Lactate dehydrogenase 1

nPM collection and transfer into aqueous suspension. We collected nPM with a high-volume ultrafine particle (HVUP) sampler (Misra et al. 2002 ) at 400 L/min flow in Los Angeles City near the CA-110 Freeway. These aerosols represent a mix of fresh ambient PM mostly from vehicular traffic nearby this freeway (Ning et al. 2007 (link)). The HVUP sampler consists of an ultrafine particle slit impactor, followed by an after-filter holder. The nPM (diameter < 200 nm) was collected on pretreated Teflon filters (20 × 25.4 cm, polytetrafluoroethylene, 2 μm pore; Pall Life Sciences, Covina, CA). We transferred the collected nPM into aqueous suspension by 30 min soaking of nPM-loaded filters in Milli-Q deionized water (resistivity, 18.2 MW; total organic compounds < 10 ppb; particle free; bacteria levels < 1 endotoxin units/mL; endotoxin-free glass vials), followed by vortexing (5 min) and sonication (30 min). As a control for in vitro experiments with resuspended nPM, fresh sterile filters were sham extracted. Aqueous nPM suspensions were pooled and frozen as a stock at –20°C, which retains chemical stability for ≥ 3 months (Li N et al. 2003; Li R et al. 2009). For in vitro experiments, nPM suspensions were diluted in culture medium, vortexed, and added directly to cultures.
Animals and exposure conditions. The nPM suspensions were reaerosolized by a VORTRAN nebulizer (Vortran Medical Technology 1 Inc., Sacramento, CA) using compressed particle-free filtered air [see Supplemental Material, Figure S1 (doi:10.​1289/ehp.1002973)]. Particles were diffusion dried by passing through silica gel; static charges were removed by passing over polonium-210 neutralizers. Particle sizes and concentrations were continuously monitored during exposure at 0.3 L/min by a scanning mobility particle sizer (SMPS model 3080; TSI Inc., Shoreview, MN). The nPM mass concentration was determined by pre- and postweighing the filters under controlled temperature and relative humidity. Inorganic ions [ammonium (NH₄⁺), nitrate (NO₃^–), sulfate (SO₄^2–)] were analyzed by ion chromatography. PM-bound metals and trace elements were assayed by magnetic-sector inductively coupled plasma mass spectroscopy. Water-soluble organic carbon was assayed by a GE-Sievers liquid analyzer (GE-Sievers, Boulder, CO). Analytic details for nPM-bound species are given by Li R et al. (2009). Samples of the reaerosolized nPM were collected on parallel Teflon filters for electron paramagnetic resonance (EPR) analysis.
Mice (C57BL/6J males, 3 months of age) were maintained under standard conditions with ad libitum Purina Lab Chow (Newco Purina, Rancho Cucamonga, CA) and sterile water. Just before nPM exposure, mice were transferred from home cages to exposure chambers that allowed free movement. Temperature and airflow were controlled for adequate ventilation and to minimize buildup of animal-generated contaminants [skin dander, carbon dioxide (CO₂), ammonia]. Reaerosolized nPM or ambient air (control) was delivered to the sealed exposure chambers for 5 hr/day, 3 days/week, for 10 weeks. Mice did not lose weight or show signs of respiratory distress. Mice were euthanized after isoflurane anesthesia, and tissue was collected and stored at –80°C. All rodents were treated humanely and with regard for alleviation of suffering; all procedures were approved by the University of Southern California Institutional Animal Care and Use Committee.
EPR spectroscopy of nPM. The reaerosolized nPM was collected on filters (described above), which were inserted directly in the EPR quartz tube (Bruker EPR spectrometer; Bruker, Rheinstetten, Germany); spectra were measured at 22°C. The g-value was determined following calibration of the EPR instrument using DPPH (2,2-diphenyl-1-picrylhydrazyl) as a standard. The EPR signal for DPPH was measured and the corresponding g-value was calculated. The difference from the known g-value of 2.0036 for DPPH was then used to adjust the observed g-value for the sample.
