Pulmonary changes induced by trans,trans-2,4-decadienal, a component of cooking oil fumes

Animal study protocol and design

In this study, 54 6-week-old male CD-1® (ICR) mice (Charles River Laboratories, Wilmington, DE, USA) mice were used. All animals were purchased from BioLASCO (Taiwan) and were housed at the Animal Facility Center at the National Health Research Institutes in Taiwan in accordance with standard and approved protocols at the facility (23±1°C, 39–43% relative humidity; water and food were available ad libitum).

ICR mice were used in this study because the general information on their pulmonary status (anatomy, histology and pathology) is well established 17, and we have previous experience using this strain of mouse. In the present study, mice were divided into three groups: 21 mice in the vehicle group (controls), 10 in the low-dose (8 mg·kg⁻¹ body weight tt-DDE) and 23 in the high-dose group (24 mg·kg⁻¹ body weight tt-DDE). Mice in the control group were intratracheally instilled with 50 μL vehicle solution, which was prepared as 7.5% v/v tricaprylin (Sigma Chemical, St. Louis, MO, USA) dissolved in 0.9% sodium chloride. Mice in the tt-DDE-exposed groups were intratracheally instilled with either 8 or 24 mg·kg⁻¹ body weight tt-DDE per week for 8 weeks under isoflurane anaesthesia.

The selection of a working dose regimen for our present study was based on a prior dose–response study. A dosage of ≤24 mg·kg⁻¹ tt-DDE was found to be relatively safe for ICR mice during the period of 8-week exposure. All animals survived with relatively good health with good body weight gain compared to the controls. The duration of exposure for our present study was determined in accordance with our previous in vitro studies, which lasted ∼8 weeks 15, 16. In the present in vivo study, we aimed to expose the animals for approximately the same duration of time in order to compare the in vitro and in vivo studies, if needed. To ensure that the final dosage was biologically effective, animals were sacrificed 1 week after final intratracheal administration (ninth week of the experiment). The overall experimental design and treatment scheme are shown in figure 1⇓.

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Fig. 1—

Experimental design and schedule for intratracheal instillation treatment in the animal study. All animals (vehicle (n = 21) and trans,trans-2,4-decadienal (tt-DDE) at a dose of 8 mg·kg⁻¹ (n = 10) and 24 mg·kg⁻¹ (n = 23)) were treated weekly for 8 weeks. Animals were sacrificed 1 week (ninth week) after the last treatment.

Preparations and evaluations of bronchoalveolar lavage fluids and necropsy specimens

Animals were sacrificed via isoflurane inhalation to ensure no undue suffering. At necropsy, whole lung was dissected out surgically and was lavaged with 1 mL saline. The recovered amount of lavagate was recorded and saved in individually labelled bottles.

To assess the inflammatory response in lung tissues induced by tt-DDE treatment, the total cell numbers and cell types in the bronchoalveolar lavage fluid (BALF) from the animals were determined with a cell counter (Coulter Inc., Miami, FL, USA). The BALF was cytospun at 450×g for 15 min using a Shandon Cytospin 4 (Thermo Scientific, Waltham, MA, USA). The cytospin smear was then prepared and Liu’s staining (Tonyar Biotech, Tao Yuan, Taiwan) was performed to distinguish different cell types. Cells in the BALF were classified into two main categories: epithelial cells and leukocytes (including macrophages, neutrophils and lymphocytes). The cytology was separately evaluated and scored by two independent pathologists. Data presented represent the mean of two scorings.

