Vol. 284, Issue 2, L298-L306, February 2003
9-Tetrahydrocannabinol disrupts mitochondrial
function and cell energetics
Theodore A.
Sarafian,
Shaghig
Kouyoumjian,
Farnaz
Khoshaghideh,
Donald P.
Tashkin, and
Michael D.
Roth
Department of Medicine, Division of Pulmonary and Critical
Care, Center for Health Sciences, University of California, Los
Angeles, Los Angeles, California 90095
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ABSTRACT |
We have observed rapid and extensive
depletion of cellular energy stores by
9-tetrahydrocannabinol (THC) in the pulmonary
transformed cell line A549. ATP levels declined dose dependently with
an IC50 of 7.5 µg/ml of THC after 24-h exposure. Cell
death was observed only at concentrations >10 µg/ml. Studies using
JC-1, a fluorescent probe for mitochondrial membrane potential,
revealed diminished mitochondrial function at THC concentrations as low
as 0.5 µg/ml. At concentrations of 2.5 or 10 µg/ml of THC, a
decrease in mitochondrial membrane potential was observed as early as
1 h after THC exposure. Mitochondrial function remained diminished
for at least 30 h after THC exposure. Flow cytometry studies on
cells exposed to particulate smoke extracts indicate that JC-1 red
fluorescence was fivefold lower in cells exposed to marijuana smoke
extract relative to cells exposed to tobacco smoke extract. Comparison
with a variety of mitochondrial inhibitors demonstrates that THC
produced effects similar to that of carbonyl cyanide
p-trifluoromethoxyphenylhydrazone, suggesting uncoupling of
electron transport. Loss of red JC-1 fluorescence by THC was suppressed
by cyclosporin A, suggesting mediation by the mitochondrial
permeability transition pore. This disruption of mitochondrial function
was sustained for at least 24 h after removal of THC by extensive
washing. These results suggest that exposure of the bronchopulmonary
epithelium to THC may have important health and physiological consequences.
adenosine 5'-triphosphate; JC-1; marijuana; flow cytometry; mitochondrial membrane potential
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INTRODUCTION |
RECREATIONAL AND MEDICINAL
USE of marijuana remains prevalent despite numerous reports of
adverse and toxic consequences (1). The epithelial lining
of the lung acts as the primary site exposed to the inhaled components
of marijuana smoke, including the high concentrations of cannabinoids
associated with the particulate tar fraction. Histopathological
alterations in the lung resulting from marijuana smoking include
proximal airway inflammation, reserve and goblet cell hyperplasia,
squamous cell metaplasia, loss of epithelial microvilli, and cellular
atypia (13, 35). These changes have been observed by
bronchoscopic visualization and by microscopic inspection of human
airway mucosal biopsy specimens. The mechanisms underlying these
effects, however, are poorly understood and have not been investigated extensively.
We previously reported that exposure to marijuana smoke in vitro
results in significant oxidative stress and suppression of fas-induced
apoptosis (42, 43). We have also observed that agents that compromise cell energetics, including antioxidants, enhance
the cytotoxic effects produced by 9-tetrahydrocannabinol
(THC) (41). There have been several reports suggesting
that THC alters mitochondrial function in brain and muscle tissue
(4, 7, 8, 10). Effects of THC on glucose metabolism have
also been described (38, 39) that may be a consequence of
primary mitochondrial effects. Because direct effects of THC on
pulmonary cell energetics have not been reported, we used the lung
epithelial tumor cell line A549 to study in vitro responses to THC
exposure. We observed numerous effects suggesting mitochondrial injury,
which may help explain previously observed cytopathology.
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MATERIALS AND METHODS |
Materials.
Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP),
cyclosporin A, propidium iodide, digitonin, and oligomycin were purchased from Sigma (St. Louis, MO). Reagents for ATP measurements include firefly lantern extract (FLE-50) and disodium ATP (Sigma). 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was purchased from Molecular Probes (Eugene, OR). FCS,
BSA, PBS, RPMI 1640 medium, and trypsin-EDTA solution were purchased
from Irvine Scientific (Irvine, CA). Marijuana cigarettes containing
either 2.8% THC or 0% THC (placebo) were obtained from the National
Institute on Drug Abuse (NIDA; Bethesda, MD). Placebo marijuana
cigarettes were prepared at NIDA from marijuana leaves that had been
extracted with ethanol to remove cannabinoids. Tobacco cigarettes were
purchased commercially (Marlboro Red hard-pack filtered cigarettes, 10 cm, 1-1.05 g). Purified THC at a stock concentration of 50 mg/ml
in ethanol was obtained from NIDA.
