Animals
Time-mated, nulliparous mice (C57BL/6BomTac, Taconic Europe, Ejby, Denmark) arrived at gestation day (GD) 3 and were randomly grouped 5 or 6 in polypropylene cages with bedding and enrichment (removed during nursing). Animals were housed under controlled environmental conditions, with 12 hour light from 6.00 a.m. and access to food (Altromin 1324) and tap water ad libitum (further information in Additional file 1). On GD4, animals were weighed and assigned to two groups of 22 and 23 animals, respectively, with similar weight distributions. For cross-over mating, naïve CBA/J mice (Charles River Wiga, Sulzfeld, Germany) were supplied at nine weeks of age. Procedures complied with EC Directive 86/609/EEC and Danish regulations on experiments with animals (Permission 2006/561-1123).
Material characterization
This study used UV-titan L181 (Kemira, Pori, Finland), a rutile modified with unspecified amounts of zirconium (Zr), silicon (Si), aluminum (Al) and coated with polyalcohols.
Physical particle size, morphology and general state of agglomeration/aggregation were determined by analysis of particles suspended on holey carbon-coated Cu TEM-grids using a 200 kV Transmission Electron Microscope (TEM) (Tecnai G20, FEI Company, Hillsboro, Oregon, USA). Sample preparation for TEM analysis is described in Additional file 1.
Crystalline phases and crystallite sizes were determined by powder X-ray diffraction (XRD) with a Bruker D8 Advance diffractometer equipped with a Lynxeye CCD detector (Bruker AXS Inc., Madison, WI 53711-5373, USA), using monochromated CuKα1 (1.540598 Å) rays. Results were obtained by Rietveld refinement of the X-ray diffractograms using Bruker TOPAS V4.1 software. Elongation was determined by analysis of reflections from principal crystallographical axis using the Scherrer equation.
Specific surface area was determined on a Quantachrome Autosorp-1 (Quantachrome GmbH & Co. KG, Odelzhausen, Germany) using multipoint Brunauer, Emmett, and Teller (BET) nitrogen adsorption method after 1 h degassing at 300°C. Analysis was completed according to DIN ISO 9277 as a commercial service by Quantachrome GmbH & Co. KG.
Elemental composition was analyzed by X-ray Fluorescence analysis on a Philips PW-2400 spectrometer as a commercial service by the Department of Earth Sciences, University of Aarhus, Denmark. Elemental concentrations were determined using their standard protocol using rock standards for calibration.
The organic coating of the UV-titan particles was extracted with methanol by Pressurized Liquid Extraction (PLE) at 2000 psi and 200°C, followed by centrifugation at 4000 rpm (3310 g) for 10 min. Chemical composition of the supernatant was analyzed by laser desorption ionization and time of flight MS (MALDI-TOF without matrix) on a stainless steel ground target with a Bruker AutoFlex II (Bruker Daltonics, Inc., Bremen Germany). Acurate mass determination (1 ppm) was performed with electrospray-MS (ESI-MS) on a Bruker microQ-TOF (Bruker Daltonics, Inc., Bremen Germany) with direct injection. The masses are reported as mass to charge ratios (m/z) of the protonated compounds ([M+H]+).
Exposure
Mice were exposed to filtered clean air or a target concentration of 40 mg UV-Titan/m3 on GD8-18, one hr/day as described [13, 14]. Airflow in the exposure chamber was dynamic (20 L/min) with evenly distributed exposure atmosphere. A microfeeder aerosolized powder particles through a dispersion nozzle at a pressure of 5 bar (Fraunhofer Institute für Toxicologie und Aerosolforschung, Hannover, Germany). The dose from one hour exposure to 40 mg TiO2/m3 corresponds to the 8-hr time weighted average (TWA) occupational exposure limit according to Danish Regulations [15]. Animals were placed separately in rooms of a "twelve-room-pie"; a cylindrical wire mesh cage (∅ 29 cm, height 9 cm) with radical partitions. Females were observed for signs of toxicity and returned to cages less than 5 min after exposure. Body weight was recorded before exposure on GD9, 11, 14, and 18.