Measurement of reduced glutathione and glutathione disulfide in mouse lungs

Glutathione (GSH) was quantified by reacting with o-phthalaldehyde, which produced a fluorescence product with excitation and emission at 365 and 430 nm, respectively 18. In brief, lung tissues were homogenised with homogenisation buffer (154 mM KCl, 5 mM diethylenetriaminepentaacteic acid and 0.1 M potassium phosphate buffer, pH 6.8), diluted with equal volume of redox quenching buffer (10% trichloroacetic acid, 40 mM HCl, 10 mM diethylenetriaminepentaacteic acid and 20 mM ascorbic acid) and centrifuged at 14,000×g for 15 min at 4°C. For quantification of GSH, the supernatant was incubated with 2.5 mg·mL⁻¹ o-phthalaldehyde in redox quenching buffer. For quantification of total GSH, the supernatant was incubated with 2.5 mg·mL⁻¹ o-phthalaldehyde in redox quenching buffer containing 100 mM dithionite. Samples were incubated at room temperature for 30 min and the fluorescence was measured at excitation and emission at 365 and 430 nm, respectively 18. The amount of GSH disulfide (GSSG) was calculated according to the following formula: GSSG  =  (total glutathione−GSH)/2.

Quantifications of pathological changes at the bronchioloalveolar junctions induced by tt-DDE

Formalin-fixed and paraffin-embedded lung tissues were serially sectioned at 3-μm thickness. The first, sixth and eleventh consecutive sections from each mouse lung were randomly selected and stained with haematoxylin and eosin for histological examinations. Masson’s trichrome stain was also performed to demonstrate and confirm fibrotic status on bronchioloalveolar junctions (BAJ) lesions.

The BAJ lesions in each group were assessed in two parameters: frequency (percentage of occurrence) and extent of involvements (size of the lesions). The fibrotic status on BAJ lesions was quantified as the percentage of BAJ lesions with increased fibrous (collagen) deposit. A minimum of three tissue sections from each animal per study group were examined and evaluated. The percentage of BAJ lesions was calculated as the average number of BAJ lesions observed versus the total number of BAJs found in each study group (BAJ lesion/BAJ×100). The extensiveness or severity of the BAJ lesions were estimated by the “size” of the BAJ lesions that developed. The perimeters (area of each lesion) were traced and auto-evaluated quantitatively via a computer-assisted morphometric microscope with MetaMorph software (Molecular Devices, Downington, PA, USA). The percentage of the BAJ lesions with increased collagen deposit versus the total number of BAJ lesions found in each study group was calculated as the number of BAJ lesions with increased collagen deposit/total BAJ lesions×100. Statistical analysis was then performed via standard statistical methods as described in the Statistical analysis section.

Immunohistochemistry

In order to identify the specific cell type and protein expression involved in the BAJ pathology, immunohistochemistry was performed as previously described 19. The Universal LSAB2 kit and Chromogen DAB+ system (DakoCytomation, Glostrup, Denmark) was used for detecting the immunoreactivity with a single antibody. The following specific antibodies were used: anti-cytokeratin (CK; Chemicon, Huissen, the Netherlands) for bronchiolar epithelial cells; anti-Clara cell secretory protein (CCSP; Millipore, Billerica, MA, USA) for bronchiolar Clara cells; and anti-prosurfactant protein C (proSP-C; Chemicon) for alveolar type II cells. Cells involved in proliferation or growth (hyperplasia) were assessed by the immunostaining of proliferating cell nuclear antigen (PCNA; BD Transduction Laboratories, Lexington, KT, USA). The immunostaining of phosphorylated signal transducer and activator of transcription 3 (pSTAT3; Cell Signaling, Danvers, MA, USA) was used to detect the localisation of pSTAT3. Normal sera instead of primary antibodies were used as negative controls.

Double immunostaining

Double immunostainings were performed to determine the associations of proSP-C (alveolar type II cells) and PCNA/pSTAT3, or CCSP (Clara cells) and PCNA/pSTAT3 in the BAJ lesions. A multivision polymer detection system (Thermo Fisher Scientific, Fremont, CA, USA) was used in our study. The double immunostaining protocol was based on manufacturer’s procedures with minor adjustments on incubation conditions for primary antibodies. The incubation times for proSP-C, or CCSP and pSTAT3 were adjusted to 2 h and 16 h at room temperature, respectively, for optimal results for mice lung tissues.

