MUTAGENICITY OF STEREOCHEMICAL CONFIGURATIONS OF 1,3-BUTADIENE EPOXY METABOLITES IN HUMAN CELLS
Impact/Purpose:
The objective of this research was to evaluate the cytotoxic and mutagenic potency of each stereoisomer of key BD epoxy metabolites (BDO, BDO2, and BDO-diol) in human cells. The original aims included synthesis of stereoisomers of BDO, BDO2, and BDO-diol, measurement of the HPRT and TKMfs in TK6 cells exposed to each isomer of the BD metabolites, and characterization and comparison of the mutational spectra of large-scale genetic alterations caused by the three stereoisomers of BDO2.
The specific aims were as follows:
1. To synthesize eight stereoisomers of BDO, BDO2, and BDO-diol, meso-BDO2 was synthesized in a previous HEI-funded study in our laboratory (Walker et al. 2009).
2. To evaluate the cytotoxicity and mutagenicity of each stereoisomer of BDO2 and to determine the frequencies and types of large-scale changes in the HPRT gene in mutant clones obtained from TK6 cells treated with each stereoisomer of BDO2, compared with control TK6 cells.
3. To evaluate the cytotoxicity and mutagenicity of each stereoisomer of BDO in TK6 cells, as in Aim 2.
4. To evaluate the cytotoxicity and mutagenicity of each stereoisomer of BDO-diol in TK6 cells, as in Aim 2.
During this study, no marked differences in cytotoxicity and mutagenicity among the three stereoisomers of BDO2 in TK6 cells were found. Preliminary results of molecular analyses of HPRT mutants demonstrated similar mutational spectra for each stereoisomer of BDO2. Therefore, with the approval of the HEI Research Committee, analysis of additional HPRT and TK mutants was not conducted.
Description:
The synthesis of the stereoisomers was conducted at the University of Texas Medical Branch in Galveston, Texas. The investigators assessed the purity of each compound by evaluating various analytic parameters such as gas chromatography mass spectrometry and nuclear magnetic resonance spectra, melting point, and optical rotation. Stereoisomers result from the asymmetric bonding of some atoms to a carbon atom, resulting in “left hand” (S) and “right hand” (R) forms, which differ in their three-dimensional configuration and optical activity but are chemically identical. BDO has two stereoisomers: (2R) and (2S). BDO2 has three stereoisomers: (2R, 3R), (2S,3S), and meso (a compound that is not optically active). BDO-diol has four stereoisomers: (2R,3R), (2R, 3S), (2S,3R), and (2S,3S).
The cytotoxicity and mutagenicity of each stereoisomer was evaluated in the TK6 human lymphoblastoid cell line. Cells were exposed in culture to different doses of each stereoisomer for 24 hours. Cytotoxicity, or percent survival, at each dose was calculated as the percentage of cells that form colonies relative to control cells (with the control value set at 100%). Mutations were examined in the HPRT and the TK genes. Cells with mutations at the HPRT gene grew in a medium containing 6-thioguanine, while cells with mutations at the TK gene were grown in the presence of trifluorothymidine; normal cells cannot grow in these media. The frequency of mutations was calculated as the percentage of cells that grew in the presence of the selection agent relative to the percentage that grew in normal medium (without the agent). In separate experiments, Meng and colleagues determined the spectrum of the mutations at the HPRT gene induced by BDO2 using amplification of the HPRT DNA and analysis of the DNA by ultraviolet light. This method primarily detects large deletions.