Cigarette smoke-induced cell death of a spermatocyte cell line can be prevented by inactivating the Aryl hydrocarbon receptor

Abstract

Cigarette smoke exposure causes germ cell death during spermatogenesis. Our earlier studies demonstrated that cigarette smoke condensate (CSC) causes spermatocyte cell death in vivo and growth arrest of the mouse spermatocyte cell line (GC-2spd(ts)) in vitro via the aryl hydrocarbon receptor (AHR). We hypothesize here that inactivation of AHR could prevent the CSC-induced cell death in spermatocytes. We demonstrate that CSC exposure generates oxidative stress, which differentially regulates mitochondrial apoptosis in GC-2spd(ts) and wild type (WT) and AHR knockout (AHR-KO) mouse embryonic fibroblasts (MEFs). SiRNA-mediated silencing of Ahr augments the extent of CSC-mediated cellular damage while complementing the AHR-knockout condition. Pharmacological inhibition using the AHR-antagonist (CH223191) modulates the CSC-altered expression of apoptotic proteins and significantly abrogates DNA fragmentation though the cleavage of PARP appears AHR independent. Pretreatment with CH223191 at concentrations above 50 μM significantly prevents the CSC-induced activation of caspase-3/7 and externalization of phosphatidylserine in the plasma membrane. However, MAPK inhibitors alone or together with CH223191 could not prevent the membrane damage upon CSC addition and the caspase-3/7 activation and membrane damage in AHR-deficient MEF indicates the interplay of multiple cell signaling and cytoprotective ability of AHR. Thus the data obtained on one hand signifies the protective role of AHR in maintaining normal cellular homeostasis and the other, could be a potential prophylactic therapeutic target to promote cell survival and growth under cigarette smoke exposed environment by receptor antagonism via CH223191-like mechanism. Antagonist-mediated inactivation of the aryl hydrocarbon receptor blocks downstream events leading to cigarette smoke-induced cell death of a spermatocyte cell line.

Figure 1

CSC induces oxidative stress but does not alter mitochondrial membrane potential in spermatocytes. (a, c, and e) Representative flow cytometric analyses of spermatocytes exposed to DMSO (0.1% for 5 h) or CSC (40 μg/ml) for 1 and 5 h, then stained with (a) cellROX deep red reagent (BluFL1) and counter-stained with viable dye SYBR 14 (BluFL4), (c) mitoSOX superoxide indicator (BluFL1) and counter-stained with viable nuclear dye TO-PRO-3 (RedFL1), or (e) mitoProbe (Green, BluFl1 on y axis; Orange, BluFL4 on x axis). Percentages of double-positive cells are indicated in the upper right quadrants. (b, d and f). Histograms present the mean percentages of double-positive spermatocytes from three independent experiments, each assayed in triplicate,±S.E.M.

Figure 2

CSC modulates the expression of antiapoptotic proteins. (a, c, e, and g) Representative flow cytometric analyses of (a and e) spermatocytes transfected with scr-siRNA or Ahr-siRNAs and (c and g) WT and AHR-KO MEF cells treated with DMSO or CSC for 18 h and then stained with (a and c) anti-BCL2L1 (BluFL1) or (e and g) anti-BCL2 (BluFL1) antibody and counter-stained with TO-PRO-3 (RedFL1). Percentages of double-positive cells are indicated in the upper right quadrants. (b, d, f and h). Histograms present the mean percentages of double-positive spermatocytes and MEFs from three independent experiments, each assayed in triplicate,±S.E.M.

Figure 3

Regulation of proapoptotic proteins by CSC. (a, c, e, and g) Representative flow cytometric analyses of (a and e) spermatocytes transfected with scr-siRNA or Ahr-siRNAs and (c and g) WT and AHR-KO MEF cells treated with DMSO or CSC for 18 h and then stained with (a and c) anti-BAX (BluFL1) or (e and g) anti-BAD (BluFL1) antibody and counter-stained with TO-PRO-3 (RedFL1). Percentages of double-positive cells are indicated in the upper right quadrants. (b, d, f and h). Histograms present the mean percentages of double-positive spermatocytes and MEFs from three independent experiments, each assayed in triplicate,±S.E.M.

