Formation of Hemoglobin and Albumin Adducts of Benzene Oxide in Mouse, Rat, and Human Blood

Lindstrom, Andrew B.; Yeowell-O'Connell, Karen; Waidyanatha, Suramya; McDonald, Thomas A.; Golding, Bernard T.; Rappaport, Stephen M.

doi:10.1021/tx9701788

Cited by 47 publications

(32 citation statements)

References 45 publications

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“…1) Isolation of HSA from Blood-Blood was obtained with informed consent from healthy subjects according to human-subject protocols approved by the University of North Carolina. After removing blood cells, ammonium sulfate was added to a final concentration of 60% to precipitate immunoglobulins and the remaining proteins were exhaustively dialyzed against water, lyophilized, and dissolved in water at 50 mg/ml (14). The current investigation of these archived proteins was conducted following 13 y of storage at Ϫ80°C.…”

Section: Profiling Hsa-cys34 Adductsmentioning

confidence: 99%

Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring

Grigoryan

Funk

et al. 2011

Molecular & Cellular Proteomics

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118

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A method is described for profiling putative adducts (or other unknown covalent modifications) at the Cys 34 locus of human serum albumin (HSA), which represents the preferred reaction site for small electrophilic species in human serum. By comparing profiles of putative HSACys 34 adducts across populations of interest it is theoretically possible to explore environmental causes of degenerative diseases and cancer caused by both exogenous and endogenous chemicals. We report a novel application of selected-reaction-monitoring (SRM) mass spectrometry, termed fixed-step SRM (FS-SRM), that allows detection of essentially all HSA-Cys 34 modifications over a specified range of mass increases (added masses). After tryptic digestion, HSA-Cys 34 adducts are contained in the third largest peptide (T3), which contains 21 amino acids and an average mass of 2433.87 Da. The FS-SRM method does not require that exact masses of T3 adducts be known in advance but rather uses a theoretical list of T3-adduct m/z values separated by a fixed increment of 1.5. In terms of added masses, each triply charged parent ion represents a bin of ؎2.3 Da between 9.1 Da and 351.1 Da. Synthetic T3 adducts were used to optimize FS-SRM and to establish screening rules based upon selected band y-series fragment ions. An isotopically labeled T3 adduct is added to protein digests to facilitate quantification of putative adducts. We used FS-SRM to generate putative adduct profiles from six archived specimens of HSA that had been pooled by gender, race, and smoking status. An average of 66 putative adduct hits (out of a possible 77) were detected in these samples. Putative adducts covered a wide range of concentrations, were most abundant in the mass range below 100 Da, and were more abundant in smokers than in nonsmokers. With minor modifications, the FS-SRM methodology can be applied to other nucleophilic sites and proteins. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.004606, 1-13, 2011.Reactive electrophiles, particularly aldehydes, epoxides, quinones, and short-lived oxygen and nitrogen species, have long been implicated as causes of major human diseases (1-5). These electrophiles enter the blood following metabolism of exogenous compounds, from oxidation of lipids and other natural molecules, from ionizing radiation, and from inflammation associated with infections and disease processes. Because of their short life spans in vivo, it is rarely possible to measure reactive electrophiles in body fluids. However, adducts produced by reactions with blood nucleophiles, notably hemoglobin (Hb) 1 and human serum albumin (HSA), reflect the presence of reactive electrophiles in the systemic circulation (6, 7). Levels of targeted Hb and/or HSA adducts have been investigated in human blood for several exogenous or endogenous toxicants that are either electrophilic carcinogens or their precursors, notably, acrylamide, aflatoxin B1, benzene, 1,3-butadiene, ethylene oxide, as well as assorted aromatic amines, polycyclic aromatic hydrocarbons, and aldehyde...

