Habitual marijuana smoking is associated with inflammation and atypia of airway epithelium accompanied by symptoms of chronic bronchitis. We hypothesized that Δ9-tetrahydrocannabinol (THC), the primary psychoactive component of marijuana, might contribute to these findings by impairing cellular energetics and mitochondrial function. To test this hypothesis, we examined particulate smoke extracts from marijuana cigarettes, tobacco cigarettes, and placebo marijuana (0% THC) cigarettes for their effects on the mitochondrial function of A549 cells in vitro. Only extracts prepared from marijuana cigarettes altered mitochondrial staining by the potentiometric probe JC-1. With the use of a cross-flow, nose-only inhalation system, rats were then exposed for 20 min to whole marijuana smoke and examined for its effects on airway epithelial cells. Inhalation of marijuana smoke produced lung tissue concentrations of THC that were 8–10 times higher than those measured in blood (75 ± 38 ng/g wet wt tissue vs. 9.2 ± 2.0 ng/ml), suggesting high local exposure. Intratracheal infusion of JC-1 immediately following marijuana smoke exposure revealed a diffuse decrease in lung cell JC-1 red fluorescence compared with tissue from unexposed or placebo smoke-exposed rats. Exposure to marijuana smoke in vivo also decreased JC-1 red fluorescence (54% decrease, P < 0.01) and ATP levels (75% decrease, P < 0.01) in single-cell preparations of tracheal epithelial cells. These results suggest that inhalation of marijuana smoke has deleterious effects on airway epithelial cell energetics that may contribute to the adverse pulmonary consequences of marijuana smoking.
- adenosine 5′-triphosphate
the toxicological consequences of marijuana smoking on the lung, brain, immune system, and other organs is a subject of considerable importance and debate (1, 3, 14, 19, 31–33). Most of the toxic compounds found in tobacco smoke also are present in similar quantities in marijuana smoke, except that marijuana contains high concentrations of Δ9-tetrahydrocannabinol (THC) and other cannabinoids in place of nicotine (21). Furthermore, because of differences in the way that it is smoked, a single marijuana cigarette may deposit 5–10 times as many smoke-related particulates into the lungs as does a regular tobacco cigarette (35). Even though habitual marijuana smokers may consume only a few marijuana cigarettes per day, they demonstrate symptoms of chronic bronchitis and signs of airway inflammation and mucosal epithelial injury similar to those observed in moderate to heavy tobacco smokers (35). Despite this evidence, there is a common perception that exposure to marijuana smoke poses no danger to humans and has little effect on the lung (1, 12).
Several early reports suggested that THC, the major psychoactive component of marijuana smoke, can disrupt normal mitochondrial function and cell energetics in a variety of tissues and biological systems. In vitro studies using isolated tissues and mitochondria described decreased utilization of O2 (5) and glucose (10) in response to THC. Mahoney and Harris (15) performed detailed studies on the interaction between THC and isolated mitochondria from rat liver and found deleterious effects on respiration, energetics, and organelle swelling. Uncoupling of state IV respiration was suggested as a possible mechanism, although further characterization was not performed.
Using A549 lung adenocarcinoma cells as a model for human airway epithelial cells, we observed that exposure to 0.5–10 μg/ml THC decreased cell ATP levels and disrupted mitochondrial membrane potential as measured using the fluorescent probe JC-1 (28). These events were independent of effects on cell viability. Flow cytometric analysis revealed that particulate marijuana smoke extract produced effects similar to those of THC, whereas tobacco smoke produced distinctly different JC-1 fluorescence staining characteristics. Effects resulting from exposure to THC were partially prevented by pretreatment with cyclosporin A, suggesting a role for the mitochondrial permeability transition pore.
Similar effects of THC on the mitochondrial activity of primary cultures of human lung epithelial cells were recently reported (27), raising the question as to whether or not marijuana smoke exerts these effects on lung cells in vivo. The airway epithelium is directly exposed to inhaled marijuana smoke and may be at the highest risk for exposure-related toxicity.
To address this question, we established a nose-only exposure model in which rats are exposed to controlled amounts of marijuana smoke and evaluated for effects on airway epithelial cells in vivo. This approach identifies an acute and deleterious impact from marijuana smoke that is attributable to the intrapulmonary exposure to THC.
