Pulmonary toxicity of sodium dichloroisocyanurate after intratracheal instillation in sprague-dawley rats

Abstract

In water, sodium dichloroisocyanurate (NaDCC), a source for chlorine gas generation, releases free available chlorine in the form of hypochlorous acid, a strong oxidizing agent. NaDCC has been used as a disinfectant in humidifiers; however, its inhalation toxicity is a concern. Seven-week-old rats were exposed to NaDCC doses of 100, 500, and 2500 μg·kg⁻¹ body weight by intratracheal instillation (ITI) to investigate pulmonary toxicity. The rats were sacrificed at 1 d (exposure group) or 14 d (recovery group) after ITI. Despite a slight decrease in body weight after exposure, there was no statistically significant difference between the control and NaDCC-treated groups. A significant increase in the total protein level of the bronchoalveolar lavage fluid (BALF) was observed in the exposure groups. Lactate dehydrogenase leakage into the BALF increased significantly (p < 0.01) in the exposure groups; however, recovery was observed after 14 d. The measurement of cytokines in the BALF samples indicated a significant increase in interleukin (IL)-6 in the exposure group and IL-8 in the recovery group. Histopathological examination revealed inflammatory foci and pulmonary edema around the terminal bronchioles and alveoli. This study demonstrated that ITI of NaDCC induced reversible pulmonary edema and inflammation without hepatic involvement in rats.

Keywords

Sodium dichloroisocyanurate disinfectant intratracheal instillation bronchoalveolar lavage fluid pulmonary inflammation

Introduction

Sodium dichloroisocyanurate (NaDCC; NaCl₂(NCO)₃) is widely used as a disinfectant and source of free available chlorine through the generation of hypochlorous acid (HOCl). In detail, when NaDCC (usually produced in granular form) is dissolved in water, HOCl and sodium cyanurate (NaH₂(NCO)₃) are formed. HOCl emits free available chlorine, which is antimicrobial.¹ Within its effective antimicrobial concentration range, HOCl is nonirritating and non-sensitizing and exhibits lower cytotoxicity to mammalian cells than sodium hypochlorite (NaOCl) or hydrogen peroxide (H₂O₂).² Due to its lower toxicity, NaDCC has been substituted for NaOCl to stabilize chlorine for the disinfection of swimming pools, in the food industry, and recently, in household point-of-use water treatment.^3,4 In addition, NaDCC has been reported as one of the active ingredients in humidifier disinfectants (HDs) in South Korea.⁵ Besides NaDCC, several substances, such as polyhexamethylene guanidine phosphate (PHMG-P), oligo (2-(2-ethoxy) ethoxyethyl guanidinium (PGH), chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT), didecyldimethylammonium chloride (DDAC), and benzalkonium chloride (BAC) have been used as HDs.⁵ Most previous studies on NaDCC toxicity have been on its sterilization function as a disinfectant for drinking or household water using free available chlorine from hypochlorite ions and HOCL produced by dissolving NaDCC in water, in which cases, pulmonary toxicity has been rare.^6–8

However, in 2011, severe and ultimately fatal respiratory failure occurred in several young women admitted to hospital in Seoul, Korea, with no previously reported medical conditions.^9,10 The patients had regressed to severe pneumonia without fever, similar to acute interstitial pneumonia or idiopathic acute respiratory distress syndrome.¹¹ Numerous reports of HD victims prompted several case-controlled and disinfectant inhalation animal studies using various HDs.^12,13 A previous report indicated that intratracheally instilled PHMG-P induced lung inflammation and pulmonary fibrosis in mice.¹⁴ Inhalation studies on HDs containing PHMG-P or PGH indicated that they caused pulmonary fibrosis (determined by biopsy) and dyspnea.¹⁵ DDAC, used in many biocidal products, and reported to be widely used in HDs, has been a cause of severe lung injury.⁵ The intratracheal instillation (ITI) of 1500 µg·kg⁻¹ DDAC resulted in severe morbidity with pulmonary congestive edema, and 150 µg·kg⁻¹ caused low-grade inflammation.¹⁶ Furthermore, the no-observed-adverse-effect level of 13-week whole-body exposure of Sprague-Dawley (SD) rats to DDAC was 0.11 mg·m⁻³, with a slight increase in lung weight, inflammatory cell infiltration, and interstitial pneumonia.¹⁷

