Critical Reviews in Toxicology
ISSN: 1040-8444 (Print) 1547-6898 (Online) Journal homepage: http://www.tandfonline.com/loi/itxc20
Mercapturic Acids, Protein Adducts, and DNA Adducts as Biomarkers of Electrophilic Chemicals Ronald T. H. van Welie, Rob G. J. M. van Dijck, Nico P. E. Vermeulen & Nico J. van Sittert To cite this article: Ronald T. H. van Welie, Rob G. J. M. van Dijck, Nico P. E. Vermeulen & Nico J. van Sittert (1992) Mercapturic Acids, Protein Adducts, and DNA Adducts as Biomarkers of Electrophilic Chemicals, Critical Reviews in Toxicology, 22:5-6, 271-306 To link to this article: http://dx.doi.org/10.3109/10408449209146310
Published online: 25 Sep 2008.
Submit your article to this journal
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Citing articles: 13 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=itxc20 Download by: [Australian National University]
Date: 05 November 2015, At: 15:49
Critical Reviews in Toxicology, 22(5/6):27 1-306 ( 1 992)
Mercapturic Acids, Protein Adducts, and DNA Adducts as Biomarkers of Electrophilic Chemicals Ronald T. H. van Welie, Rob G. J. M. van Duck, and Nico P. E. Vermeulen*
Downloaded by [Australian National University] at 15:49 05 November 2015
Department of Pharmacochernistry, Division of Molecular Toxicology, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Nico J. van Sittert Health Safety and Environment Division, Shell International Petroleum Maatschappij, P.O. Box 162, 2501 AN The Hague, The Netherlands
*
To whom all correspondence should be addressed
ABSTRACT: The possibilities and limitations of using mercapturic acids and protein and DNA adducts for the assessment of internal and effective doses of electrophilic chemicals are reviewed. Electrophilic chemicals may be considered as potential mutagens and/or carcinogens. Mercapturic acids and protein and DNA adducts are considered as selective biomarkers because they reflect the chemical structure of the parent compounds or the reactive electrophilic metabolites formed during biotransformation. In general, mercapturic acids are used for the assessment of recent exposure, whereas protein and DNA adducts are used for the assessment of semichronic or chronic exposure. 2-Hydroxyethyl mercapturic acid has been shown to be the urinary excretion product of five different reactive electrophilic intermediates. Classification of these electrophiles according to their acidbase properties might provide a tool to predict their preference to conjugate with either glutathione and proteins or with DNA. Constant relationships appear to exist in the cases of 1,2-dibromoethane and ethylene oxide between urinary mercapturic acid excretion and DNA and protein adduct concentrations. This suggests that mercapturic acids in some cases may also play a role as a biomarker of effective dose. It is concluded that simultaneous determination of mercapturic acids, protein and DNA adducts, and other metabolites can greatly increase our knowledge of the specific roles these biomarkers play in internal and effective dose assessment. If the relationship between exposure and effect is known, similar to protein and DNA adducts, mercapturic acids might also be helpful in (individual) health risk assessment. KEY WORDS: biological monitoring, electrophilic chemicals, pesticides, risk assessment, thioethers, mercapturic acids, macromolecular adducts.
ABBREVIATIONS AFB BE1 B(a)P CI DCP DBE
Aflatoxin B, Biological exposure index Benzo(a)pyrene Chemical ionization 7,3-Dichloropropene 1,2-Dibromoethane
DNA ECD E-DCP EI ELISA EO FID FPD
Deoxyribonucleic acid Electron capture detection E- 1,3-Dichloropropene Electron impact Enzyme-linked immunosorbent assay Ethylene oxide Flame ionization detection Flame photometric detection
1040-8444/92/$.50
0 1992 by CRC Press, Inc
271
Downloaded by [Australian National University] at 15:49 05 November 2015
Gas chromatography GC 2-GEMA N-Acetyl-S-(2-[N-7-guanyl]ethyl)-~cy steine Glutathione GSH Glutathione S-transferase GST Hemoglobin Hb 2-HEMA N-Acetyl-S-(2-hydroxyethyl)-~cy steine High-performance liquid HPLC chromatography Immuno slot blot technique ISB Mass spectrometry MS Nuclear magnetic resonance NMR Nitrogen phosphorus detection NPD Occupational exposure limit OEL Polycyclic aromatic hydrocarbon PAH Radioimmunoassay RIA Sui I"ny dry i SH Time-weighted average TWA USERIA Ultrasensitive enzymatic radioimmunoassay Ultraviolet uv Z-DCP Z- 1,3-Dichloropropene
c chcmical
I
t
ToxiWcauon ( I ) sponlanmus (2) oxidation (3) conjugation
I Covalent binbng
Directly gcnoloxlc Chunlcal
Alkylrled nucluc acid baes. dkylalcd amim sdr
wirh cellular macromolecules
R N A adducu
DNA addrrls
P m m adducls
Cellular lesions. toxicity
FIGURE 1. Possible metabolic pathways of (geno)toxic chemicals. (Adapted and modified from Van Sittert, N. J. and De Jong, G., Food Chem. Toxicol.,
23,23,1985.)