Tar extracts.
Tar-containing extracts from tobacco or marijuana smoke were prepared
by passing whole smoke from a single cigarette through a Cambridge
filter (Performance Systematic, Caledonia, MI), according to a
standardized protocol (29). Filters were thoroughly dried and weighed, and the deposited tar was extracted with DMSO. Tar concentrations in these extracts were normalized based on standard curves for absorbance at 400 nm as described previously
(43).
Cell culture.
A549 cells, a human lung cancer cell line with alveolar epithelial
characteristics (CCL-185; American Type Culture Collection, Bethesda,
MD), were cultured in RPMI 1640 medium containing 10% FCS and 1%
penicillin-streptomycin-fungizone (Irvine Scientific, Irvine, CA).
Cells were passaged every 3-4 days and plated in 12-well plates at
1 × 105 per well or in 24-well plates at 5 × 104 per well, for assays.
ATP and ADP assay.
A549 cells were grown in 24-well plates and exposed to various agents
for 24 h, and an extract was prepared for measurement of the
effects on cellular ATP. Cells were washed twice with 1 ml of PBS
before addition of 100 µl of 0.6 M perchloric acid. After 15 min on
ice with shaking, extracts were transferred to microcentrifuge tubes.
After a second extraction with 100 µl of perchloric acid for 5 min,
extracts were combined. Fifty microliters of 2 M
K2CO3 were added, and the samples were mixed
rapidly and centrifuged at 12,000 g for 1.5 min. We assayed
supernatant (5 µl) for ATP as described previously (23,
40) using 100 µl of firefly luciferase suspension (Sigma) with
a BG-1 Bacterial Systems luminometer. We assayed ADP levels as
described previously (40) by adding 100 units of rabbit
muscle pyruvate kinase (EC2.7.1.40, Sigma Type VII; 482 U/mg) to 100 µl of cell extract supernatant in 5 mM
Tris · HCl, pH 7.4. Samples were incubated for 10 min at 37°C and assayed for ATP as above. ADP was calculated as the difference in ATP level between pyruvate kinase-treated and untreated samples. We determined protein levels in the supernatants using the
Bio-Rad Coomassie protein assay using bovine gamma-globulin as protein
standard. Data were expressed as picomoles of ATP or ADP per microgram
of protein.
Mitochondrial membrane potential.
Mitochondrial membrane potential was measured by two methods using the
fluorescent probe JC-1, which produces green fluorescence in the
cytoplasm and red-orange fluorescence when concentrated in respiring
mitochondria having a negative internal potential.
For flow cytometry, cells treated overnight with various agents in
12-well plates were incubated for 30 min with 2 µg/ml of JC-1 in
culture medium. The adherent cell layer was then washed three times
with PBS and dislodged with 250 µl of trypsin-EDTA. Cells were
collected in PBS-2% BSA, washed twice by centrifugation, and
resuspended in 0.3 ml of PBS-2% BSA for analysis using a FACS Caliber
Analytic Flow Cytometer (BD Biosciences, San Jose, CA). Cytometer
settings were optimized for green (FL-1) and red (FL-2) fluorescence,
and data were analyzed with Cell Quest Software (BD Biosciences).
Alternatively, a Cytofluor 2300 fluorescence plate reader (PerSeptive
Biosystems, Framingham, MA) was used to perform time course and
reversibility studies on the effects of THC on cellular JC-1
fluorescence. For time course studies, THC or control medium containing
the matching concentration of ethanol was added to A549 cells cultured
in 48-well plates at the indicated times. At the end of incubation, a
prewarmed solution of JC-1 was added to wells to give a final
concentration of 2 µM. Fluorescence measurements were taken
at 1, 30, and 60 min using 530 excitation (Ex)/590 emission (Em),
sensitivity = 2 settings for red J-aggregate fluorescence and 485 Ex/530 Em, sensitivity = 3 for green cytoplasmic fluorescence. After subtraction of background values obtained from wells containing JC-1 but devoid of cells, red/green fluorescence ratios were
calculated. For studies on the reversibility of THC-induced
mitochondrial injury, adherent A549 cells were treated with THC or
control medium for 3 h and washed three times with PBS-2% FBS.
