Animal Model: Female, Sprague-Dawley (SD rats) were purchased from Hilltop Laboratories (Scottdale, PA), and housed in an Assessment and Accreditation of Laboratory Animal Care (AAALAC) approved facility at West Virginia University (WVU) under a regulated temperature and 12:12 hour light-dark cycle. Rats were randomly assigned to either the sham-control or nano-TiO2 exposure groups and acclimated for 48-72-hours before mating. Rats had ad libitum access to food and water throughout the acclimation period. To increase the likelihood of viable progeny, pregnant rats were exposed to nano-TiO2 aerosols on or after implantation gestational day 10 (GD 10) as prior indications of inhalation exposure results in near to total loss of pregnancy. Weights of the pregnant dams were recorded weekly. Dams were allowed to deliver pups naturally. Pups were housed with dams for 21 days and weaned based on sex. Once female pups (F1 females) reached sexual maturity (6-8 weeks of age) they were mated to control males to establish pregnancy and euthanized on GD 20. All procedures were approved by the Institutional Animal Care and Use Committee of West Virginia University.
Engineered Nanomaterial: Nano-TiO2 powder was obtained from Evonik (Aeroxide TiO2, Parsippany, NJ). It is a mixture composed of anatase (80%) and rutile (20%) TiO2. Particle characteristics have been determined including the primary particle size (21 nm), the specific surface area (48.08 m /g), and the Zeta potential (−56.6 mV) .
Aerosol size distributions were determined in the exposure chamber while the target mass concentration was being maintained at 12 ± 0.13 mg/m3 with: 1) a high-resolution electrical low-pressure impactor (ELPI+; Dekati, Tampere, Finland), 2) a scanning particle mobility sizer (SMPS 3938; TSI Inc., St. Paul, MN), and 3) an aerodynamic particle sizer (APS 3321; TSI Inc., St. Paul, MN), and a Nano Micro-Orifice Uniform Deposit Impactor (MOUDI 115R, MSP Corp, Shoreview, MN).
Inhalation Exposure: Nano-TiO2 aerosols were generated using a high-pressure acoustical generator (HPAG, IEStechno, Morgantown, WV). The output of the generator was fed into a Venturi pump (JS-60M, Vaccon, Medway, MA) which further de-agglomerated the particles. The nano-TiO2 aerosol/air mix then entered the whole-body exposure chamber. A personal DataRAM (pDR-1500; Thermo Environmental Instruments Inc., Franklin, MA) was utilized to sample the exposure chamber air to determine the aerosol mass concentration in real-time. Feedback loops within the software automatically adjusted the acoustic energy to maintain a stable mass concentration during the exposure. Gravimetric measurements were conducted on Teflon filters concurrently with the DataRAM measurements to obtain a calibration factor. The gravimetric measurements were also conducted during each exposure to calculate the mass concentration measurements reported in the study. Bedding material soaked with water was used in the exposure chamber to maintain humidity (30-70%) during exposures. Sham-control animals were exposed to HEPA filtered air only with similar temperature and humidity chamber conditions.
Inhalation exposures in F0 dams lasted for 6 days after GD 10 to decrease animal stress. The pregnant rats were exposed to an average target concentration of 12 mg/m3. This concentration was chosen to match our previous late gestation inhalation exposure studies [10,64] To estimate lung dose with nano-TiO2 aerosols  we used the equation: D=F⋅V⋅C⋅T, where F is the deposition fraction (10%), V is the minute ventilation (208.3 cc), C equals the mass concentration (mg/m3), and T equals the exposure duration (minutes) . This exposure paradigm (12 mg/m3, 6 h/exposure, 6 days) produced an estimated target lung dose of 525 ± 16 µg with the last exposure conducted 24 h prior to sacrifice and experimentation. These calculations represent total lung deposition and do not account for clearance (MPPD Software v 2.11, Arlington, VA).
Enzyme linked immunosorbent assay (ELISA): F1 Dams were anesthetized with isoflurane gas (5% induction, 2–3.5% maintenance). The animals were placed on a heating pad to maintain a 37 °C rectal temperature. The trachea was intubated to ensure an open airway and the right carotid artery was cannulated to allow for blood sampling of ~3 mL. Plasma and serum were collected on GD 20 and ELISAs were performed for estrogen, progesterone (P4), luteinizing hormone (LH), and follicle stimulating hormone (FSH). All kits were performed according to the recommendation of the manufacturer (CalBiotech, Spring Valley, CA). Additionally, plasma was collected at 8 weeks of age in F1 males and females was assayed for IL-6 (R&D Systems, Minneapolis, MN), glucose (Cayman, Ann Arbor, MI), and plasma protein carbonyls (Abcam, Cambridge, MA).
