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. 2019 Jul 9;16(1):29.
doi: 10.1186/s12989-019-0303-7.

Quantitative biokinetics over a 28 day period of freshly generated, pristine, 20 nm titanium dioxide nanoparticle aerosols in healthy adult rats after a single two-hour inhalation exposure

Wolfgang G Kreyling  1   2 , Uwe Holzwarth  3 , Carsten Schleh  4   5 , Stephanie Hirn  4 , Alexander Wenk  4   6 , Martin Schäffler  4 , Nadine Haberl  4 , Manuela Semmler-Behnke  4 , Neil Gibson  3
Affiliations

Quantitative biokinetics over a 28 day period of freshly generated, pristine, 20 nm titanium dioxide nanoparticle aerosols in healthy adult rats after a single two-hour inhalation exposure

Wolfgang G Kreyling et al. Part Fibre Toxicol. .

Abstract

Background: Industrially produced quantities of TiO2 nanoparticles are steadily rising, leading to an increasing risk of inhalation exposure for both professionals and consumers. Particle inhalation can result in inflammatory and allergic responses, and there are concerns about other negative health effects from either acute or chronic low-dose exposure.

Results: To study the fate of inhaled TiO2-NP, adult rats were exposed to 2-h intra-tracheal inhalations of 48V-radiolabeled, 20 nm TiO2-NP aerosols (deposited NP-mass 1.4 ± 0.5 μg). At five time points (1 h, 4 h, 24 h, 7d, 28d) post-exposure, a complete balance of the [48V]TiO2-NP fate was quantified in organs, tissues, carcass, lavage and body fluids, including excretions. After fast mucociliary airway clearance (fractional range 0.16-0.31), long-term macrophage-mediated clearance (LT-MC) from the alveolar region is 2.6-fold higher after 28d (integral fraction 0.40 ± 0.04) than translocation across the air-blood-barrier (integral fraction 0.15 ± 0.01). A high NP fraction remains in the alveoli (0.44 ± 0.05 after 28d), half of these on the alveolar epithelium and half in interstitial spaces. There is clearance from both retention sites at fractional rates (0.02-0.03 d- 1) by LT-MC. Prior to LT-MC, [48V]TiO2-NP are re-entrained to the epithelium as reported earlier for 20 nm inhaled gold-NP (AuNP) and iridium-NP (IrNP).

Conclusion: Comparing the 28-day biokinetics patterns of three different inhaled NP materials TiO2-NP, AuNP and IrNP, the long-term kinetics of interstitial relocation and subsequent re-entrainment onto the lung-epithelium is similar for AuNP and Ir-NP but slower than for TiO2-NP. We discuss mechanisms and pathways of NP relocation and re-entrainment versus translocation. Additionally, after 28 days the integral translocated fractions of TiO2-NP and IrNP across the air-blood-barrier (ABB) are similar and become 0.15 while the translocated AuNP fraction is only 0.04. While NP dissolution proved negligible, translocated TiO2-NP and IrNP are predominantly excreted in urine (~ 0.1) while the urinary AuNP excretion amounts to a fraction of only 0.01. Urinary AuNP excretion is below 0.0001 during the first week but rises tenfold thereafter suggesting delayed disagglomeration. Of note, all three NP dissolve minimally, since no ionic radio-label release was detectable. These biokinetics data of inhaled, same-sized NP suggest significant time-dependent differences of the ABB translocation and subsequent fate in the organism.

Keywords: Accumulation in secondary organs and tissues; Characterization of physicochemical particle properties; Intratracheal inhalation of freshly generated aerosols; Long-term alveolar macrophage-mediated nanoparticle clearance; Nanoparticle relocation into the interstitium; Re-entrainment from interstitium back to lung epithelium for clearance towards the larynx; Spark ignition generated titanium dioxide nanoparticle aerosols; Translocation across air-blood-barrier.

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Conflict of interest statement

The authors declare that they have no competing interests. The authors alone are responsible for the content and writing of the manuscript.

