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Evaluation of the SOS/umu-test post-treatment assay for the detection of genotoxic activities of pure compounds and complex environmental mixtures.

This study presents an evaluation of the SOS/umu-test after introducing an additional dilution and incubation in the post-treatment assay. This treatment reduces the influence of coloured test compounds that otherwise affect the colorimetric determination of the beta-galactosidase activity and the bacterial growth measurement during the testing of complex environmental samples. The post-treatment assay significantly increased the beta-galactosidase activity and consequently the enzyme induction ratios at higher doses of model genotoxins 4-nitroquinoline-N-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminoanthracene, benzo(a)pyrene with low or no effect on the sensitivity of the test itself. On the other hand tests of environmental extracts indicated significant increases in sensitivity after additional incubation. 4-Nitroquinoline-N-oxide treatments of bacteria in the test affected cell division and caused filamentous growth. The size of filamentous bacteria and incidence rate of the length categories was positively correlated with the concentrations of genotoxins. Presence of filamentous tester bacteria proved induction of SOS response and genotoxic activity of environment samples in SOS/umu-test.

4-Nitroquinoline-1-oxide↗

Capillary electrophoresis of proteins for proteomic studies.

Analyses of proteins in complex mixtures such as cell lyzates are presently performed mainly by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. For structural analysis, each protein in a spot is digested with proteases and the fragment peptides are subjected to Edman sequencing and/or mass spectrometry. These works aim at the total analysis of proteins in a complex mixture and reconstruction of their cooperative functions. Genomic studies are now being combined with these proteomic studies. This review article focuses on the application of capillary electrophoresis aiming at the total analysis of complex protein systems or structural analysis of each separated protein. From this viewpoint, articles on capillary zone electrophoresis, capillary isoelectric focusing, and sieving SDS capillary electrophoresis are reviewed. Since these techniques of capillary electrophoresis have been thoroughly reviewed previously, papers published in 1997 and 1998 are mainly covered.

Electrophoresis, Capillary↗

Mammalian social odours: attraction and individual recognition.

Mammalian social systems rely on signals passed between individuals conveying information including sex, reproductive status, individual identity, ownership, competitive ability and health status. Many of these signals take the form of complex mixtures of molecules sensed by chemosensory systems and have important influences on a variety of behaviours that are vital for reproductive success, such as parent-offspring attachment, mate choice and territorial marking. This article aims to review the nature of these chemosensory cues and the neural pathways mediating their physiological and behavioural effects. Despite the complexities of mammalian societies, there are instances where single molecules can act as classical pheromones attracting interest and approach behaviour. Chemosignals with relatively high volatility can be used to signal at a distance and are sensed by the main olfactory system. Most mammals also possess a vomeronasal system, which is specialized to detect relatively non-volatile chemosensory cues following direct contact. Single attractant molecules are sensed by highly specific receptors using a labelled line pathway. These act alongside more complex mixtures of signals that are required to signal individual identity. There are multiple sources of such individuality chemosignals, based on the highly polymorphic genes of the major histocompatibility complex (MHC) or lipocalins such as the mouse major urinary proteins. The individual profile of volatile components that make up an individual odour signature can be sensed by the main olfactory system, as the pattern of activity across an array of broadly tuned receptor types. In addition, the vomeronasal system can respond highly selectively to non-volatile peptide ligands associated with the MHC, acting at the V2r class of vomeronasal receptor. The ability to recognize individuals or their genetic relatedness plays an important role in mammalian social behaviour. Thus robust systems for olfactory learning and recognition of chemosensory individuality have evolved, often associated with major life events, such as mating, parturition or neonatal development. These forms of learning share common features, such as increased noradrenaline evoked by somatosensory stimulation, which results in neural changes at the level of the olfactory bulb. In the main olfactory bulb, these changes are likely to refine the pattern of activity in response to the learned odour, enhancing its discrimination from those of similar odours. In the accessory olfactory bulb, memory formation is hypothesized to involve a selective inhibition, which disrupts the transmission of the learned chemosignal from the mating male. Information from the main olfactory and vomeronasal systems is integrated at the level of the corticomedial amygdala, which forms the most important pathway by which social odours mediate their behavioural and physiological effects. Recent evidence suggests that this region may also play an important role in the learning and recognition of social chemosignals.