The ITI of 400 µg·kg⁻¹ BAC (an active ingredient in HDs and preservatives) is toxic to pulmonary tissue. Additionally, a mixture of nontoxic doses of BAC (200 µg·kg⁻¹) and triethylene glycol (TEG) (1000 µg·kg⁻¹) induced significant lung injury in rats.¹⁸ The BAC/TEG mixture induced inflammation with the recruitment of polymorphonuclear leukocytes in terminal bronchioles and increased cytokine levels, tumor necrosis factor α and interleukin-6, in bronchoalveolar lavage fluid (BALF).¹⁸ Swiercz et al. reported that BAC aerosol inhaled by rats during two periods exhibited strong inflammatory responses, primarily in the respiratory tract.¹⁹

The toxic effects caused by HDs have been confirmed for multiple chemicals, including PHMG-P and CMIT/MIT.²⁰ However, the toxic effects of NaDCC inhalation are not yet clearly known. In contrast to inhalation exposure studies, ITI has the advantages of lower cost and not requiring large facilities. ITI exposes the chemical directly to the lungs through the trachea; therefore, it is useful in elucidating dose-response relations and lung disorders induced by chemical substances.²¹ A case of a 5-year-old child who presented with acute lung injury due to unintentional inhalation of the mist formed from the reaction of NaDCC tablets with water has been reported,²² indicating that the toxicity due to the inhalation of NaDCC is a serious concern. To our knowledge, no studies on NaDCC lung toxicity using ITI have been conducted. Therefore, this study is the first to report the adverse effects of ITI exposure of rat lungs to water-dissolved NaDCC.

Materials and methods

Chemicals

NaDCC (CAS number: 2893-78-9) used in this study was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

Animals

Six-week-old specific-pathogen-free male SD rats were purchased from Orient Bio Inc. (Seongnam, Korea) and maintained in our laboratory animal facility at 22 ± 3°C, with a relative humidity of 50 ± 20%, under a 12-h light/dark cycle. In this study, a total of 20 rats were randomized into four groups (5 rats in each group), including the control group. The acclimation period was one week before the beginning of the exposure. The body weights of the rats and food consumption were measured twice a week from the beginning of the study until the animals were sacrificed, and general signs and symptoms were monitored during the exposure period. The experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the National Institute of Environmental Research (Republic of Korea).

Intratracheal instillation (ITI) experimental design

ITI was performed on 7-week-old rats to investigate the pulmonary toxicity of NaDCC. NaDCC, dissolved in sterile saline, was instilled into the tracheae of rats under inhalation anesthesia with isoflurane. The instillation volume was 1 mL·kg⁻¹ body weight. A total of 20 rats were randomized into four groups (5 rats in each group) and exposed to 0 (treated with sterile saline, control group), 100 (low-exposure group), 500 (middle-exposure group), or 2500 µg·kg⁻¹ body weight (high-exposure group) of NaDCC by ITI. The rats were sacrificed at 1 or 14 d after a single ITI of NaDCC. The exposure groups sacrificed at 1 d were termed low-, middle-, and high-exposure groups, and those sacrificed at 14 d were termed low-, middle-, and high-exposure recovery groups. The experiments were performed according to the National Institute of Environmental Research (Incheon, Republic of Korea) guidelines for care and use.