Analysis of real-time RT-PCR array

RNA was extracted from lung tissues preserved in TriReagent (Life Technologies, Rockville, MD, USA). Synthesis of cDNA was performed using the high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Six representative lung cDNA samples were selected from vehicle- and 24 mg·kg⁻¹ tt-DDE-exposed mice. Between both groups, the relative expression of inflammatory genes was measured using mouse inflammatory cytokine and receptor PCR array (PAMM-011; Superarray, Frederick, MD, USA). The pathway analysis of the inflammatory gene expression was performed using Metacore™ (Genego, St Joseph, MI, USA).

Statistical analysis

Cell numbers/populations in BALF from vehicle, 8 mg·kg⁻¹ tt-DDE or 24 mg·kg⁻¹ tt-DDE-treated mice were compared via one way ANOVA. An unpaired t-test was used to compare data of PCR array from vehicle and 24 mg·kg⁻¹ tt-DDE-exposed groups. The linear regression analysis method was used to analyse the dose correlations on the severity or size of the BAJ lesions induced by tt-DDE. All statistical analyses were performed using the SPSS Base 10.0 software (SPSS, Chicago, IL, USA).

Changes in total cell numbers and differential cell populations in the BALF from animals exposed to tt-DDE

In the BALF, the total cell counts in mice exposed to 24 mg·kg⁻¹ tt-DDE were significantly increased by approximately two-fold when compared to that of controls (fig. 2a⇓). Differential cell counts of the BALF also showed that both epithelial and nonepithelial components (leukocytes) in 24 mg·kg⁻¹ tt-DDE-exposed mice were 2.3-fold of those in controls (fig. 2b⇓). The BALF of 24 mg·kg⁻¹ tt-DDE-treated mice showed a 2.5-fold increase in macrophages without significant elevation in neutrophil (polymorphonuclear cells) and lymphocyte counts (fig. 2c⇓). These parameters were not found to be significantly changed in the BALF of 8 mg·kg⁻¹ tt-DDE-exposed mice (fig. 2⇓).

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Fig. 2—

Changes in cell numbers among different cell populations in the bronchoalveolar lavage fluids (BALF) from mouse lungs at the ninth week of trans,trans-2,4-decadienal (tt-DDE) exposure. a) Total cell numbers in the BALF. b) Leukocytes and epithelial cells represented the two major populations of cells increased in the BALF. c) Among the leukocytic cell populations (macrophages, neutrophils and lymphocytes) in the BALF, only macrophages showed the most significant elevation in number. □: vehicle (n = 17); ▓;: 8 mg·kg⁻¹ tt-DDE (n = 11); ▪: 24 mg·kg⁻¹ tt-DDE (n = 23). *: p<0.05, represents significance and was analysed by the one way ANOVA when compared with vehicle group.

The oxidative stress status determined by the GSH/GSSG ratio

The GSH/GSSG ratio was used as a criterion to reflect the general oxidative stress status in mouse lungs. In vehicle groups, the GSH/GSSG ratio was 1.33±0.68. In 8 mg·kg⁻¹ and 24 mg·kg⁻¹ tt-DDE exposed groups, the GSH/GSSG ratios were 1.21±0.81 and 1.28±0.62, respectively. No significant alternation in the GSH/GSSG ratio was found in lung tissues of tt-DDE-exposed animals.

Pulmonary pathological changes induced by tt-DDE exposure

Two prominent lesions were found in the lungs of mice exposed to tt-DDE: epithelial hyperplasia and granulomatous nodules (fig. 3b⇓ and c). Both lesions were located at the BAJ and were much more remarkable in the high dose (24 mg·kg⁻¹) treated mice rather than the low dose (8 mg·kg⁻¹) treated animals (table 1⇓). Careful examination of these lesions revealed that the BAJ hyperplasia was characterised by nonciliated bronchiolar epithelial hyperplasia, lymphocytic infiltration and mild peri-lesional interstitial thickening possibly representing fibrosis (fig. 3b⇓ inset). Some of the BAJ hyperplasia could be seen to extend and develop into focal tissue mass or granulomatous nodules, which primarily comprised epithelial hyperplasia, lympohocytic infiltrates, epitheloid cell and macrophage aggregations, and fibroblasts (fig. 3c⇓). Special histochemical stain (Masson’s trichrome) also confirmed increased collagen deposits (mild fibrosis) in the interstitial areas of these BAJ hyperplastic sites and in the granulomatous nodules (fig. 3d⇓ and e). Linear regression analysis of the collected data revealed dose-correlated increases in collagen deposit, as well as in number and size of the BAJ lesions (hyperplasia and granulomatous nodules) in mice exposed to tt-DDE (table 1⇓).