1. INTRODUCTION The annual world production of man-made chemicals such as pesticides, drugs, plastics, fibers, and petroleum products continues to rise year after year. Man and his environment are likely to be exposed to many of these body-foreign chemicals or xenobiotics. I Special attention must be paid to genotoxic chemicals possessing or acquiring (by biotransformation processes) electrophilic properties capable of altering DNA integrity. Genotoxic chemicals may exert not only mutagenic and carcinogenic, but also neurotoxic, teratogenic, reproductive-toxic, and other toxic effects on organs such as the liver, kidney, spleen, testes, heart, and lung. The possible metabolic pathways of direct- and indirect-acting mutagens and carcinogens are shown in Figure 1. Human exposure needs to be kept under surveillance. In order to assess the health risk associated with exposure to potentially toxic chemicals, biological monitoring methods can be very useful. However, most assays in biological monitoring are only concerned with exposure assessment, not with risk assessment. Effects of exposure also can differ between individuals due to differences in toxicokinetics and -dynamics. In272
dividual dose monitoring is therefore playing an increasingly important role in occupational toxicology. Biomarkers, the end points used in biological monitoring, can generally be subdivided into biomarkers of dose, response, and susceptibility (e.g., i n h e r e n ~ e ) .This ~ . ~ review is restricted to biomarkers of dose, of which two types are recognized, i.e., biomarkers of internal dose and those of effective or target dose. The internal dose is the total amount of a xenobiotic taken up by the organism. It is assessed by measuring the concentration of the parent compound or its metabolite(s) in body fluids or tissues. The target dose is the dose that evades metabolic detoxification and penetrates to the biologically significant sites in DNA.4 The interaction with DNA is presumed to be mechanistically relevant by being an etiological determinant in the process of mutagenicity and/or carcinogenicity .5 The biologically effective dose is assessed by determining the amount of the parent compound or metabolite(s) interacting with cellular macromolecules, e.g., DNA, RNA, and proteins. Exposure to potentially genotoxic and other toxic chemicals may result in the formation of
Downloaded by [Australian National University] at 15:49 05 November 2015
products that may be biomarkers of internal or effective dose. The level of formation of either type of biomarker depends on many factors and has to be (experimentally) determined. Figure 2 gives a schematic view of the reactions of either “hard” or “soft” electrophilic xenobiotics and/ or metabolites reacting covalently with DNA/ RNA and/or protein/glutathione (GSH) (for further details on the classification of electrophiles see Section IV.B.2). Whether a chemical tends more to the top or the bottom of the distinct fields in Figure 2 not only determines the level of formation of certain types of biomarkers but, even more importantly, also determines the level of DNA adduct formation, i.e., toxification, and GSH conjugation, i.e., detoxification. During the last decade, there has been increased research interest in the potential for GSH conjugation and macromolecular adduct formation.6.7
Covalent binding
DNA
i
Protein and GSH
FIGURE 2. Schematic relationship between biotransformation of potentially electrophilic chemicals, the classification of reactive intermediates according to “hard” and “soft” acid-base properties, the cellular targets alkylated, the type of biomarkers formed, and the possible toxic effects. The horizontal arrow demonstrates a possible sequence of events in time; vertical arrows show the continuous scale of each box.
The aim of this review is to compare biomarkers of internal and effective dose and to evaluate their possibilities and limitations in occupational monitoring studies. Special attention is paid to biomarkers of internal dose derived from GSH conjugation; the biomarkers of effective dose discussed are DNA and protein adducts.
