Biomedical subjects

Patrick G Hatcher

Publications and source records attributed to Patrick G Hatcher.

At least 19 recordsLinked to original sources

PubMed2006

Mechanism of arylating quinone toxicity involving Michael adduct formation and induction of endoplasmic reticulum stress.

Quinones permeate our biotic environment, contributing to both homeostasis and cytotoxicity. All quinones generate reactive oxygen species through redox cycling, while partially substituted quinones also undergo arylation (Michael adduct formation) yielding covalent bonds with nucleophiles such as cysteinyl thiols. In contrast to reactive oxygen species, the role of arylation in quinone cytotoxicity is not well understood. We found that the arylating quinones, including unsubstituted 1,4-benzoquinone (1,4-BzQ) and partially substituted vitamin E congener gamma-tocopherol quinone (gamma-TQ), were cytotoxic, with gamma-TQ > 1,4-BzQ, whereas the fully substituted nonarylating vitamin E congener alpha-tocopherol quinone was not. In vitro, both arylating quinones formed Michael adducts with the thiol nucleophile N-acetylcysteine (NAC) at rates where 1,4-BzQ > gamma-TQ. In cultured cells, concurrent addition of NAC eliminated 1,4-BzQ caused toxicity, but preincubation was required for the same NAC detoxification effect on gamma-TQ. These data clearly established the role of arylation in quinone toxicity and revealed that arylating quinone structure affects cytotoxicity by governing detoxification through the rate of adduct formation. Furthermore, arylating quinones induced endoplasmic reticulum (ER) stress by activating the pancreatic ER kinase (PERK) signaling pathway including elF2alpha, ATF4, and C/EBP homologous protein (CHOP). Detoxification by NAC greatly attenuates CHOP induction in arylating quinone-treated cells, suggesting that ER stress is a cellular mechanism for arylating quinone cytotoxicity.

3T3 Cells↗

PubMed2005

On the electrophilicity of hydroxyl radical: a laser flash photolysis and computational study.

The rate coefficients for reactions of hydroxyl radical with aromatic hydrocarbons were measured in acetonitrile using a novel laser flash photolysis method. Comparison of kinetic data obtained in acetonitrile with those obtained in aqueous solution demonstrates an unexpected solvent effect on the reactivity of hydroxyl radical. In particular, reactions of hydroxyl radical with benzene were faster in water than in acetonitrile, and by a significant factor of 65. Computational studies, at the B3LYP and CBS-QB3 levels, have confirmed the rate enhancement of hydroxyl radical addition to benzene via calculation of the transition states in the presence of explicit solvent molecules as well as a continuum dielectric field. The origin of the rate enhancement lies entirely in the structures of the transition states and not in the pre-reactive complexes. The calculations reveal that the hydroxyl radical moiety becomes more anionic in the transition state and, therefore, looks more like hydroxide anion. In the transition states, solvation of the incipient hydroxide anion is more effective with water than with acetonitrile and provides the strong energetic advantage for a polar solvent capable of hydrogen bonding. At the same time, the aromatic unit looks more like the radical cation in the transition state. The commonly held view that hydroxyl radical is electrophilic in its reactions with DNA bases is, therefore, strongly dependent on the ability of the organic substrate to stabilize the resulting radical cation.

Journal Article↗

PubMed2005

Molecular characterization of biodegradable dissolved organic matter using bioreactors and [12C/13C] tetramethylammonium hydroxide thermochemolysis GC-MS.

Little is known about the molecular composition of the biodegradable fraction of dissolved organic matter (BDOM) in stream ecosystems. We combined plug-flow biofilm reactors, tetramethylammonium hydroxide (TMAH) thermochemolysis GC-MS, and 13C-labeled TMAH thermochemolysis GC-MS to study the molecular composition of BDOM from two stream ecosystems. TMAH products derived from fatty acids, lignin, and other aromatic molecules were quantified using an internal standard approach. We applied the 13C-TMAH thermochemolysis procedure to differentiate between compounds in dissolved organic matter (DOM) that had natural methoxyl groups from those that acquired methoxyl groups during the TMAH reaction. In Rio Tempisquito, a stream draining a tropical evergreen forest, and White Clay Creek, a stream draining a temperate deciduous woodlands, carbohydrates, fatty acids, and lignin contributed to the DOM and BDOM molecular composition. We observed 97 different peaks in the chromatograms of streamwater, with 57% of the peaks common to both streams. The DOM and BDOM pools from each site also contained a unique suite of compounds. Our combined use of TMAH and 13C-TMAH thermochemolysis revealed that heterotrophic bacteria can selectively degrade and demethylate different types of compounds in the lignin residues of DOM. This demonstration of bacterial demethylation of lignin, an abundant and refractory plant molecule, has potential implications for global carbon cycling.

Biodegradation, Environmental↗

PubMed2005

Comparison of polycyclic aromatic hydrocarbon distributions and sedimentary organic matter characteristics in contaminated, coastal sediments from Pensacola Bay, Florida.

In this study, we examined the distribution of polycyclic aromatic hydrocarbons (PAHs) in a contaminated coastal area and the characteristics of the natural organic matter in tandem. We present a detailed study of PAH concentration, distribution, and organic matter characteristics of three core samples from Pensacola Bay, Florida. Solid-state 13C Nuclear Magnetic Resonance (NMR), pyrolysis gas chromatography coupled with mass spectrometry (GC-MS), and tetramethyl ammonium hydroxide (TMAH) thermochemolysis GC-MS were applied to obtain structural details about the sedimentary organic matter. Elemental compositions (carbon and nitrogen) and estimates of black carbon contents are also reported. These coastal sediments were found to contain more PAHs in the upper 15 cm layers than in the bottom 15-25 cm samples. The samples that contained the most PAHs also contained the least amount of aromatic carbon and contained a significant amount of paraffinic carbon. Lignin-derived pyrolysis and TMAH thermochemolysis products were abundant and generally higher in all of the samples in comparison to those reported for modern coastal sediments, indicating a large flux of terrestrial carbon. The black carbon contents were found to range from 4.3% to 6.8%, which are significantly lower than other reports of black carbon in sediments, which represent as much as 65% of the total organic carbon content. The low black carbon content suggests that this type of refractory carbon may not be as responsible for regulating PAH distribution as indicated by other researchers.

Carbon Isotopes↗

PubMed2005

Tocopherol metabolism using thermochemolysis: chemical and biological properties of gamma-tocopherol, gamma-carboxyethyl-hydroxychroman, and their quinones.

Identification and quantitative estimation of quinone metabolites of gamma-tocopherol (gamma-T) and its derivative gamma-carboxyethyl hydroxychroman (gamma-CEHC) are complicated by their functions as arylating electrophiles. We hypothesize that their biological properties are expressed through arylating quinone electrophile addition (Michael reaction) with thiol nucleophiles in cells and tissues. Glutathione (GSH) reacted with gamma-tocopheryl quinone (gamma-TQ) to form the hydroquinone adduct, which was identified by electrospray time-of-flight MS (ESI-TOF-MS). Tetramethylammonium hydroxide (TMAH) thermochemolysis reduced and methylated quinones and cleaved and methylated thioether adducts. These relatively nonpolar derivatives were readily separated by GC and identified by MS fragmentation patterns. gamma-CEHC was synthesized and oxidized to a product identified as the quinone lactone (gamma-CEHC-QL). TMAH methylated both gamma-CEHC-QL and its GSH adduct without opening the lactone ring, and these products were separated by GC and identified by MS fragmentation patterns. gamma-CEHC-QL reacted with both the cysteinyl enzyme papain and fetal bovine serum, and TMAH thermochemolysis showed that each product mixture contained unreacted precursor and thioether adduct. Cytotoxicities of phenolic precursors, gamma-T and gamma-CEHC, and their quinones, gamma-TQ and gamma-CEHC-QL, respectively, were compared in COS1, NT2, 3T3, and N2a cell lines. Phenolic precursor gamma-T had a small effect only with NT2 and 3T3 cells while gamma-CEHC had no effect in any cell line. Arylating quinones were highly cytotoxic in all cell lines with gamma-TQ showing a significantly greater cytotoxicity than gamma-CEHC-QL. These data are consistent with our arylating electrophile hypothesis as an explanation for some biological activities of Ts through their quinone metabolites.

3T3 Cells↗

PubMed2004

Solid-state NMR characterization of pyrene-cuticular matter interactions.