Hematology and blood biochemistry

Blood samples were collected from the abdominal aorta into a blood-collecting tube containing ethylenediaminetetraacetic acid anticoagulant. The following parameters of the whole blood samples were measured: red blood cell count, mean corpuscular hemoglobin, mean plasma volume, white blood cell count, hemoglobin, mean corpuscular hemoglobin concentration, procalcitonin, mean corpuscular volume, hematocrit, red blood cell distribution width, platelet distribution width, and platelets. The following parameters of the serum samples were measured: triglyceride, total cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), glucose, alkaline phosphatase, total bilirubin, albumin, creatinine, and blood urea nitrogen. Hematologic analyses were performed using an automated blood cell counter (Microsemi LC-660, HORIBA, Kyoto, Japan), and the blood serum was analyzed using a DRI-CHEM 4000i chemistry analyzer (FUJIFILM Co., Tokyo, Japan).

Bronchoalveolar lavage fluid analysis

The rat BALF was examined to evaluate the toxicity caused by ITI of NaDCC. The rat lungs were lavaged four times with 5 mL calcium- and magnesium-free phosphate-buffered saline (PBS, pH 7.4). The BALF was centrifuged at 1500 rpm for 10 min, and the supernatants were collected and stored at −80°C. Total protein (TP) was quantified using a bicinchoninic acid protein assay kit (iNtRON Biotechnology, Seongnam, Korea), and LDH assay was conducted using a cytotoxicity assay kit (Daeillab Service Co., Seoul, Korea). Analyses of the tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-8, and IL-1β expression were conducted using enzyme-linked immunosorbent assay kits provided by R&D Systems (Minneapolis, MN, USA) to evaluate the variation in inflammatory cytokine levels. After centrifugation, BALF pellets were collected, and the number of cells in the BALF samples was counted using a Vi-Cell^® XR analyzer (Beckman Coulter, Brea, CA, USA). Fifty microliters of the ready-made samples were diluted to 2 × 10⁵ cells·mL⁻¹ and injected into the slide set of a Shandon Cytospin (Shandon, Pittsburgh, PA, USA). The slides were stained with Diff-Quick (Fisher Scientific, Swedesboro, NJ, USA).

Histopathological analysis

Following a single ITI of NaDCC, the animals were sacrificed under anesthesia, and after gross examination, the excised liver, lungs, and nasal cavities of each animal were fixed in 10% neutral buffered formalin. The nasal cavities were decalcified, then cross-sectioned at three levels: section-level I (immediately posterior to upper incisor teeth), section-level II (between the incisive papillae and first palatal ridge), and section-level III (middle of molar teeth). Following the routine tissue processing, the tissues were embedded in paraffin and sectioned in 3-μm thickness. The tissue sections were then stained with hematoxylin and eosin (H&E) for histological examination under a light microscope (Olympus BX41, Tokyo, Japan). The lesions were individually graded depending on their severity by the pathologist.

Statistical analysis

The results are expressed as the mean ± standard error (SE). Nonparametric statistical tests were used to compare the NaDCC-treated groups with the control groups. The values were analyzed using one-way multiple variances of analysis (Tukey’s multiple comparison test) or by two-tailed unpaired Student’s t-test using GraphPad Prism Version 5.01 (GraphPad Software Inc., USA). Statistical analysis was used to compare the exposure groups with the control group to determine whether the p-value was less than 0.05, 0.01, or 0.001.

Results

Body weight and blood biochemistry

The weights of the rats and food consumption were measured throughout the experiment; however, no significant differences were observed between the intratracheally exposed and control groups. No differences in clinical signs were observed between the NaDCC-instilled rats and controls. Blood samples were collected, and the serum and hemocyte samples were analyzed using indicator markers. The results showed no significant differences between the control and the NaDCC exposure group in most indicator markers. However, blood urea nitrogen, the marker most frequently used to evaluate renal function, was altered in the low-exposure group at 1 d after exposure compared with the control group (results not shown). This change was observed only in the low-exposure group, indicating no dose-dependent relationship between the groups; thus, it may be concluded that the change was not caused by exposure to NaDCC.

