* Department of Pharmacology and Toxicology,
Department of Animal Resources, and
Center for Human Toxicology, University of Utah, Salt Lake City, Utah 84112-5820
Received November 15, 2002; accepted January 20, 2003
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ABSTRACT |
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Key Words: capsaicinoids; vanilloid receptors; TRPV1; cytokines; pepper sprays; inflammation; bronchiolar epithelial cells; BEAS-2B cells.
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INTRODUCTION |
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The recent cloning and characterization of a capsaicin-sensitive receptor from animal (Caterina et al., 1997) and human (Hayes et al., 2000
) tissues has provided a long-awaited molecular target for the capsaicinoids. Structure-activity studies of capsaicin (and structural variants) have demonstrated a strict requirement for both the 4-hydroxy-3-methoxybenzylamide (vanilloid ring pharmacophore) and acyl chain moieties for pharmacologic activity (Caterina and Julius, 2001
; Szallasi and Blumberg, 1999
). Similarly, a variety of other receptor ligands (resiniferatoxin, olvanil, capsazepine, phorbol 20-homovanillates, etc.) required the presence of the vanilloid ring. Because of this apparent structural requirement, the capsaicin receptor has been named the vanilloid receptor type-1 (VR1). A new nomenclature has recently been suggested for the superfamily of transient receptor potential (TRP) cation channels (Montell et al., 2002
). This nomenclature renames vanilloid receptor type-1 (VR1) as TRPV1, and this new designation is used hereafter. TRPV1 was the first member of the growing family of vanilloid receptors to be characterized (Caterina and Julius, 2001
; Montell et al., 2002
; Szallasi, 2001
) and has been described as a cell membrane-bound ligand-gated calcium channel, with high selectivity for capsaicin and other vanilloid-like compounds. TRPV1 has also been shown to be activated by acidic pH and temperatures >42°C (Caterina and Julius, 2001
; Montell et al., 2002
; Szallasi, 2001
).
Since the discovery of TRPV1, a variety of other vanilloid receptor-like proteins (e.g., VRL-1, VRL-2, VR-OAC, SIC, TRPM8, and VR.5'sv) have been identified (Caterina et al., 1999; Delany et al., 2001
; Schumacher et al., 2000
). Recent data have placed the vanilloid receptors in an ever-expanding family (Montell et al., 2002
) of TRP ion channels that includes not only ligand-, heat-, and pH-activated calcium channels, but receptors that are activated by cold (McKemy et al., 2002
), extracellular osmolarity (Liedtke et al., 2000
; Strotmann et al., 2000
), and cell volume (Suzuki et al., 1999
). Some of these receptors (i.e., VR.5'sv and VRL-2) do not have known functions and/or agonists (Delany et al., 2001
; Schumacher et al., 2000
). This intriguing family of genes presents the scientific community with a cornucopia of receptors that appear to respond to an amazing variety of environmental stimuli, including noxious irritants, environmental pollutants, and temperature (Caterina and Julius, 2001
).
Perhaps the most intriguing facet of the identification of the vanilloid receptor family of ion channels has been that their functions do not appear to be limited only to the perception of noxious stimuli (i.e., capsaicin, pH, or heat) through activation of nerve fibers, but that several of the vanilloid receptors (e.g., the VRL-2 and VR.5'sv receptors) are highly expressed in non-neuronal cells (Hayes et al., 2000; Inoue et al., 2002
; Sanchez et al., 2001
) including epithelial cells of the kidney and respiratory tissues (Delany et al., 2001
; Hayes et al., 2000
). To date, however, a physiological role for these receptors in non-neuronal tissues has not been established.
