Complementary Square-Wave Voltammetry and LC-MS/MS Analysis to Elucidate Induced Damaged and Mutated Mitochondrial and Nuclear DNA from in Vivo Knockdown of the 1 Gene in the Mouse Skeletal Muscle.

Breast cancer 1 gene (BRCA1) DNA mutations impact skeletal muscle functions. Inducible skeletal muscle specific Brca1 homozygote knockout (Brca1KO, KO) mice accumulate mitochondrial DNA (mtDNA) mutations resulting in loss of muscle quality. Complementary electrochemical andmass spectrometry analyses were utilized to rapidly assess mtDNA or nuclear DNA (nDNA) extracted directly from mouse skeletal muscles.

Skeletal Muscle Function Is Dependent Upon BRCA1 to Maintain Genomic Stability.

Breast Cancer gene 1 (BRCA1) is a large, multifunctional protein that regulates a variety of mechanisms in multiple different tissues. Our work established that Brca1 is expressed in skeletal muscle and localizes to the mitochondria and nucleus. Here, we propose BRCA1 expression is critical for the maintenance of force production and mitochondrial respiration in skeletal muscle.

Synthesis of redox-active fluorinated 5-hydroxytryptophans as molecular reporters for biological electron transfer.

Fluorinated 5-hydroxytryptophans (F-5HOWs) were synthesized in gram scale quantities and incorporated into a β-hairpin peptide and the protein azurin. The redox-active F-5HOWs exhibit unique radical spectroscopic signatures that expand the function of as probes for biological electron transfer.

Impact of Local Electrostatics on the Redox Properties of Tryptophan Radicals in Azurin: Implications for Redox-Active Tryptophans in Proton-Coupled Electron Transfer.

Tyrosine and tryptophan play critical roles in facilitating proton-coupled electron transfer (PCET) processes essential to life. The local protein environment is anticipated to modulate the thermodynamics of amino acid radicals to achieve controlled, unidirectional PCET. Herein, square-wave voltammetry was employed to investigate the electrostatic effects on the redox properties of tryptophan in two variants of the protein azurin.

Electrochemical Detection of Small Molecule Induced Biofilm Dispersion.

A simple electrochemical assay to monitor the dispersion of PA01 biofilm is described. Pyrolytic graphite (PG) electrodes were modified with PA01 using layer-by-layer (LbL) methods. The presence of the bacteria on the electrodes was directly monitored using square wave voltammetry (SWV) via the electrochemical reduction of electroactive phenazine compounds expressed by the bacteria, which indicate the presence of biofilm.

Modern Approaches to Chemical Toxicity Screening.

Chemical toxicity has a serious impact on public health, and toxicity failures of drug candidates drive up drug development costs. Many bioassays exist for toxicity screening, and newer versions of these tend to be high throughput or high content assays, some of which rely on electrochemical detection. Toxicity very often results from metabolites of the chemicals we are exposed to, so it is important that assays feature metabolic conversion.

State-of-the-Art Metabolic Toxicity Screening and Pathway Evaluation.

Routine in vitro bioassays and animal toxicity studies of drug and environmental chemical candidates fail to reveal toxicity in ∼30% of cases. This Feature article addresses research on new approaches to in vitro toxicity testing as well as our own efforts to produce high-throughput genotoxicity arrays and LC-MS/MS approaches to reveal possible chemical pathways of toxicity.

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.

Electrochemical study on the effects of epigenetic cytosine methylation on anti-benzo[a]pyrene diol epoxide damage at TP53 oligomers.

Anti-benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (anti-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 anti-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.

Metabolic toxicity screening using electrochemiluminescence arrays coupled with enzyme-DNA biocolloid reactors and liquid chromatography-mass spectrometry.

New chemicals or drugs must be guaranteed safe before they can be marketed. Despite widespread use of bioassay panels for toxicity prediction, products that are toxic to a subset of the population often are not identified until clinical trials. This article reviews new array methodologies based on enzyme/DNA films that form and identify DNA-reactive metabolites that are indicators of potentially genotoxic species.

