Development of a novel site-specific mutagenesis assay using MALDI-ToF MS (SSMA-MS).

We have developed and validated a novel site-specific mutagenesis assay, termed SSMA-MS, which incorporates MALDI-ToF mass spectrometry (MALDI-MS) analysis as a means of determining the mutations induced by a single DNA adduct. The assay involves ligating an adducted deoxyoligonucleotide into supF containing pSP189 plasmid. The plasmid is transfected into human Ad293 kidney cells allowing replication and therefore repair or a mutagenic event to occur. Escherichia coli indicator bacteria are transformed with recovered plasmid and plasmids containing the insert are identified colormetrically, as they behave as frameshift mutations. The plasmid is then amplified and digested using a restriction cocktail of Mbo11 and Mnl1 to yield 12 bp deoxyoligonucleotides, which are characterized by MALDI-MS. MALDI-MS takes advantage of the difference in molecular weight between bases to identify any induced mutations. This analysis method therefore provides qualitative and quantitative information regarding the type and frequency of mutations induced. This assay was developed and validated using an O(6)-methyl-2'-deoxyguanosine adduct, which induced the expected GC-->AT substitutions, when replicated in human or bacterial cells. This approach can be applied to the study of any DNA adduct in any biologically relevant gene sequence (e.g. p53) in human cells and would be particularly amenable to high-throughput analysis.

Pharmacokinetics and tissue disposition of indole-3-carbinol and its acid condensation products after oral administration to mice.

Indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) are promising cancer chemopreventive agents in rodent models, but there is a paucity of data on their pharmacokinetics and tissue disposition. The disposition of I3C and its acid condensation products, DIM, [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LTr(1)), indolo[3,2b]carbazole (ICZ) and 1-(3-hydroxymethyl)-indolyl-3-indolylmethane (HI-IM) was studied, after oral administration of I3C (250 mg/kg) to female CD-1 mice. Blood, liver, kidney, lung, heart, and brain were collected between 0.25 and 24 h after administration and the plasma and tissue concentrations of I3C and its derivatives determined by high-performance liquid chromotography. I3C was rapidly absorbed, distributed, and eliminated from plasma and tissues, falling below the limit of detection by 1 h. Highest concentrations of I3C were detected in the liver where levels were approximately 6-fold higher than those in the plasma. Levels of DIM, LTr(1), and HI-IM were much lower, although they persisted in plasma and tissues for considerably longer. DIM and HI-IM were still present in the liver 24 h after I3C administration. Tissue levels of DIM and LTr(1) were found to be in equilibrium with plasma at almost every time point measured. In addition to acid condensation products of I3C, a major oxidative metabolite (indole-3-carboxylic acid) and a minor oxidative metabolite (indole-3-carboxaldehyde) were detected in plasma of mice after oral administration of I3C. ICZ was also tentatively identified in the liver of these mice. This study shows for the first time that, after oral administration to mice, I3C, in addition to its acid condensation products, is absorbed from the gut and distributed systemically into a number of well-perfused tissues, thus allowing the possibility for some pharmacological activity of the parent compound in vivo.

Liquid chromatographic assay for the simultaneous determination of indole-3-carbinol and its acid condensation products in plasma.

A high-performance liquid chromatographic method was developed for the simultaneous determination of indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM), [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LTr(1)), and indolo[3,2b]carbazole (ICZ). Compounds were extracted from mouse plasma using tert.-butyl methyl ether, incorporating 4-methoxy-indole as internal standard. Chromatographic separation utilized a Waters Symmetry RP18 in tandem with a Thermoquest BDS C(18) column, an acetonitrile-water gradient and UV (280 nm) in series with fluorescence (ex. 335 nm; em. 415 nm) detection. Calibration curves were linear (r(2)>0.99) between 50 and 15,000 ng/ml for I3C; 150 and 15,000 ng/ml for LTr(1); and 0.15 and 37.5 ng/ml for ICZ and the method was reproducible and precise (within-day and between-day coefficients of variation below 9.7 and 13%, respectively). The method described is suitable for comprehensive pharmacokinetic studies with indole-3-carbinol.

