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Bioassay by intratracheal instillation for detection of lung toxicity due to fine particles in F344 male rats
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EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Bioassay by intratracheal instillation for detection of lung toxicity due to fine particles in F344 male rats Masanao Yokohiraa,b, Hijiri Takeuchia,b, Keiko Yamakawaa, Kousuke Saooa, Yoko Matsudaa, Yu Zenga, Kyoko Hosokawaa, Katsumi Imaidaa, a
Onco-Pathology, Department of Pathology and Host-Defense, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan b Gastrointestinal Surgery, Faculty of Medicine, Kagawa University, Japan Received 8 June 2006; accepted 5 October 2006
Abstract We have established and documented an in vivo bioassay for detection of hazards with intratracheally instilled fine particles, which can be used for risk assessment of toxicity of materials inhaled into deep lung tissue of humans (Yokohira et al. Establishment of a bioassay system for detection of lung toxicity due to fine particle instillation: sequential histopathological changes with acute and subacute lung damage due to intratracheal instillation of quartz in F344 male rats. J Toxicol Pathol 2005;18:13–8). For validation we here examined toxicity of fine particles from quartz, hydrotalcite, potassium octatitanate, palladium oxide and carbon black with this bioassay. A total of 108, 10-week-old F344/DuCrj male rats were randomly divided into 8 groups. Groups 1 to 5 underwent intratracheal instillation of the 5 test particles (4 mg/rat) suspended in 0.2 ml vehicle (saline or 10% propylene glycol and 1% sodium carboxymethyl cellulose in saline: PG-CMC) with a specially designed aerolizer, and subgroups of 7 rats were killed on Days 1 and 28 thereafter. Groups 6 and 7 similarly were exposed to saline and PG-CMC, respectively, as vehicle controls, while group 8 was maintained untreated. Using histopathological changes and immunohistochemically assessed bromodeoxyuridine (BrdU) labeling indices, inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-3 (MMP-3) levels as end points, the quartz treated group exhibited high toxicity, while the values for the other particle-treated groups pointed to only slight effects. Although additional efforts are needed to establish advantages and disadvantages with our bioassay, models featuring intratracheal instillation clearly can be useful for detection of acute or subacute lung toxicity due to inhaled fine particles by using histopathological scoring and markers like BrdU and iNOS for screening purposes in short-term studies. r 2006 Elsevier GmbH. All rights reserved. Keywords: Intratracheal instillation; Fine particles; Lung toxicity; Respiratory system; Quartz; Bioassay
Introduction Corresponding author. Tel.: +81 87 891 2109; fax: +81 87 891 2112. E-mail address: [email protected] (K. Imaida).
0940-2993/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2006.10.001
There are many toxicants in our environment, including air pollutants, and human investigations focusing on concentrated ambient particles have shown acute lung inflammation and changes in both blood
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indices and heart rate after exposure (Ghio and Huang, 2004). We have established an in vivo bioassay for detection of hazards due to fine particles by intratracheal instillation (Yokohira et al., 2005), which can be used for risk assessment of materials inhaled into deep lung tissue of human beings. Sequential analysis of the effects of quartz (DQ-12, 4 mg/rat), a typical lung toxic agent, revealed Days 1 and 28 after instillation as most appropriate for detection of acute and subacute inflammatory changes, respectively, with bromodeoxyuridine (BrdU) on Day 1 and inducible nitric oxide synthase (iNOS) on Day 28 as suitable end-point markers. For further validation, toxicity of fine particles from various materials (quartz, hydrotalcite, potassium octatitanate, palladium oxide and carbon black) was here examined with this bioassay. These examples were selected for their variety of characteristics, including particle diameters, in one case in the nanometer order. Intratracheal instillation of quartz into rats produces inflammatory reactions followed by histological changes characteristic of lung fibrosis (Benson et al., 1986) and this agent was therefore chosen as the positive reference for the present study. Hydrotalcite is a protective agent for the gastric mucosa (Holtermuller et al., 1992; Rankin et al., 2001), neutralizing gastric acid (Yu et al., 2003), which to our knowledge has not been previously assessed for lung toxicity. Potassium octatitanate forms fibers that are widely used as reinforcing, antifrictional, heat insulation, or filtration material for common thermoplastic resins, engineering plastics, and a variety of matrices (Ikegami et al., 2004). Exposure limited to 1 fiber/cm3 does not pose a significant hazard to human health in the workplace based on animal experiments and medical surveys of workers (Ikegami et al., 2004). Palladium oxide acts as a catalyst in chemical engineering, but there are no data on its influence on the lung in vivo. Carbon black is employed primarily in rubber products, mainly tyres and other automotive products, but is also commonly present in inks, paints, and toners (Brockmann et al., 1998; Rosenkranz et al., 1980). Highdoses can promote oxidative deoxyribonucleic acid (DNA) damage that is consistent with the hypothesis that inflammatory cell-derived oxidation may play a role in the pathogenesis of rat lung tumors following longterm exposure to carbon black (Gallagher et al., 2003). In the present experiment, toxicity of fine particles from these various types of material was examined with intratracheal instillation in our in vivo bioassay with an especial focus on correlations between immunohistochemical and histopathological findings. Antibodies specific for BrdU provide a sensitive method for detecting DNA replication for DNA repair and cell proliferation in situ (Gratzner, 1982) while iNOS is temporally and anatomically associated with the development of lung damage, inflammation, granulomas and
fibrosis induced by inhalation of silica (Castranova et al., 2002). Matrix metalloproteinase-3 (MMP-3) cleaves proteoglycans, collagens (type II, IX, XI), gelatin, laminin, and fibronectin (Basset et al., 1990; Murphy et al., 1991) and may degrade cell matrix materials in areas with active inflammation (Kirkegaard et al., 2004). These 3 parameters were therefore chosen for analysis in the present comparative study.
