Toxicology Letters 282 (2018) 1–7
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8-Isoprostane is an early biomarker for oxidative stress in chlorine-induced acute lung injury
MARK
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Linda Elfsmarka, , Lina Ågrena, Christine Akfura, Anders Buchta,b, Sofia Jonassona a b
Swedish Defence Research Agency, CBRN Defence and Security, Umeå, Sweden Department of Public Health and Clinical Medicine, Unit of Respiratory Medicine, University Hospital, Umeå, Sweden
A R T I C L E I N F O
A B S T R A C T
Keywords: Chlorine Biomarkers Inflammation Acute lung injury 8-Isoprostane
Inhalation of chlorine (Cl2) may cause oxidative acute lung injury (ALI) characterized by pulmonary edema, pneumonitis, and hyperreactive airways. The aim of the study was to identify possible biomarkers for Cl2induced ALI. Female BALB/c mice were exposed to Cl2 for 15 min using two protocols 1) concentration-dependent response (25–200 ppm) and 2) time-kinetics (2h–14 days post-exposure). Exposure to 50–200 ppm Cl2 caused a concentration-dependent inflammatory response with increased expression of IL-1β, IL-6 and CXCL1/KC in bronchoalveolar lavage fluid 2–6 h after exposure which was followed by increased lung permeability and a neutrophilic inflammation 12–24 h post-exposure. The early inflammatory cytokine response was associated with a clear but transient increase of 8-isoprostane, a biomarker for oxidative stress, with its maximum at 2 h after exposure. An increase of 8-isoprostane could also be detected in serum 2 h after exposure to 200 ppm Cl2, which was followed by increased levels of IL-6 and CXCL1/KC and signs of increased fibrinogen and PAI-1. Melphalan, a non-oxidizing mustard gas analog, did not increase the 8-isoprostane levels, indicating that 8-isoprostane is induced in airways through direct oxidation by Cl2. We conclude that 8-isoprostane represents an early biomarker for oxidative stress in airways and in the blood circulation following Cl2-exposure.
1. Introduction Chlorine (Cl2) is a powerful oxidizing gas extensively used as bleaching agent, disinfectant, and in the manufacture of industrial chemicals. Due to its high toxicity, Cl2 has historically been used as a chemical weapon and is still considered a terrorist threat and used in armed conflicts globally (United Nations General Assembly 2014; Hemstrom et al., 2016). Inhalation of Cl2 can produce a range of acute pulmonary effects including impaired lung function, inflammatory reactions, increase of epithelial permeability, and airway hyperresponsiveness (AHR) (Evans, 2005; Koohsari et al., 2007; Morris et al., 2005; Tuck et al., 2008; White and Martin, 2010; Williams, 1997). In some individuals the acute lung injury (ALI) evolves into long-term respiratory manifestations, collectively named reactive airway dysfunction syndrome (RADS), that are characterized by persistent cough, wheezing, chest tightness and dyspnea (Brooks et al., 1985). Not all exposed individuals develop long-standing effects like RADS and pulmonary fibrosis, and the biological mechanisms behind the pathological changes still remain unsolved (Lemiere et al., 1997). However,
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predicting the outcome of long-standing effects, like RADS, in a patient at an early stage is not possible. Therefore, there is a need of an early non-invasive specific diagnostic tool for ALI that enables monitoring the prognosis of delayed effects after exposure and predicting the individual need of early medical treatment to avoid severe consequences such as RADS. We have previously demonstrated that mice and rats exposed to 200–300 ppm Cl2 develop a neutrophilic pulmonary injury manifested by e.g. edema, cardiovascular effects, pulmonary fibrosis and longstanding AHR in close agreement with clinical findings in humans exposed to Cl2 (Jonasson et al., 2013a, 2013b; Wigenstam et al., 2016, 2015). The acute symptoms of inhaled Cl2 are generally limited to the exposed tissues (Evans, 2005; Kales and Christiani, 2004) but there is also evidence that Cl2 induces systemic inflammatory responses (induced IL-8/KC and IL-6) and cardiovascular effects, e.g. alterations in blood coagulation and fibrinolysis (Luo et al., 2014; Wang et al., 2006; Wigenstam et al., 2015; Yadav et al., 2011; Zarogiannis et al., 2014). From a medical point-of-view, interference with the early response is of significance since treatment with corticosteroids within the first hours
Corresponding author at: CBRN Defence and Security, Swedish Defence Research Agency, SE-901 82, Umeå, Sweden. E-mail address: [email protected] (L. Elfsmark).
http://dx.doi.org/10.1016/j.toxlet.2017.10.007 Received 10 May 2017; Received in revised form 4 October 2017; Accepted 6 October 2017 Available online 07 October 2017 0378-4274/ © 2017 Elsevier B.V. All rights reserved.
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after a Cl2-exposure is shown to reduce long-term lung injury (Jonasson et al., 2013b; Wigenstam et al., 2015). Based on previous results, a mouse model of Cl2-induced lung injury was used to investigate early markers of oxidative stress, inflammatory response, disruption of lung barrier integrity, and blood coagulation. As a marker of lipid peroxidation we used the arachidonic acid derivate 8isoprostane (8-isoPGF2α), which is recognized to be a reliable marker of oxidative stress in airways (Roberts and Morrow, 2000). As early markers of inflammatory response the expression of cytokines associated with induction of acute inflammation was analyzed in bronchoalveolar lavage fluid (BALF) and serum (Hosseinian et al., 2015; Ware et al., 2013). The degree of ALI was determined using markers of lung barrier integrity and counting of inflammatory cells in airways. To assess effects on blood coagulation and fibrinolysis we analyzed plasminogen activator inhibitor (PAI-1) and fibrinogen, previously reported by us to be increased in serum of mice exposed for high concentrations of Cl2 (Wigenstam et al., 2015). By using a concentration range of Cl2 (25–200 ppm), concentration-dependencies of the biomarker expression was assessed e.g. whether the increased formation and their appearance in BALF and serum could be associated with ALI and disease severity. The high-level concentration of Cl2, 200 ppm, was considered to be relevant for a scenario of a chemical disaster at an industrial plant or during transportation, resulting in life-threatening lung injuries. Such chemical disaster would also include humans exposed to lower concentrations displaying no observable acute symptoms during the first 24 h although having a potential risk to develop delayed effects. To study the specificity of the oxidative and inflammatory markers, the responses in the Cl2-induced model were compared with the responses in models of chemical-induced ALI induced by the nitrogen mustard analog, melphalan. Similar to Cl2, melphalan induces an acute airway inflammation when exposed to the lung but without a prominent direct chemical-induced oxidation in target tissues. Studies have shown that melphalan is a possible surrogate for nitrogen mustard causing a neutrophilic airway inflammation with AHR in the acutephase (1–2 days post-exposure) which turns into a lymphocytic inflammation in absence of AHR in the sub-acute phase (after 14 days) (Ekstrand-Hammarstrom et al., 2011; Wigenstam et al., 2012, 2009). By the use of these mouse models, the present study was designed to determine the specificity of these biomarkers with the aim to identify markers in serum, BALF and exhaled breath condensate (EBC) that can potentially be used as a diagnostic tools to predict the risk of ALI and long-standing symptoms such as RADS.
Fig. 1. Collection of EBC. Experimental setup for collection of exhaled breath condensates (EBC) using a cold trap. Expired breath was sampled during 45 min from anesthetized and tracheostomized rats mechanically ventilated with a small animal ventilator.
range of Cl2 concentrations that were believed to cause mild irritation (no evident airway inflammation) to severe sub-lethal lung injury. In the concentration-response study, the mice were exposed to 25 ppm (6.3 ppm h), 50 ppm (12.5 ppm h), 100 ppm (25 ppm h) or 200 ppm (50 ppm h) Cl2 for 15 min (n = 5–7/group) and samples were collected at 2, 6 and 24 h after exposure. In the time-kinetic study the animals were exposed to 200 ppm Cl2 for 15 min (n = 5-7/group) and samples were collected at 2, 6, 12, 24, 48, and 72 h up to 14 days post-exposure. Rats were exposed to 200 ppm (50 ppm h) Cl2 for 15 min according to previously described protocol (Wigenstam et al., 2016) and EBC was collected 24 h after exposure (Fig. 1). Age-matched controls exposed to room air for 15 min were included at each time-point analyzed. 2.3. Collection of EBC Exhaled breath condensate was sampled from Cl2-exposed and control rats anesthetized with pentobarbital sodium (50 mg/kg body weight (b.w)). The rats were tracheostomized with a 15-gauge cannula and mechanically ventilated with a small animal ventilator (flexiVent™, SCIREQ®) at a frequency of 90 breaths/min and a tidal volume of 10 ml/kg b.w. A positive end-expiratory pressure of 3 cmH2O was applied and the rats were paralyzed with pancuronium (0.1 mg/kg b.w). Expired air from the ventilated animal was lead through a cold trap consisting of a pear-shaped glass flask for distillation with two necks immersed in ice (Fig. 1). The connection of the ventilator tubing to the glass flask was carefully sealed and EBC was collected during 45 min, yielding approximately 150 μl of condensate from each individual rat (n = 4–5/group). The condensates were immediately frozen and stored at −80 °C until analysis.
