Osteoblasts and adipocytes originate from common mesenchymal precursors. With aging, there is a decrease in osteoprogenitor cells that parallels an increase of adipocytes in bone marrow. We observed that rabbit serum (RS) induces adipocyte-like differentiation in human osteosarcoma SaOS-2/B10 and MG-63 cell lines, in rat ROS17/2.8 cells, and in mouse calvaria-derived osteoblastic MB1.8 cells, as evidenced by the accumulation of Oil Red O positive lipid vesicles and the decrease in alkaline phosphatase expression. Both SaOS-2/B10 and MG-63 cells, but not ROS17/2.8 nor MB1.8 cells, express significant levels of PPARgamma mRNA, a member of the peroxisome proliferator activated receptor (PPAR) family that has been implicated in the control of adipocyte differentiation. However, both ROS17/2.8 and MG-63 cells express significant levels of the adipocyte selective marker, aP2 fatty acid binding mRNA, which can be further increased by RS. These cell types express PPARdelta/NUC-1 but not PPARalpha, indicating that cells that do not express either PPARgamma or PPARalpha are capable of differentiating into adipocyte-like cells. Transfection experiments in COS cells showed that compared with fetal bovine serum (FBS), RS is rich in agents that stimulate PPAR-dependent transcription. The stimulatory activity was ethyl acetate extractable and was 35-fold more abundant in RS than in FBS. Purification and analysis revealed that the major components of this extract are free fatty acids. Furthermore, the same fatty acids, a mixture of palmitic, oleic, and linoleic acids, activate the PPARs and induce adipocyte-like differentiation of both ROS17/2.8 and SaOS-2/B10 cells. These findings suggest that fatty acids or their metabolites can initiate the switch from osteoblasts to adipocyte-like cells.

No abstract

International guidelines for cytotoxicity limits for the in vitro chromosomal aberration assay require reductions in cell growth of greater than 50%. This sets no upper limit on toxicity and there is concern about the number of false or irrelevant results obtained in the aberration assay, i.e., positive results at toxic dose levels only, with no evidence for primary DNA damaging ability and with negative results in the other genotoxicity tests. We have previously proposed that no truly genotoxic compound would be missed if the toxicity of the highest dose did not exceed 50%. Cell growth measured by cell counts as a percentage of controls can underestimate toxicity. For example, if we seed half a million cells per culture, and the controls double to 1 million during the experiment, a culture that truly has no growth will still have a cell count 50% of the control. Measurement of population doublings (PDs) more accurately assesses cell growth. To assess the use of PD in dose selection, we examined previous data from this lab and data from new experiments with "true," primary DNA damaging clastogens, and with clastogens, including drugs, thought to act indirectly, through cytotoxicity-associated mechanisms. We compared aberration results where the highest doses scored were based on 50% reductions in final cell counts with results obtained when the highest doses were based on PD. The PD method allows detection of true clastogens, including those that are active in a range with some toxicity, and reduces the number of toxicity-related "false"-positive results.

No abstract

Drug induced liver injury (DILI) is a major reason for drug candidate attrition from development, denied commercialization, market withdrawal, and restricted prescribing of pharmaceuticals. The metabolic bioactivation of drugs to chemically reactive metabolites (CRM) contribute to liver-associated adverse drug reactions (ADR) in humans that often goes undetected in conventional animal toxicology studies. A challenge for pharmaceutical drug discovery has been reliably selecting drug candidates with a low liability of forming CRM and reduced DILI potential, at projected therapeutic doses, without falsely restricting the development of safe drugs. We have developed an in vivo rat liver transcriptional signature biomarker reflecting the cellular response to drug bioactivation. Measurement of transcriptional activation of integrated Nuclear factor erythroid 2-related factor 2 (NRF2)/Kelch-like ECH-associated protein 1 (Keap1) electrophilic stress, and Nuclear factor erythroid 2-related factor 1 (NRF1) proteasomal endoplasmic reticulum (ER) stress responses, is described for discerning estimated clinical doses of drugs with potential for bioactivation-mediated hepatotoxicity. The approach was established using well benchmarked CRM forming test agents from our company. This was subsequently tested using curated lists of commercial drugs and internal compounds, anchored in the clinical experience with human hepatotoxicity, while agnostic to mechanism. Based on results with 116 compounds in short-term rat studies, with consideration of the maximum recommended daily clinical dose, this CRM mechanism-based approach yielded 32% sensitivity and 92% specificity for discriminating safe from hepatotoxic drugs. The approach adds new information for guiding early candidate selection and informs structure activity relationships (SAR) thus enabling lead optimization and mechanistic problem solving. Additional refinement of the model is ongoing. Case examples are provided describing the strengths and limitations of the approach.