ABSTRACT
Breast cancer 1 gene (BRCA1) DNA mutations impact skeletal muscle functions. Inducible skeletal muscle specific Brca1 homozygote knockout (Brca1KOsmi, KO) mice accumulate mitochondrial DNA (mtDNA) mutations resulting in loss of muscle quality.1 Complementary electrochemical andmass spectrometry analyses were utilized to rapidly assess mtDNA or nuclear DNA (nDNA) extracted directly from mouse skeletal muscles. Oxidative peak currents (Ip) from DNA immobilized layer by layer (LbL) were monitored using square-wave voltammetry (SWV) via Ru(bpy)32+ electrocatalysis. Ip significantly decreased (p < 0.05) for KO mtDNA compared to heterozygous KO (Het) or wild type (WT), indicative of decreases in the guanine content. nDNA Ip significantly increased in KO compared to WT (p < 0.05), suggesting an accumulation of damaged nDNA. Guanine or oxidatively damaged guanine content was monitored via appropriate m/z mass transitions using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Guanine in both KO mtDNA and nDNA was significantly lower, while oxidatively damaged guanine in KO nDNA was significantly elevated versus WT. These data demonstrate a loss of guanine content consistent with mtDNA mutation accumulation. Oxidative damage in KO nDNA suggests that repair processes associated with Brca1 are impacted. Overall, electrochemical and LC-MS/MS analysis can provide chemical-level answers to biological model phenotypic responses as a rapid and cost-effective analysis alternative to established assays.
Subject(s)
Genes, BRCA1 , Tandem Mass Spectrometry , Animals , Chromatography, Liquid , DNA, Mitochondrial/genetics , Mice , Muscle, SkeletalABSTRACT
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.
Subject(s)
Breast Neoplasms , Muscle, Skeletal , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Breast Neoplasms/genetics , Female , Genomic Instability , Humans , Mitochondria , Muscle, Skeletal/metabolismABSTRACT
Fluorinated 5-hydroxytryptophans (Fn-5HOWs) were synthesized in gram scale quantities and incorporated into a ß-hairpin peptide and the protein azurin. The redox-active Fn-5HOWs exhibit unique radical spectroscopic signatures that expand the function of as probes for biological electron transfer.
Subject(s)
5-Hydroxytryptophan/chemistry , 5-Hydroxytryptophan/chemical synthesis , Halogenation , Chemistry Techniques, Synthetic , Electron Transport , Models, Molecular , Molecular ConformationABSTRACT
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. Each variant contains a single redox-active tryptophan, W48 or W108, in a unique and buried protein environment. These tryptophan residues exhibit reversible square-wave voltammograms. A Pourbaix plot, representing the reduction potentials versus pH, is presented for the non-H-bonded W48, which has potentials comparable to those of tryptophan in solution. The reduction potentials of W108 are seen to be increased by more than 100 mV across the same pH range. Molecular dynamics shows that, despite its buried indole ring, the N-H of W108 hydrogen bonds with a water cluster, while W48 is completely excluded from interactions with water or polar groups. These redox properties provide insight into the role of the protein in tuning the reactivity of tryptophan radicals, a requirement for controlled biological PCET.
Subject(s)
Azurin/chemistry , Electrons , Free Radicals/chemistry , Tryptophan/chemistry , Molecular Dynamics Simulation , Oxidation-Reduction , Static ElectricityABSTRACT
A simple electrochemical assay to monitor the dispersion of Pseudomonas aeruginosa PA01 biofilm is described. Pyrolytic graphite (PG) electrodes were modified with P. aeruginosa 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. Upon treatment of bacteria-modified electrodes with a 2-aminoimidazole (2-AI) derivative with known Pseudomonas anti-biofilm properties, the bacteria-related electrochemical reduction peaks decreased in a concentration dependent manner, indicating dispersal of the biofilm on the electrode surface. A similar 2-AI compound with negligible anti-biofilm activity was used as a comparative control and produced muted electrochemical results. Electrochemical responses mirrored previously established bioassay-derived half maximal inhibition concentration (IC50) and half maximal effective concentration (EC50) values.. Biofilm dispersal detection via the electrochemical response was validated by monitoring crystal violet absorbance after its release from electrode confined P. aeruginosa biofilm. Mass spectrometry data showing multiple redox active phenazine compounds are presented to provide insight into the surface reaction complexity. Overall, we present a very simple assay to monitor the anti-biofilm activity of compounds of interest.
