Objective-The continued development and use of engineered nanomaterials (ENM) has given rise to concerns over the potential for human health effects. While the understanding of cardiovascular ENM toxicity is improving, one of the most complex and acutely demanding "special" circulations is the enhanced maternal system to support fetal development. The "Barker Hypothesis" proposes that fetal development within a hostile gestational environment may predispose/program future sensitivity. Therefore, the objective of this study was two-fold: 1) to determine if maternal ENM exposure alters uterine and/or fetal microvascular function and 2) test the Barker Hypothesis at the microvascular level. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.The author(s) report(s) no conflict of interest.An abstract has been accepted to present this work at the 52 nd Annual Meeting of the Society of Toxicology, San Antonio TX, to be held March 10-14, 2013. NIH Public Access Author ManuscriptAm J Obstet Gynecol. Author manuscript; available in PMC 2014 September 01. NIH-PA Author ManuscriptStudy Design-Pregnant (gestation day 10) Sprague-Dawley rats were exposed to nanotitanium dioxide aerosols (11.3±0.039 (mg/m 3 )*hour, 5 hours/day, 8.2±0.85 days) to evaluate the maternal and fetal microvascular consequences of maternal exposure. Microvascular tissue isolation (gestation day 20) and arteriolar reactivity studies (<150μm passive diameter) of the uterine premyometrial and fetal tail arteries were conducted.Results-ENM exposures led to significant maternal and fetal microvascular dysfunction which presented as robustly compromised endothelium-dependent and -independent reactivity to pharmacologic and mechanical stimuli. Isolated maternal uterine arteriolar reactivity was consistent with a metabolically impaired profile and hostile gestational environment, impacting fetal weight. The fetal microvessels isolated from exposed dams demonstrate significant impairments to signals of vasodilation specific to mechanistic signaling and shear stress.Conclusion-To our knowledge, this is the first report providing evidence that maternal ENM inhalation is capable of influencing fetal health, thereby supporting that the Barker Hypothesis is applicable at the microvascular level.

1 Acetylcholine (ACh) elicits an endothelium-dependent relaxation and hyperpolarization in the absence of nitric oxide (NO) and prostaglandin synthesis in the guinea-pig coronary artery (GPCA). This response has been attributed to a factor termed endothelial-derived hyperpolarizing factor (EDHF). Recently it has been suggested that EDHF may be a cytochrome P450 product of arachidonic acid (AA) i.e., an epoxyeicosatrienoic acid (EET). The present study investigated whether this pathway could account for the response to ACh observed in the GPCA in the presence of 100 mM N o -nitro-L-arginine and 10 mM indomethacin. 2 ACh, AA and 11,12-EET each produced concentration-dependent relaxations in arteries contracted with the H 1 -receptor agonist AEP (2,2-aminoethylpyridine). The AA-induced relaxation was signi®cantly enhanced in the presence of the cyclo-oxygenase/lipoxygenase inhibitor, eicosatetranynoic acid (30 mM). 3 The cytochrome P450 inhibitors proadifen (10 mM) and clotrimazole (10 mM) inhibited ACh, lemakalim (LEM) and AA-induced relaxation, whereas 17-octadecynoic acid (100 mM) and 7-ethoxyresoru®n (10 mM) were without eect on all three vasodilators. Proadifen and clotrimazole also inhibited ACh (1 mM) and LEM (1 mM)-induced hyperpolarization. 4 The ability of various potassium channel blockers to inhibit relaxation responses elicited with ACh, AA and 11,12-EET was also determined. Iberiotoxin (IBTX; 100 nM) was without eect on responses to ACh but signi®cantly reduced responses to both AA and 11,12-EET. In contrast, 4-aminopyridine (4-AP; 5 mM) signi®cantly reduced response to ACh but not responses to AA and 11,12-EET. Combined IBTX plus (4-AP) inhibited the ACh-induced relaxation to a greater extent than 4-AP alone. Apamin (1 mM), glibenclamide (10 mM) and BaCl 2 (50 mM) had no signi®cant eect on responses to ACh, AA and 11,12-EET. 5 IBTX (100 nM) signi®cantly reduced both 11,12-EET (33 mM) and AA (30 mM) hyperpolarization without aecting the ACh (1 mM)-induced hyperpolarization. In contrast, 4-AP signi®cantly reduced the ACh-induced hyperpolarization without aecting either AA or 11,12-EET-induced hyperpolarizations. 6 In summary, our results suggest that the coronary endothelium releases a factor upon application of AA which hyperpolarizes the smooth muscle. The similarity of pharmacology between AA and 11,12-EET suggests that this factor is an EET. However, the disparity of pharmacology between responses to ACh versus responses to 11,12-EET do not support the hypothesis that EETs represent the predominant factor which ACh releases from the endothelium that leads to NO-and prostaglandin-independent hyperpolarization and relaxation in the GPCA.