Animals↗

Use of bioassay methods to evaluate mutagenicity of ambient air collected near a municipal waste combustor.

An ambient air sampling study was conducted around a municipal waste combustor; a primary goal was to develop procedures and methods to evaluate the emissions of organic mutagens resulting from incomplete combustion of municipal waste. The products of incomplete combustion from incineration include complex mixtures of organics, particularly polycyclic aromatic compounds, which are present after atmospheric dilution and cooling in emissions as semi-volatile or particle bound organic compounds. Combustion emissions are generally recognized as a potential cancer risk since they contain many carcinogenic and mutagenic polycyclic aromatic hydrocarbons. Analyzing such a complex mixture for the presence of even a few selected chemicals is difficult and provides risk information on only a fraction of the chemicals present. Bioassay methods, however, may be directly applied to evaluate the mutagenic and potential carcinogenic activity of the complex organics from combustion emissions. The Salmonella (Ames) assay was used to determine the mutagenicity associated with particles from ambient air collected near a municipal waste combustor. Dose-response data was generated, and mutagenicity concentrations were calculated to demonstrate the utility of bioassay in assessing the potential impact of emissions from municipal waste combustion. This phase of study quantified mutagenicity concentrations in ambient air but did not detect organic mutagens that could be attributed to incinerator emissions.

Air Pollutants, Occupational↗

Multivariate selectivity as a metric for evaluating comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry subjected to chemometric peak deconvolution.

Two-dimensional gas chromatography (GC x GC) coupled to time-of-flight mass spectrometry (TOFMS) [GC x GC-TOFMS)] is a highly selective technique well suited to analyzing complex mixtures. The data generated is information-rich, making it applicable to multivariate quantitative analysis and pattern recognition. One separation on a GC x GC-TOFMS provides retention times on two chromatographic columns and a complete mass spectrum for each component within the mixture. In this report, we demonstrate how GC x GC-TOFMS combined with trilinear chemometric techniques, specifically parallel factor analysis (PARAFAC) initiated by trilinear decomposition (TLD), results in a powerful analytical methodology for multivariate deconvolution. Using PARAFAC, partially resolved components in complex mixtures can be deconvoluted and identified without requiring a standard data set, signal shape assumptions or any fully selective mass signals. A set of four isomers (iso-butyl, sec-butyl, tert-butyl, and n-butyl benzenes) is used to investigate the practical limitations of PARAFAC for the deconvolution of isomers at varying degrees of chromatographic resolution and mass spectral selectivity. In this report, multivariate selectivity was tested as a metric for evaluating GC x GC-TOFMS data that is subjected to PARAFAC peak deconvolution. It was found that deconvolution results were best with multivariate selectivities over 0.18. Furthermore, the application of GC x GC-TOFMS followed by TLD/PARAFAC is demonstrated for a plant metabolite sample. A region of GC x GC-TOFMS data from a complex natural sample of a derivatized metabolic plant extract from Huilmo (Sisyrinchium striatum) was analyzed using TLD/PARAFAC, demonstrating the utility of this analytical technique on a natural sample containing overlapped analytes without selective ions or peak shape assumptions.

Evaluation Studies as Topic↗

Overview of testing methods used in inhalation toxicity: from facts to artifacts.

For smaller rodent species, homogenous in size and growth, small head or nose-only chambers are commonly used up to subchronic exposure durations, whereas larger whole-body exposure chambers are used for long-term exposures or exposure paradigms exceeding the normal working day. The advantages and disadvantages of each different technique have already been identified and published in detail. It is often believed best that whole-body inhalation chambers simulate potential human exposure to environmental chemicals or pesticides and this serves as a justification for preferring this mode of exposure. However, real-life exposure conditions of humans cannot be readily duplicated. A comparable mode of exposure may be employed rather than duplicating both the exposure regimens and atmospheres similar to those present in real-world settings. Especially in inhalation studies with complex mixtures, in which atmosphere generation is difficult to control, non-homogenous exposure atmospheres and artifacts are more likely to occur in larger whole-body chambers than in the smaller nose-only inhalation chambers. Inhalation studies with complex mixtures not only face all the challenges of traditional inhalation toxicity testing, but also they are frequently subject to artifacts not readily detected. Thus, a disproportionation of volatile and particulate constituents might occur in inhalation chambers depend on selected technical features, i.e., whether a dynamic or (quasi)static mode of exposure is chosen. Inappropriate timing of the sampling of biological specimens may lead to the underestimation of effects, especially in whole-body exposed animals.