2. Materials and methods 2.1. Animals Female BALB/c mice and Sprague-Dawley rats (Envigo RMS B.V, Netherlands) were used in this study. After arrival, the animals were allowed to acclimatize for at least one week. All mice were between 10 and 11 weeks old when experiments were initiated and the rats were between 9 and 10 weeks old. The animals were housed under standard laboratory conditions (12 h daylight cycle, 22 °C, 30% relative humidity) and permitted free access to both food (R36, Lantmännen, Sweden) and water. The animals were weighted to monitor their health condition before the experiments and following exposure. The care of the animals and the experimental protocols were approved by the regional ethics committee on animal experiments in Umeå, Sweden.
2.4. Melphalan-exposure and sampling protocol Melphalan (4-[bis(2-chloroethyl)amino]-1-phenylalanine) (SigmaAldrich, St Louis, MO) was dissolved in phosphate-buffered saline (PBS, Sigma-Aldrich) according to the protocol described by Wigenstam et al. (Wigenstam et al., 2012). Melphalan (50 μl) was administered by intratracheal (i.t.) instillation to anesthetized mice at a dose of 1 mg/kg b.w (n = 6–7/group). Control mice received vehicle alone (n = 5–7 at each time-point analyzed). Samples were collected and evaluated at different time-points (2, 6, 12, 24, 48, and 72 h) after exposure.
2.2. Cl2-exposure and sampling protocol Animals were placed in a Battelle inhalation exposure tower for noseonly exposure (EMMS, UK) and were exposed to Cl2 (Air Liquide, Germany; mixture crystal, 0.1 mol-% Cl2, 99.9 mol-% nitrogen) according to previously described protocols (Jonasson et al., 2013a, 2013b; Wigenstam et al., 2016). Based on our previous studies we selected a
2.5. Serum sampling and collection of BALF Mice were anesthetized using isoflurane and blood was collected through orbital punction. After retrieving blood samples the animals 2
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2.10. Analysis of fibrinogen and PAI-1 in serum
were euthanized through cervical dislocation. Samples were kept in room temperature (RT) for 1 h before centrifugation (15 min, 2500 rpm) and the serum was stored at −80 °C for later analysis. Mice were then tracheostomized and BALF was collected by injecting and withdrawing 4 × 1 ml of ice cold Hank’s balanced salt solution (HBSS, Sigma-Aldrich). The BALF was centrifuged (1500 rpm, 10 min, 4 °C) and the supernatant was stored at −80 °C for later analysis. The number of leukocytes was counted in a Bürker chamber using trypan blue staining. From the BALF of each animal 20 000 cells were fixed on duplicate slides using a Cytospin® centrifuge (Shandon© cytospin 3 cyto-centrifuge, cell preparation system, Runcorn, UK) and stained with May-Grünwald-Giemsa reagents (Merck Millipore, VWR International, Sweden) before a differential count of total leukocytes (including macrophages, neutrophils, eosinophils and lymphocytes) was performed in a blinded manner, counting 300 cells per slide.
Fibrinogen was measured in serum (diluted 1:1000) using a Matched-Pair Antibody Set (Enzyme Research Laboratories, South Bend, IN) according to the manufacturer’s description. The absorbance that was read at 450 nm (Thermo Scientific Multiscan FC) was proportional to the concentration of fibrinogen in the sample. PAI-1 was quantified in serum (diluted 1:20) using a sandwich PAI-1 DuoSet ELISA Development kits (R & D Systems) according to the manufacturer’s description. 2.11. Statistical analysis All results were expressed as mean values ± standard error of the mean (SEM) (n = 5–7). Controls from each time-point were pooled when appropriate. Using the GraphPad Prism Version 6.0 (GraphPad Software, La Jolla, CA) data were analyzed by one-way-ANOVA followed by Dunnettś post hoc-test. Analysis of correlation between data sets were performed using Pearson correlation calculations. A statistical result of p < 0.05 was considered significant (*p < 0.05, ** p < 0.01 and ***p < 0.001).
2.6. Analysis of 8-isoprostane in BALF and serum 8-isoprostane was analyzed using a competitive 8-isoprostane (8-iso PGF2α) EIA Kit (Cayman Chemical Company, Ann Arbor, MI, USA), 50 μl of the first 2 ml collected BALF samples from each animal were analyzed and the assay was performed according to the manufacturer’s description. The absorbance were read at 405 nm using an ELISA reader (Thermo Scientific Multiskan FC, Thermo Fischer Scientific Oy,Vantaa, Finland). Results were analyzed in GraphPad Prism 6 as (% sample or standard bound/maximum bound) according to the manufacturer’s recommendations and the amount of 8-isoprostane was calculated from a standard curve. Serum samples were purified before analysis. In brief, diluted serum (1:5 in eicosanoid affinity column buffer) were run through an affinity 8-isoprostane sorbent column (Cayman Chemical Company). Bound 8-isoprostane was then eluted with ethanol and frozen. In connection with analysis the ethanol was evaporated and the samples were diluted to the original serum volume (200 μl) in HBSS according to the manufacturer’s description.
3. Results 3.1. Cl2-induced ALI 3.1.1. Inflammatory response in BALF A 15-min exposure to 25–200 ppm Cl2 triggered an early and a concentration-dependent release of pro-inflammatory cytokines in airways but did not cause any lethal effects. A significant increase of IL-6 was evident in BALF at 2 h after inhalation of 50, 100 and 200 ppm Cl2 (Fig. 2A). The neutrophil-chemoattractant CXCL1/KC was significantly increased at the early time-points for all Cl2 groups (Fig. 2B) while IL1β was significantly increased only in the highest exposure groups (100 and 200 ppm Cl2 groups) (Fig. 2C). In addition to IL-1β, all other cytokines investigated were also significantly increased at 6 h post-exposure in BALF in the 100 and 200 ppm Cl2 groups. In the 50 ppm Cl2 group, CXCL1/KC and IL-1β were also increased 6 h after exposure. A significant increase of neutrophils was observed in BALF within 6 h in the 50, 100 and 200 ppm Cl2 groups as compared to non-exposed animals. There were no significant differences in the extent of neutrophilic inflammation between the Cl2 groups (Fig. 3A). The increased number of neutrophils was sustained in BALF for 24 h for the 200 ppm group (Fig. 3D), while the number of neutrophils were reduced to control levels for both the 50 ppm and the 100 ppm Cl2 group at 24 h post-exposure (data not shown).
2.7. Analysis of SP-D and CC16 in serum Surfactant Protein D (SP-D) levels in serum (diluted 1:5) were determined using Mouse Pulmonary surfactant associated protein type D (SP-D) ELISA Kit (BlueGene Biotech, Shanghai, China) and Clara cell 16 levels in serum (diluted 1:50) were measured with Mouse Clara Cell specific protein (CC16) ELISA kit (BlueGene Biotech). Both assays were performed according to the manufacturer’s description. The absorbance were read at 450 nm using an ELISA reader (Thermo Scientific Multiskan FC, Thermo Fischer Scientific Oy,Vantaa, Finland). The concentration were calculated using standards provided by the manufacturer. 2.8. Analysis of albumin in BALF
3.1.2. Inflammatory responses in serum and lung/serum-specific proteins In association with the increased pulmonary infiltration of inflammatory cells there was also an increased permeability of the lungblood barrier as indicated by leakage of serum albumin into the alveolar space at 6–24 h post-exposure (Fig. 3B and E). Alteration in the integrity of lung-blood barrier was also evaluated through analysis of the lung-specific proteins SP-D and CC16 in serum but with inconsistent results, although a decrease in serum levels 24 h post-exposure was observed to be negatively correlated with increasing Cl2 concentrations (data not shown). Inhalation of Cl2 caused a concomitant early inflammatory response in serum with increased IL-6 and CXCL1/KC at 6 h in the two highexposure groups (Fig. 2D–E). At this time-point, IL-1β was the only analyzed cytokine upregulated in the 50 ppm group (Fig. 2F). A concentration-dependent alteration in the balance between thrombosis and fibrinolysis, with significant increases in both fibrinogen and PAI-1 was observed in serum 24 h after inhalation of 100 and 200 ppm Cl2 respectively (Fig. 3C).