One- and two-dimensional nuclear magnetic resonance (NMR) experiments were performed on Agave americana cutan and tomato cutin to examine the interactions between a hydrophobic pollutant, pyrene, and cuticular material. Variable-temperature NMR experiments show that cutan, an acid- and base-resistant cuticular biopolymer, undergoes the characteristic melting behavior of "polyethylene-like" crystallites, while the tomato cutin does not. The melting point of A. americana cutan was found to be approximately 360 K, which is consistent with the thickness of the polyethylene crystallites of 30-40 methylene units. Sorption models predict that the sorption behavior of hydrophobic pollutants should depend on the phase of the cuticular material. 13C NMR experiments on labeled pyrene were performed. The 13C T1 of pyrene decreases significantly from that of crystalline pyrene upon sorption to both tomato fruit cutin and A. americana cutan, indicating that the pyrene is mobile upon sorption. Magic angle spinning experiments at low spinning frequencies (2-4 kHz) provided the chemical shift anisotropy (CSA) parameters delta, the anisotropy, and eta, the asymmetry parameter, for crystalline and sorbed pyrene. For crystalline pyrene, two types of crystallographically distinctive pyrenes were observed. The first had delta = -97.4+/-0.5 ppm and eta = 0.934+/-0.006, while the second had delta = -98.1+/-0.5 ppm and eta = 0.823+/-0.008. After sorption to cutan, these CSA parameters were found to be delta = -78.9+/-5.3 ppm and eta < 0.70 independent of the length of time since completion of the sorption procedure. In tomato cutin, the CSA parameters were found to be dependent upon the time since completion of the sorption procedure. One and one-half months after sorption, delta was found to have a value of -30.4 ppm < delta < 0.0 ppm and eta was undeterminable, while after 22 months these values become delta = -80.0 +/-3.3 ppm and eta< 0.42. These changes in the CSA parameters demonstrate that upon sorption of pyrene to cutan, the pyrene undergoes anisotropic motion, while in cutin pyrene initially can tumble isotropically, but after 22 months this motion also becomes anisotropic. 2D heteronuclear correlation experiments indicate that pyrene is in close proximity to aliphatic cuticular materials after sorption. This work is directly relevant toward understanding the physical and chemical mechanisms of pollutant sorption to soil organic matter and, thus, help develop improved sorption models and pollution remediation techniques.

Adsorption↗

PubMed2004

Overestimates of black carbon in soils and sediments.

Several recent reports suggest that black carbon (BC), which broadly encompasses charcoal, soot, and other forms of pyrogenic carbon, may constitute a significant proportion of the refractory carbon in soil and sedimentary organic matter. BC is a sink for biospheric and atmospheric carbon dioxide, and is intimately tied to the biogeochemical cycling of both carbon and oxygen through its role in organic matter cycling. Additionally, BC may represent a large fraction of the "missing carbon sink" in global carbon accounting. Here, we demonstrate that documented measurements of BC may be the result of methodological artifacts, which inadvertently overestimate the amount of BC. We found that a widely used thermal oxidative method can create a residue that falls under the operational definition of BC in samples that are relatively BC-free. Moreover, during this procedure, labile organic matter constituents are condensed into pyrogenic carbon, implying that the labile components are present in lesser quantities. These methodological deficiencies are promoting overestimates in the amount of refractory carbon in soil and sedimentary organic matter and may endorse inaccuracies in the rates of carbon fluxes, the mean residence times of terrestrial carbon, and organic matter burial rates in oceanic environments.