Simultaneous detection of five different 2-hydroxyethyl-DNA adducts formed by ethylene oxide exposure, using a high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry assay.

A method has been developed for the simultaneous detection and quantitation of five different 2-hydroxyethyl-DNA (HE-DNA) adducts that could be formed as a result of exposure to ethylene oxide (EO). In addition to the major N7-HE-guanine (N7-HEG) adducts this assay can also measure the less prevalent but potentially more biologically significant N1-HE-2'-deoxyadenosine (N1-HEdA), O(6)-HE-2'-deoxyguanosine (O(6)-HEdG), N(6)-HE-2'-deoxyadenosine (N(6)-HEdA) and N3-HE-2'-deoxyuridine adducts (N3-HEdU). The method involves the isolation of HE adducts from the unmodified nucleosides by either neutral thermal hydrolysis or enzymatic digestion, followed by high-performance liquid chromatographic (HPLC) purification, before detection and quantification by liquid chromatography tandem mass spectrometry (LC/MS/MS) using selective reaction monitoring (SRM). The limits of detection were in the range 0.5-25 fmol for each individual adduct, making this one of the most sensitive assays available for the detection of N7-HEG. To illustrate the possible applications of the assay, it has been employed in the measurement of endogenous/background and EO-induced HE adducts in a variety of DNA samples.

Improving the diagnostic accuracy of N-terminal B-type natriuretic peptide in human systolic heart failure by plasma profiling using mass spectrometry.

We have combined the measurement of N-terminal pro-B type natriuretic peptide (NTproBNP) with plasma peptide profiling to evaluate the effect on sensitivity and specificity of systolic heart failure (SHF) diagnosis. Plasma NTproBNP levels were measured from 100 SHF patients and 100 age/gender matched controls and plasma protein profiles obtained using MALDI-MS. Sixty-seven m/z peaks were significantly different between SHF and normals, and following logistic regression analysis with NTproBNP values, 6 peaks retained independent predictive value. Receiver operating characteristic (ROC) curves for SHF diagnosis had areas of 0.91 for NTproBNP, improving to 0.99 with the model. In a separate validation test set (32 SHF, 20 normals), the model remained highly accurate (ROC area 0.995). An artificial neural network with these 6 peak intensities and NTproBNP produced ROC areas of 0.99 in both training and test sets. The sensitivity and specificity of SHF diagnosis using NTproBNP in training (85, 85%) and test (93, 75%) sets was improved in the model for both training (96, 96%) and test (100, 95%) sets. The accuracy of SHF diagnosis using NTproBNP is improved by the use of a plasma profile of 6 peptide peaks, reducing the uncertainty in the diagnostic gray zone of using NTproBNP alone.

Determination of endogenous and exogenously derived N7-(2-hydroxyethyl)guanine adducts in ethylene oxide-treated rats.

Ethylene oxide (EO) is one of the most widely used intermediates in the chemical industry. It is also formed endogenously as a result of cytochrome P450-mediated metabolism of ethylene, which is ubiquitous in the environment. Additionally, ethylene is generated in vivo during normal physiological processes such as methionine oxidation and lipid peroxidation; therefore, humans are continually exposed to EO. EO is classed by the IARC as carcinogenic to humans and reacts with DNA, primarily forming N7-(2-hydroxyethyl)guanine adducts (N7-HEG), which can be used as biomarkers of exposure and potential cancer risk. To assess the risks to humans associated with occupational exposure to low EO concentrations, it is necessary to establish the relative contribution of DNA damage arising from endogenous and exogenously derived EO. Using a newly developed highly sensitive LC-MS/MS assay with selected reaction monitoring that offers a limit of detection of 0.1 fmol of N7-HEG on column, we have established background levels of N7-HEG (1.1-3.5 adducts/10(8) nucleotides) in tissues of rats. Following intraperitoneal administration of a single dose or three daily doses of EO (0.01-1.0 mg/kg), N7-HEG adducts generally increased with dose, except at the lowest concentration where total N7-HEG levels were no different to that detected in control animals, indicating that any increase was negligible as compared to the endogenous damage already present. In the 3 day study, the kinetics of adduct removal were also investigated and in comparing N7-HEG formation in the two studies, DNA damage did not appear to accumulate with repeated administration.