Materials and methods Chemicals Quartz dust (DQ-12) was obtained from Douche Montan Technologie (GmbH, Germany) with a particle diameter less than 7 mm. Hydrotalcite (trade name Kyoward 500, PL-1686) was obtained from Kyowa Chemical Industry Co., Ltd. (Kagawa, Japan) with an average diameter of 7.8171.52 mm. Potassium octatitanate fibers (trade name TISMO) were supplied by Otsuka Chemical Co., Ltd. (Osaka, Japan) with dimensions mostly shorter than 50 mm in length and less than 2 mm in width. Palladium oxide was broken into a 0.7 mm peak grain diameter after being obtained from Kanto Kagaku Co., Ltd (Tokyo, Japan) to give an average diameter of 0.5471.11 mm. Carbon black (conductible) was obtained from Mitsubishi Kasei Co., Ltd. (Tokyo, Japan) with a particle diameter of 28 nm, but could not been suspended evenly in saline because of its light weight, so that a special vehicle, PG-CMC (10% propylene glycol and 1% sodium carboxymethyl cellulose in saline), with an appropriate degree of viscosity, was used.
Animals Male F344/DuCrj rats (8 weeks of age), purchased from Japan Charles River (Atsugi, Japan), were maintained in a specific pathogen-free (SPF) room in the Kagawa University Animal Facility according to our institutional animal care guidelines. The animals were housed in polycarbonate cages with white wood chips for bedding, and given free access to drinking water and a basal diet, CE-2 (CLEA Japan Inc., Tokyo, Japan), under controlled conditions of humidity (60710%), lighting (12 h light/dark cycle) and temperature (2472 1C). After a 2 weeks acclimation period, the total of 108, 10-week-old rats, were randomly separated into 8 groups. Seventy rats (Groups 1–5) were exposed by intratracheal instillation to the 5 kinds of test particles (4 mg/rat) suspended in 0.2 ml saline using a specially designed aerolizer (Penn Century, Philadelphia, USA), and subgroups of 7 rats were killed on Days 1 and 28
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thereafter under ether anesthesia. Group 6 and 7 animals (14 each) were respectively exposed to an intratracheal instillation of 0.2 ml saline and PG-CMC as vehicle controls, while the remaining 10 rats were maintained as an untreated group. Subgroups of half of the rats were sacrificed as with the test groups. All rats received an intraperitoneal injection of BrdU (100 mg/ 5 ml saline/kg rat) (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan), 1 h before sacrifice. At autopsy, the lungs, bronchi, liver, adrenal glands, spleen, pulmonary lymph nodes and kidneys were removed. The lungs and bronchi were rinsed in 10% neutral buffered formalin after excision and weighed and then infused with 10% neutral buffered formalin. Six hours later, the fixative solution was changed to 100% ether to preserve antigens. Other organs were immersed in 10% neutral buffered formalin for a week. Slices of organs were routinely processed for embedding in paraffin for histopathological examination of H&E stained sections and immunohistochemistry.
Histopathological analysis Each lung lobe was examined histopathologically for neutrophil infiltration in the walls and spaces of the alveoli, pulmonary edema, pulmonary fibrosis, histiocyte infiltration in the alveoli, restructuring of walls and microgranulation. Severity for each parameter was assessed as follows: 0, no change; 1, weak; 2, moderate; 3, severe. These scores were used for histopathological assessment.
Immunohistochemical analysis Lungs were immunostained for BrdU and iNOS by the avidin-biotin complex (ABC) method, all staining Table 1.
Quartz Hydrotalcite Potassium octatitanate Palladium oxide Carbon black Saline control PG-CMC control Untreated control
Image analysis Each section stained immunohistochemically was examined with the assistance of an image analyzer (IPAP; Image Processor for Analytical Pathology, Sumika Technoservice Co., Hyogo, Japan). More than 20 microscope images ( 400) from all lung lobes of the rats were assessed for numbers and areas of immunohistochemically positive cells. Numbers and areas were indicated as follows: %No (labeling indices); (number of positive cells/total number of lung cells) 1000, %Area; (total area of positive cells/total area of lung)
Day 1a
Day 28
No.b
%c
No.
%
7 7 7 7 7 7 7 5
4.4970.38d 4.2270.34d 5.0670.82d 4.2970.27 4.7370.39 3.8370.38 4.1970.38 4.0270.27
7 7 7 5 7 7 7 5
5.0370.49e 3.9970.20e 4.4170.38e 3.8270.14e 4.2670.40f 3.5370.19 3.7470.23 3.5870.58
Days after intratracheal instillation. Effective number of rats. c Lung weight/Body weight 1000. d Po0.05 vs. saline control group Day 1. e Po0.05 vs. saline control group Day 28. f Po0.05 vs. PG-CMC control group Day 28. b
processes from deparaffinization to counterstaining with hematoxylin being performed automatically using the Ventana DiscoveryTM staining system (Ventana Medical Systems, AZ, USA). Antimouse BrdU monoclonal antibody, code No. M0744 purchased from DAKO, Glostrup, Denmark, and antirabbit inducible nitric oxide synthase (iNOS) polyclonal antibody, NOS2 (N-20), sc-650 purchased from SANTACRUZ, CA, USA, were used at 1:100 dilution. For mouse matrix metalloproteinase-3 (MMP-3) immunostaining, the routine avidin-biotin complex (ABC) method, with 3,30 -diaminobenzidine as the substrate, was applied, together with Gill’s hematoxylin counterstaining to facilitate orientation. For the antimouse matrix metalloproteinase-3 (MMP-3) monoclonal antibody, code no. 551117 from BD Biosciences, NJ, USA, antigen retrieval was performed by heating to 121 1C for 5 min in an autoclave in citrate buffer solution. Blocking of endogenous peroxidase activity and background staining with horse serum were performed and sections were incubated with the antibody (diluted 1:10) for 12 h at 4 1C.
Relative weights of lungs (lung weight/body weight) from fine particle treated and control rats
Group
a
213
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Fig. 1. Histopathological changes in lungs from test fine particles and saline (as a control) treated rats. H&E stained sections, 100.
1000. In this experiment, data for ‘area’ as well as ‘number’ were used in order to indicate positive parameters. Since an image analyzer was used for counting positive cells, adjacent positive cells sometimes were counted as only 1. Therefore, %area was also calculated to indicate positive lesions for each parameter. At least, 2310, 3200 and 2200 cells per group were
counted to generate labeling indices of BrdU, iNOS and MMP-3, respectively.
