title: The carcinogen safrole increases intracellular free Ca2+ levels and causes death in MDCK cells.
abstract: The effect of the carcinogen safrole on intracellular Ca2+ movement in renal tubular cells has not been explored previously. The present study examined whether safrole could alter Ca2+ handling in Madin-Darby canine kidney (MDCK) cells. Cytosolic free Ca2+ levels ([Ca2+]i) in populations of cells were measured using fura-2 as a fluorescent Ca2+ probe. Safrole at concentrations above 33 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 400 microM. The Ca2+ signal was reduced by 90% by removing extracellular Ca2+, but was not affected by nifedipine, verapamil, or diltiazem. Addition of Ca2+ after safrole had depleted intracellular Ca(2+)-induced dramatic Ca2+ influx, suggesting that safrole caused store-operated Ca2+ entry. In Ca(2+)-free medium, after pretreatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release more Ca 2+. Inhibition of phospholipase C with 2 microM U73122 did not affect safrole-induced Ca2+ release. Trypan blue exclusion assays revealed that incubation with 650 microM safrole for 30 min did not kill cells, but killed 70% of cells after incubation for 60 min. Collectively, the data suggest that in MDCK cells, safrole induced a [Ca2+] increase by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent fashion, and by inducing Ca2+ influx via store-operated Ca2+ entry. Furthermore, safrole can cause acute toxicity to MDCK cells.
authors.affiliation: Department of Surgery, Ping-Tung Christian Hospital, Ping-Tung 900, Taiwan, ROC.
authors.forename: Wei-Chuan
authors.initials: WC
authors.lastname: Chen
authors.forename: He-Hsiung
authors.initials: HH
authors.lastname: Cheng
authors.forename: Chun-Jen
authors.initials: CJ
authors.lastname: Huang
authors.forename: Yih-Chau
authors.initials: YC
authors.lastname: Lu
authors.forename: I-Shu
authors.initials: IS
authors.lastname: Chen
authors.forename: Shiuh-Inn
authors.initials: SI
authors.lastname: Liu
authors.forename: Shu-Shong
authors.initials: SS
authors.lastname: Hsu
authors.forename: Hong-Tai
authors.initials: HT
authors.lastname: Chang
authors.forename: Jong-Khing
authors.initials: JK
authors.lastname: Huang
authors.forename: Jin-Shyr
authors.initials: JS
authors.lastname: Chen
authors.forename: Chung-Ren
authors.initials: CR
authors.lastname: Jan
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003470:Culture Media / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology; D045604:Extracellular Fluid / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D009994:Osmolar Concentration; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D012451:Safrole / Q000494:pharmacology*; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
chemical_list: D002121:Calcium Channel Blockers; D002273:Carcinogens; D003470:Culture Media; D004963:Estrenes; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D019284:Thapsigargin; D010738:Type C Phospholipases; D012451:Safrole; D002118:Calcium