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PubMed1983

Recent developments in mass spectrometry for the analysis of complex mixtures.

Analytical chemists faced with complex problems such as food or drug analysis, chemical dump site analysis, or incorporation of xenobiotics and natural toxins into the food chain, require increasingly sophisticated analytical tools. Recent developments in mass spectrometry may be applied to some of these analytical problems. Negative chemical ionization mass spectrometry and to a lesser extent tandem mass spectrometry have passed the stage of expensive curiosities and are now vital screening tools. Negative chemical ionization is a powerful new method for the analysis of complex environmental samples for trace levels of both oxidizing and alkylating agents. Since these compounds comprise a large number of substances known to cause cancer or other environmental health problems, it seems likely that use of NCIMS will continue to grow. Tandem mass spectrometry has several advantages for the analysis of specific organic compounds in complex mixtures. Target compounds can be isolated and identified almost instantaneously at detection limits comparable and identified almost instantaneously at detection limits comparable to GC-MS with minimum sample preparation. A major deterrent to MS/MS is price ($400,000 or more). Also, the operator must know something about the sample and what to look for. Complete characterization of a sample by MS/MS is impractical. In the past, application of mass spectrometry to the determination of molecular weight and structure of polar (or thermally labile) compounds was severely limited. The limitations are due to inability to vaporize samples or to prevent thermal decomposition. The development of desorption chemical ionization, fast atom bombardment, secondary ionization mass spectrometry, and 252Cf plasma desorption mass spectrometry was in an attempt to rectify this situation. Each of the ionization techniques has advantages and limitations. With continued research into actual ionization/desorption process and continued instrumentation development (perhaps with a lower price tag), many of these techniques will become commonplace.

Mass Spectrometry↗
PubMed1993

Molecular analysis of mutations induced at the hisD3052 allele of Salmonella by single chemicals and complex mixtures.

More single chemicals and complex environmental mixtures have been evaluated for mutagenicity at the hisD3052 allele of Salmonella, primarily in strain TA98, than in any other mutation assay. The development of colony probe hybridization procedures and the application of the polymerase chain reaction and direct DNA sequencing has permitted rapid molecular access to this allele. We discuss these techniques and the resulting mutation spectra that have been induced by a variety of environmental mutagens and complex mixtures. A common GC or CG deletion within a hot-spot region of the sequence dominates most of the spectra. In addition to this two-base deletion, we have recovered about 200 other types of mutations within the 72-base target for reversion of the hisD3052 allele. These include a variety of deletions (as large as 35 bases), duplications (as large as 46 bases), and complex mutations involving base substitutions. The quasipalindromic nature of the target sequence and its potential to form DNA secondary structures and slippage mismatches appear to be an important basis for the mutability of this allele.

Alleles↗
PubMed1996

Assessing the estrogenic and dioxin-like activities of chemicals and complex mixtures using in vitro recombinant receptor-reporter gene assays.

In vitro recombinant receptor-reporter gene assays have been used to assess and rank the potency of chemicals and complex mixtures suspected of possessing estrogen and (or) aryl hydrocarbon receptor (AhR) mediated activity. The environmental estrogen (E2) bioassay consists of a Gal4-human estrogen receptor chimeric construct (Gal4-HEGO) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc) that have been stably integrated into HeLa cells. The assay exhibits 10-fold induction in luciferase reporter gene activity following treatment with 1 nM 17 beta-estradiol and has a detection limit of approximately 5 pg of 17 beta-estradiol/mL. The AhR bioassay uses Hepa 1c1c7 wild-type cells transiently transfected with a dioxin response element regulated luciferase reporter gene. These assays were used to assess the estrogen and dioxin-like activities of naringenin, atrazine, and simazine and complex mixtures such as pulp and paper mill black liquor and urban air particulates. The activities of these chemicals and complex mixtures are confirmed using the pure antiestrogen ICI 164,384 and in in vitro gel retardation assays. Results of this study demonstrate the utility of in vitro recombinant receptor-reporter gene assays in identifying and assessing the estrogenic and dioxin-like activities of chemicals and complex mixtures.

Air Pollutants↗
PubMed2005

Long-term effects of a standardized complex mixture of urban dust particulate on the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons in human cells in culture.