Mouse Albumin ELISA kits were purchased from Bethyl Laboratories, (Montgomery, AL, US) and albumin content was determined in BALF (diluted 1:10.000) from each animal according to the manufacturer’s description. The absorbance were read at 450 nm using an ELISA reader (Thermo Scientific Multiskan FC). After generating a four-parameter logistic standard curve the albumin content could be quantified. 2.9. Analysis of cytokines in BALF and serum The concentration of IL-1β, IL-6 and CXCL1/KC in BALF and serum (diluted 1:2 and 1:3 respectively) from each animal was measured using specific DuoSet ELISA Development kits (R & D Systems) for each cytokine according to the manufacturer’s description. The absorbance were read at 450 nm using an ELISA reader (Thermo Scientific Multiskan FC) at 450 nm and the amount of each cytokine was quantified using a four-parameter logistic standard curve. 3
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Fig. 2. Pro-inflammatory markers. Early concentration-dependent activation of inflammatory markers after chlorine (Cl2)-exposure. Concentrations in bronchoalveolar lavage fluid (BALF) are shown for (A) IL-6, (B) CXCL1/KC and (C) IL-1β 2 h and 6 h post exposure and (D-F) concentrations in serum 6 h post-exposure from mice exposed to Cl2: (●) 25 ppm, (■) 50 ppm, (▲) 100 ppm or (□) 200 ppm. Values indicate means ± SEM. Statistical significant differences compared to control group are shown, *p < 0.05, ** p < 0.01 and *** p < 0.001.
to the large amount of serum required for the analysis. An increased amount of 8-isoprostane was also found in the EBC collected from rats 24 h after exposure to 200 ppm Cl2 (Fig. 4E).
3.1.3. Expression of 8-isoprostane in BALF, serum and EBC Inhalation of 100 and 200 ppm Cl2 caused a concentration-dependent release of 8-isoprostane in BALF, with significantly increased levels at 2 h, the earliest time-point for sampling (Fig. 4A). The amount of 8-isoprostane remained increased at 6 h after exposure to 50–200 ppm Cl2 (Fig. 4B). Analysis of 8-isoprostane during a time-course up to 14 days showed that maximum levels were detected 2 h after exposure (100 ± 11.3 pg/ml) and slowly declined to concentrations corresponding to non-exposed mice (6.3 ± 0.7 pg/ml) after 48 h (Fig. 4C). Formation of 8-isoprostane correlated with severity of the lung-blood barrier damage, measured as leakage of serum albumin (r2 = 0.48, p = 0.002) into the BALF. Increased concentrations of 8-isoprostane 2 h after 200 ppm Cl2 exposure could also be detected in serum (Fig. 4D). Further investigations of the time-course and the Cl2-exposure-response of 8-isoprostane in serum could not be performed due
3.2. Expression of 8-isoprostane in melphalan-induced ALI Intratracheal instillation of melphalan induced a pulmonary inflammation dominated by neutrophils (Fig. 5A). Analysis of IL-6, CXCL1/ KC and IL-1β revealed an early immune activation similar to the response after Cl2-exposure (data not shown). The short-term inflammatory response reached its maximum at 12–24 h after exposure to melphalan and was preceded by changes in vascular permeability 6 h post-instillation as indicated by significantly higher amount of serum albumin than in nonexposed animals (Fig. 5B). At 6 h post-instillation, melphalan induced a significant increase in PAI-1 but the amount of fibrinogen in serum was
Fig. 3. Chlorine-induced ALI. Chlorine (Cl2)-induced (A) infiltration of inflammatory cells and (B) serum albumin leakage in bronchoalveolar lavage fluid (BALF) from mice 6 h after exposure to 25, 50, 100 or 200 ppm Cl2. (C) Concentration-dependent changes of fibrinogen and PAI-1 levels in serum 24 h after Cl2-exposure. (D) Alterations in cellular infiltration and (E) serum albumin leakage in BALF from 2 h to 14 days post-exposure to 200 ppm Cl2. Values indicate means ± SEM. Statistical significant differences compared to control group are shown, * p < 0.05, ** p < 0.01 and *** p < 0.001.
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Fig. 4. Expression of 8-isoprostane. Concentration-dependent induction of 8-isoprostane (8-isoPGF2α) in mice exposed to 25–200 ppm chlorine (Cl2) in bronchoalveolar lavage fluid (BALF) (A) 2 h and (B) 6 h after exposure. (C) Time-kinetics of 8-isoprostane expression was analyzed in BALF from 2 h to 14 days after exposure to 200 ppm Cl2 and (D) the concentration of 8-isoprostane in BALF was compared with the concentration in serum from mice at the 2 h time-point. (E) The level of 8-isoprostane in exhaled breath condensate (EBC) from rats exposed to 200 ppm Cl2 24 h after exposure. Values indicate means ± SEM. Statistical significant differences compared to control group are shown, ** p < 0.01 and *** p < 0.001.
stress, inflammation and coagulation. Mice were exposed to different concentrations of Cl2 (20–200 ppm), thereby the concentration-dependency of the biomarkers could be assessed and correlated with disease severity. In addition, the time-kinetics for the appearance of the biomarkers were investigated by performing analyses in the interval between 2 h and 14 days post-exposure. Furthermore, the specificity of the biomarkers for Cl2-induced ALI was evaluated by comparing the
not elevated (Fig. 5C). Melphalan-exposure did not increase the levels of 8-isoprostan during a period of 72 h post-exposure (Fig. 5D). 4. Discussion The aim of this study was to identify predictive biomarkers of disease severity after Cl2-exposure by using early indicators of oxidative
Fig. 5. Melphalan-induced ALI. Melphalan-induced (1 mg/kg i.t.) immune activation and lung damage in mice 2 h to 72 h post-exposure. (A) The number of inflammatory cells in bronchoalveolar lavage fluid (BALF), (B) serum albumin leakage in BALF, (C) fibrinogen and PAI-1 in serum and (D) 8-isoprostane (8-isoPGF2α) in BALF. Values indicate means ± SEM. Statistical significant differences compared to control group are shown, * p < 0.05, ** p < 0.01 and *** p < 0.001.
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isoprostanes with GC/NICI-MS in lung tissue to assess lipid peroxidation in rats after exposure to 400 ppm Cl2 for 30 min. They observed a two-fold increase of F2-isoprostanes that was generated during Cl2-exposure and did not increase further after finalizing the Cl2-exposure (remained elevated at 6 h after exposure) (Yadav et al., 2011). One of their hypothesis was that Cl2 itself and/or its hydrolysis product HOCl is the likely mediator of arachidonic acid peroxidation. Another possibility is Cl2-induced dysfunction or down-regulation of enzymes that normally is responsible for the breakdown of F2-isoprostanes. In association with increased infiltration of inflammatory cells there was also an increased permeability in the lung-blood barrier, as indicated by leakage of serum albumin into the alveolar space. Both Cl2induced inflammation and alveolar-vascular permeability of serum albumin in BALF increased concentration-dependently in mice exposed to 25–200 ppm for 15 min. A linkage between Cl2-induced oxidation and vascular leak was supported in our study by the significant correlation between 8-isoprostane levels and leakage of serum albumin into the alveolar space. Alteration in the integrity of lung-blood barrier was also evaluated through analysis of the lung-specific proteins SP-D and CC16 in serum but with inconclusive results, although a decrease in serum levels 24 h post-exposure was found to be negatively correlated with increasing Cl2-concentrations. The inconsistent results could be explained by a modification by Cl2 or its reactive derivatives of the antibody epitopes that were used to detect the lung specific SP-D and CC16 by ELISA. Previous studies have shown that oxidation mediated by e.g. Cl2, HOCl or ROS may affect both the structure and function of pulmonary surfactants which together with increased vascular leak is proposed to be one underlying physiological mechanism to the respiratory dysfunction following Cl2-exposure (Crouch et al., 2010; Ingenito et al., 2001; Massa et al., 2014; Matalon et al., 2009; Rodríguez-Capote et al., 2006). We have previously demonstrated indirect effects in the blood circulation of inhaled Cl2, manifested by an increase in fibrinogen and PAI1 in serum between 12 and 24 h post-exposure (Wigenstam et al., 2015). These two proteins are important in maintaining hemostasis in the body, i.e. fibrinogen by contributing to thrombosis and PAI-1 as down-regulator of the fibrinolytic pathway. In this study we confirm increases in both fibrinogen and PAI-1 24 h after inhalation of 100 and 200 ppm Cl2, providing further support for Cl2-induced disturbances in the balance between formation and resolution of blood clots. This effect was preceded by oxidative and inflammatory responses in the blood circulation as indicated by increased expression of 8-isoprostane, and the pro-inflammatory cytokines IL-6, CXCL1/KC and IL-1β. Such response might be a sign of ARDS in humans as a retrospective case control study identified abnormal levels of five biomarkers among which IL-8, the human CXCL1/KC homolog, and IL-6 were found to be selective for diagnosis of ARDS in patients with severe sepsis (Ware et al., 2013).
