Carroll R. McBride

Microvascular and mitochondrial dysfunction in the female F1 generation after gestational TiO2 nanoparticle exposure.

Abstract 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.

A New Ion Mobility–Linear Ion Trap Instrument for Complex Mixture Analysis

A new instrument that couples a low-pressure drift tube with a linear ion trap mass spectrometer is demonstrated for complex mixture analysis. The combination of the low-pressure separation with the ion trapping capabilities provides several benefits for complex mixture analysis. These include high sensitivity, unique ion fragmentation capabilities, and high reproducibility. Even though the gas-phase separation and the mass measurement steps are each conducted in an ion filtering mode, detection limits for mobility-selected peptide ions are in the tens of attomole range. In addition to ion separation, the low-pressure drift tube can be used as an ion fragmentation cell yielding mobility-resolved fragment ions that can be subsequently analyzed by multistage tandem mass spectrometry (MS(n)) methods in the ion trap. Because of the ion trap configuration, these methods can be comprised of any number (limited by ion signal) of collision-induced dissociation (CID) and electron transfer dissociation (ETD) processes. The high reproducibility of the gas-phase separation allows for comparison of two-dimensional ion mobility spectrometry (IMS)-MS data sets in a pixel-by-pixel fashion without the need for data set alignment. These advantages are presented in model analyses representing mixtures encountered in proteomics and metabolomics experiments.

Whole-body nanoparticle aerosol inhalation exposures.

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle inhalation toxicology studies, the aerosols in a chamber housing the experimental animals must have: 1) a steady concentration maintained at a desired level for the entire exposure period; 2) a homogenous composition free of contaminants; and 3) a stable size distribution with a geometric mean diameter < 200 nm and a geometric standard deviation σg < 2.5 (5). The generation of aerosols containing nanoparticles is quite challenging because nanoparticles easily agglomerate. This is largely due to very strong inter-particle forces and the formation of large fractal structures in tens or hundreds of microns in size (6), which are difficult to be broken up. Several common aerosol generators, including nebulizers, fluidized beds, Venturi aspirators and the Wright dust feed, were tested; however, none were able to produce nanoparticle aerosols which satisfy all criteria (5). A whole-body nanoparticle aerosol inhalation exposure system was fabricated, validated and utilized for nano-TiO2 inhalation toxicology studies. Critical components: 1) novel nano-TiO2 aerosol generator; 2) 0.5 m(3) whole-body inhalation exposure chamber; and 3) monitor and control system. Nano-TiO2 aerosols generated from bulk dry nano-TiO2 powders (primary diameter of 21 nm, bulk density of 3.8 g/cm(3)) were delivered into the exposure chamber at a flow rate of 90 LPM (10.8 air changes/hr). Particle size distribution and mass concentration profiles were measured continuously with a scanning mobility particle sizer (SMPS), and an electric low pressure impactor (ELPI). The aerosol mass concentration (C) was verified gravimetrically (mg/m(3)). The mass (M) of the collected particles was determined as M = (Mpost-Mpre), where Mpre and Mpost are masses of the filter before and after sampling (mg). The mass concentration was calculated as C = M/(Q*t), where Q is sampling flowrate (m(3)/min), and t is the sampling time (minute). The chamber pressure, temperature, relative humidity (RH), O2 and CO2 concentrations were monitored and controlled continuously. Nano-TiO2 aerosols collected on Nuclepore filters were analyzed with a scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis. In summary, we report that the nano-particle aerosols generated and delivered to our exposure chamber have: 1) steady mass concentration; 2) homogenous composition free of contaminants; 3) stable particle size distributions with a count-median aerodynamic diameter of 157 nm during aerosol generation. This system reliably and repeatedly creates test atmospheres that simulate occupational, environmental or domestic ENM aerosol exposures.

Maternal Engineered Nanomaterial Exposure Disrupts Progeny Cardiac Function and Bioenergetics

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/m3, 130- to 150-nm count median aerodynamic diameter) for 7-8 nonconsecutive days, beginning at gestational day 5-6 Physiological and bioenergetic effects on heart function and cardiomyocytes across three time points, fetal (gestational day 20), 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.NEW & NOTEWORTHY 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-TiO2) during gestation negatively impacts cardiac function and mitochondrial respiration and bioenergetics. We conclude that maternal nano-TiO2 inhalation contributes to adverse cardiovascular health effects, lasting into adulthood.

