Use of DNA adducts to identify human health risk from exposure to hazardous environmental pollutants: the increasing role of mass spectrometry in assessing biologically effective doses of genotoxic carcinogens

The carcinogens to which humans are exposed are normally in the form of complex mixtures, and much effort has gone into determining the nature of the most significant carcinogenic components in these mixtures and their mechanisms of action. Essential to achieving this aim in exposed populations is the use of biomarkers, which can characterize the chemical nature of the carcinogens involved and identify key biological effects that result from the exposure. DNA adducts are particularly appropriate as biomarkers in the case of genotoxic carcinogens as they indicate the biologically effective dose of the genotoxin in the target tissue under study. This review considers in particular the use of mass spectrometry (MS), which is having an increasing role in the determination of DNA adducts. Compared to other existing DNA damage detection methods, such as 32P-postlabeling, HPLC-fluorescence or electrochemical detection, immunoassay-based techniques and modified Comet assays, MS provides improved structural characterization of adducts. Greater selectivity in the analyses is achieved by the use of tandem MS with selected reaction monitoring or constant neutral loss of ions. Use of capillary/nano liquid chromatography and micro/nano electrospray ionization improves the analytical sensitivity and higher throughput may be obtained by the use of online-column switching. The application of microfluidics technology offers exciting new possibilities for interfacing sample preparation to the mass spectrometer. Despite these improvements in the use of MS for adduct detection, the main current requirement is to validate these methods both analytically and in molecular epidemiology studies. More knowledge of the stability of stored samples is required. Development of sensitive mass spectrometric DNA adductomic screening systems, and of long-term biomarkers (e.g., phosphotriester adducts that are not repaired efficiently) seems important areas for the future assessment of the effects of human exposure to environmental genotoxins, together with studies of dose-response relationships at low doses.     

Studies using specific biomarkers for human exposure assessment to exogenous and endogenous chemical agents.

The extent of formation of carcinogen adducts with DNA and protein may be used to assess the biologically effective dose of these carcinogens in the tissue under study. In normal human tissues, such carcinogen adducts arise in part from exposures to exogenous genotoxic compounds, although it has been shown that endogenously formed carcinogens also make a significant contribution to the observed DNA and protein damage. Although, highly sensitive analytical methods, such as immunoassay, 32P-postlabelling and mass spectrometry have been developed and successfully applied to measure carcinogen adducts, further methodological advances are making these methods more amenable to molecular epidemiological studies. Thus, the use of immunoslot blot assays allows a higher sample throughput for adduct quantification. Liquid chromatographic separations of adducts, either for their radiochemical detection following 32P-postlabelling or for their determination by mass spectrometry, improves the specificity and applicability of these techniques. In this review, the sensitivities and specificities of the analytical methods used for adduct detection are compared and the limitations of these methods described.     

Polycyclic nitroarenes (nitro-PAHs) as biomarkers of exposure to diesel exhaust

Diesel exhaust contains numerous genotoxic carcinogens. It is essentially unknown to which extent this source contributes to the total load of these chemicals in humans. One possible approach to the problem is to find suitable biomarkers. To this end five polycyclic mononitroarenes (nitro-PAH) were selected and methods developed to determine the sulfinic acid-type hemoglobin adducts they form in vivo. The nitro-PAHs are: 1-nitropyrene, 2-nitrofluorene, 3-nitrofluoranthene, 9-nitrophenanthrene, and 6-nitrochrysene. Hydrolysis of the hemoglobin adducts yields the respective arylamines which were analyzed by gas chromatography/mass spectrometry. The detection limit was 0.01-0.08 pmol/g Hb. Blood samples were analyzed from 29 bus garage workers, occupationally exposed to diesel exhaust, and from 20 urban hospital workers and 14 rural council workers as controls. Hb adducts above the detection limit were found in most blood samples. The most abundant cleavage products were 1-aminopyrene and 2-aminofluorene with levels ranging from 0.01 to 0.68 pmol/g Hb. However, there was no significant difference between the groups for 1-nitropyrene and 2-nitrofluorene supporting the conclusion that both are widespread environmental contaminants resulting in significant background exposures. A significant difference on a group from individuals from urban and rural areas was found only if all five adducts were added, this may indicate an additional exposure from traffic. The new specific nitro-PAH Hb adducts are proposed to be used as biomarkers to trace the sources and to identify above-background exposures.     