inflammatory patterns both in BALF and serum with pulmonary injury induced by melphalan. Previous studies have shown that after exposure to 200 ppm Cl2, mice developed airway inflammation with increased total number of leukocytes mainly involving neutrophils and macrophages. Exposed mice also showed alterations in AHR, increased edema and pulmonary fibrosis with similarities to the pathological changes noted in humans accidently exposed to the gas (Jonasson et al., 2013a; Jonasson et al., 2013b; Martin et al., 2003; Tian et al., 2008). The data from this study combined with the previous findings in our laboratory (Jonasson et al., 2013a, 2013b; Wigenstam et al., 2015) clearly demonstrate that our mouse model for Cl2-induced lung injury is fulfilling the criteria for experimental ALI in animals defined by the American Thoracic Society, i.e. maximal lung injury evident within 24 h, decreased lung compliance, increased permeability in the alveolar vascular barrier with signs of both edema and leakage of serum albumin into the alveolar space together with pathological changes in histology showing neutrophil accumulation, epithelial cell hyperplasia and interstitial fibrosis (Matute-Bello et al., 2011). The main finding in this study is the high expression of 8-isoprostane, both in BALF and serum from mice with Cl2-induced ALI. 8isoprostane is produced in a non-cyclooxygenase dependent manner (Morrow et al., 1992, 1990) and is associated to diseases where oxidative stress is a prominent feature, e.g. interstitial fibrotic lung diseases and ALI/acute respiratory distress syndrome (ARDS) (Carpenter et al., 1998; Montuschi et al., 1998). 8-isoprostane has been proposed to affect both the lung by smooth muscle contraction, plasma exudation and release of the pro-fibrotic mediator TGFβ, and the cardiovascular system by promoting platelet activation and aggregation (Kang et al., 1985; Kawikova et al., 1996; Minuz et al., 1998; Montero et al., 2000; Okazawa et al., 1997; Rolin et al., 2007). The levels of 8-isoprostane were significantly elevated 2 h after Cl2-exposure in a concentrationdependent manner indicating a process of ongoing oxidative stress. Furthermore, it showed that the fold-increase of 8-isoprostane in serum was as pronounced as in BALF 2 h after Cl2-exposure and that the amount formed in airways were sufficient enough to be detected in EBC from Cl2-exposed rats at 24 h post-exposure. The amount of 8-isoprostane was successively reduced and after 48 h the levels of 8-isoprostane was similar as control animals. The reduction of 8-isoprostane may possibly be due to efficient activation of major enzymes, e.g. 15prostaglandin dehydrogenase (15-PGDH), prostaglandin Δ13-reductase and/or peroxisomal β-oxidation, which are responsible for the metabolism of 8-isoprostane (Basu, 1997, 1998; Stafforini et al., 2006). The activation and recruitment of inflammatory cells, mainly neutrophils, into the alveolar spaces is central to the pathophysiology of ALI. Activation of these cells is a result of early expression of pro-inflammatory and chemotactic cytokines produced mainly by alveolar macrophages that may lead to further release of mediators that promote inflammation and generate reactive oxygen species (ROS). From our data it is clear that neutrophil inflammation was preceded by early and transient induction of IL-1β, IL-6 and CXCL1/KC in BALF. In parallel to the transient increase in cytokine levels at lower concentrations there was also a more rapid pattern in the time kinetics of pulmonary neutrophil infiltration and vascular leakage. In Cl2-exposed animals the levels of 8-isoprostane were elevated already after 2 h, a time-point when we could not detect any inflammatory cells in BALF, indicating that increased 8-isoprostane is not a result of neutrophil-derived ROS. A non-inflammatory mechanism for 8-isoprostane induction was further supported by the lack of induction after exposure for the non-oxidizing nitrogen mustard analog melphalan. Exposure to melphalan resulted in a severe ALI without any detectable signs of increased 8-isoprostane in BALF at any investigated time-point. Therefore, we consider it likely that 8-isoprostane in airways is mainly formed by direct oxidation by inhaled Cl2 rather than a consequence of neutrophil-induced ROS production. Such mechanism is consistent with a report by Yadav and colleagues (Yadav et al., 2011) measuring the total level of F2-
5. Conclusions We have demonstrated that inhalation of high concentrations of Cl2 induce a prominent inflammatory response dominated by neutrophils, increased lung permeability and markers of blood coagulation. This response is preceded by an early and transient increases of pro-inflammatory cytokines and 8-isoprostanse in airways and blood circulation. The induction of 8-isoprostane was almost nonexistent in ALI induced by a non-oxidizing toxic chemical indicating that mechanism of early 8-isoprostane formation is through direct oxidation of arachidonic acid by Cl2. Moreover, the early expression of 8-isoprostane in airways together with the pro-inflammatory cytokines IL-1β, IL-6 and CXCL1/ KC offer further contribution to the understanding of the mechanisms by which exposure to chemical compounds causes lung injury and AHR. Finally, we conclude that 8-isoprostane represents a promising high sensitivity biomarker for Cl2-induced oxidative lung injury that potentially can be used as a diagnostic tool, and that the role of this marker for long-standing effects such as RADS needs further attention. 6
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Pharmacol. 309, 44–54. Williams, J.G., 1997. Inhalation of chlorine gas. Postgrad. Med. J. 73, 697–700. Yadav, A.K., Doran, S.F., Samal, A.A., Sharma, R., Vedagiri, K., Postlethwait, E.M., Squadrito, G.L., Fanucchi, M.V., Roberts, L.J., Patel, R.P., Matalon, S., 2011. Mitigation of chlorine gas lung injury in rats by postexposure administration of sodium nitrite. Am. J. Physiol. Lung Cell. Mol. Physiol. 300, L362–369. Zarogiannis, S.G., Wagener, B.M., Basappa, S., Doran, S., Rodriguez, C.A., Jurkuvenaite, A., Pittet, J.F., Matalon, S., 2014. Postexposure aerosolized heparin reduces lung injury in chlorine-exposed mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 307, L347–354.