Administration, Inhalation↗

Examination of the additivity assumption using the spiral and standard Salmonella assays to evaluate binary combinations of mutagens.

Binary combinations of pure chemicals and complex mixtures were evaluated for their ability to produce additive mutagenicity responses in the spiral and standard Salmonella mutagenicity assays. Single chemicals were selected that were representative of the primary chemical class responsible for much of the mutagenic activity of each complex mixture. The following agents were evaluated in the absence of S9: 1-nitropyrene, diesel exhaust extract, and the chlorinated drinking water mutagen 3-chloro-4-dichloromethyl-5-hydroxy-2-[5H]-furanone (MX). In the presence of S9, the following agents were evaluated: 4-aminobiphenyl, benzo[a]pyrene, and an organic extract from the particulate emissions resulting from the combustion of polyethylene in a rotary kiln incinerator. Binary combinations of the agents within each S9 group were tested. The results were analyzed for additivity by determining whether the difference between the expected response of the binary mixture was significantly different from the observed response. In general, the combinations of mutagens produced additive responses at low doses in both assays. However, at high doses, the results generally departed from additivity, especially for combinations of indirect-acting mutagens. A requirement for exogenous metabolic activation imposes conditions, such as different S9 optima for each mutagen, that complicate the application of the additivity assumption to binary combinations of indirect-acting mutagens. Although the two bioassays generally gave similar results for each binary combination of mutagens, the spiral assay permitted a gradient of mutagen and/or S9 concentrations to be evaluated, and it allowed the mutagens to be evaluated in the absence of solvents, eliminating any potential solvent-mutagen interactions. These features of the spiral assay may be advantageous when studying chemical interactions.

Drug Interactions↗

Spin-column isolation of DNA-protein interactions from complex protein mixtures for AFM imaging.

Applications of atomic force microscopy (AFM) to investigate structural-functional interactions between DNA and proteins, at the molecular level, should prove valuable for gaining a better understanding of gene expression. Specific genomic DNA-protein interactions occur within a sea of intracellular proteins. Successful AFM imaging requires isolating the specific DNA-protein complex free of background protein contamination. Using spin-column chromatography, we report the successful isolation and AFM imaging of transcription factor DNA complexes from DNA molecules incubated with crude cell lysates. This method should be applicable for the isolation and imaging of other specific DNA-protein complexes pertinent to functional genomic research.

Chromatography, Gel↗

Comprehensive two-dimensional gas chromatography of volatile and semi-volatile components using a diaphragm valve-based instrument.

A high-temperature configuration for a diaphragm valve-based gas chromatography (GCXGC) instrument is demonstrated. GCxGC is a powerful instrumental tool often used to analyze complex mixtures. Previously, the temperature limitations of valve-based GCxGC instruments were set by the maximum operating temperature of the valve, typically 175 degrees C. Thus, valve-based GCxGC was constrained to the analysis of mainly volatile components; however, many complex mixtures contain semi-volatile components as well. A new configuration is described that extends the working temperature range of diaphragm valve-based GCxGC instruments to significantly higher temperatures, so both volatile and semi-volatile compounds can be readily separated. In the current investigation, separations at temperatures up to 250 degrees C are demonstrated. This new design features both chromatographic columns in the same oven with the valve interfacing the two columns mounted in the side of the oven wall so the valve is both partially inside as well as outside the oven. The diaphragm and the sample ports in the valve are located inside the oven while the temperature-restrictive portion of the valve (containing the O-rings) is outside the oven. Temperature measurements on the surface of the valve indicate that even after a sustained oven temperature of 240 degrees C, the portions of the valve directly involved with the sampling from the first column to the second column track the oven temperature to within 1.2% while the portions of the valve that are temperature-restrictive remain well below the maximum temperature of 175 degrees C. A 26-component mixture of alkanes, ketones, and alcohols whose boiling points range from 65 degrees C (n-hexane) to 270 degrees C (n-pentadecane) is used to test the new design. Peak shapes along the first column axis suggest that sample condensation or carry-over in the valve is not a problem. Chemometric data analysis is performed to demonstrate that the resulting data have a bilinear structure. After over 6 months of use and temperature conditions up to 265 degrees C, no deterioration of the valve or its performance has been observed.