Figures

Fig. 1
Fig. 1
Fractional retention of intratracheally inhaled [48V]TiO2-NP in the total lungs and lavaged lungs between 1 h and 28 days p.e. Besides lung retention data, recoveries in BALC, BALF and in the trachea sample (trachea with main bronchi) are also presented. Panel a: linear y-axis for lung retention and BALC; panel b: logarithmic y-axis for BALF and in the trachea sample. The data are given as fractions of IPLD; i.e. corrected for fast [48V]TiO2-NP clearance from airways (MCC); the mean IPLD in mass (number) of [48V]TiO2-NP of all five retention time points is 1.01 ± 0.55 μg (2.67 ± 1.16•1010 #). Fractional data are given as mean ± SEM, n = 4 rats/time point. Data in both panels are corrected for [48V]TiO2-NP retained in the residual blood volume of the lungs. Statistical one-way ANOVA analysis with the post-hoc Bonferoni test in between all time points are given in the table belowformula image
Fig. 2
Fig. 2
Daily fecal excretion as 48V fractions of intratracheally inhaled [48V]TiO2-NP. Panel a: Daily fecal excretion of intratracheally inhaled [48V]TiO2-NP normalized to the total deposited [48V]TiO2-NP lung dose (ILD) present in the lungs 2 h p.e., representing MCC from the conducting thoracic airways during the first two days after inhalation and thereafter LT-MC from the alveolar region. The mean ILD in mass (number) of [48V]TiO2-NP of all five retention time points is 1.36 ± 0.45 μg (3.59 ± 1.08•1010 #). Data of the groups of rats analyzed 7 days and 28 days p.e. are presented. For the 28 days group, fecal excretions sampled over 3–4 days are divided by the number of sampling days and associated with the mean day of the sampling period. Panel b: Cumulative fecal excretion without MCC of the 7 days and 28 days group. Panel c: The excretion rates are normalized to contemporary lung retention (CLR) at each dissection time point. The CLR was obtained from an exponential fit to the lung fractions (sum of lavaged lungs, BALF and BALC) at each dissection time point (see Eq. (1a) and (1c) of the Additional file 1). Mean ± SEM, n = 4 rats per time point
Fig. 3
Fig. 3
Daily urinary excretion as 48V fractions of intratracheally inhaled [48V]TiO2-NP. Panels a + b: Daily urinary excretion as 48V fractions of intratracheally inhaled [48V]TiO2-NP of the 7 days and 28 days group. Data are given as fractions of IPLD. The mean IPLD in mass (number) of [48V]TiO2-NP of all five retention time points is 1.01 ± 0.55 μg (2.67 ± 1.16•1010 #). For the 28 days group, the urine sampled over 3–4 days are divided by the number of sampling days and associated with the mean day of the sampling period. Mean ± SEM, n = 4 rats per time point. Panel c: Translocated [48V]TiO2-NP across the ABB presented as stacked columns with accumulation in major secondary organs, soft tissue, skeleton, and cumulative urine. While translocated [48V]TiO2-NP accumulated predominantly in soft tissue up to 24 h the fraction released in urine increases rapidly from day 7 to day 28 p.e. Mean ± SEM, n = 4 rats per time point
Fig. 4
Fig. 4
Translocated and retained fractions of intratracheally inhaled [48V]TiO2-NP in the organism. Retained fractions of intratracheally inhaled [48V]TiO2-NP investigated up to 28 days p.e. a: total translocation and blood; b: liver, spleen, and kidneys; c: heart, brain, and uterus (at 1 h p.e. heart and uterus fractions were < 0.00001); d: carcass, soft tissue, and skeleton. The mean IPLD in mass (number) of [48V]TiO2-NP of all five retention time points is 1.01 ± 0.55 μg (2.67 ± 1.16•1010 #). [48V]TiO2-NP retention is given as fractions of the initial peripheral lung dose (IPLD). Data in all panels correspond to the third line (all corr.) of each organ in Table 3. Mean ± SEM, n = 4 rats per time point. Statistical one-way ANOVA analysis with the post-hoc Bonferoni test in between all time points are given in the table below: formula image
Fig. 5
Fig. 5
Kinetics of [48V]TiO2-NP concentrations per weight of organ or tissue: (a): total translocation and blood, (b): liver, spleen, and kidneys, (c): heart, uterus, and brain, (d): carcass, skeleton, and soft tissue. The mean IPLD in mass (number) of [48V]TiO2-NP of all five retention time points is 1.01 ± 0.55 μg (2.67 ± 1.16•1010 #). Data are corrected for [48V]TiO2-NP retained in the residual blood volume of organs and tissues; data are presented as mean ± SEM; n = 4 rats per time point. Statistical one-way ANOVA analysis with the post-hoc Bonferoni test in between all time points are given in the table below: formula image
Fig. 6
Fig. 6
Comparison of daily fecal excretion for three types of inhaled, 20-nm sized nanoparticles. Mean daily fecal excretion (± SEM) for n = 4 rats of each group analyzed at different retention days. a: [48V]TiO2-NP; b: [192Ir]IrNP; c: [195Au]AuNP
Fig. 7
Fig. 7
Comparison of the daily urinary excretion for three types of inhaled, 20-nm sized nanoparticles. Mean daily urinary excretion (± SEM) for n = 4 rats of each group analyzed at different retention days. a: [48V]TiO2-NP; b: [192Ir]IrNP; c: [195Au]AuNP
Fig. 8
Fig. 8
Stacked retention fractions for three types of inhaled, 20-nm sized nanoparticles at each time point. Stacked fractions of three different NP in various secondary organs, tissues, blood, and cumulative excretion in urine over time p.e., respectively. a: [48V]TiO2-NP; b: [192Ir]Ir-NP; c: [195Au]AuNP. Panel d: blood concentrations of all three NP types over time p.e
Fig. 9
Fig. 9
Comparison of interstitial relocation and epithelial re-entrainment for nano- and micron-sized particles. Estimated [48V]TiO2-NP retention fractions for the total lung surface macrophage pool [26] (for the definition of the AM pool, see the Additional file 1) derived from the experimentally determined kinetics of lavageable [48V]TiO2-NP fractions associated with BALC (downward triangles). Additionally, the [48V]TiO2-NP fractions found in the interstitium, estimated from [48V]TiO2-NP fractions of lavaged lungs (diamonds) are shown. Fractions are normalized to the contemporary lung burden at 1 h to 28 days after intratracheal inhalation. Additionally, the fractions of free [48V]TiO2-NP in BALF are presented, which rapidly decline p.e. (upward triangles). These data are compared with averaged retention fractions for 20 nm [192Ir] IrNP and [195Au] AuNP in the total AM pool [23, 25, 26] and with those of 1.3 μm fused aluminum-silicate particles (FAP) particles and 2.1 μm polystyrene (PSL) particles in the total AM pool [45, 46]. Data points are mean ± SEM; n = 4 rats per time point
Fig. 10
Fig. 10
Mechanisms and pathways of NP relocation, NP re-entrainment, and translocation across the ABB. Graphical sketch of mechanisms and pathways of NP relocation into the epithelium and the interstitium, subsequent re-entrainment back onto the alveolar epithelium and translocation across the ABB into blood circulation for distribution throughout the entire organism
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