Acknowledgements We wish to thank Bo Koch, Elisabeth Wigenstam and Karin Wallgren for excellent technical assistance and for invaluable help with the animal experiments. This work was supported by grants from the Swedish Ministry of Defence and the Swedish Center for Disaster Toxicology at the National Board of Health and Welfare. References Basu, S., 1997. Metabolism of 8-iso-prostaglandin F2α in the rabbit. Prostaglandins Leuk. Essent. Fatty Acids 57, 267. Basu, S., 1998. Metabolism of 8-iso-prostaglandin F2alpha. FEBS Lett. 428, 32–36. Brooks, S.M., Weiss, M.A., Bernstein, I.L., 1985. Reactive airways dysfunction syndrome (RADS): Persistent asthma syndrome after high level irritant exposures. Chest 88, 376–384. Carpenter, C.T., Price, P.V., Christman, B.W., 1998. Exhaled breath condensate isoprostanes are elevated in patients with acute lung injury or ARDS. Chest 114, 1653–1659. Crouch, E.C., Hirche, T.O., Shao, B., Boxio, R., Wartelle, J., Benabid, R., McDonald, B., Heinecke, J., Matalon, S., Belaaouaj, A., 2010. Myeloperoxidase-dependent inactivation of surfactant protein D in vitro and in vivo. J. Biol. Chem. 285, 16757–16770. Ekstrand-Hammarstrom, B., Wigenstam, E., Bucht, A., 2011. Inhalation of alkylating mustard causes long-term T cell-dependent inflammation in airways and growth of connective tissue. Toxicology 280, 88–97. Evans, R.B., 2005. Chlorine: state of the art. Lung 183, 151–167. Hemstrom, P., Larsson, A., Elfsmark, L., Astot, C., 2016. L-alpha-phosphatidylglycerol chlorohydrins as potential biomarkers for chlorine gas exposure. Anal. Chem. 88, 9972–9979. Hosseinian, N., Cho, Y., Lockey, R.F., Kolliputi, N., 2015. The role of the NLRP3 inflammasome in pulmonary diseases. Ther. Adv. Respir. Dis. 9, 188–197. Ingenito, E.P., Mora, R., Cullivan, M., Marzan, Y., Haley, K., Mark, L., Sonna, L.A., 2001. Decreased surfactant protein-B expression and surfactant dysfunction in a murine model of acute lung injury. Am. J. Respir. Cell Mol. Biol. 25, 35–44. Jonasson, S., Koch, B., Bucht, A., 2013a. Inhalation of chlorine causes long-standing lung inflammation and airway hyperresponsiveness in a murine model of chemical-induced lung injury. Toxicology 303, 34–42. Jonasson, S., Wigenstam, E., Koch, B., Bucht, A., 2013b. Early treatment of chlorineinduced airway hyperresponsiveness and inflammation with corticosteroids. Toxicol. Appl. Pharmacol. 271, 168–174. Kales, S.N., Christiani, D.C., 2004. Acute chemical emergencies. N. Engl. J. Med. 350, 800–808. Kang, K.H., Morrow, J.D., Roberts, L.J., Newman, J.H., Banerjee, M., 1985. 1993: Airway and vascular effects of 8-epi-prostaglandin F2 alpha in isolated perfused rat lung. J. Appl. Physiol. 74, 460–465. Kawikova, I., Barnes, P.J., Takahashi, T., Tadjkarimi, S., Yacoub, M.H., Belvisi, M.G., 1996. 8-Epi-PGF2 alpha, a novel noncyclooxygenase-derived prostaglandin, constricts airways in vitro. Am. J. Respir. Crit. Care Med. 153, 590–596. Koohsari, H., Tamaoka, M., Campbell, H.R., Martin, J.G., 2007. The role of gamma delta T cells in airway epithelial injury and bronchial responsiveness after chlorine gas exposure in mice. Respir. Res. 8, 21. Lemiere, C., Malo, J.L., Boutet, M., 1997. Reactive airways dysfunction syndrome due to chlorine: sequential bronchial biopsies and functional assessment. Eur. Respir. J. 10, 241–244. Luo, S., Trübel, H., Wang, C., Pauluhn, J., 2014. Phosgene- and chlorine-induced acute lung injury in rats: comparison of cardiopulmonary function and biomarkers in exhaled breath. Toxicology 326, 109–118. Martin, J.G., Campbell, H.R., Iijima, H., Gautrin, D., Malo, J.L., Eidelman, D.H., Hamid, Q., Maghni, K., 2003. Chlorine-induced injury to the airways in mice. Am. J. Respir. Crit. Care Med. 168, 568–574. Massa, C.B., Scott, P., Abramova, E., Gardner, C., Laskin, D.L., Gow, A.J., 2014. Acute chlorine gas exposure produces transient inflammation and a progressive alteration in surfactant composition with accompanying mechanical dysfunction. Toxicol. Appl. Pharmacol. 278, 53–64. Matalon, S., Shrestha, K., Kirk, M., Waldheuser, S., McDonald, B., Smith, K., Gao, Z., Belaaouaj, A., Crouch, E.C., 2009. Modification of surfactant protein D by reactive oxygen-nitrogen intermediates is accompanied by loss of aggregating activity, in vitro and in vivo. FASEB J. 23, 1415–1430. Matute-Bello, G., Downey, G., Moore, B.B., Groshong, S.D., Matthay, M.A., Slutsky, A.S., Kuebler, W.M., Group, A.L.I.i.A.S., 2011. An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. Am. J. Respir. Cell Mol. Biol. 44, 725–738.
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
8-Isoprostane is an early biomarker for oxidative stress in chlorine-induced acute lung injury
MARK
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Linda Elfsmarka, , Lina Ågrena, Christine Akfura, Anders Buchta,b, Sofia Jonassona a b
Swedish Defence Research Agency, CBRN Defence and Security, Umeå, Sweden Department of Public Health and Clinical Medicine, Unit of Respiratory Medicine, University Hospital, Umeå, Sweden
A R T I C L E I N F O
A B S T R A C T
Keywords: Chlorine Biomarkers Inflammation Acute lung injury 8-Isoprostane
Inhalation of chlorine (Cl2) may cause oxidative acute lung injury (ALI) characterized by pulmonary edema, pneumonitis, and hyperreactive airways. The aim of the study was to identify possible biomarkers for Cl2induced ALI. Female BALB/c mice were exposed to Cl2 for 15 min using two protocols 1) concentration-dependent response (25–200 ppm) and 2) time-kinetics (2h–14 days post-exposure). Exposure to 50–200 ppm Cl2 caused a concentration-dependent inflammatory response with increased expression of IL-1β, IL-6 and CXCL1/KC in bronchoalveolar lavage fluid 2–6 h after exposure which was followed by increased lung permeability and a neutrophilic inflammation 12–24 h post-exposure. The early inflammatory cytokine response was associated with a clear but transient increase of 8-isoprostane, a biomarker for oxidative stress, with its maximum at 2 h after exposure. An increase of 8-isoprostane could also be detected in serum 2 h after exposure to 200 ppm Cl2, which was followed by increased levels of IL-6 and CXCL1/KC and signs of increased fibrinogen and PAI-1. Melphalan, a non-oxidizing mustard gas analog, did not increase the 8-isoprostane levels, indicating that 8-isoprostane is induced in airways through direct oxidation by Cl2. We conclude that 8-isoprostane represents an early biomarker for oxidative stress in airways and in the blood circulation following Cl2-exposure.
1. Introduction Chlorine (Cl2) is a powerful oxidizing gas extensively used as bleaching agent, disinfectant, and in the manufacture of industrial chemicals. Due to its high toxicity, Cl2 has historically been used as a chemical weapon and is still considered a terrorist threat and used in armed conflicts globally (United Nations General Assembly 2014; Hemstrom et al., 2016). Inhalation of Cl2 can produce a range of acute pulmonary effects including impaired lung function, inflammatory reactions, increase of epithelial permeability, and airway hyperresponsiveness (AHR) (Evans, 2005; Koohsari et al., 2007; Morris et al., 2005; Tuck et al., 2008; White and Martin, 2010; Williams, 1997). In some individuals the acute lung injury (ALI) evolves into long-term respiratory manifestations, collectively named reactive airway dysfunction syndrome (RADS), that are characterized by persistent cough, wheezing, chest tightness and dyspnea (Brooks et al., 1985). Not all exposed individuals develop long-standing effects like RADS and pulmonary fibrosis, and the biological mechanisms behind the pathological changes still remain unsolved (Lemiere et al., 1997). However,
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predicting the outcome of long-standing effects, like RADS, in a patient at an early stage is not possible. Therefore, there is a need of an early non-invasive specific diagnostic tool for ALI that enables monitoring the prognosis of delayed effects after exposure and predicting the individual need of early medical treatment to avoid severe consequences such as RADS. We have previously demonstrated that mice and rats exposed to 200–300 ppm Cl2 develop a neutrophilic pulmonary injury manifested by e.g. edema, cardiovascular effects, pulmonary fibrosis and longstanding AHR in close agreement with clinical findings in humans exposed to Cl2 (Jonasson et al., 2013a, 2013b; Wigenstam et al., 2016, 2015). The acute symptoms of inhaled Cl2 are generally limited to the exposed tissues (Evans, 2005; Kales and Christiani, 2004) but there is also evidence that Cl2 induces systemic inflammatory responses (induced IL-8/KC and IL-6) and cardiovascular effects, e.g. alterations in blood coagulation and fibrinolysis (Luo et al., 2014; Wang et al., 2006; Wigenstam et al., 2015; Yadav et al., 2011; Zarogiannis et al., 2014). From a medical point-of-view, interference with the early response is of significance since treatment with corticosteroids within the first hours
Corresponding author at: CBRN Defence and Security, Swedish Defence Research Agency, SE-901 82, Umeå, Sweden. E-mail address: [email protected] (L. Elfsmark).
http://dx.doi.org/10.1016/j.toxlet.2017.10.007 Received 10 May 2017; Received in revised form 4 October 2017; Accepted 6 October 2017 Available online 07 October 2017 0378-4274/ © 2017 Elsevier B.V. All rights reserved.
