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Title: Development of an immunoassay to detect benzene adducts in hemoglobin

Abstract

The purpose of this project was to develop an immunoassay to detect the adducts formed in hemoglobin after exposure to benzene, which is known to cause bone marrow degeneration and acute myelogenous leukemia. The use of benzene-adduct detection as a biological monitoring method would permit measurement of low exposures and exposures sustained weeks earlier. The reactivity of hydroquinone, an important benzene metabolite, with blood proteins and amino acids was investigated in order to decide which antigens and analytes were likely to be suitable for immunoassay development. The second section determined the combination of benzene-metabolite and antigen need to produce an immunoassay with the requisite low detection limit and specificity. The immunoassays with the best performance were tested on hemoglobin from benzene-exposed mice. In vitro studies showed that hydroquinone efficiently formed adducts with erythrocyte membranes and hemoglobin but not with albumin. Adduction efficiency was greater in incubations using purified hemoglobin than whole blood. Cysteine accounted for 15 to 27% of the adducts formed by hydroquinone. The site of the other adducts were not identified although there was evidence that the hemoglobin heme was adducted. Adducts were found on only 1 of the 2 globin chains. Tryptic digestion of the globin failedmore » to associate the adducts with a specific peptide. Antigens made from hydroquinone-adducted hemoglobin but not hydroquinone-adducted cysteines coupled to carrier proteins effectively elicited adduct-specific antibodies. Interference due to reactivity to hemoglobin was controlled by using uniform quantities of hemoglobin in all wells. The mid-range of the best assays were approximately 12 pmoles HQ per well. Antibodies directed toward hemoglobin adducted with the benzene metabolites phenol, catechol and 1,2,4-trihydroxybenzene were also made. The performance of the anti-1,2,4-trihydroxybenzene were suitable for quantitative immunoassays.« less

Authors:
Publication Date:
Research Org.:
California Univ., Berkeley, CA (United States)
OSTI Identifier:
7026118
Resource Type:
Miscellaneous
Resource Relation:
Other Information: Thesis (Ph.D.)
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; 59 BASIC BIOLOGICAL SCIENCES; ADDUCTS; DETECTION; BENZENE; BIOLOGICAL INDICATORS; ENZYME IMMUNOASSAY; FEASIBILITY STUDIES; HEMOGLOBIN; ANTIGEN-ANTIBODY REACTIONS; BONE MARROW; CYTOCHEMISTRY; LEUKEMIA; ANIMAL TISSUES; AROMATICS; BIOASSAY; BIOCHEMISTRY; BODY; CARBOXYLIC ACIDS; CHEMISTRY; DISEASES; GLOBINS; HEMATOPOIETIC SYSTEM; HETEROCYCLIC ACIDS; HETEROCYCLIC COMPOUNDS; HYDROCARBONS; IMMUNE SYSTEM DISEASES; IMMUNOASSAY; NEOPLASMS; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANS; PIGMENTS; PORPHYRINS; PROTEINS; TISSUES 560300* -- Chemicals Metabolism & Toxicology; 550200 -- Biochemistry