II. BIOMARKERS OF INTERNAL DOSE In principle, GHS conjugation-derived metabolites can be used as biomarkers of internal dose. GSH, a tripeptide consisting of the amino acids glycine, cysteine, and y-glutamine, plays an important role in the detoxification of potentially electrophilic chemicals or metabolities.6.’2 In contrast, toxification via GSH conjugation, e . g . , of 1,2-dibromoethane, hexachlorobutadiene,8 benzyl- and allylis~thiocyanate,~ has also been reported, P-Lyase-dependent bioactivation of cysteine conjugates, derived from the initially formed GSH conjugates, sometimes results in the formation of new reactive intermediates that are responsible for carcinogenic, mutagenic, and other toxicological effects. ‘‘*’I The initial step in GSH conjugation is reaction of the nucleophilic sulfhydryl with electrophilic centers of a chemical. GSH conjugation is catalyzed by a family of GSH S-transferase (GST) enzymes. A wide range of chemicals can be handled by this enzyme system due to the existence of a large number of isoenzymes with different, though overlapping, substrate selectivity. An organism’s ultimate detoxification capacity through GSH and GST enzymes depends on endogenous factors such as tissue distribution, genetic deficiencies, aging and hormonal influences, and on exogenous factors such as sensitivity to inhibition and induction of GSTs.I2 GSH conjugates are not normally excreted unchanged in urine or feces. Catabolism of GSH conjugates results in the formation and excretion of a variety of sulfur-containing metabolites, among which mercapturic acids (S-substituted N acetyl-cysteine conjugates) are some of the most important.I3 The first step in catabolism of the GSH conjugate formed is removal of the glutamyl moiety by y-glutamyltranspeptidase of which the kidney possesses the highest activity, followed by the pancreas. l 4 Cysteinylglycinase and aminopeptidases catalyze the removal of glycine of the cysteinylglycine S-conjugates to form the corresponding cysteine conjugate. The highest activity of these peptidases is found in the liver and kidney.” The final step in mercapturic acid formation is N-acetylation of the cysteine S-conjugates by N-acetyltransferases, notably located
273
in the liver and the kidney. The mercapturic acid pathway is shown in Figure 3.
C0N HCH, COOH
R X + HS-CH~-
ISSN: 1040-8444 (Print) 1547-6898 (Online) Journal homepage: http://www.tandfonline.com/loi/itxc20
Mercapturic Acids, Protein Adducts, and DNA Adducts as Biomarkers of Electrophilic Chemicals Ronald T. H. van Welie, Rob G. J. M. van Dijck, Nico P. E. Vermeulen & Nico J. van Sittert To cite this article: Ronald T. H. van Welie, Rob G. J. M. van Dijck, Nico P. E. Vermeulen & Nico J. van Sittert (1992) Mercapturic Acids, Protein Adducts, and DNA Adducts as Biomarkers of Electrophilic Chemicals, Critical Reviews in Toxicology, 22:5-6, 271-306 To link to this article: http://dx.doi.org/10.3109/10408449209146310
Published online: 25 Sep 2008.
Submit your article to this journal
Article views: 76
View related articles
Citing articles: 13 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=itxc20 Download by: [Australian National University]
Date: 05 November 2015, At: 15:49
Critical Reviews in Toxicology, 22(5/6):27 1-306 ( 1 992)
Mercapturic Acids, Protein Adducts, and DNA Adducts as Biomarkers of Electrophilic Chemicals Ronald T. H. van Welie, Rob G. J. M. van Duck, and Nico P. E. Vermeulen*
Downloaded by [Australian National University] at 15:49 05 November 2015
Department of Pharmacochernistry, Division of Molecular Toxicology, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Nico J. van Sittert Health Safety and Environment Division, Shell International Petroleum Maatschappij, P.O. Box 162, 2501 AN The Hague, The Netherlands
*
To whom all correspondence should be addressed
ABSTRACT: The possibilities and limitations of using mercapturic acids and protein and DNA adducts for the assessment of internal and effective doses of electrophilic chemicals are reviewed. Electrophilic chemicals may be considered as potential mutagens and/or carcinogens. Mercapturic acids and protein and DNA adducts are considered as selective biomarkers because they reflect the chemical structure of the parent compounds or the reactive electrophilic metabolites formed during biotransformation. In general, mercapturic acids are used for the assessment of recent exposure, whereas protein and DNA adducts are used for the assessment of semichronic or chronic exposure. 2-Hydroxyethyl mercapturic acid has been shown to be the urinary excretion product of five different reactive electrophilic intermediates. Classification of these electrophiles according to their acidbase properties might provide a tool to predict their preference to conjugate with either glutathione and proteins or with DNA. Constant relationships appear to exist in the cases of 1,2-dibromoethane and ethylene oxide between urinary mercapturic acid excretion and DNA and protein adduct concentrations. This suggests that mercapturic acids in some cases may also play a role as a biomarker of effective dose. It is concluded that simultaneous determination of mercapturic acids, protein and DNA adducts, and other metabolites can greatly increase our knowledge of the specific roles these biomarkers play in internal and effective dose assessment. If the relationship between exposure and effect is known, similar to protein and DNA adducts, mercapturic acids might also be helpful in (individual) health risk assessment. KEY WORDS: biological monitoring, electrophilic chemicals, pesticides, risk assessment, thioethers, mercapturic acids, macromolecular adducts.