Humans are exposed to complex mixtures of polycyclic aromatic hydrocarbons in the atmosphere. We examined the long term effects of a standard reference material (SRM) 1649a over time on the metabolic activation and DNA adduct formation by two carcinogenic PAHs, benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) in the human mammary carcinoma derived cell line MCF-7. PAH-DNA adduct analysis, cytochrome P450 (CYP) enzyme activity, CYP1A1 and CYP1B1 protein expression were determined in cells treated with SRM 1649a alone or SRM 1649a with either BP or DBP for 24 to 120 h. Averaging over time, significantly higher levels of DNA adducts were observed in cells treated with BP or DBP alone than in co-treatments with SRM 1649a and BP or DBP. Ethoxyresorufin O-deethylase assay indicated a significant increase in activity in cells treated with BP alone and co-treated with SRM1649a in comparison to other treatment groups. Induction of CYP1A1 and CYP1B1 protein expression was observed by immunoblots in cells treated with BP alone or in co-treatments of SRM 1649a and BP or DBP. These data demonstrate the influence of the components of SRM 1649a in inhibiting the activation of BP or DBP by CYP enzymes in the formation of DNA adducts. It also suggests that the carcinogenic activity of PAH within a complex mixture may vary with composition and activation of the components present in the complex mixture.

Aryl Hydrocarbon Hydroxylases↗
PubMed1993

Comparison of DNA adducts from exposure to complex mixtures in various human tissues and experimental systems.

DNA adducts derived from complex mixtures of polycyclic aromatic compounds emitted from tobacco smoke are compared to industrial pollution sources (e.g., coke ovens and aluminum smelters), smoky coal burning, and urban air pollution. Exposures to coke oven emissions and smoky coal, both potent rodent skin tumor initiators and lung carcinogens in humans, result in high levels of DNA adducts compared to tobacco smoke in the in vitro calf thymus DNA model system, in cultured lymphocytes, and in the mouse skin assay. Using tobacco smoke as a model in human studies, we have compared relative DNA adduct levels detected in blood lymphocytes, placental tissue, bronchoalveolar lung lavage cells, sperm, and autopsy tissues of smokers and nonsmokers. Adduct levels in DNA isolated from smokers were highest in human heart and lung tissue with smaller but detectable differences in placental tissue and lung lavage cells. Comparison of the DNA adduct levels resulting from human exposure to different complex mixtures shows that emissions from coke ovens, aluminum smelters, and smoky coal result in higher DNA adduct levels than tobacco smoke exposure. These studies suggest that humans exposed to complex combustion mixtures will have higher DNA adduct levels in target cells (e.g., lung) as compared to nontarget cells (e.g., lymphocytes) and that the adduct levels will be dependent on the genotoxic and DNA adduct-forming potency of the mixture.

Adult↗
PubMed1993

Epidemiological studies of neurotoxic, reproductive, and carcinogenic effects of complex mixtures.

Neurotoxic, reproductive, and carcinogenic effects are potentially important health end points in epidemiological studies of complex mixtures, particularly when such mixtures contain volatile organic compounds or trace metals. Epidemiological studies of neurotoxicity often will require direct clinical, behavioral, and/or physiological testing of study subjects, because these effects are likely to be subtle and not identifiable as clearly defined diseases. Peripheral nervous system toxicity can be assessed by clinical neurologic examinations, by electrophysiological tests of nerve conduction, and by physiological tests of thresholds for neurosensory perception, though these tests require considerable standardization for use outside the clinical setting, and most of the available tests have not been assessed for their utility in detecting effects of neurotoxic exposures. Neurobehavioral effects of exposures to solvents, as examples of complex mixtures, have been studied widely; but batteries of tests are often used, and these have not been well standardized and are generally unfamiliar to most research investigators in this area. Recently standardized neurobehavioral test systems developed by the World Health Organization and by a U.S. group for use in field studies, show promise in detecting neurobehavioral effects at relatively low environmental exposures. Similarly, new and sensitive measures of disturbed reproductive function, such as time-to-conception and biochemical indices of early pregnancy loss, are affected by some low-concentration environmental agents; but those measures have not yet been applied to studies of complex mixtures.(ABSTRACT TRUNCATED AT 250 WORDS)

Air Pollutants↗
PubMed1989

Comparative antimutagenicity of 5 compounds against 5 mutagenic complex mixtures in Salmonella typhimurium strain TA98.