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after a Cl2-exposure is shown to reduce long-term lung injury (Jonasson et al., 2013b; Wigenstam et al., 2015). Based on previous results, a mouse model of Cl2-induced lung injury was used to investigate early markers of oxidative stress, inflammatory response, disruption of lung barrier integrity, and blood coagulation. As a marker of lipid peroxidation we used the arachidonic acid derivate 8isoprostane (8-isoPGF2α), which is recognized to be a reliable marker of oxidative stress in airways (Roberts and Morrow, 2000). As early markers of inflammatory response the expression of cytokines associated with induction of acute inflammation was analyzed in bronchoalveolar lavage fluid (BALF) and serum (Hosseinian et al., 2015; Ware et al., 2013). The degree of ALI was determined using markers of lung barrier integrity and counting of inflammatory cells in airways. To assess effects on blood coagulation and fibrinolysis we analyzed plasminogen activator inhibitor (PAI-1) and fibrinogen, previously reported by us to be increased in serum of mice exposed for high concentrations of Cl2 (Wigenstam et al., 2015). By using a concentration range of Cl2 (25–200 ppm), concentration-dependencies of the biomarker expression was assessed e.g. whether the increased formation and their appearance in BALF and serum could be associated with ALI and disease severity. The high-level concentration of Cl2, 200 ppm, was considered to be relevant for a scenario of a chemical disaster at an industrial plant or during transportation, resulting in life-threatening lung injuries. Such chemical disaster would also include humans exposed to lower concentrations displaying no observable acute symptoms during the first 24 h although having a potential risk to develop delayed effects. To study the specificity of the oxidative and inflammatory markers, the responses in the Cl2-induced model were compared with the responses in models of chemical-induced ALI induced by the nitrogen mustard analog, melphalan. Similar to Cl2, melphalan induces an acute airway inflammation when exposed to the lung but without a prominent direct chemical-induced oxidation in target tissues. Studies have shown that melphalan is a possible surrogate for nitrogen mustard causing a neutrophilic airway inflammation with AHR in the acutephase (1–2 days post-exposure) which turns into a lymphocytic inflammation in absence of AHR in the sub-acute phase (after 14 days) (Ekstrand-Hammarstrom et al., 2011; Wigenstam et al., 2012, 2009). By the use of these mouse models, the present study was designed to determine the specificity of these biomarkers with the aim to identify markers in serum, BALF and exhaled breath condensate (EBC) that can potentially be used as a diagnostic tools to predict the risk of ALI and long-standing symptoms such as RADS.
Fig. 1. Collection of EBC. Experimental setup for collection of exhaled breath condensates (EBC) using a cold trap. Expired breath was sampled during 45 min from anesthetized and tracheostomized rats mechanically ventilated with a small animal ventilator.
range of Cl2 concentrations that were believed to cause mild irritation (no evident airway inflammation) to severe sub-lethal lung injury. In the concentration-response study, the mice were exposed to 25 ppm (6.3 ppm h), 50 ppm (12.5 ppm h), 100 ppm (25 ppm h) or 200 ppm (50 ppm h) Cl2 for 15 min (n = 5–7/group) and samples were collected at 2, 6 and 24 h after exposure. In the time-kinetic study the animals were exposed to 200 ppm Cl2 for 15 min (n = 5-7/group) and samples were collected at 2, 6, 12, 24, 48, and 72 h up to 14 days post-exposure. Rats were exposed to 200 ppm (50 ppm h) Cl2 for 15 min according to previously described protocol (Wigenstam et al., 2016) and EBC was collected 24 h after exposure (Fig. 1). Age-matched controls exposed to room air for 15 min were included at each time-point analyzed. 2.3. Collection of EBC Exhaled breath condensate was sampled from Cl2-exposed and control rats anesthetized with pentobarbital sodium (50 mg/kg body weight (b.w)). The rats were tracheostomized with a 15-gauge cannula and mechanically ventilated with a small animal ventilator (flexiVent™, SCIREQ®) at a frequency of 90 breaths/min and a tidal volume of 10 ml/kg b.w. A positive end-expiratory pressure of 3 cmH2O was applied and the rats were paralyzed with pancuronium (0.1 mg/kg b.w). Expired air from the ventilated animal was lead through a cold trap consisting of a pear-shaped glass flask for distillation with two necks immersed in ice (Fig. 1). The connection of the ventilator tubing to the glass flask was carefully sealed and EBC was collected during 45 min, yielding approximately 150 μl of condensate from each individual rat (n = 4–5/group). The condensates were immediately frozen and stored at −80 °C until analysis.
2. Materials and methods 2.1. Animals Female BALB/c mice and Sprague-Dawley rats (Envigo RMS B.V, Netherlands) were used in this study. After arrival, the animals were allowed to acclimatize for at least one week. All mice were between 10 and 11 weeks old when experiments were initiated and the rats were between 9 and 10 weeks old. The animals were housed under standard laboratory conditions (12 h daylight cycle, 22 °C, 30% relative humidity) and permitted free access to both food (R36, Lantmännen, Sweden) and water. The animals were weighted to monitor their health condition before the experiments and following exposure. The care of the animals and the experimental protocols were approved by the regional ethics committee on animal experiments in Umeå, Sweden.
2.4. Melphalan-exposure and sampling protocol Melphalan (4-[bis(2-chloroethyl)amino]-1-phenylalanine) (SigmaAldrich, St Louis, MO) was dissolved in phosphate-buffered saline (PBS, Sigma-Aldrich) according to the protocol described by Wigenstam et al. (Wigenstam et al., 2012). Melphalan (50 μl) was administered by intratracheal (i.t.) instillation to anesthetized mice at a dose of 1 mg/kg b.w (n = 6–7/group). Control mice received vehicle alone (n = 5–7 at each time-point analyzed). Samples were collected and evaluated at different time-points (2, 6, 12, 24, 48, and 72 h) after exposure.
2.2. Cl2-exposure and sampling protocol Animals were placed in a Battelle inhalation exposure tower for noseonly exposure (EMMS, UK) and were exposed to Cl2 (Air Liquide, Germany; mixture crystal, 0.1 mol-% Cl2, 99.9 mol-% nitrogen) according to previously described protocols (Jonasson et al., 2013a, 2013b; Wigenstam et al., 2016). Based on our previous studies we selected a
2.5. Serum sampling and collection of BALF Mice were anesthetized using isoflurane and blood was collected through orbital punction. After retrieving blood samples the animals 2
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2.10. Analysis of fibrinogen and PAI-1 in serum
were euthanized through cervical dislocation. Samples were kept in room temperature (RT) for 1 h before centrifugation (15 min, 2500 rpm) and the serum was stored at −80 °C for later analysis. Mice were then tracheostomized and BALF was collected by injecting and withdrawing 4 × 1 ml of ice cold Hank’s balanced salt solution (HBSS, Sigma-Aldrich). The BALF was centrifuged (1500 rpm, 10 min, 4 °C) and the supernatant was stored at −80 °C for later analysis. The number of leukocytes was counted in a Bürker chamber using trypan blue staining. From the BALF of each animal 20 000 cells were fixed on duplicate slides using a Cytospin® centrifuge (Shandon© cytospin 3 cyto-centrifuge, cell preparation system, Runcorn, UK) and stained with May-Grünwald-Giemsa reagents (Merck Millipore, VWR International, Sweden) before a differential count of total leukocytes (including macrophages, neutrophils, eosinophils and lymphocytes) was performed in a blinded manner, counting 300 cells per slide.
Fibrinogen was measured in serum (diluted 1:1000) using a Matched-Pair Antibody Set (Enzyme Research Laboratories, South Bend, IN) according to the manufacturer’s description. The absorbance that was read at 450 nm (Thermo Scientific Multiscan FC) was proportional to the concentration of fibrinogen in the sample. PAI-1 was quantified in serum (diluted 1:20) using a sandwich PAI-1 DuoSet ELISA Development kits (R & D Systems) according to the manufacturer’s description. 2.11. Statistical analysis All results were expressed as mean values ± standard error of the mean (SEM) (n = 5–7). Controls from each time-point were pooled when appropriate. Using the GraphPad Prism Version 6.0 (GraphPad Software, La Jolla, CA) data were analyzed by one-way-ANOVA followed by Dunnettś post hoc-test. Analysis of correlation between data sets were performed using Pearson correlation calculations. A statistical result of p < 0.05 was considered significant (*p < 0.05, ** p < 0.01 and ***p < 0.001).
2.6. Analysis of 8-isoprostane in BALF and serum 8-isoprostane was analyzed using a competitive 8-isoprostane (8-iso PGF2α) EIA Kit (Cayman Chemical Company, Ann Arbor, MI, USA), 50 μl of the first 2 ml collected BALF samples from each animal were analyzed and the assay was performed according to the manufacturer’s description. The absorbance were read at 405 nm using an ELISA reader (Thermo Scientific Multiskan FC, Thermo Fischer Scientific Oy,Vantaa, Finland). Results were analyzed in GraphPad Prism 6 as (% sample or standard bound/maximum bound) according to the manufacturer’s recommendations and the amount of 8-isoprostane was calculated from a standard curve. Serum samples were purified before analysis. In brief, diluted serum (1:5 in eicosanoid affinity column buffer) were run through an affinity 8-isoprostane sorbent column (Cayman Chemical Company). Bound 8-isoprostane was then eluted with ethanol and frozen. In connection with analysis the ethanol was evaporated and the samples were diluted to the original serum volume (200 μl) in HBSS according to the manufacturer’s description.
