Lung tissue bioenergetics and caspase activity in rodents
© Alsuwaidi et al.; licensee BioMed Central Ltd. 2013
Received: 26 July 2012
Accepted: 10 January 2013
Published: 12 January 2013
This study aimed to establish a suitable in vitro system for investigating effects of respiratory pathogens and toxins on lung tissue bioenergetics (cellular respiration and ATP content) and caspase activity. Wistar rats and C57Bl/6 mice were anesthetized by sevoflurane inhalation. Lung fragments were then collected and incubated at 37°C in a continuously gassed (with 95% O2:5% CO2) Minimal Essential Medium (MEM) or Krebs-Henseleit buffer. Phosphorescence O2 analyzer that measured dissolved O2 concentration as a function of time was used to monitor the rate of cellular mitochondrial O2 consumption. Cellular ATP content was measured using the luciferin/luciferase system. The caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspase activity; cleaved AMC moieties (reflecting caspase activity) were separated on HPLC and detected by fluorescence. Lung histology and immunostaining with anti-cleaved caspase-3 antibody were also performed.
For Wistar rats, the values of k c and ATP for 0 < t ≤ 7 h (mean ± SD) were 0.15 ± 0.02 μM O2 min-1 mg-1 (n = 18, coefficient of variation, Cv = 13%) and 131 ± 69 pmol mg-1 (n = 16, Cv = 53%), respectively. The AMC peak areas remained relatively small despite a ~5-fold rise over 6 h. Good tissue preservation was evident despite time-dependent increases in apoptotic cells. Lung tissue bioenergetics, caspase activity and structure were deleterious in unoxygenated or intermittently oxygenated solutions. Incubating lung tissue in O2 depleted MEM for 30 min or anesthesia by urethane had no effect on lung bioenergetics, but produced higher caspase activity.
Lung tissue bioenergetics and structure could be maintained in vitro in oxygenated buffer for several hours and, thus, used as biomarkers for investigating respiratory pathogens or toxins.
KeywordsIn vitro Cytotoxicity Apoptosis
In vitro systems for studying tissue mitochondrial O2 consumption (cellular respiration) and morphology in BALB/c mice are recently described [1–3]. These parameters, however, are insufficient for fully assessing cellular viability. For example, cells may respire (consume O2) at a higher rate due to uncoupling oxidative phosphorylation (short-circuiting the inner mitochondrial membrane), an event that results from increased caspase activities . Therefore, measuring cellular ATP and caspase activity are essential biomarkers for a more comprehensive assessment of tissues in vitro.
Lung toxicity is a major health concern in the drug development. The US Food and Drug Administration (FDA) has issued several post-marketing drug recalls due to adverse events related to mitochondrial dysfunctions . Thus, developing in vitro systems to assess adverse effects of drugs on lung tissue bioenergetics is important. Drug-induced mitochondrial disturbances have been investigated using different methods, including fluorescence-based oxygen sensitive probes [6–8].
In this study, the previously described method [1–3, 7] is expanded to include measurements of lung tissue ATP content and caspase activity in Wistar rats and C57Bl/6 mice. The work aimed to establish an in vitro system suitable for investigating respiratory pathogens and toxins. The impacts of experimental conditions (e.g., anesthetic agents, incubation solutions, and oxygenation) are also addressed.
Pd(II) complex of meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin ( Pd phosphor) was purchased from Porphyrin Products (Logan, UT). A lyophilized powder of caspase inhibitor I [N-benzyloxycarbonyl-val-ala-asp(O-methyl)-fluoromethylketone; zVAD-fmk; m.w. = 467.5; pan-caspase inhibitor] was purchased from Calbiochem (La Jolla, CA). Ac-DEVD-AMC (N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin; m.w. = 675.64; caspase-3 substrate) was purchased from Axxora LLC (San Diego, CA). Minimum Essential Medium (MEM Alpha Modification) was purchased from Gibco (labeled here as MEM). Dactinomycin (actinomycin D, MW ≈ 1255) was purchased from Merck (Whitehouse Station, NJ). Glucose (anhydrous) and remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO).