Carbon↗

PubMed2004

Identification of black carbon derived structures in a volcanic ash soil humic acid by Fourier transform ion cyclotron resonance mass spectrometry.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), coupled with cross-polarization magic angle spinning 13C nuclear magnetic resonance (NMR) spectroscopy and Kendrick mass defect analysis, was used to study the molecular composition of an aromatic carbon-rich humic acid extracted from a dark black soil from Iwata, Japan. Black carbon, produced by the incomplete combustion of fossil fuels and organic matter, has been suggested as a major component of humic acids having intense peaks in the aromatic and carboxyl regions of the 13C NMR spectrum. Taking advantage of the high resolving power of FT-ICR MS to make precise formula assignments, three different types of highly carboxylated polycyclic aromatic compounds were identified in the sample: linearly fused aromatic structures, aromatic structures linked by carbon-carbon single bonds, and highly condensed aromatic structures. These carboxylated aromatic structures have a low mass defect in their mass spectra due to their abundance of oxygen and deficiency of hydrogen. This mass defect is observed in the vast majority of peaks present in the entire mass spectrum, differentiating them from structures that are hydrogen-rich (e.g., fatty acids, proteins, carbohydrates). Thus, we conclude that the bulk of the sample analyzed is comprised of these heavily carboxylated, hydrogen-deficient, condensed aromatic structures, features believed to be characteristic of black carbon-like material.

Carbon↗

PubMed2004

Noncovalent interactions between aromatic compounds and dissolved humic acid examined by nuclear magnetic resonance spectroscopy.

We examined the molecular-level interactions of aromatic compounds with a humic acid that was extensively characterized with one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Interactions of target compounds were evaluated by monitoring their NMR relaxation properties. Solid-state NMR revealed that the humic sample contains paraffinic carbon (31%), substituted aliphatic carbon (30%), and aromatic carbon (28%). The liquid-state experiments further identified amino acids, a range of carbohydrates, methoxylated aromatics (likely derived from lignin), and a series of aliphatic chains. The 13C spin-lattice relaxation time (T1) of site-specifically labeled naphthalene, 1-naphthol, and quinoline was found to decrease with increasing additions of humic acid. The 1H T1 values also were measured and demonstrated that the association with the humic acid was not specific because all the protons acquired the T1 value of the humic acid at the same rate. The lack of a chemical shift change and an increase in signal line broadening indicates that the interaction between these compounds and humic acid is noncovalent. These interactions were detected at low humic concentrations (5 mg C/L) and suggest that low concentrations of humic material, which are prevalent in both aquatic and terrestrial systems, will significantly affect the fate and transport of contaminants in the environment.

Carbon Isotopes↗

PubMed2003

Graphical method for analysis of ultrahigh-resolution broadband mass spectra of natural organic matter, the van Krevelen diagram.

Electrospray ionization mass spectrometry (ESI-MS) is becoming an important tool in the study of natural organic matter (NOM) at the molecular level. Ultrahigh-resolution ESI-MS analyses of NOM often produce very complicated spectra; therefore, visual presentation and structural interpretations of the spectra are difficult. To meet this analytical challenge, we herein propose and demonstrate an approach using the van Krevelen diagram. With this approach, complicated mass spectra can be visualized in a way that allows for (1) possible reaction pathways to be identified and presented, and (2) qualitative analyses on major classes of compounds that comprise ultrahigh-resolution spectra. The qualitative analyses are in a good agreement with results obtained from analyses by other analytical techniques. Additionally, the van Krevelen diagram can be expanded to a 3D plot by using peak intensities or relative intensities as the z-axis. The 3D van Krevelen diagram allows for an evaluation of the relative significance of structurally related compounds. The 3D plot can also be a useful tool for compositional differentiation among samples.

Coal↗

PubMed2003

Solid-state and multidimensional solution-state NMR of solid phase extracted and ultrafiltered riverine dissolved organic matter.

In this study we used multidimensional solution-state NMR to elucidate the differences in the chemical composition of solid phase extracted and ultrafiltered DOM isolates. DOM was isolated from water sampled from an oligotrophic river, the River Tagliamento (Italy). The recovery of total DOM was up to 42% with both isolation techniques. In addition to 1- and 2-D solution-state NMR, we also applied 1-D solid-state 13C NMR spectroscopy for DOM characterization. 13C NMR spectroscopy only produced broad overlapping resonances, thus allowing a bulk characterization of DOM composition. However, it demonstrated that the bulk chemical composition of the two DOM fractions exhibited minor spatial-temporal changes. The 2-D experiments (TOCSY, HMQC) showed that the solid phase extracted hydrophobic DOM contained predominantly aliphatic esters, ethers, and hydroxyl groups, whereas the ultrafiltered DOM was comprised partially of peptides/protein, with further evidence for a small amount of aliphatic/fatty acid material. Sugars were present in both DOM fractions. The results show the two isolation techniques selected for different suites of compounds within the bulk DOM pool.