Fresh culture medium was added, and JC-1 fluorescence was measured on
the Cytofluor plate reader at the indicated time. Cultures were
returned to the 37°C CO2 incubator in between measurements.
Fluorescence microscopy.
Fluorescence microscopy was performed under a Zeiss Axiophot 2 fluorescent microscope equipped with rhodamine/fluoresceine dual filter
set and a Spot 2 digital camera and imaging system (Diagnostic
Instruments). Cells treated with THC, marijuana tar extract, or FCCP as
above were treated with JC-1 for 30 min in a 37°C CO2
incubator. Cells were then washed twice with PBS and examined
immediately under a ×40 aqueous immersible objective. We developed
images using Spot 2 Software with the camera shutter set at 0.3 s
for red light and 0.5 s for green light.
Cell viability.
Cell viability was measured as described previously (44).
After various exposures, 50 µM propidium iodide was added. After 15 min at room temperature, fluorescence measurements were taken with the
Cytofluor 2300 plate reader at Ex = 546 and Em = 590 (sensitivity = 3). Background measurements (blank) were obtained from cell-free wells containing media and propidium iodide. Digitonin (160 µM) was then added, and, after 20 min at room temperature, fluorescence measurements were repeated to obtain Fmax, a
function of total cell number. Percentage viability was calculated as
100 
(F blank/Fmax blank) × 100, where F is the measured propidium iodide fluorescence.
Statistical methods.
Statistical analysis was performed by ANOVA and Fisher's post hoc test
using the Statview software program version 5.0 (SAS Institute).
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RESULTS |
We previously reported that a synergistic cytotoxicity occurred
when butylated hydroxyanisole (BHA) and THC were added together to A549
cells, an effect that may have been mediated by their combined impact
on cellular ATP levels (41). To characterize the capacity
for THC to disturb energetics in these cells, we set up a dose-response
study in which cellular ATP levels and viability were measured after
24-h exposure to various concentrations of THC (0-15 µg/ml). THC
provided a dose-dependent effect on lowering ATP with an
IC50 value of 7.5 µg/ml (Fig.
1). Consistent with previous studies
(41, 43), 24-h exposure to 10 µg/ml of THC resulted in
<15% cell death, indicating that reductions in ATP levels did not
result in immediate cytotoxicity and were not simply due to cell death.
At 15 µg/ml, ATP levels were <5% those of control, and cell
viability declined by 20%. Cellular ADP levels were also found to
decline by 28 ± 2.3%, 55 ± 1.8%, and 68 ± 3% (SE)
after 24-h exposure to 5, 10, and 15 µg/ml of THC, respectively (data not shown), indicating further decrement in cellular energy charge.
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Fig. 1.
Dose-dependent effect of
9-tetrahydrocannabinol (THC) on cell ATP level and
viability. A549 cells were exposed to THC for 24 h, extracted with
0.3 M perchloric acid, and assayed for ATP by a firefly luciferase
assay as described in MATERIALS AND METHODS. Values were
expressed as pmol ATP/µg protein. Cell viability was determined using
a separate parallel culture plate using propidium iodide to identify
dead cells. Values represent means of 4-8 determinations ± SE. *P < 0.01 compared with ethanol-treated controls
using ANOVA and Fisher's post hoc test.
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To examine mitochondrial function, we incubated cells treated with THC
for 24 h with the fluorescent probe JC-1. Examination of ethanol
vehicle control cells by fluorescent microscopy revealed bright
orange-red punctate staining in cytoplasmic regions in virtually all
cells, consistent with formation of JC-1 "J-aggregates" within
actively functioning mitochondria (Fig.
2). Nuclei were unstained as expected.
Variable and faint green staining was observed in most cells. THC
produced a concentration-dependent decrease in number and intensity of
orange spots, similar to results produced by FCCP, an electron
transport chain uncoupler. Analysis of cells by flow cytometry revealed
a concentration-dependent decrease in red fluorescence detectable at
0.5 µg/ml of THC with an IC50 between 1 and 2.5 µg/ml
of THC (Fig. 3). This decrease in red J-aggregate fluorescence is indicative of loss of mitochondrial membrane potential. Green fluorescence, reflecting cytoplasmic staining, increased only at THC concentrations >5 µg/ml. The
THC-induced effects on JC-1 fluorescence provided a more sensitive
measure of the mitochondrial function than did measurements of changes in ATP levels (Fig. 4). The fluorescent
changes in JC-1 produced by THC exposure closely resembled that of the
uncoupling agent FCCP, with decreased red fluorescence and enhanced
green fluorescence (Fig. 5). In contrast,
the ATP synthase inhibitor oligomycin increased the percentage of cells
with high red fluorescence (upper quadrants) from 66 to 94%.