MATERIALS AND METHODS
Unfiltered marijuana cigarettes weighing 700–800 mg and containing either 0% THC (placebo marijuana, ethanol extracted) or 2.7–3% THC (active marijuana) were obtained from the National Institute on Drug Abuse (NIDA, Bethesda, MD). Synthetic THC and deuterated THC ([2H3]THC; 1 mg/ml ethanol) also were obtained from NIDA. Tobacco cigarettes were purchased commercially (Marlboro Red hard-pack filtered cigarettes, 10 cm, 1–1.05 g). Tar-containing extracts were prepared by passing whole smoke through a Cambridge filter (Performance Systematic, Caledonia, MI) according to a standardized protocol, followed by solubilization in DMSO and final dilution in culture medium (29). DMSO, in the absence of tar extract, was diluted in culture medium as a control. Purified THC (50 mg/ml in ethanol) was serially diluted in culture medium. 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was purchased from Molecular Probes (Eugene, OR), and fetal calf serum (FCS), BSA, PBS, and RPMI 1640 medium were purchased from Irvine Scientific (Irvine, CA). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was obtained from Sigma-Aldrich (St. Louis, MI).
In vitro cell cultures and mitochondrial assays.
A549 cells (CCL-185; American Tissue Culture Collection, Bethesda, MD) were cultured in RPMI 1640 containing 10% FCS and 1% penicillin/streptomycin/fungizone (Irvine Scientific). Cells were passaged every 3–4 days and plated in 12-well plates at 1 × 105 cells per well or in 24-well plates at 0.5 × 104 cells per well for assays. Mitochondrial function was assessed using the fluorescent probe JC-1, which produces green fluorescence in the cell cytoplasm and red/orange fluorescence when it aggregates in respiring mitochondria. Cells were incubated for 30 min with 2 μg/ml JC-1 in culture medium and washed once with medium. Fluorescence measurements were taken with a fluorescence plate reader (Cytofluor 2300; PerSeptive Biosystems, Farmingham, MA) using the 530-nm excitation/590-nm emission setting for J aggregates (red/orange fluorescence) and the 485-nm excitation/530-nm emission setting for cytoplasmic fluorescence (green). Alternatively, fluorescence microscopy was performed using a Zeiss Axiophot 2 fluorescent microscope equipped with a dual rhodamine/fluorescein filter set, ×40 aqueous immersible objective, and digital camera imaging system (Diagnostic Instruments). Mounted fresh lung tissue and isolated tracheal epithelial cells were examined with an oil-immersion ×40 objective.
Animals and smoke exposure model.
Male Sprague-Dawley rats weighing 200–350 g were used for all studies (Charles River) and handled according to protocols approved by the UCLA Animal Research Committee. Drinking water and standard laboratory chow were available ad libitum, and animals were housed in a pathogen-free, barrier-confined vivarium with alternating 12-h periods of light and dark until 1 day before the experiment. Rats were briefly introduced to nose-only exposure cylinders before experiments and demonstrated no adverse reaction to the confinement. Exposure to marijuana, placebo, or tobacco smoke was achieved via a nose-only manifold with rats held at a 90° angle relative to smoke flow. Rats were held in appropriate-sized tubes fitted with an aluminum nose cone connected via a double O-ring seal to the exposure manifold (23). Puffing was performed with the use of a Hamilton syringe delivering 50-ml puffs at 20-s intervals into a 5-liter dilution chamber. Smoke was pulled from the dilution chamber through the nose-only manifold at 0.5 l/min with a calibrated rotameter. Smoke that did not deposit in the animal or the nose-only exposure manifold was collected on a Cambridge filter pad for spectrophotometric quantification. Smoke generated from one cigarette was passed into the dilution chamber over a period of 5 min and mixed with air flowing through a separate intake port at 0.5 l/min. The gradually diluted smoke from the mixing chamber was administered to the animal for a total exposure time of 20 min. After smoke exposure, rats were immediately anesthetized with a lethal intraperitoneal injection of pentobarbital sodium (100 mg/kg).
Characterization of marijuana and tobacco smoke.
