Maternal engineered nanomaterial (ENM) inhalation is associated with uterine vascular impairments and endocrine disruption that may lead to altered gestational outcomes. We have shown that nano-titanium dioxide (nano-TiO 2 ) inhalation impairs endothelium-dependent uterine arteriolar dilation in pregnant rats. However, the mechanism underlying this dysfunction is unknown. Due to its role as a potent vasoconstrictor and essential reproductive hormone, we examined how kisspeptin is involved in nano-TiO 2 -induced vascular dysfunction and placental efficiency. Pregnant Sprague Dawley rats were exposed (gestational day [GD] 10) to nano-TiO 2 aerosols (cumulative dose ¼ 525 6 16 lg; n ¼ 8) or sham exposed (n ¼ 6) and sacrificed on GD 20. Plasma was collected to evaluate estrogen (E 2 ), progesterone (P4), prolactin (PRL), corticosterone (CORT), and kisspeptin. Pup and placental weights were measured to calculate placental efficiency (grams fetus/gram placental). Additionally, pressure myography was used to determine uterine artery vascular reactivity. Contractile responses were assessed via cumulative additions of kisspeptin (1 Â 10 À9 to 1 Â 10 À4 M). Estrogen was decreased at GD 20 in exposed (11.08 6 3 pg/ml) versus sham-control rats (66.97 6 3 pg/ml), whereas there were no differences in P4, PRL, CORT, or kisspeptin. Placental weights were increased in exposed (0.99 6 0.03 g) versus sham-control rats (0.70 6 0.04 g), whereas pup weights (4.01 6 0.47 g vs 4.15 6 0.15 g) and placental efficiency (4.5 6 0.2 vs 6.4 6 0.5) were decreased in exposed rats. Maternal ENM inhalation exposure augmented uterine artery vasoconstrictor responses to kisspeptin (91.2%62.0 vs 98.6%60.10). These studies represent initial evidence that pulmonary maternal ENM exposure perturbs the normal gestational endocrine vascular axis via a kisspeptin-dependent mechanism, and decreased placental, which may adversely affect health outcomes.

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-TiO 2) exposure on the placenta and umbilical vascular reactivity. We hypothesized that pulmonary nano-TiO 2 inhalation exposure increases placental vascular resistance and impairs umbilical vascular responsiveness. Pregnant Sprague-Dawley rats were exposed via whole-body inhalation to nano-TiO 2 with an aerodynamic diameter of 188 ± 0.36 nm. On gestational day (GD) 11, rats began inhalation exposures (6h/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 (Ca 2+-free). Mean outflow pressure was measured in placental units. ACh increased outflow pressure to 53 ± 5 mm Hg in sham-controls but only to 35 ± 4 mm Hg in exposed subjects. ANGII decreased outflow pressure in placentas from exposed animals (17 ± 7 mm Hg) compared to sham-controls (31 ± 6 mm Hg). Ca 2+-free superfusate yielded maximal outflow pressures in sham-control (63 ± 5 mm Hg) and exposed (30 ± 10 mm Hg) rats. Umbilical artery endothelium-dependent dilation was decreased in nano-TiO 2 exposed fetuses (30 ± 9%) compared to sham-controls (58 ± 6%), but ANGII sensitivity was increased (−79 ± 20% vs −36