Searching for biomarkers of heart failure in the mass spectra of blood plasma.

We have developed a technique for analysing blood plasma using MALDI-MS with subsequent data analysis to identify significant and specific differences between heart failure (HF) patients and healthy individuals. A training dataset comprising 100 HF patients and 100 healthy individuals was used to search for biomarkers (m/z range 1000-10,000). EWP cartridges when used in tandem with microcon centrifugal filters were found to give the best results. A data management chain including event binning, background subtraction and feature extraction was developed to reduce the data, and statistical analysis was used to map feature intensities on to a common scale. Various mathematical approaches including a simple cumulative score, support vector machines (SVM) and genetic algorithms (GAs) were then used to combine the results from individual features and provide a robust classification algorithm. The SVM gave the most promising results (accuracy 95%, receiver operating characteristic (ROC) score of 0.997 using 18 selected features). Finally, a test dataset comprising a further 32 HF patients and 20 controls was used to verify that the 18 putative biomarkers and classification algorithms gave reliable predictions (accuracy 88.5%, ROC score 0.998).

A novel 14C-postlabeling assay using accelerator mass spectrometry for the detection of O6-methyldeoxy-guanosine adducts.

Accelerator mass spectrometry (AMS) is currently one of the most sensitive methods available for the trace detection of DNA adducts and is particularly valuable for measuring adducts in humans or animal models. However, the standard approach requires administration of a radiolabeled compound. As an alternative, we have developed a preliminary 14C-postlabeling assay for detection of the highly mutagenic O6-methyldeoxyguanosine (O6-MedG), by AMS. Procedures were developed for derivatising O6-MedG using unlabeled acetic anhydride. Using conventional liquid chromatography/mass spectrometry (LC/MS) analysis, the limit of detection (LOD) for the major product, triacetylated O6-MedG, was 10 fmol. On reaction of O6-MedG with 14C-acetic anhydride, using a specially designed enclosed system, the predominant product was 14C-di-acetyl O6-MedG. This change in reaction profile was due to a modification of the reaction procedure, introduced as a necessary safety precaution. The LOD for 14C-di-acetyl O6-MedG by AMS was determined as 79 amol, approximately 18,000-fold lower than that achievable by liquid scintillation counting (LSC). Although the assay has so far only been carried out with labeled standards, the degree of sensitivity obtained illustrates the potential of this assay for measuring O6-MedG levels in humans.

The human apoptosis-inducing protein AMID is an oxidoreductase with a modified flavin cofactor and DNA binding activity.

AMID (apoptosis-inducing factor-homologous mitochondrion-associated inducer of death; also known as PRG3 (p53-responsive gene 3)) is a human caspase-independent pro-apoptotic protein with some similarity to apoptosis-inducing factor. AMID was purified from a recombinant bacterial host, enabling biochemical analysis of the protein. AMID is a flavoprotein; possesses NAD(P)H oxidase activity; and catalyzes NAD(P)H-dependent reduction of cytochrome c and other electron acceptors, including molecular oxygen. NADPH binds approximately 10-fold tighter than NADH. AMID binds 6-hydroxy-FAD (a cofactor that accumulates only adventitiously and at low abundance in other flavoprotein enzymes) to form a stoichiometric cofactor.protein complex. AMID has a distinctive electronic spectrum due to the modified flavin. NAD(P)+ binding perturbed the spectrum, enabling determination of K(d) values for these coenzymes. 6-Hydroxy-FAD could be removed from AMID and the apoprotein reconstituted with FAD. FAD was converted to 6-hydroxy-FAD in reconstituted AMID during aerobic turnover with NADPH. AMID is a DNA-binding protein that lacks apparent DNA sequence specificity. Formation of the protein.DNA complex (i) effected a major protein conformational change and (ii) was prevented in the presence of nicotinamide coenzyme. Apo-AMID retains DNA binding activity. Our studies establish a link between coenzyme and DNA binding that likely impacts on the physiological role of AMID in cellular apoptosis.

