Safrole-induced cellular Ca²⁺ increases and death in human osteosarcoma cells

The effect of the carcinogen safrole on intracellular Ca²⁺ movement has not been explored in osteoblast-like cells. This study examined whether safrole could alter Ca²⁺ handling and viability in MG63 human osteosarcoma 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 130 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 450 μM. The Ca²⁺ signal was reduced by 30% by removing extracellular Ca²⁺. Addition of Ca²⁺ after safrole had depleted intracellular Ca²⁺ induced Ca²⁺ influx, suggesting that safrole caused Ca²⁺ entry. In Ca²⁺-free medium, after pretreatment with 650 μM safrole, 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) failed to release more Ca²⁺; and pretreatment with thapsigargin inhibited most of the safrole-induced [Ca²⁺]_i increases. Inhibition of phospholipase C with U73122 did not affect safrole-induced Ca²⁺ release; whereas activation of protein kinase C with phorbol ester enhanced safrole-induced [Ca²⁺]_i increase. Trypan exclusion assays revealed that incubation with 65 μM safrole for 30 min did not kill cells, but incubation with 650 μM safrole for 10-30 min nearly killed all cells. Flow cytometry demonstrated that safrole evoked apoptosis in a concentration-dependent manner. Safrole-induced cytotoxicity was not reversed by chelation of Ca²⁺ with BAPTA. Collectively, the data suggest that in MG63 cells, safrole induced a [Ca²⁺]_i increase by causing Ca²⁺ release mainly from the endoplasmic reticulum in a phospholipase C-independent manner. The safrole response involved Ca²⁺ influx and is modulated by protein kinase C. Furthermore, safrole can cause apoptosis in a Ca²⁺-independent manner.