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

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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.

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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.
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fig1: 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.

Mentions: We previously used microscopy to demonstrate that GC-2spd(ts) cells (hereafter referred to as spermatocytes) accumulate reactive oxygen species after six hours of CSC exposure.13 To better quantitate this effect, we used flow cytometry to assess the percentage of cells that stained with cellROX, an indicator of cytoplasmic oxidative stress. We found that the percentage of cellROX-positive cells increased significantly upon exposure to 40 μg/ml CSC (Figures 1a and b). In addition, flow cytometric analysis revealed that the percentage of cells positive for the mitochondrial superoxide indicator mitoSOX increased significantly upon CSC exposure (Figures 1c and d). We next assessed mitochondrial membrane potential by staining control and CSC-exposed cells with MitoProbe DiOC2(3). As a monomer, DiOC2(3) emits green fluorescence and, in a reaction driven by the mitochondrial membrane potential, converts to a red-fluorescence-emitting dimer. Here we found that CSC did not alter the membrane potential of mitochondria either at one or five hours of exposure (Figures 1e and f). Thus, CSC at 40 μg/ml induces oxidative stress in both the cytoplasm and mitochondria of spermatocytes, but does not alter mitochondrial membrane potential.


Cigarette smoke-induced cell death of a spermatocyte cell line can be prevented by inactivating the Aryl hydrocarbon receptor
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.
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fig1: 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.
Mentions: We previously used microscopy to demonstrate that GC-2spd(ts) cells (hereafter referred to as spermatocytes) accumulate reactive oxygen species after six hours of CSC exposure.13 To better quantitate this effect, we used flow cytometry to assess the percentage of cells that stained with cellROX, an indicator of cytoplasmic oxidative stress. We found that the percentage of cellROX-positive cells increased significantly upon exposure to 40 μg/ml CSC (Figures 1a and b). In addition, flow cytometric analysis revealed that the percentage of cells positive for the mitochondrial superoxide indicator mitoSOX increased significantly upon CSC exposure (Figures 1c and d). We next assessed mitochondrial membrane potential by staining control and CSC-exposed cells with MitoProbe DiOC2(3). As a monomer, DiOC2(3) emits green fluorescence and, in a reaction driven by the mitochondrial membrane potential, converts to a red-fluorescence-emitting dimer. Here we found that CSC did not alter the membrane potential of mitochondria either at one or five hours of exposure (Figures 1e and f). Thus, CSC at 40 μg/ml induces oxidative stress in both the cytoplasm and mitochondria of spermatocytes, but does not alter mitochondrial membrane potential.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus