Expression of osteoblast marker genes in rat calvarial osteoblast-like cells, and effects of the endocrine disrupters diphenylolpropane, benzophenone-3, resveratrol and silymarin

Compelling evidence indicates that some endocrine disrupters (EDs), acting as selective estrogen-receptor modulators, interfere with osteoblast differentiation and function. Hence, we investigated whether four EDs [bisphenol-A (BSP), benzophenone-3 (BP3), resveratrol and silymarin] affect differentiation and growth of rat calvarial osteoblast-like (ROB) cells in primary in vitro culture. ROB cells were cultured for up 30 days in a medium supplemented with fetal calf serum (FCS), and conventional RT-PCR detected the expression of collagen-1alpha and osteonectin mRNAs through the entire culture period. Real time-PCR demonstrated that at days 2 and 7 of culture the expressions of collagen-1alpha and osteonectin were very low, and underwent a 192- and a 334-fold increase, respectively, at day 21 of culture. In contrast, osteocalcin expression remained unchanged from days 2 to 21 of culture. EIA showed that ROB cells secreted sizeable amounts of osteocalcin and osteopontin between days 13 and 15 of culture. EDs were added at day 13 of culture at concentrations ranging from 10(-10) to 10(-6) M, being the culture medium deprived of FCS, and their effects were tested 48 h later. None of EDs was found to affect osteocalcin and osteopontin secretion from ROB cells, suggesting that their effects were tested at a relatively earlier stage of culture, when ROB cell differentiation into osteoblats is not fully accomplished, and/or the presence of estrogens contained in FCS is needed for EDs to exert their osteoblast-differentiation modulating action. BSP and BP3, but not resveratrol and silymarin, decreased proliferative activity of cultured ROB cells, a cytotoxic effect conceivably independent of their estrogen-receptor modulating activity.