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2 DNA-Adduct Technology The consensus of several recent meetings on DNA-adduct research has been that new assay systems for detecting and measuring DNA adducts and protein adducts have the potential to improve markedly the biologic bases for estimating the risks of human exposure to several important classes of environmental pollutants (Bartsch et al., 1988; Berlin et al., 1984; de Serres, 1988; de Serres et al., 1985; Farmer et al., 19871. This chapter describes and evaluates some of the currently used assays and discusses how the information they provide can lead to the improvement of risk assessment and epidemiological studies. New DNA-adduct technology embodies striking technologic improvements in sensitivity and specificity and permits measurement of mammalian re- sponse to small, intermittent environmental exposures. For example, molec- ular dosimetric assays and mutation analysis in mammalian cells in tissue culture suggest that low exposures to genetic toxicants produce DNA lesions at approximately 2,000 per cell in the lung after exposure to aromatic amines and 100,000 per cell in the upper layer of skin after exposure to the ultraviolet component of sunlight (Lohman et al., 1985~. Those figures correspond to about 1 adduct per 10-7 bases in the lung and at least 1 adduct per 10-4 bases per day in the upper layer of skin. The new methods of measuring DNA adducts are in many instances rel- atively inexpensive, fast, and reproducible. They can be applied to readily available samples of body fluids, such as blood, semen, and urine, and to small samples of cells, such as buccal mucosa or skin biopsy specimens. 38
DNA-Adduct Technology 39 TECHNIQUES FOR DETECTING DNA ADDUCTS Chromatographic and Spectrometric Methods Chromatography has many variations, but all involve the flow of test material through tubing containing stationary material designed to adsorb the components of the test mixture selectively, and thus create different flow rates and a series of bands (chromatograms) by which their identity can be determined. In spectrometry, the sample Interacts with light or particles to yield dis- tinctive spectral signals. One version is mass spectrometry, the most accurate technique for trace organic-chemical analysis, according to the National Bu- reau of Standards. The polarity and size of DNA adducts, or fragments of DNA adducts, can markedly influence the separation power and thus the sensitivity of these techniques. Each analytic method possesses potentially good to high resolving power, but the suitability and sensitivity of any method or combination of methods commonly depends on the physicochemical properties of the adduct or the class of adducts to be tested. . . · . - . . ~ LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY (LC/MS) In this method, DNA adducts are separated from the test sample by ad- sorption on an activated surface. They are then put into a mass spectrometer for final analysis. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) This method combines speed and high-resolution power to fractionate DNA adducts by column chromatography of modified DNA bases. It can detect fluorescence. ATOMIC-ABSORPTION SPECTROMETRY (AAS) This quantitative analytic technique detects adducts containing metals by absorbing light of specific wavelengths from excited atoms. TANDEM MASS SPECTROMETRY (MS/MS) Two mass spectrometers are used in sequence to detect fragments (ions) of specific molecules, such as DNA adducts. An ion from the mixture is selected and focused through the first mass spectrometer; thereafter, it is .
40 DRINKING WATER AND HEALTH fragmented into smaller portions for further, high-resolution analysis in the second mass spectrometer (Farmer et al., 1988~. FLUORESCENCE LINE-NARROWING SPECTROMETRY (FLNS) Particularly useful for fluorescent adducts of polycyclic aromatic hydro- carbons, FENS uses low temperature and laser excitation to improve sen- sitivity in the quantitative fluorometric analysis of DNA adducts (Jankowiak et al., 1988). ULTRAVIOLET RADIATION/HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (UV/HPEC) Because all adducts absorb ultraviolet radiation, testing for UV absorption is useful for large, bulky adducts, such as those formed by aflatoxin, but is rarely sensitive enough for human monitoring. Sensitive HPLC can be added to detect fluorescence after hydrolysis of particular carcinogen-DNA ad- ducts. For example' synchronous fluorescence spectrometry (SFS) scans a , ~ · - ^ ^ · . · . . - · · - sample with a flxecl wavelength oliterence between excitation and emission. Three-dimensional plots of fluorescence intensity, emission, and excitation- emission wavelength difference should be able to help identify some unknown adducts (Farmer et al., 19871. GAS CHROMATOGRAPHY/ELECTRON CAPTURE NEGATIVE TON MASS SPECTROMETRY (GC/ECNIMS) In this technique, the test material is derivatized and volatilized into a carrier gas stream whose components pass through a chromatography column at different rates, where the adducts are separated. The sample then enters the mass spectrometer, where distinctive ions are formed and detected with high sensitivity and specificity. A related, less specific method is GC with electron capture detection (GC/ECD). Quantitative Immunoassays Monoclonal or polyclonal antisera specific for carcinogen-DNA adducts or carcinogen-modified DNA are used in immunoassays to quantify the bind- ing of known carcinogens with DNA in biologic samples of nucleic acid (Poirier, 19841. These immunoassays are simple to perform, inexpensive, and thus appropriate for human samples; but the antisera are chemical-spe- cific, and thus different antisera must be developed for each adduct of interest (Santella, 19881. The most sensitive immunoassays are run in a competitive mode, in which