Electrochemical detection of anti-benzo[a]pyrene diol epoxide DNA damage on TP53 codon 273 oligomers.

DNA damage from (+/-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) at a hotspot TP53 gene sequence was electrochemically detected. BPDE was exposed to gold electrode immobilized double-stranded DNA oligomers followed by voltammetric measurements in the presence of redox-active C(12)H(25)V(2+)C(6)H(12)V(2+)C(12)H(25) (V(2+) = 4,4'-bipyridyl or viologen, C12-viologen). Square wave voltammograms from BPDE-exposed DNA-modified electrodes showed the emergence of a C12-viologen-DNA complex at -0.

Recent advances in electrochemical DNA hybridization sensors.

Even with the advent of industry produced electrochemical DNA analysis chips, electrochemical DNA hybridization detection continues to be an intensive research focus area. The advantages of electrochemical detection continue to inspire efforts to improve selectivity and sensitivity. Here, we summarize the landscape of recent efforts in electrochemical DNA hybridization detection.

Characterization of mismatched DNA hybridization via a redox-active diviologen bound in the PNA-DNA minor groove.

Diviologen molecules of the general formula CH3(CH2)11V2+(CH2)6V2+(CH2)11CH3 (C12VC6VCI2, V2+ = 4,4'-bipyridinium or viologen) were employed to electrochemically assay DNA hybridization to PNA probes immobilized at Au electrodes. Immobilized 15-mer PNA probes were exposed to 25-mer DNA oligonucleotides containing either complementary or single base mismatched sequences. In the presence of complementary PNA-DNA hybrids, the V2+/+ redox couple of C12VC6VC12 exhibited a unique double-wave cyclic voltammogram, with a formal potential shifted -100 mV from the E(f) in the presence of single base mismatched DNA hybrids or PNA probes alone.

Human cyt P450 mediated metabolic toxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) evaluated using electrochemiluminescent arrays.

Electrochemiluminescent (ECL) arrays containing polymer ([Ru(bpy)(2)(PVP)(10)](2+), PVP = polyvinylpyridine), DNA, and selected enzymes were employed to elucidate cytochrome (cyt) P450 dependent metabolism of the tobacco specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Bioactivated NNK metabolites formed upon H(2)O(2)-enzymatic activation were captured as DNA adducts and detected simultaneously from 36 spot arrays by capturing and quantifying emitted ECL with an overhead CCD camera. Increased ECL emission was dependent on NNK exposure time.

Folding control and unfolding free energy of yeast iso-1-cytochrome c bound to layered zirconium phosphate materials monitored by surface plasmon resonance.

The free energy change (Delta G degrees ) for the unfolding of immobilized yeast iso-1-cytochrome c (Cyt c) at nanoassemblies was measured by surface plasmon resonance (SPR) spectroscopy. Data show that SPR is sensitive to protein conformational changes, and protein solid interface exerts a major influence on bound protein stability. First, Cyt c was self-assembled on the Au film via the single thiol of Cys-102.

Synergistic metabolic toxicity screening using microsome/DNA electrochemiluminescent arrays and nanoreactors.

Platforms based on thin enzyme/DNA films were used in two-tier screening of chemicals for reactive metabolites capable of producing toxicity. Microsomes were used for the first time as sources of cytochrome (cyt) P450 enzymes in these devices. Initial rapid screening involved electrochemiluminescent (ECL) arrays featuring spots containing ruthenium poly(vinylpyridine), DNA, and rat liver microsomes or bicistronically expressed human cyt P450 2E1 (h2E1).

Enzyme-DNA biocolloids for DNA adduct and reactive metabolite detection by chromatography-mass spectrometry.

Silica microbead bioreactors (0.5 microm diameter) coated with DNA and enzymes were fabricated to measure reactive metabolite and DNA-adduct formation rates relevant to genotoxicity screening. Cytochrome (cyt) P450 2E1, cyt P450(cam), and myoglobin (Mb) were incorporated into thin films with DNA using the electrostatic layer-by-layer (LbL) method.

Electrochemical genotoxicity screening for arylamines bioactivated by N-acetyltransferase.