Citation Formats

Grassman, J.A. Development of an immunoassay to detect benzene adducts in hemoglobin. United States: N. p., 1993. Web.
Grassman, J.A. Development of an immunoassay to detect benzene adducts in hemoglobin. United States.
Grassman, J.A. 1993. "Development of an immunoassay to detect benzene adducts in hemoglobin". United States. doi:.
@article{osti_7026118,
title = {Development of an immunoassay to detect benzene adducts in hemoglobin},
author = {Grassman, J.A.},
abstractNote = {The purpose of this project was to develop an immunoassay to detect the adducts formed in hemoglobin after exposure to benzene, which is known to cause bone marrow degeneration and acute myelogenous leukemia. The use of benzene-adduct detection as a biological monitoring method would permit measurement of low exposures and exposures sustained weeks earlier. The reactivity of hydroquinone, an important benzene metabolite, with blood proteins and amino acids was investigated in order to decide which antigens and analytes were likely to be suitable for immunoassay development. The second section determined the combination of benzene-metabolite and antigen need to produce an immunoassay with the requisite low detection limit and specificity. The immunoassays with the best performance were tested on hemoglobin from benzene-exposed mice. In vitro studies showed that hydroquinone efficiently formed adducts with erythrocyte membranes and hemoglobin but not with albumin. Adduction efficiency was greater in incubations using purified hemoglobin than whole blood. Cysteine accounted for 15 to 27% of the adducts formed by hydroquinone. The site of the other adducts were not identified although there was evidence that the hemoglobin heme was adducted. Adducts were found on only 1 of the 2 globin chains. Tryptic digestion of the globin failed to associate the adducts with a specific peptide. Antigens made from hydroquinone-adducted hemoglobin but not hydroquinone-adducted cysteines coupled to carrier proteins effectively elicited adduct-specific antibodies. Interference due to reactivity to hemoglobin was controlled by using uniform quantities of hemoglobin in all wells. The mid-range of the best assays were approximately 12 pmoles HQ per well. Antibodies directed toward hemoglobin adducted with the benzene metabolites phenol, catechol and 1,2,4-trihydroxybenzene were also made. The performance of the anti-1,2,4-trihydroxybenzene were suitable for quantitative immunoassays.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = 1993,
month = 1
}