Previous research has demonstrated that the activation of TRPV1 expressed by cultured neurons isolated from rat dorsal root ganglia promoted cell death (Szallasi and Blumberg, 1999). The cytotoxic properties of capsaicinoids in peripheral sensory (A
and C-fiber) neurons have been well documented (Szallasi and Blumberg, 1999
; Wood et al., 1988
) and are exploited for the treatment of chronic pain (McMahon et al., 1991
). The mechanism of neurotoxicity by vanilloid receptor ligands has been shown to be calcium-dependent, inhibited by capsazepine and ruthenium red, and, thus, mediated by TRPV1. The role of TRPV1 in the cytotoxicity of capsaicin in non-neuronal cell lines has also been investigated, but not fully elucidated. For example, HEK293 cells engineered to overexpress rat TRPV1 demonstrated enhanced calcium flux and cell death that was inhibited by capsazepine, ruthenium red, and by removal of calcium from the media (Caterina et al., 1997
; Jordt et al., 2000
). However, a variety of other cell lines, including monkey kidney (Vero; Creppy et al., 2000
), human neuroblastoma (SHSY-5Y; Richeux et al., 1999
), and human endothelial (ECV340; Richeux et al., 2000
) were not protected from cytotoxicity by capsazepine or modulators of calcium flux unless rat TRPV1 was transfected into these cells. Similarly, human glioblastoma (A172) cells (Lee et al., 2000
) were not protected by capsazepine or modulators of calcium flux, despite the apparent endogenous expression of TRPV1. Therefore, a general mechanism to explain the TRPV1-mediated cytotoxicity of capsaicin (and other vanilloid compounds) has not been established. Rather, it appears that different cell lines respond in unique manner to TRPV1-mediated signaling induced by ligand binding.
Capsaicinoids have also been used to study the cough reflex and neurogenic inflammation in respiratory tissues. In neurogenic inflammation, capsaicin promotes the calcium- and TRPV1-dependent release of Substance P, and other neuropeptides from neurons in the airway tissues (Veronesi et al., 1999, 2000
), to stimulate inflammatory responses to potentially harmful stimuli, including particulate material. Recent work at the United States Environmental Protection Agency has demonstrated that capsaicin, particulate matter, and neuropeptides acted synergistically to promote the production of inflammatory mediators (IL-6, IL-8, and tumor necrosis factor-
[TNF-
]) by human respiratory epithelial cells: human bronchiolar epithelial cells (BEAS-2B), human lung adenocarcinoma cell line (A549), and normal human bronchiolar epithelial cells (Quay et al., 1998
; Veronesi et al., 1999
, 2000
). Cytokine production by BEAS-2B cells was ameliorated by capsazepine and by removal of calcium from the treatment solutions (Veronesi et al., 1999
, 2000
). Similar cytokine responses were also observed in rats (intratracheal instillation) and humans (bronchoscope) treated with concentrated ambient particulate material (Carter et al., 1997
; Lay et al., 1999
). Thus, direct activation of TRPV1 in these cells by various stimuli can cause calcium-dependent cytokine production and acute respiratory inflammation.
Although these data provided evidence for the expression of functional TRPV1 in these cells, direct evidence of TRPV1 expression was not provided. Also, the influence of TRPV1 on cellular susceptibility to cytotoxicity by these substances was not investigated, despite observation in vivo that demonstrated increases in lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluid of treated animals and humans (Carter et al., 1997; Lay et al., 1999
). Therefore, it seems likely that activation of vanilloid receptors, presumably TRPV1, in respiratory epithelial cells by capsaicinoids initiates the production of proinflammatory cytokines to mount a host-defense response to protect against potentially harmful inhaled cytotoxic substances including capsaicin and particulate material. Unfortunately, this response may lead to cell death.
Thus, a hypothesis is formulated that capsaicinoids, which are present in pepper spray products, induce acute inflammation and respiratory epithelial cell injury through activation of TRPV1 in rat and human respiratory tissues. Activation of TRPV1 may induce cell death through the production of cytokines that are toxic to the same cells that have produced them; alternatively, cell death may be independent of cytokine effects. This hypothesis was addressed by nose-only inhalation exposure of rats to pepper sprays (capsaicinoids), by in vitro studies with human lung epithelial (BEAS-2B and A549) or liver cells: the human hepatoma cell line (HepG2), and by the production and characterization of a TRPV1 overexpressing human lung epithelial cell line.
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MATERIALS AND METHODS |
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Nose-only inhalation.