Chromatography, Gas↗

Probing proteins on functionalized silicon surfaces using matrix-assisted laser desorption/ionization mass spectrometry.

Flat H-terminated Si(111) substrates modified with alkyl monolayers terminated with hydrophobic and hydrophilic functional groups were prepared using known surface functionalization methods and characterized by FTIR, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The surfaces were then used for the study of non-specific binding of proteins from complex mixtures (using standard mixture of proteins with average molecular weight approximately 6-66 kDa) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protein adsorption on these surfaces (following on-probe fractionation of the mixture) was found to be dependent on the nature of surface functional groups, and nature and pH of rinsing solutions used. The results obtained in this work demonstrate that simple silicon-based surface modifications can be effective for direct analysis of complex mixtures by MALDI-MS. Preliminary results obtained using similarly functionalized porous silicon substrates proved that such substrates are (due to their increased surface areas) better performing than flat silicon.

Adsorption↗

Deconvolution of a complex target using DNA aptamers.

In vitro selection of single-stranded nucleic acid aptamers from large random sequence libraries is now a straightforward process particularly when screening with a single target molecule. These libraries contain considerable shape diversity as evident by the successful isolation of aptamers that bind with high affinity and specificity to chemically diverse targets. We propose that aptamer libraries contain sufficient shape diversity to allow deconvolution of a complex mixture of targets. Using unfractionated human plasma as our experimental model, we aim to develop methods to obtain aptamers against as many proteins as possible. To begin, it is critical that we understand how aptamer populations change with increasing rounds of in vitro selection when using complex mixtures. Our results show that sequence representation in the selected population changes dramatically with increasing rounds of selection. Certain aptamer families were apparent after only three selection rounds. Two additional cycles saw a decline in the relative abundance of these families and the emergence of yet another family that accounted for more than 60% of sequences in the pool. To overcome this population convergence, an aptamer-based target depletion method was developed, and the library screen was repeated. The previous dominant family effectively disappeared from the selected populations but was replaced by other aptamer families. Insights gained from these initial experiments are now being applied in the creation of second generation plasma protein screens and also to the analysis of other complex biological targets.

Aptamers, Nucleotide↗

Strategies for the enrichment and identification of basic proteins in proteome projects.

Two-dimensional gel electrophoresis (2-DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2-DE technology, even when complex mixtures are separated using commercially available pH 6-11 or pH 7-10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6-11 and novel pH 9-12 immobilized pH gradients and 65 protein spots were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI > 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography (LC) - tandem mass spectrometry post-tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI > 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC 'dimensions' may be a useful complement to 2-DE for 'near-to-total' proteome coverage in the alkaline pH range.

Bacterial Proteins↗

Assessment of the trace organic molecular composition of industrial and municipal wastewater effluents by capillary gas chromatography/real-time high-resolution mass spectrometry: a preliminary report.

In summary, the use of HRGC/HRMS and ECC can provide valuable information on the organic constituents of refinery wastewater samples, even when present in extremely complex mixtures. In many cases, the low-resolution (nominal mass) spectra would not have been interpretable without the availability of the corresponding high-resolution (accurate mass and elemental composition) information, since, even with the use of capillary columns, the components of these complex mixtures were not chromatographically resolved. It is apparent that additional components in these fractions could be identified if additional data processing followed by detailed analysis of the ECC data set is carried out incorporating the retention index information derivable from the series of n-alkanes present, although the incomplete chemical fractionation of the samples is a complicating factor in these particular wastewater extracts.

California↗

Standard reference materials for chemical and biological studies of complex environmental samples.

Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST) are often used in methods development and interlaboratory comparison studies since they are homogeneous and readily available to the scientific community. SRM 1649 (urban dust/organics), SRM 1650 (diesel particulate matter), and SRM 1597 (complex mixture of polycyclic aromatic hydrocarbons from coal tar) are three environmental samples which have been used by the scientific community for these purposes. These SRMs were originally developed to assist laboratories in validating analytical procedures for the determination of polycyclic organic compounds in complex mixtures. In addition, these SRMs have been valuable for the comparison of methodologies for bacterial bioassays and the development of bioassay-directed fractionation and bioassay-directed chemical analysis techniques. Most recently these SRMs were chosen for use as test samples in a collaborative study coordinated by the World Health Organization--International Programme on Chemical Safety. This paper provides a summary of much of the work to date (published and unpublished) on the chemical and biological characterization of these three SRMs. Information regarding the availability of other NIST SRMs that might be useful for these types of studies are provided also.

