Prioritizing Chemical Constituents of Tobacco for Screening of Skeletal Developmental Toxicity using ToxPI

Though it is well known that tobacco related products can cause prenatal maldevelopment, very little is known on how tobacco products affect bone tissue as it develops in the embryo. Identifying which chemicals can induce the greatest harm to the prenatal skeletal system is an improbable task as there are over 7,000 chemicals in tobacco smoke alone. In order to assess what chemicals out of the thousands can cause maldevelopment the Toxicological Priority Index (ToxPI) program was used to rank osteogenic cytotoxic potential. Assay on various pathways and genes were found by accessing the Environmental Protection Agency’s Interactive Chemical Safety for Sustainability (iCCS) dashboard and then integrated into the ToxPI system. In order to assess the ability of ToxPI to predict in vivo maldevelopment based on our parameters, two compounds were tested for their potency to inhibit osteogenic differentiation in vitro. A ToxPI predicted negative control (nicotine) and a known osteogenic inhibitor (cyclopamine) were selected as a positive control. Human embryonic stem cells were differentiated into osteoblasts and exposed to various concentrations of each compound. Cell viability was measured via MTT assay in conjunction with a calcium assay to measure osteogenic differentiation yield. In accordance with its predicted ToxPI score of 2.3, cyclopamine was cytotoxic in vitro and inhibited differentiation at similar concentrations. In contrast, results revealed nicotine, although not cytotoxic in vitro, to cause differentiation inhibition. Together our data suggests that ToxPi might be useful to identify strongly inhibitory chemicals based on their cytotoxicity, by might also give false negative results for chemicals that cause differentiation inhibition at sub-toxic levels