Physical/Chemical/Immunologic Analytical Methods Jia-Sheng Wang and John D

unit 2. biomarkers: practical aspects

chapter 4. Physical/chemical/immunologic analytical methods Jia-Sheng Wang and John D. Groopman Unit 2 Chapter 4 Chapter

Summary Introduction

Biomarkers can be used to measure Over the past 25 years, the validated biomarkers to traditionally the presence of a wide variety of development, validation and descriptive epidemiological studies parent compounds and metabolites application of molecular helps to: delineate the continuum in body fluids and excreta, and biomarkers that reflect events from of events between an exposure and serve as biomarkers of internal environmental exposure to the resulting disease; identify smaller dose. Chemical-macromolecular formation of clinical disease (e.g. exposures to specific xenobiotics; adducts formed in blood and cancer) has rapidly expanded our indicate earlier events in the natural tissue or excreted in urine serve knowledge of the mechanisms history of diseases and reduce as biomarkers of exposure as well, of pathogenesis and provided misclassification of dependent and and in many instances reflect both opportunities for devising improved independent variables; enhance exposure and additional relevant tools for disease treatment and individual and group risk monitoring biological processes. An assortment prevention. Molecular epidemiology and assessments; and reveal of analytical techniques have been and its evolving paradigm refers toxicologic mechanisms by which developed to identify and measure to the use of biomarkers in an exposure and a disease are parent compounds, metabolites, epidemiological research (i.e. related (5,6). A unique feature of chemical-DNA and protein incorporation of molecular, molecular epidemiologic studies is adducts. This chapter will discuss cellular and other biochemical the interdisciplinary collaboration many analytical techniques that measurements into epidemiological between population and field measure biomarkers in molecular studies of the etiology, prevention, scientists and laboratory scientists epidemiologic studies, including and control of health risks faced from various disciplines, such as biological, physical, chemical and by human populations)(1–4) epidemiology, toxicology, molecular immunological methods. (Figure 4.1). The application of biology, genetics, immunology,

Unit 2 • Chapter 4. Physical/chemical/immunologic analytical methods 43

Figure 4.1. Molecular epidemiology paradigm

biochemistry, pathology and clinical disease (Figure 4.1). Any step or an altered state recognized as analytical chemistry. The analytic in this process may be modified by impairment or disease. A biomarker measurement of biomarkers is host-susceptibility factors including of susceptibility is an indicator or a critical to molecular epidemiologic genetic traits and effect modifiers (e.g. metric of an inherent or acquired studies and requires special diet or environmental exposures). ability of an organism to respond attention to the collection, handling Therefore, biomarkers are indicators to the challenge of exposure to a and storage of biologic specimens, of events for physiologic, cellular, specific xenobiotic substance or as well as development and subcellular and molecular alterations other toxicant. Such a biomarker may validation of analytical methods (7). in the multistage development of be the unusual presence or absence specific diseases (9). of an endogenous component, or Biomarker paradigm Molecular biomarkers are an abnormal functional response and validation strategies typically used as indicators of to an administered challenge exposure, effect or susceptibility. (9). Molecular epidemiology and As adapted from Perera and A biomarker of exposure refers molecular dosimetry thus have Weinstein (1) and the Committee to measurement of the specific great utility in addressing the on Biological Markers of the agent of interest, its metabolite(s), relationships between exposure National Research Council (8), the or its specific interactive products to environmental agents and development of disease as a result in a body compartment or fluid, development of clinical diseases, of exposure to an environmental which indicates the presence and in identifying those individuals agent or other toxicant is multistage: (and magnitude) of current and at high risk for the diseases (2,6,10). it starts with exposure and past exposure. A biomarker of Collectively, these data also help to progresses to internal dose (e.g. effect indicates the presence inform the risk assessment process, deposited body dose), biologically (and magnitude) of a biological where regulations can be tested effective dose (e.g. dose at the response to exposure to an against biological measurements of site of toxic action), early biological environmental agent. Such a exposure to determine the efficacy effect (e.g. at the subcellular level), biomarker may be an endogenous of policies. altered structure or function (e.g. component, a measure of the The development and subclinical changes) and finally to functional capacity of the system, application of molecular biomarkers

44 for environmental chemical agents Extensive efforts have been made and experimental samples. should be based upon specific to identify these high-risk individuals Conceptually, as shown in Figure knowledge of their metabolism, using various genetic and metabolic 4.2, an appropriate animal model is interactive product formation and susceptibility markers (e.g. used to determine the associative general mechanisms of action measurement of polymorphism of or causal role of the biomarker (11,12). Examples in the field are genotype and phenotype of various in the disease or effect pathway, studies on the relationships between enzymes involved in transformative and to establish relations between tobacco smoking and lung cancer metabolic reactions of certain dose and response. The putative (13–16) and between aflatoxin known carcinogens and the DNA biomarker can then be validated exposure and liver cancer (17,18). repair process)(20–23). Although in pilot human studies, where A specific application of biomarker this strategy has not yet proven to sensitivity, specificity, accuracy technology to human cancer is the be broadly applicable to many other and reliability parameters can be study of the variation in response human diseases, progress is being established. Data obtained in these

among individuals following made for many types of cancers studies can then be used to assess Unit 2 exposures to tobacco. For example, (24). intraindividual or interindividual even in heavy tobacco smokers, less The validation of any biomarker- variability, background levels, 4 Chapter than 15% of these exposed people effect link requires sequential relationship of the biomarker to develop lung cancer (19); thus, or parallel experimental and external dose or to disease status, intrinsic susceptibility factors must human studies (12). Following the as well as feasibility for use in larger affect the time course of disease development of the hypothesis of population-based studies. It is development and eventual outcome. an exposure-disease linkage, there important to establish a connection The identification of those at highest is the need to develop the analytical between the biomarker and risk for developing cancers should methodology necessary to measure exposure, effect or susceptibility. be facilitated by biomarker studies. these biological markers in human To fully interpret the information that

Figure 4.2. Validation scheme for molecular biomarker research

Unit 2 • Chapter 4. Physical/chemical/immunologic analytical methods 45 the biomarker provides, prospective toxicity can vary by both charge interindividual variation in dose– epidemiological studies may be state (e.g. tri- and penta-valent state response of environmental necessary to demonstrate the role of arsenic) (30) and methylation (e.g. agents, the central focus in risk that the biomarker plays in the methyl mercury) (31). Measurement assessment. While biomarkers overall pathogenesis of the disease of parent organic compounds in have been most widely applied in or effect. To date, few biomarkers biological samples, although still in the area of clinical pharmacology, have been rigorously validated practice, is less favoured because they are increasingly being used to using this entire process. most organic toxic/carcinogenic document interindividual variation compounds undergo metabolism and in the context of the much lower Techniques and strategies for exert their toxicologic/carcinogenic exposures found in the environment measuring parent compounds effects through metabolic activation (32,33). There are many examples and metabolites (22). Collectively, the problem of variation in the pharmacologic faced in these investigations is the action of drugs in people; in general, Many analytical techniques, including relationship between exposure the response varies about 10-fold biological, physical, chemical, and and dose. Unlike most organic in the general population for most immunological methods, have compounds, metals’ measurements pharmaceutics. At issue with the been developed and standardized often reflect both exposure and extrapolation of these findings to by regulating agencies (e.g. US dose. Figure 4.3 illustrates a environmentally occurring toxicants Environmental Protection Agency) nomograph relating the increase is the recognition that a limited for biohazard identification and risk in analytical sensitivity by various variance in response is selected assessment in various exogenous instrument methods of the past for during drug development, settings, such as environmental 10–15 years. As these technologies whereas in an environmental and occupational arenas. For have advanced, the number of non- setting, there may be a much wider establishment of a quantitative detects in human epidemiological range of toxicologic outcomes. relationship with exposure, analytical studies has dramatically decreased. This is further complicated by the methods have been extended to Incorporation of biomarkers age, gender and health status measure these parent compounds in in molecular epidemiology can of the general population when biological samples; they could serve provide critical information on compared to people being treated very well as a biomarker of exposure.

