The carcinogen safrole increases intracellular free Ca ²⁺ levels and causes death in MDCK cells

The effect of the carcinogen safrole on intracellular Ca ²⁺ movement in renal tubular cells has not been explored previously. The present study examined whether safrole could alter Ca ²⁺ handling in Madin-Darby canine kidney (MDCK) cells. Cytosolic free Ca ²⁺ levels ([Ca ²⁺ ] _i ) in populations of cells were measured using fura-2 as a fluorescent Ca ²⁺ probe. Safrole at concentrations above 33 μM increased [Ca ²⁺ ] _i in a concentration-dependent manner with an EC ₅₀ value of 400 μM. The Ca ²⁺ signal was reduced by 90% by removing extracellular Ca ²⁺ , but was not affected by nifedipine, verapamil, or diltiazem. Addition of Ca ²⁺ after safrole had depleted intracellular Ca ²⁺ -induced dramatic Ca ²⁺ influx, suggesting that safrole caused store-operated Ca ²⁺ entry. In Ca ²⁺ -free medium, after pretreatment with 650 μM safrole, 1 μM thapsigargin (an endoplasmic reticulum Ca ²⁺ pump inhibitor) failed to release more Ca ²⁺ . Inhibition of phospholipase C with 2 μM U73122 did not affect safrole-induced Ca ²⁺ release. Trypan blue exclusion assays revealed that incubation with 650 μM 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 [Ca ²⁺ ] _i increase by causing Ca ²⁺ release from the endoplasmic reticulum in a phospholipase C-independent fashion, and by inducing Ca ²⁺ influx via store-operated Ca ²⁺ entry. Furthermore, safrole can cause acute toxicity to MDCK cells.