Statistics Percentage data from immunohistochemistry were analyzed by the Tukey–Kramer post-hoc test.
7 7 7 7 5 7 7 7 5 7 7 7 5
1 1 1 1 1 28 28 28
28 28 28 28 28
1.670.54a 0.970.38b 0.970.38 1.170.38 0.870.45 2.170.38c 1.070.00 1.070.00 1.070.00 1.370.49d 0.970.38 0.370.49 0.470.55
1.870.45c 1.670.54d 0.770.49 0.0 0.0
2.370.76 1.170.38 2.170.69a
1.970.38a 1.470.54b 0.370.49 0.470.54 0.0 2.970.38c 1.670.54c 1.370.49c
2.070.57 0.670.54 2.370.49a
a
a
2.870.45c 1.770.49d 1.370.49 0.170.38 0.670.55
1.070.58 1.170.69 0.970.38 1.170.38 0.470.55 2.370.49c 1.370.49 1.370.49
0.370.49 0.670.54 0.970.38
a
Edema
In walls
In alveolar spaces
Pulmonary
Neutrophil infiltration
Histopathological changes
*Mean7SD. Severity of each parameter was represented as follows: 0, no change; 1, weak; 2, moderate; 3, severe a Po0.05 vs. saline control of Day 1. b Po0.05 vs. PG-CMC control of Day 1. c Po0.05 vs. saline control of Day 28. d Po0.05 vs. PG-CMC control of Day 28.
7 7 7
1 1 1
Quartz Hydrotalcite Potassium Octatitanate Palladium Oxide Carbon Black Saline Control PG-CMC Control Untreated Control Quartz Hydrotalcite Potassium Octatitanate Palladium Oxide Carbon Black Saline Control PG-CMC Control Untreated Control
No. of rats
Days after treatment
Scoring indices for parameters of histopathological inflammatory change
Chemical
Table 2.
1.470.55c 1.070.00 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 2.970.38c 1.070.00 1.370.49c
0.0 0.0 0.0
Fibrosis
1.870.45c 1.470.54d 0.0 2.370.49 0.470.55
0.770.76a 0.770.49 0.0 1.370.49 0.0 3.070.00 1.770.76c 1.470.54c
0.770.49a 0.170.38 0.770.76a
Histiocyte infiltration in alveoliin
1.470.55c 1.170.38d 0.370.49 0.470.54 0.0
0.0 0.0 0.0 0.0 0.0 2.970.38c 1.070.00c 1.170.38c
0.0 0.0 0.0
Restructuringof alveolar walls
2.470.89c 1.470.54d 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 2.370.49c 1.070.82c 1.470.54c
0.0 0.0 0.470.54
Microgranulation
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Comparative analysis In order to assess toxicity to lungs for each fine particle material and to screen them for hazard in a relatively simple way, effects were classified into 4 groups (high, moderate, slight and low) as follows. (1) Histopathological change: average points for the 7 parameters were summed for each test particle group and ratios relative to the respective control value were calculated. BrdU: and iNOS: ratios were calculated from the %number of positive cells. MMP-3: ratios were calculated from %Areas of positive areas, since positive staining of MMP-3 was mainly in cytoplasm. (2) Criteria to give scales of 0 to 10 points were applied (Table 3). The maximum scale for each parameter was configured as average of the highest ratio in the group— 0.5 standard deviation. (3) All points on a scale of 0 to 10 points from histopathological changes and immunohistochemistry (BrdU, iNOS, MMP-3) were summed (Table 4). Toxicity was rank from these points as follows: more than 50 points, high toxicity; 50–40, moderate; 40–30, slight; and less than 30, low.
Results After intratracheal instillation, though some rats receiving carbon black or palladium oxide experienced
breathing difficulty, they soon recovered. The general condition of the rats in all groups demonstrated no remarkable change during the experimental period. However, at bleeding, 2 rats of the palladium oxide treated group died because of fortuitous accidents.
Lung weights Relative lung weights (lung weight/body weight) for fine particle and solution control treated rats are shown in Table 1. On Day 1, lung weights of the quartz, hydrotalcite or potassium octatitanate treated groups were significantly increased. On Day 28, lung weights of all fine particle treated groups were elevated.
Macroscopic findings Representative lungs of rats sacrificed on Days 1 and 28 and treated with palladium oxide or carbon black showed surface discoloration and were partially black on Day 1. This change was found to be diminished on Day 28. The bronchi, liver, adrenal glands, spleen, pulmonary lymph nodes and kidneys demonstrated no remarkable changes (data not shown).
Fig. 2. Labeling indices from BrdU immunohistochemistry. *Po0.05 vs. the respective control group.
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Histopathological analysis
Immunohistochemical analysis
Sequential histopathological changes of lungs in rats treated with test fine particles and saline are illustrated in Fig. 1. Main findings were neutrophil infiltration in the walls and spaces of the alveoli, pulmonary edema, pulmonary fibrosis, histiocyte infiltration in the alveoli, restructuring of alveolar walls and microgranulation. Lungs of rats in the potassium octatitanate, palladium oxide and carbon black treated groups sacrificed on Day 1 demonstrated deposits of fine particles. In addition, lungs of rats in the quartz treated group killed on Day 28 demonstrated marked granulation like change with giant cells and macrophages in the alveoli. In the other groups changes were milder, scoring indices for each lesion being summarized in Table 2. Lungs of rats in the quartz treated group killed on Day 1 exhibited the strongest values for neutrophil infiltration, followed closely by potassium octatitanate and palladium oxide, and on Day 28 for all parameters (Table 3). The bronchi, liver, adrenal glands, spleen, pulmonary lymph nodes and kidneys did not demonstrate significant histopathological alteration.
Numbers and areas of BrdU positive cells were highest overall in lungs of rats in the quartz treated group. In the potassium octatitanate treated group values were high on Day 1, whereas on Day 28 they were the lowest of all groups treated with fine particles (Fig. 2). Data for iNOS immunohistochemistry are also shown in Fig. 3. In the quartz treated group on Days 1 and 28, numbers and areas of iNOS positive cells were higher than in the other fine particle treated groups. A similar result was obtained for MMP-3 staining, but here the differences were much smaller (Fig. 4).