A good example is the measurement Figure 4.3. Nomograph for analytical methods of various heavy metals (e.g. lead, arsenic, cadmium and mercury) in human biospecimens, such as urine, blood, hair and tissues (25). In general, most heavy metal measurements have been in environmental samples and technologies including atomic absorption (AA) (26), inductively coupled plasma-optical emission spectrometer (ICP-OES) (27) and inductively coupled plasma-mass spectrometer (ICP-MS)(28). In addition to the measurement of environmental sources of heavy metals, methods such as X-ray fluorescence (XRF) permit the assessment of body burden of lead through its deposition in bone (29). Of importance to molecular epidemiology is the need to speciate the heavy metal compound, since

46 for a clinically diagnosed disease. confirmatory analytical techniques chemical carcinogen, have been Further, exposures to drugs tend and other quality control methods. detected and quantified in the urine to be of shorter duration then the Each sample matrix poses varied of smokers; these metabolites lifetime exposures to environmental challenges for clean-up. Thus, as were not found in the urine of non- compounds that can have wide the complexity of sample clean-up smokers (40). Intraindividual and day-to-day variations in dose. increases, the effective number of interindividual variations in these While the ability of environmental samples that can be analysed in a metabolites of NNK in smokers’ exposures to reach the level given day diminishes. The largest urine were noted and might prove where Km of an enzyme becomes automated, robotically driven to be important in disease risk limiting is debatable, the effective clean-up strategy, The National (41). Two metabolites of NNK, concentration of an agent might well Report on Human Exposure to 4-(methylnitrosamino)-1-(3-pyridyl)- exceed this level at the cellular site Environmental Chemicals (http:// 1-butanol (NNAL) and NNAL-β-O- of action (34). www.cdc.gov/exposurereport/), is D-glucosiduronic acid (NNAL-Gluc),

A very significant consideration based upon the NHANES survey excreted in the urine of smokeless Unit 2 in the design of the molecular samples. This repository has been tobacco users, were found to be epidemiologic investigation is the used to explore many environmental associated with the presence of oral 4 Chapter balance between the analytical exposures to low molecular weight leukoplakia (42). Other examples of sensitivity of the chosen method and chemicals (35,36). However, most internal dose markers include the the operational sample throughput individual laboratories lack this measurement of blood and serum per day. Our infrastructure capacity type of infrastructure for large-scale levels of DDE (1,1-dichloro-2,2- to collect, annotate and store large sample preparation for chemical bis(p-chlorophenyl)-ethylene), the numbers of samples far outpaces analysis; hence, these operational major metabolite of DDT (2,2-bis(p- the number of samples that can be considerations impinge upon the chlorophenyl)-1,1,1-trichloroethane), analysed. Most environmental and size and scope of the molecular which have been used as biomarkers occupational toxicants of concern epidemiology study. in breast cancer studies in women exist at trace levels in human A variety of metabolites of (43,44). samples. Thus, while the biological toxicants/carcinogens found in An example of the use of agent- impact may be substantial, both the body fluids and excreta (e.g. blood, specific biomarkers to identify sample size available and analytical urine, feces, hair and milk) have etiologic factors in human cancer technology employed conspire to the potential for use as biomarkers is the study of aristolochic acid reduce the number of tests that can of internal dose. These measures (45,46). This compound has been be run in a given day. Further, as could provide information about the associated with Balken endemic analytical sensitivity increases, the actual concentration of toxicants/ nephropathy (BEN), a chronic contribution of noise to an analysis carcinogens that have been renal tubulointerstitial disease that also increases, necessitating more absorbed and distributed in the body. often is accompanied by upper extensive clean-up methods to Measurement of these metabolites urinary tract urothelial cancer. maintain an appropriate signal-to- has been incorporated into several Using 32P-postlabelling/PAGE and noise ratio. For most quantitative human epidemiologic studies. For authentic standards, both adenine analyses, reliable measurements example, excretion of aflatoxin M1 and guanine aristolactam DNA can be made only with signal- (AFM1), one of the major metabolites adducts were detected in the renal to-noise ratios that exceed 3:1. of aflatoxin B1 (AFB1), has been used cortex of patients with BEN, but not Unfortunately, judgement about as a biomarker for the evaluation of in patients with other chronic renal the amount of material that will be human exposure to aflatoxin and diseases. In addition, urothelial used for an analysis results in many was found to be associated with the cancer tissue was obtained from samples being at this borderline level risk of liver cancer (37,38). Reflecting residents of endemic villages with of detection. Since the background its complex pharmacokinetics, this upper urinary tract malignancies. from a urine (or other biological) metabolite has also been measured The AmpliChip p53 microarray sample can be very heterogeneous in both human urine and milk samples was then used to sequence exons from person-to-person, many (39). Specific metabolites of one of 2–11 of the p53 gene where 19 pilot studies have inadvertently the tobacco-specific nitrosamines, base substitutions were identified. generated false-positives when 4-(methylnitrosamino)-1-(3-pyridyl)- Mutations at A:T pairs accounted for they do not employ multiple 1-butanone (NNK), a potent 89% of all p53 mutations, with 78% of

Unit 2 • Chapter 4. Physical/chemical/immunologic analytical methods 47 these being A:T→T:A transversions. specific biomarkers in biological measurement. Further, several It was concluded that DNA adducts samples. Many of the studies to investigations have employed hybrid derived from aristolochic acid measure these biomarkers employ methods using HPLC separation with are present in renal tissues of single or multiple chromatographic the collection of fractions followed by patients with documented BEN. step(s) to facilitate biomarker analysis using immunoassays (52). These adducts can be detected detection. In a preparative mode, An obvious advantage of clean- in transitional cell cancers, and this chromatography usually consists up using chromatography before A:T→T:A transversions dominate of high-capacity chromatographic an immunoassay is the removal of the p53 mutational spectrum in the columns using reverse phase, normal potential cross-reactive materials upper urinary tract malignancies phase and ion-exchange resins. that contribute to a false-positive found in this population. These columns are generally gravity result. It is also important to note that The process of toxicant/ or low-pressure devices and provide any clean-up before an analytical carcinogen metabolism produces a crude first stage enrichment measurement lowers the number a variety of reactive electrophilic method. Analytical chromatography of samples per day and potentially intermediates that constitute is used as a low-capacity, but highly- introduces other artefacts, such as biologically effective forms of the selective, technology to separate cross-contamination of samples and ultimate chemicals, such as aflatoxin many of the compounds in a complex stability of the biomarker.

