Pregnancy requires metabolic and endocrine changes that must occur to support fetal growth and development. Aberrations in these necessary modifications can impact maternal health and fetal growth. Exposure to toxicants during pregnancy can negatively affect fetal health and development, but studies on electronic cigarette (e-cig) exposure is limited. We hypothesized that maternal e-cig exposure during gestation leads to hormonal and redox imbalance and negatively impacts fetal development. Pregnant Sprague–Dawley rats were exposed to e-cig aerosols (1227 ± 131 mg/m3) or HEPA-filtered air for 90 min from gestational day (GD) 10–19 for a total of 6 days. Dams were euthanized on GD 20 and dam serum, liver, lung, ovaries, and placental tissue were collected for analysis. Fetal mass, placental mass, and sex ratios were assessed. Fetal and placental mass were significantly decreased in e-cig exposed compared to sham-control (2.61 ± 0.19 g vs. 3.37 ± 0.09 g and 0.62 ± 0.03 g vs. 0.70 ± 0.02 g, respectively). Placental xanthine oxidase (XO) activity was significantly increased in e-cig exposed compared to sham-control (5.29 ± 0.27 µU/mL vs. 4.28 ± 0.36 µU/mL). Circulating prolactin (PRL) levels of e-cig exposed dams were significantly decreased compared to sham-control (2.40 ± 0.06 ng/mL of plasma vs. 3.83 ± 0.64 ng/mL of plasma). Maternal e-cig inhalation exposure during gestation negatively impacted fetal growth, increased placental XO activity, and decreased circulating PRL levels. These data demonstrate two potential mechanisms that could lead to the observed reduction in fetal growth following maternal exposure: potential redox imbalance within the placenta and/or hormonal imbalance directly affects fetal growth and potentially influences growth later in life.

Alarmins are classified by their release from damaged or ruptured cells. Many alarmins have been found to increase vascular tone and oppose endothelium-dependent dilatation (EDD). Interleukin (IL)-33 plays a prominent role in lung injury and can be released during vascular injury and in chronic studies found to be cardioprotective. Our recent work has implicated IL-33 in acute vascular dysfunction following inhalation of engineered nanomaterials (ENM). However, the mechanisms linking IL-33 to vascular tone have not been interrogated. We therefore aimed to determine whether IL-33 directly influenced microvascular tone and endothelial function. Isolated feed arteries and in vivo arterioles from male and female Sprague-Dawley rats were used to determine direct vascular actions of IL-33. Mesenteric feed arteries and arterioles demonstrated reduced intraluminal diameters when treated with increasing concentrations of recombinant IL-33. IL-33 activated extracellular signal regulated kinase (ERK)1/2 of rat aortic smooth muscle cells but not phosphorylation of myosin light chain kinase. This suggested IL-33 may sensitize arterioles to Ca2+-mediated responses. Indeed, IL-33 augmented the myogenic- and phenylephrine-induced vasoconstriction. Additionally, incubation of arterioles with 1 ng IL-33 attenuated ACh-mediated EDD. Mechanistically, in human aortic endothelial cells, we demonstrate that IL-33-mediated ERK1/2 activation leads to inhibitory phosphorylation of serine 602 on endothelial nitric oxide synthase. Finally, we demonstrate that IL-33-ERK1/2 contributes to vascular tone following two known inducers of IL-33; ENM inhalation and the rupture endothelial cells. The present study provides novel evidence that IL-33 increases vascular tone via canonical ERK1/2 activation in microvascular smooth muscle and endothelium. Altogether, it is suggested IL-33 plays a critical role in microvascular homeostasis following barrier cell injury. KEY POINTS: Interleukin (IL)-33 causes a concentration-dependent reduction in feed artery diameter. IL-33 acts on vascular smooth muscle cells to augment Ca2+-mediated processes. IL-33 causes inhibitory phosphorylation of endothelial nitric oxide synthase and opposes endothelium-dependent dilatation. Engineered nanomaterial-induced lung injury and endothelial cell rupture in part act through IL-33 to mediate increased vascular tone.