ABBREVIATIONS AFB BE1 B(a)P CI DCP DBE
Aflatoxin B, Biological exposure index Benzo(a)pyrene Chemical ionization 7,3-Dichloropropene 1,2-Dibromoethane
DNA ECD E-DCP EI ELISA EO FID FPD
Deoxyribonucleic acid Electron capture detection E- 1,3-Dichloropropene Electron impact Enzyme-linked immunosorbent assay Ethylene oxide Flame ionization detection Flame photometric detection
1040-8444/92/$.50
0 1992 by CRC Press, Inc
271
Downloaded by [Australian National University] at 15:49 05 November 2015
Gas chromatography GC 2-GEMA N-Acetyl-S-(2-[N-7-guanyl]ethyl)-~cy steine Glutathione GSH Glutathione S-transferase GST Hemoglobin Hb 2-HEMA N-Acetyl-S-(2-hydroxyethyl)-~cy steine High-performance liquid HPLC chromatography Immuno slot blot technique ISB Mass spectrometry MS Nuclear magnetic resonance NMR Nitrogen phosphorus detection NPD Occupational exposure limit OEL Polycyclic aromatic hydrocarbon PAH Radioimmunoassay RIA Sui I"ny dry i SH Time-weighted average TWA USERIA Ultrasensitive enzymatic radioimmunoassay Ultraviolet uv Z-DCP Z- 1,3-Dichloropropene
c chcmical
I
t
ToxiWcauon ( I ) sponlanmus (2) oxidation (3) conjugation
I Covalent binbng
Directly gcnoloxlc Chunlcal
Alkylrled nucluc acid baes. dkylalcd amim sdr
wirh cellular macromolecules
R N A adducu
DNA addrrls
P m m adducls
Cellular lesions. toxicity
FIGURE 1. Possible metabolic pathways of (geno)toxic chemicals. (Adapted and modified from Van Sittert, N. J. and De Jong, G., Food Chem. Toxicol.,
23,23,1985.)
1. INTRODUCTION The annual world production of man-made chemicals such as pesticides, drugs, plastics, fibers, and petroleum products continues to rise year after year. Man and his environment are likely to be exposed to many of these body-foreign chemicals or xenobiotics. I Special attention must be paid to genotoxic chemicals possessing or acquiring (by biotransformation processes) electrophilic properties capable of altering DNA integrity. Genotoxic chemicals may exert not only mutagenic and carcinogenic, but also neurotoxic, teratogenic, reproductive-toxic, and other toxic effects on organs such as the liver, kidney, spleen, testes, heart, and lung. The possible metabolic pathways of direct- and indirect-acting mutagens and carcinogens are shown in Figure 1. Human exposure needs to be kept under surveillance. In order to assess the health risk associated with exposure to potentially toxic chemicals, biological monitoring methods can be very useful. However, most assays in biological monitoring are only concerned with exposure assessment, not with risk assessment. Effects of exposure also can differ between individuals due to differences in toxicokinetics and -dynamics. In272
dividual dose monitoring is therefore playing an increasingly important role in occupational toxicology. Biomarkers, the end points used in biological monitoring, can generally be subdivided into biomarkers of dose, response, and susceptibility (e.g., i n h e r e n ~ e ) .This ~ . ~ review is restricted to biomarkers of dose, of which two types are recognized, i.e., biomarkers of internal dose and those of effective or target dose. The internal dose is the total amount of a xenobiotic taken up by the organism. It is assessed by measuring the concentration of the parent compound or its metabolite(s) in body fluids or tissues. The target dose is the dose that evades metabolic detoxification and penetrates to the biologically significant sites in DNA.4 The interaction with DNA is presumed to be mechanistically relevant by being an etiological determinant in the process of mutagenicity and/or carcinogenicity .5 The biologically effective dose is assessed by determining the amount of the parent compound or metabolite(s) interacting with cellular macromolecules, e.g., DNA, RNA, and proteins. Exposure to potentially genotoxic and other toxic chemicals may result in the formation of
Downloaded by [Australian National University] at 15:49 05 November 2015