B1-8,9-epoxide, benzo(a)pyrene- mixture. Liquid chromatography Internal standard development 7,8-diol-9,10-epoxide (BPDE), and is the most common mode for is an area of considerable N-acetoxy-2-acetylaminofluorene separation, but many compounds, if importance that has received far (47–49). The actual concentration they are intrinsically or derivatisable less attention than it should. All of the products formed by these to a volatile agent, can be used quantitative measurements require ultimate forms of toxicants/ in gas chromatography (GC). GC the use of an internal standard carcinogens in the human body has a much higher capacity to to account for sample-to-sample or target tissues should serve as resolve different chemical species. recovery variations. In the case biomarkers for biologically effective All chromatography methods are of mass spectrometry, internal dose. Great efforts have been made coupled with a form of spectroscopy standards generally employ an over many years to develop methods for selective detection. For example, isotopically labelled material that is to identify and detect these active GC and high performance liquid physicochemically identical to the metabolic products. These ultimate chromatography (HPLC) coupled chemical that is being measured. toxicants/carcinogens can be online with UV, fluorescence and Obtaining such isotopically labelled directly monitored in various in vitro electron-capture detectors and mass materials does require chemical models and in vivo animal systems spectrometry (MS), are necessary synthesis (if they are not commercially by a variety of sensitive analytical techniques in these studies. available), which has impeded the techniques. These approaches are In general, modern analytical application of internal standards often difficult to apply in human instrumentation can lead to a limit in many studies. In the case of populations simply because of the of detection of chemical biomarkers immunoassays internal standards extremely short half-lives of these in the femptomole (fmol) to low pose a different challenge, since the ultimate toxicants/carcinogens, and picomole (pmol) range. As a caution, addition of an internal standard that the high background of interfering many studies report sensitivity and is recognized by an antibody results substances. Alternative methods limits of detection using standards; in a positive value contribution. The for measuring the formation of DNA however, the operational limits of dynamic range is usually less than and protein adducts in human blood detection are between 10 to 100- 100 in immunoassays; therefore and tissues include measuring their fold higher when measuring real great care must be taken to spike a further metabolites, such as diols, biological samples. sample with an internal standard to conjugates (including glucuronide Immunoassays, such as obtain a valid result (53). In contrast, and mercapturic acids) or nucleic radioimmunoassay (RIA), enzyme- for example, most chromatographic acid base adducts in human urine. linked immunosorbent assay methods result in dynamic ranges of As discussed previously, clean- (ELISA) and immunoaffinity analyses that can be over a 10 000- up methods are required to lower chromatography (IAC), are also used fold range of levels. the noise and enhance the signal for in biomarker analysis (50,51) for both the detection of any of the chemical- preparative methods and analytical

48 Techniques for measuring in single exposure experimental and pancreas were harvested from DNA adducts systems and as a means of each animal, and DNA was isolated elucidating the metabolic activation from each tissue. Adducts were The metabolically activated ultimate of previously uninvestigated detectable in the bladder and liver form of carcinogens can covalently potential carcinogens. 32P-post- DNA samples from every animal interact with cellular DNA, which labelling can give an impression of at every time point, at levels that is a critical step in the process total adduct burden, but it is rarely ranged from three per 109 to 1.5 of carcinogenesis (23,54–56). possible to quantify specific adducts per 107 nucleotides. Adduct levels Measurement of carcinogen-DNA accurately in human samples. were highest in animals given 3,5- adducts has an important role in Advances may lie in the use of better DMA and lowest in those given human biomonitoring and molecular chemical standards, more advanced 3-EA. Taken together, the results epidemiologic studies, as they are preparative techniques, and in strongly suggest that these three specific biomarkers that provide a connection with MS techniques alkylanilines are metabolized in vivo

way to measure human exposure to (3,22). Carcinogen-DNA adduct to electrophilic intermediates that Unit 2 chemical carcinogens and provide detection by fluorescence has been covalently bind to DNA, and that information about a specific dose applied to compounds that lead to adducts are formed in the DNA of 4 Chapter to a carcinogen target site (DNA). either highly fluorescent products bladder, which is a putative target Moreover, it has been possible to or adducts that can subsequently organ for these alkylanilines (68). establish a correlation between be derived to highly fluorescent Many analytical techniques have tumour incidence and exposure by chemical species. Physicochemical been used to measure composite measuring the level of these adducts methods, including MS, offer and specific DNA adducts in cellular (see (57) as an example). the advantage of high chemical DNA isolated from peripheral Many different analytical specificity. Major improvements lymphocytes, bladder, breast, techniques have been developed to in sensitivity have allowed the lung and colonic tissues, as well identify and measure carcinogen- measurement of increasingly smaller as excreted DNA adducts in urine DNA adducts, including: amounts of adducted species in (3,50,60). These techniques have immunoassays: ELISA, RIA, IAC, biological matrices. The sensitivities also been applied in the clinical and immunohistochemical staining of individual methods vary and often setting to examine carcinogen- assay (IHC); radiometric postlabelling depend on the amount of DNA that macromolecular adducts of people methods: 32P-post-labelling; and can be analysed. Detection limits for undergoing chemotherapy with various physicochemical methods: quantitative assays are typically in alkylating agents, in an attempt to GC, HPLC, GC-MS, LC-MS, the range of one adduct in 107 or 109 associate adduct levels with clinical electrochemical detection (ECD), nucleotides. However, accelerator outcome (70,71). Recently, these fluorescence and phosphorescence mass spectrometry (AMS), which is methods have also been applied spectroscopy, or a combination of highly sophisticated and involved in to human clinical trials to validate these methods (4,10,20,50,58– use of low levels of 3H- or 14C-labelled various intervention tools for the 63). Capillary electrophoresis and compound, has a detection limit assessment of chemopreventive other new separation techniques of one adduct in 1012 nucleotides agents in modulating various have improved the sensitivity (66,67). A recent application of intermediate biomarkers (17,72). and specificity of these methods. this technology has been in the Nuclear magnetic resonance (NMR) identification of the fate of a variety Measurement of DNA adducts spectrometry has also been used of alkylanilines in experimental for studying complex to determine stereospecificity and models (68). In this investigation, mixtures of carcinogen three-dimensional structure (64,65). the 14C-labelled 2,6-dimethyl- exposure The 32P-post-labelling assay, (2,6-DMA), 3,5-dimethyl- (3,5- which radioactively labels adducts DMA), and 3-ethylaniline (3-EA) Many studies have used DNA digested from sample DNA, has compounds, associated with adducts to assess potential sources been widely applied because of its human bladder cancer (69), were of carcinogen exposure. One classic high sensitivity and the requirement administered to C57BL/6 mice, study has examined a spectrum of for only microgram amounts of DNA. which were subsequently sacrificed molecular biomarkers to assess This assay has been especially 2, 4, 8, 16 and 24 hours post-dosing. human exposure to complex mixtures useful for detection of adducts Bladder, colon, kidney, liver, lung of environmental pollution in Poland