Nanomaterial production is expanding as new industrial and consumer applications are introduced. Nevertheless, the impacts of exposure to these compounds are not fully realized. The present study was designed to determine whether gestational nano-sized titanium dioxide exposure impacts cardiac and metabolic function of developing progeny. Pregnant Sprague-Dawley rats were exposed to nano-aerosols (~10 mg/m, 130- to 150-nm count median aerodynamic diameter) for 7-8 nonconsecutive days, beginning at gestational Physiological and bioenergetic effects on heart function and cardiomyocytes across three time points, fetal (gestational), neonatal (4-10 days), and young adult (6-12 wk), were evaluated. Functional analysis utilizing echocardiography, speckle-tracking based strain, and cardiomyocyte contractility, coupled with mitochondrial energetics, revealed effects of nano-exposure. Maternal exposed progeny demonstrated a decrease in E- and A-wave velocities, with a 15% higher E-to-A ratio than controls. Myocytes isolated from exposed animals exhibited ~30% decrease in total contractility, departure velocity, and area of contraction. Bioenergetic analysis revealed a significant increase in proton leak across all ages, accompanied by decreases in metabolic function, including basal respiration, maximal respiration, and spare capacity. Finally, electron transport chain complex I and IV activities were negatively impacted in the exposed group, which may be linked to a metabolic shift. Molecular data suggest that an increase in fatty acid metabolism, uncoupling, and cellular stress proteins may be associated with functional deficits of the heart. In conclusion, gestational nano-exposure significantly impairs the functional capabilities of the heart through cardiomyocyte impairment, which is associated with mitochondrial dysfunction. Cardiac function is evaluated, for the first time, in progeny following maternal nanomaterial inhalation. The findings indicate that exposure to nano-sized titanium dioxide (nano-TiO) during gestation negatively impacts cardiac function and mitochondrial respiration and bioenergetics. We conclude that maternal nano-TiO inhalation contributes to adverse cardiovascular health effects, lasting into adulthood.

Fused deposition modeling (FDM™), or three-dimensional (3D) printing has become routine in industrial, occupational and domestic environments. We have recently reported that 3D printing emissions (3DPE) are complex mixtures, with a large ultrafine particulate matter component. We and others have reported that inhalation of xenobiotic particles in this size range is associated with an array of cardiovascular dysfunctions. Sprague-Dawley rats were exposed to 3DPE aerosols via nose-only exposure for ~3 hours. Twenty-four hours later, intravital microscopy was performed to assess microvascular function in the spinotrapezius muscle. Endothelium-dependent and -independent arteriolar dilation were stimulated by local microiontophoresis of acetylcholine (ACh) and sodium nitroprusside (SNP). At the time of experiments, animals exposed to 3DPE inhalation presented with a mean arterial pressure of 125±4 mm Hg, and this was significantly higher than that for the sham-control group (94±3 mm Hg). Consistent with this pressor response in the 3DPE group, was an elevation of ~12% in resting arteriolar tone. Endothelium-dependent arteriolar dilation was significantly impaired after 3DPE inhalation across all iontophoretic ejection currents (0–27±15%, compared to sham-control: 15–120±21%). Endothelium-independent dilation was not affected by 3DPE inhalation. These alterations in peripheral microvascular resistance and reactivity are consistent with elevations in arterial pressure that follow 3DPE inhalation. Future studies must identify the specific toxicants generated by FDM™ that drive this acute pressor response.

Due to the ongoing evolution of nanotechnology, there is a growing need to assess the toxicological outcomes in under-studied populations in order to properly consider the potential of engineered nanomaterials (ENM) and fully enhance their safety. Recently, we and others have explored the vascular consequences associated with gestational nanomaterial exposure, reporting microvascular dysfunction within the uterine circulation of pregnant dams and the tail artery of fetal pups. It has been proposed (via work derived by the Barker Hypothesis) that mitochondrial dysfunction and subsequent oxidative stress mechanisms as a possible link between a hostile gestational environment and adult disease. Therefore, in this study, we exposed pregnant Sprague-Dawley rats to nanosized titanium dioxide aerosols after implantation (gestational day 6). Pups were delivered, and the progeny grew into adulthood. Microvascular reactivity, mitochondrial respiration and hydrogen peroxide production of the coronary and uterine circulations of the female offspring were evaluated. While there were no significant differences within the maternal or litter characteristics, endothelium-dependent dilation and active mechanotransduction in both coronary and uterine arterioles were significantly impaired. In addition, there was a significant reduction in maximal mitochondrial respiration (state 3) in the left ventricle and uterus. These studies demonstrate microvascular dysfunction and coincide with mitochondrial inefficiencies in both the cardiac and uterine tissues, which may represent initial evidence that prenatal ENM exposure produces microvascular impairments that persist throughout multiple developmental stages.