32P-adduct assay: Short- and long-term persistence of 2-acetylaminofluorene-DNA adducts and other applications of the assay

³²P-Postlabeling techniques have been developed to detect and measure adducts formed by covalent binding of carcinogens of Known or unknown origin with DNA (and RNA). The assay is applicable to various classes of chemical carcinogens and permits detection of many adducts at attomole (10^–18 mol) level using microgram amounts of DNA. Here, we demonstrate the application of the assay for the analysis of short- and long-term persistence of 2-acetylaminofluorene-DNA adducts in rat liver in vivo and also outline examples illustrating the applicability of the procedure to different experimental problems.Abbreviations AAF 2-acetylaminofluorene - N-OH-AAF N-hydroxy-2-acetylaminofluorene     

Monitoring human exposure to carcinogens by DNA adduct measurement

Sensitive immunologic techniques are now available for the detection and quantitation of carcinogen-DNA adducts. We have developed a number of specific monoclonal antibodies which recognize DNA modified by particular carcinogens, including benzo[a]-py-rene, aminopyrene, 8-methoxypsoralen plus UVA light and vinyl chloride. These antibodies can be used in competitive enzyme-linked immunosorbent assays to quantitate adducts in DNA isolated from biological samples. Samples from treated animals as well as from humans with occupational or environmental exposure to carcinogens have been studied. In addition, antibodies can be used in indirect immunohistochemical studies to localize adduct formation in various tissues or cell types.Abbreviations BPDE-1 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - CTCL Cutaneous T cell lymphoma - ELISA enzyme linked immunosorbent assay - 8-MOP 8-methoxypsoralen - PUVA 8-MOP plus UVA irradiation     

Analysis of DNA and protein adducts of benzo[a]pyrene in human tissues using structure-specific methods

We review studies which investigate the presence, using structure-specific analytical methods, of DNA or protein adducts of the carcinogen benzo[a]pyrene (BaP) in human tissues. The analytical methods include high performance liquid chromatography with fluorescence detection and gas chromatography-mass spectrometry. Although, for DNA detection these methods are somewhat less sensitive than non-specific techniques such as 32P-postlabeling and immunoassay, they have the distinct advantage of providing reliable structural information. In order to achieve adequate sensitivity, these methods often require the use of fairly large amounts of DNA (>100 microg) or protein (50-100mg). Most studies reviewed here measured tetraols released from DNA or protein by hydrolysis of adducts derived from (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), a major ultimate carcinogen of BaP. BPDE-DNA adducts were detected in 39% of 705 samples analyzed. BPDE-protein adducts were found in 59% of 772 samples. There was no single exposure situation that led to an overwhelming presence of detectable adducts. For example, BPDE-DNA adducts were detected in 45% of smokers, 33% of former smokers, 52% of non-smokers, 39% of occupationally exposed individuals, and 34% of environmentally exposed people. Adduct levels were influenced by polymorphisms in carcinogen metabolizing genes such as GSTM1, the presence of which was frequently protective. The relatively high occurrence of non-detectable adducts may result from low levels of BaP exposure and host factors such as genetic polymorphisms. Our analysis demonstrates that the presence of BaP adducts in human tissues cannot be assumed, even in situations where exposure to BaP is relatively high.     

