Lisbeth Knudsen received an MSc in Biochemistry in 1980 and PhD in Biomedicine 1993. Her present position is professor of experimental toxicology at the Department of Occupational and Environmental Health, The Institute of Public Health, University of Copenhagen. She was until 2009 a national member of the Scientific Advisory Committee (ESAC) for the European Centre for the validation of Alternative Methods (ECVAM) and is currently an active member of the EU Implementation Group on Biomonitoring. At the national level Lisbeth has coordinated several major biomonitoring studies within occupational health. She is the current Coordinator of the European Network on Children’s Susceptibility and Exposure to Environmental Genotoxicants. She is an active participant in a number of EU-projects: COPHES (Consortium to Perform Human Biomonitoring on a European Scale), DEMOCOPHES (Demonstration of a Study to Coordinate and Perform Human Biomonitoring on a European Scale) and LIFE+ 09 ENV/BE00410. Lisbeth is also an active member of the Research Council of the Faculty of Health Sciences at the University of Copenhagen and the Danish Consensus Platform for 3R Alternatives to Animal Experimentation (DACOPA). In 2006 Lisbeth received the Nordic Alternative Price for promoting the development of alternatives to animal testing and she is the current coordinator of the Danish in vitro toxicology network and treasurer in the European Consensus Platform for 3R Alternatives to Animal Experimentation (ECOPA). Professor Domenico Franco Merlo received his Ph.D. in Environmental Health, Epidemiology from the University of Cincinnati, Ohio, USA in 1988. He is currently Director of the Unit of Epidemiology, Biostatistics and Clinical Trials, Department of Cancer Epidemiology and Prevention of the National Cancer Institute of Genoa, Italy. He coordinated and contributed to European and national studies on cancer risk associated with environmental and occupational exposure to crystalline silica, benzene, 2,3,7,8-TCDD, pesticides as well as studies including biomarkers of exposure, early biological effects, and individual susceptibility.

Biomarkers and Human Biomonitoring

Volume 2: Selected Biomarkers of Current Interest

By Lisbeth E. Knudsen, Domenico Franco Merlo

The Royal Society of Chemistry

Copyright © 2012 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84973-242-0

Contents

Volume 1,
Chapter 1 Introduction to Human Biomonitoring Lisbeth E. Knudsen, Nanna Hundebøll and Domenico Franco Merlo, 1,
Chapter 2 Examples of Ongoing Studies,
Chapter 3 Biomarkers of Exposure,
Subject Index, 446,
Volume 2,
Chapter 4 Biomarkers of Exposure: Hemoglobin Adducts H. von Stedingk, S. Osterman-Golkar and M. Törnqvist, 1,
Chapter 5 Biomarkers of Human Exposure to Environmental Tobacco Smoke (ETS) David H. Phillips, 23,
Chapter 6 Biomarkers of Exposure: Mycotoxins – Aflatoxin, Deoxynivalenol and Fumonisins Paul C Turner, Liana Van Der Westhuizen and Andre Nogueira Da Costa, 50,
Chapter 7 Biological Measures and the Psychosocial Working Environment Åse Marie Hansen, Anne Helene Garde and Marie Aarrebo Jensen, 87,
Chapter 8 Micronuclei for Human Biomonitoring Micheline Kirsch-Volders and Ilse Decordier, 115,
Chapter 9 Biomarkers of Individual Susceptibility: Genetic Polymorphisms and their Interplay with Micronucleus Frequencies Ilse Decordier, Kim Vande Loock and Micheline Kirsch-Volders, 129,
Chapter 10 Biomarkers of Effects on Hormone Functions Mandana Ghisari, Tanja Krüger, Manhai Long and Eva Cecilie Bonefeld-Jørgensen, 142,
Chapter 11 Biomarkers of Exposure: Oxidative Stress to DNA and Lipids – Relation to Air Pollution Steffen Loft and Peter Møller, 160,
Chapter 12 Male and Female Germ Cell Biomarkers Diana Anderson, Julian Laubenthal and Adolf Baumgartner, 174,
Chapter 13 Biomarkers of Exposure and Effect: Ionizing Radiation A. Fucic and A. Sorokin, 199,
Chapter 14 Ethics and Data Protection in Human Biomarker Studies B. Dumez, K. Van Damme and L. Casteleyn, 216,
Subject Index, 227,

CHAPTER 1

Biomarkers of Exposure: Hemoglobin Adducts

H. VON STEDINGK, S. OSTERMAN-GOLKAR AND M. TÖRNQVIST

4.1 Introduction

Chemical compounds that are electrophilically reactive or form reactive intermediates in the metabolism are potentially toxic owing to their ability to react with electron-dense atoms in proteins and DNA. The reactivity makes these compounds difficult to measure in vivo, as a result of their fast detoxification and short half-lives.