Cells in bronchoalveolar lavage fluid

The number of cells in bronchoalveolar lavage fluid (BALF) dose-dependently increased 1 d after NaDCC instillation, indicating a 1.7-fold increase in the high-exposure group, although the rate of increase was reduced 14 d after instillation (Figure 1(b)). The percentage of polymorphonuclear leukocytes (PMN) also dose-dependently increased 1 d after instillation, indicating a 33-fold increase in the high-exposure group, with a slight increase at 14 d; however, no statistical differences were observed in the high-exposure recovery group compared to those present in the control group (Figure 1(a) and (c)).

Figure 1.

ulmonary inflammation induced by sodium dichloroisocyanurate (NaDCC) in rats. (a) Diff-quick staining of cells in BALF. (b) Total cell count in BALF. (c) Percentage of PMNs in BALF. Male rats were exposed to NaDCC at 0, 100, 500, and 2500 μg·kg⁻¹ body weight by intratracheal instillation. Values are reported as means ± SE (Student’s t-test, *, **, ***p < 0.05, 0.01, 0.001, respectively, vs control). BALF: bronchoalveolar lavage fluid; NaDCC: sodium dichloroisocyanurate; PMN: polymorphonuclear leukocyte.

Effect of NaDCC on BALF biochemical parameters

BALF was collected, and the TP levels were determined (Figure 2(a)). The TP level 1 d after NaDCC exposure increased significantly in the high-exposure group (6.2-fold compared with the control group). The low- and middle-exposure groups also showed significantly increased expression. However, although the TP level increased 14 d after exposure, near full-recovery was attained in the high-exposure group, compared to 1 d after exposure. LDH increased significantly 1 d after instillation, indicating a 2.2-fold increase in the high-exposure group, and the recovery was confirmed at 14 d after instillation, similar to the trend observed with TP level (Figure 2(b)).

Figure 2.

Changes in pulmonary injury biomarker levels in the broncheoalveolar lavage fluid (BALF) following intratracheal instillation (ITI) of sodium dichloroisocyanurate (NaDCC) in rats. (a) Total protein in BALF. (b) LDH activity in BALF. Male rats exposed to NaDCC at 0, 100, 500, and 2500 μg·kg⁻¹ body weight by intratracheal instillation. Values are reported as means ± SE (Student’s t-test, *, **, ***p < 0.05, 0.01, 0.001, vs. control). BALF: bronchoalveolar lavage fluid; LDH: lactate dehydrogenase.

The cytokines in BALF were measured, and the levels in the exposure group were compared to those in the control group. No statistical differences were observed in IL-1β and TNF-α levels (Figure 3, (c)) between any exposure or recovery groups. A significant increase in IL-6 was observed in the high-exposure group in which BALF samples were collected 1 d after treatment; in contrast, the level of IL-8 was increased in the middle- and high-exposure recovery groups (Figure 3(b) and (d)). There was a dose-dependent increase in IL-8, a well-known cytokine that recruits and activates neutrophils during inflammation.^23–25 Significant increases were observed in the middle- and high-exposure recovery groups (samples collected at 14 d after treatment), although the increase was less than 10% compared to the levels in the control group (Figure 3(b)).

Figure 3.

Variations in pulmonary cytokines in broncheoalveolar lavage fluid (BALF), induced by sodium dichloroisocyanurate (NaDCC) in rats. (a) Tumor necrosis factor-alpha (TNF-α), (b) Interleukin-8 (IL-8), (c) Interleukin-1beta (IL-1β), (D) Interleukin-6 (IL-6). Male rats exposed to NaDCC at 0, 100, 500, and 2500 μg·kg-1 body weight by intratracheal instillation. Means ± SE (Student’s t-test, *, **p < 0.05, 0.01 vs control). BALF: bronchoalveolar lavage fluid; NaDCC: sodium dichloroisocyanurate.

Histopathological observation

After the autopsy, rat livers and lungs were collected, and a histopathological examination was performed. There were no specific abnormal findings in the livers of the exposure group. In a few livers of the recovery group, multifocal infiltration of mononuclear cells or lymphocytes was observed around the portal area or central veins or in the portal triad (Figure 4). However, the lesions were not related to NaDCC instillation because they were randomly observed in the control group also, irrespective of NaDDC exposure (Table 2).