Unit 2 • Chapter 4. Physical/chemical/immunologic analytical methods 49 (73). Measurement of genotoxic detected in oral mucosa cells of higher levels of 4-ABP-Hb adducts damage in peripheral blood samples smokers and non-smokers. Levels at baseline and after smoking from residents of high-exposure of DNA damage were elevated in cessation. These results show that regions indicated that environmental each of 16 smokers compared to 16 PAH-DNA and 4-ABP-Hb adducts pollution is associated with age-, race-, and sex-matched non- can be useful as intermediate significant increases in carcinogen- smokers. There was about a three- biomarkers by verifying smoking DNA adducts (polynuclear aromatic fold range between smokers and cessation and possibly identifying hydrocarbon (PAH)-DNA and non-smokers (75). persons who are at increased other aromatic adducts), sister Measurement of carcinogen- risk of cancer from exposure to chromatic exchanges, chromosomal DNA adducts in target tissues can cigarette smoke, due to high levels aberrations and frequency of offer useful information related to of carcinogen binding. increased ras oncogene expression. the mechanism of carcinogenesis; In other reports, anti-BPDE- The presence of aromatic adducts however, the limitation of availability DNA adducts were detected in four on DNA was found to be significantly of these specimens in humans is an of seven colon mucosa samples, correlated with chromosomal impediment to extensive studies. but not in any of 11 human pancreas mutation, providing a possible link An alternative method is to use samples from smokers and non- between environmental exposure the DNA isolated from peripheral smokers. Adduct levels in human and genetic alterations relevant to white blood cells (WBC). One colon samples varied between disease. example is the detection of PAH- 0.2–1.0 adducts/108 nucleotides Tobacco smoke, the primary DNA adducts in specific subsets (78). DNA adducts have also been cause of lung cancer, contains of WBC. It was observed that DNA detected in biopsy samples of several types of known carcinogens. combined from lymphocyte and human urinary bladder tissue. Total The most abundant of these are monocyte fractions of smokers, PAH-DNA adduct levels, and the PAHs, arylamines and the tobacco- exhibited detectable levels of DNA average levels of several specific specific nitrosamines, including adducts with a mean of 4.38 ± 4.29 adducts, were significantly elevated the lung-specific carcinogen adducts/108 nucleotides, while the in samples from current smokers, NNK. These carcinogens are corresponding values were 1.35 ± compared to never-smokers and metabolically activated to reactive 0.78/108 (P < 0.001) in non-smokers ex-smokers who had abstained species which form specific DNA (76). The elevated levels of PAH- from smoking for at least five years adducts. Smokers are usually found DNA adducts in DNA obtained from (79). Putative aromatic amine to have significantly elevated levels WBC of smokers compared to non- adducts were detected, one of of aromatic and/or hydrophobic smokers suggested that only certain which displayed chromatographic adducts as compared with non- subsets of WBC are a valid, readily behaviour identical to the smokers, and some studies found accessible source for monitoring predominant adduct induced by the that DNA-adduct levels are linearly genotoxicity from cigarette smoke. human urinary bladder carcinogen, related to total smoking exposure The decline of PAH-DNA and 4-ABP, which is present in cigarette (74). One investigation measured 4-aminobiphenyl-haemoglobin smoke. Immunohistochemical the level of bulky, hydrophobic (4-ABP-Hb) adducts in peripheral quantitation of 4-ABP-DNA adducts DNA adducts in lung parenchyma blood following smoking cessation and p53 nuclear overexpression of smokers and ex-smokers by the in serial samples from 40 heavy in T1 bladder cancer of smokers 32P-postlabelling method. Smokers smokers (≥ 1 pack/day for ≥ 1 year) and non-smokers was described had five-fold higher levels of DNA was described (77). The substantial (80). Mean relative staining adducts than did ex-smokers. A reduction (50–75%) of PAH-DNA intensity for 4-ABP-DNA adducts positive linear correlation between and 4-ABP-Hb adduct levels after was significantly higher in current bulky adduct levels and CYP1A1 quitting indicates that they are smokers compared to non-smokers. (AHH) activity was found in smokers. reflective of smoking exposure, There was a linear relationship A statistically significant correlation which is essential information in between mean level of relative was determined comparing the validation of biomarkers (77). staining and number of cigarettes pulmonary microsomal AHH activity The estimated half-life of the PAH- smoked, with lower levels in the and the level of BPDE-DNA adducts DNA adducts in leukocytes was 1–19 cig/day group, compared to (r = 0.91; P < 0.01) (71). Additionally, 9–13 weeks; for 4-ABP-Hb adducts, the 20–40 and the >40 cig/day BPDE-DNA adducts have been it was 7–9 weeks. Women had groups. Nuclear overexpression of

50 p53 was observed in 27 (59%) of workplace, cigarette smoke, and well documented. Since the initial the 45 stage T1 tumours analysed. through endogenous formation. reports in aniline dye workers, Nuclear staining of p53 was These compounds alkylate DNA, many laboratories have applied correlated with smoking status, cig/ leading to formation of various types technologies for measuring these day, and 4-ABP-DNA adducts. In of DNA adducts, such as 7-alkyl-2’- carcinogens and their metabolites to another study, 4-ABP-DNA adducts deoxyguanosine (dG) (e.g. 7-methyl- confirm exposure in the workplace in 11 human lung and eight urinary dGp and 7-ethyl-dGp). Several (56). The occurrence of DNA bladder mucosa specimens were investigations have focused on the adducts in exfoliated urothelial cells analysed by alkaline hydrolysis and levels of 7-methyl-dG adducts in of a worker exposed to the aromatic negative chemical ionization GC- human lung tissue, where higher amine 4,4’-methylene-bis(2- MS. Adduct levels were found to be levels have been found in smokers chloroaniline) (MOCA), an agent 0.32–49.5 adducts/108 nucleotides in compared to non-smokers (84–87). that induces lung and liver tumours the lung and 0.32–3.94 adducts/108 Separately, 7-methyl-dG levels in in rodents and urinary bladder

nucleotides in the bladder samples lung tissues have been associated tumours in dogs, was reported (91). Unit 2 (81). with cytochrome P4502D6 and 2E1 32P-postlabelling analysis revealed