Gestational Outcomes in F0 and F1 Dams
Liter size, sex ratio, implantation sites, and uterine weights were recorded in F0 and F1 dams. Once trunk blood was collected from F1 dams, F2 pups and placenta were carefully dissected away from the uterine wall and weighed individually immediately after sacrifice (wet weight) and after desiccation (dry weight). F2 pup and placental weights were measured to calculate placental efficiency (grams fetus/gram placental).
AML12 hepatocytes were grown in DMEM/F12 (ThermoFisher Scientific, Waltham, MA) media supplemented with dexamethasone, (ThermoFisher Scientific, Waltham, MA) growth supplement (ThermoFisher Scientific, Waltham, MA), and 10% FBS (ThermoFisher Scientific, Waltham, MA). For plasma exposure AML12 cells were seeded in a clear bottom black sided 96-well plate (ThermoFisher Scientific, Waltham, MA) and allowed to adhere for 24 hours. At ~90% confluency the media was replaced with plasma samples mixed with 2 parts culture media and incubated for 1 hour prior to H2O2 measurements. In other experiments AML12 cells were reverse transfected using lipofectamine 3000 with 10nM of either scrambled or p65 targeted siRNA (ThermoFisher Scientific, Waltham, MA) when plated in the 96-well plates. Cells were grown in these conditions for 48 hours. Then media was replaced with plasma spiked culture media as above and left overnight before assessing H2O2 measurements.
Rat Aortic smooth muscle cells (RASMC) were grown in Lonza smooth muscle complete media (Lonza, Basel, Switzerland). For experiments RASMC were plated in 6-well or clear bottom black sided 96-well plate. Cells were then exposed to 1 ng/ml of Kiss for the times indicated (0, 5, or 10 minutes) and 96-well plates fixed for in-cell western.
Coumarin Boronic Acid Assay
Coumarin Boronic acid (CBA) was conducted as previously published [52,67–70]. Tissue was homogenized in PBS (Fisher Scientific, Waltham, MA) containing a protease and phosphatase cocktail (Thermofisher Scientific, Waltham, MA). 10 µl of equal concentration sample homogenate were loaded into a black sided 96 well plate along with 90 µl of CBA buffer (containing PBS, L-Name (Sigma-Aldrich, St. Louis, MO), Taurine (Sigma-Aldrich, St. Louis, MO), and 500 µM CBA probe (Cayman, Ann Arbor, MI) +/- 1 KU catalase (Sigma-Aldrich, St. Louis, MO). For 96-well plates cell culture media was removed and 100 µl of CBA buffer (again containing 500 µM of CBA probe) was added +/- 1 KU catalase. Plates were then run in a 37 °C plate reader and fluorescence measured (ex: 350, em: 450) every minute over 2 hours. Signal from the negative control catalase wells were subtracted out from the sample wells and only the catalase inhibitable signal was analyzed. The rate of the relative fluorescent units per minute was calculated for all samples and fold change from control treatment calculated.
Samples were prepared and according to the kit protocol (ab239709, Abcam, Cambridge, MA). In brief, aorta and liver were homogenized in assay buffer and each sample was run with and without glutathione reductase to measure total GSH, reduced GSH, and calculate the oxidized to reduced ratio.