Toxicity classification Conclusions for fine particle toxicity are detailed in Table 4. Only quartz was classified into the highly toxic group, hydrotalcite, potassium octatitanate and palladium oxide demonstrating slight and carbon black only low toxicity.
Fig. 3. Labeling indices from iNOS immunohistochemistry. *Po0.05 vs. the respective control group. #Po0.05 vs. the untreated group.
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Discussion The lung weight is generally one of the important parameters for objective examination of the degree of lung toxicity. In the present study, on Day 1, weights of lungs from quartz, hydrotalcite or potassium octatitanate treated groups were increased significantly. On Day 28, increase was seen in all fine particle treated groups, the results being totally consistent with histopathological findings for inflammation. Lung weight increase on Days 1 and 28 is indicative of acute lung toxicity and continuing reactions in vivo against lung damage, respectively. With the comparative histopathological assessment (see Table 4 and Fig. 5), the quartz treated group demonstrated severe toxicity while the other particle treated groups all exhibited relatively mild toxicity. Biochemical analyses at different time points following instillation of different materials earlier demonstrated all exposed groups to develop granulomatous pneumonia. Alveolar lipoproteinosis and pulmonary fibrosis was most severe in the quartz treated lungs and progressed with time (Renne et al., 1980). The present results for BrdU immunohistochemistry demonstrated that, in all groups, proliferation was enhanced due to particle exposure at Day 1 but had returned to almost
normal values by Day 28. In contrast, iNOS values were higher at the latter time point in the quartz and hydrotalcite groups. This time course of change in iNOS expression may be important in terms of toxicity assessment and it may be necessary to examine a later time point (estimated about 8 months) after intratracheal instillation to clarify influence of change with time. MMP-3 may degrade cell matrix materials in the areas with active inflammation and fibrosis (Kirkegaard et al., 2004). However, in this experiment, there was no association with any of the subchronic changes, such as granulation, collagenization or fibrosis after particle instillation. Another marker for fibrosis is therefore needed. Generally, bronchoalveolar lavage (BALF) with markers of inflammation is often used to assess lung toxicity of test particles instilled in rats (Ernst et al., 2002). In our experiment, histopathological findings and 3 different immunohistochemical markers (BrdU, iNOS and MMP-3) were selected and were scored in order to provide an objective assessment. Compared to BALF this approach has the advantage of allowing detailed investigation of lung damage but there are disadvantages, for example, in that rats must be sacrificed at each time point and sequential changes cannot be followed at the individual animal level.
Fig. 4. Labeling indices from MMP-3 immunohistochemistry. *Po0.05 vs. the respective control group.
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Fig. 5. Scores for each parameter relative to the respective control group value. *Po0.05 vs. the respective Day 1 value.
Table 3. Points 0 1 2 3 4 5 6 7 8 9 10 a
Criteria for scoring Histopathological findings 0.0–0.5 0.5–1.0 1.0–1.5 1.5–2.0 2.0–2.5 2.5–3.0 3.0–3.5 3.5–4.0 4.0–4.5 4.5–5.0 5.0– b
a
BrdU
iNOS
MMP-3
0.0–1.3 1.3–2.6 2.6–3.9 3.9–5.2 5.2–6.5 6.5–7.8 7.8–9.1 9.1–10.4 10.4–11.7 11.7–13.0 13.0–
0.0–6.0 6.0–12.0 12.0–18.0 18.0–24.0 24.0–30.0 30.0–36.0 36.0–42.0 42.0–48.0 48.0–54.0 54.0–60.0 60.0–
0.0–0.3 0.3–0.6 0.6–0.9 0.9–1.2 1.2–1.5 1.5–1.8 1.8–2.1 2.1–2.4 2.4–2.7 2.7–3.0 3.0–
Values of ratios. Average of the highest ratio in the group 0.5 standard deviation.
b
Table 4.
Scoring indices
Groups
Quartz Hydrotalcite Potassium octatitanate Palladium oxide Carbon black
Total (Day 1+Day 28) Histopathological findings
BrdU(Number)
iNOS(Number)
MMP-3(Area)
Total score
13 7 11 13 8
12 9 11 10 5
12 2 2 1 0
20 14 14 10 9
57 32 38 34 22
(3+10) (2+5) (6+5) (5+8) (2+6)
(10+2) (8+1) (10+1) (8+2) (3+2)
(2+10) (0+2) (2+0) (1+0) (0+0)
(10+10) (10+4) (9+5) (4+6) (7+2)
(25+32) (20+12) (27+11) (18+16) (12+10)
Scoring indices for each parameter were calculated from the values of ratios in Fig. 5. (See Materials and Methods.)
The present study featured comparison of 5 different particles in one experiment using an in vivo bioassay model. There have hitherto been no data for instillation
or inhalation of hydrotalcite and palladium oxide. TISMO was evaluated by inhalation (Ikegami et al., 2004) and similarly quartz and carbon black after
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inhalation and instillation (Albrecht et al., 2004; Driscoll et al., 1996; Friemann et al., 1994; Li et al., 1999). In this experiment, quartz exerted much stronger toxicity than the other test particles, which did not greatly differ in their effects. One of the reasons is the dose configuration. The dose of 4 mg/rat of quartz instilled directly into the trachea in this experiment was selected from data reported previously (Mercer et al., 2003) and dose dependence at higher levels is now under investigation in our laboratory. There are biologically different responses to inhalation and instillation (Osier and Oberdo¨rster, 1997), but given the rapidity with which different particles can enter the lung, acute effects may be most important. Assessment of particle toxicity by using in vivo bioassays such as intratracheal instillation has been stressed (Mohr et al., 2006; Pott and Sto¨ber, 1983). Though additional efforts are needed to further establish advantages and disadvantages of our bioassay, the intratracheal instillation method can be useful for detection of acute and subacute pulmonary particle effects by using a histopathological scoring system and markers like BrdU and iNOS.
Acknowledgments We thank Koji Kato (Nagoya City University Graduate School of Medical Sciences) for technical assistance and Dr. Malcolm A. Moore for help in the preparation and critical reading of the manuscript.