Nano-titanium dioxide (nano-TiO2) is an engineered nanomaterial (ENM), which is widely utilized in diverse products like surface coatings, cosmetics, medical devices, and filters. Inhaled nano-TiO2 has been demonstrated to induce pulmonary inflammation, impair microvascular function, and hinder generational reproductive endpoints. These outcomes indicate why it is important to explore gestation, as it requires timely vascular adaptations to support maternal and fetal health and development. Therefore, the central hypothesis of my dissertation is: maternal nano-TiO2 inhalation exposure will impact maternal microvascular function and fetoplacental hemodynamics and function 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.

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.

Cardiovascular toxicology is the study of chemicals that cause untoward effects on the heart or vasculature. This is an ongoing course of study because the toxicants that humans are exposed to is constantly changing, however many of the mechanisms of toxicity remain largely unchanged over hundreds of years. Therefore, this chapter introduces the concept of cardiovascular homeostasis, and how fundamental anatomical and physiological elements make this possible. Lists of common toxicants that target cardiac and vascular mechanisms, and examples of how such agents alter cardiovascular health are discussed. Upon completion of this chapter, the reader should have a general understanding of how certain toxicants influence cardiovascular health, and to distinguish between therapeutic dose/benefit vs untoward outcome.

Abstract Disclosure: E. Bowdridge: None. A. Dunn: None. J. Griffith: None. E. DeVallance: None. E. Kelley: None. S. Lewis: None. Maternal inhalation exposure to nano-TiO2 during gestation impacts litter size, pup and placental mass, circulating estrogen concentration, and uterine microvascular reactivity. In addition, we have recently shown that maternal inhalation of nano-TiO2 during gestation results in redox imbalance in dams during late gestation. However, the mechanism linking these dysfunctions with exposure has yet to be explored. Therefore, we hypothesized that elevated xanthine oxidoreductase (XOR), a critical source of oxidants in numerous inflammatory processes, is at least partially responsible for the increased oxidant production observed. The objective of this study was to assess if treatment with a XOR inhibitor, febuxostat (Uloric®), prevents the poor microvascular, reproductive, and endocrine outcomes induced by gestational nano-TiO2 exposure. Female Sprague Dawley rats, 6-8 weeks of age, received febuxostat treated water (50 mg/L) one week prior to being mated in-house and throughout gestation until sacrifice on gestational day (GD) 20. Once pregnant, dams were randomly assigned to either sham-control (N = 6) or nano-TiO2 (N = 6) groups. Dams were exposed (nano-TiO2 concentration = 12 mg/m3; HEPA-filtered air 25 ml/min) for 6 hrs/d for 6 d between GD 10-19. Dam and litter characteristics as well as placental and fetal weights were recorded on GD 20. Dam blood samples were obtained at the time of sacrifice to determine xanthine oxidase and estrogen concentrations. No significant differences were observed between sham-control and nano-TiO2 groups for litter size (8.8±2.1 versus 9.8±2.1), or fetal mass (3.9±0.1 g versus 4.0±0.1 g). However, placental mass (0.72±0.03 g versus 0.62±0.02 g) was significantly reduced in the nano-TiO2 group. There was no significant difference in circulating estrogen between control rats given febuxostat water (29.8±2.70 pg) and nano-TiO2 rats given febuxostat water (36.7±1.29 pg). However, nano-TiO2 rats treated with febuxostat water saw a significant increase in estrogen levels (36.7±1.29 pg) compared to nano-TiO2 rats without the febuxostat water (19.9±2.39 pg). Additionally, uterine arteries were isolated, and reactivity was assessed ex vivo. Uterine arteries from exposed females treated with febuxostat showed similar vasoconstriction to kisspeptin (97.6%±1.95) as control females given febuxostat (99.8%±0.61). Additionally, there was no difference in dam liver mass (11.5% ± 0.80 g control versus 13.9% ± 0.60 g nano-TiO2), which we have previously shown to be increased due to nano-TiO2 exposure. Taken together, these observations indicate that reproductive, endocrine, liver, and microvascular functions are protected, at least in part, from nano-TiO2 inhalation exposure induced dysfunction in pregnant dams by XOR inhibition. Presentation: Saturday, June 17, 2023