Figure 4.

Histopathology of rat livers in recovery groups.

Multifocal mononuclear cell infiltration (arrows) was noted around the portal area (p) or central veins (c). Note the lymphocytic cell infiltration (arrow) in the portal triad (p) in Recovery-Middle group. Those lesions were not related to the NaDCC. Male rats were exposed to NaDCC at 0, 100, 500, and 2500 μg·kg⁻¹ body weight by intratracheal instillation. H&E. Bars = 50 μm for Recovery-Low and Recovery-Middle, 25 μm for Recovery-Control and Recovery-High. NaDCC: sodium dichloroisocyanurate.

Summary of histopathological lesions in the lungs of the exposure and recovery groups are demonstrated in Table 1 and Table 2.

Table 1.

Summary of histopathological lesions in the lungs of the exposure group.

Histopathology	Control	Low	Middle	High
No. examined (%)	5	5	5	5
No specific lesion	1(20)	1(20)	3(60)	3(60)
Inflammatory foci, bronchioloalveolar, necrotizing	1(20)	0(0)	2(40)	1(20)
Grades: minimal	1	0	0	0
mild	0	0	1	0
moderate	0	0	1	1
Pulmonary edema, diffuse	0(0)	0(0)	0(0)	1(20)
Grades: severe	0	0	0	1
Foamy macrophage aggregates, multifocal	0(0)	1(20)	0(0)	0(0)
Grades: minimal	0	1	0	0
Hemoglobin pneumonia, alveolitis, focal	1(20)	0(0)	0(0)	0(0)
Grades: minimal	1	0	0	0
Cell infiltration, mononuclear or mixed cells, perivascular	2(40)	0(0)	0(0)	1(20)
Grades: minimal	2	0	0	1
Vascular mineralization, (multi)focal	1(20)	3(60)	0(0)	0(0)
Grades: minimal	1	3	0	0

Table 2.

Summary of histopathological lesions in the lungs and livers of the recovery group.

Organ/Histopathology	Group	Control	Low	Middle	High
Lung	No. examined	5	5	5	5
No specific lesion		2(40.0)	5(100)	2(40.0)	4(80.0)
Desquamation of bronchiolar epithelial cells, focal		0(0.00)	0(0.00)	0(0.00)	1(20.0)
Grades: minimal		0	0	0	1
Foamy macrophage aggregates, multifocal		0(0.00)	0(0.00)	1(20.0)	0(0.00)
Grades: minimal		0	0	1	0
Hemoglobin pneumonia, alveolitis, focal		1(20.0)	0(0.00)	0(0.00)	0(0.00)
Grades: minimal		1	0	0	0
Cell infiltration, mononuclear cells or mixed cells, perivascular		2(40.0)	0(0.00)	2(40.0)	1(20.0)
Grades: minimal		2	0	2	1
Vascular mineralization, (multi)focal		1(20.0)	0(0.00)	1(20.0)	0(0.00)
Grades: minimal		1	0	1	1
Liver	No. examined	5	5	5	5
No specific lesion		3(60.0)	2(40.0)	3(60.0)	1(20.0)
Cell infiltration, mononuclear cells, multifocal		2(40.0)	2(40.0)	0(0.00)	1(20.0)
Grades: minimal		2	2	0	1
Cell infiltration, lymphocytic, portal		1(20.0)	1(20.0)	2(40.0)	3(60.0)
Grades: minimal		1	1	2	3

Inflammatory foci were observed around the terminal bronchioles and alveoli in a few rats with the NaDCC instillation (Figure 5). In the rat lungs of the middle-exposure group, bronchiolar epithelial cells were hypertrophic, and some cells showed mitotic features. The inflammation in the lungs indicated bronchopneumonia, extending from the airways exposed to NaDCC. The epithelial cells of the bronchioles affected by the NaDCC instillation were necrotized and exfoliated to obliterate their lumina, and inflammatory cells, mainly mononuclear cells, were heavily infiltrated in the pulmonary parenchyma around the necrotizing airways (Figure 5).