Cyclosporin A, which stabilizes membrane pore transition, increased red
fluorescence when added to cells alone and attenuated the loss of red
fluorescence produced by THC. In response to 4 µM cyclosporin A, the
percentage of gated cells in the upper quadrants increased from 12%
for cells treated with 5 µg/ml of THC alone to 37% from cells
cotreated with THC and cyclosporin A.
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Fig. 2.
THC alters the pattern of
5,5',6,6',-tetrachloro-1,1',3,3',-tetrabenzimidazolylcarbocyanine
iodide (JC-1) staining in A549 cells. Cells were cultured in 35-mm
dishes and exposed for 24 h to 0.1% ethanol (control,
A), 2 µg/ml of THC (B), 4 µg/ml of THC
(C), 6 µg/ml of THC (D), 50 µg/ml marijuana
tar extract (E), or 50 µM carbonyl cyanide
p-trifluoromethoxyphenylhydrazone (FCCP, F).
Photos reveal punctate nonnuclear red-orange staining characteristic of
JC-1 accumulation in respiring mitochondria. THC, marijuana tar
extract, and FCCP caused a decrease in number and intensity of
red-stained mitochondria while slightly increasing the green intensity
of cytoplasmic JC-1. Magnification = ×1,200.
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Fig. 3.
Flow cytometric analysis of THC effects on mitochondrial
membrane potential as detected by JC-1 fluorescence. A549 cells were
treated with THC for 24 h, washed, and stained with 2 µg/ml of
JC-1 for 30 min at 37°C. Cells were trypsinized to provide a single
cell suspension and analyzed for green (FL-1) and red (FL-2)
fluorescence by flow cytometry. A: flow cytometry data were
analyzed using Cell Quest software and dot-plot generated to track
fluorescence in response to treatment. Con, control. B: bar
graphs indicating the percentage of gated cells falling in upper
quadrants (Upper Quads) or lower quadrants (Lower Quads) as shown in
A. This experiment was repeated twice with similar
results.
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Fig. 4.
Simultaneous analysis of the effects of THC on A549
mitochondrial membrane potential, cellular ATP, and cell viability.
JC-1 fluorescence was measured both with a flow cytometer (FACS JC-1)
measuring the percentage of cells gated in the upper quadrants (see
Fig. 3) and with a Cytofluor 2300 fluorescence plate reader measuring
the ratio of red/green fluorescence (Cyto JC-1). ATP levels expressed
as pmol/µg protein were assayed using a luminescence firefly
luciferase assay, and % viability was assayed with propidium iodide
staining. All values are expressed as a percentage of vehicle-treated
controls and represent means of duplicate assays. The experiment was
repeated twice with similar results.
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Fig. 5.
Mitochondrial inhibitors alter JC-1 fluorescence and
alter the response to THC. A549 cells were pretreated for 24 h
with 0.2% ethanol [control (Con)], 5 µg/ml of THC, 25 µM FCCP, 5 µM cyclosporin A (Cys A), 50 µM oligomycin (Oligo), or 5 µg/ml of
THC and 5 µM Cys A and analyzed for the impact on JC-1 by flow
cytometry. Data are presented as dot plots (A) and as bar
graphs representing the percentage of gated cells in upper or lower
quadrants (B). This experiment was repeated twice with
similar results.
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We evaluated the ability of THC to affect mitochondrial membrane
potential in the presence of cigarette smoke components by comparing
the effects of particulate tar extracts from different cigarettes (Fig.
6). Extracts from Marlboro Red cigarettes
added to culture medium for 24 h at 50 µg/ml of tar caused an
increase in the mean red fluorescence intensity in the upper quadrants from 732 in solvent-treated control to 1,662. Exposure to the same
concentration of marijuana tar extract, however, reduced red
fluorescence to only 293. To further characterize the specific role of
THC in the decrease in red fluorescence, we examined the effect of
placebo marijuana tar extract lacking THC. A 24-h exposure to 50 µg/ml of tar produced a red fluorescence intensity of 1,578, similar
to that of tobacco extract. When 5 µg/ml of THC was added to the
placebo tar extract, Y-mean red fluorescence declined to 299. No
increase in green fluorescence was observed.
