Mutagenicity of Stereochemical Configurations of 1,3-Butadiene Epoxy Metabolites in Human CellsEPA Grant Number: R834677C150
Subproject: this is subproject number 150 , established and managed by the Center Director under grant R834677
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Health Effects Institute (2010 — 2015)
Center Director: Greenbaum, Daniel S.
Title: Mutagenicity of Stereochemical Configurations of 1,3-Butadiene Epoxy Metabolites in Human Cells
Investigators: Meng, Ryan Q
Institution: Battelle , Health Effects Institute (HEI)
EPA Project Officer: Hunt, Sherri
Project Period: April 1, 2010 through March 31, 2015
RFA: Health Effects Institute (2010) RFA Text | Recipients Lists
Research Category: Health Effects , Air Quality and Air Toxics , Air
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.
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). BDO 2 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.
The Investigators successfully synthesized the nine stereoisomers with sufficient yield to conduct the proposed assays. The purity of the stereoisomers was determined to be at least 98% to 99.9%. A key and novel finding was that the (2R,3S)-BDO-diol stereoisomer was about 30-fold more cytotoxic and mutagenic than the other three BDO-diol stereoisomers. The dose-related mutagenic responses for the stereoisomers of BDO and BDO2 were not statistically different. (2R,3S)-BDO-diol was 5-to-10-fold less mutagenic than any of the BDO2 stereoisomers, but 10-to-20-fold more mutagenic than the BDO stereoisomers.
The analysis of the types of mutation induced by the stereoisomers of BDO2 showed a higher frequency of deletions in cells exposed to each stereoisomer of BDO2 relative to unexposed cells. While appropriately carried out, these experiments did not provide any new insights relating to the mechanism of action of BD. A more detailed analysis of the sizes of the deletions would be helpful to determine whether the deletion spectra of the mutants differ.
The study’s key finding that one of the four stereoisomers of BDO-diol was responsible for most of the mutagenicity of this metabolite is interesting and could explain some of the possible species differences in the mutagenicity of BD. The study also suggests that the species differences in susceptibility to BD that have been noted in other work are not likely to be related to the stereochemistry of BDO and BDO2. The caveat to these conclusions is that the normal target cells for BD in rodents may detoxify the different BD metabolites at very different rates, thus leading to steady-state levels of one metabolite or its stereoisomers that are higher than those of others. The results of this study using a cell line and individual stereoisomers provide justification for follow-up studies that consider the kinetics of the formation and distribution of BDO-diol stereoisomers in rodents and humans and that have endpoints relating to both mutagenicity and carcinogenicity
Publications and Presentations:Publications have been submitted on this subproject: View all 9 publications for this subproject | View all 69 publications for this center
Journal Articles:Journal Articles have been submitted on this subproject: View all 2 journal articles for this subproject | View all 5 journal articles for this center
Supplemental Keywords:Health Effects, Air Toxics, epidemiology, carcinogens, exposure models, 1,3-Butadiene, inhalation exposure, cytotoxic, mutagenic
Progress and Final Reports:Final Report
Main Center Abstract and Reports:R834677 Health Effects Institute (2010 — 2015)
Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
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