Studies on biomarkers in cancer etiology and prevention: a summary and challenge of 20 years of interdisciplinary research

Sensitive, specific methods have been developed that allow quantitative measurements of the metabolites of carcinogen metabolites and of DNA and protein adducts in humans exposed occupationally, environmentally and endogenously to genotoxic agents. The interrelationship between exposure to carcinogens, host risk factors and the responses of biomarkers has been examined in cross-sectional, ecological and case-control studies which provided new insights into the causes of cancer and the mechanisms of carcinogenesis. The identification of hitherto unknown DNA-reactive chemicals formed in the human body from dietary precursors and of carcinogenic components of complex mixtures has increased the possibility of establishing causal relationships in etiology. The identification of individuals and subgroups heavily exposed to carcinogens has led to the development of measures for avoiding or decreasing exposure to carcinogenic risk factors. New, ultrasensitive methods for measuring DNA adducts allow the quantification and structural elucidation of specific DNA damage in humans arising from oxidative stress and lipid peroxidation (LPO), which have been found to be the driving forces in several human malignancies. Background DNA damage in "unexposed" individuals has been shown unequivocally to be due to LPO products, and a significant interindividual variation in adduct levels has been shown in individuals with comparable exposure to carcinogens. Thus, pharmacogenetic variants with higher susceptibility to carcinogenic insults, due to genetic polymorphism in xenobiotic-metabolizing enzymes, have been characterized by a combination of genotyping and measurements of macromolecular adducts. Dosimetry has been used in human studies to evaluate the efficacy of interventions with chemopreventive agents like ascorbic acid, dietary phenols and green tea. Advances in the application of selected biomarkers in human studies are reviewed and illustrated by examples from the author's research conducted during the past two decades.     

The organ-specific induction of DNA adducts in 2-acetylaminofluorene-treated rats, studied by means of a sensitive immunochemical method

Exposure of cells to chemical carcinogens and mutagens may result in the formation of DNA adducts, which can give rise to mutations in the genome and to cellular transformation. Methods to measure DNA-adduct formation may be useful for ‘biomonitoring’, to establish exposure of laboratory animals or humans to DNA-damaging agents. For such purposes, immunochemical methods appear to be suitable, because they allow sensitive detection and quantification of DNA adducts in small amounts of sample in a non-radiolabelled form. We have worked out optimal conditions for the detection of DNA adducts by means of competitive enzyme-linked immunosorbent assay (ELISA). This technique involves interaction of soluble antigen, immobilized antigen and antibody. It appeared that the sensitivity of the competitive assay can be improved by lowering the amount of immobilized antigen, adsorbed to the wall of the plastic reaction vessel. On the basis of these observations, suitable conditions were selected for a sensitive quantitative assay of adducts in DNA isolated from various organs of rats, treated (p.o.) with the liver carcinogen 2-acetylaminofluorene (2-AAF). Under the conditions of these experiments, the available rabbit antiserum recognizes the guanosine-AAF adduct with high specificity. A time- and dose-dependent induction of AAF adducts could be measured in liver DNA from exposed rats, whereas the amount of adducts in DNA from spleen and nucleated blood cells remained below the detection limit (1 adduct/10⁸ nucleotides). The implications of these findings with respect to the relevance of blood cell biomonitoring for target cell exposure are discussed.     

DNA adducts in humans as dosimeters of exposure to environmental, occupational, or dietary genotoxins.

The quantitation of adducts of genotoxins with DNA is probably one of the best indicators of genetic damage due to exposure to toxins or carcinogens. It is generally believed that such adducts can lead to mutations, which in turn can trigger the initiation of the carcinogenic process. DNA adducts have been quantitated in white blood cells and in various tissues of smokers, persons in certain high-exposure occupations, and persons consuming foods contaminated with certain carcinogens. The feasibility of this approach for biochemical epidemiologic studies has been demonstrated using methods such as 32P-postlabeling, enzyme-linked immunosorbent assay, and synchronous fluorescence spectrophotometry. Relatively large interindividual differences in DNA adducts have been observed in both exposed and nonexposed persons. As a result, there are only a few studies in which clear quantitative and qualitative differences between these two groups have been observed. In addition, it appears that in some studies the 32P-postlabeling method does not detect the presence of the polycyclic aromatic hydrocarbon DNA adducts that are detectable by immunoassays. More extensive studies in additional populations at risk should shed further light on the utility of DNA adduct analysis in biochemical monitoring, especially if further refinements in methodology would result in increased sensitivity and specificity.     