zVAD-fmk solution (2.14 mM) was made by dissolving 1.0 mg in 1.0 mL dimethyl sulfoxide and stored at -20°C in small aliquots. Ac-DEVD-AMC solution (7.4 mM) was made by dissolving 5.0 mg in 1.0 mL dimethyl sulfoxide and stored at -20°C. Pd phosphor solution (2.5 mg/ml = 2 mM) was prepared in dH2O and stored at -20°C in small aliquots. Dactinomycin solution was made fresh in dH2O; its concentration was determined by absorbance at 440 nm, using an extinction coefficient of 24,450 M-1 · cm-1. Sodium cyanide (NaCN) solution (1.0 M) was prepared in dH2O; the p H was adjusted to ~7.0 with 12N HCl and stored at -20°C. Glucose oxidase (10 mg/mL) was dissolved in dH2O and stored at -20°C. Krebs-Henseleit (KH) buffer (115 mM NaCl, 25 mM NaHCO3, 1.23 mM NaH2PO4, 1.2 mM Na2SO4, 5.9 mM KCL, 1.25 mM CaCl2, 1.18 mM MgCl2 and 10 mM glucose, p H ~7.4) was made fresh.
Wistar rats (weighing about 215 grams) and C57Bl/6 mice (weighing about 32.5 grams) and BALB/c mice (Additional file 1: Table S2) used in this study were maintained at the animal facility that was in compliance with NIH guidelines (http://grants.nih.gov/grants/olaw/references/phspol.htm). All animals were housed in rooms maintained at 22°C with ~60% relative humidity and a 12-hr light/dark cycle. They had ad libitum access to standard rodent chow and filtered water. The study was approved by the Animal Research Ethics Committee for care and use of laboratory animals at the College of Medicine and Health Sciences, UAE University.
The animals were anesthetized by sevoflurane inhalation (100 μL per 10 g) and sacrificed as outlined [2, 3]. Lung specimens were immediately immersed in continuously gassed with 95% O2:5% CO2 ice-cold MEM or KH buffer. For O2 measurements, specimens were placed in 1.0 mL MEM or KH buffer containing 0.5% fat-free bovine albumin and 3 μM Pd phosphor. Specimens were also processed for histology and measurements of caspase activity and ATP content as described below.
Specimens were fixed in 10% buffered formalin solution and embedded in paraffin wax blocks. Sections of the fixed lung pieces (of 3 μm thickness) were stained with haematoxylin and eosin (H&E) and examined under a light microscope.
Staining for apoptosis was performed using avidin-biotin immunoperoxidase method that detects activated caspase-3 (Cell Signaling Technology, Boston, MA). The procedure was performed on 3-μm paraffin sections using rabbit anti-cleaved caspase-3 antibody. Positive and negative control sections for apoptosis were used.
Intracellular caspase activity
Lung fragments (about 20 mg each) were collected from Wistar rats and C57Bl/6 mice as described . The samples were incubated in vitro at 37°C in 50 mL KH buffer or MEM (continuously gassed with 95% O2:5% CO2) for up to 6 h. At specific time points, samples were incubated in oxygenated KH buffer or MEM with 32 μM zVAD-fmk or 15 μL DMSO for 20 min (f/v = 1.0 mL). Ac-DEVD-AMC (37 μM) was then added and the incubation continued for additional 20 min. At the end of incubation, the tissue was disrupted by vigorous homogenization for 2 min, sonication for 3 min and 10 passages through a 27-G needle. This disruption procedure quenched the Ac-DEVD-AMC cleavage reaction due to dilution. The supernatants were collected by centrifugation (~16,300g for 90 min) through Microcentrifuge Filter (nominal molecular weight limit = 10,000 Dalton, Sigma©), separated on HPLC, and analyzed for the fluorogenic AMC moiety.
The analysis was performed on a Waters reversed-phase HPLC system, which consisted of a manual injector, a pump and a fluorescent detector. The excitation wavelength was 380 nm and the emission wavelength 460 nm. Solvents A and B were HPLC-grade methanol:dH2O 1:1 (isocratic). The column, 4.6 × 250 mm Beckman Ultrasphere IP column, was operated at 25°C at 1.0 ml/min. The run time was 10 min and the injection volume was 20 μL.