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EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Bioassay by intratracheal instillation for detection of lung toxicity due to fine particles in F344 male rats Masanao Yokohiraa,b, Hijiri Takeuchia,b, Keiko Yamakawaa, Kousuke Saooa, Yoko Matsudaa, Yu Zenga, Kyoko Hosokawaa, Katsumi Imaidaa, a
Onco-Pathology, Department of Pathology and Host-Defense, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan b Gastrointestinal Surgery, Faculty of Medicine, Kagawa University, Japan Received 8 June 2006; accepted 5 October 2006
Abstract We have established and documented an in vivo bioassay for detection of hazards with intratracheally instilled fine particles, which can be used for risk assessment of toxicity of materials inhaled into deep lung tissue of humans (Yokohira et al. Establishment of a bioassay system for detection of lung toxicity due to fine particle instillation: sequential histopathological changes with acute and subacute lung damage due to intratracheal instillation of quartz in F344 male rats. J Toxicol Pathol 2005;18:13–8). For validation we here examined toxicity of fine particles from quartz, hydrotalcite, potassium octatitanate, palladium oxide and carbon black with this bioassay. A total of 108, 10-week-old F344/DuCrj male rats were randomly divided into 8 groups. Groups 1 to 5 underwent intratracheal instillation of the 5 test particles (4 mg/rat) suspended in 0.2 ml vehicle (saline or 10% propylene glycol and 1% sodium carboxymethyl cellulose in saline: PG-CMC) with a specially designed aerolizer, and subgroups of 7 rats were killed on Days 1 and 28 thereafter. Groups 6 and 7 similarly were exposed to saline and PG-CMC, respectively, as vehicle controls, while group 8 was maintained untreated. Using histopathological changes and immunohistochemically assessed bromodeoxyuridine (BrdU) labeling indices, inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-3 (MMP-3) levels as end points, the quartz treated group exhibited high toxicity, while the values for the other particle-treated groups pointed to only slight effects. Although additional efforts are needed to establish advantages and disadvantages with our bioassay, models featuring intratracheal instillation clearly can be useful for detection of acute or subacute lung toxicity due to inhaled fine particles by using histopathological scoring and markers like BrdU and iNOS for screening purposes in short-term studies. r 2006 Elsevier GmbH. All rights reserved. Keywords: Intratracheal instillation; Fine particles; Lung toxicity; Respiratory system; Quartz; Bioassay
Introduction Corresponding author. Tel.: +81 87 891 2109; fax: +81 87 891 2112. E-mail address: [email protected] (K. Imaida).
0940-2993/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2006.10.001
There are many toxicants in our environment, including air pollutants, and human investigations focusing on concentrated ambient particles have shown acute lung inflammation and changes in both blood
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indices and heart rate after exposure (Ghio and Huang, 2004). We have established an in vivo bioassay for detection of hazards due to fine particles by intratracheal instillation (Yokohira et al., 2005), which can be used for risk assessment of materials inhaled into deep lung tissue of human beings. Sequential analysis of the effects of quartz (DQ-12, 4 mg/rat), a typical lung toxic agent, revealed Days 1 and 28 after instillation as most appropriate for detection of acute and subacute inflammatory changes, respectively, with bromodeoxyuridine (BrdU) on Day 1 and inducible nitric oxide synthase (iNOS) on Day 28 as suitable end-point markers. For further validation, toxicity of fine particles from various materials (quartz, hydrotalcite, potassium octatitanate, palladium oxide and carbon black) was here examined with this bioassay. These examples were selected for their variety of characteristics, including particle diameters, in one case in the nanometer order. Intratracheal instillation of quartz into rats produces inflammatory reactions followed by histological changes characteristic of lung fibrosis (Benson et al., 1986) and this agent was therefore chosen as the positive reference for the present study. Hydrotalcite is a protective agent for the gastric mucosa (Holtermuller et al., 1992; Rankin et al., 2001), neutralizing gastric acid (Yu et al., 2003), which to our knowledge has not been previously assessed for lung toxicity. Potassium octatitanate forms fibers that are widely used as reinforcing, antifrictional, heat insulation, or filtration material for common thermoplastic resins, engineering plastics, and a variety of matrices (Ikegami et al., 2004). Exposure limited to 1 fiber/cm3 does not pose a significant hazard to human health in the workplace based on animal experiments and medical surveys of workers (Ikegami et al., 2004). Palladium oxide acts as a catalyst in chemical engineering, but there are no data on its influence on the lung in vivo. Carbon black is employed primarily in rubber products, mainly tyres and other automotive products, but is also commonly present in inks, paints, and toners (Brockmann et al., 1998; Rosenkranz et al., 1980). Highdoses can promote oxidative deoxyribonucleic acid (DNA) damage that is consistent with the hypothesis that inflammatory cell-derived oxidation may play a role in the pathogenesis of rat lung tumors following longterm exposure to carbon black (Gallagher et al., 2003). In the present experiment, toxicity of fine particles from these various types of material was examined with intratracheal instillation in our in vivo bioassay with an especial focus on correlations between immunohistochemical and histopathological findings. Antibodies specific for BrdU provide a sensitive method for detecting DNA replication for DNA repair and cell proliferation in situ (Gratzner, 1982) while iNOS is temporally and anatomically associated with the development of lung damage, inflammation, granulomas and
fibrosis induced by inhalation of silica (Castranova et al., 2002). Matrix metalloproteinase-3 (MMP-3) cleaves proteoglycans, collagens (type II, IX, XI), gelatin, laminin, and fibronectin (Basset et al., 1990; Murphy et al., 1991) and may degrade cell matrix materials in areas with active inflammation (Kirkegaard et al., 2004). These 3 parameters were therefore chosen for analysis in the present comparative study.