One approach used for measurement of exposure or the in vivo dose of electrophilic compounds is based on measurement of their stable adducts with biomacromolecules. The macromolecules used for this purpose are mostly DNA, serum albumin and hemoglobin (Hb) in blood. An adduct has been defined as a complex that forms when a chemical binds to a biological molecule. In the present context it has also been found practical to use the term adduct for a moiety covalently bound to the macromolecule as a consequence of a reaction (Figure 4.1).

The emphasis of this chapter is on the application of adducts with Hb as a biomarker of occupational, environmental and lifestyle exposures to carcinogenic compounds. Some applications are illustrated with examples from the authors’ own work. The usefulness of adduct studies is by no means restricted to mutagens and carcinogens. The chemical reactivity of compounds and/or of their metabolic intermediates may, even at very low concentrations, alter tissue constituents in such a way as to give rise to various harmful conditions or diseases.

As discussed later in more detail the Hb adduct level reflects the dose (AUC; area under the concentration vs. time curve) of an electrophilic compound in blood. The AUC is determined by the absorption (or formation) of the compound, distribution, metabolism and excretion (Figure 4.2).

4.1.1 Early Studies/Milestones

By the middle of the twentieth century, adducts with proteins, including Hb, were used to measure the bioavailability of reactive compounds in experimental animals. Notably, studies with aromatic amines contributed to this field of research. In 1953, Jackson and Thompson demonstrated that a radiolabeled derivative of phenylhydroxylamine bound strongly to Hb in erythrocytes and that this bound material was eliminated only in the course of erythrocyte degradation. The usefulness of monitoring Hb adducts from aromatic amines was later supported by studies of occupational exposure.

In 1974, Ehrenberg and co-workers suggested that in vivo doses from exposure to electrophilic agents might be determined through the measurement of the adducts they form with tissue proteins, leading to the exploration of Hb as a monitor molecule. In experiments with mice the approach was tested with the directly reactive agent ethylene oxide, and a compound that requires metabolic activation, namely dimethylnitrosamine. The early development of analytical methods for the isolation of adducts, and for their analysis by gas chromatography–mass spectrometry (GC-MS), was important for the application of Hb adduct measurement to exposure to potentially genotoxic and cancer risk increasing agents. Adducts with Hb in humans were used for the first time by Calleman et al. to calculate doses in blood explicitly following occupational exposure to ethylene oxide.

4.2 Mechanisms and Kinetics of Adduct Formation

4.2.1 Hemoglobin and Reactive Sites in Hemoglobin

Hemoglobin (Hb) is synthesized in the erythrocytes during their development in the bone marrow. The normal human adult Hb molecule, α2β2 (HbA), is a tetramer consisting of two α-chains and two β-chains, each containing a heme group. In “normal” adults hemoglobin HbA is the dominating component, making up ~97% of the total Hb substance.

The major sites of adduct formation are cysteine-S, histidine-N, the N-terminal NH2 group, and the carboxyl groups of aspartic and glutamic acid and of the C-terminal amino acid. Further, serine, threonine, tyrosine, lysine, arginine, methionine, and tryptophan residues may react with electrophilic compounds. Valine is the N-terminal amino acid of both the α- and the β-chain of adult Hb.

The pattern of binding of an electrophilic compound to the various nucleophilic sites in a protein may, at least to some extent, be predicted. The reactivity of nucleophilic atoms towards alkylating agents generally decreases in the order S > N > O. Alkylating agents have been assigned s-values that describe their ability to react selectively with these atoms. Alkylating agents with high s-values, such as ethylene oxide, have a strong propensity for reactions with cysteine-S. Agents with a low s-value, such as the reactive intermediates of alkylnitrosamines, show less selectively and give relatively high levels of adducts with reactive oxygen atoms.

