Haemopoietic and osteogenic toxicity testing in vitro using murine bone marrow cultures

Bone marrow and the surrounding bone with its high storage capacity for inorganic compounds may accumulate various lipophilic and electrophilic substances that enter the bloodstream. In bone marrow a few stem cells are responsible for the continuous production of blood cells and bone cells during the entire life of the organism. Damage to these cells may result in haemopoietic failure, blood disorders or bone diseases. Therefore bone marrow needs to be considered as one of the major targets of chemicals that enter the circulation. A battery of different in vitro bone marrow assays is established in which interference of chemicals with proliferation and differentiation of marrow cells with haemopoietic, stromal or bone forming marrow commitment may be screened routinely. Stromal cells form the network of extracellular matrix and growth factors that is needed by the haemopoietic cells to proliferate and differentiate. If stromal marrow cells are cultured in the presence of ascorbic acid and beta-glycerophosphate, bone-specific proteins and an extracellular matrix are produced, which calcifies within 3 wk. To evaluate the specificity of the effects on marrow cells, a general cytotoxicity assay is included using 3T3-fibroblasts. Various concentrations of xenobiotics were added over the course of 3 days to the different asssays. Lead nitrate inhibited proliferation of stromal stem cells and their calcification in the bone-forming assay at much lower concentrations than those which were inhibitory to the proliferation of 3T3 cells. The benzene metabolite hydroquinone was equally inhibitory in all the marrow assays, but 3T3 cells needed 10 times more hydroquinone to reach the same degree of inhibition. Catechol, which is another benzene metabolite, was highly toxic but was equally effective in all the assays and showed no specific effects on the marrow cells. As in vivo, benzene itself and phenol showed hardly any effect in the in vitro assays. Not only pollutants but also cytokines may be screened with these assays. Differential effects on marrow cells could be demonstrated for interleukin 10.