Materials and methods Chemicals Quartz dust (DQ-12) was obtained from Douche Montan Technologie (GmbH, Germany) with a particle diameter less than 7 mm. Hydrotalcite (trade name Kyoward 500, PL-1686) was obtained from Kyowa Chemical Industry Co., Ltd. (Kagawa, Japan) with an average diameter of 7.8171.52 mm. Potassium octatitanate fibers (trade name TISMO) were supplied by Otsuka Chemical Co., Ltd. (Osaka, Japan) with dimensions mostly shorter than 50 mm in length and less than 2 mm in width. Palladium oxide was broken into a 0.7 mm peak grain diameter after being obtained from Kanto Kagaku Co., Ltd (Tokyo, Japan) to give an average diameter of 0.5471.11 mm. Carbon black (conductible) was obtained from Mitsubishi Kasei Co., Ltd. (Tokyo, Japan) with a particle diameter of 28 nm, but could not been suspended evenly in saline because of its light weight, so that a special vehicle, PG-CMC (10% propylene glycol and 1% sodium carboxymethyl cellulose in saline), with an appropriate degree of viscosity, was used.
Animals Male F344/DuCrj rats (8 weeks of age), purchased from Japan Charles River (Atsugi, Japan), were maintained in a specific pathogen-free (SPF) room in the Kagawa University Animal Facility according to our institutional animal care guidelines. The animals were housed in polycarbonate cages with white wood chips for bedding, and given free access to drinking water and a basal diet, CE-2 (CLEA Japan Inc., Tokyo, Japan), under controlled conditions of humidity (60710%), lighting (12 h light/dark cycle) and temperature (2472 1C). After a 2 weeks acclimation period, the total of 108, 10-week-old rats, were randomly separated into 8 groups. Seventy rats (Groups 1–5) were exposed by intratracheal instillation to the 5 kinds of test particles (4 mg/rat) suspended in 0.2 ml saline using a specially designed aerolizer (Penn Century, Philadelphia, USA), and subgroups of 7 rats were killed on Days 1 and 28
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thereafter under ether anesthesia. Group 6 and 7 animals (14 each) were respectively exposed to an intratracheal instillation of 0.2 ml saline and PG-CMC as vehicle controls, while the remaining 10 rats were maintained as an untreated group. Subgroups of half of the rats were sacrificed as with the test groups. All rats received an intraperitoneal injection of BrdU (100 mg/ 5 ml saline/kg rat) (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan), 1 h before sacrifice. At autopsy, the lungs, bronchi, liver, adrenal glands, spleen, pulmonary lymph nodes and kidneys were removed. The lungs and bronchi were rinsed in 10% neutral buffered formalin after excision and weighed and then infused with 10% neutral buffered formalin. Six hours later, the fixative solution was changed to 100% ether to preserve antigens. Other organs were immersed in 10% neutral buffered formalin for a week. Slices of organs were routinely processed for embedding in paraffin for histopathological examination of H&E stained sections and immunohistochemistry.
Histopathological analysis Each lung lobe was examined histopathologically for neutrophil infiltration in the walls and spaces of the alveoli, pulmonary edema, pulmonary fibrosis, histiocyte infiltration in the alveoli, restructuring of walls and microgranulation. Severity for each parameter was assessed as follows: 0, no change; 1, weak; 2, moderate; 3, severe. These scores were used for histopathological assessment.
Immunohistochemical analysis Lungs were immunostained for BrdU and iNOS by the avidin-biotin complex (ABC) method, all staining Table 1.
Quartz Hydrotalcite Potassium octatitanate Palladium oxide Carbon black Saline control PG-CMC control Untreated control
Image analysis Each section stained immunohistochemically was examined with the assistance of an image analyzer (IPAP; Image Processor for Analytical Pathology, Sumika Technoservice Co., Hyogo, Japan). More than 20 microscope images ( 400) from all lung lobes of the rats were assessed for numbers and areas of immunohistochemically positive cells. Numbers and areas were indicated as follows: %No (labeling indices); (number of positive cells/total number of lung cells) 1000, %Area; (total area of positive cells/total area of lung)
Day 1a
Day 28
No.b
%c
No.
%
7 7 7 7 7 7 7 5
4.4970.38d 4.2270.34d 5.0670.82d 4.2970.27 4.7370.39 3.8370.38 4.1970.38 4.0270.27
7 7 7 5 7 7 7 5
5.0370.49e 3.9970.20e 4.4170.38e 3.8270.14e 4.2670.40f 3.5370.19 3.7470.23 3.5870.58
Days after intratracheal instillation. Effective number of rats. c Lung weight/Body weight 1000. d Po0.05 vs. saline control group Day 1. e Po0.05 vs. saline control group Day 28. f Po0.05 vs. PG-CMC control group Day 28. b
processes from deparaffinization to counterstaining with hematoxylin being performed automatically using the Ventana DiscoveryTM staining system (Ventana Medical Systems, AZ, USA). Antimouse BrdU monoclonal antibody, code No. M0744 purchased from DAKO, Glostrup, Denmark, and antirabbit inducible nitric oxide synthase (iNOS) polyclonal antibody, NOS2 (N-20), sc-650 purchased from SANTACRUZ, CA, USA, were used at 1:100 dilution. For mouse matrix metalloproteinase-3 (MMP-3) immunostaining, the routine avidin-biotin complex (ABC) method, with 3,30 -diaminobenzidine as the substrate, was applied, together with Gill’s hematoxylin counterstaining to facilitate orientation. For the antimouse matrix metalloproteinase-3 (MMP-3) monoclonal antibody, code no. 551117 from BD Biosciences, NJ, USA, antigen retrieval was performed by heating to 121 1C for 5 min in an autoclave in citrate buffer solution. Blocking of endogenous peroxidase activity and background staining with horse serum were performed and sections were incubated with the antibody (diluted 1:10) for 12 h at 4 1C.
Relative weights of lungs (lung weight/body weight) from fine particle treated and control rats
Group
a
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Fig. 1. Histopathological changes in lungs from test fine particles and saline (as a control) treated rats. H&E stained sections, 100.
1000. In this experiment, data for ‘area’ as well as ‘number’ were used in order to indicate positive parameters. Since an image analyzer was used for counting positive cells, adjacent positive cells sometimes were counted as only 1. Therefore, %area was also calculated to indicate positive lesions for each parameter. At least, 2310, 3200 and 2200 cells per group were
counted to generate labeling indices of BrdU, iNOS and MMP-3, respectively.