The particle size distribution of marijuana, placebo marijuana, and tobacco smoke delivered by the nose-only exposure manifold, as well as the content of THC within particles of different sizes, was measured by collecting smoke with a seven-stage cascade impactor (RJR model; In-Tox Products, Albuquerque, NM). Impactor stages were soaked in 2 ml of acetonitrile for a minimum of 30 min, and eluted material was assayed for tar content by absorbance at 400 nm (17, 29). THC content of each fraction also was measured using gas chromatography-mass spectroscopy (GC-MS) on samples purified by solid-phase chromatography according to the United Chemical Technologies protocol for the ZSDAU020 extraction column. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the particulates within each type of smoke and for the THC within marijuana smoke particulates were used in conjunction with a rat airway dosimetry program [multiple path particle dosimetry (MPPD) model (24)] to estimate their regional deposition within the lung (extrathoracic, tracheobronchial, and alveolar). Default respiratory values were used in these calculations (nasal breathing, functional residual capacity = 4.0 ml, tidal volume = 2.1 ml, breathing frequency = 102 breaths/min).
Determination of THC concentrations in whole blood and lung tissue.
After death, a thoracotomy was performed and blood samples were collected by direct cardiac puncture. The lungs were then lavaged with warm PBS, and the vascular circulation was perfused with PBS (37°C) via a right cardiac ventricular catheter until the lung tissue was completely blanched. The left diaphragmatic lobe was removed and minced with scissors, and a preweighed piece (∼1 g) homogenized with a glass homogenizer in 2 ml of PBS containing 200 ng of [2H3]THC as an internal standard for determination of THC content by GC-MS.
Assessing mitochondrial activity in whole lung tissue.
The impact of smoke exposure on mitochondrial function in situ was assessed by staining with JC-1. Immediately after death, the trachea was cannulated and 5 ml of prewarmed RPMI 1640 (37°C) containing 10% FCS and JC-1 (1 μg/ml) was instilled and allowed to dwell for 15 min. Lungs were then lavaged with several 5-ml aliquots of warm PBS and removed from the animals for processing. The lung right cardiac lobe was sectioned into 100-μm thick slices and placed onto a glass slide with 10 μl of PBS. The tissue was compressed using a coverslip, sealed with paraffin, and immediately examined by fluorescence microscopy using a ×40 oil-immersion objective. Images captured from 15–20 randomly selected fields were analyzed for red and green fluorescence intensity (Adobe Photoshop). Red/green fluorescence ratios were determined after background subtraction.
Recovery and assay of rat tracheal epithelial cells.
Fresh viable tracheal epithelial cells were rapidly recovered from control and smoke-exposed animals by a modification of the method of Davidson et al. (6). Rat tracheae were excised, extraneous tissue was dissected away, and tracheal sections were incubated for 1 h (37°C) with a digestion buffer consisting of 44 mM NaCO3, 55 mM KCl, 110 mM NaCl, 0.9 mM NaH2PO4, 0.25 mM FeN3O9, 42 μM phenol red, 1.4 mg/ml pronase (Streptomyces griseus), and 0.1 mg/ml pancreatic DNase I, pH 7.5 (all from Sigma-Aldrich). After digestion and mild agitation, the tracheal husk was removed and cells were isolated and washed by centrifugation in DMEM/Ham’s F-12 (50:50). More than 90% of cells recovered from the digestion medium stained with a pan-cytokeratin antibody (catalog no. 250400; Calbiochem) consistent with their epithelial cell origin.
In assays evaluating mitochondrial membrane potential by JC-1 fluorescence, l μg/ml JC-1 was included during the digestion procedure to simultaneously load the cells with this potentiometric probe. Single-cell suspensions were washed, and cytospins were prepared onto glass slides (3 × 104 cells, 5 min at 500 rpm). For some experiments, a portion of these tracheal epithelial cells were suspended in PBS and exposed for 10 min to 10 μg/ml THC or to 50 μM FCCP before preparing cytospins. Cells were counterstained with Hoechst 33342 dye (10 μM; Molecular Probes), and a wet mount was immediately examined using fluorescence microscopy. Captured images were analyzed for red and green fluorescence intensity as described above. ATP levels in the single cell preparations were determined using the Promega CellTiter-Glo Luminescent assay kit (catalog no. G7571) according to the manufacturer's protocol.