Impacts of prenatal nanomaterial exposure on male adult Sprague-Dawley rat behavior and cognition.

ABSTRACT It is generally accepted that gestational xenobiotic exposures result in systemic consequences in the adult F1 generation. However, data on detailed behavioral and cognitive consequences remain limited. Using our whole-body nanoparticle inhalation facility, pregnant Sprague-Dawley rats (gestational day [GD] 7) were exposed 4 d/wk to either filtered air (control) or nano-titanium dioxide aerosols (nano-TiO2; count median aerodynamic diameter of 170.9 ± 6.4 nm, 10.4 ± 0.4 mg/m3, 5 h/d) for 7.8 ± 0.5 d of the remaining gestational period. All rats received their final exposure on GD 20 prior to delivery. The calculated daily maternal deposition was 13.9 ± 0.5 µg. Subsequently, at 5 mo of age, behavior and cognitive functions of these pups were evaluated employing a standard battery of locomotion, learning, and anxiety tests. These assessments revealed significant working impairments, especially under maximal mnemonic challenge, and possible deficits in initial motivation in male F1 adults. Evidence indicates that maternal engineered nanomaterial exposure during gestation produces psychological deficits that persist into adulthood in male rats.

Uterine microvascular sensitivity to nanomaterial inhalation: An in vivo assessment.

With the tremendous number and diverse applications of engineered nanomaterials incorporated in daily human activity, exposure can no longer be solely confined to occupational exposures of healthy male models. Cardiovascular and endothelial cell dysfunction have been established using in vitro and in situ preparations, but the translation to intact in vivo models is limited. Intravital microscopy has been used extensively to understand microvascular physiology while maintaining in vivo neurogenic, humoral, and myogenic control. However, a tissue specific model to assess the influences of nanomaterial exposure on female reproductive health has not been fully elucidated. Female Sprague Dawley (SD) rats were exposed to nano-TiO2 aerosols (171 ± 6 nm, 10.1 ± 0.39 mg/m(3), 5h) 24-hours prior to experimentation, leading to a calculated deposition of 42.0 ± 1.65 μg. After verifying estrus status, vital signs were monitored and the right horn of the uterus was exteriorized, gently secured over an optical pedestal, and enclosed in a warmed tissue bath using intravital microscopy techniques. After equilibration, significantly higher leukocyte-endothelium interactions were recorded in the exposed group. Arteriolar responsiveness was assessed using ionophoretically applied agents: muscarinic agonist acetylcholine (0.025 M; ACh; 20, 40, 100, and 200 nA), and nitric oxide donor sodium nitroprusside (0.05 M; SNP; 20, 40, and 100 nA), or adrenergic agonist phenylephrine (0.05 M; PE; 20, 40, and 100 nA) using glass micropipettes. Passive diameter was established by tissue superfusion with 10(-4)M adenosine. Similar to male counterparts, female SD rats present systemic microvascular dysfunction; however the ramifications associated with female health and reproduction have yet to be elucidated.

Comparative plasma proteomic studies of pulmonary TiO2 nanoparticle exposure in rats using liquid chromatography tandem mass spectrometry.

Mounting evidence suggests that pulmonary exposure to nanoparticles (NPs) has a toxic effect on biological systems. A number of studies have shown that exposure to NPs result in systemic inflammatory response, oxidative stress, and leukocyte adhesion. However, significant knowledge gaps exist for understanding the key molecular mechanisms responsible for altered microvasculature function. Utilizing comprehensive LC-MS/MS and comparative proteomic analysis strategies, important proteins related to TiO2 NP exposure in rat plasma have been identified. Molecular pathway analysis of these proteins revealed 13 canonical pathways as being significant (p ≤ 0.05), but none were found to be significantly up or down-regulated (z>|2|). This work lays the foundation for future research that will monitor relative changes in protein abundance in plasma and tissue as a function of post-exposure time and TiO2 NP dosage to further elucidate mechanisms of pathway activation as well as to decipher other affected pathways.