Male Sprague-Dawley rats (125 g) were purchased from Charles River Laboratories (Wilmington, MA). Prior to exposure to the capsaicinoids, the rats were anesthetized by ip injection of 80 mg/kg ketamine and 5 mg/kg acetopromazine. The animals were placed inside a nose-only exposure apparatus (In-Tox Products, Albuquerque, NM) and exposed (30 min) to aerosols generated from ethanolic solutions of capsaicinoids that were collected from pepper spray canisters (Reilly et al., 2001a). Aerosols were generated using a Lovelace nebulizer operated at a flow rate of approximately 0.5 l/min. The chamber flow was approximately 10 l/min and was maintained under a vacuum of -0.5 psi (approximately 26 mm Hg). Using this protocol, approximately 85% of the generated aerosol particles were between 1.7 and 0.2 µm median mass aerodynamic diameter, determined using a seven-stage cascade impactor. An estimate of the delivered dose was achieved by quantitative analysis (Reilly et al., 2001a
,b
) of a paper filter (0.2 µm) that collected aerosol from a sampling orifice. The sampling rate for the filter was approximately 0.5 l/min. The delivered dose was calculated using a minute volume of 0.2 l/min and an approximate deep lung deposition factor of 10% (Edward Barr, Lovelace Respiratory Research Institute, Albuquerque, NM, personal communication). Prior to each experiment, all gas flows were calibrated. The concentrations of capsaicinoids in lung and blood of rats exposed to capsaicinoids using this protocol was recently published (Reilly et al., 2002
).
Following exposure, the animals were removed from the chamber and permitted to recover. Following the predetermined recovery periods, the animals were sacrificed by CO2-induced asphyxiation and the nasal passages, trachea, and lungs were excised. A catheter-tipped syringe was used to manually infuse approximately 250500 µl of 10% neutral-buffered formalin into the trachea and lungs until they increased approximately two-fold in volume. This degree of inflation was sufficient to observe small airway architecture. The respiratory tissues were treated for 23 days in fixative prior to sample preparation and sectioning through the major bronchiolar and distal alveolar regions. Nasal samples were obtained by sectioning through the first and second palatal ridges of decalcified heads (Young, 1981), prepared by treating the heads for approximately 36 h in CalExII-decalcifying/fixing solution (Fisher Scientific, Fairlawn, NJ). The samples were sectioned, stained with hematoxylin and eosin, and evaluated using both qualitative and semiquantitative criteria (i.e., a numerical score was used to represent the frequency and/or severity of the lesions). For example, the degree of inflammation (congestion and edema) was scored 04 based on the size of the lesion (i.e., the entity was not present versus an entity greater than >0.5 mm). Cellular infiltrate was scored 04 by counting the number and type of cells observed in four different fields at 45X while epithelial loss was scored 04 based on the presence or absence of the lesion, its continuity, and its frequency. Necrosis, epithelial dysplasia, squamous metaplasia, inflammation (congestion and edema), epithelial loss, cellular infiltrate (lymphocytes, plasma cells, macrophages, neutrophils, and eosinophils), fibroendothelial proliferation, fibrosis, giant cells, goblet cell hyperplasia, hemorrhage, and alveolar emphysema were all evaluated by semiquantitative criteria. Lesions that appeared at a high frequency and at moderate to marked severity (a score of 14) in at least 50% of the treated population were identified and described below.
Cell culture.
Immortalized human bronchiolar epithelial (BEAS-2B), human lung adenocarcinoma (A549), and human hepatoma (HepG2) cell lines were obtained from American Type Culture Collection (Rockville, MD). BEAS-2B cells were cultured in Lechner and LaVeck media (LHC-9), containing retinoic acid (33 nM) and epinephrine (2.75 µM), using plastic cell culture dishes pre-coated with LHC-basal medium containing BSA (100 µg/ml) collagen (30 µg/ml) and fibronectin (10 µg/ml) for 4 h at 37°C. A549 cells were cultured using DMEM:F12 media containing 10% FBS. HepG2 cells were cultured in Eagles MEM (Gibco BRL) supplemented with 1 mM sodium pyruvate, 2 mM sodium bicarbonate, and 10% FBS. All cells were maintained in 75-cm2 flasks at 37°C in an air ventilated and humidified incubator maintained at 5% CO2. Culture media was renewed every 23 days and cells were subcultured every 56 days using 0.25% trypsin.
Cytotoxicity assays.