For in vitro multiwell JC-1 studies on smoke particulate extracts, data were analyzed using two-way ANOVA with Fisher's post hoc test. All other data were analyzed using the Student's t-test. P values <0.05 were considered significant.
A549 cells, a tumor line with alveolar epithelial cell characteristics, were used to evaluate different smoke extracts and purified THC for their in vitro effects on mitochondria. The ratio of JC-1 in its aggregated intramitochondrial form (red/orange fluorescence) to its soluble cytoplasmic form (green fluorescence) was used as a quantitative index of mitochondrial function (26, 30). As demonstrated in Fig. 1A, exposure of A549 cells to marijuana smoke extract for 1 h produced a concentration-dependent decrease in red/green fluorescence ratio with an ∼50% reduction, compared with vehicle-treated control cells, at a tar extract concentration of 15 μg/ml. By contrast, when A549 cells were exposed to tar generated from tobacco or placebo marijuana cigarettes (<0.01% THC), the red/green fluorescence ratio modestly increased compared with control cells. As a proof of principle, the addition of THC (8 μg/ml) to control cells (not shown) or to tobacco tar extracts recapitulated the effects observed with marijuana tar extract. Examination of these cells by fluorescence microscopy confirmed the presence of punctate red/orange staining within the mitochondria of control cells and cells exposed to tobacco or placebo marijuana tar but the obvious loss of these mitochondrial aggregates by cells exposed to marijuana tar extract or to pure THC (Fig. 1B).
A small-animal smoke exposure system was developed to assess whether whole marijuana smoke had a similar effect on epithelial cells in vivo when inhaled into the lung (Fig. 2). With this model, mainstream smoke was generated from the proximal end of lit cigarettes by puffing with a syringe, and the smoke was expelled into a mixing chamber. The smoke was diluted with fresh air and then delivered as a continuous flow to rats held within with nose-only exposure cylinders inserted at a 90° angle to the smoke exposure manifold. A multistage cascade impactor was used to evaluate the smoke delivered by this system and to predict particle deposition within the rat lung based on particle size distribution (Table 1). The MMAD for particulates contained within marijuana and placebo marijuana smoke were similar (0.82 and 0.81 μm, respectively) but significantly larger than the MMAD for tobacco smoke (0.46 μm). THC was relatively equally distributed among the particles. A computer-based prediction model was then employed to estimate the relative deposition of tar throughout the respiratory tract of the rats, assuming normal ventilatory parameters (Table 2). With the larger particle size, a modestly higher percentage of marijuana tar, compared with tobacco tar, was predicted to deposit in the upper airway (nasopharynx/hypopharynx; 16.9 vs. 7.4%, respectively). Despite this, deposition within the trachea and bronchioles, and within the alveoli of different regions of the lung, was predicted to be relatively similar. Based on these determinations and on average breath parameters, THC deposition within the lung during the 20-min smoke exposure was estimated at 39.5 μg, with 15.1 μg predicted to be deposited within alveoli.
Because of the high levels of local deposition, we hypothesized that lung tissue concentrations of THC might be considerably higher than those measured in the blood. Animals killed after breathing smoke from a single marijuana cigarette (2.7–3.0% THC) for 20 min had lung THC levels on average 75 ± 38 ng THC/g wet tissue weight (measured from left diaphragmatic lobe; Fig. 3). After animals breathed smoke from two marijuana cigarettes (over 50 min), we measured an average THC content of 114 ± 34 ng THC/g wet tissue weight. These levels ranged from 5 to 10 times higher than THC levels measured simultaneously in whole blood from the same animals (9.2 and 14 ng/ml for 1 and 2 cigarettes, respectively).