Background Pregnancy is associated with many rapid biological adaptations that support healthy development of the growing fetus. One of which is critical to fetal health and development is the coordination between maternal liver derived substrates and vascular delivery. This crucial adaptation can be potentially derailed by inhalation of toxicants. Engineered nanomaterials (ENM) are commonly used in household and industrial products as well as in medicinal applications. As such, the potential risk of exposure remains a concern, especially during pregnancy. We have previously reported that ENM inhalation leads to upregulation in the production of oxidative species. Therefore, we aimed to determine if F0 dam maternal nano-TiO2 inhalation exposure (exclusively) resulted in altered H2O2 production capacity and changes in downstream redox pathways in the F0 dams and subsequent F1 pups. Additionally, we investigated whether this persisted into adulthood within the F1 generation and how this impacted F1 gestational outcomes and F2 fetal health and development. We hypothesized that maternal nano-TiO2 inhalation exposure during gestation in the F0 dams would result in upregulated H2O2 production in the F0 dams as well as her F1 offspring. Additionally, this toxicological insult would result in gestational vascular dysfunction in the F1 dams yielding smaller F2 generation pups. Results Our results indicate upregulation of hepatic H2O2 production capacity in F0 dams, F1 offspring at 8 weeks and F1 females at gestational day 20. H2O2 production capacity was accompanied by a twofold increase in phosphorylation of the redox sensitive transcription factor NF-κB. In cell culture, naïve hepatocytes exposed to F1-nano-TiO2 plasma increased H2O2 production. Overnight exposure of these hepatocytes to F1 plasma increased H2O2 production capacity in a partially NF-κB dependent manner. Pregnant F1- nano-TiO2 females exhibited estrogen disruption (12.12 ± 3.1 pg/ml vs. 29.81 ± 8.8 pg/ml sham-control) and vascular dysfunction similar to their directly exposed mothers. F1-nano-TiO2 uterine artery H2O2 production capacity was also elevated twofold. Dysfunctional gestational outcomes in the F1-nano-TiO2 dams resulted in smaller F1 (10.22 ± 0.6 pups vs. sham-controls 12.71 ± 0.96 pups) and F2 pups (4.93 ± 0.47 g vs. 5.78 ± 0.09 g sham-control pups), and fewer F1 male pups (4.38 ± 0.3 pups vs. 6.83 ± 0.84 sham-control pups). Conclusion In conclusion, this manuscript provides critical evidence of redox dysregulation across generations following maternal ENM inhalation. Furthermore, dysfunctional gestational outcomes are observed in the F1-nano-TiO2 generation and impact the development of F2 offspring. In total, this data provides strong initial evidence that maternal ENM exposure has robust biological impacts that persists in at least two generations.

Pregnancy requires rapid adaptations in the uterine microcirculation to support fetal development. Nanomaterial inhalation is associated with cardiovascular dysfunction, which may impair gestation. We have shown that maternal nano-titanium dioxide (nano-TiO2) inhalation impairs microvascular endothelial function in response to arachidonic acid (AA) and thromboxane (TXA2) mimetics. However, the mechanisms underpinning this process are unknown. Therefore, we hypothesize that maternal nano-TiO2 inhalation during gestation results in uterine microvascular prostacyclin (PGI2) and TXA2 dysfunction. Pregnant Sprague-Dawley rats were exposed from gestational day (GD) 10-19 to nano-TiO2 aerosols (12.17 mg/m3 ± 1.67) or filtered air (sham-control). Dams were euthanized on GD20, and serum, uterine radial arterioles, implantation sites and lungs were collected. Serum was assessed for PGI2 and TXA2 metabolites. TXB2, the stable TXA2 metabolite, was significantly decreased in nano-TiO2 exposed dams (597.3 ± 84.4 pg/mL vs 667.6 ± 45.6 pg/mL), whereas no difference was observed for 6-keto-PGF1α, the stable PGI2 metabolite. Radial arteriole pressure myography revealed that nano-TiO2 exposure caused increased vasoconstriction to the TXA2 mimetic, U46619, compared to sham-controls (-41.3 ± 4.3% vs -16.8 ± 3.4%). Nano-TiO2 exposure diminished endothelium-dependent vasodilation to carbaprostacyclin, a PGI2 receptor agonist, compared to sham-controls (30.0 ± 9.0% vs 53.7 ± 6.0%). Maternal nano-TiO2 inhalation during gestation decreased nano-TiO2 female pup weight when compared to sham-control males (3.633 ± 0.064 g vs 3.995 ± 0.124 g). Augmented TXA2 vasoconstriction and decreased PGI2 vasodilation may lead to decreased placental blood flow and compromise maternofetal exchange of waste and nutrients, which could ultimately impact fetal health outcomes.