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  • Immunoassay methods have become available for environmental applications. Their simplicity, reliability, and ability to provide information rapidly and on-site is enhancing the efficiency of many field and laboratory programs. Immunoassay methods rely upon antibody molecules to provide the sensitivity and specificity characteristics they exhibit, but many molecules are either insufficiently immunogenic or structurally unremarkable to induce an appropriate antibody response. Such compounds are usually considered to be incompatible with the development of an immunoassay method. An immunoassay method for the detection of benzene in water would have utility in detecting contamination from spills and leaking underground storage tanks. Benzene, however,more » is frequently considered to be in the class of compounds considered to be incompatible with antibody, and therefore immunoassay, development. The authors have developed an immunoassay for the detection of benzene in water by developing both sample processing and immunochemical procedures and reagents that overcome the technical limitations frequently encountered.« less
  • This work describes the use of protein binding to study the tissue doses of two classes of DNA-reactive metabolites resulting from administration of benzene to rats and mice. A desulfurization catalyst (Raney{reg_sign} nickel) formed the basis of a method to measure adducts of benzoquinones (BQ) with cysteinyl residues of proteins. Sulfur-bound adducts were cleaved by Raney{reg_sign} nickel, extracted, derivatized and analyzed by GC-ECD or GC-MS. Reactions of 1,4-BQ with whole blood of humans, rats and mice, in vitro, were undertaken to compare dose-related formation of BQ adducts. Rate constants for reactions of BQ with blood and blood proteins were estimated.more » Binding with hemoglobin (Hb) and bone-marrow proteins was used to compare in vivo reactions of four electrophilic metabolites of benzene, benzene oxide, 1,2-BQ, 1,4-BQ and 4,4{prime}-diphenoquinone, in F344 rats and B6C3F{sub 1} mice. Animals were given a single oral administration of [{sup 13}C{sub 6}] benzene and/or [{sup 14}C]benzene. The proportions of total protein binding were estimated for each reactive metabolite. Dose-related production of adducts of benzene oxide, 1,2-BQ and 1,4-BQ with Hb and bone-marrow proteins was seen in both species. Interestingly, large differences in adduct formation were observed between species and tissues. In the rat, benzene-oxide adducts of Hb predominated in the blood, whereas 1,2-BQ adducts were more prevalent in the marrow. In the mouse, adducts of 1,4-BQ were of greatest abundance with both Hb and bone-marrow proteins. The average blood concentrations of 1,4-BQ, estimated from the adduct levels and reaction-rate constants, were 2- to 4-fold higher in the mouse than in the rat. No adducts of 4,4{prime}-diphenoquionone were detected, in vivo. Background levels of 1,2-BQ and 1,4-BQ adducts were observed with proteins from humans, rats and mice, stemming from the dietary and endogenous sources of BQ precursors.« less
  • Benzene is a myelotoxin and a human leukemogen. Humans are exposed to this compound, both occupationally and environmentally. This study was conducted to determine whether formation of benzene-derived adducts with blood hemoglobin (Hb) can be used as a biomarker of exposure to benzene. B6C3F1 mice and F344/N rats were given 0.1 to 10,000 mumol (14C)benzene/kg body wt, orally. Twenty-four hours later, animals were euthanized, and globin was isolated from blood samples. The globin was analyzed by liquid scintillation spectrometry for the presence of (14C)benzene-derived adducts. Hb adduct formation was linear with respect to dose for amounts of up to 500more » mumol (14C)benzene/kg body wt, for both rodent species. Within this linear dose-response range, mice formed adducts from (14C)benzene approximately 3.5 times less efficiently (0.022 +/- 0.010 (pmol adducts/mg globin)/(mumol/kg body wt dose)) than did rats (0.076 +/- 0.014 (pmol adducts)/(mumol/kg body wt dose)). Benzene-derived Hb adducts also accumulated linearly when mice and rats were given up to three daily doses of 500 mumol (14C)benzene/kg body wt. These data were used to develop a physiological model for benzene-derived Hb adduct formation. Both first-order and saturable pathways for adduct formation were incorporated. The results showed that the model simulated the levels of Hb adducts in both mice and rats after oral exposures to benzene and predicted the levels of Hb adducts present after inhalation exposure. These studies suggest that Hb adducts might be useful biomarkers for human exposures to benzene.« less
  • Human Hb, the monomeric Hb of Glycera dibranchiata and horse Mb were modified by replacement of the protoheme with 2,4-dibromodeuteroheme. Following neutron capture by [sup 79]Br and [sup 81]Br, the locations of radioactive Br were determined. Although human Hb had approximately four times the mass and volume of the other proteins, about 9% of the activated Br was inserted into each of the three globins. These results suggest that the insertion is short-range (within 15 [angstrom]) and that this method could be used to label target sites in various proteins and other biological structures. Carp Hb's containing proto-, meso-, deutero-more » and dibromoheme were prepared. Kinetic and thermodynamic parameters for oxygen and CO binding were determined at Ph 6 (+IHP) (T-state, low-affinity protein) and Ph 9 (R-state, high-affinity protein). Parameters for the binding of oxygen and CO were related to the properties of the four hemes to estimate the inductive and steric factors in the ligation process. The results suggest that the steric factors are more important for the T-state than for the R-state. The T-state carp Hbs were very readily oxidized. Two new procedures were developed for the rapid determination of oxygen equilibrium isotherms for the T-state carp Hbs. The kinetic and thermodynamic parameters for ligation of oxygen and CO with the isolated carp [alpha]-chains were determined. Carp [alpha]-chains are the only hemoglobin chains isolated to date that can be classified as T-state. The secondary thermodynamic parameter ([delta]H[degrees]) was found to be essential for classifying hemoglobins as T- or R-state.« less
  • The purpose of this research was to investigate the feasibility of using high performance liquid chromatography (HPLC) and enzyme linked immunosorbent assays (ELISA) as sensitive techniques for monitoring polycyclic aromatic hydrocarbon (PAH) metabolites in human urine. The method was tested using synthesized PAH conjugates as positive markers. Results showed that a PAH conjugate, S-(9,10-dihydro-9-hydroxy-10-phenanthryl)N-acetyl cysteine (PHONAC), present in HPLC effluent could be detected by ELISA at picomole levels, well below the sensitivity of the HPLC UV detector. Analyses of urine from mice dosed with phenanthrene demonstrated that a substance detected by HPLC which was not detected in ELISA tests wasmore » the principal phenanthrene metabolite. This substance was not hydrolysed by Beta-glucuronidase. PHONAC was detected by ELISA in mouse urine extracts subjected to HPLC.« less