TY - JOUR
T1 - Mutagenicity of DNA adducts derived from ethylene oxide exposure in the pSP189 shuttle vector replicated in human Ad293 cells
AU - Tompkins, Elaine M.
AU - McLuckie, Keith I.E.
AU - Jones, Donald J.L.
AU - Farmer, Peter B.
AU - Brown, Karen
N1 - Funding Information:
We thank Dr. Michael Festing for help with the statistical analysis and Dr Didier Gasparutto (Laboratoire des Lésions des Acides Nucléiques, CEA, Grenoble, France) for kindly supplying the O 6 -HedG standard. This work was funded by the Lower Olefins Sector Group and the Ethylene Oxide and Derivatives Sector Group of the European Chemical Industry Council (Cefic) and the Olefins Panel of the American Chemistry Council.
PY - 2009/8/2
Y1 - 2009/8/2
N2 - Ethylene oxide (EO) is a widely used chemical intermediate also formed endogenously from ethylene metabolism. Despite conflicting epidemiological evidence, EO is classified by the IARC as a human carcinogen. The mutagenicity and carcinogenicity of EO is attributed to direct reaction with DNA and formation of multiple 2-hydroxyethyl (HE) DNA adducts. However, the actual lesions responsible for the reported mutagenicity of EO have not been established. This study used the supF mutation assay to investigate the biological relevance of low levels of EO-induced DNA adducts in human Ad293 cells, with respect to the type and level of each HE adduct present. Initial experiments were conducted using pSP189 plasmid containing up to 290 N7-HEGuanine (N7-HEG) adducts/106 nucleotides, which far exceeds that typically detected in human DNA. No other HE-lesions were detectable using our validated LC-MS/MS assay. Replication in cells failed to produce a statistically significant increase in relative mutation frequency, above background rates in the solvent control. Furthermore, the mutation spectrum compiled for EO-treated plasmid (10-2000 μM) did not differ significantly from the spontaneous distribution, suggesting EO is not strongly mutagenic in this system. Under refined reaction conditions using higher EO concentrations capable of inducing detectable levels of N1-HEdA, O6-HEdG and N3-HEdU along with N7-HEG, there was a significant dose-related increase in relative mutation frequency above background (3.76- and 5.30-fold at 10 and 30 mM, respectively). EO treatment appeared associated with an elevated frequency of GC → CG mutations and the occurrence of substitutions at AT base pairs. Additionally, there was a distinct GC → TA mutational hotspot in the 10 mM EO spectrum. Overall, the results suggest a certain level of promutagenic adducts must be attained before mutations become detectable above background, indicating that N7-HEG is not a promutagenic lesion, and support a role for the minor products of DNA hydroxyethylation in the generation of base substitutions by EO.
AB - Ethylene oxide (EO) is a widely used chemical intermediate also formed endogenously from ethylene metabolism. Despite conflicting epidemiological evidence, EO is classified by the IARC as a human carcinogen. The mutagenicity and carcinogenicity of EO is attributed to direct reaction with DNA and formation of multiple 2-hydroxyethyl (HE) DNA adducts. However, the actual lesions responsible for the reported mutagenicity of EO have not been established. This study used the supF mutation assay to investigate the biological relevance of low levels of EO-induced DNA adducts in human Ad293 cells, with respect to the type and level of each HE adduct present. Initial experiments were conducted using pSP189 plasmid containing up to 290 N7-HEGuanine (N7-HEG) adducts/106 nucleotides, which far exceeds that typically detected in human DNA. No other HE-lesions were detectable using our validated LC-MS/MS assay. Replication in cells failed to produce a statistically significant increase in relative mutation frequency, above background rates in the solvent control. Furthermore, the mutation spectrum compiled for EO-treated plasmid (10-2000 μM) did not differ significantly from the spontaneous distribution, suggesting EO is not strongly mutagenic in this system. Under refined reaction conditions using higher EO concentrations capable of inducing detectable levels of N1-HEdA, O6-HEdG and N3-HEdU along with N7-HEG, there was a significant dose-related increase in relative mutation frequency above background (3.76- and 5.30-fold at 10 and 30 mM, respectively). EO treatment appeared associated with an elevated frequency of GC → CG mutations and the occurrence of substitutions at AT base pairs. Additionally, there was a distinct GC → TA mutational hotspot in the 10 mM EO spectrum. Overall, the results suggest a certain level of promutagenic adducts must be attained before mutations become detectable above background, indicating that N7-HEG is not a promutagenic lesion, and support a role for the minor products of DNA hydroxyethylation in the generation of base substitutions by EO.
KW - DNA adduct
KW - Ethylene oxide
KW - Mutation
KW - supF
UR - http://www.scopus.com/inward/record.url?scp=69449088524&partnerID=8YFLogxK
U2 - 10.1016/j.mrgentox.2009.05.011
DO - 10.1016/j.mrgentox.2009.05.011
M3 - Article
C2 - 19477295
AN - SCOPUS:69449088524
SN - 1383-5718
VL - 678
SP - 129
EP - 137
JO - Mutation Research - Genetic Toxicology and Environmental Mutagenesis
JF - Mutation Research - Genetic Toxicology and Environmental Mutagenesis
IS - 2
ER -