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Fig. 6.
Marijuana tar extract produces effects similar to THC.
A549 cells were pretreated 24 h with a 50 µg/ml concentration of
cigarette smoke extracts prepared from the particulate phase of tobacco
(Tob Extr), marijuana (MJ Extr), or placebo [Plac (0% THC)]
marijuana smoke. A: FL-1 (green) fluorescence intensity is
plotted on the x-axis and FL-2 (red) fluorescence on the
y-axis. B: bar graphs depicting the mean red
fluorescence intensity (y-axis) for cells in upper quadrants
or lower quadrants under the conditions shown. Exposure of A549 cells
to cigarette tar extract resulted in a nonspecific general increase in
red fluorescence compared with control cells (see Fig. 3). This
experiment was repeated 3 times with similar results.
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JC-1 fluorescence was also examined in situ using a Cytofluor 2300 plate reader. Time course studies on the effect of THC revealed a rapid
decrease in mitochondrial membrane potential measured as the ratio of
red/green fluorescence (Fig. 7). Within 1 h of exposure to 2.5 µg/ml or 10 µg/ml of THC, the red/green ratio declined by 24 and 69%, respectively. At 2.5 µg/ml of THC, this value declined to 49% of control by 3 h and remained low for
up to 30 h without detectable loss of cell viability. At 10 µg/ml of THC, the red/green fluorescence ratio remained at
~20-25% of control for up to 30 h with ~25% loss of
cell viability observed at 30 h.
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Fig. 7.
Time course measurements of the impact of THC on
mitochondrial membrane potential using JC-1 fluorescence (A)
and cell viability using propidium iodide (B). All assays
were performed using a Cytofluor 2300 fluorescent plate reader to
measure changes in red and green fluorescence. Either 2.5 µg/ml of
THC, 10 µg/ml of THC, or vehicle 0.5% ethanol (EtOH) was added to
wells at the indicated times before incubation with JC-1 (30 min) or
propidium iodide (15 min). JC-1 fluorescence values represent means of
quadruplicate measurements ± SE. Values for THC-treated wells
were significantly different (P < 0.01) from
EtOH-treated control wells and from 0-h exposed controls at all time
points. Viability assays were performed in duplicate. This experiment
was repeated twice with similar results.
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Studies on the reversibility of THC-induced mitochondrial injury
demonstrated that the rate and degree of recovery varied as a function
of THC concentration (Fig. 8). After
3 h of exposure to 5 µg/ml of THC, the red/green fluorescence
ratio decreased by 74% compared with ethanol-treated control cells.
Upon removal of THC by extensive washing with PBS containing 2% fetal
calf serum, the fluorescence ratio increased significantly
(P < 0.01) yet remained 30-60% lower than
similarly washed control cells for up to 24 h. THC at 10 µg/ml
initially decreased fluorescence ratio by 95%, and values following
THC removal were never higher than 50% of control. Similar results
were obtained following 24-h exposure to THC (data not shown). However,
exposure to THC for only 1 h produced lower initial decrements in
JC-1 fluorescence ratio, and complete recovery to control levels was
observed 4 h after washing (data not shown). However, exposure to
5 µg/ml of THC for only 1 h produced lower initial decrements in
JC-1 fluorescence ratio and complete recovery to control levels by 2 h after washing (Fig. 8B).
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Fig. 8.
Effect of incubation time on recovery of mitochondrial
membrane potential following removal of THC. A549 cells were treated
with the indicated concentration of THC or 0.2% EtOH (control) for
3 h (A) or for 1 h (B). THC was then
removed by extensive washing of cells, which were subsequently
incubated in culture medium in a 37°C CO2 incubator for
0, 2, or 24 h. Cells were then labeled with 2 µg/ml of JC-1 for
1 h, as were unwashed control cells. Red and green JC-1
fluorescence measurements were then taken, background-subtracted ratios
were calculated, and values were expressed as percentage of control
EtOH-treated cells. Bar graph values represent means of 4 determinations ± SE, and the experiment was repeated 3 times with
similar results. Red/green ratio values for unwashed EtOH-treated cells
was 32.9 ± 2.3 (SE). *P < 0.01 compared with
time-matched control by ANOVA with Fisher's post hoc test.
