Electrochemical Study on the Effects of Epigenetic Cytosine Methylation on <i>Anti</i>-Benzo[<i>a</i>]pyrene Diol Epoxide Damage at TP53 Oligomers

<i>Anti</i>-benzo­[<i>a</i>]­pyrene-<i>r</i>-7,<i>t</i>-8-dihydrodiol-<i>t</i>-9,10-epoxide (<i>anti</i>-BPDE) is a known carcinogen that damages DNA, and this damage is influenced by the DNA sequence and epigenetic factors. The influence of epigenetic cytosine methylation on the reaction with <i>anti</i>-BPDE at a known hotspot DNA damage site was studied electrochemically. Gold electrodes were modified with thiolated DNA oligomers spanning codons 270–276 of the TP53 gene. The oligomers exhibited 5-carbon cytosine methylation at the codon 273 location on the bound probe, the acquired complementary target, or both. Redox active diviologen compounds of the form C₁₂H₂₅V²⁺C₆H₁₂V²⁺C₁₂H₂₅ (V²⁺ = 4,4′-bipyridyl or viologen, C12-Viologen) were employed to detect <i>anti</i>-BPDE damage to DNA. DNA was exposed to racemic (±)- or enantiomerically pure (+)-<i>anti</i>-BPDE solutions followed by electrochemical interrogation in the presence of C12-Viologen. Background subtracted square wave voltammograms (SWV) showed the appearance of two peaks at approximately −0.38 V and −0.55 V vs Ag/AgCl upon <i>anti</i>-BPDE exposure. The acquired voltammetry is consistent with singly reduced C12-Viologen dimers bound at two different DNA environments, which arise from BPDE damage and are influenced by cytosine methylation and BPDE stereochemical considerations. UV spectroscopic and mass spectrometric methods employed to validate the electrochemical responses showed that (+)-<i>anti</i>-BPDE primarily adopts a minor groove bound orientation within the oligomers while selectively targeting the nontranscribed ssDNA sequence within the duplexes

Dual Electrochemical and Physiological Apoptosis Assay Detection of in Vivo Generated Nickel Chloride Induced DNA Damage in Caenorhabditis elegans

Environmental nickel exposure is known to cause allergic reactions, respiratory illness, and may be responsible for some forms of cancer in humans. Nematodes are an excellent model organism to test for environmental toxins, as they are prevalent in many different environments. Nickel exposure has previously been shown to impact nematode life processes. In this study, Caenorhabditis elegans nematodes exposed to NiCl₂ featured high levels of programmed cell death (PCD) in a concentration-dependent manner as measured by counting apoptotic corpses in the nematode germ line. A green fluorescent protein (GFP) reporter transgene was used that highlights cell corpse engulfment by fluorescence microscopy. Analysis of the reporter in a <i>p53</i> mutant strain putatively indicates that the PCDs are a result of genomic DNA damage. In order to assay the potential genotoxic actions of NiCl₂, DNA was extracted from nematodes exposed to increasing concentrations of NiCl₂ and electrochemically assayed. In vivo damaged DNA was immobilized on pyrolytic graphite electrodes using the layer-by-layer (LbL) technique. Square-wave voltammograms were obtained in the presence of redox mediator, ruthenium trisbipyridine (Ru­(bpy)₃²⁺), that catalytically oxidizes guanines in DNA. Oxidative peak currents were shown to increase as a function of NiCl₂ exposure, which further suggests that the extracted DNA from nematodes exposed to the nickel was damaged. This report demonstrates that our electrochemical biosensor can detect damage at lower Ni concentrations than our physiological PCD assay and that the results are predictive of physiological responses at higher concentrations. Thus, a biological model for toxicity and animal disease can be assayed using an electrochemical approach