ABSTRACT
Chemical toxicity has a serious impact on public health, and toxicity failures of drug candidates drive up drug development costs. Many in vitro 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. Combining bioassays, computational predictions, and accurate chemical pathway elucidation presents our best chance for reliable toxicity prediction. Employing electrochemical and electrochemiluminescent approaches, cell-free microfluidic arrays can measure relative rates of formation of DNA-metabolite adduct formation (a measure of genotoxicity) as well as DNA oxidation levels resulting from enzyme-generated metabolites. Enzymes for several organ types can be studied simultaneously. These arrays can be used to identify the most reactive metabolites, and subsequent mechanistic details can then be investigated with high throughput LC-HPLC using enzyme/DNA-coated magnetic beads.
ABSTRACT
Routine in vitro bioassays and animal toxicity studies of drug and environmental chemical candidates fail to reveal toxicity in â¼30% 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.
Subject(s)
High-Throughput Screening Assays/methods , Toxicity Tests/methods , Animals , Chromatography, High Pressure Liquid , Drug-Related Side Effects and Adverse Reactions , Humans , Molecular Structure , Pharmaceutical Preparations , Tandem Mass SpectrometryABSTRACT
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.
Subject(s)
Apoptosis/drug effects , Caenorhabditis elegans/cytology , Caenorhabditis elegans/drug effects , DNA Damage/drug effects , Mutagens/toxicity , Nickel/toxicity , Animals , Biosensing Techniques/methods , Caenorhabditis elegans/genetics , DNA/genetics , DNA/isolation & purification , Electrochemical Techniques/methodsABSTRACT
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. 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 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) were employed to detect anti-BPDE damage to DNA. DNA was exposed to racemic (±)- or enantiomerically pure (+)-anti-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 anti-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 (+)-anti-BPDE primarily adopts a minor groove bound orientation within the oligomers while selectively targeting the nontranscribed ssDNA sequence within the duplexes.
Subject(s)
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/pharmacology , Cytosine/chemistry , DNA Methylation , Electrochemical Techniques , Epigenesis, Genetic , Genes, p53/genetics , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/chemistry , Cytosine/metabolism , DNA Damage , DNA Methylation/drug effects , DNA Methylation/genetics , Electrodes , Epigenesis, Genetic/genetics , Gold/chemistry , Molecular StructureABSTRACT
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. This molecularly based methodology is designed in a rapid screening array that utilizes electrochemiluminescence (ECL) to detect metabolite-DNA reactions, as well as biocolloid reactors that provide the DNA adducts and metabolites for liquid chromatography-mass spectrometry (LC-MS) analysis. ECL arrays provide rapid toxicity screening, and the biocolloid reactor LC-MS approach provides a valuable follow-up on structure, identification, and formation rates of DNA adducts for toxicity hits from the ECL array screening. Specific examples using this strategy are discussed. Integration of high-throughput versions of these toxicity-screening methods with existing drug toxicity bioassays should allow for better human toxicity prediction as well as more informed decision making regarding new chemical and drug candidates.
Subject(s)
DNA/genetics , Luminescent Measurements/methods , Mass Spectrometry/methods , Mutagenicity Tests/instrumentation , Mutagenicity Tests/methods , Mutagens/toxicity , Animals , Chromatography, Liquid/methods , DNA/analysis , DNA/metabolism , DNA Adducts/analysis , DNA Adducts/genetics , DNA Adducts/metabolism , Enzymes, Immobilized/metabolism , Equipment Design , Humans , Luminescent Measurements/instrumentation , Microarray Analysis/instrumentation , Microarray Analysis/methods , Models, Molecular , Mutagens/metabolismABSTRACT
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 -0.37 V versus Ag/AgCl. The peak current intensity of this redox wave was dependent on both BPDE concentration and exposure time. Controls with alternate xenobiotics and DNA sequences showed this redox wave to be primarily due to BPDE damage at the wild-type DNA sequence. The detection limit was determined to be approximately 170 nM BPDE. Mass spectrometry and UV thermal melting experiments provided insight into the BPDE reaction and mirrored the sensor results. This report demonstrates that an electrochemical hybridization sensor can be used to detect sequence-related xenobiotic DNA damage.
Subject(s)
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/toxicity , Carcinogens/toxicity , Codon/genetics , DNA Damage/genetics , Electrochemistry/methods , Oligodeoxyribonucleotides/genetics , Tumor Suppressor Protein p53/genetics , Base Sequence , Genomics , Kinetics , Mass Spectrometry , Models, Molecular , Nucleic Acid Conformation , Nucleic Acid Denaturation , Oligodeoxyribonucleotides/chemistry , Transition TemperatureABSTRACT
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. We specifically focus on some main areas from where novel work continues to originate: redox active molecules designed for specific interaction with double stranded DNA, DNA mimics to eliminate background electrochemical signals, external nanoparticle or enzyme modifications for sensitivity enhancements, split and self-hybridizing single stranded DNA probe modifications, and novel catalytic oxidation techniques. Additionally, we touch on the use of DNA hybridization sensors to monitor alternative biochemical (non-DNA hybridization) processes.