Using the Ames Salmonella/microsome assay, we compared the antimutagenic activities of chlorophyllin, retinol, beta-carotene, vitamin C, and vitamin E against solvent extracts of coal dust, diesel emission particles, airborne particles, fried beef, and tobacco snuff. The results show that chlorophyllin inhibited 69% of the mutagenic activity of tobacco snuff and over 90% of that of the other 4 complex mixtures. Retinol inhibited 29-48% of the mutagenic activity of all 5 complex mixtures. beta-Carotene, vitamin C, and vitamin E inhibited, if any, less than 39% of the activity of the complex mixtures studied. Vitamin C enhanced the mutagenicity of airborne particles. These results indicate that for these dietary and environmental complex mixtures chlorophyllin is a more effective antimutagen than retinol, beta-carotene, vitamin C, and vitamin E.

Air↗
PubMed2002

Gas-phase concentration, purification, and identification of whole proteins from complex mixtures.

Five proteins present in a relatively complex mixture derived from a whole cell lysate fraction of E. coli have been concentrated, purified, and dissociated in the gas phase, using a quadrupole ion trap mass spectrometer. Concentration of intact protein ions was effected using gas-phase ion/ion proton-transfer reactions in conjunction with mass-to-charge dependent ion "parking" to accumulate protein ions initially dispersed over a range of charge states into a single lower charge state. Sequential ion isolation events interspersed with additional ion parking ion/ion reaction periods were used to "charge-state purify" the protein ion of interest. Five of the most abundant protein components present in the mixture were subjected to this concentration/purification procedure and then dissociated by collisional activation of their intact multiply charged precursor ions. Four of the five proteins were subsequently identified by matching the uninterpreted product ion spectra against a partially annotated protein sequence database, coupled with a novel scoring scheme weighted for the relative abundances of the experimentally observed product ions and the frequency of fragmentations occurring at preferential cleavage sites. The identification of these proteins illustrates the potential of this "top-down" protein identification approach to reduce the reliance on condensed-phase chemistries and extensive separations for complex protein mixture analysis.

Amino Acid Sequence↗
PubMed1986

Application of modified Salmonella/microsome prescreen to petroleum-derived complex mixtures and polynuclear aromatic hydrocarbons (PAH)

In some cases, the Salmonella mutagenicity assay may fail to predict the carcinogenic potential of PAH (and of complex mixtures containing PAH) because of nonoptimal in vitro metabolic activation parameters. In this study, 7 petroleum-derived complex mixtures, as well as a number of individual PAH which were representative constituents of such mixtures, were tested in a Salmonella prescreen using quadrant plates with rat or hamster S9 at concentrations approximately 2-8 times those used in the standard assay. Some PAH (perylene, quinoline, benzo[b]chrysene, phenanthrene, anthracene) were optimally activated to mutagens by S9 at 400 microliters/plate. Rat S9 was similar to hamster S9 for most tested PAH, but anthracene and quinoline mutagenicity was enhanced by hamster S9. All 7 complex mixtures were more mutagenic with 200-400 microliters/plate S9; rat was generally slightly more efficient than hamster. Modifying this assay to include a prescreen using a range of S9 concentrations (and perhaps from species other than rat) may improve prediction of the potential carcinogenicity of complex petroleum-derived mixtures.

Animals↗
PubMed1993

Introduction and recommendations: working group on indoor air and other complex mixtures.

Air in indoor and outdoor environments typically contains many gaseous and particulate pollutants that may affect adversely any individual at sufficiently high concentrations and more sensitive individuals at lower concentrations. The public health relevance of addressing the effects of mixtures is becoming increasingly evident as we improve the concept of total personal exposure to pollution and obtain more data from personal monitoring. The papers within this volume represent the deliberations of a working group assembled with the goal of improving the epidemiologic approach to investigating the health effects of indoor air pollution and other complex mixtures. The group, composed of epidemiologists, human and animal toxicologists, and experts on biomarkers, comprehensively reviewed the methodologic issues involved in investigating complex mixtures. Members noted the deficiencies of current epidemiologic methodology for studying complex mixtures and called for broad-based advances in study design, exposure assessment, outcome assessment, and data analysis and interpretation. Understanding the health effects of complex mixtures will require multidisciplinary research using not only epidemiologic studies incorporating the new methods of exposure assessment but animal and clinical toxicology.

Air Pollutants↗
PubMed1988

Genotoxicity of complex mixtures: strategies for the identification and comparative assessment of airborne mutagens and carcinogens from combustion sources.