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-TiO 2 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-TiO 2 aerosols (12.17 ± 1.69 mg/m 3 ) 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-TiO 2 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-TiO 2 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-TiO 2 exposed fetal females only had a significant decrease in outflow pressure in the presence of the thromboxane (TXA 2 ) mimetic, U46619, compared to sham-control fetal females (3.97 ± 1.30 mm Hg vs 9.10 ± 1.07 mm Hg) and nano-TiO 2 fetal males (9.96 ± 0.66 mm Hg). Maternal nano-TiO 2 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-TiO 2 inhalation exposure on zone specific mechanisms.

Abstract 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.

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.

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 (TXA(2)) mimetics. However, the mechanisms underpinning this process are unknown. Therefore, we hypothesize that maternal nano-TiO2 inhalation during gestation results in uterine microvascular prostacyclin (PGI(2)) and TXA(2) dysfunction. Pregnant Sprague-Dawley rats were exposed from gestational day 10-19 to nano-TiO2 aerosols (12.17 +/- 1.67 mg/m(3)) 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 PGI(2) and TXA(2) metabolites. TXB2, the stable TXA(2) 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-PGF(1 alpha), the stable PGI(2) metabolite. Radial arteriole pressure myography revealed that nano-TiO2 exposure caused increased vasoconstriction to the TXA(2) 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 PGI(2) 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 TXA(2) vasoconstriction and decreased PGI(2) vasodilation may lead to decreased placental blood flow and compromise maternofetal exchange of waste and nutrients, which could ultimately impact fetal health outcomes.

Objective Chronic multisymptom illness (CMI) is an idiopathic disease affecting thousands of U.S. Veterans exposed to open-air burn pits emitting aerosolized particulate matter (PM) while serving in Central and Southwest Asia and Africa. Exposure to burn pit PM can result in profound biologic consequences including chronic fatigue, impaired cognition, and respiratory diseases. Dysregulated or unresolved inflammation is a possible underlying mechanism for CMI onset. We describe a rat model of whole-body inhalation exposure using carbon black nanoparticles (CB) as a surrogate for military burn pit-related exposure. Using this model, we measured biomarkers of inflammation in multiple tissues. Results Male Sprague Dawley rats were exposed to CB aerosols by whole body inhalation (6 ± 0.83 mg/m 3 ). Proinflammatory biomarkers were measured in multiple tissues including arteries, brain, lung, and plasma. Biomarkers of cardiovascular injury were also assayed in plasma. CB inhalation exposure increased CMI-related proinflammatory biomarkers such as IFN-γ and TNFα in multiple tissue samples. CB exposure also induced cardiovascular injury markers (adiponectin, MCP1, sE-Selectin, sICam-1 and TIMP1) in plasma. These findings support the validity of our animal exposure model for studies of burn pit-induced CMI. Future studies will model more complex toxicant mixtures as documented at multiple burn pit sites.

Abstract 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 maternal nano-TiO2 inhalation exposure resulted in altered H2O2 production capacity and changes in downstream redox pathways. 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 would result in upregulated H2O2 in the F0 dams as well as her F1 offspring, resulting 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 2-fold 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 (29.81 ± 8.8 pg/ml vs. 12.12 ± 3.1 pg/ml sham-control) and vascular dysfunction similar to their directly exposed mothers. F1-nano-TiO2 uterine artery H2O2 production capacity was also elevated 2-fold. Dysfunctional gestational outcomes in the F1-nano-TiO2 dams resulted in smaller F2 pups (4.93 ± 0.47 g vs. 5.78 ± 0.09 g sham-control pups). Conclusion In conclusion, this manuscript provides critical evidence of redox dysregulation across generations following maternal ENM inhalation. Furthermore, the dysfunctional gestational outcomes observed in the F1-nano-TiO2 generation 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.

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.