Cell culture and nPM exposure. Hippocampal slices from postnatal day 10–12 rats were cultured 2 weeks in a humidified incubator (35°C/5% CO₂) (Jourdi et al. 2005 (link)) with nPM suspensions added for 24–72 hr of exposure. Primary neurons from embryonic day 18 rat cerebral cortex were plated at 20,000 neurons/cm² on cover slips coated with poly-d-lysine/laminin and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with B27, at 37°C in 5% CO₂ atmosphere (Rozovsky et al. 2005 (link)). Primary glial cultures from cerebral cortex of neonatal day 3 rats (F344) were plated at 200,000 cells/cm² in DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% l-glutamine and incubated as described above (Rozovsky et al. 1998 (link)). For conditioned medium experiments, glial cultures were treated with 10 mg nPM/mL; after 24 hr, media were transferred by pipette to neuron cultures.
Neurite outgrowth and toxicity assays. After treatments, neurons were fixed in 4% paraformaldehyde and immunostained with anti–β-III-tubulin (1:1,000, rabbit; Sigma Chemical Co., St. Louis, MO); F-actin was stained by rhodamine phalloidin (1:40; Molecular Probes, Carlsbad, CA). A neurite was defined as a process extending from the cell soma of the neuron that was immunopositive for both β-III-tubulin (green) and F-actin (red). The length of neurites was measured using NeuronJ software (Meijering et al. 2004 (link)). Growth cones were defined by the presence of actin-rich filopodia and lamellipodia (Kapfhammer et al. 2007 ). Collapsed growth cones were defined as actin-rich neuritic endings in which filopodia and lamellipodia were indistinguishable. In neurite outgrowth and growth cone collapse assays, individual neurons were selected from two cover slips per condition; n is the total number of neurons analyzed per treatment. Cytotoxicity in slice cultures was assayed by lactate dehydrogenase (LDH) release to media and by cellular uptake of propidium iodide (PI) (Jourdi et al. 2005 (link)). Neuronal viability was assayed by Live/Dead Cytotoxicity Kit (Invitrogen, Carlsbad, CA) by computer-assisted image analysis of fluorescent images. Mitochondrial reductase was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 585 nm in undifferentiated PC12 cells (Mosmann 1983 (link)). For viability assays, n is the total number of hippocampal slices analyzed (LDH release and PI uptake) or the total number of cell culture wells analyzed per condition.
Immunoblotting. Mouse hippocampi were homogenized using a glass homogenizer in cold lysis buffer as described by Jourdi et al. (2005) (link). After sample preparation, 20 μg protein was electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gels, followed by transfer to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% bovine serum albumin for 1 hr and probed with primary antibodies overnight at 4°C: anti-GluA1 (glutamate receptor subunit 1; 1:3,000, rabbit; Abcam, Cambridge, MA), anti-GluA2 (1:2,000, rabbit; Millipore, Billerica, MA), anti-PSD95 (1:1,000, mouse; Abcam), anti-synaptophysin (1:5,000, mouse; Stressgene; Enzo, Plymouth Meeting, PA), and anti-β-III tubulin (loading control; 1:15,000, rabbit; Sigma), followed by incubation with secondary antibodies (1:10,000) conjugated with IRDye 680 (rabbit, LI-COR Biosciences, Lincoln, NE) and IRDye 800 (mouse, LI-COR). Immunofluorescence was detected by infrared imaging (Odyssey, LI-COR).
Quantitative polymerase chain reaction (qPCR). Total cellular RNA was extracted from cerebral cortex of nPM-exposed mice and rat primary glia (Tri Reagent; Sigma), and cDNA (2 μg RNA; Superscript III kit; Invitrogen) was analyzed by qPCR, with primers appropriate for mouse (in vivo) or rat (in vitro). Genes examined by qPCR were CD14, CD68, CD11b, CD11c, GFAP (glial fibrillary acidic protein), IFN-γ (interferon-γ), IL-1α, IL-1, IL-6, and TNFα. Data were normalized to β-actin.
Statistical analysis. Data are expressed as mean ± SE. The numbers of individual measurements (n) are described above and listed in the figure legends. Single and multiple comparisons used Student’s t-test (unpaired) and one-way analysis of variance (ANOVA)/Tukey’s honestly significant difference, with statistical significance defined as p < 0.05.

