title: Dual electrochemical and physiological apoptosis assay detection of in vivo generated nickel chloride induced DNA damage in Caenorhabditis elegans.
abstract: 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 p53 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)3(2+)), 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.
authors.affiliation: Department of Biology, and ‡Department of Chemistry, East Carolina University , Greenville, North Carolina 27858, United States.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Huffnagle
authors.forename: Alyssa
authors.initials: A
authors.lastname: Joyner
authors.forename: Blake
authors.initials: B
authors.lastname: Rumble
authors.forename: Sherif
authors.initials: S
authors.lastname: Hysa
authors.forename: David
authors.initials: D
authors.lastname: Rudel
authors.forename: Eli G
authors.initials: EG
authors.lastname: Hvastkovs
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015374:Biosensing Techniques / Q000379:methods; D017173:Caenorhabditis elegans / Q000166:cytology* / Q000187:drug effects* / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004249:DNA Damage / Q000187:drug effects*; D055664:Electrochemical Techniques / Q000379:methods; D009153:Mutagens / Q000633:toxicity*; D009532:Nickel / Q000633:toxicity*
chemical_list: D009153:Mutagens; C022838:nickel chloride; D009532:Nickel; D004247:DNA