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Fig. 3—

Pathological changes induced by intratracheal trans,trans-2,4-decadienal (tt-DDE) instillation in mouse lungs. a) Controls. Normal lung histology with bronchioloalveolar junctions (BAJs; arrows). b) Lung section from animal treated with tt-DDE (24 mg·kg⁻¹). Increased cellularity around many BAJs was noted (arrowheads). Inset: higher magnification of a BAJ from demonstrating BAJ hyperplasia, with increased cellular proliferation extending from the bronchiolar site into the alveolar area. Lymphocytic infiltration and mild fibrosis were also evident. c) A granulomatous nodule at the BAJ. This lesion can be seen to be physically continuous with the BAJ and was probably a tissue outgrowth from the BAJ hyperplasia. Careful examination revealed epithelial hyperplasia, epithelioid cell/macrophage aggregations, lymphocytic infiltration and fibroblasts in this lesion. d, e) Increased collagen deposits (blue colour) in the BAJ hyperplasia area (d) and in a bronchioalveolar granulomatous nodule (e) were demonstrated confirming mild fibrosis was present in these lesions. a–c) Haematoxylin and eosin stain; d, e) Masson’s trichrome stain. Scale bars: a, b) 500 μm; inset, d) 100 μm; c, e) 50 μm.

It is also important to affirm the occurrence of hyperplastic activity and to characterise the specific cell types of involvement in the BAJ lesions. Special histochemical stains were employed for such affirmation and characterisation. Immunohistochemically, the cells at BAJ hyperplasia demonstrated both CK and PCNA reactivities (fig. 4a⇓ and b), verifying that they were indeed of epithelial origin (CK positive) and hyperplastic (proliferative) in nature (PCNA positive). By means of CCSP (Clara cell marker) and proSP-C (alveolar type II cell marker) staining, both Clara cells and alveolar type II cells could be identified in the BAJ lesions (fig. 4c⇓ and d). However, by means of double staining with PCNA antibody (a proliferating cell marker), together with the other two cell markers (CCSP or pro-SP-C), only Clara cells, but not alveolar type II cells, were found to be actively proliferative in these BAJ lesions (fig. 4e⇓ and f). It is interesting to note that similar cell marker staining procedures revealed that only alveolar type II cells, not Clara cells, were present in the BAJ granulomatous nodules (fig. 5c⇓ and d).

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Fig. 4—

Identification of specific epithelial cell types involved in the trans,trans-2,4-decadienal (tt-DDE)-induced bronchioloalveolar junctions (BAJ) hyperplasia using immunohistochemical techniques. a) Cytokeratin immunostaining is a specific stain for epithelial cells. It demonstrates that all the hyperplastic cells involved in the BAJ hyperplasia were epithelial cells (arrows). b) Proliferating cell nuclear antigen (PCNA) immunostaining is a specific stain in nuclei of cells with hyperplastic activity. We have demonstrated that the increased epithelial cell mass observed in the BAJ were indeed hyperplastic (arrows). c) Clara cell secretory protein (CCSP) immunostaining is a specific stain for Clara cells. It demonstrates that many Clara cells (a member of bronchiolar epithelium) were involved in the BAJ hyperplasia. d) Prosurfactant protein C (proSP-C) immunostaining is a specific stain for alveolar type II cells. Proliferation of alveolar type II cells, which were more heavily stained than normal alveolar area and tended to form rows or clusters of cell aggregations (arrow), were demonstrated in the BAJ hyperplasia lesions. e) Double immunostaining with CCSP (marker for Clara cells) and PCNA (marker for proliferating cells) in a BAJ hyperplasia lesion showed that many cells with blue cytoplasm (Clara cells) also demonstrated positive PCNA staining (brownish stained nuclei; arrows), indicating that many Clara cells in the BAJ hyperplasia were actively proliferative. f) Double immunostaining for proSP-C and PCNA in a BAJ granulomatous nodule did not show an association of PCNA positivity (proliferating activity) and alveolar type II cells (cells with blue cytoplasm). This finding indicates that although alveolar type II cells were involved in BAJ granulomatous development, they were not actively proliferating in the granulomas. a–d) Counterstaining was performed with haematoxylin. Scale bars: a–d) 100 μm; e, f) 20 μm.