Cells were subcultured into 96-well cell culture plates at ~ 75% confluency and permitted to adhere for 812 h at 37°C. The cells were washed once with sterile phosphate-buffered saline and treated with increasing concentrations of capsaicin (0200 µM; prepared in 100% ethanol and maintained at 0.5% v/v in the treatment solutions), or other TRPV1 ligands, in serum-free cell culture medium (minus FBS) for 24 h at 37°C. Where specified, inhibitors of TRPV1 were added 30 min prior addition of the treatment solutions. Cell viability was assessed using the Dojindo Cell Counting Kit-8 (Dojindo Laboratories, Gaithersburg, MD), according to the supplier recommendations. Cell viability was expressed as the percentage of viable cells relative to untreated cells using the absorbance at 450 nm. All experiments were performed in triplicate on three separate occasions.
ELISA assays for IL-6.
BEAS-2B cells were subcultured into 24-well coated cell culture dishes at ~50% confluency and permitted to adhere for 812 h at 37°C. Prior to treatment, the cells were washed once with fresh media. Treatments were performed in cell culture media containing capsaicin and various modulators of TRPV1 function for 24 h at 37°C. After 24 h, the media was collected, clarified by centrifugation, and stored at -20°C until assayed for IL-6 content. To ensure consistent results, the cell viability for each well was determined using the Dojindo Cell Counting Kit-8. Samples exhibiting unusual values for cell viability were discarded. Cytokine production was assessed using commercial ELISA kits for IL-6 (R&D Systems, Minneapolis, MN) and performed as outlined by the manufacturer. All experiments were repeated on three separate occasions.
RT/PCR screening for TRPV1 expression.
RT-PCR was used to assess the expression of TRPV1 mRNA in BEAS-2B, A549, and HepG2 cells. Total RNA was isolated from cultured cells (approximately 1.0 x 107 cells) using the RNeasy total RNA isolation kit (Qiagen, Valencia, CA) as described by the manufacturer protocols. Total RNA was quantified using the ultraviolet-absorbance ratio (A280/A260) and 5 µg were used as a template for cDNA synthesis using Superscript II-reverse transcriptase (InVitrogen, Carlsbad, CA) and PolyT as a primer. Five µl of the first-strand synthesis reaction were used as a template for PCR. Primers specific for the published human TRPV1 sequence (Hayes et al., 2000; GenBank number XM_008512; Sense: 5'-GCAAGAACATCTGGAAGCTGC-3' and Antisense: 5'-CTTCTCCCCGGAAGCGGCAGG-3') were used to amplify a 436 nucleotide (nt) 3'-fragment of TRPV1. ß-Actin (180 nt) was also amplified by PCR as an internal control. Following an initial 2.5-min melting step, PCR was performed using a PTC-100 Programmable Thermal Controller (MJ Research Inc., Watertown, MA) and the following cycling program: 94°C for 1 min, 53°C for 1 min, and 72°C for 1.5 min. PCR was performed for 34 cycles and was followed by a 20-min incubation at 72°C. PCR products were resolved using a 1% Tris-acetate-EDTA (TAE)-agarose gel containing ethidium bromide. ß-Actin was used to normalize the PCR product band intensity during scanning densitometry using a Bio-Rad Gel-Doc 1000 System (Hercules, CA). The relative intensities for the PCR products were linear in relation to cycle number. The TRPV1 fragment was also cloned into a TOPO-TA vector (Invitrogen, Carlsbad, CA), the sequence determined, and verified by comparison to published sequences.
Cloning and overexpression of TRPV1.
