Role of poly(ADP-ribosyl)ation in a 'two-hit' model of hypoxia and oxidative stress in human A549 epithelial cells in vitro

Abstract

A preceding hypoxic insult can sensitize the cells or the organism to a subsequent, second insult. The aim of the present study was to investigate the molecular mechanism of this phenomenon (often termed 'two-hit' injury paradigm), in an in vitro model of hypoxia/oxidative stress injury in A549 epithelial cells, with special emphasis on the role of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) in the process. Pre-exposure of the cells to 24 h hypoxia significantly reduced intracellular glutathione (GSH) levels, reduced mitochondrial activity and adenosine triphosphate (ATP) levels. However pre-exposure to hypoxia failed to induce any change in PARP-1 expression and activation, DNA single‑strand breaks or plasma membrane integrity. Pre-exposure to hypoxia markedly increased the sensitivity of the cells to subsequent oxidative stress-induced DNA damage. Hydrogen peroxide (H2O2) induced a concentration-dependent increase in DNA breakage, PARP activation, depletion of intracellular ATP, inhibition of mitochondrial activity and two distinct parameters that quantify the breakdown of plasma membrane integrity (propidium iodide uptake or lactate dehydrogenase release). PARP-1 activation played a significant role in the H2O2-induced cell death response because PARP activation, depletion of intracellular ATP, inhibition of mitochondrial activity, and the breakdown of plasma membrane integrity were attenuated in cells with permanently silenced PARP-1. Based on measurement of the endogenous antioxidant GSH, we hypothesized that the mechanism of hypoxia-mediated enhancement of H2O2 involves depletion of the GSH during the hypoxic period, which renders the cells more sensitive to a subsequent DNA single‑strand break elicited by H2O2. DNA strand breakage then activates PARP-1, leading to the inhibition of mitochondrial function, depletion of ATP and cell necrosis. PARP-1 deficiency protects against the cytotoxicity, to a lesser degree, by protecting against GSH depletion during the hypoxic period, and, to a larger degree, by maintaining mitochondrial function and preserving intracellular ATP levels during the subsequent oxidative stress period.

Figure 1

(A) Experimental design: an in vitro model of ‘two-hit’ injury in cultured A549 human lung epithelial cells. Control and shPARP-1 cells were grown to form an 80% confluent monolayer and then subjected to either hypoxia or normoxia for 24 h. Immediately after hypoxia, the cellular glutathione (GSH) content and poly(ADP-ribose) polymerase-1 (PARP-1) protein level was measured or cells were treated with H₂O₂ for 10 min, followed by measuring of the poly(ADP-ribose) polymer (PAR) formation by western blotting or the sensitivity to single-strand DNA breakage by comet assay. The prolonged effect of oxidative stress on cells undergoing hypoxia was examined by cell viability and toxicity assays at 24 h after H₂O₂ exposure. (B) Effect of hypoxia on intracellular GSH levels in cultured A549 human lung epithelial cells. Cells were incubated in normoxia or in a hypoxic chamber for 24 h, followed by measurement of cellular GSH content. A significant reduction of cellular GSH content was measured in both the control and shPARP-1 cells. Data are shown as mean ± SEM (n=6). ^*P<0.05 shows a significant difference in the response of cells in hypoxia compared to normoxia; ^#P<0.05 shows a significant difference between wild-type vs. shPARP-1 cells.

Figure 2

Effect of 24 h of hypoxia on poly(ADP-ribose) polymerase-1 (PARP-1) protein expression in cultured A549 human lung epithelial cells. Following 24 h of hypoxia, PARP-1 content was determined by western blotting. Actin was used as an invariant control. The protein level of PARP-1 was not affected in the control cells after 24 h of hypoxia. shPARP-1 cells exhibited markedly reduced levels of PARP-1. Representative western blots of determination are shown (n=3).