Subject(s)
Biosensing Techniques/methods , DNA/chemistry , Electrochemistry , Nucleic Acid HybridizationABSTRACT
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. Integration of the CVs demonstrated that C12VC6VC12 exhibited binding cooperativity to the complementary PNA-DNA hybrids and saturated at a ratio of 2:1 (C12VC6VC12:hybrid). Reduced C12VC6VC12 (V+) absorption spectra showed a significant lambda(max) blue shift (22 nm) in the presence of complementary hybrids compared to the lambda(max) in the presence of PNA or mismatched DNA hybrids. Chronocoulometry was employed to assay surface populations and obtain thermodynamics for C12VC6VC12 binding. These data are consistent with C12VC6VC12 bound in the minor groove of complementary hybrids as face-to-face pi-dimers. This approach to distinguishing complementary hybrids from mismatched hybrids is novel, with potential applications involving detection of DNA damage or single nucleotide polymorphism (SNP) analysis.
Subject(s)
DNA/chemistry , Nucleic Acid Hybridization , Oxidation-Reduction , Peptide Nucleic Acids/chemistry , Absorption , Base Pair Mismatch , DNA Damage , Dimerization , Electrochemistry/methods , Electrodes , Models, Chemical , Polymorphism, Single Nucleotide , Pyridinium Compounds/chemistry , Surface Properties , ThermodynamicsABSTRACT
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. Of the enzymes tested, the activity toward NNK bioactivation was cyt P450 1A2 > 2E1 > 1B1 approximately chloroperoxidase (CPO) > myoglobin (Mb) in accordance with reported in vivo studies. Cyt P450/polyion films were also immobilized on 500 nm diameter silica nanospheres for product analysis by LC-MS. Analysis of the nanosphere film reaction media provided ECL array validation and quantitation of the bioactivated NNK hydrolysis product 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) confirming production of reactive metabolites in the films. Chemical screening in this fashion allows rapid clarification of enzymes responsible for genotoxic activation as well as offering insight into cyt P450-related toxicity and mechanisms.
Subject(s)
Cytochrome P-450 Enzyme System/physiology , Electrochemistry/methods , Nitrosamines/toxicity , Butanones/toxicity , Carcinogens , Chloride Peroxidase/chemistry , Chromatography, Liquid/methods , Cytochrome P-450 Enzyme System/metabolism , DNA Adducts , Genetic Techniques , Humans , Hydrolysis , Luminescence , Mass Spectrometry/methods , Models, Chemical , Myoglobin/chemistry , Pyridines/toxicityABSTRACT
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. Then, crystalline sheets of layered alpha-Zr(O(3)POH)(2).H(2)O (alpha-ZrP) or Zr(O(3)PCH(2)CH(2)COOH)(2).xH(2)O (alpha-ZrCEP) were adsorbed to construct alpha-ZrP/Cyt c/Au or alpha-ZrCEP/Cyt c/Au nanoassemblies. The construction of each layer was monitored by SPR, in real time, and the assemblies were further characterized by atomic force microscopy and electrochemical studies. Thermodynamic stability of the protein nanoassembly was assessed by urea-induced unfolding. Surprisingly, unfolding is reversible in all cases studied here. Stability of Cyt c in alpha-ZrP/Cyt c/Au increased by approximately 4.3 kJ/mol when compared to the unfolding free energy of Cyt c/Au assembly. In contrast, the protein stability decreased by approximately 1.5 kJ/mol for alpha-ZrCEP/Cyt c/Au layer. Thus, OH-decorated surfaces stabilized the protein whereas COOH-decorated surfaces destabilized it. These data quantitate the role of specific functional groups of the inorganic layers in controlling bound protein stability.
Subject(s)
Cytochromes c/metabolism , Fungal Proteins/metabolism , Protein Folding , Zirconium/metabolism , Cysteamine/chemistry , Electrochemistry , Gold/metabolism , Microscopy, Atomic Force , Protein Binding , Protein Denaturation , Spectrum Analysis , Surface Plasmon Resonance , ThermodynamicsABSTRACT
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). Cyt P450 enzymes were activated via the NADPH/reductase cycle. When bioactivation of substrates in the films gives reactive metabolites, they are trapped by covalent attachment to DNA bases. The rate of increase in ECL with enzyme reaction time reflects relative DNA damage rates. "Toxic hits" uncovered by the array were studied in structural detail by using enzyme/DNA films on silica nanospheres as "nanoreactors" to provide nucleobase adducts from reactive metabolites. The utility of this synergistic approach was demonstrated by estimating relative DNA damage rates of three mutagenic N-nitroso compounds and styrene. Relative enzyme turnover rates for these compounds using ECL arrays and LC-UV-MS correlated well with TD 50 values for liver tumor formation in rats. Combining ECL array and nanoreactor/LC-MS technologies has the potential for rapid, high-throughput, cost-effective screening for reactive metabolites and provides chemical structure information that is complementary to conventional toxicity bioassays.