Carcinogen-DNA adducts in genetic polymorphisms (84). One the presence of a single, major DNA 4 Chapter human breast tissue samples study analysed N7-alkylguanine adduct that cochromatographed have been reported (82). A total of adduct levels in DNA in a group of with the major N-hydroxy-MOCA- 31 breast tissue samples, which 46 patients with larynx tumours by DNA adduct, N-(deoxyadenosin-8- included tumour and tumour- the 32P-postlabelling method. The yl)-4-amino-3-chlorobenzyl alcohol, adjacent tissues from 15 women average level of N7-alkylguanines formed in vitro. PAH-DNA adducts in with breast cancer and normal was 26.2/107 nucleotides in tumour WBC and 1-hydroxypyrene in urine tissue samples from four women cells, 22.7/107 in non-tumour cells, were examined in a group of 105 undergoing breast reduction, and 13.1/107 in blood leukocytes. workers from a primary aluminum were analysed. Among the breast Males and smokers had significantly plant with different PAH exposures cancer cases, the mean aromatic/ higher levels of adducts than females (92). Exposure was measured by hydrophobic-DNA adduct level and non-smokers (88). In another personal monitoring and ranged assayed was 5.3 ± 2.4 adducts/108 study, 7-alkyl-2’-deoxyguanosine from 0.4–150 fg/m3. High exposure nucleotides, compared to 2.3 ± adducts were measured in eight to PAH in the work atmosphere 1.5/108 nucleotides from the non- separate lung segments of 10 was associated with increased cancer patients. Five of 15 tissues autopsy specimens (89). Levels of concentration of 1-hydroxypyrene from the cases displayed a pattern 7-methyl-dGp were detected in all in the urine. PAH-DNA adducts of adducts associated with tobacco eight samples (ranging from 0.3– were detected in 93% of the worker smoke exposure; all of these positive 11.5 adducts/107 dG; mean 2.5 ± samples. Workers with a high PAH samples were from current smokers. 2.3). In all but five of the samples, exposure had significantly higher Tissue samples from the eight 7-ethyl-dGp levels were detected adduct levels than did those with a low non-smoking cases did not exhibit (ranging from <0.1–7.1 adducts/107 PAH exposure. A good correlation this pattern. This study indicated dG; mean 1.6 ± 1.7). 7-methyl-dG was found between PAH exposure that biomarkers may be useful in levels were approximately 1.5-fold and the average PAH-adduct values investigating specific environmental higher than 7-ethyl-dG, and were in blood. A statistically significant exposures that could contribute to positively correlated with each correlation was also found between breast cancer. In another study, other in most individuals. There the average adduct values and the BPDE-DNA and other adducts were was no consistent pattern of adduct concentration of 1-hydroxypyrene in also found in the smooth muscle distribution in the different lung lobar the urine of smokers. layer of atherosclerotic lesions in segments (90). The lymphocyte bulky PAH-DNA abdominal aorta specimens by adduct levels have been examined various analytical methods (83). Measurement of DNA adducts in workers exposed to ambient air Alkylating agents, such for studying occupational pollution (93). A significantly higher as N-nitroso compounds, are carcinogen exposure adduct level was found in bus drivers potential human carcinogens. working in central Copenhagen Humans are known to be exposed Occupational exposure to many compared with those driving in to N-nitrosoamines from diet, chemical carcinogens has been suburban areas. The urban drivers

Unit 2 • Chapter 4. Physical/chemical/immunologic analytical methods 51 had higher adduct levels than rural biomarkers in humans. The urinary Among the many oxidatively controls. There was no observation excretion of aflatoxin metabolites damaged DNA bases formed, of significant influence on adduct in an area of China with a high 8-hydroxy-2’-deoxyguanosine, level by potential confounders incidence of liver cancer was or 8-oxo-7, 8-dihydro-2’- including smoking and diet, studied (97). Total 24-hour urine deoxyguanosine (oxo8dG) is a GSTM1, or NAT2. A separate study samples were collected and lesion that can be sensitively measured BPDE-DNA adducts in analysed by an IAC-HPLC analysis measured. Several techniques 39 coke oven workers (exposed to to determine individual aflatoxins in have been developed and applied PAH) and 39 non-exposed controls the urine samples. The aflatoxins to determine this damage product (each group consisting of smokers most commonly detected were in biofluids and tissue samples 7 and non-smokers) (94). Adducts AFB-N -Gua, AFM1, AFP1 and AFB1; from animals and humans. These were detected in 51% of workers and however, only AFB-N7-Gua and methods include HPLC-EC, GC/ in 18% of controls. The mean level AFM1 showed a dose-dependent MS, immunoassay, fluorescence in workers (15.7x108 nucleotides) relationship between aflatoxin intake postlabelling, 3H-postlabelling and was 15 times higher than in non- and urinary levels, which indicates 32P-postlabelling (100). IA column exposed controls. Although large that these two metabolites might methods have also been described interindividual variations were noted, be useful biomarkers of exposure. for the analysis of oxidative damage smoking workers had 3.5 times Interestingly, these studies also products of nucleic acids excreted more adducts than non-smokers. demonstrated that the kinetics of in urine (101). Quantitative analysis formation and excretion of AFB-N7- of these adducts in the urine of rats Measurement of DNA adducts Gua in urine is almost identical in fed a nucleic acid-free diet suggests in excretion for studying the F344 rat and humans, thereby that 8-oxo-7, 8-dihydroguanine is carcinogen exposure enhancing the value of rodent the principal repair product from studies for assessing risk to humans. oxo8dG in DNA. In addition to In addition to monitoring carcinogen- Modification of N-3 at the these reports, excretion of oxidative DNA adducts in situ in DNA, the position of adenine is a major DNA damage products in urine has excised products of these adducts route of DNA adduct formation for also been correlated with dietary can be determined in urine samples. many alkylating carcinogens. The antioxidant consumption in humans These urinary biomarkers have resulting 3-alkyldeoxyadenosines (102,103). Thus, these markers been especially amenable to are unstable, rapidly depurinating may eventually be used to assess comprehensive validation studies to give the corresponding protection status as well as risk of (95). One example is the examination 3-alkyladenines (3-alkAde) that are disease in people. of the dose-dependent excretion of excreted in urine. These can then Malonaldehyde (MA) is the major urinary aflatoxin biomarkers in the be quantitated by immunochemical reactive aldehyde resulting from the rat following a single exposure to and/or GC-MS methods. In a peroxidation of polyunsaturated

AFB1 (96). The relationship between study of cancer patients receiving fatty acids (PUFA) constituents

AFB1 dose and the excretion of the methylnitrosourea (MNU) as part of biological membranes, and major nucleic acid adduct, AFB1- of combination chemotherapy, 24- is a by-product of prostaglandin N7-Guanine (AFB-N7-Gua), over hour urine samples were collected. biosynthesis (104). MA has been the initial 24-hour period following An analysis of urinary 3-MeAde proven to be carcinogenic in rats, exposure, showed an excellent in smokers showed increased mutagenic in several bacterial and linear correlation between dose excretion of this biomarker. Overall, mammalian mutation assays, and and excretion in urine. In contrast, a dose-dependent excretion of readily reacts with DNA to produce other oxidative metabolites, such 3-MeAde was observed (98). several adducts. Relatively high as aflatoxin P1 (AFP1), revealed no endogenous levels of MA-DNA linear excretion characteristic. Measurement for endogenous adducts have been detected in One approach for the DNA damage healthy individuals (1–10 adducts/107 development and validation of nucleotides). Thus, MA is considered aflatoxin adduct biomarkers has Endogenous oxidative DNA damage an important endogenously entailed parallel experiments in may play an important role in the produced genotoxic agent that may animal models with the systematic formation of chronic degenerative contribute to the development of evaluation of these molecular diseases, including cancer (99). some human cancers, particularly to

