Significant gaps exist in our knowledge of how cellular redox status, sometimes referred to as oxidative stress, impacts placental trophoblasts. The present study used tert-butyl hydroperoxide (TBHP) as a known generator of reactive oxygen species (ROS) in the extravillous trophoblast cell line HTR-8/SVneo to examine the role of cellular redox disruption of prostaglandin E2 (PGE2) and the cytokine IL-6 in cell death. Cells were exposed to 0, 12.5, 25, or 50 μM TBHP for 4, 8, and 24 h to ascertain effects on cell viability, caspase 3/7 activity, PGE2 release, PTGS2 mRNA expression, and IL-6 release. Experiments with inhibitors included the cyclooxygenase inhibitor indomethacin, mitogen-activated protein kinase inhibitors (PD169316, U0126, or SP600125), or treatments to counter expected consequences of TBHP-stimulated generation of ROS (deferoxamine [DFO], butylated hydroxyanisole [BHA], and N,N′-diphenyl-1,4-phenylenediamine [DPPD]) using 24-h exposure to 50 μM TBHP. Cell viability, measured by ATP content, decreased 24% relative to controls with a 24-h exposure to 50 μM TBHP, but not at lower TBHP concentrations nor at earlier time points. Exposure to 50 μM TBHP increased caspase 3/7 activity, an indicator of apoptosis, after 8 and 24 h. Antioxidant treatment markedly reduced TBHP-stimulated caspase 3/7 activity, PGE2 release, and IL-6 release. TBHP-stimulated IL-6 release was blocked by PD169316 but unaltered by indomethacin. These data suggest that TBHP-stimulated IL-6 release and caspase 3/7 activation were independent of PGE2 yet were interrupted by treatments with known antioxidant properties, providing new insight into relationships between PGE2, IL-6, and apoptosis under conditions of chemically induced cellular oxidation.
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Waclawik A, Kaczynski P, Jabbour HN. Autocrine and paracrine mechanisms of prostaglandin E(2) action on trophoblast/conceptus cells through the prostaglandin E(2) receptor (PTGER2) during implantation. Endocrinology. 2013;154(10):3864–76. https://doi.org/10.1210/en.2012-2271.
Kaczynski P, Kowalewski MP, Waclawik A. Prostaglandin F2alpha promotes angiogenesis and embryo-maternal interactions during implantation. Reproduction. 2016;151(5):539–52. https://doi.org/10.1530/REP-15-0496.
McElrath TF, Hecht JL, Dammann O, Boggess K, Onderdonk A, Markenson G, et al. Pregnancy disorders that lead to delivery before the 28th week of gestation: an epidemiologic approach to classification. Am J Epidemiol. 2008;168(9):980–9. https://doi.org/10.1093/aje/kwn202.
Burton GJ, Fowden AL, Thornburg KL. Placental origins of chronic disease. Physiol Rev. 2016;96(4):1509–65. https://doi.org/10.1152/physrev.00029.2015.
Schoots MH, Gordijn SJ, Scherjon SA, van Goor H, Hillebrands J-L. Oxidative stress in placental pathology. Placenta. 2018;69:153–61. https://doi.org/10.1016/j.placenta.2018.03.003.
Jones DP. Redefining oxidative stress. Antioxid Redox Signal. 2006;8(9–10):1865–79. https://doi.org/10.1089/ars.2006.8.1865.
Murata M, Fukushima K, Takao T, Seki H, Takeda S, Wake N. Oxidative stress produced by xanthine oxidase induces apoptosis in human extravillous trophoblast cells. J Reprod Dev. 2013;59(1):7–13.
Rao H, Bai Y, Li Q, Zhuang B, Yuan Y, Liu Y, et al. SATB1 downregulation induced by oxidative stress participates in trophoblast invasion by regulating beta-catenin. Biol Reprod. 2018;98(6):810–20. https://doi.org/10.1093/biolre/ioy033.
Chen CP, Chen CY, Wu YH, Chen CY. Oxidative stress reduces trophoblast FOXO1 and integrin beta3 expression that inhibits cell motility. Free Radic Biol Med. 2018;124:189–98. https://doi.org/10.1016/j.freeradbiomed.2018.06.006.
Banu SK, Stanley JA, Taylor RJ, Sivakumar KK, Arosh JA, Zeng L, et al. Sexually dimorphic impact of chromium accumulation on human placental oxidative stress and apoptosis. Toxicol Sci. 2018;161(2):375–87. https://doi.org/10.1093/toxsci/kfx224.