Subject(s)
DNA/analysis , DNA/drug effects , Drug Evaluation, Preclinical/methods , Luminescent Measurements/methods , Microsomes, Liver/metabolism , Nanospheres , Toxicity Tests/methods , Animals , Cytochrome P-450 CYP2E1/metabolism , DNA/metabolism , DNA Damage/drug effects , Drug Evaluation, Preclinical/instrumentation , Drug Synergism , Drug-Related Side Effects and Adverse Reactions , Electrochemistry , Humans , Microsomes, Liver/drug effects , Molecular Structure , Rats , Tandem Mass SpectrometryABSTRACT
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. The utility of these biocolloids was demonstrated by oxidation of guaiacol, styrene, and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). Enzyme turnover rates for formation of reactive metabolites were monitored using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC-MS). Capillary LC-MS/MS was employed to determine DNA nucleobase adducts after catalyzing the reactive metabolite formation with DNA-enzyme biocolloids and then using neutral thermal hydrolysis on the biocolloids. Dramatic improvements in surface area to volume ratio over similar films on macroscopic surfaces opens new avenues for genotoxicity screening and enabled the first use of pure cyt P450 enzymes in enzyme-DNA films to produce DNA adducts. The method makes possible identification and formation rate measurements of major and minor DNA adducts as well as the metabolites themselves in <5 min of reaction time using relevant human liver enzymes.
Subject(s)
DNA Adducts/analysis , DNA/chemistry , Liver/enzymology , Camphor 5-Monooxygenase/metabolism , Catalysis , Chromatography, Liquid/methods , Cytochrome P-450 CYP2E1/metabolism , Epoxy Compounds/chemistry , Humans , Mass Spectrometry/methods , Myoglobin/metabolism , Static Electricity , Time FactorsABSTRACT
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. DNA damage in the films from exposure to reactive 2-AF metabolites was measured subsequent to the enzyme reaction using catalytic voltammetric oxidation with Ru(bpy)32+. Square wave voltammetric (SWV) peaks increased with enzyme reaction time, and relative DNA damage rates at pH 5.8 were measured within 2 min. Control incubations of DNA/NAT films without AcCoA gave no significant sensor response. CapLC-MS/MS analysis of 2-AAF/DNA reaction products was consistent with 2-AF-guanine adducts formed in the films. DNA damage occurred more rapidly under weakly acidic conditions (pH 5.5-5.8) than at neutral pH, suggesting that genotoxicity from arylamine metabolism by NAT could be more significant in slightly acidic environments.
Subject(s)
2-Acetylaminofluorene/toxicity , Acetyl Coenzyme A/metabolism , Arylamine N-Acetyltransferase/metabolism , Biosensing Techniques/methods , DNA Damage , DNA/drug effects , Mutagenicity Tests/methods , Acetylation , Catalysis , Chromatography, Liquid , Electrochemistry , Electrodes , Graphite/chemistry , Hydrogen-Ion Concentration , Kinetics , Mass SpectrometryABSTRACT
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. Such films have bright prospects for chiral synthesis and biofuel cells. Excellent bioactivity and retention of enzyme structure in these films facilitates their use in detailed kinetic studies. Biosensors and arrays employing DNA-enzyme films show great promise in predicting genotoxicity of new drug and chemical product candidates. These devices combine metabolic biocatalysis, reactive metabolite-DNA reactions, and DNA damage detection. Catalytic voltammetry or electrochemiluminescence (ECL) can be used for high throughput arrays utilizing multiple LbL "spots" of DNA, enzyme and metallopolymer. DNA-enzyme films can also be used to produce nucleobase adduct toxicity biomarkers for detection by LC-MS. These approaches provide valuable high throughput tools for drug and chemical product development and toxicity prediction.
Subject(s)
DNA/chemistry , DNA/metabolism , Enzymes/chemistry , Enzymes/metabolism , Biochemical Phenomena , Biochemistry , Biosensing Techniques , Catalysis , DNA/genetics , Humans , Ions/chemistryABSTRACT
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). Subsequent to the enzyme reaction, DNA damage in the sensor films was measured simultaneously using a simple apparatus combining a standard voltammetry cell coupled with an optical fiber and photomultiplier tube. The model enzyme reaction converted styrene to styrene oxide, which reacts with DNA nucleobases. ECL and SWV signals increased with enzyme reaction time on the scale of several min, and provided relative enzyme turnover rates for DNA damage suitable for toxicity screening applications. Within 1 min, the sensor detects approximately 3 damaged bases per 10,000 DNA bases using this simultaneous detection.