Statistics Percentage data from immunohistochemistry were analyzed by the Tukey–Kramer post-hoc test.
7 7 7 7 5 7 7 7 5 7 7 7 5
1 1 1 1 1 28 28 28
28 28 28 28 28
1.670.54a 0.970.38b 0.970.38 1.170.38 0.870.45 2.170.38c 1.070.00 1.070.00 1.070.00 1.370.49d 0.970.38 0.370.49 0.470.55
1.870.45c 1.670.54d 0.770.49 0.0 0.0
2.370.76 1.170.38 2.170.69a
1.970.38a 1.470.54b 0.370.49 0.470.54 0.0 2.970.38c 1.670.54c 1.370.49c
2.070.57 0.670.54 2.370.49a
a
a
2.870.45c 1.770.49d 1.370.49 0.170.38 0.670.55
1.070.58 1.170.69 0.970.38 1.170.38 0.470.55 2.370.49c 1.370.49 1.370.49
0.370.49 0.670.54 0.970.38
a
Edema
In walls
In alveolar spaces
Pulmonary
Neutrophil infiltration
Histopathological changes
*Mean7SD. Severity of each parameter was represented as follows: 0, no change; 1, weak; 2, moderate; 3, severe a Po0.05 vs. saline control of Day 1. b Po0.05 vs. PG-CMC control of Day 1. c Po0.05 vs. saline control of Day 28. d Po0.05 vs. PG-CMC control of Day 28.
7 7 7
1 1 1
Quartz Hydrotalcite Potassium Octatitanate Palladium Oxide Carbon Black Saline Control PG-CMC Control Untreated Control Quartz Hydrotalcite Potassium Octatitanate Palladium Oxide Carbon Black Saline Control PG-CMC Control Untreated Control
No. of rats
Days after treatment
Scoring indices for parameters of histopathological inflammatory change
Chemical
Table 2.
1.470.55c 1.070.00 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 2.970.38c 1.070.00 1.370.49c
0.0 0.0 0.0
Fibrosis
1.870.45c 1.470.54d 0.0 2.370.49 0.470.55
0.770.76a 0.770.49 0.0 1.370.49 0.0 3.070.00 1.770.76c 1.470.54c
0.770.49a 0.170.38 0.770.76a
Histiocyte infiltration in alveoliin
1.470.55c 1.170.38d 0.370.49 0.470.54 0.0
0.0 0.0 0.0 0.0 0.0 2.970.38c 1.070.00c 1.170.38c
0.0 0.0 0.0
Restructuringof alveolar walls
2.470.89c 1.470.54d 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 2.370.49c 1.070.82c 1.470.54c
0.0 0.0 0.470.54
Microgranulation
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Comparative analysis In order to assess toxicity to lungs for each fine particle material and to screen them for hazard in a relatively simple way, effects were classified into 4 groups (high, moderate, slight and low) as follows. (1) Histopathological change: average points for the 7 parameters were summed for each test particle group and ratios relative to the respective control value were calculated. BrdU: and iNOS: ratios were calculated from the %number of positive cells. MMP-3: ratios were calculated from %Areas of positive areas, since positive staining of MMP-3 was mainly in cytoplasm. (2) Criteria to give scales of 0 to 10 points were applied (Table 3). The maximum scale for each parameter was configured as average of the highest ratio in the group— 0.5 standard deviation. (3) All points on a scale of 0 to 10 points from histopathological changes and immunohistochemistry (BrdU, iNOS, MMP-3) were summed (Table 4). Toxicity was rank from these points as follows: more than 50 points, high toxicity; 50–40, moderate; 40–30, slight; and less than 30, low.
Results After intratracheal instillation, though some rats receiving carbon black or palladium oxide experienced
breathing difficulty, they soon recovered. The general condition of the rats in all groups demonstrated no remarkable change during the experimental period. However, at bleeding, 2 rats of the palladium oxide treated group died because of fortuitous accidents.
Lung weights Relative lung weights (lung weight/body weight) for fine particle and solution control treated rats are shown in Table 1. On Day 1, lung weights of the quartz, hydrotalcite or potassium octatitanate treated groups were significantly increased. On Day 28, lung weights of all fine particle treated groups were elevated.
Macroscopic findings Representative lungs of rats sacrificed on Days 1 and 28 and treated with palladium oxide or carbon black showed surface discoloration and were partially black on Day 1. This change was found to be diminished on Day 28. The bronchi, liver, adrenal glands, spleen, pulmonary lymph nodes and kidneys demonstrated no remarkable changes (data not shown).
Fig. 2. Labeling indices from BrdU immunohistochemistry. *Po0.05 vs. the respective control group.
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Histopathological analysis
Immunohistochemical analysis
Sequential histopathological changes of lungs in rats treated with test fine particles and saline are illustrated in Fig. 1. Main findings were neutrophil infiltration in the walls and spaces of the alveoli, pulmonary edema, pulmonary fibrosis, histiocyte infiltration in the alveoli, restructuring of alveolar walls and microgranulation. Lungs of rats in the potassium octatitanate, palladium oxide and carbon black treated groups sacrificed on Day 1 demonstrated deposits of fine particles. In addition, lungs of rats in the quartz treated group killed on Day 28 demonstrated marked granulation like change with giant cells and macrophages in the alveoli. In the other groups changes were milder, scoring indices for each lesion being summarized in Table 2. Lungs of rats in the quartz treated group killed on Day 1 exhibited the strongest values for neutrophil infiltration, followed closely by potassium octatitanate and palladium oxide, and on Day 28 for all parameters (Table 3). The bronchi, liver, adrenal glands, spleen, pulmonary lymph nodes and kidneys did not demonstrate significant histopathological alteration.
Numbers and areas of BrdU positive cells were highest overall in lungs of rats in the quartz treated group. In the potassium octatitanate treated group values were high on Day 1, whereas on Day 28 they were the lowest of all groups treated with fine particles (Fig. 2). Data for iNOS immunohistochemistry are also shown in Fig. 3. In the quartz treated group on Days 1 and 28, numbers and areas of iNOS positive cells were higher than in the other fine particle treated groups. A similar result was obtained for MMP-3 staining, but here the differences were much smaller (Fig. 4).