Exposure monitoring
Mass-concentrations of total suspended dust was controlled periodically by filter sampling and adjusted to maintain a concentration of ~40 mg/m3. Exposure air was sampled on pre-weighed Millipore Fluoropore filters (∅ 2.5 cm; pore size 0.45 μm) at an airflow of 2 L/min using Millipore cassettes, for 10 min. Filters were weighed immediately on a Sartorius Microscale (Type M3P 000V001). Final gravimetric data were obtained on acclimatized filters (50%RH and 20°C).
Particle number and size distribution in the exposure atmosphere were monitored using a GRIMM Sequential (Stepping) Mobility Particle Sizer (SMPS) system for sub-μm particles (12.8 to 486 nm; based on the rutile density of 4.25 g/cm3[16]) and a GRIMM Dustmonitor (Model 1.106) for coarse particles (0.75 to > 15 μm). The SMPS consisted of a Long Electrostatic Classifier (Model No. 5.521) and a GRIMM Condensation Particle Counter (Model 5.400). The time resolution was 218 and 6 s for the SMPS and Dustmonitor data, respectively (see Additional file 1 for further explanation on the on-line particle exposure monitoring).
Parturition and lactation
After exposure on GD18, females were singly housed. Delivery was expected on GD20, and designated postnatal day (PND) 0. Pups were counted and sexed on PND1. Dams and individual pups were weighed at PND1, 8, 11, 16, 19, and 22. On PND2, one pup from litters with at least 5 pups, and on PND23-24 one male and one female per litter, were sacrificed by decapitation. Lungs, liver, heart, brain, and on PND2, stomachs containing milk, were dissected, weighed, snap frozen in liquid N2 and stored at -80°C. At weaning (PND22), one male and female per litter were randomly chosen for behavioral testing and housed as described.
Non-pregnant time-mated females without implantations ("NP females") were euthanized on PND3 (i.e. 5 days post exposure) and subjected to bronchoalveolar lavage (BAL), as were dams with litters at PND24-25 ("P females"; 26-27 days post exposure). Females were anaesthetized with Hypnorm and Dormicum and sacrificed by withdrawal of heart blood (stabilized in 0.17 mol/l K2EDTA). BAL was performed as described below, followed by determination of uterine implantation sites and dissection of organs as described for offspring.
Titanium in tissue and milk
Approximately 25-75 mg tissue (lung and liver for adults, liver for offspring) and 110-140 mg (milk) were weighed and analyzed for content of titanium (Ti). For PND2 pups, milk and liver samples were pooled from 4-5 animals. Maternal lung was included to determine remaining TiO2 and liver to assess systemic distribution in adults [17, 18] and fetal animals [19]. Tissues were digested in concentrated nitric acid (PlasmaPure, SCP Science, Quebec, Canada) in a microwave oven (Multiwave, Anton Paar, Graz, Austria), and Ti content determined by quadrupole-based inductively coupled plasma mass spectrometer (ICPMS 7500ce, Agilent Technologies, Tokyo, Japan) equipped with a collision/reaction cell (CRC). The CRC was pressurized with helium as collision gas to reduce polyatomic interferences on Ti isotopes. Settings for ICPMS measurements are given in (Additional file 1, Table S1). Sulphur-containing polyatomics (e.g. 32S16O+) strongly interfered with the most abundant Ti isotope, 48Ti (abundance 73.8%). There was less interference with 49Ti and 50Ti (abundance 5.5 and 5.4%, respectively), which were selected for quantitative analysis. The limit of detection (LOD) for Ti in tissues, based on three times the standard deviation of repeated blank measurements, was estimated to be 0.2-5 mg/kg depending on sample intake and dilution.
BAL preparation and analyses
We used BAL cell composition and neutrophil influx to indicate lung inflammation. This has proven to be a relevant and sensitive marker of pulmonary inflammation (e.g. [20, 21]). BAL was performed four times with 0.8 ml 0.9% sterile saline ([20]; further information in Additional file 1). The total number of cells and of dead cells in BAL samples was determined in cell suspension B by NucleoCounter. Differential counts of macrophages, neutrophils, lymphocytes, eosinophils, and epithelial cells were determined by counting 200 cells in cell supernatant fixed with 96% ethanol and stained with May-Grünwald-Giemsa stain. All slides from both time points were randomized, blinded and scored on the same day. Total number of cells was calculated by combining data from differential cell counts with the total number of cells in BAL.