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Fig. 5—

The immunohistochemical staining of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) in the trans,trans-2,4-decadienal (tt-DDE)-induced bronchioloalveolar junctions (BAJ) hyperplasia. a) Some cells in the BAJ hyperplasia displayed positive immunoreactivity (light brown colour) of pSTAT3 in the nuclei (arrows). The counterstain was haematoxylin. b) Double immunostainings for Clara cell secretory protein (CCSP) (Clara cells) and pSTAT3. All the Clara cells (dark blue-brown cytoplasm) were shown to have no brownish nuclear staining (arrows) indicating that they were not associated with pSTAT3 accumulation. pSTAT3 stained cells appeared to be isolated light brown nuclei without dark blue-brown cytoplasmic staining (circles). c) Double immunostainings for prosurfactant protein C (proSP-C) (alveolar type II cells) and pSTAT3. Many alveolar type II cells (dark blue cytoplasm) in a BAJ granulomatous nodule showed brown staining nuclei (arrows) indicating enhanced pSTAT3 in these cells. d) Double immunostainings for CCSP (Clara cells) and pSTAT3 in a BAJ granulomatous nodule. All pSTAT3 stained cells only showed brownish nuclei without dark blue cytoplasmic staining indicating that cells with enhanced pSTAT3 accumulation were not Clara cells. The pSTAT3-positive cells without cytoplasmic staining were probably alveolar type II cells (as shown in c). No Clara cell (cells with positive CCSP staining) was in fact identified in the BAJ granulomatous nodule. Scale bars: a, b) 50 μm; c, d) 20 μm.

Effects of tt-DDE exposure on gene expression of inflammatory cytokines and chemokines

Utilising the real-time RT-PCR array, we further identified modulation in several inflammatory cytokines and chemokines in lung tissues of animals exposed to tt-DDE. Chemokine (C-C motif) ligand (CCL)1, CCL11, CCL12, chemokine (CXC motif) ligand (CXCL)13 and cytokine IL-10 were significantly upregulated, whereas CCL2 and IL-1β were downregulated in the tt-DDE-treated mice (table 2⇓). In contrast, three receptors, CCLR1, IL-R1-II and IL-8R were downregulated by tt-DDE exposure (table 2⇓). To further clarify the potential downstream regulator or factor that could be affected by these tt-DDE-induced inflammatory genes, we used Metacore™ (Genego) to perform pathway analysis on these inflammatory genes. pSTAT3 was identified as the common downstream factor activated by CCL1, CCL11, CCL12, CXCL13 and IL-10, which were all significantly elevated by tt-DDE.

Enhancement of pSTAT3 in cells in the BAJ lesions

To confirm the involvement of pSTAT3 in the BAJ lesions, we further performed an immunohistochemical staining for pSTAT3 protein in lung tissues from tt-DDE-treated mice. Strong nuclear staining of pSTAT3 was demonstrated in many cells in the BAJ lesions (fig. 5a⇑). Via double immunostainings of pSTAT3 with CCSP (marker for Clara cells) or proSP-C (marker for alveolar type II cells), we further demonstrated that the enhanced pSTAT3 accumulation was not associated with Clara cells (fig. 5b⇑), but was strongly associated with alveolar type II cells (fig. 5c⇑) in the BAJ lesions. It is also important to note that, although Clara cells were abundant in the BAJ hyperplasia lesion (fig. 5b⇑), they were not found in the BAJ granulomatous nodules (fig. 5d⇑).