Figure 1.
Ethanol induces cell damage in dog PDEC.
(A) Cells were treated with the indicated ethanol concentrations for 1, 4, or 24 h, respectively. After the treatment, cell viability was determined with MTS assay. The values were calculated relative to the control group. The results are mean ± SEM and representative of three independent experiments (n = 4 - 6 for each condition). (B) After 1, 4, or 24 h exposure to the indicated ethanol, LDH activity in extracellular culture medium was measured. The values are presented as relative to LDH release by 0.5% Triton-X 100.
Figure 2.
(A) Cells were treated with the indicated ethanol concentrations for 4 h, and then stained with annexin V-FITC (green, apoptotic cells), ethidium homodimer III (red), and Hoechst 33342 (blue, nucleus staining) for 15 min. Staurosporine was a positive control to trigger apoptosis. Bar is 20 μm. (B) Percentage of apoptotic cells from two independent experiments. (C) Cells treated with 500 or 750 mM ethanol for 4 h were analyzed by Western blot to detect activated caspase-3 and actin proteins. (D) Activated caspase-3 was calibrated by actin level of the samples and presented as relative to the control. Two experiments. ** P < 0.01 and *** P < 0.001 compared to control group.
Figure 3.
Ethanol evokes ROS generation and mitochondrial depolarization, which are blocked by an antioxidant, NAC.
(A and B) ROS measured with CM-H2DCFDA. The areas under the curves and above the 1.0 level were calculated during drug treatments (3 to 15 min) and normalized to that of the control. (C and D) Mitochondrial membrane potential (MMP) measured with JC-1 dye. Decrease of ratio (F561/F488) indicates MMP depolarization. For the bar graph (D), MMP was evaluated as averages during drug treatments. Note that 3 mM NAC reversed ROS generation and MMP depolarization induced by ethanol. N = 7 - 23 for each condition, ## P < 0.01 and ### P < 0.001 compared to the control. ** P < 0.01 compared to the ethanol-treated group.
Figure 4.
NAC reduces ethanol-induced cell damage.
PDEC were treated with 750 mM ethanol for 4 h with or without 30 min pretreatment of indicated NAC concentrations, and then cell viability was measured. n = 4 - 6 for each condition, ### P < 0.001 compared to the control. ** P < 0.01 compared to the cells treated with ethanol alone.
Figure 5.
Acetaldehyde induces cell damage that is inhibited by NAC.
(A) PDEC were treated with indicated concentrations of acetaldehyde for 4 h, and then cell viability was measured. (B) PDEC were treated with 1 mM acetaldehyde for 4 h with or without 30 min pretreatment of 3 or 10 mM NAC. n = 4 - 13 for each condition, ### P < 0.001 compared to control. ** P < 0.01 and *** P < 0.001 compared to the cells treated acetaldehyde alone. (C and D) MMP measured with JC-1 dye. For the bar graph, MMP was evaluated as averages during drug treatments. n = 5 - 6 for each condition, ## P < 0.01 compared to control and *** P < 0.001 compared to the cells treated with acetaldehyde alone. (E and F) Caspase-3 activation estimated with Western blot analysis. Cells treated with 1 mM acetaldehyde for 4 h were analyzed to detect activated caspase-3. For the summary bar graph, activated caspase-3 was calibrated by actin level of the samples and presented as relative to untreated control. n = 3 for each condition, *P < 0.05 compared to control.
Figure 6.
Ethanol is oxidized in PDEC and its metabolite, acetaldehyde, is involved in ethanol-induced cell damage.
(A) RT-PCR analysis on expression of oxidative metabolic genes of ethanol in control dog PDEC. Representative gels from at least three independent experiments. (B) Cells were treated with 500 mM ethanol for 4 h with or without 30 min pretreatment with disulfiram (ALDH1 inhibitor), daidzin (ALDH2 inhibitor), or both. n = 6 - 18 for each condition, # P < 0.05 compared to the control. ** P < 0.01 compared to the cells treated ethanol alone. Submaximal 500 mM ethanol was used in this experiment to test the further damage by acetaldehyde accumulation. (C) NMR analysis for the detection of cellular acetaldehyde production. Arrows indicate two peaks for acetaldehyde. Estimated acetaldehyde concentrations in the samples are 0, 1.7, 2.1, and 48.4 μM for control cells, cells treated with ethanol, cells treated with ethanol plus ALDH inhibitors, and standard solution including ethanol and acetaldehyde, respectively.
Figure 7.
Ethanol increases the leakage of PDEC monolayers.
(A) After ethanol treatment of PDEC monolayers for 4 h, their permeability was determined by perfusing fluorescein into the luminal chamber. Coated disk membranes (N = 6) without cells represent the maximum permeability, while untreated control monolayers (N = 4, overlapped with 500 mM ethanol and therefore invisible) and the monolayers treated with 500 mM ethanol (N = 4) had low permeabilities, i.e. tight monolayers. Monolayers treated with 750 mM ethanol (N = 6) had a higher permeability to fluorescein. Treatment of PDEC monolayers with 10 mM NAC 30 min before and during 750 mM ethanol (N = 3) prevented monolayer leakage. (B) Average rate of fluorescence intensity crossing the monolayers. The rate was estimated from the initial slop between 2 and 8 min. ## p < 0.01 compared with the control group. ** p < 0.01 compared with 750 mM ethanol-treated monolayers. AU, arbitrary unit. The average and error for control are very small at this scale and therefore invisible.
Figure 8.
A schematic model for ethanol-induced apoptosis in PDEC.
Ethanol can be metabolized through oxidative and nonoxidative pathways. Ethanol and its metabolites (i.e. acetaldehyde and FAEE, fatty acid ethyl ester) induce ROS generation, mitochondrial depolarization, and cytochrome C release that finally activates caspase-3 enzyme to trigger apoptosis.