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Section: Profiling Hsa-cys34 Adductsmentioning

confidence: 99%

Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring

Grigoryan

Funk

et al. 2011

Molecular & Cellular Proteomics

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118

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“…For example, benzene oxide, a reactive species and the first oxidation product of benzene (Figure 1), has been recently shown to be somewhat stable in blood (half-life of 8 min). 97 Moreover, benzene oxide adducts with proteins have been measured in rat and mouse bone marrow following administration of benzene. 68 The relative importance of benzene oxide to other metabolites is not known at this time.…”

Section: Predictions and Critiques Of The Hypothesismentioning

confidence: 99%

Hypothesis: Phenol and hydroquinone derived mainly from diet and gastrointestinal flora activity are causal factors in leukemia

et al. 2001

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High background levels of phenol and hydroquinone are present in the blood and urine of virtually all individuals, but vary widely. Phenol and hydroquinone have been strongly implicated in producing leukemia associated with benzene exposure, because they reproduce the hematotoxicity of benzene, cause DNA and chromosomal damage found in leukemia, inhibit topoisomerase II, and alter hematopoiesis and clonal selection. The widely varying background levels of phenol and hydroquinone in control individuals stem mainly from direct dietary ingestion, catabolism of tyrosine and other substrates by gut bacteria, ingestion of arbutin-containing foods, cigarette smoking, and the use of some over-the-counter medicines. We hypothesize that these background sources of phenol and hydroquinone and associated adducts play a causal role in producing some forms of de novo leukemia in the general population. This hypothesis is consistent with recent epidemiological findings associating leukemia with diets rich in meat and protein, the use of antibiotics (which change gastrointestinal flora make-up), lack of breastfeeding, and low activity of NAD(P)H quinone oxidoreductase which detoxifies quinones derived from phenol and hydroquinone and protects against benzene hematotoxicity. An attractive feature of our hypothesis is that it may explain why many people who have no known occupational exposures or significant smoking history develop leukemia. The hypothesis predicts that susceptibility to the disease would be related to diet, medicinal intake, genetics and gut-flora composition. The latter two of these are largely beyond our control, and thus dietary modification and reduced use of medicines that elevate phenol levels may be the best intervention strategies for lowering leukemia risk. Leukemia (2001) 15, 10-20.

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“…It has been demonstrated that, in most cases, one of the most reactive site of hemoglobin is the cysteine 93 of the b-chain (30,56). Methods based on Raney Nickel procedure are based on the cleavage of the carbon-sulphur bond in the electrophile-cysteine adduct to form alcohols, which are extracted, suitably derivatised and analysed by gas chromatography/electron capture detection (GC/ECD) or gas chromatography/negative chemical ionizationmass spectrometry (GC/NICI-MS) (54,57,58). These techniques are useful in the evaluation of the exposure to aromatic toxicants, as styrene, benzene and their metabolites, but are not applied to non aromatic electrophiles.…”

Section: Discussionmentioning

confidence: 99%

New perspectives in hemoglobin adducts analysis: selective digestion with Calpain I

Pieri¹

2014

PER

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Background: hemoglobin adducts are considered suitable biomarkers to evaluate human exposure to electrophile agents. One of the procedures used for the molecular dosimetry of hemoglobin adducts is the enzymatic digestion of the protein and the subsequent quantification of modified peptides by mass spectrometry. The aim of the study is to evaluate the ability of Calpain to discriminate between normal and alkylated globin chains. Methods: digestions of pure hemoglobin sample and of an alkylated one with Calpain I at 200:1 and 25:1 weight ratios, for 1, 5 and 18 hrs were carried out. Only the standard deviations (SD) of the ratios between alkylated globins with respect to the unmodified ones (αECH/α and bECH/b) were estimated because the aim of the study is to assess the ability of Calpain I to selectively digest the samples and, above all, to find the optimal digestion conditions and characterize obtained peptides. Results: by using a 200:1 weight ratio only peptides α(1-137), α(1-138) and b(1-142) were produced, pointing out that the first proteolytic sites are located at the C-terminus ends of both α-and b-chains. The identification of peptides obtained from the 25:1 digestions allowed the determination of internal secondary proteolytic sites, not reported in literature. In both cases, a non-negligible proteolysis was observed only when a reaction time of 18 hrs was used. Discussion: results showed a progressive enrichment of modified hemoglobin with respect to the unmodified one, particularly for the α-chain and represent a promising initial step in the development of a selective purification procedure to be applied to protein adducts analysis.

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Formation of Hemoglobin and Albumin Adducts of Benzene Oxide in Mouse, Rat, and Human Blood

Cited by 47 publications

References 45 publications

Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring

Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring

Hypothesis: Phenol and hydroquinone derived mainly from diet and gastrointestinal flora activity are causal factors in leukemia

New perspectives in hemoglobin adducts analysis: selective digestion with Calpain I

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