Toxicity classification Conclusions for fine particle toxicity are detailed in Table 4. Only quartz was classified into the highly toxic group, hydrotalcite, potassium octatitanate and palladium oxide demonstrating slight and carbon black only low toxicity.
Fig. 3. Labeling indices from iNOS immunohistochemistry. *Po0.05 vs. the respective control group. #Po0.05 vs. the untreated group.
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Discussion The lung weight is generally one of the important parameters for objective examination of the degree of lung toxicity. In the present study, on Day 1, weights of lungs from quartz, hydrotalcite or potassium octatitanate treated groups were increased significantly. On Day 28, increase was seen in all fine particle treated groups, the results being totally consistent with histopathological findings for inflammation. Lung weight increase on Days 1 and 28 is indicative of acute lung toxicity and continuing reactions in vivo against lung damage, respectively. With the comparative histopathological assessment (see Table 4 and Fig. 5), the quartz treated group demonstrated severe toxicity while the other particle treated groups all exhibited relatively mild toxicity. Biochemical analyses at different time points following instillation of different materials earlier demonstrated all exposed groups to develop granulomatous pneumonia. Alveolar lipoproteinosis and pulmonary fibrosis was most severe in the quartz treated lungs and progressed with time (Renne et al., 1980). The present results for BrdU immunohistochemistry demonstrated that, in all groups, proliferation was enhanced due to particle exposure at Day 1 but had returned to almost
normal values by Day 28. In contrast, iNOS values were higher at the latter time point in the quartz and hydrotalcite groups. This time course of change in iNOS expression may be important in terms of toxicity assessment and it may be necessary to examine a later time point (estimated about 8 months) after intratracheal instillation to clarify influence of change with time. MMP-3 may degrade cell matrix materials in the areas with active inflammation and fibrosis (Kirkegaard et al., 2004). However, in this experiment, there was no association with any of the subchronic changes, such as granulation, collagenization or fibrosis after particle instillation. Another marker for fibrosis is therefore needed. Generally, bronchoalveolar lavage (BALF) with markers of inflammation is often used to assess lung toxicity of test particles instilled in rats (Ernst et al., 2002). In our experiment, histopathological findings and 3 different immunohistochemical markers (BrdU, iNOS and MMP-3) were selected and were scored in order to provide an objective assessment. Compared to BALF this approach has the advantage of allowing detailed investigation of lung damage but there are disadvantages, for example, in that rats must be sacrificed at each time point and sequential changes cannot be followed at the individual animal level.
Fig. 4. Labeling indices from MMP-3 immunohistochemistry. *Po0.05 vs. the respective control group.
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Fig. 5. Scores for each parameter relative to the respective control group value. *Po0.05 vs. the respective Day 1 value.
Table 3. Points 0 1 2 3 4 5 6 7 8 9 10 a
Criteria for scoring Histopathological findings 0.0–0.5 0.5–1.0 1.0–1.5 1.5–2.0 2.0–2.5 2.5–3.0 3.0–3.5 3.5–4.0 4.0–4.5 4.5–5.0 5.0– b
a
BrdU
iNOS
MMP-3
0.0–1.3 1.3–2.6 2.6–3.9 3.9–5.2 5.2–6.5 6.5–7.8 7.8–9.1 9.1–10.4 10.4–11.7 11.7–13.0 13.0–
0.0–6.0 6.0–12.0 12.0–18.0 18.0–24.0 24.0–30.0 30.0–36.0 36.0–42.0 42.0–48.0 48.0–54.0 54.0–60.0 60.0–
0.0–0.3 0.3–0.6 0.6–0.9 0.9–1.2 1.2–1.5 1.5–1.8 1.8–2.1 2.1–2.4 2.4–2.7 2.7–3.0 3.0–
Values of ratios. Average of the highest ratio in the group 0.5 standard deviation.
b
Table 4.
Scoring indices
Groups
Quartz Hydrotalcite Potassium octatitanate Palladium oxide Carbon black
Total (Day 1+Day 28) Histopathological findings
BrdU(Number)
iNOS(Number)
MMP-3(Area)
Total score
13 7 11 13 8
12 9 11 10 5
12 2 2 1 0
20 14 14 10 9
57 32 38 34 22
(3+10) (2+5) (6+5) (5+8) (2+6)
(10+2) (8+1) (10+1) (8+2) (3+2)
(2+10) (0+2) (2+0) (1+0) (0+0)
(10+10) (10+4) (9+5) (4+6) (7+2)
(25+32) (20+12) (27+11) (18+16) (12+10)
Scoring indices for each parameter were calculated from the values of ratios in Fig. 5. (See Materials and Methods.)
The present study featured comparison of 5 different particles in one experiment using an in vivo bioassay model. There have hitherto been no data for instillation
or inhalation of hydrotalcite and palladium oxide. TISMO was evaluated by inhalation (Ikegami et al., 2004) and similarly quartz and carbon black after
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inhalation and instillation (Albrecht et al., 2004; Driscoll et al., 1996; Friemann et al., 1994; Li et al., 1999). In this experiment, quartz exerted much stronger toxicity than the other test particles, which did not greatly differ in their effects. One of the reasons is the dose configuration. The dose of 4 mg/rat of quartz instilled directly into the trachea in this experiment was selected from data reported previously (Mercer et al., 2003) and dose dependence at higher levels is now under investigation in our laboratory. There are biologically different responses to inhalation and instillation (Osier and Oberdo¨rster, 1997), but given the rapidity with which different particles can enter the lung, acute effects may be most important. Assessment of particle toxicity by using in vivo bioassays such as intratracheal instillation has been stressed (Mohr et al., 2006; Pott and Sto¨ber, 1983). Though additional efforts are needed to further establish advantages and disadvantages of our bioassay, the intratracheal instillation method can be useful for detection of acute and subacute pulmonary particle effects by using a histopathological scoring system and markers like BrdU and iNOS.
Acknowledgments We thank Koji Kato (Nagoya City University Graduate School of Medical Sciences) for technical assistance and Dr. Malcolm A. Moore for help in the preparation and critical reading of the manuscript.
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