Nano-titanium dioxide inhalation exposure during gestation drives redox dysregulation and vascular dysfunction across generations.

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.

Using nitrous acid-modified de Man, Rogosa, and Sharpe medium to selectively isolate and culture lactic acid bacteria from dairy foods.

Nitrous acid was used to modify traditional de Man, Rogosa, Sharpe medium to evaluate whether the addition of sodium nitrite to MRS medium could improve the rate of growth and density of various lactic acid bacteria and nontarget species. Yogurt and Cheddar cheese were inoculated with individual bacterial species followed by the recovery and enumeration of the species using the pour plate method to compare the sensitivity between nitrous acid-modified MRS (mMRS) and traditional MRS. Lactobacillus delbrueckii ssp. bulgaricus were recovered at significantly higher counts from cheese in nitrous acid mMRS than MRS, whereas no significant difference was observed for other species and food systems. Growth curves were also generated for multiple lactic acid bacteria as well as nonstarters in both mMRS and MRS to measure the selectivity of nitrous acid mMRS. The selectivity evaluation of nitrous acid mMRS demonstrated that 5 of the tested lactic acid bacterial species (Bifidobacterium longum, Streptococcus salivarius, Lactococcus lactis, Lactobacillus acidophilus, and Lactobacillus delbrueckii ssp. bulgaricus) grew to significantly higher densities more rapidly in mMRS broth than in traditional MRS. Nontarget bacteria Enterococcus faecalis and Bacillus cereus revealed a more prolific growth rate and higher optical density readings in traditional MRS compared with mMRS. It was determined that nitrous acid mMRS is a viable alternative medium for culturing selected lactic acid bacteria, and offers an improved formulation of MRS for use in standard evaluation methods and optimization of probiotic and other dairy cultures.

Maternal nano-titanium dioxide inhalation exposure alters placental cyclooxygenase and oxidant balance in a sexually dimorphic manner.

The placenta plays a critical role in nutrient-waste exchange between the maternal and fetal circulation, and thus impacts fetal growth and development. We have previously shown that nano-titanium dioxide (nano-TiO2) inhalation exposure during gestation decreased fetal female pup and placenta mass [1], which persists in the following generation [2]. In utero exposed females, once mated, their offspring's placentas had increased capacity for H2O2 production. Generation of oxidants such as hydrogen peroxide (H2O2), have been shown to impact cyclooxygenase activity, specifically metabolites such as prostacyclin (PGI2) or thromboxane (TXA2). Therefore, we hypothesized that maternal nano-TiO2 inhalation exposure during gestation results in alterations in placental production of prostacyclin and thromboxane mediated by enhanced H2O2 production in a sexually dimorphic manner. Pregnant Sprague-Dawley rats were exposed to nano-TiO2 aerosols or filtered air (sham--control) from gestational day (GD) 10-19. Dams were euthanized on GD 20, and fetal serum and placental tissue were collected based on fetal sex. Fetal placental zones (junctional zone (JZ) and labyrinth zone (LZ)) were assessed for xanthine oxidoreductase (XOR) activity, H2O2, and catalase activity, as well as 6-keto-PGF1α and TXB2 levels. Nano-TiO2 exposed fetal female LZ demonstrated significantly greater XOR activity compared to exposed males. Exposed fetal female LZ also demonstrated significantly diminished catalase activity compared to sham-control females. Exposed fetal female LZ had significantly increased abundance of 6-keto-PGF1α compared to sham-control females and increased TXB2 compared to exposed males. In the aggregate these data indicate that maternal nano-TiO2 inhalation exposure has a greater impact on redox homeostasis and PGI2/TXA2 balance in the fetal female LZ. Future studies need to address if treatment with an XO inhibitor during gestation can prevent diminished fetal female growth during maternal nano-TiO2 inhalation exposure.

Distinct profiles of oxylipid mediators in liver, lung, and placenta after maternal nano-TiO2 nanoparticle inhalation exposure.