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DISCUSSION |
Prior studies in this laboratory characterized oxidative stress
associated with exposure of cultured cells to marijuana smoke (42). This oxidative stress was found to increase as a
function of THC content of the cigarette, suggesting a direct role for THC. Removal of the THC-containing particulate smoke fraction exacerbated the oxidative stress, however, indicating that THC was not
acting as a primary oxidant. This conclusion is consistent with
voltametry studies revealing a weak reducing potential for THC.
Nonetheless, studies with A549 cells revealed that THC exerts a
cytotoxic effect and that this effect was potentiated by the antioxidant food additive BHA (41). Because BHA is known
to be a mitochondrial toxicant, a possible mechanism for the observed synergistic toxicity of THC and BHA was suggested on the basis of
aggravated mitochondrial injury.
To investigate this hypothesis, we have examined the effects of THC on
cell energetics. THC was found to decrease A549 ATP levels in a time-
and dose-dependent manner with effects observed at concentrations <1
µg/ml. A decline in ATP >80% was observed without significant loss
of cell viability, indicating that ATP loss was not a secondary
consequence of death-associated cell permeability. This decline in ATP
would be expected to affect numerous cellular functions, including
membrane ion transport, macromolecule synthesis, and cell signaling.
When marijuana cigarettes are smoked, milligram quantities of THC
are deposited directly onto the respiratory epithelium in the form of
THC-laden smoke particulates (36, 54). Although local
pulmonary concentrations of THC have not been measured, they likely
exceed the range of 5-250 ng/ml that has been observed in the
peripheral blood of subjects smoking one or more marijuana cigarettes
(5, 30, 33, 47). Exposing mice to marijuana smoke
similarly produced peak blood levels of 400 ng/ml (25), whereas intravenous administration of 10 mg/kg THC resulted in a blood
level of 720 ng/ml after 20 min (25). These systemic THC
concentrations in human studies and animal models are near the range
that begin to disrupt mitochondrial membrane potential and ATP levels.
The effects on respiratory epithelial cells, exposed to even higher
local concentrations during smoking, are likely to be significant.
Such severe loss in cellular ATP suggested impairment of mitochondrial
function. To investigate this possibility, we used the fluorescent
probe JC-1 (2, 32, 37, 52). JC-1 is a lipophilic cationic
molecule that is readily taken up by cells and produces a green
fluorescent signal when present in the cytosolic fraction. In actively
respiring mitochondria, the electronegative inner membrane potential of
these mitochondria promote accumulation of JC-1 and formation of the
J-aggregate form that fluoresces, producing a red-orange color. Control
A549 cultures displayed >90% cells with bright orange punctate
staining within a faint green cytosolic background. Exposure of cells
to THC caused a dose-dependent decrease in red fluorescence, indicating
loss of mitochondrial membrane potential. Both uncoupling agents, such as FCCP, and inhibitors of electron transport, such as antimycin A, are
known to produce similar effects (46). At higher
concentrations of THC, green cytosolic fluorescence increased,
suggesting release of JC-1 monomer from mitochondria to cytoplasm.
Decreased red fluorescence induced by THC was not due to direct
interaction of THC with JC-1, since cell-free incubation with these
compounds resulted in no decrease in red fluorescence (data not shown). In fact, red fluorescence was enhanced at [THC] >10 µg/ml under these conditions. THC-induced loss of mitochondrial membrane potential and cellular ATP was partially prevented by cyclosporin A, suggesting that mitochondrial injury was mediated by the permeability transition pore complex. Cyclosporin A acts by binding to the mitochondrial matrix
protein cyclophilin D and subsequently preventing interaction between
adenine nucleotide translocator and the voltage-dependent anion channel
(11, 20). This channel is regulated by vicinal thiol
groups and damaged by oxidative stress, ganglioside GD3, and FCCP
(9, 26, 31). It should be noted that the binding of
cyclosporin A to cyclophilin D also results in alterations in
Ca2+-mediated cell signaling pathways (21)
that could, in turn, be responsible for the partial attenuation of the
observed THC-induced mitochondrial effects. Nevertheless, the
mitochondrial permeability transition is an important regulator for
both apoptotic and necrotic cell death (19).
