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The carcinogen safrole increases intracellular free Ca2+ levels and causes death in MDCK cells
Wei-Chuan Chen1, He-Hsiung Cheng, Chun-Jen Huang
1Department of Surgery, Ping-Tung Christian Hospital, Ping-Tung 900, Taiwan, ROC.
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.
Insights
The carcinogen safrole increases intracellular calcium ([Ca2+]i) in kidney cells by releasing it from endoplasmic reticulum stores and promoting influx via store-operated calcium entry. Safrole also induces acute toxicity in these renal tubular cells.
Area of Science:
- Cellular Biology
- Toxicology
- Renal Physiology
Background:
- Intracellular calcium (Ca2+) homeostasis is crucial for renal tubular cell function.
- The toxicological effects of the carcinogen safrole on cellular Ca2+ handling remain largely unexplored.
- Madin-Darby canine kidney (MDCK) cells are a widely used model for studying renal tubular cell physiology.
Purpose of the Study:
- To investigate the impact of safrole on intracellular Ca2+ dynamics in MDCK cells.
- To elucidate the mechanisms by which safrole affects Ca2+ handling.
- To assess the acute cytotoxicity of safrole in this cell model.
Main Methods:
- Measurement of cytosolic free Ca2+ levels ([Ca2+]i) using fura-2 fluorescence.
- Pharmacological manipulation of extracellular and intracellular Ca2+ stores.
- Assessment of cell viability using trypan blue exclusion assays.
Main Results:
- Safrole increased [Ca2+]i in a concentration-dependent manner (EC50 = 400 microM).
- Safrole-induced Ca2+ increase involved both release from endoplasmic reticulum stores (independent of phospholipase C) and store-operated Ca2+ entry.
- High concentrations of safrole (650 microM) induced significant cell death after 60 minutes of incubation.
Conclusions:
- Safrole disrupts Ca2+ homeostasis in MDCK cells through ER Ca2+ release and store-operated Ca2+ entry.
- These alterations in Ca2+ handling contribute to the acute toxicity of safrole in renal tubular cells.
- The findings highlight a novel mechanism of safrole-induced cellular damage relevant to its carcinogenic potential.
