Cigarette smoke inhibits endometrial epithelial cell proliferation through a nitric oxide– mediated pathway Omid Khorram, M.D., Ph.D., Guang Han, M.D.,

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Cigarette smoke inhibits endometrial epithelial cell proliferation through a nitric oxide– mediated pathway Omid Khorram, M.D., Ph.D., Guang Han, M.D., Thomas Magee, Ph.D. Fertility and Sterility Volume 93, Issue 1, Pages 257-263 (January 2010) DOI: 10.1016/j.fertnstert.2008.09.074 Copyright © 2010 Terms and Conditions

Figure 1 (A) Effects of various concentration of aqueous cigarette smoke–saturated solution (CS) on HES cell line proliferation. Results in this and subsequent figures are expressed as mean ± SEM. Each bar represents n = 8 in this and subsequent panels of this figure. ∗P<.05 vs. control. (B) Effects of hemoglobin (Hb) on CS-induced effect on HES cell proliferation. ∗P<.05 vs. control.+P=NS vs. control. (C) Effect of L-arginine (L-Arg), LNMMA, and combination of these drugs on HES cell proliferation. ∗P<.05 vs. control; +P<.05 vs. CS; CS+L-Arg was not different than CS+LNMMA+L-Arg. (D) Effect of the NO donor drug SNAP on HES cell proliferation. ∗P<.05 vs. control. Fertility and Sterility 2010 93, 257-263DOI: (10.1016/j.fertnstert.2008.09.074) Copyright © 2010 Terms and Conditions

Figure 2 Effects of cigaret smoke (CS), nicotine (NIC, 10−5 mol/L) and benzo[a]pyrene (BP, 10−5mol/L) on eNOS (A) and iNOS (B) expression and NOx secretion in vitro (C) from HES cells. (A) Lanes 1–3 = control; 4–6 = CS; 7–9 = nicotine; 10–12 = acetone control for BP; 13–15 = BP. (B) Lane 1 = positive control (stimulated mouse macrophages); 2–3 = control; 4–6 = CS; 7–9 = nicotine (10−5 mol/L), 10–12 = acetone control for BP; 13–15 = BP (10−5 mol/L). ∗P<.05; ∗∗P<.01 vs. control. (C) Effect of CS, NIC, and BP on NOx secretion from HES cells in vitro. eNOS = endothelial nitric oxide synthase; iNOS = inducible nitric oxide synthase; NOx = nitrite, nitrate, and nitric oxide (NO). Fertility and Sterility 2010 93, 257-263DOI: (10.1016/j.fertnstert.2008.09.074) Copyright © 2010 Terms and Conditions

Figure 3 Time dependence of cigarette smoke (CS) and its major components, nicotine (NIC, 10−5 mol/L) and benzo[a]pyrene (BP, 10−5 mol/L) on eNOS expression in HES cells (n = 6 samples per bar). Cigarette smoke (A), NIC (B), and BP (C) significantly stimulated expression of eNOS at 24 h, whereas at 48 h of culture CS (A) and BP (C) were without an effect and NIC (B) significantly inhibited eNOS expression in HES cells. ∗P<.05; ∗∗∗P<.001 vs. control. Abbreviations as in Figure 2. Fertility and Sterility 2010 93, 257-263DOI: (10.1016/j.fertnstert.2008.09.074) Copyright © 2010 Terms and Conditions

Figure 4 Effect of cigarette smoke (CS) on eNOS (A) and iNOS (B) expression in primary endometrial cells derived from reproductive aged women (n = 6 samples per group). Abbreviations as in Figure 2. Fertility and Sterility 2010 93, 257-263DOI: (10.1016/j.fertnstert.2008.09.074) Copyright © 2010 Terms and Conditions

Figure 5 Effect of antioxidants ascorbic acid (AA) and glutathione (Glu) on cigarette smoke (CS)–induced eNOS/iNOS expression in HES cells (n = 6 samples per group). Abbreviations as in Figure 2. Fertility and Sterility 2010 93, 257-263DOI: (10.1016/j.fertnstert.2008.09.074) Copyright © 2010 Terms and Conditions