Monitoring occupational exposure to carcinogens: detection by 32P-postlabelling of aromatic DNA adducts in white blood cells from iron foundry workers

Blood samples were volunteered by workers in a Finnish iron foundry who were occupationally exposed to polycyclic aromatic hydrocarbons and from control subjects not known to be occupationally exposed to this class of chemical carcinogens. DNA was isolated from peripheral white blood cells and digested with micrococcal nuclease, spleen phosphodiesterase and nuclease P1. The DNA digest was then incubated with [gamma-32P]ATP and polynucleotide kinase. Aromatic adducts present in the digest that were resistant to nuclease P1 were thus 32P-labelled while unmodified nucleotides were not. The 32P-labelled adducts were resolved by t.l.c. and detected by autoradiography. Foundry workers were classified as belonging to high, medium or low exposure groups according to their exposure to airborne benzo[a]pyrene (high greater than 0.2, medium 0.05-0.2, low less than 0.05 microgram BP/m3 air). Aromatic adducts were found to be present in DNA from 3/4 samples from the high exposure group, 8/10 samples from the medium exposure group. 4/18 samples from the low exposure group and 1/9 samples from the unexposed controls. The levels of adducts found in the high and medium group samples ranged up to 1 adduct in 10(7) nucleotides but the levels formed in the low exposure group samples were not significantly different from those in unexposed controls. No differences related to the smoking habits of the subjects were observed. Most of the DNA adducts detected had chromatographic mobilities distinct from those formed when the 7,8-diol 9,10-oxide of BP reacted with DNA. The results indicate that highly-exposed individuals are more likely to contain aromatic DNA adducts in their white blood cells, but large interindividual variations were evident. In addition, multiple samples from the same subjects indicate that qualitative and quantitative changes in adduct patterns occur with time. This pilot study suggests that 32P-postlabelling may be useful in monitoring human exposure to known and to previously unidentified environmental genotoxic agents.     

High-performance liquid chromatographic analysis of 32P-postlabeled DNA-aromatic carcinogen adducts

The technique of 32P postlabeling of DNA-carcinogen adducts is a useful and extremely sensitive method of detecting and quantitating DNA damage by carcinogens. We have adapted the 32P method to analysis by high-pressure liquid chromatography, making the procedure more rapid and convenient than when thin-layer chromatography is used. Following DNA isolation and hydrolysis, nucleotide-carcinogen adducts are enhanced relative to normal nucleotides by solvent extraction and then labeled with high-specific-activity [gamma-32P]ATP. The resulting 32P-postlabeled nucleotides are resolved by reverse-phase ion-pair HPLC. After as little as 3 h of exposure to carcinogens, DNA adducts can be demonstrated from 1 microgram or less of mouse hepatic DNA. Acetylated and nonacetylated adducts can be resolved from hepatic DNA of mice treated with 2-aminofluorene. Differences in DNA damage as measured by adduct formation were demonstrated between "rapid" and "slow" acetylator mouse strains. Rapid-acetylator C57BL/6J mice had three times the amount of hepatic DNA adducts as slow-acetylator A/J mice 3 h after a 60 mg/kg dose of 2-aminofluorene. 4-Aminobiphenyl and 2-naphthylamine each showed an adduct peak with retention time similar to that of the nonacetylated 2-aminofluorene adduct, while benzidine gave a major adduct that eluted somewhat earlier as would be expected for an acetylated adduct. The alkenylbenzenes, safrole and methyleugenol, also formed DNA adducts detectable by this method. DNA prepared from skin of mice painted with benzo[a]pyrene also contained carcinogen-DNA adducts detectable and resolvable by HPLC analysis following 32P postlabeling. The combination of HPLC with 32P postlabeling appears to be a useful technique for the rapid detection and quantitation of DNA damage caused by several classes of aromatic carcinogens.     