A phosphorescence oxygen analyzer was used to monitor O2 consumption by lung specimens [2, 3]. Briefly, O2 detection was performed with the aid of Pd phosphor (absorption maximum at 625 nm and phosphorescence maximum at 800 nm). Samples were exposed to light flashes (600 per min) from a pulsed light-emitting diode array with peak output at 625 nm (OTL630A-5-10-66-E, Opto Technology, Inc., Wheeling, IL). Emitted phosphorescent light was detected by a Hamamatsu photomultiplier tube after passing through an interference filter centered at 800 nm. The amplified phosphorescence decay was digitized at 1.0 MHz by a 20-MHz A/D converter (Computer Boards, Inc., Mansfield, MA).
A program was developed using Microsoft Visual Basic 6, Microsoft Access Database 2007, and Universal Library components (Universal Library for Measurements Computing Devices; http://www.mccdaq.com/daq-software/universal-library.aspx). It allowed direct reading from the PCI-DAS 4020/12 I/O Board (PCI-DAS 4020/12 I/O Board; http://www.mccdaq.com/pci-data-acquisition/PCI-DAS4020-12.aspx). The pulse detection was accomplished by searching for 10 phosphorescence intensities >1.0 volt (by default). Peak detection was accomplished by searching for the highest 10 data points of a pulse and choosing the data point closest to the pulse decay curve .
The phosphorescence decay rate (1/τ) was characterized by a single exponential; I = Ae-t/τ, where I = Pd phosphor phosphorescence intensity. The values of 1/τ were linear with dissolved O2: 1/τ = 1/τo + k q [O2, where 1/τ = the phosphorescence decay rate in the presence of O2, 1/τo = the phosphorescence decay rate in the absence of O2, and kq = the second-order O2 quenching rate constant in s-1 · μM-1.
Lung tissue respiration was measured at 37°C in 1-mL sealed vials. Mixing was with the aid of parylene-coated stirring bars. In vials sealed from air, [O2] decreased linearly with time, indicating the kinetics of mitochondrial O2 consumption was zero-order. The rate of respiration (k, in μM O2 min-1) was thus the negative of the slope d[O2]/dt. Sodium cyanide (NaCN) inhibited respiration, confirming O2 was being consumed in the mitochondrial respiratory chain.
The calibration reaction contained PBS with 3 μM Pd phosphor, 0.5% fat-free albumin, 50 μg/mL glucose oxidase and various concentrations of β-glucose . [O2 was calculated using, 1/τ = 1/τo + k q [O2.
Cellular ATP contents
Lung tissue fragments were homogenized in 0.5 mL of ice-cold 2% trichloroacetic acid for 2 min. The supernatants were collected by centrifugation (1000xg at 4°C for 5 min) and stored at -20°C until analysis. Immediately before ATP measurement, the samples were thawed and neutralized with 0.5 mL 100 mM Tris-acetate, 2 mM EDTA (final p H, 7.75). ATP concentration was determined using the Enliten ATP Assay System (Bioluminescence Detection Kit, Promega, Madison, WI). Briefly, 2.5 μL of the supernatant was added to 25 μL of the luciferin/luciferase reagent. The luminescence intensity was then measured at 25°C using Glomax Luminometer (Promega, Madison, WI). The ATP standard curve ranged from 10 pM to 100 nM (R 2 >0.9999).
The need for continuous gassing with O2:CO2 was then investigated. Figure 2A–D show Wistar rat lung tissue respiration, ATP content and caspase activity in KH buffer without gassing. The values of k c (1.5 ≤ t ≤ 8 h) were 0.118 ± 0.041 (n = 6; Cv = 35%), which were lower than those in Figure 1A (p = 0.181). The values of cellular ATP (Figure 2B, done in triplicates for 0 ≤ t ≤ 8 h) was 2.9 ±1.4 pmol mg-1 (n = 8, Cv = 48%), which were significantly less (p = 0.001) than in Figure 1B. Figure 2D shows the AMC moiety was highest at 2 h (area = 5.6 ± 1.8), while the AMC peak area for Ac-DEVD-AMC incubated without lung specimen (substrate alone) for 6 h was relatively small. Thus, continuous tissue oxygenation was necessary for maintaining cellular bioenergetics and preventing caspase activation. Furthermore, the incubation in room air (21% O2) invoked deleterious effects on cellular bioenergetics with increased caspase activity.