Environmental Monitoring↗

PubMed2003

3-D structural modeling of humic acids through experimental characterization, computer assisted structure elucidation and atomistic simulations. 1. Chelsea soil humic acid.

This paper describes an integrated experimental and computational framework for developing 3-D structural models for humic acids (HAs). This approach combines experimental characterization, computer assisted structure elucidation (CASE), and atomistic simulations to generate all 3-D structural models or a representative sample of these models consistent with the analytical data and bulk thermodynamic/structural properties of HAs. To illustrate this methodology, structural data derived from elemental analysis, diffuse reflectance FT-IR spectroscopy, 1-D/2-D 1H and 13C solution NMR spectroscopy, and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI QqTOF MS) are employed as input to the CASE program SIGNATURE to generate all 3-D structural models for Chelsea soil humic acid (HA). These models are subsequently used as starting 3-D structures to carry out constant temperature-constant pressure molecular dynamics simulations to estimate their bulk densities and Hildebrand solubility parameters. Surprisingly, only a few model isomers are found to exhibit molecular compositions and bulk thermodynamic properties consistent with the experimental data. The simulated 13C NMR spectrum of an equimolar mixture of these model isomers compares favorably with the measured spectrum of Chelsea soil HA.

Humic Substances↗

PubMed2003

The identification of plant derived structures in humic materials using three-dimensional NMR spectroscopy.

Here we demonstrate the application of 3-D NMR spectroscopy to structural studies of humic substances, the most abundant of organic compounds on earth. The increased spectral dispersion provided by the additional dimension is proven to be highly advantageous in separating the overlapping signals observed in 2-D spectra. Assignments of the major aliphatic structures and selected aromatic moieties are given as examples. We find that in a forest soil fulvic acid the major aliphatic materials are likely derived from leaf cuticles and further demonstrate that lignin signatures can be identified among the aromatic species. Once identified from the 3-D spectra, these structures can be assigned using the partial information available in 2-D, and in some cases, in the 1-D spectra. These signals are demonstrated to be characteristic to given samples of natural organic matter, and the case is made for their use as indicators of terrestrial biomarkers in mixtures of compounds with unknown origins.

Benzopyrans↗

PubMed2003

Electrophile tocopheryl quinones in apoptosis and mutagenesis: thermochemolysis of thiol adducts with proteins and in cells.

Electrophile tocopheryl quinones from the phenolic antioxidants gamma-tocopherol and delta-tocopherol form Michael adducts with the thiol nucleophile glutathione. These tocopheryl quinones are involved in cytotoxicity, apoptosis, and mutagenesis, and their biologic properties are associated with the depletion of intracellular thiols. We now show that both proteins and tissues treated with the electrophile gamma-tocopheryl quinone (gamma-TQ) form thiol adducts. The monoglutathion-S-yl derivative of gamma-TQ was subjected to thermochemolysis with the strong methylating base tetramethylammonium hydroxide. GC/MS showed four signature peaks and a fragmentation pattern characteristic of the thiol adduct. Similarly, pure monoglutathion-S-yl and diglutathion-S-yl derivatives of delta-TQ were subjected to thermochemolysis, and GC/MS showed characteristic fragmentation patterns for thiol adducts. The four signature peaks were identified when pure proteins with accessible thiol groups (hemoglobin and histone), FBS, and tissue culture medium and cell preparations were treated with gamma-TQ. Signature peaks in both complete medium and washed cells showed the presence of both soluble and insoluble thiol adducts. The effective or free arylating electrophile concentration in complete medium should always be evaluated in tissue culture studies. gamma-TQ is a mutagen but not a genotoxin; therefore, the histone adduct may be a previously unrecognized histone modification involved in chromatin dynamics leading to mutagenesis.

Apoptosis↗

PubMed2003

New insights on the structure of algaenan from Botryoccocus braunii race A and its hexane insoluble botryals based on multidimensional NMR spectroscopy and electrospray-mass spectrometry techniques.

