well as the rapid development of engineered NPs, raises the potential for inhalation and systemic distribution of NPs across the pulmonary air-blood barrier. Particle translocation from the lung has been demonstrated in real-world human exposures [5,6] and controlled animal studies [7,8].
Pregnancy complications (e.g., miscarriage, fetal growth restriction) associated with PM exposure manifest in both human and animal studies which suggests a link between pulmonary NP exposure and adverse pregnancy outcomes [[9], [10], [11]]. Recent work gives further cause for concern over maternal inhalation of ambient NPs during pregnancy and their distribution [12]. Human placentas collected after delivery show that inhaled NPs can travel to the placenta [12]. Using electron microscopy and energy dispersive X-ray spectroscopy, PM consistent with the morphology, clustering, and chemical construct of PM0.1 from combustion-associated processes were found on the fetal side of the placenta after real-world exposure throughout pregnancy [12]. Additional evidence from animal studies reveal that pulmonary exposures to nano-polystyrene [9] or engineered nano-silver [10] led to placental and fetal distribution. Altogether, this evidence supports the theory that NPs inhaled during pregnancy can disseminate through the body, reaching the placenta and fetal tissues. Therefore, consideration is growing for underrepresented individuals, such as pregnant women and their children, who may be more vulnerable to these exposures.
Characterizing absorption or uptake and distribution are critical components to understanding toxicokinetics of NPs that enters the body. Nose-only exposure to titanium dioxide nanoparticles (nano-TiO2) in healthy male rats identified systemic distribution to the filter organs within 3-h of exposure [7]. During pregnancy, the drastic physiological changes associated with gestation may alter the absorption and distribution to secondary tissues, including 30–40% increase in tidal volume, 45% increase in maternal blood volume, and increased blood perfusion of the uterus, liver, and kidneys [[13], [14], [15]]. Together, this suggests that the maternal reproductive tissues, liver, and kidneys may be key sites of NP distribution and accumulation in a pregnancy model; however, this has not been well characterized in the literature.
The placenta is a transient but critical tissue that embeds into the maternal uterus. Given the increase in blood flow to the uteroplacental tissues during pregnancy, the placenta may be a direct target for NP deposition. Maternal blood bathes the placental syncytium cell layer, a 2 or 3 cell layer thick tissue in a human or rodent, respectively, that separates the maternal and fetal circulations. The syncytium is the most important cell type of the placenta performing the critical functions of: 1) fetal barrier protection, 2) expression of plasma membrane receptors that regulate protein synthesis, 3) production and secretion of peptide hormones (e.g., human chorionic gonadotropin), and 4) transport of substances across to the fetal blood circulation. Chemicals or pollutants such as NPs within maternal blood can directly encounter the syncytium. In vitro experiments have shown primary human syncytial cells to internalize and accumulate metallic NPs [16]. Once internalized, NPs mainly distribute to lysosomal vesicles while some remain freely suspended in cytoplasm [17]. Other intracellular consequences include NP-protein aggregation, increased endoplasmic reticulum stress, mitophagy, and increased intracellular ROS [17]. Cellular damage to the placenta may cause serious organ dysfunction. NP-mediated decreases in uterine invasion [18] and increases in uterine vascular resistance [19] have been empirically shown to lead to poor placental perfusion. Additional studies have shown decreased placental growth [20], barrier integrity [21], and hormone secretion [22] after NP exposure. Increased inflammatory cytokine secretion [10] and trophoblast shedding [21] from NP exposure have also been identified, all of which can result in attenuated barrier and nutrient provision for the fetus. While these findings are critical to the field, there are inherent limitations. Studies utilize in vitro or ex vivo models may apply NP doses significantly higher than physiological transport would permit of NP to elicit effects. Those employing short and direct lung instillation exposure schemes (e.g., single or acute exposure timelines and direct intratracheal instillation) often make for difficult extrapolation to the human population who often experience chronic, lower dose exposures to ambient NPs. Therefore, it is critical to utilize a recurring whole-body exposure scheme throughout pregnancy to evaluate inhaled ambient NP aerosol translocation from the lung to the placenta and syncytium internalization.