Figure 5.

Histopathology of rat lungs in the NaDCC exposure groups. Note the inflammatory foci (circles in the left figures) around terminal bronchioles (b) and alveoli (a), characterized by infiltration of inflammatory cells. In the high exposure group, note the necrotic cell debris mass within a terminal bronchiole in the inflammatory foci (n). Male rats were exposed to NaDCC at 0, 100, 500, and 2500 μg·kg⁻¹ bodyweight by intratracheal instillation. H&E. Bars = 50 μm for Middle-1(a), Middle-4(a), and High(a) and 25 μm for Middle-1(b), Middle-4(b), and High(b). NaDCC: sodium dichloroisocyanurate.

Pulmonary edema was also observed in an animal of the high-exposure group of NaDCC. Those lesions were not evident in the non-treated rat lungs and have not been defined as background lesions in laboratory animals. The minimum grade of perivascular infiltration of mononuclear cells, which is often noted in untreated rat lungs, was observed in a few rats of the control group and the high-exposure group of NaDCC. Multifocal histiocytosis, characterized by aggregations of foamy macrophages and focal mineralization in an arterial wall, was observed as background lesions without a relation to the exposure to NaDCC (Table 2).

In Recovery-Control, note the mononuclear cell infiltration (thick arrow) around arteries (v). In Recovery-Middle, note focal aggregation of foamy macrophages (arrows). In Recovery-High(a) and Recovery-High(b), focal chronic inflammation with thickened alveolar walls is visible around terminal bronchioles (circles). In Recovery-High(a), note desquamated bronchiolar epithelial cells within terminal bronchiole (arrow). Male rats were exposed to NaDCC at 0, 100, 500, and 2500 μg·kg⁻¹ body weight by intratracheal instillation. a, alveolus; b, bronchiole. H&E. Bars = 50 μm for all. NaDCC: sodium dichloroisocyanurate.

In the recovery group, multifocal chronic inflammation was evident around the terminal bronchioles in a rat lung of the high-exposure group of NaDCC, characterized by the thickened alveolar walls with mononuclear cell infiltration. Foamy cells, presumably desquamated bronchiolar epithelial cells or macrophages, were often present, attached to the bronchiolar walls, or detached within the lumina of the bronchioles (Figure 6). Those lesions could be associated with the chronic recovery phase of the bronchioloalveolar inflammation with necrotizing bronchiolitis that occurred by exposure to NaDCC. A minimum grade of perivascular infiltration of mononuclear cells and focal mineralization on an arterial wall were also noted as other lesions, but these were not related to the exposure to NaDCC.

Figure 6.

Histopathology of rat lungs in the recovery group.

Discussion

Chlorine-releasing disinfectants such as NaDCC are characterized by fast microbiocidal activity, cost-effectiveness, and durable effects; they oxidize membrane proteins of bacteria, fungi, and viruses. Therefore, NaDCC is widely used as an antibacterial in homes, offices, industries, and hospitals.^3,26–28 It is also used as a typical, effective disinfectant, especially for drinking water sterilization⁴; however, its use is controversial in South Korea because vapor inhalation from the chemical solution used in the disinfection of humidifiers has caused severe pulmonary disorders. A two-week study on NaDCC inhalation in rats documented that the main target organ, the upper respiratory tract, showed the degeneration of the transitional epithelium in the nasal cavity, as well as inflammation, and squamous metaplasia in the larynx, due to acute irritation. However, adverse effects were not observed in the recovery groups, indicating that the effects were reversible.²⁹

In this study, SD rats were exposed to NaDCC by ITI. Although this method has limitations in that the experimental chemical may not be uniformly injected and instilled into the lungs and could show varying results compared with inhalation, it may be useful as a substitute for inhalation in laboratories that do not have inhalation chambers.³⁰ Compared with the inhalation method, ITI has the advantage of measuring how much test material enters the lungs, and due to its simpler exposure process, it reduces potential harm to laboratory workers handling toxic test substances.³¹

In a previous report, SD rats exposed to DDAC for 13 weeks in a whole-body exposure chamber showed an increase in lung weight, and inflammatory cell infiltration and interstitial pneumonia were partially observed in the middle- and high-exposure groups.¹⁷ However, consistent with our observations in this study, severe histopathological symptoms such as proteinosis or fibrosis were not observed.