3. Results 3.1. Cl2-induced ALI 3.1.1. Inflammatory response in BALF A 15-min exposure to 25–200 ppm Cl2 triggered an early and a concentration-dependent release of pro-inflammatory cytokines in airways but did not cause any lethal effects. A significant increase of IL-6 was evident in BALF at 2 h after inhalation of 50, 100 and 200 ppm Cl2 (Fig. 2A). The neutrophil-chemoattractant CXCL1/KC was significantly increased at the early time-points for all Cl2 groups (Fig. 2B) while IL1β was significantly increased only in the highest exposure groups (100 and 200 ppm Cl2 groups) (Fig. 2C). In addition to IL-1β, all other cytokines investigated were also significantly increased at 6 h post-exposure in BALF in the 100 and 200 ppm Cl2 groups. In the 50 ppm Cl2 group, CXCL1/KC and IL-1β were also increased 6 h after exposure. A significant increase of neutrophils was observed in BALF within 6 h in the 50, 100 and 200 ppm Cl2 groups as compared to non-exposed animals. There were no significant differences in the extent of neutrophilic inflammation between the Cl2 groups (Fig. 3A). The increased number of neutrophils was sustained in BALF for 24 h for the 200 ppm group (Fig. 3D), while the number of neutrophils were reduced to control levels for both the 50 ppm and the 100 ppm Cl2 group at 24 h post-exposure (data not shown).
2.7. Analysis of SP-D and CC16 in serum Surfactant Protein D (SP-D) levels in serum (diluted 1:5) were determined using Mouse Pulmonary surfactant associated protein type D (SP-D) ELISA Kit (BlueGene Biotech, Shanghai, China) and Clara cell 16 levels in serum (diluted 1:50) were measured with Mouse Clara Cell specific protein (CC16) ELISA kit (BlueGene Biotech). Both assays were performed according to the manufacturer’s description. The absorbance were read at 450 nm using an ELISA reader (Thermo Scientific Multiskan FC, Thermo Fischer Scientific Oy,Vantaa, Finland). The concentration were calculated using standards provided by the manufacturer. 2.8. Analysis of albumin in BALF
3.1.2. Inflammatory responses in serum and lung/serum-specific proteins In association with the increased pulmonary infiltration of inflammatory cells there was also an increased permeability of the lungblood barrier as indicated by leakage of serum albumin into the alveolar space at 6–24 h post-exposure (Fig. 3B and E). Alteration in the integrity of lung-blood barrier was also evaluated through analysis of the lung-specific proteins SP-D and CC16 in serum but with inconsistent results, although a decrease in serum levels 24 h post-exposure was observed to be negatively correlated with increasing Cl2 concentrations (data not shown). Inhalation of Cl2 caused a concomitant early inflammatory response in serum with increased IL-6 and CXCL1/KC at 6 h in the two highexposure groups (Fig. 2D–E). At this time-point, IL-1β was the only analyzed cytokine upregulated in the 50 ppm group (Fig. 2F). A concentration-dependent alteration in the balance between thrombosis and fibrinolysis, with significant increases in both fibrinogen and PAI-1 was observed in serum 24 h after inhalation of 100 and 200 ppm Cl2 respectively (Fig. 3C).
Mouse Albumin ELISA kits were purchased from Bethyl Laboratories, (Montgomery, AL, US) and albumin content was determined in BALF (diluted 1:10.000) from each animal according to the manufacturer’s description. The absorbance were read at 450 nm using an ELISA reader (Thermo Scientific Multiskan FC). After generating a four-parameter logistic standard curve the albumin content could be quantified. 2.9. Analysis of cytokines in BALF and serum The concentration of IL-1β, IL-6 and CXCL1/KC in BALF and serum (diluted 1:2 and 1:3 respectively) from each animal was measured using specific DuoSet ELISA Development kits (R & D Systems) for each cytokine according to the manufacturer’s description. The absorbance were read at 450 nm using an ELISA reader (Thermo Scientific Multiskan FC) at 450 nm and the amount of each cytokine was quantified using a four-parameter logistic standard curve. 3
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Fig. 2. Pro-inflammatory markers. Early concentration-dependent activation of inflammatory markers after chlorine (Cl2)-exposure. Concentrations in bronchoalveolar lavage fluid (BALF) are shown for (A) IL-6, (B) CXCL1/KC and (C) IL-1β 2 h and 6 h post exposure and (D-F) concentrations in serum 6 h post-exposure from mice exposed to Cl2: (●) 25 ppm, (■) 50 ppm, (▲) 100 ppm or (□) 200 ppm. Values indicate means ± SEM. Statistical significant differences compared to control group are shown, *p < 0.05, ** p < 0.01 and *** p < 0.001.
to the large amount of serum required for the analysis. An increased amount of 8-isoprostane was also found in the EBC collected from rats 24 h after exposure to 200 ppm Cl2 (Fig. 4E).
3.1.3. Expression of 8-isoprostane in BALF, serum and EBC Inhalation of 100 and 200 ppm Cl2 caused a concentration-dependent release of 8-isoprostane in BALF, with significantly increased levels at 2 h, the earliest time-point for sampling (Fig. 4A). The amount of 8-isoprostane remained increased at 6 h after exposure to 50–200 ppm Cl2 (Fig. 4B). Analysis of 8-isoprostane during a time-course up to 14 days showed that maximum levels were detected 2 h after exposure (100 ± 11.3 pg/ml) and slowly declined to concentrations corresponding to non-exposed mice (6.3 ± 0.7 pg/ml) after 48 h (Fig. 4C). Formation of 8-isoprostane correlated with severity of the lung-blood barrier damage, measured as leakage of serum albumin (r2 = 0.48, p = 0.002) into the BALF. Increased concentrations of 8-isoprostane 2 h after 200 ppm Cl2 exposure could also be detected in serum (Fig. 4D). Further investigations of the time-course and the Cl2-exposure-response of 8-isoprostane in serum could not be performed due
3.2. Expression of 8-isoprostane in melphalan-induced ALI Intratracheal instillation of melphalan induced a pulmonary inflammation dominated by neutrophils (Fig. 5A). Analysis of IL-6, CXCL1/ KC and IL-1β revealed an early immune activation similar to the response after Cl2-exposure (data not shown). The short-term inflammatory response reached its maximum at 12–24 h after exposure to melphalan and was preceded by changes in vascular permeability 6 h post-instillation as indicated by significantly higher amount of serum albumin than in nonexposed animals (Fig. 5B). At 6 h post-instillation, melphalan induced a significant increase in PAI-1 but the amount of fibrinogen in serum was
Fig. 3. Chlorine-induced ALI. Chlorine (Cl2)-induced (A) infiltration of inflammatory cells and (B) serum albumin leakage in bronchoalveolar lavage fluid (BALF) from mice 6 h after exposure to 25, 50, 100 or 200 ppm Cl2. (C) Concentration-dependent changes of fibrinogen and PAI-1 levels in serum 24 h after Cl2-exposure. (D) Alterations in cellular infiltration and (E) serum albumin leakage in BALF from 2 h to 14 days post-exposure to 200 ppm Cl2. Values indicate means ± SEM. Statistical significant differences compared to control group are shown, * p < 0.05, ** p < 0.01 and *** p < 0.001.
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Fig. 4. Expression of 8-isoprostane. Concentration-dependent induction of 8-isoprostane (8-isoPGF2α) in mice exposed to 25–200 ppm chlorine (Cl2) in bronchoalveolar lavage fluid (BALF) (A) 2 h and (B) 6 h after exposure. (C) Time-kinetics of 8-isoprostane expression was analyzed in BALF from 2 h to 14 days after exposure to 200 ppm Cl2 and (D) the concentration of 8-isoprostane in BALF was compared with the concentration in serum from mice at the 2 h time-point. (E) The level of 8-isoprostane in exhaled breath condensate (EBC) from rats exposed to 200 ppm Cl2 24 h after exposure. Values indicate means ± SEM. Statistical significant differences compared to control group are shown, ** p < 0.01 and *** p < 0.001.
stress, inflammation and coagulation. Mice were exposed to different concentrations of Cl2 (20–200 ppm), thereby the concentration-dependency of the biomarkers could be assessed and correlated with disease severity. In addition, the time-kinetics for the appearance of the biomarkers were investigated by performing analyses in the interval between 2 h and 14 days post-exposure. Furthermore, the specificity of the biomarkers for Cl2-induced ALI was evaluated by comparing the
not elevated (Fig. 5C). Melphalan-exposure did not increase the levels of 8-isoprostan during a period of 72 h post-exposure (Fig. 5D). 4. Discussion The aim of this study was to identify predictive biomarkers of disease severity after Cl2-exposure by using early indicators of oxidative
Fig. 5. Melphalan-induced ALI. Melphalan-induced (1 mg/kg i.t.) immune activation and lung damage in mice 2 h to 72 h post-exposure. (A) The number of inflammatory cells in bronchoalveolar lavage fluid (BALF), (B) serum albumin leakage in BALF, (C) fibrinogen and PAI-1 in serum and (D) 8-isoprostane (8-isoPGF2α) in BALF. Values indicate means ± SEM. Statistical significant differences compared to control group are shown, * p < 0.05, ** p < 0.01 and *** p < 0.001.