Immunological detection of lesions in DNA

The purpose of this paper is to show that the antibodies to nucleic acids, to nucleosides or to DNA damaged by a physical or a chemical agent, are useful tools in the study of DNA damage and repair. The results obtained with antibodies to nucleosides, antibodies to nucleosides and DNA modified by chemical carcinogens emphasize the potential of immunological methods in three main areas, a) the sensitive detection and quantitation of adducts; b) the visualization of adducts in tissues, individual cells, and along the DNA double helix; c) the study of conformational changes of DNA induced by adducts.     

Haemoglobin adducts as biomarkers of exposure to tobacco-related nitrosamines

A sensitive gas chromatography/mass spectrometry (GC/MS) method was developed to measure nitrosamine-haemoglobin adducts (HPB-Hb) (4-hydroxy-3-pyridinyl-1-butanone) at trace levels in red blood cells of smoking and non-smoking mothers and their newborn babies. GC/MS methods with chemical ionization (CI) of methane reagent gas in both positive and negative ion mode as well as electron ionization (EI) were studied to determine differences in sensitivity among the various ionization methods. Detection limits using both positive and negative chemical ionization modes were found to be 30 fmol HPB, whereas detection using electron impact modes yielded a detection limit of 80 fmol HBP. In order to apply the various methods of detection to tobacco-exposed samples from human populations, we characterized adduct levels in maternal as well as paired fetal samples obtained from mothers exposed to tobacco smoke during pregnancy. Maternal samples were characterized using serum cotinine levels and were classified as non-smokers, passively smoke-exposed women, less than one pack per day smokers and greater than one pack per day smokers. Paired maternal and fetal blood samples were obtained at delivery for qualitative and qualitative analysis of nitrosamine adducts. Comparative derivatization of HPB released under alkaline hydrolysis conditions was performed using O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) and 2,3,4,5,6-pentafluorobenzoylchloride (PFBC). Both negative CI and positive CI modes of analysis were compared to the more widely accepted EI modes of mass spectrometric analysis. These results suggest that both NICI and PICI modes of detection offer a greater sensitivity of adduct characterization when compared with EI ionization techniques and that either NICI or PICI modes are preferably applicable towards the detection of human biomarker assessment of tobacco-related nitrosamines.     

In vitro evolution of RNA aptamers recognizing carcinogenic aromatic amines

The modification of cellular DNA by environmental substances is thought to be a crucial event in chemical induced carcinogenesis. Among the environmental carcinogens, aromatic amines are known for the fact that they can induce several types of cancers through the formation of so-called DNA adducts. We took advantage of the potential of the SELEX method to select for highly specific RNA ligands that recognize specific genotoxic aromatic amines. The aromatic amine 4,4'-methylenedianiline (MDA) was used as a target. Following in vitro selection, we obtained specific MDA-binding RNA molecules based on an affinity chromatography assay. These results open the possibility of using the SELEX technique to generate RNA molecules as diagnostic tools for the detection of DNA damaging compounds and ultimately DNA adducts.     

Correlation of DNA adduct levels with tumor incidence: carcinogenic potency of DNA adducts

The quantitative relationship between DNA adducts and tumor incidence is evaluated in this review. All available data on DNA adduct levels determined after repeated administration of a carcinogen to rats or mice have been compiled. The list comprised 27 chemicals, of all major structural classes of carcinogens. For the correlation with tumor incidence, the DNA adduct levels measured at the given dose were normalized to the dose which resulted in a 50% tumor incidence under the conditions of a 2-year bioassay (TD50 dose). In rat liver, the calculated adduct concentration 'responsible' for a 50% hepatocellular tumor incidence spanned from 53 to 2083 adducts per 108 nucleotides, for aflatoxin B1, tamoxifen, IQ, MeIQx, 2,4-diaminotoluene, and dimethylnitrosamine (in this order). In mouse liver, the respective figures were 812 to 5543 adducts per 108 nucleotides, for ethylene oxide, dimethylnitrosamine, 4-aminobiphenyl, and 2-acetylaminofluorene. The observed span (40-fold in rats, 7-fold in mice) reflects differences between the various DNA adducts to lead to critical mutations. If additional carcinogens fit in with this astonishingly narrow range, the measurement of DNA adduct levels in target tissue has the potential to be not only an exposure marker but an individual cancer risk marker. For toremifen and styrene, low levels of DNA adducts were detected in rat liver at the end of a negative long-term bioassay. This shows that the limit of detection of DNA adducts can be well below the limit of detection of an increased tumor incidence. For a cancer risk assessment at low levels of DNA damage, treatment-related adducts must be discussed in relation to the background DNA damage and its inter- and intraindividual variability.     