Having documented that animals were exposed to smoke and that THC was deposited in the lung at high concentrations, we proceeded to evaluate the biological impact of these exposures on mitochondrial function. The intratracheal instillation of JC-1 was used as a method for rapidly loading lung epithelial cells with this potentiometric dye. Analysis of fresh viable tissue sections by fluorescence microscopy revealed diffuse punctate red/orange staining of mitochondria similar to that observed for A549 cells loaded with JC-1 in vitro (Fig. 4A). This red staining, indicating respiring, polarized mitochondria, appeared against a background of variable green cytoplasmic fluorescence. No red/orange punctate staining was observed in lung tissue in the absence of JC-1 exposure (not shown), and similar to the effect of marijuana smoke extract on mitochondrial staining in vitro, there was an obvious decrease in mitochondrial staining when lung sections were examined from marijuana smoke-exposed animals. Quantification of red fluorescence by image analysis revealed a dose-dependent decrease in the ratio of JC-1 red/green fluorescence when lung sections from animals exposed to marijuana smoke were compared with those from control animals (Fig. 4B). These effects were specific for marijuana smoke because an equivalent exposure to tobacco smoke had no significant effect on JC-1 red fluorescence (Fig. 5). As a positive control for these experiments, some of the control animals were exposed to an intratracheal infusion of FCCP (50 μM), a mitochondrial electron transport uncoupler, for 10 min before loading with JC-1. FCCP significantly decreased JC-1 red fluorescence to levels similar to those observed after marijuana smoke exposure.
To more precisely relate the smoke exposure to effects on airway epithelium, we recovered single-cell preparations of airway epithelial cells from tracheal sections. More than 90% of the cells isolated by pronase digestion stained with a pan-cytokeratin antibody and appeared characteristic for airway epithelial cells by microscopy. Similar to the results obtained with whole lung sections, tracheal epithelial cells from control animals displayed red/orange punctate staining (Fig. 6). To verify the sensitivity of mitochondria in the isolated tracheal epithelial cells, we exposed cells obtained from control untreated rats for 30 min to either control medium or medium containing 10 μg/ml THC or 50 μM FCCP. When these cells were loaded with JC-1 and viewed under the microscope, cells treated with THC or FCCP displayed 40 and 52% reduction, respectively, in red staining relative to control cells (Fig. 6).
Tracheal epithelial cells recovered from untreated control, placebo, and marijuana smoke-exposed animals were compared for their red/green fluorescence ratios and intracellular ATP content (Fig. 7). Animals exposed to smoke from a single marijuana cigarette exhibited red/green fluorescence ratios that were 46% that of matching cells from an unexposed control rat (Fig. 7B). Similarly, ATP levels in tracheal epithelial cells from marijuana smoke-exposed rats were only 25% those of unexposed control cells (0.71 ± 0.10 vs. 2.57 ± 0.53 pmol/μg protein, respectively, P < 0.01) (Fig. 7C). These parameters were not significantly altered by placebo marijuana smoke.
The objective in establishing this animal model was to have a controlled, reproducible inhalation system that resulted in measurable levels of THC in lung and blood, comparable to those observed in human marijuana smokers. Although exact characteristics of human marijuana smoking in terms of breathholding volume and duration cannot be reproduced in the rat, the desired objectives were achieved. With MMAD of 0.82 μm for marijuana and 0.46 μm for tobacco, the smoke generated in our model had particle characteristics comparable to those reported previously (2, 9, 18). The slightly larger MMAD of marijuana smoke may have resulted from the presence of the cellulose filter of the tobacco cigarette, which would hinder flow of larger particles, or from the dissimilar composition of nicotine and cannabinoids (13). The fact that placebo marijuana smoke had a nearly identical MMAD value to that of marijuana smoke suggests that cannabinoid chemical composition did not influence particle size. THC was found to be distributed fairly uniformly among the particulate fractions with a slightly higher concentration within larger particles.
Modeling studies of airway flow dynamics indicate that smoke with the described properties would penetrate throughout all levels of the tracheobronchial tree, including the alveoli (16, 22, 25). These models predict that somewhat higher levels of smoke particulates and THC would be deposited in the left apical and right diaphragmatic lobes of the lung relative to the other lobes. Our measurements of lung and blood levels of THC confirm the predictions from the air flow and particle deposition model. The fact that lung levels of THC on a per gram wet weight tissue basis were higher than blood levels 20 min after cessation of smoke inhalation suggests that substantial amounts of THC might be retained in the lung for extended periods. This retained THC is likely contained in insoluble tar particulates manifesting slow diffusion rates with some being taken up by alveolar macrophages. Thus epithelial cells of the lung and trachea would be exposed to THC for prolonged periods and subjected to potential toxic injury similar to that observed in vitro.