The full length cDNA encoding TRPV1 was amplified by PCR from total RNA isolated from human fetal brain (Stratagene, La Jolla, CA) using pwo proofreading DNA polymerase and the following primers: Forward: 5'-CACCATGAAGAAATGGAGCAGCAC-3', containing a 5'-CACC Kozac sequence followed by the translation start-site, and Reverse: 5'-CTTCTCCCCGGAAGCGGCAGG-3'. The antisense primer was designed to amplify TRPV1, but omit the stop codon. This strategy permitted the fusion of the V5 epitope/His6 tag, contained within the expression vector sequence, to the recombinant TRPV1 protein. The amplified cDNA (2517 nt) was purified by agarose gel electrophoresis (1% TAE-agarose gel containing ethidium bromide) and cloned into the pcDNA 3.1 D-V5/His6 TOPO mammalian expression vector, as directed by the manufacturer (Invitrogen). Positive bacterial clones were selected by ampicillin resistance and screened for the presence and orientation of TRPV1 by Apa1 restriction digestion. Plasmid DNA was isolated from positive clones and the sequence of the construct verified. Vector containing the TRPV1 insert was transformed into BEAS-2B cells using FuGene6 Transfection Reagent (Roche Molecular Biochemicals, Indianapolis, IN; 3:1 FuGene6:DNA) for 8 h at 37°C in DMEM:F12 media containing 10% FBS. Transfected cells were selected by resistance to Geneticin® (400 µg/ml). BEAS-2B colonies originating from single-cell clones were readily visible about 23 weeks post-transfection. These colonies were harvested using trypsin-treated filter disks ("cloning disks"), subcultured, expanded, and screened for overexpression of TRPV1 by RT-PCR using the following primers to amplify a 510 nt fragment corresponding to the 5'end of TRPV1 as well as primers selective for the presence of the V5 epitope fusion protein (364 nt): TRPV1 Forward 5'-CACCATGAAGAAATGGAGCAGCAC-3', TRPV1 Reverse 5'-CCGTCATGCAGGTTGAGCATG-3', TRPV1-V5 Forward 5'-CTGGACCACCTGGAACACCAA-3', and TRPV1-V5 Reverse 5'-GAGGGTTAGGGATAGGCTTAC-3'. A single clone, that overexpressed TRPV1 and the mRNA for the V5-fusion protein (from approximately 26 colonies screened) was identified. Additional Geneticin-resistant colonies that did not overexpress TRPV1 or V5-epitope mRNA were also used as controls in experiments that were designed to assess the influence of TRPV1 on cellular responses to capsaicin. Since suitable antibodies for the detection of human TRPV1 protein are not available, functional overexpression of TRPV1 in BEAS-2B, TRPV1 overexpressing cells, and Geneticin-resistant (but not TRPV1 overexpressing) cells was assessed using capsaicin-induced cobalt and calcium flux, that was blocked by capsazepine.
Enhanced capsaicin-induced calcium flux was subjectively monitored with the intracellular calcium chelator Fluo-4-AM (Molecular Probes, Eugene, OR), as described by the manufacturer, and microscopic evaluation of cellular fluorescence. Quantitative assays for cobalt influx was achieved by treating cells (6 well plate; 1.0 x 106 cells) with 1.0 µM capsaicin for 10 min at 37°C in calcium- and magnesium-free Hanks Balanced Salt Solution (HBSS) containing 2.5 mM CoCl2. After incubation, the cells were placed on ice, washed twice in HBSS, and solubilized in 0.5 mL HBSS containing 2% SDS. Cellular cobalt concentration was determined using ICP-MS, performed by the Veterinary Diagnostic Laboratory at Utah State University (Logan, UT). The use of cobalt as a measure for calcium flux through TRPV1 has previously been described by Wood et al.(1988).
Analysis of apoptosis and necrosis by flow cytometry.
Differentiation between apoptosis and necrosis was assessed using the Vybrant apoptosis assay kit (Molecular Probes) containing fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide and monitoring for cellular fluorescence due to the exposure of phosphatidylserine on extracellular membrane surfaces (FITC-Annexin V binding to assess apoptosis) and nuclear staining due to loss of membrane integrity (propidium iodide staining to assess necrosis) using flow cytometry, fluorescence microscopy, and an ELISA assay measuring histone-associated DNA strand breaks. For flow cytometry and fluorescence microscopy, human bronchiolar epithelial cells (BEAS-2B) or TRPV1 overexpressing cells were treated with various concentrations of capsaicin for up to 24 h, washed once with calcium- and magnesium-free phosphate-buffered saline, harvested by trypsinization and centrifugation, and resuspended in 50 mM HEPES, pH 7.4, containing 700 mM NaCl and 12.5 mM CaCl2 (annexin binding buffer). The cells were washed once by centrifugation at 500 g for 5 min and resuspended in the same buffer. Aliquots of approximately 1 x 105 cells/ml were prepared, pelleted by centrifugation, and resuspended in 5 µl FITC-Annexin V (as provided in the assay kit) and 1 µl propidium iodide (100 µg/ml), and incubated at room temperature for 15 min. After 15 min, 400 µl of the HEPES buffer was added and the cells placed on ice until assayed by flow cytometry using a Becton-Dickenson FACScanTM fluorescence activated cell sorter and established methods for the analysis of Annexin V/propidium iodide staining. A total of 10,000 events (cells) were counted for each sample. The ELISA assay that was used to measure histone-associated DNA strand breaks was performed according to the manufacturers protocols (Roche Molecular Biochemicals).