The same experiment was repeated in room air supplemented with 5% CO2. Briefly, lung fragments from a Wistar rat were incubated at 37°C in 50 mL MEM in a CO2 tissue culture incubator. Samples were removed from the incubation medium at 0, 3 and 6 h and processed for measurements of O2 consumption, ATP content and caspase activity. The values of k c (μM O2 min-1 mg-1) increased with time and the values of ATP (pmol mg-1) decreased with time, confirming uncoupling of oxidative phosphorylation (Additional file 1: Figure S2). Thus, the incubation in 21% O2:5% CO2 also invoked deleterious effects on cellular bioenergetics.
Additional file 1: Tables S1–S3 summarize the impacts of oxygen supply and anesthetic agents on the measured parameters; these results clearly demonstrate the need for tissue oxygenation and sevoflurane anesthesia.
The term cellular respiration implies delivery of O2 and metabolic fuels to the mitochondria, oxidation of reduced metabolic fuels with passage of electrons to O2, and ATP synthesis. Impaired respiration thus entails an interference with any of these processes. O2 consumption is a highly sensitive parameter in detecting cellular injury, since an intact biological system is required for consuming O2 and producing ATP. Interruptions at any level, such as the cell membrane, mitochondria and enzymes lead to impaired energy conversion and ATP production . For example, mitochondria isolated from hamster lungs were inhibited by amiodarone (an antidysrhythmic agent with potent pulmonary toxicities) . Similarly, inefficient oxidative phosphorylation was reported in lung mitochondria after exposure to environmental pollutants, such as dioxins and furans .
Cells are expected to repair damages if sufficient ATP is present. ATP depletion, on the other hand, inevitably leads to cell death. Thus, the fate of the cell, for most part, is determined by its bioenergetic status . This fact reflects the inherent dependency of our biologic system on high rates of aerobic metabolism. Cancer cells, on the other hand, are capable of surviving on anaerobic metabolism (commonly referred to as: “aerobic glycolysis” or Warburg effect) .
One study suggested that the mitochondrial electron transport chain could be responsible for indirect DNA damages in lung tissue . Therefore, monitoring cellular O2 consumption by mitochondrial cytochrome oxidase is important for assessing cytotoxicity.
The main purpose of this work was to develop an in vitro method to study the impact of respiratory pathogens and toxins on metabolic biomarkers (cellular respiration, ATP content and caspase activity) in lung tissue. The procedure described in Figure 1 resulted in reasonable preservation of lung tissue bioenergetics and structure (Figure 6B–C) for up to 6 h with minimum caspase activation. The success of this method relied on three critical procedural steps. The first step involved anesthesia and tissue collection. Anesthesia with sevoflurane and rapid (within 20 sec) removal of thin lung fragments from the animal were essential. It was vital to avoid organ ischemia or hypoxia before specimen collection, i.e., the organ should be well perfused during specimens’ collection. The second step involved tissue processing which required immediate immersion of collected tissues in oxygenated ice-cold MEM or KH buffer. The specimens were then cut into small slices using a sharp blade or punch biopsy device, while remaining in ice cold buffer or MEM continuously gassed with 95% O2:5% CO2. The third step involved sample incubation in a large volume solution (e.g., 50 mL) with continuous gassing with 95% O2: 5% CO2. Having adhered to these procedural steps, a coefficient of variation (Cv) for rates of respiration of 13% and for ATP of 53% over 6 h was achieved. The larger variation in ATP was likely reflecting inability to maintain a tissue perfusion comparable to that of in vivo and induction of apoptosis (Figure 7B–C).
