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.
Highlights
Habitual smokers have 13% lower sperm count than nonsmokers.[1]
In this study, we tested the hypothesis that inactivation of aryl hydrocarbon receptor (AHR) could prevent Cigarette smoke condensate (CSC)-mediated cell death in the spermatocyte cell line GC-2spd(ts)
Mitochondrial hyperpolarization (Δψm) is an early apoptotic event and it has been shown that the activated AHR can regulate mitochondrial function through ATP5α1.21 In this study, the lack of change in Δψm by 4% CSC up to 6 h indicated that the magnitude of mitochondrial oxidative stress was not sufficient enough to alter the membrane potential at the given time point and concentration (40 μg)
Summary
Habitual smokers have 13% lower sperm count than nonsmokers.[1]. This is because the 7000 constituents, including 69 proven carcinogens, of cigarette smoke (CS) accumulate in the systemic circulation and seminal plasma and irreversibly impair both the quality and quantity of human spermatozoa.[2,3] Chronic exposure to CS is toxic to germ cells[4,5] and results in excessive generation of free oxygen radicals.[6]. We found that in MEFs, Ahr was not required for changes in the percentage of cells positive for BCL2L1, BCL2, BAX, and BAD upon CSC exposure (Figures 2c, d, g, h and 3c, d, g, h) These results suggest that CSC-induced oxidative stress activates the mitochondrial pathway of apoptosis in spermatocytes by differentially modifying the expression of apoptotic proteins in an AHR-independent manner. WT and AHR-KO MEFs treated with CSC had equivalent percentages of annexin-V-positive cells (Figures 6e and f) These data indicate that CSC structurally modifies the spermatocyte cell membrane during apoptosis, and that, whereas this event is not affected by loss of AHR expression, it is abrogated by inactivation of the AHR pathway. These data ruled out the possibility of direct participation of MAPKs in membrane damage while implicating the specificity of AHR pathway in apoptosis
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