The reactivity of a nucleophilic site in Hb is also dependent on its pKa; the base in an acid–base equilibrium being far more reactive than the acid (reviewed by Törnqvist et al.). This influences the rate of adduct formation. Thus, because of the low pKa of the N-terminal amino groups in hemoglobin (6.8–7.8), histidines (5.6–7), and cysteines (7.9–8.5) these sites have a com- paratively high reactivity towards, for example, alkylating agents. Lysines have high pKa-values (9.5–12.5) and consequently have a relatively low reactivity towards this type of electrophilic compound. However, there is also a correlation between alkalinity (as measured by pKa) and nucleophilic strength, counteracting the effect of protonation. Thus, certain types of electrophilic compound, such as acylating agents, may give relatively high levels of adducts with lysine. A majority of the applications are based on determination of adducts with cysteine and N-terminal valine.

For high-molecular-weight compounds, it may be difficult to predict the sites of reaction. It has been suggested that the tertiary structure of the protein may be playing a more important role in adduct formation for these compounds.

Neonates: At birth, HbF (α2γ2) comprises a major part of the child’s Hb. These levels decline and after 6 months adult Hb (α2β2) takes over as the predominant form of Hb in normal children. In HbF the N-terminal amino acid of the γ-chain is glycine. Such differences in Hb should be considered in biomonitoring of Hb adducts in neonates.

Other species: There is a high degree of homology in the amino acid sequences in Hb of different mammalian species. Valine is the N-terminal amino acid in the α- and β-chain of several species. This amino acid residue has a similar reactivity in mouse, rat, dog and human Hb as has been shown in in vitro experiments with various low-molecular-weight epoxides and with acrylonitrile and acrylamide. Thus, besides a correction for species differences in the life span of erythrocytes in the laboratory animals used, comparisons of the dose of reactive compounds could be based on measurements of adducts with N-terminal valine, with only minor correction for differences in reactivity. However, the Hb from some other species studied, for instance bovine Hb, has N-terminal valines in two of the four chains.

The Hb of the rat and some but not all mouse strains contains a cysteine residue with a particularly high reactivity. Segerbäck compared the in vitro rates of reaction of ethylene oxide with Hb and found 170 and 12 times higher reactivity of cysteine in rat and mouse Hb, respectively, than in human Hb. Species differences in Hb binding in vivo that are attributed to differences in cysteine reactivity have been demonstrated in several studies, for instance for acrylonitrile in rats and mice. This difference in reactivity has to be accounted for in interspecies comparisons.

4.2.2 Accumulation of Adducts through Formation and Removal

The life span of erythrocytes with their content of Hb is generally considered to be about 4 months in humans. Thus, the level of Hb adducts observed in a blood sample is the result of adduct formation and adduct removal during the 4 months prior to sampling [Scheme 4.1, Equation (4.1)].

The yield of adduct formation in Hb depends on the concentration and the persistence (AUC) of the electrophilic compound within the erythrocytes. Further, the yield depends on the chemical reactivity of the electrophile towards the nucleophilic sites involved in the adduct formation. The relationship between adduct yield and AUC for a single exposure of relatively short duration is shown in Scheme 4.1, Equation (4.2).

The turnover of erythrocytes is the main cause of adduct removal. Although most adducts studied to date have been shown to be chemically stable, some adducts, for example those with carboxyl groups, have been shown to be eliminated faster than would be predicted based on erythrocyte turnover. In special cases there might be other parameters that have to be considered in comparisons of Hb adduct levels. In humans, changes in body weight and blood volume may become relevant during pregnancy and in neonates. In experimental animals exposed to carcinogens the dilution of Hb adducts due to increased body weight during the course of the experiment has to be taken into account. Further, exposures at high levels to certain chemicals may cause hemolysis, and increased altitude does increase Hb concentrations. Approximately 20% of the Hb content is lost from the circulating erythrocyte during its life time. This process is most pronounced in old cells and has a marginal effect only on adduct elimination (reviewed by Osterman-Golkar and Vesper). Recent studies have indicated an interindividual variation in the erythrocyte life span. Furne et al. found a life span of 122 ± 23 days in 40 healthy volunteers. It has been believed that fetal red blood cells have a considerably shorter life span than in adults. Later literature however strongly indicates that the fetal erythrocyte life span is about the same as in adults.