Statistical analysis.
Statistical analysis was performed using the Microsoft Excel software package. Statistical differences between samples were established using the two-sample t-test and a 95% confidence interval (p < 0.025).
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RESULTS |
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The precise molecular mechanisms by which the pepper sprays (capsaicinoids) caused inflammation and cell damage in vivo were further investigated in vitro using various cell lines derived from human lung and liver tissues. BEAS-2B cells are an SV-40-transfected, immortalized, human bronchiolar epithelial cell line that has frequently been used to study the mechanisms of airway toxicants in vitro. Similarly, A549 cells are derived from human adenocarcinoma and serve as an additional model for studying airway toxins. The A549 cells serve as a surrogate for human alveolar epithelial cells. Human hepatoma, HepG2 cells, were used to represent liver cell responses. BEAS-2B cells treated with increasing concentrations of capsaicin (0200 µM) for 24 h exhibited a dose-dependent decrease in cell viability (Fig. 2A). The approximate LC50 value was 100 µM. Approximately 80% of the decrease in cell viability was observed within 8 h (data not shown); however, a 24-h exposure period was used to ensure the complete loss of cell viability and to minimize variability. Similar decreases in cell viability were also observed for A549 and HepG2 cells (Fig. 2A
). The LC50 value for capsaicin in A549 and HepG2 cells was approximately 110 and 200 µM, respectively. To ensure that very brief exposures to capsaicin would also cause cell death, cells were treated for 30 min, washed extensively to remove capsaicin, and the cell viability determined 24 h later. BEAS-2B cells, treated with 100 µM capsaicin in this manner, demonstrated an approximate 4050% loss in cell viability (data not shown). Thus, the LC50 values did not significantly change even with very short exposures to capsaicin, provided that cytotoxicity was assessed 24 h after the initial treatment.
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Overexpression of TRPV1 resulted in an approximate 100-fold increase in the susceptibility to cytotoxicity (Fig. 5B). Similarly, the cytotoxicity of several other TRPV1 ligands was increased in TRPV1 overexpressing cells (Table 2
). The LC50 values for olvanil and RTX decreased 15-fold and 75,000-fold, respectively, although the LC50 values for various other TRPV1 ligands decreased a mere 12-fold, suggesting that different mechanisms are involved in the cytotoxicity of these diverse compounds.
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DISCUSSION |
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The doses used for animal inhalation experiments (approximately 1.01.2 mg/kg in a 30-min exposure) are similar to doses that humans would receive during a 510 s exposure of pepper spray. Pepper sprays contain approximately 1 to 32 µg total capsaicinoids per µl of condensed spray, depending upon the product and formulation (Reilly et al., 2001a,b
). If one assumes that the pepper spray canisters contain between 5 and 50 ml of condensed spray (unpublished observations), then each canister would contain approximately 5 mg to 1.6 g total capsaicinoids. Therefore, it is reasonable to predict that a dose of 1 mg/kg (or more) could be inhaled by people exposed to large amounts of pepper spray. Our previous research (Reilly et al., 2002
) has shown that the concentrations of capsaicinoids in the blood of rats exposed to 0.57 mg/kg (approximately half of the dose used in the current studies) were as high as 125 ng/ml in blood and 174 ng/mg for lung tissues. Extrapolation of these values to the dose used in the current studies (1.01.2 mg/kg) would predict a concentration of approximately 1 µM (vide supra), which happens to be the LC50 of capsaicin in the TRPV1 overexpressing cell line. Concentrations much higher than 1 µM (concentration in blood) would be expected at the site of delivery (particle deposition) in respiratory cells after an inhalation exposure. Thus, it is also reasonable to assume that humans exposed to pepper sprays could have nasal, tracheal, bronchiolar, and/or alveolar capsaicinoid exposures similar to the concentrations that elicited cellular death and/or cytokine release in these cell culture studies, even if the subject did not receive a total dose of 1 mg/kg. Since capsaicinoids can produce significant cell death and IL-6 production in very short time periods (0.52 h), it is also reasonable to predict that inhaled doses of pepper sprays in humans could cause adverse respiratory inflammatory responses similar to those characterized in this study.