ENMs and reagents. The CPPs obtained ZnO from Meliorum Technologies Inc. (Rochester, NY). TiO₂-P25 (81% anatase and 19% rutile) was purchased from Evonik (Parsippany, NJ); TiO₂-A was provided by P. Biswas (Washington University, St. Louis, MO); and the CPPs prepared the TiO₂-NBs as previously described (Hamilton et al. 2009 (link)). The CPPs obtained the O-MWCNT stock in powder form from Cheap Tubes Inc. (Brattleboro, VT); obtained the P-MWCNT by treating O-MWCNT with dilute acids, chelating agents, and mild conditions to minimize oxidized or damaged tubes; and created F-MWCNT through further acid treatment of P-MWCNT, which introduced carboxyl groups on 5.27% of the carbon backbone (on a per weight basis) (Chen and Mitra 2008 ; Wang et al. 2011 (link)).
The CPPs purchased low-endotoxin bovine serum albumin (BSA) from Gemini Bio-Products (West Sacramento, CA); dipalmitoylphosphatidylcholine, phorbol 12-myristate, 13-acetate (PMA), and lipopolysaccharide (LPS from Escherichia coli 0127:B8) from Sigma-Aldrich (St. Louis, MO); and 1,25-dihydroxy-vitamin D₃ from EMD Millipore (Billerica, MA). The CPPs purchased the cytotoxicity assays CellTiter 96 (MTS assay) and CytoTox 96 [LDH (lactate dehydrogenase) assay] from Promega (Madison, WI).
Preparation of ENMs in cell culture media. The CPPs prepared ENM stock solutions (5 mg/mL) from dry powder using endotoxin-free sterile water and then prepared all ENM suspensions in cell culture media using the stock solutions as needed. Briefly, the CPPs vortexed and then sonicated ENM stock solutions (with the exception of TiO₂-NB, which was stirred to prevent mechanical shear) using a water bath sonicator or cup horn sonicator (depending on laboratory availability) immediately before diluting the solutions into complete cell culture media.
Cell culture and co-incubation with EMN. The CPPs grew all cells at 37°C in a 5% CO₂ atmosphere. RLE-6TN cells, a rat alveolar type II epithelial cell line, from American Type Culture Collection (ATCC; Manassas, VA) were cultured in Ham’s F12 medium (ATCC) supplemented with l-glutamine, bovine pituitary extract (BPE), insulin, insulin growth factor (IGF)-1, transferrin, and epithelial growth factor (EGF), supplemented with 10% fetal bovine serum (FBS). THP-1 cells, a human acute monocytic leukemia cell line (ATCC) were cultured in HEPES-buffered RPMI 1640 supplemented with l-glutamine (Mediatech, Corning, NY), 0.05 mM β-mercaptoethanol, and 10% FBS (PAA Laboratories, Dartmouth, MA). BEAS-2B cells (ATCC) were cultured in bronchial epithelial growth medium (BEGM) obtained from Lonza Inc. (Walkersville, MD) supplemented with BPE, insulin, hydrocortisone, human EGF, epinephrine, triiodothyronine, transferrin, gentamicin/amphotericin-B, and retinoic acid. For the THP-1 differentiation performed in the first series of experiments (phase I), the CPPs pretreated cells with 1.62 µM (1 µg/mL) PMA for 18 hr. However, the CPPs identified excessive cell clumping and cell death during the phase I studies. Therefore, the CPPs alternatively pretreated THP-1 cells with vitamin D₃ at 150 nM overnight and then 5 nM PMA in order to obtain the differentiated macrophage-like cells used during the second series of experiments (phase II). For the IL-1β release, co-culturing THP-1 cells with 10 ng/mL LPS was necessary to initiate transcription of pro-IL-1β. The CPPs initiated aggressive phagocytic activity by adding PMA just before particle exposure.
Before ENM exposure, the CPPs cultured aliquots of 1.5 × 10⁴ cells (for THP-1 cells, 10⁵ cells were seeded into each well of a 96-well plate) in 0.2 mL of the cell culture media in 96-well plates (Costar, Corning, NY) at 37°C for 24 hr. The CPPs freshly prepared all of the ENM suspensions at final concentrations of 10, 25, 50, and 100 µg/mL in the cell culture media. After exposure of the cells to the ENMs for 24 hr at 37°C, the CPPs collected supernatants to measure LDH and IL-1β production then used the remaining cells to test cellular viability by MTS assay.