Yang C, Lim W, Bazer FW, Song G. Butyl paraben promotes apoptosis in human trophoblast cells through increased oxidative stress-induced endoplasmic reticulum stress. Environ Toxicol. 2018;33(4):436–45. https://doi.org/10.1002/tox.22529.
Park HR, Kamau PW, Korte C, Loch-Caruso R. Tetrabromobisphenol A activates inflammatory pathways in human first trimester extravillous trophoblasts in vitro. Reprod Toxicol. 2014;50:154–62. https://doi.org/10.1016/j.reprotox.2014.10.005.
Park HR, Kamau PW, Loch-Caruso R. Involvement of reactive oxygen species in brominated diphenyl ether-47-induced inflammatory cytokine release from human extravillous trophoblasts in vitro. Toxicol Appl Pharmacol. 2014;274(2):283–92. https://doi.org/10.1016/j.taap.2013.11.015.
Tetz LM, Cheng AA, Korte CS, Giese RW, Wang P, Harris C, et al. Mono-2-ethylhexyl phthalate induces oxidative stress responses in human placental cells in vitro. Toxicol Appl Pharmacol. 2013;268(1):47–54. https://doi.org/10.1016/j.taap.2013.01.020.
Hassan I, Kumar AM, Park HR, Lash LH, Loch-Caruso R. Reactive oxygen stimulation of interleukin-6 release in the human trophoblast cell line HTR-8/SVneo by the trichlorethylene metabolite S-(1,2-dichloro)-l-cysteine. Biol Reprod. 2016;95(3):66. https://doi.org/10.1095/biolreprod.116.139261.
Elkin ER, Harris SM, Loch-Caruso R. Trichloroethylene metabolite S-(1,2-dichlorovinyl)-l-cysteine induces lipid peroxidation-associated apoptosis via the intrinsic and extrinsic apoptosis pathways in a first-trimester placental cell line. Toxicol Appl Pharmacol. 2018;338:30–42. https://doi.org/10.1016/j.taap.2017.11.006.
Menon R, Fortunato SJ, Yu J, Milne GL, Sanchez S, Drobek CO, et al. Cigarette smoke induces oxidative stress and apoptosis in normal term fetal membranes. Placenta. 2011;32(4):317–22. https://doi.org/10.1016/j.placenta.2011.01.015.
Jin J, Richardson L, Sheller-Miller S, Zhong N, Menon R. Oxidative stress induces p38MAPK-dependent senescence in the feto-maternal interface cells. Placenta. 2018;67:15–23. https://doi.org/10.1016/j.placenta.2018.05.008.
Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N, et al. Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res. 1993;206(2):204–11.
Rice-Evans C, Baysal E, Pashby DP, Hochstein P. t-butyl hydroperoxide-induced perturbations of human erythrocytes as a model for oxidant stress. Biochim Biophys Acta. 1985;815(3):426–32.
Masaki N, Kyle ME, Farber JL. Tert-butyl hydroperoxide kills cultured hepatocytes by peroxidizing membrane lipids. Arch Biochem Biophys. 1989;269(2):390–9. https://doi.org/10.1016/0003-9861(89)90122-7.
Öllinger K, Brunk UT. Cellular injury induced by oxidative stress is mediated through lysosomal damage. Free Radic Biol Med. 1995;19(5):565–74. https://doi.org/10.1016/0891-5849(95)00062-3.
van de Water B, Zoeteweij JP, de Bont HJ, Mulder GJ, Nagelkerke JF. Role of mitochondrial Ca2+ in the oxidative stress-induced dissipation of the mitochondrial membrane potential. Studies in isolated proximal tubular cells using the nephrotoxin 1,2-dichlorovinyl-L-cysteine. J Biol Chem. 1994;269(20):14546–52.
Timmins GS, Davies MJ. Free radical formation in isolated murine keratinocytes treated with organic peroxides and its modulation by antioxidants. Carcinogenesis. 1993;14(8):1615–20. https://doi.org/10.1093/carcin/14.8.1615.
Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat New Biol. 1971;231(25):232–5. https://doi.org/10.1038/newbio231232a0.
Johnson JL, Wimsatt J, Buckel SD, Dyer RD, Maddipati KR. Purification and characterization of prostaglandin H synthase-2 from sheep placental cotyledons. Arch Biochem Biophys. 1995;324(1):26–34. https://doi.org/10.1006/abbi.1995.9934.