Figure 3

Effect of pre-exposure to hypoxia on the sensitivity of cultured A549 human lung epithelial cells to exhibit DNA single-strand break in response to H₂O₂. DNA single-strand break was measured with the comet assay. After 24 h of hypoxia or normoxia, wild-type cells were subjected to 50 or 100 μM H₂O₂ for 10 min. Single cells were then exposed to an electric field in agarose gel and stained with SYBR-Green. Labeled DNA was visualized under fluorescence microscopy. (A) Representative images. (B) Main tail moments as an index of DNA damage. Hypoxia rendered the cells significantly more sensitive to 50 μM H₂O₂-induced single-strand DNA breakage. Data are shown as mean ± SEM (n=6). ^*P<0.05 shows a significant difference in the response of cells in hypoxia compared to normoxia.

Figure 4

Effect of pre-exposure to hypoxia on the sensitivity of cultured A549 human lung epithelial cells to exhibit poly(ADP-ribose) polymer (PAR) formation in response to H₂O₂. H₂O₂-induced PAR formation in control and shPARP-1 cells either in normoxia or in 24 h hypoxia was measured by immunoblotting. After 24 h of hypoxia or normoxia, the cells were treated with increasing concentrations of H₂O₂ for 10 min. The cells that were pre-exposed to hypoxia exhibited significantly higher amounts of PAR. Reduced PAR synthesis was measured in shPARP-1 cells, compared to wild-type cells. (A) Representative western blot analysis. (B) Results evaluated by densitometry and analyzed statistically. In part A, mean ± SEM values of n=3 are shown. ^*P<0.05 shows significant difference in the response of cells in hypoxia compared to normoxia; ^#P<0.05 shows a significant difference between wild-type vs. shPARP-1 cells.

Figure 5

Effect of pre-exposure to hypoxia on the H₂O₂-induced changes in cellular adenosine triphosphate (ATP) content in cultured A549 human lung epithelial cells. After 24 h of hypoxia or normoxia, the cells were treated with increasing concentrations of H₂O₂ for 24 h. Oxidative stress decreased the cellular ATP content in wild-type and shPARP-1 cells. Cells that were pre-exposed to hypoxia exhibited significantly higher decreases in ATP level after H₂O₂, compared to cells that were not pre-exposed to hypoxia. shPARP-1 cells were significantly protected against an H₂O₂-induced decrease in ATP, compared to wild-type cells. Data are shown as mean ± SEM (n=3). ^*P<0.05 shows a significant difference in the response of cells in hypoxia compared to normoxia; ^#P<0.05 shows significant difference between wild-type vs. shPARP-1 cells.

Figure 6

Effect of pre-exposure to hypoxia on the H₂O₂-induced changes in (A) mitochondrial MTT conversion and (B) cell viability in cultured A549 human lung epithelial cells. (A) Mitochondrial MTT conversion and (B) decreases in cell viability were evaluated by the measurement of lactate dehydrogenase (LDH) release into the cell culture supernatant. After 24 h of hypoxia or normoxia, the cells were treated with increasing concentrations of H₂O₂ for 24 h. Oxidative stress decreased the MTT activity and increased LDH release in wild-type and shPARP-1 cells. Cells that were pre-exposed to hypoxia exhibited significantly more pronounced responses to H₂O₂, compared to cells that were not pre-exposed to hypoxia. shPARP-1 cells were significantly protected against H₂O₂-induced alterations, compared to wild-type cells. Data are shown as mean ± SEM (n=3). ^*P<0.05 shows a significant difference in the cell response in hypoxia compared to normoxia. ^#P<0.05 shows a significant difference between wild-type and shPARP-1 cells.

Figure 7

Effect of pre-exposure to hypoxia on the H₂O₂-induced plasma membrane injury in cultured A549 human lung epithelial cells. Plasma membrane injury was assessed by propidium iodide (PI) staining. After 24 h of hypoxia or normoxia, the cells were treated with increasing concentrations of H₂O₂ for 24 h. Oxidative stress induced plasma membrane injury in wild-type and shPARP-1 cells. Cells that were pre-exposed to hypoxia exhibited significantly more pronounced responses to H₂O₂, compared to cells that were not pre-exposed to hypoxia. shPARP-1 cells were significantly protected against H₂O₂-induced alterations, compared to wild-type cells. Data are shown as mean ± SEM (n=3). ^*P<0.05 shows a significant difference in the cell response in hypoxia compared to normoxia; ^#P<0.05 shows a significant difference between wild-type and shPARP-1 cells.