products that may be biomarkers of internal or effective dose. The level of formation of either type of biomarker depends on many factors and has to be (experimentally) determined. Figure 2 gives a schematic view of the reactions of either “hard” or “soft” electrophilic xenobiotics and/ or metabolites reacting covalently with DNA/ RNA and/or protein/glutathione (GSH) (for further details on the classification of electrophiles see Section IV.B.2). Whether a chemical tends more to the top or the bottom of the distinct fields in Figure 2 not only determines the level of formation of certain types of biomarkers but, even more importantly, also determines the level of DNA adduct formation, i.e., toxification, and GSH conjugation, i.e., detoxification. During the last decade, there has been increased research interest in the potential for GSH conjugation and macromolecular adduct formation.6.7
Covalent binding
DNA
i
Protein and GSH
FIGURE 2. Schematic relationship between biotransformation of potentially electrophilic chemicals, the classification of reactive intermediates according to “hard” and “soft” acid-base properties, the cellular targets alkylated, the type of biomarkers formed, and the possible toxic effects. The horizontal arrow demonstrates a possible sequence of events in time; vertical arrows show the continuous scale of each box.
The aim of this review is to compare biomarkers of internal and effective dose and to evaluate their possibilities and limitations in occupational monitoring studies. Special attention is paid to biomarkers of internal dose derived from GSH conjugation; the biomarkers of effective dose discussed are DNA and protein adducts.
II. BIOMARKERS OF INTERNAL DOSE In principle, GHS conjugation-derived metabolites can be used as biomarkers of internal dose. GSH, a tripeptide consisting of the amino acids glycine, cysteine, and y-glutamine, plays an important role in the detoxification of potentially electrophilic chemicals or metabolities.6.’2 In contrast, toxification via GSH conjugation, e . g . , of 1,2-dibromoethane, hexachlorobutadiene,8 benzyl- and allylis~thiocyanate,~ has also been reported, P-Lyase-dependent bioactivation of cysteine conjugates, derived from the initially formed GSH conjugates, sometimes results in the formation of new reactive intermediates that are responsible for carcinogenic, mutagenic, and other toxicological effects. ‘‘*’I The initial step in GSH conjugation is reaction of the nucleophilic sulfhydryl with electrophilic centers of a chemical. GSH conjugation is catalyzed by a family of GSH S-transferase (GST) enzymes. A wide range of chemicals can be handled by this enzyme system due to the existence of a large number of isoenzymes with different, though overlapping, substrate selectivity. An organism’s ultimate detoxification capacity through GSH and GST enzymes depends on endogenous factors such as tissue distribution, genetic deficiencies, aging and hormonal influences, and on exogenous factors such as sensitivity to inhibition and induction of GSTs.I2 GSH conjugates are not normally excreted unchanged in urine or feces. Catabolism of GSH conjugates results in the formation and excretion of a variety of sulfur-containing metabolites, among which mercapturic acids (S-substituted N acetyl-cysteine conjugates) are some of the most important.I3 The first step in catabolism of the GSH conjugate formed is removal of the glutamyl moiety by y-glutamyltranspeptidase of which the kidney possesses the highest activity, followed by the pancreas. l 4 Cysteinylglycinase and aminopeptidases catalyze the removal of glycine of the cysteinylglycine S-conjugates to form the corresponding cysteine conjugate. The highest activity of these peptidases is found in the liver and kidney.” The final step in mercapturic acid formation is N-acetylation of the cysteine S-conjugates by N-acetyltransferases, notably located
273
in the liver and the kidney. The mercapturic acid pathway is shown in Figure 3.
C0N HCH, COOH
R X + HS-CH~-