52 the carcinogenic effects associated were found to contain the MDA- serum albumin are the proteins with high dietary fat intake. Recent DNA adducts at the level of >1/107 of choice, although efforts have reviews on this field can be found in nucleotides. Age and body mass did been made to validate histone (105,106). not significantly influence the levels and collagen adducts, because The effect of dietary fatty acid of these adducts. However, the they are readily accessible, more composition on the endogenous presence of a previously detected abundant than DNA, and have formation of MA-DNA adducts BP-DNA adduct in the breast known rates of turnover. The was investigated in a group of 59 tissues was associated with higher lifespan of haemoglobin is ~60 healthy men and women (107). levels of the MDA-dA adducts days in rodents and 120 days in They were initially fed a milk fat- in cancer patients. Interestingly, humans, and the half-life of serum based diet (rich in saturated fatty the level of MA-dA adducts was albumin in humans is 23 days. acids) for two weeks to induce a significantly lower in smokers and Because protein adducts are stable homogeneous dietary background. ex-smokers compared to non- and are not removed by active

Following this period, the subjects smokers. Tumor tissues (n = 11) repair processes, they constitute Unit 2 were randomly divided into two also displayed significantly lower a much more precise dosimetry subgroups: 30 people were given a levels of MA adducts than their tool when compared with DNA 4 Chapter sunflower oil-based (SO) diet (rich corresponding normal adjacent adducts. Interaction of a carcinogen in polyunsaturated fatty acid), and tissues. These results suggest that with a protein typically occurs by the remaining 29 people were fed a lipid peroxidation products can substitution at a nucleophilic amino low erucic acid rapeseed oil-based accumulate in human breast tissues acid. For alkylating agents, the most (RO) diet (rich in monounsaturated and reach relatively high levels in commonly substituted amino acid is fatty acid) for 25 days. At the end of the breast tissues of women with cysteine, but modifications for other the study, the fatty acid composition breast cancer. carcinogens have been reported of plasma lipids and the level of Estrogen is also known to be a at lysine, aspartate, glutamate, MA-DNA adduct in total WBC were major risk factor in breast cancer, and tryptophan, histidine and valine determined. A higher concentration its biological effects are mediated by (3,112). of PUFA in plasma triglycerides and both receptor and metabolism (109). Formation of haemoglobin or higher levels of MA-DNA adducts Estrogen can form DNA adducts, serum albumin adducts has been were found in the subjects of the SO which have been measured in reported in experimental animals diet group as compared with those human samples (110). Formation of and humans for many categories in the RO diet group. The average the 4-hydroxyestradiol-N7-guanine of carcinogens, including AFB1, adduct level (7.4 ± 8.7 adducts/107 (4-OHE2-N7-guanine) adduct from aromatic amines, B(a)P, benzene, nucleotides) in the SO group was the reaction of estradiol-3,4-quinone dimethylnitrosamine, ethylene oxide, 3.6-fold higher than that in the RO with DNA and its detection in vivo, 2-amino-3-methylimidazo(4,5-f) group, although large interindividual has been established. Therefore, quinoline, methylmethane sulfonate, variation was noted. the development and application NNK, propylene oxide, styrene, and Malondialdehyde (MDA)-DNA of methods to measure estrogen- workplace and medicinal (psoriasis) adducts were analysed in surgical guanine adducts will, in the future, PAHs (3,50,113). Techniques for specimens of normal breast tissues explore the biological implications of measuring carcinogen-protein of 51 breast cancer patients, while these compounds to determine their adducts include immunoassays normal breast tissue samples from contribution to estrogen toxicology. (ELISA, RIA, and IAC) and 28 non-cancer patients served analytical chemical methods (GC, as controls (108). Two previously Techniques for measuring GC-MS, HPLC, LC-MS, and AMS). characterized MDA-deoxyadenosine carcinogen-protein adducts Several combinative methods, (dA) and one MDA-deoxyguanosine such as IAC-HPLC with fluorescent (dG) adducts were detected in all Formation of carcinogen-protein detection and isotope dilution MS, tissue samples examined. Normal adducts is considered to be a have been applied to measure breast tissues from cancer patients valuable surrogate for DNA adducts, protein adducts (114). Sensitivity exhibited significantly higher levels since many chemical carcinogens of these methods typically can be than those found in non-cancer bind to both DNA and protein in within the pmol and fmol range. For controls. Ten of the 51 cancer blood with similar dose–response detection of haemoglobin or albumin patients and one of the 28 controls kinetics (3,111). Haemoglobin and adducts in humans, samples must

Unit 2 • Chapter 4. Physical/chemical/immunologic analytical methods 53 be enriched for adducts, or adducts of 4-ABP-Hb adducts increased provide a better prediction and must be removed from the protein, with increasing ETS level. This assessment of human cancer risk. before analysis (3,60,111). This is relationship between ETS exposure It was found that mean 3- accomplished by either chemical and 4-ABP-Hb adduct levels and 4-ABP-Hb adduct levels in or enzymatic release of the adduct supports the concept that ETS is a 151 subjects were statistically or carcinogen from the protein or probable hazard during pregnancy. significantly higher in cigarette digestion of the protein into peptides Metabolic polymorphism, both smokers compared to non-smokers, and amino acids. Solvent extraction in NAT and in CYP1A2, is also and that the level increased with or IAC purification may then be expected to affect the formation increasing number of cigarettes used for partial purification before of 4-ABP-DNA- and Hb-adducts. smoked per day (118). Again, undergoing analysis with GC-MS, Levels of DNA adducts in bladder slow acetylators consistently HPLC, or LC-MS. cells and 4-ABP-Hb adducts in exhibited higher mean levels of 79 individuals, together with the ABP-Hb adducts compared to Measurement of haemoglobin acetylator phenotype and genotype, rapid acetylators. The mean level adducts for studying were determined (117). Among the of 4-ABP-Hb adduct was higher in carcinogen exposure slow acetylators, levels of 4-ABP- subjects possessing the GSTM1- Hb adducts were significantly higher null versus GSTM1-non-null A wide variety of aromatic amines compared to those present in rapid genotype (46.5 versus 36.0 pg/g and PAHs have been found to acetylators. This study indicated that Hb; P = 0.037). In another study, bind at high levels to haemoglobin clearance of low-dose carcinogens a polymorphic distribution of the (115). A brief summary showing is decreased in the slow acetylator CYP1A2 and NAT2 phenotypes the range of the different chemical phenotype. Since the highest was examined in relation to ABP-Hb haemoglobin adducts that have levels of adducts were found in adduct formation in 97 healthy males been detected in human non- individuals with rapid N-oxidation (119). Rapid oxidizers and subjects smokers is found in Table 4.1. One (CYP1A2) and slow N-acetylation with the combined slow acetylator- carcinogen-Hb adduct that has been (NAT2) phenotype, determination rapid oxidizer phenotype showed well characterized is formed by the of phenotypes and genotypes may the highest ABP-Hb adduct levels at potent urinary bladder carcinogen 4-ABP, and has been reported in Table 4.1. Haemoglobin adducts in human non-smokers human blood specimens (113). It has been concluded that 4-ABP- Compound fmole/g Haemoglobin Hb adduct is closely associated with three major risk factors for HPB from NNK 29.3 ± 25.9 bladder cancer: cigarette smoking, 2-Aminonapthalene 40 ± 20 the type of tobacco smoked and acetylator phenotype. The relation 4-ethylaniline 99 ± 10 between exposure to environmental tobacco smoke (ETS) and levels of 2,6-dimethylaniline 157 ± 50 4-ABP-Hb adducts in non-smoking 4-Aminobiphenyl 166 ± 77 pregnant women compared to adduct levels in those women who 3,5-dimethylaniline 220 ± 20 smoked during pregnancy has been o-Toluidine 320 ± 90 reported (116). A questionnaire on smoking and exposure to ETS was p-Toluidine 640 ± 370 administered to pregnant women. Samples of maternal blood and cord m-Toluidine 6400 ± 1900 blood were collected during delivery N-(2-carbamoylethyl)valine 19000 ± 12000 and analysed for 4-ABP-Hb adducts by GC-MS. The mean adduct level Aniline 41000 ± 22000 in smokers was approximately nine- N-(2-Hydroxyethyl)valine 58000 ± 25000 fold higher than that in non-smokers. Among non-smokers, the levels Table compiled from (69,115,120).