Air Pollutants↗

Amino acid synthesis from CO-N2 and CO-N2-H2 gas mixtures via complex organic compounds.

Reaction among hydrogen cyanide (HCN), formaldehyde (H2CO) and ammonia (NH3) are generally considered an important reaction in amino acid synthesis by electric discharge. Precursors of glycine and aspartic acid were, however, synthesized by adding water to metastable complex compounds produced by quenching a CO-N2 high-temperature plasma. In order to investigate effects of water remaining in an experimental vacuum chamber, optical emission spectroscopic and mass spectrometric measurements were conducted with CO-N2 and CO-N2-H2 gas mixtures. Although residual hydrogen atoms were detected in the CO-N2 experiment, the amount of them was much less than that in the CO-N2-H2 experiment.

Amino Acids↗

Optimization of an HPLC method for analyses of 32P-postlabeled DNA adducts.

A further development of an HPLC method to analyze 32P-postlabeled DNA adducts is presented. The method is based on on-line detection of 32P radioactivity after separation by reversed-phase chromatography. The method has an advantage in that the postlabeling mixture can be injected directly into the HPLC system without any prior purification, with the background radioactivity on a low level. The analysis includes the whole range of substances from orthophosphate to non-polar DNA adducts, which makes it possible to analyze normal nucleotides and ATP together with DNA adducts. The analytical system has a high reproducibility and separates complex mixtures of DNA adducts. The slightly lower sensitivity compared to the TLC method is compensated for by the possibility of injecting large amounts of DNA into the system without affecting the analytical properties. The system can be applied to different DNA adducts as well as complex mixtures of DNA adducts.

2-Acetylaminofluorene↗

Element-coded affinity tags for peptides and proteins.

Isotope-coded affinity tags (ICAT) represent an important new tool for the analysis of complex mixtures of proteins in living systems [Aebersold, R., and Mann, M. (2003) Nature, 422, 198-207]. We envisage an alternative protein-labeling technique based on tagging with different element-coded metal chelates, which affords affinity chromatography, quantification, and identification of a tagged peptide from a complex mixture. As proof of concept, a synthetic peptide was modified at a cysteine side chain with either a carboxymethyl group or acetamidobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid (AcBD) chelates of terbium or yttrium. A mixture of the three modified peptides in a mole ratio of 100:1.0:0.83 carboxymethyl:AcBD-Tb:AcBD-Y was trypsinized, purified on a new affinity column that binds rare-earth DOTA chelates, and analyzed by LC-MS/MS. Chelate-tagged tryptic peptides eluted cleanly from the affinity column; the tagged peptides chromatographically coeluted during LC-MS analysis, were present in the expected ratio as indicated by MS ion intensity, and were sequence-identified by tandem mass spectrometry. DOTA-rare earth chelates have exceptional properties for use as affinity tags. They are highly polar and water-soluble. Many of the rare earth elements are naturally monoisotopic, providing a variety of simple choices for preparing mass tags. Further, the rare earths are heavy elements, whose mass defects give the masses of tagged peptides exact values not normally shared by molecules that contain only light elements.

Affinity Labels↗

Representation of natural stimuli in the rodent main olfactory bulb.

Natural odorants are complex mixtures of diverse chemical compounds. Monomolecular odorants are represented in the main olfactory bulb by distinct spatial patterns of activated glomeruli. However, it remains unclear how individual compounds contribute to population representations of natural stimuli, which appear to be unexpectedly sparse. We combined gas chromatography and intrinsic signal imaging to visualize glomerular responses to natural stimuli and their fractionated components. While whole stimuli activated up to 20 visible glomeruli, each fractionated component activated only one or few glomeruli, and most glomeruli were activated by only one component. Thus, responses to complex mixtures reflected activation by multiple components, with each contributing only a small part of the overall representation. We conclude that the population response to a complex stimulus is largely the sum of the responses to its individual components, and activation of an individual glomerulus independently signals the presence of a specific component.

Animals↗