A study on the pulmonary toxicity of a mixture of benzalkonium chloride and triethylene glycol administered to rats via ITI demonstrated that exposure increased mixture-recruited inflammatory cells such as PMNs.¹⁸ Similarly, Kwon et al. reported that rats intratracheally instilled with a mixture of DDAC and ethylene glycol showed a significant increase in BALF neutrophils 1 d after treatment, and the inflammatory response was resolved 7 d after instillation, similar to the variation of TP in BALF, a biomarker of alveolar-capillary barrier permeability.³² Variations in PMNs and TP in BALF are sensitive indicators in instillation and short-term inhalation studies.³³ For example, Lim and Chung demonstrated that after 2 weeks of inhalation exposure to DDAC, the high-exposure groups showed a significant increase in PMN count and albumin concentration in the BALF compared with the controls.³⁴ Combining the results of previous biocide toxicity-related reports with this study, we determined that the ITI of NaDCC showed a similar PMN and TP pattern. Measuring LDH leakage in the BALF is essential to identify pulmonary damage at the cellular level because it is a sensitive indicator of cellular injury. Additionally, LDH activity and its isoenzyme pattern in the BALF provide direct information on epithelial cell injury throughout the airways from the trachea to the alveoli.³⁵ Our results demonstrated that NaDCC instillation damaged lung cells, although recovery occurred after 14 d.

The level of bronchoalveolar IL-6, a pro-inflammatory cytokine, has been identified as an early biomarker of lung injury correlated with lung failure.^36,37 In this study, the significant increase in IL-6 in the high-exposure group was correlated with TP and LDH variations in the BALF. In a previous report, the level of IL-6 in the BALF in mice increased after ITI treatment with 150 μg·kg⁻¹ of DDAC, and the induced level decreased after peaking 7 d after treatment.¹⁶ This result was consistent with that of the exposure group after treatment with NaDCC, in which the induced level of IL-6 increased significantly in the high-exposure group and recovered 14 d after instillation.

Histopathological analysis revealed that ITI of NaDCC in rats did not induce any toxic effects on the liver, also supported by no changes in the liver injury-related enzymes such as ALT and AST. However, ITI of NaDCC was found to induce necrotizing bronchiolitis and bronchioloalveolar inflammation around the airways exposed to NaDCC. Future studies with repeated NaDCC instillation are necessary to confirm its chronic effects in rats.

Conclusions

Rats were intratracheally instilled with 100, 500, and 2500 μg·kg⁻¹ body weight of NaDCC in the low-, middle-, and high-exposure groups, respectively. An autopsy was performed, and samples were collected 1 d and 14 d after exposure. LDH, TP, total cell count, and PMN were significantly increased in the low-, middle-, and high-exposure groups compared with the control group. However, after the 14-d recovery period, these increases were fully reversed in the LDH and total cell count, and recovery was observed in the levels of TP and PMN to some extent. IL-6 in the exposure group and IL-8 in the recovery group increased in the BALF. Histopathological examination revealed inflamed foci, bronchioloalveolar necrosis in the middle- and high-exposure groups, and pulmonary edema in the high-exposure group. Multifocal chronic inflammation was observed in the high-exposure recovery group, accompanied by thickened alveolar walls.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

This study was conducted with the support of the National Institute of Environmental Research (NIER-2017-01-01-011), Republic of Korea, and was approved by the Institutional Animal Care and Use Committee of the National Institute of Environmental Research (NIER-17-4, October 18, 2017) (Republic of Korea).

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Ilseob Shim

Appendix

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