4.2.3 Acute Single Exposures

Single exposures to reactive compounds are frequently used in experimental animals to study adduct formation and removal. Acute human exposures may occur in connection with accidental release of chemicals, cancer therapy or anaesthesia. In a few studies volunteers have been exposed to defined low concentrations (given in mg/kg body weight) of a carcinogen in order to determine the relation between administered amount (exposure) and in vivo dose as measured by Hb adduct levels.

Following a single exposure, chemically stable adducts decline in a nearly linear fashion reaching zero or a background level after a period of time corresponding to the erythrocyte life span. The relatively well characterized and long life span of the erythrocyte is one of the major advantages of Hb adducts as biomarkers. In contrast to adducts with DNA, adducts with Hb are not subjected to repair. Figure 4.3 illustrates the time windows available for measurements of urinary metabolites (typically 1 day) and DNA adducts (the half-life is set to about 4 days in this example) as compared to months in the case of stable Hb adducts.

4.2.4 Long-term Constant Exposures

Adducts with Hb accumulate during prolonged exposure and reach a steady-state level where the rates of adduct formation and adduct removal are equal (Figure 4.4). In the case of chemically stable adducts the steady state is reached after about 122 days (ter) and the accumulated adduct level could be estimated as about a × 61, where a is the average daily adduct increment [Scheme 4.1, Equation (4.1b)]. Unstable adducts accumulate to a less degree (see Figure 4.4). If the exposure is terminated the adduct levels would decline in a curvilinear manner. Environmental and life-style exposures to carcinogenic com- pounds are generally long term and at a fairly constant level. Thus, measured adduct levels are usually assumed to represent a steady state and could be used for the estimation of the daily average adduct increment and calculation of daily dose [AUC; Scheme 4.1, Equation (4.2)].

4.3 Methodologies for Measurement of Hemoglobin Adducts

The methodologies applied today for the measurement of Hb adducts usually involve mass spectrometry (MS) as the final analytical step. A few studies have employed other detection techniques such as fluorescence detection or immunochemical approaches. Mass spectrometry techniques can offer the high sensitivity and selectivity needed for measurement of the very low levels of adducts that often are encountered. Both gas chromatography (GC) and liquid chromatography (LC) combined with MS are used. GC-MS has been the major technique used, primarily because it has been available for a longer time. Recent developments are often focused on LC-MS techniques. A few applications using capillary electrophoresis in combination with MS have also been described.

Measurement of Hb adducts in the general population from a low background exposure of potentially toxic compounds puts high demands on the analytical performance with regard to sensitivity. N-terminal adducts from, for example, epoxides can be measured down to a few pmol per gram Hb, which corresponds to about 1 modification in 10 million non-modified globin chains. To be able to measure adducts at these low levels the pre-processing of the blood samples is crucial. Which method to choose for processing of the sample and for final detection depends on both the properties of the electrophilic compound of interest and the physical and chemical properties of the adduct formed. Electrophilic compounds differ in reactivity towards different sites in the globin, as mentioned in the Introduction.

There are two principal ways to perform adduct measurements, either by analysis after detachment of the adduct from the amino acid residue in the protein, or by cleavage of peptide bonds and analysis of the modified amino acid or modified peptide. The latter approach, where the analyte includes a moiety from the protein, is advantageous because it increases the specificity of the analyte. Below are examples of frequently used principles.

4.3.1 Detachment of Adducts from the Amino Acid Residue

Aromatic amines, isocyanates and polycyclic aromatic hydrocarbons (PAHs) are examples of compounds that can be analyzed as Hb adducts using mild hydrolysis of the Hb sample. Adducts bound as sulfonamides to cysteine or esters to carboxyl groups can be detached from the nucleophilic atom in the amino acid through alkaline or acidic hydrolysis. The principle was demonstrated about 30 years ago and is applied frequently (reviewed by). Different approaches for extraction and enrichment of the free adducts have been described. Liquid–liquid extraction or solid phase extraction has been optimized for different adducts and their specific physical/chemical properties. Derivatization of detached and enriched adducts has been performed to improve chromatographic properties as well as the sensitivity of the analysis. As an example, a methodology frequently applied for biomonitoring of 4-aminobiphenyl, a known carcinogen formed in for example cigarette smoke, is illustrated in Figure 4.5. The detached 4-aminobiphenyl is derivatized with pentafluoropropionic anhydride to obtain an analyte with high response when analyzed by GC-MS.

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