DNA-Adduct Technology 41 two chemically identical haptens (in this case, DNA adducts) compete for an antibody binding site. The concentration of 1 hapten (usually an assay standard) is always kept constant, and that hapten is radioactively labeled (in radioimmunoassays) or bound to the bottom of microtiter wells (in en- zyme-linked immunosorbent assays). The other hapten is used in increasing concentrations to compete with the constant hapten for binding to the anti- body. The variable hapten can be either a standard immunogen or an unknown sample. Quantitation is based on comparison of unknowns with the inhibition curve generated by the standard immunogen. RADIOIMMUNOASSAY (RIA) In conventional RIA, a competitive technique, the antigen-antibody com- plex is separated from the whole mixture in tubes by a variety of physical or chemical methods (Poirier, 1981), and the standard hapten is radioactively labeled. ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) In ELISA a solid-phase competitive assay that uses microtiter plates, the antibody bound after competition can be measured by a second enzyme- linked antibody used to cleave a specific substrate. One of the most commonly used enzyme conjugates is alkaline phosphatase, which cleaves a variety of phosphorylated substrates into products that can be detected by spectropho- tometry or fluorescence (ELISA) (Poirier, 19811. In general, microtiter-plate assays can be more sensitive than RIAs, but they are also more often in- consistent and variable. ULTRASENSITIVE ENZYMATIC RADIOIMMUNOASSAY (USERIA) The procedure for USERIA is similar to that of ELISA, except that the substrate obtained after application of the first antibody is labeled with ra- dioactive isotopes for radiochemical measurement of enzyme interaction. Counting requires that samples be manually removed from each well (Farmer et al., 1987; Santella, 1988~. m mu noh istochem ice ~ Tech n iq ues Enzyme-staining with a fluorescence or peroxidase end point can identify the specific cell types in which DNA adducts occur (and thus the cells that are targets for carcinogens) within a complex tissue sample. Monoclonal or polyclonal antibodies are used in conjunction with other antibodies that con- tain peroxidase enzyme or a fluorescent probe. With the fluorescent probe,
42 DRINKING WATER AND H"LTH images: of the adducts can be enhanced by computer for analysis. Polyclonal antibodies are less easily identified by the most sensitive image analysis systems (Adamkiewicz et al., 1985), but they have been used with micro- fluorometry for semiquantitative comparison between samples (Huitfeldt et al., 1987) and to detect, but not quantify, binding in human tissues (den Engelse et al., 1988~. 32P-PostIabeling Technique DNA is enzymatically hydrolyzed and the digest is labeled with phosphor- ylating enzyme to incorporate radioactivity. Thin-layer chromatography (TLC) is used to separate the adducts, which can be detected by autoradiography, and a portion of the chromatogram is excised for estimation of the total count (Gupta et al., 19821. Less useful for low-molecular-weight compounds, 32p_ postlabeling is more sensitive for aromatic or bulky hydrophobic adducts and has been able to show the extent and persistence of adduct formation in animals by more than 70 compounds, including aromatic hydrocarbons, ar- omatic amines, estrogens, and methylating agents (Gupta and Randerath, 1988~. With 32P-postlabeling, it is possible to monitor human carcinogen exposure (Randerath et al., 1988~. This technique is also capable of analyzing DNA adducts formed by unknown hydrophobic compounds (Farmer et al., 19871; tandem technology might someday be developed for identifying the structure of such adducts. TECHNIQUES FOR DETECTING PROTEIN ADDUCTS Some of the same methods for detecting DNA adducts are applied to the determination of protein adducts (GC, GC-MS, immunoassay, and fluores- cence detection with HPLC). New techniques for detecting protein adducts, especially those using hemoglobin as a target molecule (Bailey et al., 1987; Osterman-Golkar, 1988; Neumann, 1984, 1988), offer high sensitivity and specificity in detecting exposure of animals to alkylating agents. Several studies have found a direct correlation between hemoglobin-adduct and DNA- adduct concentrations in exposed experimental animals (Adriaenssens et al., 1983; Pereira, et al., 1981; Shugart, 1985; Wild et al., 19861. An additional practical advantage of measuring protein adducts is that large amounts of some proteins (especially hemoglobin) can be obtained from human subjects. Thus, protein adducts might provide more reliable measurements than DNA adducts for evaluating both normal background concentrations of adducts and deviations from the normal.