As noted in Figure 2, in unoxygenated solutions cellular ATP was depleted and lung structure was altered (Figure 6E–F), while rates of respiration were progressively increasing indicating uncoupling oxidative phosphorylation. This presumption was supported by the fact that caspase activity was noted by 2 hr (also see Figure 7E–F). Activation of caspases leads to opening of the transition permeability pores in the inner mitochondrial membranes, which results in uncoupling oxidative phosphorylation. This process results in rapid cellular ATP and nutrient depletions. These two consequences contribute to further damage of lung tissue. Thus, initial toxic effects become compound by overwhelming insults resulting from mitochondrial and metabolic derangements. At this stage, inevitable cell death occurs despite the removal of the initial toxic insult, as cellular repair requires ATP. These concepts are relevant to human lung diseases, such as adult respiratory distress syndrome (ARDS). Interestingly, the findings in Figure 2 were not reproduced in later experiments when lung tissue was incubated in O2 depleted buffer for 30 min (Figure 3). This result could be explained by residual cellular ATP under our experimental conditions (rather than its total depletion), and hence the tissue was able to endure this brief hypoxia.
In one study, the susceptibility of mitochondria isolated from pneumatocytes of pigs to in vitro hypoxia was investigated . The results showed oxidative phosphorylation was unaffected by a 45-min hypoxia (as shown in Figure 3), suggesting the mitochondria were not directly responsible for damages observed after in vivo ischemia. Mitochondrial injuries were presumed to result from cellular insults, such as execution of apoptosis.
Urethane is a commonly used anesthetic agent in animal research. It is typically administered intraperitoneally. The results show that more caspases were induced in urethane compared to sevoflurane (Figure 4). Therefore, anesthesia with sevoflurane is recommended.
The mitochondrial function is best assessed in intact cells and tissues (rather than isolated organelles), using accurate determinations of cellular oxygen consumption and ATP synthesis. The conventional polarographic method for detecting oxygen is limited, especially for biological samples that require measurements over several hours . The more recent use of fluorescence-based oxygen probes has significantly improved the detection methodology [6–8].
The results here are consistent with observed energy deficits during hypoxia in kidney proximal tubule cells . Moreover, ischemia is well known to induce a mitochondrial damage that predisposes to superoxide anion production at the level of the respiratory chain . For example, nitric oxide donors to the mitochondria (mediated by S-nitrosates thiol proteins) during ischemia inhibit O2 consumption . Abnormal lung bioenergetics has been also reported in smokers (especially in patients with chronic obstructive lung disease and down-regulation of prohibitin-1) and with exposures to particulate matters of aerodynamic diameters of ≤10 μm [19, 20].
As a proof of concept, Additional file 1: Figure S1 shows induction of caspases in the presence of 8 μM dactinomycin, a well known potent inducer of apoptosis . Additional file 1: Figure S3 also shows the feasibility of utilizing the described method to monitor the effects of influenza A virus on cellular respiration and caspase induction following in vitro exposure of lung tissue to the virus.
The main limitation of the method described here is the need to collect and maintain viable lung tissue, avoiding ischemia and hypoxia. The lung should be well perfused and oxygenated throughout the procedure. Another limitation is the use of high oxygen concentration during in vitro incubation, which may promote free radical reactions. Thus, the described system is not ideal for testing pathogens, as exposure to high concentrations of O2 may produce non-physiological aberrations. The use of 95% oxygen, however, was not associated with noticeable toxicities (Figure 1). Nevertheless, this method may not be applicable to toxins that are likely to generate reactive oxygen species. Adequate buffering with a free radical scavenger (e.g., glutathione) may be helpful. The use of air-saturated buffer (Figure 2), intermittently oxygenated solution (Additional file 1: Figure S1, Panels A–C) or room air saturated with 5% CO2 (Additional file 1: Figure S2, Panel A) was associated with impaired cellular bioenergetics.
The described method is relatively simple and requires minimal tissue handling. It permits comprehensive analyses (e.g., cellular respiration, ATP and caspase activity) over several hours. These biomarkers can be used to study the effects of toxins and pathogens on lung tissue.
This work was supported by a grant from the UAE University.
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