We reported previously that anti-fas-induced apoptosis in A549
cells was suppressed by THC, as shown by a THC-induced decrease in
caspase-3 activity, phosphatidyl serine externalization, and nuclear
chromatin condensation assays (43). The inhibitory action of THC on A549 apoptosis could occur by one of several
mechanisms, including alteration of fas receptor activity or
death-inducing signaling complex protein assembly. The present findings
suggest an alternative mechanism for apoptosis inhibition,
since apoptotic cell death is known to require a minimal level of
ATP or dATP estimated to be ~25% of control levels (12, 29,
34, 48). ATP is required as a critical component of the
apoptosome in which caspase-9 complexes with cytochrome c
and Apaf-1 to activate caspase-3 (19, 50). This pathway
can be influenced by the fas receptor via activation of truncated
BH3-interacting domain, which mediates release of cytochrome
c from mitochondria (55). Although some mitochondrial inhibitors are known to induce apoptosis
(28, 53), inhibitors that deplete ATP levels severely do
not allow expression of the apoptotic pathway. Such may be the case
with THC. The observed increase in necrotic cell death caused by THC with or without fas-receptor activation may result from an inability to
maintain energy-dependent membrane transport functions.
The effects of THC on cell energetics appear to occur both when THC is
applied as a purified synthetic compound and in preparations of
marijuana smoke extract, both of which strongly diminished JC-1 red
fluorescence. Neither tobacco smoke extract nor 0% THC placebo
marijuana extract produced this effect. These results indicate that THC
exerts its mitochondrial action in the presence of the numerous
components of particulate cigarette smoke. This complex mixture by
itself in the absence of THC does not cause loss of mitochondrial
membrane potential, nor does it interfere with the action of THC. Both
flow cytometry and fluorescent microscopy studies reveal that marijuana
smoke extract stored at 15°C for up to 6 mo retains the ability to
damage A549 mitochondria (data not shown).
Further studies will be necessary to identify the mechanism underlying
THC-induced mitochondrial dysfunction. Because aromatic and phenolic
compounds are known to interfere with mitochondrial electron transport
(15, 51), this mechanism represents one possible site of
interaction for THC consistent with previous studies. Another
possibility that could account for impaired mitochondrial membrane
potential and ATP depletion would be a decline in NADH supply, either
through inhibition of Krebs cycle activity or through cytoplasmic
depletion. Such depletion is known to occur via activation of
poly(ADP-ribose) polymerase (PARP) resulting from DNA damage (27). PARP activation would be promoted in cells deficient
in caspase-3 activity, a condition previously demonstrated in
THC-treated A549 cells (43). The inactivation of PARP is
one of the primary actions of caspase-3 associated with
apoptosis (24).
Marijuana smoking is associated with increased risk of pulmonary
infection (3, 6, 45, 49) and bronchial mucosa inflammation and injury (13, 35). Disruption of cell energetic
pathways, particularly when sustained long after initial exposure to
THC, might help explain these health effects in several ways. First, immune cell-mediated defense mechanisms could be compromised due to
impairment in macromolecule synthesis by immune cells (16, 48). Second, disruption of apoptotic mechanisms in
epithelial cells has been shown to increase risk of infection in gut
lining due to the inability to eliminate and process infected cells
(18, 22). Third, pathogenic infectious agents are
routinely removed from the pulmonary epithelium by mucociliary
transport, which is driven by the coordinated action of epithelial
microvilli powered by cellular ATP. One of us (D. P. Tashkin) has
observed a dramatic loss of microvilli in the tracheobronchial lining
of marijuana smokers (14, 17). Further studies will be
required to characterize the role of THC-mediated mitochondrial injury
in pulmonary pathophysiology.
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ACKNOWLEDGEMENTS |
The authors thank Steve Young and Dr. Enrique Rozengurt for
providing access to the fluorescence microscope and Drs. Genhong Cheng
and Lee Slice for allowing use of their luminometer. We are also
grateful to Drs. Sylvia Kiertsher and Saroj Basak for assistance with
analysis of flow cytometry data and to the UCLA Jonsson Comprehensive
Cancer Center and Center for AIDS Research Flow Cytometry Core
Facility, supported by National Institutes of Health awards CA-16042
and AI-28697.
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FOOTNOTES |
This work was supported by National Institutes of Health Grant
R37DA030-18.
Address for reprint requests and other correspondence:
T. A. Sarafian, Dept. of Medicine, Div. of Pulmonary and
Critical Care, Center for Health Sciences, UCLA, Los Angeles,
California 90095 (E-mail:
tsarafian{at}mednet.ucla.edu).
The costs of publication of this
article were defrayed in part by the
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must therefore be hereby marked
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in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 25, 2002;10.1152/ajplung.00157.2002
Received 20 May 2002; accepted in final form 11 October 2002.
| |
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