Enzyme-linked immunosorbent assay (ELISA) is the gold standard method for protein biomarkers. However, scaling up ELISA for multiplexed biomarker analysis is not a trivial task due to the lengthy procedures for fluid manipulation and high reagent/sample consumption. Herein, we present a highly scalable multiplexed ELISA that achieves a similar level of performance to commercial single-target ELISA kits as well as shorter assay time, less consumption, and simpler procedures. This ELISA is enabled by a novel microscale fluid manipulation method, composable microfluidic plates (cPlate), which are comprised of miniaturized 96-well plates and their corresponding channel plates. By assembling and disassembling the plates, all of the fluid manipulations for 96 independent ELISA reactions can be achieved simultaneously without any external fluid manipulation equipment. Simultaneous quantification of four protein biomarkers in serum samples is demonstrated with the cPlate system, achieving high sensitivity and specificity (∼ pg/mL), short assay time (∼1 h), low consumption (∼5 μL/well), high scalability, and ease of use. This platform is further applied to probe the levels of three protein biomarkers related to vascular dysfunction under pulmonary nanoparticle exposure in rat’s plasma. Because of the low cost, portability, and instrument-free nature of the cPlate system, it will have great potential for multiplexed point-of-care testing in resource-limited regions.

Elevated cytosolic calcium and protein kinase C are well-established mediators of luteolytic actions of prostaglandin F2alpha (PGF2alpha). The objectives of this study were to determine 1) if calcium/calmodulin-dependent kinase kinase 2 (CAMKK2) participates in mediating PGF2alpha actions in developing (Day [d]-4) and mature (d-10) bovine corpus luteum (CL), 2) distal targets of CAMKK2, 3) developmental expression of adenosine monophosphate-activated protein kinase (AMPK), and 4) effects of AMPK activation on progesterone (P4) production. Expression of AMPK increased as the CL matured. Activation of the prostaglandin receptor (FP) induced rapid phosphorylation of AMPK, which was blocked by a CAMKK2 inhibitor. Changes in basal P4 secretion in vitro were determined in response to AMPK activation via metformin (met) or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) in d-4 and d-10 CL. Production of P4 in d-10 CL decreased with met or AICAR compared to control, similar to activation by PGF2alpha. Therefore, potential distal targets of AMPK in d-10 CL were examined during induced functional regression via exogenous PGF2alpha. Serum and luteal P4 decreased at 2 and 4 h after administration of PGF2alpha. Protein expression of LDLR decreased at 2 and 4 h, while those of ACAT1 and STAR increased 4 h after PGF2alpha. During induced regression, alterations of cholesterol transport proteins contributed to decreased luteal and serum P4. Therefore, developmental differences in signal transduction associated with FP, specifically CAMKK2 and AMPK, partially contribute to differences in the ability of PGF2alpha to induce regression in mature, but not developing, bovine CL. Multiple cholesterol transport proteins, including LDLR, were altered by PGF2alpha and could be potential AMPK targets.

