1,3-Butadiene-Induced Adenine DNA Adducts Are Genotoxic but Only Weakly Mutagenic When Replicated in Escherichia coli of Various Repair and Replication Backgrounds

Abstract

The adverse effects of the human carcinogen 1,3-butadiene (BD) are believed to be mediated by its DNA-reactive metabolites such as 3,4-epoxybut-1-ene (EB) and 1,2,3,4-diepoxybutane (DEB). The specific DNA adducts responsible for toxic and mutagenic effects of BD, however, have yet to be identified. Recent in vitro polymerase bypass studies of BD-induced adenine (BD-dA) adducts show that DEB-induced N6,N6-DHB-dA (DHB = 2,3-dihydroxybutan-1,4-diyl) and 1,N6-γ-HMHP-dA (HMHP = 2-hydroxy-3-hydroxymethylpropan-1,3-diyl) adducts block replicative DNA polymerases but are bypassed by human polymerases η and κ, leading to point mutations and deletions. In contrast, EB-induced N6-HB-dA (HB = 2-hydroxy-3-buten-1-yl) does not block DNA synthesis and is non-mutagenic. In the present study, we employed a newly established in vivo lesion-induced mutagenesis/genotoxicity assay via next-generation sequencing to evaluate the in vivo biological consequences of S-N6-HB-dA, R,R-N6,N6-DHB-dA, S,S-N6,N6-DHB-dA and R,S-1,N6-γ-HMHP-dA. In addition, the effects of AlkB-mediated direct reversal repair, MutM and MutY catalyzed base excision repair, and DinB translesion synthesis on the BD-dA adducts in bacterial cells were investigated. BD-dA adducts showed the expected inhibition of DNA replication in vivo, but were not substantively mutagenic in any of the genetic environments investigated. This result is in contrast with previous in vitro observations and opens the possibility that E. coli repair and bypass systems other than the ones studied here are able to minimize the mutagenic properties of BD-dA adducts.

Biological Evaluation of DNA Biomarkers in a Chemically Defined and Site-Specific Manner

“…N 6 -carboxymethyl-2′-deoxyadenosine ( N 6 -CMdA) minimally blocks replication in E. coli and is not mutagenic [36]. S - N 6 -HB-dA (HB = 2-hydroxy-3-buten-1-yl) and R , R - N 6 , N 6 -DHB-dA (DHB = 2,3-dihydroxybutan-1,4-diyl) do not block DNA replication and are not mutagenic in E. coli [51]. S , S - N 6 , N 6 -DHB-dA moderately inhibits replication with a 60% bypass efficiency, and causes minimal 1% A>G mutation [51].…”

“…S - N 6 -HB-dA (HB = 2-hydroxy-3-buten-1-yl) and R , R - N 6 , N 6 -DHB-dA (DHB = 2,3-dihydroxybutan-1,4-diyl) do not block DNA replication and are not mutagenic in E. coli [51]. S , S - N 6 , N 6 -DHB-dA moderately inhibits replication with a 60% bypass efficiency, and causes minimal 1% A>G mutation [51]. R , S -1, N 6 - γ -HMHP-dA (HMHP = 2-hydroxy-3-hydroxymethylpropan-1,3-diyl) strongly inhibits DNA replication but causes only 2% A>T mutation [51].…”

“…S , S - N 6 , N 6 -DHB-dA moderately inhibits replication with a 60% bypass efficiency, and causes minimal 1% A>G mutation [51]. R , S -1, N 6 - γ -HMHP-dA (HMHP = 2-hydroxy-3-hydroxymethylpropan-1,3-diyl) strongly inhibits DNA replication but causes only 2% A>T mutation [51].…”

Chemical Analysis of DNA Damage

“…155,156 When coupled with the aforementioned CRAB assay, NGS enables a multiplexed site-specific mutagenesis assay and facilitates the quantification of insertion and deletion mutations readily (Figure 10). 149,157–159 Briefly, individual lesion-containing or lesion-free control shuttle vector, i.e., M13 bacteriophage, is specifically barcoded. These barcoded genomes are all mixed together and transfected into cells with specific repair/replication background.…”

Diepoxybutane-induced apoptosis is mediated through the ERK1/2 pathway

“…106-99-0) is a regulated hazardous air pollutant, and a known human carcinogen and mutagen. 1 –5 Human exposure to BD occurs mainly in occupational settings, although low-level exposures have been reported in nonoccupational settings. 5,6 BD exposure in rodents and humans results in various toxic responses that include multisite carcinogenesis, bone marrow depletion, ovarian, spleen, and thymic atrophy.…”