Fetal sex plays a major role in the outcomes of placental mediation of maternal stress [23], uptake of xenobiotics [11], and likelihood of developing gestational disorders such as preterm birth [24]. A sex-dependent placental accumulation of metals has been identified, where placentas from male fetuses accumulated higher concentrations of soluble silver (Ag) and titanium (Ti) compared to females [25]. This sexual dimorphism is likely due to the contribution of X- and Y- linked genes leading to differential sex-specific regulatory pathways of the placenta. Another emerging and potentially significant contributor to pregnancy outcomes includes intrauterine positioning of the placenta and fetus [11,26]. We identified differences in fetal weight between uterine horns and specific horn locations after maternal nano-TiO2 inhalation [11]. Therefore, these factors may also play a role in placental uptake of NPs.
Here we employ whole-body inhalation exposure to nano-TiO2 aerosols, a widely used engineered nanomaterial and common surrogate for PM0.1 exposures, throughout gestation to simulate environmental or occupational workplace exposures as previously identified [27]. The hypothesis of this study is that nano-TiO2 will translocate from the lungs after maternal inhalation in late-stage pregnancy, Ti will be quantified in all secondary maternal, utero-placental, and fetal tissues, and NPs will be visualized in the syncytium. Furthermore, we expect sex- or intrauterine position- specific Ti accumulation. Our aims were to 1) describe the Ti distribution from the pulmonary space to maternal, placental, and fetal tissues, 2) measure Ti accumulation on the maternal and fetal sides of the placenta by sex and intrauterine position, and 3) visualize intracellular localization of NP within placental syncytium.
Section snippets
Nanoparticle Characterization
Nano-TiO2 powder was purchased from Evonik (Aeroxide TiO2, Parsippany, NJ). Using dynamic light scattering (DLS) techniques with a Zetasizer Nano ZS by Malvern, the crystalline composition of the powder was previously determined to be composed of 80% anatase and 20% rutile, primary particle size 21 ± 6.1 nm and surface area 48.08 mg2/g [28].
Animal model
Time-pregnant Sprague Dawley rats were purchased from Charles River Laboratories (Kingston, NY) on GD 1 or 2 and housed in the Rutgers School of Public
Maternal lung deposition of NPs
The total, daily, and additive lung deposition of NPs were calculated for each experimental group (Table 1). After considering daily deposition and clearance from repeated exposures, the total lung deposition for the nano-TiO2 group was calculated to be 194.81 ± 1.89 μg.
Distribution of Ti to maternal, placental, and fetal tissues
The distribution of Ti was quantified in both exposed and control tissues (Fig. 2). Background Ti concentrations in the control group are consistent with previous literature evaluating nano-TiO2 biodistribution [7,32]. Upon
Discussion
This study aimed to examine the systemic distribution of inhaled nano-TiO2 aerosols during pregnancy. Previous work has identified links between inhalation exposure and perturbations to pregnancy health (e.g., miscarriage, FGR) [9,10], with a particular emphasis on the placenta and its role as a protective barrier for the fetus. We found evidence in support of NP translocation to secondary maternal organs, the placenta, and fetal tissues after maternal inhalation. Furthermore, Ti was identified
Funding sources
This work was supported by the National Institutes of Health [R01-ES031285; R00-ES27483; P30-ES005022; T32-ES007148; R25-ES020721], ASPET-SURF, and RISE at Rutgers.
Declaration of competing interest
None.
Acknowledgments
We would like to acknowledge Ms. Chelsea Cary for assisting with necropsy tissue and data collections, and for her time in collaboration with Ms. Talia Seymore for the thoughtful review of this manuscript. We would like to thank Mr. Rajesh Patel from the Core Imaging Lab in the
Department of Pathology in the Robert Wood Johnson Medical Center for the processing, sectioning, and embedding of the TEM slides and for guidance on TEM microscope usage. We would also like to thank Dr. Kenneth Reuhl for
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