An improved liquid chromatography/tandem mass spectrometry method for the determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA samples using immunoaffinity column purification.

The analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) represents an important biomarker of oxidative stress. A sensitive method for the detection of 8-oxodG in DNA samples has been developed that utilizes immunoaffinity column purification of 8-oxodG followed by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) multiple reaction monitoring (MRM) mode analysis. An internal standard of stable-isotopically labelled 8-oxodG containing [(15)N(5)] was added prior to the enzymatic digestion of DNA to deoxynucleosides, which was then subjected to immunoaffinity column purification followed by microbore positive ion LC/MS/MS MRM. The 8-oxo-7,8-dihydroguanine (8-oxoG) base product ion at m/z 168 was monitored following cleavage of the glycosidic bond of the 8-oxodG [M+H](+) ion at m/z 284. Similar determinations were made for [(15)N(5)]8-oxodG by monitoring the [(15)N(5)]8-oxoG base product ion at m/z 173 formed from the [M+H](+) ion at m/z 289. The introduction of the immunoaffinity column purification step into the method represents a significant improvement for the accurate determination of 8-oxodG since all artefactual peaks that are observed following the direct injection of digested DNA onto the LC/MS/MS system are removed. The identity of these artefactual peaks has been confirmed to be 2'-deoxyguanosine (dG), thymidine (dT) and 2'-deoxyadenosine (dA). The presence of these artefactual peaks in MRM mode analysis can be explained as a consequence of a concentration effect due to their considerably higher relative abundance in DNA compared to 8-oxodG. The highest signal intensity was observed for the artefactual peak for dA due to the fact that the adenine base formed an adduct with methanol, which is a constituent of the mobile phase. The resulting [M+H](+) ion at m/z 284 (dA m/z 252 + CH(3)OH m/z 32) gave rise to a product ion at m/z 168 following the loss of deoxyribose in MRM mode analysis. Control calf thymus DNA was digested to deoxynucleosides and unmodfied deoxynucleosides were removed by immunoaffinity column purification; the enriched 8-oxodG was determined by LC/MS/MS MRM. The level of 8-oxodG in control calf thymus DNA was determined to be 28.8 +/- 1.2 8-oxodG per 10(6) unmodified nucleotides (n = 5) using 5 microg of digested DNA. The limit of detection of the microbore LC/MS/MS MRM for 8-oxodG was determined to be 25 fmol on-column with a signal-to-noise ratio of 3.5.

Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones.

Quorum sensing relies upon the interaction of a diffusible signal molecule with a transcriptional activator protein to couple gene expression with cell population density. In Gram-negative bacteria, such signal molecules are usually N-acylhomoserine lactones (AHLs) which differ in the structure of their N-acyl side chains. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein. Previously the authors described a violacein-negative, mini-Tn5 mutant of C. violaceum (CV026) in which pigment production can be restored by incubation with supernatants from the wild-type strain. To develop this mutant as a general biosensor for AHLs, the natural C. violaceum AHL molecule was first chemically characterized. By using solvent extraction, HPLC and mass spectrometry, a single AHL, N-hexanoyl-L-homoserine lactone (HHL), was identified in wild-type C. violaceum culture supernatants which was absent from CV026. Since the production of violacein constitutes a simple assay for the detection of AHLs, we explored the ability of CV026 to respond to a series of synthetic AHL and N-acylhomocysteine thiolactone (AHT) analogues. In CV026, violacein is inducible by all the AHL and AHT compounds evaluated with N-acyl side chains from C4 to C8 in length, with varying degrees of sensitivity. Although AHL compounds with N-acyl side chains from C10 to C14 are unable to induce violacein production, if an activating AHL (e.g. HHL) is incorporated into the agar, these long-chain AHLs can be detected by their ability to inhibit violacein production. The versatility of CV026 in facilitating detection of AHL mixtures extracted from culture supernatants and separated by thin-layer chromatography is also demonstrated. These simple bioassays employing CV026 thus greatly extend the ability to detect a wide spectrum of AHL signal molecules.