Genotoxicity screening sensors that measure DNA damage from metabolism of arylamines were developed and evaluated. The sensors feature ultrathin films containing DNA and N-acetyltransferase (NAT) on pyrolytic graphite (PG) electrodes. NAT in the film catalyzed the conversion of the arylamine 2-aminofluorene (2-AF) to 2-acetylaminofluorene (2-AAF) by acetyl coenzyme A (AcCoA) dependent N-acetylation, as verified by liquid chromatography.

Biochemical applications of ultrathin films of enzymes, polyions and DNA.

This feature article summarizes recent applications of ultrathin films of enzymes and DNA assembled layer-by-layer (LbL). Using examples mainly from our own research, we focus on systems developed for biocatalysis and biosensors for toxicity screening. Enzyme-poly(L-lysine) (PLL) films, especially when stabilized by crosslinking, can be used for biocatalysis at unprecedented high temperatures or in acidic or basic solutions on electrodes or sub-micron sized beads.

Electrochemiluminescent/voltammetric toxicity screening sensor using enzyme-generated DNA damage.

Simultaneous optical and voltammetric detection of bioactivated genotoxicity is reported for the first time employing ultrathin films of DNA, model metabolic enzymes, and electrochemiluminescence (ECL) generating metallopolymer [Ru(bpy)2PVP10]2+ on pyrolytic graphite (PG) electrodes. Cytochrome P450cam and myoglobin were used as model monoxygenase enzymes to mimic in vivo processes. Sensor film growth and component amounts were monitored using a quartz crystal microbalance (QCM).

Electrochemical detection of DNA hybridization via bis-intercalation of a naphthylimide-functionalized viologen dimer.

The synthesis and DNA binding properties of a bis-naphthyl imide tetracationic diviologen compound NI(CH2)3V(2+)(CH2)6V(2+)(CH2)3NI (where V(2+) = 4,4'-bipyridinium and NI = naphthyl imide, NIV) are described. Binding to thiolated ssDNA and dsDNA immobilized at Au electrodes was characterized using the electrochemical response for reduction of the V(2+) state to the V+ (viologen radical cation) state. Isotherms and binding constants for this molecule to both forms of immobilized DNA were generated in this fashion.

Genotoxicity screening for N-nitroso compounds. Electrochemical and electrochemiluminescent detection of human enzyme-generated DNA damage from N-nitrosopyrrolidine.

We report for the first time voltammetric/electrochemiluminescent sensors applied to predict genotoxicity of N-nitroso compounds bioactivated by human cytochrome P450 enzymes.

Toxicity screening using biosensors that measure DNA damage.

Toxicity continues to be a major cause of drug development failures. An assessment of drug toxicity as early in the discovery/development cycle as possible is important to minimize the economic impact of discontinuing a drug late in development. Currently, batteries of biological testing protocols provide good assessment and predictions of toxicity in the general population; however, new cost-effective procedures based on simpler biochemical systems that are arranged in biosensor formats are emerging that may be very useful for early toxicity screening.

Electrochemiluminescent arrays for cytochrome P450-activated genotoxicity screening. DNA damage from benzo[a]pyrene metabolites.

Arrays suitable for genotoxicity screening are reported that generate metabolites from cytochrome P450 enzymes (CYPs) in thin-film spots. Array spots containing DNA, various human cyt P450s, and electrochemiluminescence (ECL) generating metallopolymer [Ru(bpy)2PVP10]2+ were exposed to H2O2 to activate the enzymes. ECL from all spots was visualized simultaneously using a CCD camera.

Minor groove binding of a novel tetracationic diviologen.

Novel tetracationic diviologen compounds of the general formula CH3(CH2)nV2+(CH2)6V2+(CH2)nCH3 (where V2+ = 4,4'-bipyridinium and n = 5 or 11) were investigated as electrochemical reporters of DNA duplex formation. These compounds bind to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) when the DNA is either present in solution or immobilized at electrode surfaces. Binding to thiolated ssDNA and dsDNA immobilized at Au electrodes was characterized using the electrochemical response for the reduction of the V2+ state to the V+ (viologen radical cation) state.