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isoprostanes with GC/NICI-MS in lung tissue to assess lipid peroxidation in rats after exposure to 400 ppm Cl2 for 30 min. They observed a two-fold increase of F2-isoprostanes that was generated during Cl2-exposure and did not increase further after finalizing the Cl2-exposure (remained elevated at 6 h after exposure) (Yadav et al., 2011). One of their hypothesis was that Cl2 itself and/or its hydrolysis product HOCl is the likely mediator of arachidonic acid peroxidation. Another possibility is Cl2-induced dysfunction or down-regulation of enzymes that normally is responsible for the breakdown of F2-isoprostanes. In association with increased infiltration of inflammatory cells there was also an increased permeability in the lung-blood barrier, as indicated by leakage of serum albumin into the alveolar space. Both Cl2induced inflammation and alveolar-vascular permeability of serum albumin in BALF increased concentration-dependently in mice exposed to 25–200 ppm for 15 min. A linkage between Cl2-induced oxidation and vascular leak was supported in our study by the significant correlation between 8-isoprostane levels and leakage of serum albumin into the alveolar space. Alteration in the integrity of lung-blood barrier was also evaluated through analysis of the lung-specific proteins SP-D and CC16 in serum but with inconclusive results, although a decrease in serum levels 24 h post-exposure was found to be negatively correlated with increasing Cl2-concentrations. The inconsistent results could be explained by a modification by Cl2 or its reactive derivatives of the antibody epitopes that were used to detect the lung specific SP-D and CC16 by ELISA. Previous studies have shown that oxidation mediated by e.g. Cl2, HOCl or ROS may affect both the structure and function of pulmonary surfactants which together with increased vascular leak is proposed to be one underlying physiological mechanism to the respiratory dysfunction following Cl2-exposure (Crouch et al., 2010; Ingenito et al., 2001; Massa et al., 2014; Matalon et al., 2009; Rodríguez-Capote et al., 2006). We have previously demonstrated indirect effects in the blood circulation of inhaled Cl2, manifested by an increase in fibrinogen and PAI1 in serum between 12 and 24 h post-exposure (Wigenstam et al., 2015). These two proteins are important in maintaining hemostasis in the body, i.e. fibrinogen by contributing to thrombosis and PAI-1 as down-regulator of the fibrinolytic pathway. In this study we confirm increases in both fibrinogen and PAI-1 24 h after inhalation of 100 and 200 ppm Cl2, providing further support for Cl2-induced disturbances in the balance between formation and resolution of blood clots. This effect was preceded by oxidative and inflammatory responses in the blood circulation as indicated by increased expression of 8-isoprostane, and the pro-inflammatory cytokines IL-6, CXCL1/KC and IL-1β. Such response might be a sign of ARDS in humans as a retrospective case control study identified abnormal levels of five biomarkers among which IL-8, the human CXCL1/KC homolog, and IL-6 were found to be selective for diagnosis of ARDS in patients with severe sepsis (Ware et al., 2013).
inflammatory patterns both in BALF and serum with pulmonary injury induced by melphalan. Previous studies have shown that after exposure to 200 ppm Cl2, mice developed airway inflammation with increased total number of leukocytes mainly involving neutrophils and macrophages. Exposed mice also showed alterations in AHR, increased edema and pulmonary fibrosis with similarities to the pathological changes noted in humans accidently exposed to the gas (Jonasson et al., 2013a; Jonasson et al., 2013b; Martin et al., 2003; Tian et al., 2008). The data from this study combined with the previous findings in our laboratory (Jonasson et al., 2013a, 2013b; Wigenstam et al., 2015) clearly demonstrate that our mouse model for Cl2-induced lung injury is fulfilling the criteria for experimental ALI in animals defined by the American Thoracic Society, i.e. maximal lung injury evident within 24 h, decreased lung compliance, increased permeability in the alveolar vascular barrier with signs of both edema and leakage of serum albumin into the alveolar space together with pathological changes in histology showing neutrophil accumulation, epithelial cell hyperplasia and interstitial fibrosis (Matute-Bello et al., 2011). The main finding in this study is the high expression of 8-isoprostane, both in BALF and serum from mice with Cl2-induced ALI. 8isoprostane is produced in a non-cyclooxygenase dependent manner (Morrow et al., 1992, 1990) and is associated to diseases where oxidative stress is a prominent feature, e.g. interstitial fibrotic lung diseases and ALI/acute respiratory distress syndrome (ARDS) (Carpenter et al., 1998; Montuschi et al., 1998). 8-isoprostane has been proposed to affect both the lung by smooth muscle contraction, plasma exudation and release of the pro-fibrotic mediator TGFβ, and the cardiovascular system by promoting platelet activation and aggregation (Kang et al., 1985; Kawikova et al., 1996; Minuz et al., 1998; Montero et al., 2000; Okazawa et al., 1997; Rolin et al., 2007). The levels of 8-isoprostane were significantly elevated 2 h after Cl2-exposure in a concentrationdependent manner indicating a process of ongoing oxidative stress. Furthermore, it showed that the fold-increase of 8-isoprostane in serum was as pronounced as in BALF 2 h after Cl2-exposure and that the amount formed in airways were sufficient enough to be detected in EBC from Cl2-exposed rats at 24 h post-exposure. The amount of 8-isoprostane was successively reduced and after 48 h the levels of 8-isoprostane was similar as control animals. The reduction of 8-isoprostane may possibly be due to efficient activation of major enzymes, e.g. 15prostaglandin dehydrogenase (15-PGDH), prostaglandin Δ13-reductase and/or peroxisomal β-oxidation, which are responsible for the metabolism of 8-isoprostane (Basu, 1997, 1998; Stafforini et al., 2006). The activation and recruitment of inflammatory cells, mainly neutrophils, into the alveolar spaces is central to the pathophysiology of ALI. Activation of these cells is a result of early expression of pro-inflammatory and chemotactic cytokines produced mainly by alveolar macrophages that may lead to further release of mediators that promote inflammation and generate reactive oxygen species (ROS). From our data it is clear that neutrophil inflammation was preceded by early and transient induction of IL-1β, IL-6 and CXCL1/KC in BALF. In parallel to the transient increase in cytokine levels at lower concentrations there was also a more rapid pattern in the time kinetics of pulmonary neutrophil infiltration and vascular leakage. In Cl2-exposed animals the levels of 8-isoprostane were elevated already after 2 h, a time-point when we could not detect any inflammatory cells in BALF, indicating that increased 8-isoprostane is not a result of neutrophil-derived ROS. A non-inflammatory mechanism for 8-isoprostane induction was further supported by the lack of induction after exposure for the non-oxidizing nitrogen mustard analog melphalan. Exposure to melphalan resulted in a severe ALI without any detectable signs of increased 8-isoprostane in BALF at any investigated time-point. Therefore, we consider it likely that 8-isoprostane in airways is mainly formed by direct oxidation by inhaled Cl2 rather than a consequence of neutrophil-induced ROS production. Such mechanism is consistent with a report by Yadav and colleagues (Yadav et al., 2011) measuring the total level of F2-
5. Conclusions We have demonstrated that inhalation of high concentrations of Cl2 induce a prominent inflammatory response dominated by neutrophils, increased lung permeability and markers of blood coagulation. This response is preceded by an early and transient increases of pro-inflammatory cytokines and 8-isoprostanse in airways and blood circulation. The induction of 8-isoprostane was almost nonexistent in ALI induced by a non-oxidizing toxic chemical indicating that mechanism of early 8-isoprostane formation is through direct oxidation of arachidonic acid by Cl2. Moreover, the early expression of 8-isoprostane in airways together with the pro-inflammatory cytokines IL-1β, IL-6 and CXCL1/ KC offer further contribution to the understanding of the mechanisms by which exposure to chemical compounds causes lung injury and AHR. Finally, we conclude that 8-isoprostane represents a promising high sensitivity biomarker for Cl2-induced oxidative lung injury that potentially can be used as a diagnostic tool, and that the role of this marker for long-standing effects such as RADS needs further attention. 6
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