An intratracheal instillation bioassay system for detection of lung toxicity due to fine particles in f344 rats

Abstract

It is an urgent priority to establish in vivo bioassays for detection of hazards related to fine particles, which can be inhaled into deep lung tissue by humans. In order to establish an appropriate bioassay for detection of lung damage after particle inhalation, several experiments were performed in rats using quartz as a typical lung toxic particle. The results of pilot experiments suggest that Days 1 and 28 after intratracheal instillation of 2 mg of fine test particles in vehicle are most appropriate for detection of acute and subacute inflammatory changes, respectively. Furthermore, the BrdU incorporation on Day 1 and the iNOS level on Day 28 proved to be suitable end-point markers for this purpose. An examination of the toxicity of a series of particles was performed with the developed bioassay. Although some materials, including nanoparticles, demonstrated toxicity that was too strong for sensitive assessment, a ranking order could be clarified. The bioassay thus appears suitable for rapid hazard identification with a possible ranking of the toxicity of various particles at single concentrations.

Keywords: bioassay; fine particles; intratracheal instillation; particulates; rat; respiratory toxicology.

Fig. 1.

Histopathologic observation of lung tissue from F344 rats treated with 4 mg/rat quartz i.t. on Day 28. Arrows in the figures indicate typical findings. A, neutrophil infiltration in the walls; B, neutrophil infiltration in the spaces of alveoli; C, pulmonary edema; D, pulmonary fibrosis; E, histiocytic macrophage infiltration; F, restructuring of walls; G, granuloma.

Fig. 2.

Immunohistochemical observation of BrdU and iNOS. A, immunostaining of BrdU on Day 1; B, immunostaining of iNOS on Day 28 after i.t. of 4 mg/rat quartz.

Fig. 3.

Experimental design for the sequential analysis study. A total of 50, 10-week-old animals were separated into two groups. , i.t. of 4 mg/rat quartz suspended in 0.2 ml saline. , i.t. of 0.2 ml saline (control). S(5), sacrifice of 5 rats.

Fig. 4.

Experimental design for validation of the sequential analysis study. A total of 108, 10-week-old F344/DuCrj male rats were randomly divided into 8 groups. , i.t. of 4 mg/rat test substances, quartz, hydrotalcite, potassium octatitanate, palladium oxide and carbon black, suspended in 0.2 ml saline or PG-CMC for carbon black. , i.t. of 0.2 ml saline or 0.2 ml PG-CMC (control). S(7) or S(5), sacrifice of 7 or 5 rats.

Fig. 5.

Scoring indices from the comparative histopathological and immunohistochemical assessment. The quartz-treated group demonstrated severe toxicity, while the other hydrotalcite, potassium octatitanate, palladium oxide and carbon black-treated groups all exhibited relatively mild toxicity. This figure is modified version of that of Yokohira et al. 2007.

Fig. 6.

Experimental design for the dose response study. A total of 40, 10-week-old male F344 rats were randomly separated into 4 groups of 10 rats. , i.t. of 4 mg, 2 mg or 1 mg/rat quartz suspended in 0.2 ml saline. , i.t. of 0.2 ml saline (control). S(5), sacrifice of 5 rats.

Fig. 7.

Experimental design for the vehicle assessment study (dry powder assessment). A total of 20, 10-week-old male F344 rats were randomly separated into 2 groups of 10 rats. , i.t. of 4 mg/rat quartz powder with 0.2 ml air using a DP-4 Insufflator (dry powder insufflator). , i.t. of 2 ml air (control). S(5), sacrifice of 5 rats.

Fig. 8.

Experimental design for the toxicity assessment of a series of different particle types (Experiments 1–3). [Experiment 1] , i.t. of 2 mg/rat quartz, titanium dioxide, hydrotalcite or β-cyclodextrin suspended in 0.2 ml saline or K₂PdCl₄ and Na₂PdCl₄, suspended in 0.2 ml distilled water. , i.t. of 0.2 ml saline or distilled water (control). S(6), sacrifice of 6 rats. [Experiment 2] , i.t. of 2 mg/rat test substances, quartz, titanium dioxide, CuO, CuO nanoparticles, MnO₂, NiO or NiO nanoparticles suspended in 0.2 ml saline. , i.t. of 0.2 ml saline (control). S(6), sacrifice of 6 rats. [Experiment 3] , i.t. of 2 mg/rat quartz, diesel standard powder, diesel powder, C₆H₁₀O₄Pd or CaCO₃ suspended in 0.2 ml saline or neutralized Na₂PdCl₄ (final pH was 6.5) in 20 mM phosphate buffer and 5 N NaCl. , i.t. of 0.2 ml saline or vehicle of Na₂PdCl₄ (control). S(6), sacrifice of 6 rats.

Fig. 9.

Results for the fine particle toxicity. *, indicates the experiment number (Experiment 1, 2 or 3). Some materials, including nanoparticles, demonstrated toxicity that was too strong for sensitive assessment. The ranking order is CuO > quartz > neutralized Na₂PdCl₄ > NiO > hydrotalcite > MnO₂ > diesel > titanium dioxide (in Experiment 2) > β-cyclodextrin > diesel standard > titanium dioxide (in Experiment 1) > CaCO₃. This figure is modified version of that of Yokohira et al. 2008.