54 a low smoking dose. However, in a adducts, with the exception of 2,6- a value very similar to that observed subset of 45 available samples, no DMA, were higher in smokers than when rats were administered 7 association was seen between the in non-smokers, and levels of all AFB1. When the data for AFB-N - ABP-Hb adduct levels and GSTM1 arylamine-haemoglobin adducts Gua adduct excretion in urine and genotype. were higher in cases than in controls. serum albumin were compared, a A wide variety of aromatic Arylamine-haemoglobin adducts statistically significant relationship amines and PAHs have been found of 2,6-DMA, 3,5-DMA, and 3-EA was seen with a correlation to bind at high levels to haemoglobin were all independently statistically coefficient of 0.73 (123). Using

(115). Tobacco-specific nitrosamine significantly (all P < 0.001) associated ELISA, AFB1-albumin adducts in binding to haemoglobin from with bladder cancer risk after human sera from several regions pyridyloxobutylation has been adjusting for cigarette smoking at of the world were investigated detected at 29.3 ± 25.9 fmole/g the time of blood collection, lifetime (124). It was found that 12–100% of haemoglobin (54). 2-Aminonapthalene, smoking history and other potential serum samples from children and risk factors. These adducts were 4-ethylaniline, 2,6-dimethylaniline, adults of various African countries Unit 2

4-Aminobiphenyl, 3,5-dimethylaniline, also independently associated with contained AFB1-albumin adducts, Chapter 4 Chapter o-Toluidine, p-Toluidine, m-Toluidine, bladder cancer risk when only non- with levels up to 350 pg AFB1-lysine N-(2-carbamoylethyl)valine, Aniline, smokers at the time of blood draw equivalent/mg albumin. In studies and N-(2-Hydroxyethyl)valine have were considered (highest quartile conducted in the Gambia, West been measured at 40 ± 20, 99 ± 10, versus lowest quartile: 2,6-DMA, Africa, a strong dose–response 157 ± 50, 166 ± 77, 220 ± 20, 320 ± relative risk (RR) of bladder cancer relationship between aflatoxin

90, 640 ± 370, 6400 ± 1900, 19 000 = 8.1, 95% confidence interval (CI) exposure and AFB1-albumin ± 12 000, 41 000 ± 22 000, and 58 = 3.6–18.0; 3,5-DMA, RR = 2.7, adducts was also seen (125), similar 000 ± 25 000 fmole/g haemoglobin, 95% CI = 1.2–6.0; 3-EA, RR = 4.3, to that previously reported in China respectively (69,115,120). One of 95% CI = 1.6–11.6). Thus, diverse (122). From a practical perspective the carcinogen-Hb adducts that has arylamine exposures are strongly pertinent to epidemiologic studies, been well characterized is formed associated with bladder cancer risk the measurement of serum AFB1- by 4-ABP, the potent urinary bladder among non-smokers (69). albumin adduct offers a rapid, facile carcinogen; 4-ABP-Hb adducts in approach that can be used to screen human blood specimens have been Measurement of albumin very large numbers of people (17). reported (113). The results indicate adducts for studying Three methods for AFB1- that the 4-ABP-Hb adduct is closely carcinogen exposure albumin adduct measurement were associated with three major risk compared using serum samples factors for bladder cancer: cigarette In addition to carcinogen-Hb from highly exposed residents of smoking, the type of tobacco adducts, carcinogen-albumin Qidong (n=88), Fushui (n=65), and smoked, and acetylator phenotype. adducts have also been investigated, Chongming Island (n=115), China,

The role of aromatic amines in particularly for AFB1 exposure and The Gambia (n=29). Although the development of bladder cancer (113,121). There are four analytical the average levels of AFB1-albumin in non-smokers in Los Angeles, techniques currently available for adducts were similar among these

USA was explored in a population- measuring AFB1-albumin adducts regions, great individual variations based case–control study involving in human blood: ELISA, RIA, IAC- were found, as evidenced by the 298 case subjects with bladder HPLC with fluorescence detection, detectable level ranging from 0.124– cancer and 308 controls. To assess and isotope dilution MS (IDMS) 25.925 pmol aflatoxin/mg albumin in arylamine exposure, levels of (50,114). Using RIA, levels of the 297 samples. High correlations arylamine-haemoglobin adducts aflatoxin-serum albumin adducts across the methods (r = 0.80– of nine selected alkylanilines in serum samples from residents 0.90) were obtained by comparing (2,3-dimethylaniline (2,3-DMA), of Guangxi, China were monitored; samples with each of the different 2,4-DMA, 2,5-DMA, 2,6-DMA, a highly significant association analytical methods. Moreover, some

3,4-DMA, 3,5-DMA, 2-ethylaniline between AFB1-albumin adduct level of these samples can be stored

(2-EA), 3-EA, and 4-EA) were and AFB1 intake was found (122). for over 10 years with insignificant measured in peripheral blood Further, it was calculated that about losses of albumin adduct levels (126). collected from study subjects. 2% of the ingested AFB1 became The ELISA and IDMS methods were Levels of all arylamine-haemoglobin covalently bound to serum albumin, compared in measurement of 20