Medial lobe liver sections were snap frozen at the time of euthanasia. Liver tissue was homogenized in RIPA buffer containing a protease and phosphatase cocktail (Thermofisher, Waltham, MA). Samples were then prepared with laemmli sample buffer and b-mercaptoethanol at a 4 µg/ µl concentration and 32 µg of total protein run down each lane of a 4-20% gradient gel (Biorad, Hercules, California) at 70 V. Samples were then transferred to 0.45 µm nitrocellulose membranes, dried, reconstituted with ddiH2O. A Li-Cor protocol was followed for in-cell westerns. In brief, 96-well plates were treated as indicated and then fixed with 3.7% Formaldehyde (Sigma-Aldrige, St. Louis, MO), permeabilized with 0.1% Triton X-100 (Sigma-Aldrige, St. Louis, MO). Both in-cell and membranes were blocked with Li-Cor (Lincoln, Nebraska) TBS blocking buffer, and incubated in primary antibody overnight at 4 °C. Primary antibodies include b-actin (Santa Cruz Biotechnology, Dallas, Texas), phospho myosin light chain (Thr18/Ser19) (Cell Signaling Technologies, Danvers, MA), phospho and total p65 (Cell Signaling Technologies, Danvers, MA), phospho and total ERK 1/2 (Cell Signaling Technologies, Danvers, MA), and catalase (Abcam, Cambridge MA). Membranes and plates were then washed with TBS containing 1% tween (TBST), incubated in Li-Cor near-infrared secondary antibodies for 1 hour at room temperature, washed again with TBST, and finally imaged with the Li-Cor Odyssey Clx. Densitometry analysis was conducted in Image-J (National Institutes of Health, Bethesda, Maryland) with phosphor-signal normalized to total-signal or target normalized to b-actin and fold change calculated from control. In-cell western signal was normalized to cell stain and fold change calculated from control.
Isolated Microvessel Protocol (Pressure Myography) After the placenta and pups were removed, uteri were placed in a dissecting dish with physiological salt solution (PSS), as previously described , and maintained at 4 °C. A uterine artery segment was isolated, removed and transferred to a vessel chamber (Living Systems Instrumentation, Burlington, VT) containing fresh oxygenated PSS, cannulated with glass pipettes, and secured using nylon suture (11-0 ophthalmic, Alcon, U.K.). Arteries were extended to their in-situ length, pressurized to 60 mmHg with PSS, superfused with warmed
(37 °C) oxygenated PSS at a rate of 10 mL/min, and allowed to develop spontaneous tone. Internal and external arteriolar diameters were measured using video calipers (Colorado Video, Boulder, CO).
Uterine Vasculature Reactivity: Uterine arteries were allowed to develop spontaneous tone, defined as the degree of constriction experienced by a blood vessel relative to its maximally dilated state. Vascular tone ranges from 0% (maximally dilated) to 100% (maximal constriction). Vessels with a spontaneous tone ≥ 20% less than initial tone were included in this study. After equilibration, parameters of arterial vasoreactivity were analyzed. Vessels that did not develop sufficient spontaneous tone were not included in the data analysis.
Assessment of vasoreactivity: Arteries were exposed to increasing concentrations of phenylephrine (PE: 10 −9-10−4 M), acetylcholine (ACh: 10−9-10−4 M), sodium nitroprusside (SNP: 10 −9-10−4 M) and kisspeptin-10 (Kiss: 10−9-10−4 M), which were each added separately to the bath. The steady state diameter of the vessel was recorded for at least 2 min after each dose. After each dose curve was completed, the vessel bath was exchanged to remove excess chemicals by carefully removing the superfusate and replacing it with fresh warmed oxygenated PSS. After all experimental treatments were complete, the PSS was replaced with Ca2+-free PSS until maximum passive diameter was established.
Pressure Myography Calculations
Spontaneous tone was calculated by the following equation:
where Dm is the maximal diameter and Di is the initial steady state diameter recorded prior to the experiment. Active responses to pressure were normalized to the maximal diameter using the following formula:
, where DSS is the steady state diameter recorded during each pressure change. The experimental responses to ACh, PE, and SNP are expressed using the following equation:
, where Dcon is the control diameter recorded prior to the dose curve, DSS is the steady state diameter at each dose of the curve. The experimental response to PE is expressed using the following equation:
Wall thickness (WT) was calculated from the measurement of both inner (ID) and outer (OD) steady state arteriolar diameters at the end of the Ca2+ free wash using the following equation:
Statistics: Data are expressed as means ± standard error pf the mean. Point-to-point differences in the dose response curves were evaluated using two-way repeated measures analysis of variance (ANOVA) with a Tukey`s post-hoc analysis when significance was found. The animal characteristics, vessel characteristics and hepatocytes transfected with siRNA were analyzed using a one-way ANOVA with a Tukey post-hoc analysis when significance was found. Student’s t-test was utilized for comparison of two groups (i.e. liver weights). All statistical analysis was completed with Graph Pad Prism (San Diego, CA) Significance was set at p<0.05, N is the number of animals per group, n is the number of vessels per group.