Two strategies for assessment of the toxicity of complex mixtures are described and illustrated with examples from genotoxicity studies of complex combustion mixtures. The first, a strategy for identifying biologically active compounds or compound classes in complex mixtures, is called bioassay-directed fractionation and characterization. The identification and assessment of mutagens and potential carcinogens in complex mixtures has been significantly advanced by the use of short-term genetic bioassays. Bioassay-directed fractionation coupled with new analytical characterization methods has provided the tools needed to more efficiently identify potential carcinogens in complex combustion emissions and urban air samples. These studies have shown that a significant portion of the mutagenicity in combustion emissions and urban air is found in fractions more polar than polynuclear aromatic hydrocarbons (PAHs). A second strategy, the comparative potency method, provides an approach to evaluating the relative toxicities of a series of mixtures. The comparative mutagenicity and carcinogenicity of a series of combustion emissions has been assessed using dose-response studies in bacteria, mammalian cells, and rodents. Comparative mutagenic and tumorigenic emission rates or emission factors provide a means to directly compare the relative hazard of the sources. This data base has also been used to develop a comparative risk assessment methodology for combustion emissions.

Air Pollutants↗
PubMed1994

Identification of phosphorylated peptides from complex mixtures using negative-ion orifice-potential stepping and capillary liquid chromatography/electrospray ionization mass spectrometry.

A rapid method for identifying and characterizing sites of phosphorylation of peptides and proteins is described. High-performance capillary liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) is used to distinguish non-phosphorylated and phosphorylated peptides originating from mixtures as complex as enzyme digests. The method relies on the ability to produce a fragment ion characteristic and unique to phosphopeptides (m/z 79, PO3) by stepping the orifice potential of the mass spectrometer as a function of mass. At low m/z values, a high orifice potential is applied to induce extensive fragmentation of the peptide, leading to the formation of the m/z 79 phosphate-derived ion. This method is analogous to that described by Carr et al. for the identification of glycopeptides from enzymatic digestion of glycoproteins (S.A. Carr, M.J. Huddleston, M.F. Bean, Protein Science 2, 183 (1993)). The method was first evaluated and validated for a mixture of non-, mono- and di-phosphorylated synthetic peptides. Both mono- and di-phosphorylated peptides were found to generate fragment ions characteristic of PO3 whereas the non-phosphorylated peptide did not. Application of the method was extended to identifying phosphopeptides generated from an endoprotease Lys-C digestion of beta-casein. Both the expected mono- and tetra-phosphorylated Lys-C peptides were observed and identified rapidly in the LC/SEI-MS analysis. The procedure was used additionally to identify the site(s) of phosphorylation of the cytosolic non-receptor tyrosine kinase, pp60(c-src).

Amino Acid Sequence↗
PubMed1996

Mutagenic potential of binary and complex mixtures using different enzyme induction systems.

A series of experiments was conducted to investigate the mutagenic potential of binary and complex mixtures in the presence and absence of inducible liver enzyme systems prepared with several different chemical inducers. Liver homogenate (S9, or 9000 x g supernatant) fractions were obtained from Sprague-Dawley rats induced with either Aroclor 1254 (AR), phenobarbital (PB), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or corn oil (UI). the mutagenic potential of test samples was measured with Salmonella typhimurium strain TA98 using each of the various S9 fractions. Test samples included benzo[a]pyrene (BaP), pentachlorophenol (PCP), a binary mixture of BaP and PCP, two five-component mixtures, a methylene chloride extract of wood preserving waste-amended soil, and a methanol extract of coal gasification waste. At a dose of 25 micrograms/plate, BaP produced 55, 83, 217, and 161 net revertants per plate with UI-, PB-, AR-, and TCDD-induced S9, respectively. The complex mixture extracted from the wood preserving waste-amended soil induced approximately equal responses with all four S9 mixes. At a dose of 250 micrograms/plate, the methanol extract of a coal gasification waste produced 56 net revertants using the uninduced S9; however, when Ar- and TCDD-induced S9 was used, 129 and 67 net revertants were observed, respectively. These data demonstrate the relative importance of the various induced cytochrome P-450 isozymes for the metabolism of mutagenic chemicals and complex mixtures.

Animals↗
PubMed2005

Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide/hydroxide affinity chromatography (MOAC).