Hydroxyl-specific fluorescence labeling of ABP-deoxyguanosine, PhIP-deoxyguanosine, and AFB1-formamidopyrimidine with BODIPY-FL

Detection and analysis of DNA adducts resulting from endogenous or exogenous exposures to carcinogens are essential not only for quantifying biologically effective doses but also for establishing relationships between exposure and cancer risk. We have developed and validated a procedure of high sensitivity and specificity based on fluorescence labeling of DNA adducts combined with high-performance liquid chromatography-laser-induced fluorescence detection. The fluorescent dye 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL) was used to label the deoxynucleoside adducts N-(2'-deoxyguanosine-8-yl)-4-aminobiphenyl and N-(2'-deoxyguanosine-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and the base adduct aflatoxin B(1)-formamidopyrimidine by acylation. The labeling reaction was carried out on adducts at 1pmol to 30nmol concentrations at 25 degrees C for 4h in dichloromethane with 200- to 5000-fold excess of BODIPY FL. BODIPY FL and its activating agents 1,3-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were used at a molar ratio of 1:2:2. Under these conditions, all of the above adducts were quantitatively converted to bis-labeled products, as confirmed by mass spectrometry. Sites of derivatization of adduct deoxynucleosides were established primarily by nuclear magnetic resonance and by collision-induced dissociation mass spectrometric analysis, which indicated that the bis-BODIPY groups were located predominantely on the 3'- and 5'-hydroxyl groups of the deoxyribose ring.     

Whole body exposure of mice to secondhand smoke induces dose-dependent and persistent promutagenic DNA adducts in the lung

Secondhand smoke (SHS) exposure is a known risk factor for lung cancer in lifelong nonsmokers. However, the underlying mechanism of action of SHS in lung carcinogenesis remains elusive. We have investigated, using the (32)P-postlabeling assay, the genotoxic potential of SHS in vivo by determining the formation and kinetics of repair of DNA adducts in the lungs of mice exposed whole body to SHS for 2 or 4 months (5h/day, 5 days/week), and an ensuing one-month recovery period. We demonstrate that exposure of mice to SHS elicits a significant genotoxic response as reflected by the elevation of DNA adduct levels in the lungs of SHS-exposed animals. The increases in DNA adduct levels in the lungs of SHS-exposed mice are dose-dependent as they are related to the intensity and duration of SHS exposure. After one month of recovery in clean air, the levels of lung DNA adducts in the mice exposed for 4 months remain significantly higher than those in the mice exposed for 2 months (P<0.0005), levels in both groups being significantly elevated relative to controls (P<0.00001). Our experimental findings accord with the epidemiological data showing that exposure to smoke-derived carcinogens is a risk factor for lung cancer; not only does the magnitude of risk depend upon carcinogen dose, but it also becomes more irreversible with prolonged exposure. The confirmation of epidemiologic data by our experimental findings is of significance because it strengthens the case for the etiologic involvement of SHS in nonsmokers' lung cancer. Identifying the etiologic factors involved in the pathogenesis of lung cancer can help define future strategies for prevention, early detection, and treatment of this highly lethal malignancy.     

IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. International Programme on Chemical Safety

The purpose of these guidelines is to provide concise guidance on the planning, performing and interpretation of studies to monitor groups or individuals exposed to genotoxic agents. Most human carcinogens are genotoxic but not all genotoxic agents have been shown to be carcinogenic in humans. Although the main interest in these studies is due to the association of genotoxicity with carcinogenicity, there is also an inherent interest in monitoring human genotoxicity independently of cancer as an endpoint.The most often studied genotoxicity endpoints have been selected for inclusion in this document and they are structural and numerical chromosomal aberrations assessed using cytogenetic methods (classical chromosomal aberration analysis (CA), fluorescence in situ hybridisation (FISH), micronuclei (MN)); DNA damage (adducts, strand breaks, crosslinking, alkali-labile sites) assessed using bio-chemical/electrophoretic assays or sister chromatid exchanges (SCE); protein adducts; and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations. The document does not consider germ cells or gene mutation assays other than HPRT or markers of oxidative stress, which have been applied on a more limited scale.     

Separation and detection methods for covalent drug-protein adducts

Covalent binding of reactive metabolites of drugs to proteins has been a predominant hypothesis for the mechanism of toxicity caused by numerous drugs. The development of efficient and sensitive analytical methods for the separation, identification, quantification of drug-protein adducts have important clinical and toxicological implications. In the last few decades, continuous progress in analytical methodology has been achieved with substantial increase in the number of new, more specific and more sensitive methods for drug-protein adducts. The methods used for drug-protein adduct studies include those for separation and for subsequent detection and identification. Various chromatographic (e.g., affinity chromatography, ion-exchange chromatography, and high-performance liquid chromatography) and electrophoretic techniques [e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE, and capillary electrophoresis], used alone or in combination, offer an opportunity to purify proteins adducted by reactive drug metabolites. Conventionally, mass spectrometric (MS), nuclear magnetic resonance, and immunological and radioisotope methods are used to detect and identify protein targets for reactive drug metabolites. However, these methods are labor-intensive, and have provided very limited sequence information on the target proteins adducted, and thus the identities of the protein targets are usually unknown. Moreover, the antibody-based methods are limited by the availability, quality, and specificity of antibodies to protein adducts, which greatly hindered the identification of specific protein targets of drugs and their clinical applications. Recently, the use of powerful MS technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight) together with analytical proteomics have enabled one to separate, identify unknown protein adducts, and establish the sequence context of specific adducts by offering the opportunity to search for adducts in proteomes containing a large number of proteins with protein adducts and unmodified proteins. The present review highlights the separation and detection technologies for drug-protein adducts, with an emphasis on methodology, advantages and limitations to these techniques. Furthermore, a brief discussion of the application of these techniques to individual drugs and their target proteins will be outlined.     

Bioactivation of xenobiotics by prostaglandin H synthase

Prostaglandin H synthase (PHS) catalyzes the oxidation of arachidonic acid to prostaglandin H2 in reactions which utilize two activities, a cyclooxygenase and a peroxidase. These enzymatic activities generate enzyme- and substrate-derived free radical intermediates which can oxidize xenobiotics to biologically reactive intermediates. As a consequence, in the presence of arachidonic acid or a peroxide source, PHS can bioactivate many chemical carcinogens to their ultimate mutagenic and carcinogenic forms. In general, PHS-dependent bioactivation is most important in extrahepatic tissues with low monooxygenase activity such as the urinary bladder, renal medulla, skin and lung. Mutagenicity assays are useful in the detection of compounds which are converted to genotoxic metabolites during PHS oxidation. In addition, the oxidation of xenobiotics by PHS often form metabolites or adducts to cellular macromolecules which are specific for peroxidase- or peroxyl radical-dependent reactions. These specific metabolites and/or adducts have served as biological markers of xenobiotic bioactivation by PHS in certain tissues. Evidence is presented which supports a role for PHS in the bioactivation of several polycyclic aromatic hydrocarbons and aromatic amines, two classes of carcinogens which induce extrahepatic neoplasia. It should be emphasized that the toxicities induced by PHS-dependent bioactivation of xenobiotics are not limited to carcinogenicity. Examples are given which demonstrate a role for PHS in pulmonary toxicity, teratogenicity, nephrotoxicity and myelotoxicity.