Nano-titanium dioxide (nano-TiO2) is a widely used nanomaterial found in several industrial and consumer products, including surface coatings, paints, sunscreens and cosmetics, among others. Studies have linked gestational exposure to nano-TiO2 with negative maternal and fetal health outcomes. For example, maternal pulmonary exposure to nano-TiO2 during gestation has been associated not only with maternal, but also fetal microvascular dysfunction in a rat model. One mediator of this altered vascular reactivity and inflammation is oxylipid signaling. Oxylipids are formed from dietary lipids through several enzyme-controlled pathways as well as through oxidation by reactive oxygen species. Oxylipids have been linked to control of vascular tone, inflammation, pain and other physiological and disease processes. In this study, we use a sensitive UPLC-MS/MS based analysis to probe the global oxylipid response in liver, lung, and placenta of pregnant rats exposed to nano-TiO2 aerosols. Each organ presented distinct patterns in oxylipid signaling, as assessed by principal component and hierarchical clustering heatmap analysis. In general, pro-inflammatory mediators, such as 5-hydroxyeicosatetraenoic acid (1.6 fold change) were elevated in the liver, while in the lung, anti-inflammatory and pro-resolving mediators such as 17-hydroxy docosahexaenoic acid (1.4 fold change) were elevated. In the placenta the levels of oxylipid mediators were generally decreased, both inflammatory (e.g. PGE2, 0.52 fold change) and anti-inflammatory (e.g. Leukotriene B4, 0.49 fold change). This study, the first to quantitate the levels of these oxylipids simultaneously after nano-TiO2 exposure, shows the complex interplay of pro- and anti-inflammatory mediators from multiple lipid classes and highlights the limitations of monitoring the levels of oxylipid mediators in isolation.

Maternal nano-titanium dioxide inhalation alters fetoplacental outcomes in a sexually dimorphic manner.

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.

Nanomaterial Inhalation During Pregnancy Alters Systemic Vascular Function in a Cyclooxygenase-Dependent Manner.

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 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 10-19 to nano-TiO2 aerosols (12.17 ± 1.67 mg/m3) or filtered air (sham-control). Dams were euthanized on gestational day 20, 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 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 with 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 with 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 with sham-control males (3.633 ± 0.064 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.

Maternal Nanomaterial Inhalation Exposure: Critical Gestational Period in the Uterine Microcirculation is Angiotensin II Dependent.

Maternal inhalation exposure to engineered nanomaterials (ENM) has been associated with microvascular dysfunction and adverse cardiovascular responses. Pregnancy requires coordinated vascular adaptation and growth that are imperative for survival. Key events in pregnancy hallmark distinct periods of gestation such as implantation, spiral artery remodeling, placentation, and trophoblast invasion. Angiotensin II (Ang II) is a critical vasoactive mediator responsible for adaptations and is implicated in the pathology of preeclampsia. If perturbations occur during gestation, such as those caused by ENM inhalation exposure, then maternal-fetal health consequences may occur. Our study aimed to identify the period of gestation in which maternal microvascular functional and fetal health are most vulnerable. Additionally, we wanted to determine if Ang II sensitivity and receptor density is altered due to exposure. Dams were exposed to ENM aerosols (nano-titanium dioxide) during three gestational windows: early (EE, gestational day (GD) 2-6), mid (ME, GD 8-12) or late (LE, GD 15-19). Within the EE group dry pup mass decreased by 16.3% and uterine radial artery wall to lumen ratio (WLR) increased by 25.9%. Uterine radial artery response to Ang II sensitivity increased by 40.5% in the EE group. Ang II receptor density was altered in the EE and LE group with decreased levels of AT2R. We conclude that early gestational maternal inhalation exposures resulted in altered vascular anatomy and physiology. Exposure during this time-period results in altered vascular reactivity and changes to uterine radial artery WLR, leading to decreased perfusion to the fetus and resulting in lower pup mass.