Nontargeted Analysis of DNA Adducts by Mass-Tag MS: Reaction of <i>p</i>‑Benzoquinone with DNA

Using a method in which DNA adducts are discovered based on their conversion in a nucleotide form to phosphorimidazolides with isotopologue benzoylhistamines (or <i>p</i>-bromobenzoylhistamine) prior to detection by MALDI-TOF-MS, we have profiled the adducts that form when calf thymus DNA is reacted in vitro with <i>p</i>-benzoquinone (BQ). We find, as relative values normalized to 100% of adducts observed, 79% BQ-dCMP, 21% BQ-methyl-dCMP (a new DNA adduct), and trace amounts of BQ-dAMP and BQ-dGMP. Because mC is 5% of C in this DNA, the reaction of BQ with DNA in vitro is about five times faster at methyl-C than C. When equal amounts of dCMP and methyl-dCMP are reacted with BQ, equal amounts of the corresponding adducts are observed. Thus, the microenvironment of methyl-C in DNA enhances its reactivity relative to C with BQ. In a prior, similar study, but based on analysis by ³²P-postlabeling, the second most abundant adduct was assigned to BQ-A, apparently because of comigration of the BQ-A and BQ-methyl-C adducts (as bisphosphates) in the chromatographic step. Because the calf thymus DNA (used as received) was contaminated with RNA, we also detected the ribonucleotide adduct, BQ-CMP

Mono-2-Ethylhexyl Phthalate Induces Oxidative Stress Responses in Human Placental Cells In Vitro

Di-2-ethylhexyl phthalate (DEHP) is an environmental contaminant commonly used as a plasticizer in polyvinyl chloride products. Exposure to DEHP has been linked to adverse pregnancy outcomes in humans including preterm birth, low birth-weight, and pregnancy loss. Although oxidative stress is linked to the pathology of adverse pregnancy outcomes, effects of DEHP metabolites, including the active metabolite, mono-2-ethylhexyl phthalate (MEHP), on oxidative stress responses in placental cells have not been previously evaluated. The objective of the current study is to identify MEHP-stimulated oxidative stress responses in human placental cells. We treated a human placental cell line, HTR-8/SVneo, with MEHP and then measured reactive oxygen species (ROS) generation using the dichlorofluorescein assay, oxidized thymine with mass-spectrometry, redox-sensitive gene expression with qRT-PCR, and apoptosis using a luminescence assay for caspase 3/7 activity. Treatment of HTR-8 cells with 180 μM MEHP increased ROS generation, oxidative DNA damage, and caspase 3/7 activity, and resulted in differential expression of redox-sensitive genes. Notably, 90 and 180 μM MEHP significantly induced mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2), an enzyme important for synthesis of prostaglandins implicated in initiation of labor. The results from the present study are the first to demonstrate that MEHP stimulates oxidative stress responses in placental cells. Furthermore, the MEHP concentrations used were within an order of magnitude of the highest concentrations measured previously in human umbilical cord or maternal serum. The findings from the current study warrant future mechanistic studies of oxidative stress, apoptosis, and prostaglandins as molecular mediators of DEHP/MEHP-associated adverse pregnancy outcomes