Kummer JL, Rao PK, Heidenreich KA. Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase. J Biol Chem. 1997;272(33):20490–4. https://doi.org/10.1074/jbc.272.33.20490.
Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, et al. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem. 1998;273(29):18623–32. https://doi.org/10.1074/jbc.273.29.18623.
Bennett BL, Sasaki DT, Murray BW, O'Leary EC, Sakata ST, Xu W, et al. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci. 2001;98(24):13681–6. https://doi.org/10.1073/pnas.251194298.
Chen X, Zhong Z, Xu Z, Chen L, Wang Y. 2′,7′-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: forty years of application and controversy. Free Radic Res. 2010;44(6):587–604. https://doi.org/10.3109/10715761003709802.
Halliwell B. Protection against tissue damage in vivo by desferrioxamine: what is its mechanism of action? Free Radic Biol Med. 1989;7(6):645–51.
Hartley A, Davies M, Rice-Evans C. Desferrioxamine as a lipid chain-breaking antioxidant in sickle erythrocyte membranes. FEBS Lett. 1990;264(1):145–8. https://doi.org/10.1016/0014-5793(90)80786-i.
Carocho M, Ferreira IC. A review on antioxidants, prooxidants and related controversy: natural and synthetic compounds, screening and analysis methodologies and future perspectives. Food Chem Toxicol. 2013;51:15–25. https://doi.org/10.1016/j.fct.2012.09.021.
National Center for Biotechnology Information. PubChem Database. N,N′-diphenyl-p-phenylenediamine. https://pubchem.ncbi.nlm.nih.gov/compound/N_N_-Diphenyl-p-phenylenediamine. Accessed Oct 23, 2019.
Sies H, Berndt C, Jones DP. Oxidative stress. Annu Rev Biochem. 2017;86:715–48. https://doi.org/10.1146/annurev-biochem-061516-045037.
Degasperi GR, Castilho RF, Vercesi AE. High susceptibility of activated lymphocytes to oxidative stress-induced cell death. An Acad Bras Cienc. 2008;80(1):137–48.
Lu Z, Nie G, Li Y, Soe-Lin S, Tao Y, Cao Y, et al. Overexpression of mitochondrial ferritin sensitizes cells to oxidative stress via an iron-mediated mechanism. Antioxid Redox Signal. 2009;11(8):1791–803. https://doi.org/10.1089/ARS.2008.2306.
Kucera O, Endlicher R, Rousar T, Lotkova H, Garnol T, Drahota Z, et al. The effect of tert-butyl hydroperoxide-induced oxidative stress on lean and steatotic rat hepatocytes in vitro. Oxidative Med Cell Longev. 2014;2014:752506–12. https://doi.org/10.1155/2014/752506.
Bhatnagar A, Srivastava SK, Szabo G. Oxidative stress alters specific membrane currents in isolated cardiac myocytes. Circ Res. 1990;67(3):535–49.
Xu F, Putt DA, Matherly LH, Lash LH. Modulation of expression of rat mitochondrial 2-oxoglutarate carrier in NRK-52E cells alters mitochondrial transport and accumulation of glutathione and susceptibility to chemically induced apoptosis. J Pharmacol Exp Ther. 2006;316(3):1175–86. https://doi.org/10.1124/jpet.105.094599.
Turner MA, Vause S, Greenwood SL. The regulation of interleukin-6 secretion by prostanoids and members of the tumor necrosis factor superfamily in fresh villous fragments of term human placenta. J Soc Gynecol Investig. 2004;11(3):141–8. https://doi.org/10.1016/j.jsgi.2003.10.007.
Korbecki J, Baranowska-Bosiacka I, Gutowska I, Chlubek D. The effect of reactive oxygen species on the synthesis of prostanoids from arachidonic acid. J Physiol Pharmacol. 2013;64(4):409–21.
Park HR, Loch-Caruso R. Protective effect of (+/−)alpha-tocopherol on brominated diphenyl ether-47-stimulated prostaglandin pathways in human extravillous trophoblasts in vitro. Toxicol in Vitro. 2015;29(7):1309–18. https://doi.org/10.1016/j.tiv.2015.05.015.
Kumar D, Moore RM, Elkhwad M, Silver RJ, Moore JJ. Vitamin C exacerbates hydrogen peroxide induced apoptosis and concomitant PGE2 release in amnion epithelial and mesenchymal cells, and in intact amnion. Placenta. 2004;25(6):573–9. https://doi.org/10.1016/j.placenta.2003.12.005.