Heterogeneous Vascular Bed Responses to Pulmonary Titanium Dioxide Nanoparticle Exposure

A growing body of research links engineered nanomaterial (ENM) exposure to adverse cardiovascular endpoints. The purpose of this study was to evaluate the impact of ENM exposure on vascular reactivity in discrete segments so that we may determine the most sensitive levels of the vasculature where these negative cardiovascular effects are manifest. We hypothesized that acute nano-TiO2 exposure differentially affects reactivity with a more robust impairment in the microcirculation. Sprague-Dawley rats (8–10 weeks) were exposed to nano-TiO2 via intratracheal instillation (20, 100, or 200 µg suspended per 250 µL of vehicle) 24 h prior to vascular assessments. A serial assessment across distinct compartments of the vascular tree was then conducted. Wire myography was used to evaluate macrovascular active tension generation specifically in the thoracic aorta, the femoral artery, and third-order mesenteric arterioles. Pressure myography was used to determine vascular reactivity in fourth- and fifth-order mesenteric arterioles. Vessels were treated with phenylephrine, acetylcholine (ACh), and sodium nitroprusside. Nano-TiO2 exposure decreased endothelium-dependent relaxation in the thoracic aorta and femoral arteries assessed via ACh by 53.96 ± 11.6 and 25.08 ± 6.36%, respectively. Relaxation of third-order mesenteric arterioles was impaired by 100 and 20 µg nano-TiO2 exposures with mean reductions of 50.12 ± 8.7 and 68.28 ± 8.7%. Cholinergic reactivity of fourth- and fifth-order mesenteric arterioles was negatively affected by nano-TiO2 with diminished dilations of 82.86 ± 12.6% after exposure to 200 µg nano-TiO2, 42.6 ± 12.6% after 100 µg nano-TiO2, and 49.4 ± 12.6% after 20 µg nano-TiO2. Endothelium-independent relaxation was impaired in the thoracic aorta by 34.05 ± 25% induced by exposure to 200 µg nano-TiO2 and a reduction in response of 49.31 ± 25% caused by 100 µg nano-TiO2. Femoral artery response was reduced by 18 ± 5%, while third-order mesenteric arterioles were negatively affected by 20 µg nano-TiO2 with a mean decrease in response of 38.37 ± 10%. This is the first study to directly compare the differential effect of ENM exposure on discrete anatomical segments of the vascular tree. Pulmonary ENM exposure produced macrovascular and microvascular dysfunction resulting in impaired responses to endothelium-dependent, endothelium-independent, and adrenergic agonists with a more robust dysfunction at the microvascular level. These results provide additional evidence of an endothelium-dependent and endothelium-independent impairment in vascular reactivity.

Estrous cycle-dependent modulation of in vivo microvascular dysfunction after nanomaterial inhalation

Preconceptive health encompasses male and female reproductive capability. In females, this takes into account each of the stages of the estrous cycle. Microvascular reactivity varies throughout the estrous cycle in response to hormonal changes and in preparation for pregnancy. Microvascular alterations in response to engineered nanomaterial (ENM) exposure have been described within 24-h of inhalation; however, the impact upon the uterine vasculature at differing estrous stages and at late-stage pregnancy is unclear. Female Sprague Dawley (SD) rats (virgin and late stage pregnancy [GD 19]) were exposed to nano-TiO aerosols (173.2 ± 6.4 nm, 10.2 ± 0.46 mg/m3, 5 h) 24-h prior to experimentation leading to a single calculated deposition of 42.2 ± 1.9 µg nano- TiO2 (exposed) or 0µg (control). Animals were anesthetized, estrous status verified, and prepared for in situ assessment of leukocyte trafficking and vascular function by means of intravital microscopy, Uterine basal arteriolar reactivity was stimulated using iontophoretically applied chemicals: acetylcholine (ACh, 0.025 M; 20, 40, 100, 200 nA), sodium nitroprusside (SNP, 0.05 M; 20, 40, 100 nA), phenylephrine (PE, 0.05 M; 20, 40, 100 nA). Finally, adenosine (ADO, 10−4 M) was superfused over the tissue to identify maximum diameter. In situ vessel reactivity after exposure was significantly blunted based on estrous stage, but not at late-stage pregnancy. Local uterine venular leukocyte trafficking and systemic inflammatory markers were also significantly affected during preparatory (proestrus), fertile (estrus), and infertile (diestrus) periods after ENM inhalation. Overall, these deficits in reactivity and increased inflammatory activity may impair female fertility after ENM exposure.

Inhalation exposure to three-dimensional printer emissions stimulates acute hypertension and microvascular dysfunction

ABSTRACT 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. Additionally, 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 h. 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.