Recent work at the USEPA has demonstrated a key role for TRPV1 in mediating inflammatory responses to capsaicin and various forms of airborne particulate material in airway tissues (Veronesi et al., 1999, 2000
). Given these data, and the knowledge that TRPV1 and other TRP receptors are expressed in respiratory tissues, including the trachea, bronchi, and alveoli, we investigated the role of TRPV1 in these pathologies by the use of human airway and hepatic cell lines. The use of RT/PCR techniques confirmed the expression of TRPV1 mRNA transcripts in human lung epithelial (A549 and BEAS-2B) and liver (HepG2) cells. Capsaicin-induced cell death was greater in the two lung epithelial cell lines than the liver hepatoma cell line. Furthermore, the relative rank order of susceptibility to cytotoxicity by capsaicinoids correlated to the relative levels of TRPV1 transcripts in the three cell lines. These data suggested that TRPV1 may be a key mediator of the cytotoxic effects of capsaicin in these cells.
The mechanism of cell death in BEAS-2B cells was shown to be necrosis, not apoptosis. Surprisingly, several experimental variables designed to block cell death, including removal and chelation of calcium and the use of functional antagonists to TRPV1, were ineffective in ameliorating cell death. In addition, other TRPV1 ligands, both prototypic agonists such as RTX, olvanil, and anandamide, as well as prototypic antagonists such as capsazepine and isovelleral, were also cytotoxic at concentrations less than capsaicin. Thus, ligand binding to TRPV1 appears to trigger key cytotoxic responses that are independent of calcium flux into the cell through TRPV1 (as required for IL-6 production).
In order to elucidate the mechanisms that produced cellular toxicities, we cloned the human TRPV1 cDNA and overexpressed this receptor in BEAS-2B cells. These engineered cells expressed much higher levels of TRPV1 transcripts than the parent cell line as well as much higher capsaicin-induced ion flux that was ameliorated by capsazepine. Additional "control" cell lines were also produced in these studies. These cell lines showed stable incorporation of the Geneticin resistance expression cassette, but did not overexpress mRNA for TRPV1 or the TRPV1-V5 fusion protein. In addition, these cells did not exhibit functional increases in ion flux (cobalt and calcium) in the presence of capsaicin. The lack of TRPV1 overexpression, despite expression of Geneticin resistance, was likely due to the incorporation of the TRPV1 expression cassette into a silent portion of the genome or from interruption of the gene during recombination. However, the cells that did overexpress TRPV1 were dramatically more susceptible to capsaicin-induced cell death (approximately 100-fold) than either normal BEAS-2B or other control cell lines. Several other TRPV1 ligands were also evaluated for enhanced cytotoxicity, but only RTX and olvanil caused marked increases in toxicity in the overexpressing cells. Surprisingly, cytotoxicity in the overexpressing cells was, again, not blocked by TRPV1 antagonists or dependent on calcium flux from extracellular media.