Through solution state NMR spectroscopy and quadrupole-time-of-flight mass spectrometry (Q-TOF MS) studies of the hexane insoluble botryal extract of the algae Botryococcus braunii race A, coupled with high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy of the algaenan from this alga, it has been possible to advance the structural understanding of this geochemically important biopolymer. It was found that the hexane insoluble botryals in this study constitute a mixture of low molecular weight unsaturated aliphatic aldehydes and unsaturated hydrocarbons with an average chain length of about 40 carbons. Exact assignments were provided for many of the structural units present and describe the average constitution of the mixture as a whole. Reticulation of the low molecular weight chains via acetal and ester links explains the primary make up of algaenan. In this study, it is concluded that the algaenan results from the reticulation of low molecular weight hexane insoluble botryal species rather than the polyaldehyde as previously observed in studies of algae at alternate stages of their growth cycle.

Aldehydes↗

PubMed2003

Evidence for cross-linking in tomato cutin using HR-MAS NMR spectroscopy.

Cutin is a polyester biopolymer component of plant leaf and fruit cuticles, most often associated with waxes and cuticular polysaccharides, and sometimes with another aliphatic biopolymer called cutan. Insolubility of these cuticular biopolymers has made it difficult to apply traditional analytical techniques for structure determination, because most techniques providing molecular level details require solubility. By using the relatively new technique of one and two-dimensional high-resolution magic angle spinning (HR-MAS) NMR spectroscopy, with added information from solid-state 13C NMR spectroscopy, detailed through-bond connectivities and assignments are made for cutin from Lycopersicon esculentum (tomato) fruit. Based on the data obtained, tomato cutin is found to be predominantly an aliphatic polyester with some olefinic and aromatic moieties, consistent with previous studies that employed various degradative approaches. Aside from esters, there are free primary and secondary alcohol groups, as well as free fatty acids. A significant finding is the presence of alpha-branched fatty acids/esters. Mid-chain hydroxyls appear to be generally unesterified, but esters of mid-chain hydroxyls have been identified. The alpha-branched fatty acids/esters and esters of mid-chain hydroxyls could point towards cross-linking.

Biopolymers↗

PubMed2003

A new approach to evaluating the extent of Michael adduct formation to PAH quinones: tetramethylammonium hydroxide (TMAH) thermochemolysis with GC/MS.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that are converted to cytotoxic and carcinogenic metabolites, quinones, by detoxifying enzyme systems in animals. PAH metabolites such as the quinones can form Michael adducts with biological macromolecules containing reactive nucleophiles, making detection of exposure to PAHs difficult using conventional techniques. A technique has been developed for detecting exposure to PAHs. Tetramethylammonium hydroxide (TMAH) thermochemolysis coupled with GC/MS is proposed as an assay method for PAH quinones that have formed Michael adducts with biological molecules. Three PAH quinones (1,4-naphthoquinone, 1,2-naphthoquinone, and 1,4-anthraquinone) and 1,4-benzoquinone were reacted with cysteine, and the TMAH thermochemolysis method was used to assay for both thiol and amine adduction between the quinones and the cysteine. Additional studies with 1,4-naphthoquinone adducts to glutathione and bovine serum albumin showed the same thiol and amine TMAH thermochemolysis products with larger peptides as was observed with cysteine adducts. The TMAH GC/MS method clearly shows great promise for detecting PAH quinones, produced by enzymatic conversion of PAHs in biological systems, that have been converted to respective Michael adducts.

Animals↗

PubMed2002

Fulvic acid oxidation state detection using fluorescence spectroscopy.

Humic substances are a heterogeneous class of moderate molecular weight, yellow-colored biomolecules present in all soils, sediments, and natural waters. Although humic substances are generally resistant to microbial degradation under anaerobic conditions, some microorganisms in soils and sediments can use quinone moieties in humic substances as electron acceptors. Laboratory experiments have shown that humic substances can act as electron shuttles in the microbial reduction of ferric iron. Field studies of electron shuttling processes have been constrained by the lack of methods to characterize the oxidation state of quinone moieties in humic substances at natural concentrations. All humic substances have fluorescent properties, and fluorescence spectroscopy can indicate differences in precursor organic source of humic substances. Here we show that the quinone moieties responsible for electron transfer reactions contribute significantly to the fluorescence of humic substances. Further we use fluorescence spectroscopy to elucidate the oxidation state of quinone moieties in humic substances at natural concentrations found in sediment interstitial waters.

Benzopyrans↗