Behavioral testing
Investigations were performed during the light period. Exposed and control animals were tested alternately. Animals were transferred to the experimental room 1 hr before the first test. Observers were blinded to exposure status of the animals, and the same observer was used throughout any specific test.
Learning and memory was tested in the Morris water maze at age 11 and 15 weeks (males), and 12 and 16 weeks (females) as described [13] with minor modifications. A stable, invisible platform was submerged 1 cm below the water surface in a circular plastic pool (∅ 100 cm). Animals were tested in four daily trials. Mice were placed at the designated starting position and completed the trial when climbing onto the platform. When failing to locate the platform within 60 s, animals were led to the platform. All animals spent 15 s on the platform before returning to the cage. The following scheme was used: Learning: Test for 5 consecutive days with platform in center of the southeastern quadrant. Memory: Three weeks later, test with platform in south-eastern quadrant, for 3 days. Reversal learning: The following day, test with platform in north-western quadrant for 4 trials. New learning: The following day, test with platform in center of pool for 4 trials. Noldus Ethovision (Version 5, Noldus Information Technology, Wageningen, The Netherlands) was used to register latency and path length, and calculated swimming velocity and relative occupancy in the each of the quadrants.
Activity was assessed for 3 min at 14 weeks of age in an open field using the dry water maze pool. Trials commenced in the center of the field and the location of the animal was registered by Noldus Ethovision XT version 5. The tracking device calculated total ambulation, which was subsequently split into three time-bins of 1 min to test for habituation. Duration in the central and the outer 9 cm peripheral zone of the field, as well as the number of crossings from the outer to the central zone were extracted.
Acoustic startle reaction (ASR) and prepulse inhibition (PPI) were tested at 4 months as described [22] in two chambers (San Diego Instruments, San Diego, USA) with 70 dB(A) white background noise. A piezoelectric accelerometer transduced displacement of test tubes (∅ 3.6 cm) in response to movements of the animal. Animals were acclimatized for 5 min in the tube before sessions started and ended with 5 startle trials of 40 ms 120 dB(A) bursts of white noise. In between, 35 trials were delivered in semi-randomized order (10 trials of 120 dB(A); 5 each of 4 prepulse + startle trials (prepulses of 72, 74, 78, and 86 dB(A)); 5 trials with only background noise). Tube movements were averaged over 100 ms following onset of the startle stimulus (AVG). The five AVGs for each prepulse intensity were averaged and used to calculate PPI, which was expressed as percent reduction in AVG compared to the average of the 10 middle startle trials: %PPI = 100*((AVG at prepulse+startle trial)/(AVG at startle trial))*100%.
Time-to-first F2 litter
At 19 weeks of age, control and exposed offspring were cross-mated to naïve CBA/J mice (12 weeks old) and time-to-first-delivery of F2 litter, litter size, and gender ratio were recorded.
Statistics
Litter was considered the statistical unit. Gestational parameters were analyzed by Mann-Whitney U-test, and time-to-first-delivery by log rank test (separately by gender). ANOVAs were applied to the remaining data when relevant with repeated measures in trials, days, or time-bins. In the analysis of weight gain in adult females, Ti in adult tissues, and BAL cell counts, the factor "Pregnancy" was added to distinguish (barren) NP females from (littering) P females. Since these groups of adult females differed with regard to both time after exposure and pregnancy, only pairwise comparisons related to exposure were explored. ANCOVA controlled for litter size in the analyses of weight gain during exposure, birth weights, and pre-weaning pup weights. Behavioral data were analyzed by two-way ANOVA, with Prenatal exposure and Gender as factors, apart from startle data, where PPI was analyzed separately for each prepulse intensity [22]. Pairwise comparisons were performed by T-test or Mann Whitney U-test (p < 0.1). Analyses were performed in SYSTAT Software Package 9, MINITAB 14, and SAS 9.1.