A novel method termed metal oxide affinity chromatography (MOAC) of enriching for phosphorylated proteins and peptides based on the affinity of the phosphate group for Al(OH)(3) is presented here. When compared to commercial phosphoprotein-enrichment kits, this method is more selective, more cost effective and easily applicable to method optimization. The use of glutamic and aspartic acid in the loading buffer significantly enhances selectivity. Standard protein mixtures and complex Arabidopsis thaliana leaf protein extracts were tested for efficacy of enrichment. The method can be applied to proteins extracted using either mild or denaturing conditions. The same Al(OH)(3) material is suitable for the enrichment of phosphopeptides out of a tryptic digest of alpha-casein. Peptide phosphorylation was revealed by beta-elimination of phosphate groups. Enrichment and in vivo phosphorylation of A. thaliana leaf proteins were confirmed with Pro-Q diamond stain. Several of the phosphoprotein candidates that were identified by MS are known to be phosphorylated in vivo in other plant species.

Aluminum Hydroxide↗
PubMed2001

Template selection during manipulation of complex mixtures by PCR.

PCR is ubiquitous in molecular biology. It is used to amplify single sequences from large genomes, or populations of sequences from complex mixtures such as cDNA libraries in mammalian cells. These cDNA libraries are often employed in subsequent labor-intensive experiments such as genetic screens, the outcome of which depends on library quality. The use of PCR to amplify diverse sequence populations raises important technical issues. One question is whether or not PCR is capable of maintaining population diversity, specifically with respect to template selection in the first rounds of the amplification process (i.e., the possibility that rare sequences in a complex mixture are lost because of amplification failure at the outset of the PCR). Here, we analyze the properties of PCR in the context of template selection in complex mixtures and show that it is an excellent method for preserving diversity.

Animals↗
PubMed2005

Analysis of complex mixtures--cigarette smoke.

Mainstream cigarette smoke is a complex mixture that is inhaled into the respiratory system. The physical characteristics and chemical composition of mainstream smoke are reviewed and briefly compared with that of sidestream smoke. Special attention is paid to ageing effects and artifact formation during the sampling and testing of cigarette smoke, with specific examples of artifact formation during sampling discussed (nitrogen dioxide, methyl nitrite, etc.). Historically, the generation of cigarette smoke for chemical and biological testing has been based on standard smoke generation procedures that are intended for product comparisons. More recently, emerging global regulations have called for alternative smoke generation methods, with emphasis on results relevant to conditions of product use, e.g., estimates of maximum smoke emissions. Strategies for establishing such alternative smoke generation methods are discussed and the potential effects of alternative smoking conditions on analytical accuracy and precision are addressed. Current regulatory requirements that include Hoffmann analyte analysis (i.e., constituents reported to be associated with the risks of cigarette smoking) are also summarized and the potential effect of alternative smoke generation methods on individual constituent yields considered. Finally, a limited critique of emerging regulation that relates to mainstream cigarette smoke measurements, including a discussion of recent WHO recommendations, is offered.

Complex Mixtures↗
PubMedDate not supplied

[Genotoxicity resulting from a complex mixture of environmental origin using 2 bacterial systems].

The genotoxicity of environmental complex mixtures was tested with Ames mutagenicity assay and with Bacillus subtilis rec assay for estimation of primary DNA damage. Soil samples obtained from an area contaminated with petrochemical wastes were sequentially extracted with ethyl ether and methanol. After evaporation to dryness the extracts were resuspended in dimethylsulfoxide for their use in biological tests. No mutagenic response was detected with the Ames test. Ether extract from parcel 1 showed an unusually high toxicity for Salmonella typhimurium TA100 and induced DNA damage in Bacillus subtilis with a clear relationship dose-genotoxic response. The analysis of the results obtained in both assays and the nature of the samples indicate that the genotoxicity detected in the complex mixture is compatible with the one caused by DNA intercalating compounds.

Bacillus subtilis↗
PubMed1993

Assessing exposures to inhaled complex mixtures.

In the course of daily activities, individuals spend varying amounts of time in different spaces where they are exposed to a complex mixture of gas, vapor, and particulate contaminants. The term complex is used in this paper to refer to binary mixtures as well as truly complex mixtures of three or more constituents. The diversity of the environments where pollution may occur, the number of pollutants that may be present, and the nature of the activity in the environment combine to pose a challenge to investigators of the health effects of air pollutants. This article discusses several methods of measuring or assessing exposure to complex mixture air contaminants that include time-activity assessments, personal monitoring, biomarkers of exposure, and microenvironmental models that can be employed singly or in combination in a protocol for exposure assessment. The use of nested designs, involving more intensive data collection from samples or subjects, is also considered.

Air Pollutants↗