Physicochemical characterization of ENMs. The CPPs identified the primary particle size and morphology of the ENMs by using a transmission electron microscope (TEM; model 100CX) and a scanning electron microscope (SEM; model JSM-7600F) (both from JEOL Ltd., Tokyo, Japan). In addition, the CPPs characterized the particle hydrodynamic size in H₂O and cell culture media using dynamic light scattering (DLS) (Ji et al. 2010 (link)). The CPPs characterized particle crystallinity and structure using X-ray diffraction measurements and measured particle surface area by Brunauer–Emmett–Teller (BET) surface area analysis. The CPPs performed zeta-potential measurements of the ENM suspensions using a ZetaSizer Nano-ZS instrument (Malvern Instruments, Worcestershire WR, UK). Finally, the CPPs determined the elemental composition of the particles as well as ZnO dissolution rate using inductively coupled plasma mass spectrometry (ICP-MS) (model SCIEX Elan DRCII; PerkinElmer, Norwalk, CT).
Endotoxin analysis of ENMs. CPPs measured the endotoxin content of ENM stock suspensions, as well as dispersions in PBS and tissue culture media, using the colorimetric Limulus amebocyte lysate assay (Lonza Inc.). The LPS content of all ENM suspensions was < 0.3 EU/mL.
Determination of cell viability. The CPPs determined cellular viability using MTS (CellTiter 96) and LDH (CytoTox 96; both from Promega) according to the manufacturer’s protocols. To avoid the interference created by ENMs while measuring formazan absorbance at 490 nm, the CPPs introduced a centrifugation (2000 × g for 10 min) procedure in phase II experiments to collect particles in the wells after incubation with the MTS reagents. CPPs then followed this centrifugation step with a brief mixing and transfer of the supernatant to a new 96-well plate before measuring the formazan absorbance at 490 nm. The CPPs eliminated interference of any residual LDH in FBS by heat-inactivation (70°C water bath for 5 min).
ELISA for IL-1β quantification. The CPPs determined IL-1β production in the THP-1 culture supernatant using a human IL-1β ELISA kit (R&D Systems Human IL-1β DuoSet™; R&D Systems, Minneapolis, MN) following the manufacturer’s instructions.
Statistical analysis. The CPPs used the two-way analysis of variance followed by Tukey or Bonferroni correction for multiple comparisons of means for statistical analysis of responses across ENMs and cell lines. In order to define interlaboratory comparisons across two harmonization rounds, the CPPs conducted a meta-analysis of LDH, MTS, and IL-1β assays across eight different laboratories for three cell lines (BEAS-2B, RLE-6TN, and THP-1) exposed to several ENMs (TiO₂-P25, TiO₂-A, TiO₂-NBs, ZnO, O-MWCNT, P-MWCNT, and F-MWCNT). The CPPs combined information within assays and cell lines using a robust two-stage hierarchical model of toxicity. For all quantities of interest, the CPPs obtained Monte Carlo inference by implementing a custom Gibbs sampler in the R computing environment (R Foundation for Statistical Computing, Vienna, Austria). To normalize data, the CPPs subtracted background negative control values (MTS, LDH, and IL-1β) and provided adjustments for positive control values in the case of LDH assays. Details about the statistical model used for analysis are provided in Supplemental Material, p. 8 (http://dx.doi.org/10.1289/ehp.1306561).

Cells were inoculated into the 96-well cell culture plate. Measurement of LDH release was conducted using the LDH release quantification cytotoxicity Assay Kit (Beyotime, Shanghai, China) as per the manufacturer’s instructions. To measure the caspase-1 activity, the provided instructions from Beyotime (Shanghai, China) were followed. PNA in the kit was diluted to create a range of standard solutions with different concentrations. A determination system was prepared by mixing the diluted PNA with the lysed cell suspension in a 1:9 ratio. A standard curve was generated using the prepared standard solutions. Following the kit instructions, a buffer system was prepared, and the absorbance at 405 nm was measured using an enzyme reader.

To quantify the cytotoxic effects of compound treatment and/or gene knock-out, the lactate dehydrogenase (LDH) activity was measured with the lactate dehydrogenase-based in vitro toxicology assay kit (Sigma-Aldrich #TOX7) according to the manufacturer’s instructions, method 2. In brief, 50 µL of culture supernatant and 100 µL of Lactate Dehydrogenase Assay Mixture were incubated for 20–30 min. The reaction was stopped by the addition of 15 µL of 1 N HCl and light absorption was detected at 690 nm and 490 nm with a BioTek Synergy 2 plate reader (Biotek Instruments). Background values from media alone were subtracted from all samples.