Unexpectedly, the overexpressing cells were killed by capsaicin through apoptotic, not necrotic, mechanisms. A shift in the mechanism of cell death may indicate that TRPV1-mediated cellular injury in the TRPV1 overexpressing cells was truly occurring through programmed cell death mediated by TRPV1. However, the cytotoxicity observed in the parent cell line by a necrotic mechanism may have been the result of a composite response of several biochemical targets. We speculate that other vanilloid receptors (e.g., VRL-2, VR.5'sv, TRPM8, VRL-1, etc.), or vanilloid receptors comprised of mixed populations of vanilloid receptor family protein subunitsTRPV1 and others exist as tetramers that appear to form in response to agonist exposure (Delany et al., 2001; Kedei et al., 2001
; Kuzhikandathil et al., 2001
; Schumacher et al., 2000
)may be activated by these structurally diverse xenobiotics. These mixed receptors may also participate in the regulation of the responses that were observed in these studies. For example, activation of these mixed receptors or other vanilloid receptors may contribute to both pro- and antiapoptotic responses in BEAS-2B cells, with anti-apoptotic responses dominating. The net result could be necrotic cell death produced primarily from non-TRPV1-mediated processes. However, overexpression of TRPV1 may "dilute" these alternate targets for cytotoxicity, either by altering the subunit composition of mixed receptor complexes, or by increasing the density of homogenous TRPV1 tetramers, such that TRPV1-mediated proapoptotic signals dominate the cellular responses to capsaicin exposure. Regardless of the mechanism, these data reinforced the concept that cell death in lung epithelial cells treated with capsaicin was calcium-independent, but related to TRPV1 expression. These results also demonstrated that the TRPV1 overexpressing cells are a valuable tool for differentiating cytotoxicities (and proinflammatory responses) that are truly mediated by TRPV1 (as observed for capsaicin, RTX, and olvanil) versus toxicities that occur as a result of other processes that are probably independent of TRPV1 binding (e.g., capsazepine, anandamide, and others).
Another significant finding was that the TRPV1 overexpressing cells were also more responsive to proinflammatory stimuli. IL-6 production by "normal" BEAS-2B cells increased dramatically (4.57-fold) in the presence of 100 µM capsaicin. Increases in IL-6 production were also observed with 7.5 µM RTX (~twofold), but not in the presence of 12.5 µM anandamide or 25 µM capsazepine. The TRPV1 overexpressing cells doubled IL-6 production in response to capsaicin concentrations that were approximately 100200-fold lower than the concentrations that produced this response in the parent cell line. Interestingly, the only ligands that increased cytokine production in BEAS-2B cells were the same ligands that exhibited enhanced cytotoxicity in the TRPV1 overexpressing cells. Cytokine production by these cells was also inhibited by capsazepine and EGTA. These data confirmed the vital role of TRPV1 and calcium flux through TRPV1 in the induction of cytokine production by lung epithelial cells that are exposed to capsaicin.
From these data, we conclude that TRPV1 activation mediated cell death and cytokine production by BEAS-2B cells treated with capsaicin and other selected TRPV1 ligands. However, these data may also suggest that the cytotoxicity observed in normal BEAS-2B cells treated with capsaicin and other TRPV1 ligands that do not exhibit enhanced cytotoxicity in TRPV1 overexpressing cells was probably caused by interaction with additional biochemical targets. Although we have not identified the alternate biochemical targets that mediated the cytotoxicity of these xenobiotics, one hypothesis is that other members of the TRP receptor superfamily, whose function has not been evaluated in human lung epithelial cells, may mediate these responses. These hypotheses may help explain the apparent discrepancies between the cytotoxicity and cytokine data, and the inability of capsazepine to block cytotoxicity, as well as provide insight into the unique nature of the cytotoxicity of capsaicin in BEAS-2B, and possibly other lung cells. For example, it may be possible that heterogeneous receptor complexes that mediate cell death are activated by capsaicin, but are not inhibited by capsazepine. Similarly, alternate receptor complexes (probably homogeneous TRPV1 tetrameric complexes) that mediate cytokine production may be activated by capsaicin and inhibited by capsazepine. Studies to characterize the other vanilloid receptors, and the potential functional significance of heteromeric complexes of vanilloid receptors, are underway in our laboratory.
In summary, these studies demonstrated that capsaicinoids produced acute pulmonary inflammation and respiratory cell injury in experimental animals and in human lung epithelial cells. These pathologies and toxicities appeared to occur through activation of TRPV1, and possibly other related vanilloid receptor proteins, through complex processes that appear, at least in part, to be mediated by unique and separate calcium-dependent and calcium-independent mechanisms. Thus, the cytotoxic and proinflammatory response mechanisms emerge as distinct processes in human lung epithelial cells that are mediated, at least in part, by TRPV1. These studies provide a fascinating foray into the precise molecular mechanisms that control respiratory responsiveness to a large number of environmental irritants, including pepper sprays and possibly other respiratory irritants and toxicants such as ambient particulate matter. Additional characterization of other TRP/vanilloid receptor proteins that may also be expressed in respiratory epithelial cells should help clarify the relative contributions made by the plethora of vanilloid receptors that control airway responsiveness to various environmental stimuli.
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