Lappas M, Permezel M, Rice GE. N-acetyl-cysteine inhibits phospholipid metabolism, proinflammatory cytokine release, protease activity, and nuclear factor-kappaB deoxyribonucleic acid-binding activity in human fetal membranes in vitro. J Clin Endocrinol Metab. 2003;88(4):1723–9.
Rodier F, Coppe JP, Patil CK, Hoeijmakers WA, Munoz DP, Raza SR, et al. Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion. Nat Cell Biol. 2009;11(8):973–9. https://doi.org/10.1038/ncb1909.
Dixon CL, Richardson L, Sheller-Miller S, Saade G, Menon R. A distinct mechanism of senescence activation in amnion epithelial cells by infection, inflammation, and oxidative stress. Am J Reprod Immunol. 2018;79(3). https://doi.org/10.1111/aji.12790.
Straszewski-Chavez SL, Abrahams VM, Mor G. The role of apoptosis in the regulation of trophoblast survival and differentiation during pregnancy. Endocr Rev. 2005;26(7):877–97. https://doi.org/10.1210/er.2005-0003.
Sharp AN, Heazell AE, Crocker IP, Mor G. Placental apoptosis in health and disease. Am J Reprod Immunol. 2010;64(3):159–69. https://doi.org/10.1111/j.1600-0897.2010.00837.x.
Kennedy TG, Gillio-Meina C, Phang SH. Prostaglandins and the initiation of blastocyst implantation and decidualization. Reproduction. 2007;134(5):635–43. https://doi.org/10.1530/REP-07-0328.
Lucaroni F, Morciano L, Rizzo G, D’Antonio F, Buonuomo E, Palombi L, et al. Biomarkers for predicting spontaneous preterm birth: an umbrella systematic review. J Matern Fetal Neonatal Med. 2018;31(6):726–34. https://doi.org/10.1080/14767058.2017.1297404.
Liu Y, Liu Y, Zhang R, Zhu L, Feng Z. Early- or mid-trimester amniocentesis biomarkers for predicting preterm delivery: a meta-analysis. Ann Med. 2017;49(1):1–10. https://doi.org/10.1080/07853890.2016.1211789.
Wei SQ, Fraser W, Luo ZC. Inflammatory cytokines and spontaneous preterm birth in asymptomatic women: a systematic review. Obstet Gynecol. 2010;116(2 Pt 1):393–401. https://doi.org/10.1097/AOG.0b013e3181e6dbc0.
Wakabayashi A, Sawada K, Nakayama M, Toda A, Kimoto A, Mabuchi S, et al. Targeting interleukin-6 receptor inhibits preterm delivery induced by inflammation. Mol Hum Reprod. 2013;19(11):718–26. https://doi.org/10.1093/molehr/gat057.
Woodruff TJ, Parker JD, Kyle AD, Schoendorf KC. Disparities in exposure to air pollution during pregnancy. Environ Health Perspect. 2003;111(7):942–6.
We thank Dr. Peter Mancuso and Dr. Jeff Martens for their constructive criticism and advice regarding this work, Joel Whitfield of the University of Michigan Immunologic Monitoring Core for cytokine analysis, and Faith Bjork for supportive laboratory assistance. Thank you to members of the Loch-Caruso lab for providing a thoughtful sounding board, especially Dr. Lauren Tetz.
This work was supported by the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), with a research project to RL-C (P42ES017198) training grant fellowship support to CSK and KAH (T32ES007062), and supplementary project support from the Michigan Center for Lifestage Environmental Exposure and Disease (P30ES017885). Additional fellowship support for KAH was from the Michigan Institute for Clinical & Health Research, funded by the National Center for Advancing Translational Sciences (NCATS), NIH (UL1TR000433). Support from a University of Michigan Rackham Graduate Student Research Grant is also gratefully acknowledged. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIEHS, NCATS, NIH, or the University of Michigan.
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Loch-Caruso, R., Korte, C.S., Hogan, K.A. et al. Tert-Butyl Hydroperoxide Stimulated Apoptosis Independent of Prostaglandin E2 and IL-6 in the HTR-8/SVneo Human Placental Cell Line. Reprod. Sci. (2020). https://doi.org/10.1007/s43032-020-00231-5
- Oxidative stress
- Reactive oxygen species