Unit 2 • Chapter 4. Physical/chemical/immunologic analytical methods 55 human serum samples collected in urinary aflatoxin biomarkers (AFB- smokers among cases and controls 7 Guinea, West Africa; high correlation N -Gua and other AFB1 metabolites) (separately and combined) after between these two methods was were detected. The RR for people adjusting for age, gender, ethnicity, found (r = 0.856, P < 0.0001) (127). who tested positive for the HBsAg and season. In an experimental study, the level was about eight, but individuals with Aflatoxin-albumin adducts have of AFB1-albumin adducts formed as both urinary aflatoxin biomarkers been used as biomarkers for liver a function of a single dose of AFB1 and positive HBsAg status had a cancer risk. A nested case-control in rodents was compared to data RR for developing liver cancer of 57. study carried out in Taiwan, China, from humans exposed to AFB1. These results show, for the first time, followed a cohort of 8068 men for This comparison yielded a value a relationship between the presence three years (132,133). Twenty-seven for the three rat strains (Fischer of carcinogen-specific biomarkers cases of hepatocellular carcinoma 344, Wistar, and Sprague-Dawley) and cancer risk. Moreover, (HCC) were identified and matched of between 0.3–0.51 pg AFB1- these findings provided the first with 120 healthy controls. Serum lysine/mg albumin/1 µg AFB1/kg demonstration of a multiplicative samples were analysed for AFB1- body weight and a value for the interaction between these two major albumin adducts by ELISA. The mouse (C57BL) of <0.025. The risk factors for liver cancer. Further, proportion of subjects with a best estimate for people from the when individual aflatoxin metabolites detectable serum AFB1-albumin Gambia and southern China was were stratified for liver cancer adduct level was higher for HCC 1.56 pg/mg albumin for the same outcome, the presence of the AFB- cases (74%) than matched controls exposure. These data suggest that N7-Gua in urine always resulted in a (66%), giving an odds ratio of 1.5. humans exposed to AFB1 form two- to three-fold elevation in risk of There was a statistically significant amounts of albumin adducts closer developing liver cancer (130). association between detectable to those observed in AFB1-sensitive A case–control study measured level of AFB1-albumin adduct and species and 1–2 orders of magnitude BPDE-DNA adducts in DNA HCC risk among men younger than higher levels than the AFB1-resistant samples from WBC of lung cancer 52 years old, showing a multivariate- species (128). patients and healthy controls. High adjusted odds ratio of 5.3, although levels of adducts were found in no association was observed

Measurement of DNA and WBC from lung cancer patients, between AFB1-albumin adduct protein adducts for studying with a range of 65–533 adducts/108 level and HBsAg carrier status. cancer risk nucleotides. In WBC-DNA samples Another prospective, nested case– from healthy controls (smokers, non- control study (134) was carried out DNA and protein adducts not only smokers), the presence of adducts in Qidong, China in 1991. Serum serve as biomarkers for exposure, was detected only in smokers, but samples from 804 healthy HBsAg- but as biomarkers for cancer risk. A at a lower level than in lung cancer positive individuals (728 male, 76 nested case-control study initiated patients (74). PAH-DNA adducts female) aged 30–65 were obtained in 1986 in Shanghai, examined the in peripheral leukocytes were and stored frozen. Between the relationship between biomarkers for investigated from 119 non-small cell years 1993–95, 38 individuals aflatoxin and hepatitis B virus (HBV) lung cancer patients and 98 controls developed liver cancer. The serum and the development of liver cancer (131). Among them, 31 cases had samples for 34 of these cases were (129,130). In this study, over 18 000 adduct measurements in leukocytes, matched by age, gender, residence urine samples were collected from lung tumour and non-tumour and time of sampling to 170 controls. healthy males between the ages specimens collected at surgery, Serum AFB1-albumin adduct levels of 45 and 64. In the subsequent and 34 had paired leukocyte and were determined by RIA. The RR for seven years, 50 of these individuals tumour specimens. After adjustment HCC among AFB1-albumin positive developed liver cancer. The urine for age, gender, ethnicity, season individuals was 2.4 (95% CI = 1.2– samples for cases were age- and smoking, DNA adducts in 4.7). matched and residence-matched leukocytes were significantly higher with controls and analysed for both in cases than controls; the odds ratio Summary and perspectives aflatoxin biomarkers and HBsAg was 7.7 (95% CI = 1.7–34; P<0.01). for the future status. A highly significant increase DNA adducts in leukocytes were in the RR of 3.5 was observed for increased significantly in smokers Over the past 25 years, the those liver cancer cases where and ex-smokers compared to non- development and application of

56 molecular biomarkers reflecting methods (32P-post-labelling) susceptibility factors in molecular events from exposure to formation and various physicochemical epidemiologic studies of human of clinical disease has rapidly methods (GC, HPLC, GC-MS, cancer, will assist in the elucidation expanded our knowledge of LC-MS, ECD, fluorescence and of human cancer risk. the mechanisms of disease phosphorescence spectroscopy, or The molecular epidemiology pathogenesis. These biomarkers will a combination of these methods). investigations of aflatoxins probably have increasing potential for early Capillary electrophoresis and represent one of the most extensive detection, treatment, interventions other new separation techniques data sets in the field, and may serve and prevention. Biomarkers have improved sensitivity and as a template for future studies of derived from toxicant/carcinogen specificity of these strategies. other environmental agents. The metabolism include a variety of NMR spectrometry has also been development of aflatoxin biomarkers parent compounds and metabolites used to determine stereospecificity has been based upon the knowledge in body fluids and excreta, which and three-dimensional structure. of their biochemistry and toxicology

serve as biomarkers of internal Molecular epidemiologic studies that gleaned from both experimental and Unit 2 dose. Carcinogen-macromolecular employ carcinogen-macromolecular human studies. These biomarkers adducts, such as DNA and protein adduct measurements are likely have subsequently been used in 4 Chapter adducts formed in blood and to be widely applied in the future. experimental models to provide tissue or excreted in urine, serve They have the potential to generate data on the modulation of these as biomarkers for: exposure to the hypotheses regarding underlying markers under different situations complex mixture of occupational basic biologic mechanisms that of disease risk. This systematic carcinogens; biologically effective subsequently can be tested in the approach provides encouragement dose or early biological effect, to laboratory. The complex nature of for preventive interventions and measure the actual dose to the gene–environment and chemical– should serve as a template for carcinogen target site; and for risk biological interactions will be better the development, validation and assessment between carcinogen understood through the use of application of other biomarkers to exposure and eventual cancer advanced techniques, such as cancer or other chronic diseases. formation. rapidly developing metabolomics Many different analytical and proteomics, which include Acknowledgements techniques have been developed NMR-MS and matrix-assisted laser to identify and measure parent desorption/ionization (MALDI)- This work was supported in part compounds, metabolites, MS, and incorporation of validated by grants P01 ES006052 and P30 carcinogen-DNA and -protein biomarkers into large-scale studies. ES003819 from the US Public adducts. These include In the future, integration of data Health Service. immunoassays (ELISA, RIA, IHC, for these biomarkers, together and IA), radiometric postlabelling with other environmental and host

Unit 2 • Chapter 4. Physical/chemical/immunologic analytical methods 57 References

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