To study the toxicological effects of acute marijuana smoke exposure, we developed a method of in vivo JC-1 staining of rat lung. By intratracheal infusion of JC-1 in nutrient-rich medium for 15 min, followed by immediate examination of freshly sectioned lung tissue, large numbers of fluorescent cells were observed with characteristics similar to the pattern of respiring mitochondria described in vitro. Bright red fluorescence was distributed throughout the cytoplasm in a punctate pattern consistent in size and shape with mitochondria. Because this red fluorescence was greatly diminished after infusion of 50 μM FCCP, this color likely reflects the membrane potential of coupled, respiring mitochondria. Nuclear counterstaining revealed that many cells were devoid of JC-1 fluorescence, indicating either absence of respiring mitochondria or inaccessibility of cells to the intratracheally applied JC-1. Quantification of red and green fluorescence by image analysis software revealed that marijuana smoke exposure caused a loss of red fluorescence throughout randomly selected regions as well as a decreased ratio of red/green staining of individual cells. Red fluorescence was reduced to levels produced by infusion of 50 μM FCCP, a potent mitochondrial toxin that acts by uncoupling electron transport and proton gradient generation. Both tobacco and placebo smoke exposure reduced JC-1 red fluorescence slightly but not significantly. These results provide evidence for unique disruption of mitochondrial function by marijuana smoke in exposed airway cells in vivo, most likely due to the THC contained within the smoke. Thus the previously demonstrated in vitro effects of THC on mitochondria and cell energetics can occur even in the presence of the more complex in vivo environment. The presence of heterogeneous cell types, surfactant proteins, mucus, and extracellular matrix did not prevent the induced mitochondrial injury. The fact that JC-1 mitochondrial staining occurs in some of the cells indicates that at least a subset of airway cells retains viability throughout the procedure despite the absence of air and reduced blood circulation during JC-1 staining.
The observed effects of marijuana smoke on ATP and mitochondrial membrane potential appeared to be greater in tracheal epithelial cells than in cells of peripheral lung tissue (compare data from Fig. 4, 1 cigarette, and Fig. 7; data not shown for lung ATP). At least three factors may have contributed to this differential sensitivity. First, although total deposition of marijuana particulates and THC was estimated to be higher in lung than in trachea (see Table 2), concentrations of absorbed particulates were likely to be greater in the trachea because of the much lower tracheal surface area relative to total alveolar surface area (20). Second, tracheal epithelial cells were examined as an essentially pure population isolated by pronase digestion. Greater than 95% of these cells expressed cytokeratin when assessed with a pan-cytokeratin antibody. The examined lung tissue, on the other hand, consisted of the full spectrum of lung cell types including large numbers of endothelial cells that would not have been directly exposed to smoke particulates in vivo. Third, although rates of absorption of organic compounds into tracheal and alveolar epithelial cells may have been similar, vascular clearance from tracheal cells is substantially lower (7, 8). This rate of vascular clearance would be at a theoretical minimum in cultured cells such as the A549 cell line used in the present studies. These cells manifested the greatest overall impact on cell energetics resulting from THC exposure.
It will be important to evaluate the time course and functional consequences of impaired energetics in airway cells after marijuana smoke exposure. Numerous cellular and physiological functions are highly dependent on the maintenance of homeostatic epithelial cell energetics, including cytokine and surfactant production, repair of cellular damage, transepithelial water and ion transport, cellular antibacterial defense, mucociliary transport, and apoptotic cell death. Disruption of these functions could have deleterious consequences for pulmonary function, host defense capability, and carcinogenesis. For example, pulmonary fibrosis has been proposed to be mediated by disruption of mitochondrial energetics following amiodarone treatment for patients with cardiac dysrhythmia (4). Pulmonary vasoconstriction also has been reported to result from exposure to mitochondrial toxins (34). However, such agents, including FCCP and rotenone, also have been reported to possess anti-inflammatory effects via inhibition of P-selectin release from lung venular capillaries (11). Future studies are necessary to identify and address the unique impact of marijuana smoke on these functions.
This work was supported by National Institute of Drug Abuse Grant R37 DA-03018-23.
We are grateful to the UCLA Microscopy and Spectroscopy core facilities for assistance with special experimental procedures and sample handling. We thank Drs. Enrique Rosengurt, Steve Young, and David Heber for use of the fluorescent microscope and the GC-MS, respectively. We also thank Laura Kurek for technical assistance, data analysis, and manuscript preparation.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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