BackgroundThe cardiovascular effects of pulmonary exposure to engineered nanomaterials (ENM) are poorly understood, and the reproductive consequences are even less understood. Inflammation remains the most frequently explored mechanism of ENM toxicity. However, the key mediators and steps between lung exposure and uterine health remain to be fully defined. The purpose of this study was to determine the uterine inflammatory and vascular effects of pulmonary exposure to titanium dioxide nanoparticles (nano-TiO2). We hypothesized that pulmonary nano-TiO2 exposure initiates a Th2 inflammatory response mediated by Group II innate lymphoid cells (ILC2), which may be associated with an impairment in uterine microvascular reactivity.MethodsFemale, virgin, Sprague-Dawley rats (8–12 weeks) were exposed to 100 μg of nano-TiO2 via intratracheal instillation 24 h prior to microvascular assessments. Serial blood samples were obtained at 0, 1, 2 and 4 h post-exposure for multiplex cytokine analysis. ILC2 numbers in the lungs were determined. ILC2s were isolated and phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) levels were measured. Pressure myography was used to assess vascular reactivity of isolated radial arterioles.ResultsPulmonary nano-TiO2 exposure was associated with an increase in IL-1ß, 4, 5 and 13 and TNF- α 4 h post-exposure, indicative of an innate Th2 inflammatory response. ILC2 numbers were significantly increased in lungs from exposed animals (1.66 ± 0.19%) compared to controls (0.19 ± 0.22%). Phosphorylation of the transactivation domain (Ser-468) of NF-κB in isolated ILC2 and IL-33 in lung epithelial cells were significantly increased (126.8 ± 4.3% and 137 ± 11% of controls respectively) by nano-TiO2 exposure. Lastly, radial endothelium-dependent arteriolar reactivity was significantly impaired (27 ± 12%), while endothelium-independent dilation (7 ± 14%) and α-adrenergic sensitivity (8 ± 2%) were not altered compared to control levels. Treatment with an anti- IL-33 antibody (1 mg/kg) 30 min prior to nano-TiO2 exposure resulted in a significant improvement in endothelium-dependent dilation and a decreased level of IL-33 in both plasma and bronchoalveolar lavage fluid.ConclusionsThese results provide evidence that the uterine microvascular dysfunction that follows pulmonary ENM exposure may be initiated via activation of lung-resident ILC2 and subsequent systemic Th2-dependent inflammation.Electronic supplementary materialThe online version of this article (10.1186/s12989-018-0280-2) contains supplementary material, which is available to authorized users.

The placenta plays a critical role in nutrient-waste exchange between the maternal and fetal circulations, thus functioning as an interface that profoundly impacts fetal growth and development. The placenta has long been considered an asexual organ, but, due to its embryonic origin it shares the same sex as the fetus. Exposures to toxicant such as diesel exhaust, have been shown to result in sexually dimorphic outcomes like decreased placental mass in exposed females. Therefore, we hypothesize that maternal nano-TiO2 inhalation exposure during gestation alters placental hemodynamics in a sexually dimorphic manner. Pregnant Sprague-Dawley rats were exposed from gestational day 10–19 to nano-TiO2 aerosols (12.17 ± 1.69 mg/m3) or filtered air (sham-control). Dams were euthanized on GD20, and fetal tissue was collected based on fetal sex: whole placentas, placental junctional zone (JZ), and placental labyrinth zone (LZ). Fetal mass, placental mass, and placental zone percent areas were assessed for sex-based differences. Exposed fetal females were significantly smaller compared to their exposed male counterparts (2.65 ± 0.03 g vs 2.78 ± 0.04 g). Nano-TiO2 exposed fetal females had a significantly decreased percent junctional zone area compared to the sham-control females (24.37 ± 1.30% vs 30.39 ± 1.54%). The percent labyrinth zone area was significantly increased for nano-TiO2 females compared to sham-control females (75.63 ± 1.30% vs 69.61 ± 1.54%). Placental flow and hemodynamics were assessed with a variety of vasoactive substances. It was found that nano-TiO2 exposed fetal females only had a significant decrease in outflow pressure in the presence of the thromboxane (TXA2) mimetic, U46619, compared to sham-control fetal females (3.97 ± 1.30 mm Hg vs 9.10 ± 1.07 mm Hg) and nano-TiO2 fetal males (9.96 ± 0.66 mm Hg). Maternal nano-TiO2 inhalation exposure has a greater effect on fetal female mass, placental zone mass and area, and adversely impacts placental vasoreactivity. This may influence the female growth and development later in life, future studies need to further study the impact of maternal nano-TiO2 inhalation exposure on zone specific mechanisms.