Figure 4

CSC exposure causes DNA fragmentation and cleavage of PARP in spermatocytes. (a and e) Representative flow cytometric analyses of spermatocytes transfected with scr-siRNA or Ahr-siRNAs and then treated with DMSO or CSC (40 μg/ml) for (a) 8 h before TUNEL (BluFL1) and propidium iodide staining (BluFL2) or (e) 17 h before staining with anti-cleaved PARP antibody (BluFL1) and TO-PRO-3 (RedFL1). (c and g) Representative flow cytometric analyses of spermatocytes treated with CSC, AHR-inh, or both for 8 h before (c) TUNEL (BlurFL1) and propidium iodide staining (BluFL2) or (g) staining with anti-cleaved PARP antibody (BluFL1) and TO-PRO-3 (RedFL1). Percentages of double-positive cells are indicated in the upper right quadrants. (b, d, f, and h) Histograms present the mean percentages of double-positive spermatocyte from three or more experiments, each assayed in triplicate,±S.E.M.

Figure 5

CSC modulated AHR mediates caspase-3/7 activation. (a, c and e). The flow cytometric data represent the distribution of GC-2spd(ts) cells (a) treated with DMSO or CSC (40 μg/ml) for 18 h, AHR-inh alone at different concentrations (25, 50, 75, 100 and 150 μM) or AHR-inh for 1 h followed by CSC, spermatocytes transfected with scr-siRNA or Ahr-siRNAs and (c) WT and AHR-KO MEF cells (e) treated with DMSO or CSC for 18 h. The treated spermatocytes were labeled by using CellEvent caspase-3/7 green detection reagent for assessing caspase activation and counter-stained with TO-PRO-3. The cell specimens of various groups were evaluated for positive cell populations based on standard compensation calculations on a BD FACSCalibur using FlowJo software (v9.7.5, FLOWJO, LLC., Ashland, OR, USA). The percent difference in caspase-3/7⁺ cells is shown within the quadrant. b, d and f). Histogram represents the mean flow cytometric data of percent of caspase-3/7⁺ (BluFL1 and RedFL1 double-positive) spermatocyte populations among various treatment groups each assayed in triplicates±S.E.M.; n=3.

Figure 6

AHR-antagonist prevents CSC-induced cell membrane damage during apoptosis. a, c and e). The flow cytometric data represent the distribution of GC-2spd(ts) cells (a) treated with DMSO or CSC (40 μg/ml) for 17 h, AHR-inh alone at different concentrations (10, 25, 50, and 150 μM) or AHR-inh for 1 h followed by CSC, spermatocytes transfected with scr-siRNA or Ahr-siRNAs and (c) WT and AHR-KO MEF cells (e) treated with DMSO or CSC for 18 h. The treated spermatocytes were labeled with green annexin V Alexa Fluor 488 conjugate and counter-stained with PI for detecting externalization of phosphatidylserine. Following staining, the cell specimens of various groups were evaluated for positive cell populations based on standard compensation calculations on a BD FACSCalibur using FlowJo software (v9.7.5, FLOWJO, LLC.). The percent difference in annexin V⁺ cells is shown within the quadrant. b, d and f). Histogram represents the mean flow cytometric data of percent of annexin V and PI⁺ (BluFL1 and BLuFL2 double-positive) spermatocyte populations among various treatment groups each assayed in triplicate±S.E.M.; n=6.

Figure 7

Inactivation of MAPKs does not prevent CSC-induced membrane damage. (a and b). Spermatocytes were exposed to DMSO (0.1%) or CSC (40 μg/ml) for 17 h. In the antagonists treatment groups, the cells were first pretreated with AHR or MAPK inhibitors or together for 1 h followed by exposure to CSC for 17 h (a). The percentage of cells segregated following co-staining with annexin V alexa fluor 488 and PI was determined by FACS analysis using FlowJo software (v9.7.5, FLOWJO, LLC.) as described under materials and methods. The representative histogram demonstrates the distribution of spermatocytes at 17 h. The percent difference in annexin V⁺ cells is shown within the quadrant. (b) Histograms represent the mean flow cytometric data from DMSO-, CSC- or antagonists pretreated samples at 17 h of more than three independent experiments, each assayed in triplicate±S.E.M. n=4.