DNA-Adduct Technology 43 SENSITIVITY AND SPECIFICITY The intrinsic sensitivity of an assay to detect the molecular effect of a given hypothetical chemical is usually expressed as femtomoles (fmol; 10- Is moles) of adduct per milligram of DNA or protein. Tables 2-1 and 2-2 give estimates of the intrinsic sensitivities of various DNA and protein binding assays. The estimates are approximations and sensitivity might vary by sev- eral orders of magnitude, depending on the physicochemical nature of the chemical or adduct. Tables 2-1 and 2-2 show that some of the immunochemical assays and the postlabeling assay have good sensitivity and do not require invasive techniques or large tissue samples. The MS/MS method, a physicochemical method, has the same advantages as the immunochemical and postiabeling assays, but its dependence on expensive and sophisticated equipment could severely limit widespread application. Other techniques, such as immuno- chemical methods for detecting DNA adducts at the single-cell level (Bean et al., 1988; Perera et al., 1988; Van Benthem et al., 1988) and the recently introduced laser-scan immunofluorescence microscopy (Bean et al., 1986), are also limited by unique instrumentation requirements. However, the MS/ MS (Farmer et al., 1988), RIA (Umbenhauer et al., 1985), and 32P-postla- beling (Randerath et al., 1988) methods can detect interactions with small amounts of unidentified alkylating agents associated with occupational and low-level environmental exposures. Both physicochemical and immunochemical methods for detecting adducts or metabolites of genetic toxicants in urine have high sensitivity and speci- f~city (Oshima and Bartsch, 1988; Shuker and Farmer, 1988; Vanderlaan et al., 1988), but still require validation as indicators of internal exposure. A high adduct concentration in urine is often assumed to indicate high internal exposure, but this assumption is not necessarily correct. Proper mass-balance evaluations are needed to measure the intake and excretion of genetically toxic agents and their metabolites. Without such determinations, it is equally justifiable to relate the presence of a high concentration of adducts in urine to detoxif~cation or to low internal exposure (Lohman et al., 1984; van Sittert, 19841. Although technologic improvements make feasible the sensitive measure- ment of exposure to genetic toxicants in animal models and humans, no generally applicable methods have been developed for estimating genetic risk (Wogan, 19881. Attempts are under way to relate target dose in humans to biologically adverse effects of small exposures to genetically toxic agents (Ehrenberg, 1988~. The use of these new, ultrasensitive analytical techniques in risk assessment will depend on an understanding of the mechanistic relationships between DNA alterations and the ultimate expression of toxic effects. Recent devel
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DNA-Adduct Technology 47 opments in the study of DNA binding and protein binding provide a useful tool for beginning to acquire that understanding. However, additional infor- mation, such as clarification of the role of background or baseline adducts that are always present in animals and humans, will be needed to make full use of the advanced technology that is currently available. APPLICATIONS OF DNA ADDUCT TECHNOLOGY General Utility in Risk Assessment DNA-adduct and protein-adduct technology is potentially useful in the processes of hazard identification and risk assessment. The NRC has per- formed comprehensive toxicological assessments for the Environmental Pro- tection Agency on chemicals found in drinking water (NRC 1977, 1980a,b, 1982, 1983, 1986, 19871. In these assessments, information on acute, sub- chronic, and chronic effects is assembled and evaluated. Considerations of exposure and pharmacokinetics play an important role in risk assessment for chemicals with identified toxicity (NRC, 19871. It appears that adduct tech- nology could be extremely valuable in estimating dosimetry and systemic distribution, in establishing possible target tissues or organs, and in deter- mining the potential for irreversible toxicity such as cancer, mutation, or developmental effects. Table 2-3 lists some potential applications of DNA-adduct analysis to the toxicologic evaluation of drinking water contaminants for risk assessment. The table is arranged as a matrix with the components of toxicity assessment on one axis and the potential contribution of DNA-adduct analysis on the other. The second column identifies when the specific method chosen to detect adducts is important. Toxic effects can be adduct-specific, and three toxicologic components relationship of adducts to toxic response, muta- genicity, and species extrapolation might require methods that identify the adducts detected. Some analytic methods (e.g., 32P-postlabeling) do not identify the DNA adduct detected. The third column specifies whether or not it is desirable to identify the DNA adducts in using the technology for toxicologic assessments. Specific adduct identification is needed to correlate toxicity with adducts and is desirable in studies of mutagenic activity. Some DNA adducts, such as N7 alkylguanines, appear to have only a small role in mutation induction. Different components of toxicologic assessment have different requirements for quantitative or qualitative test results. These are identified in the table, as is the need for other biologic data. For example, information on chronicity of exposure is important in establishing a carcin- ogenicity hazard associated with a substance that induces formation of DNA adducts. The last column of the table identifies the extent to which DNA- adduct technology can now be used routinely in toxicologic assessments;
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DNA-Adduct Technology 49 most of the technology is still~in the research and development stage, and none can yet be considered routine. The methods listed in the table are expected to evolve, so the conditions identified will also change. However, the table should facilitate an understanding of which aspects of toxicologic testing can be aided by DNA-adduct technology, which methods to consider first, and what data one should expect from a specific approach. Features of the four categories of methods now available for use in toxicity testing are summarized in Table 2-4. Immunochemical and~physical methods require considerable expertise in chemical synthesis, antibody production and radiolabeling, and analytic instrumentation. Some methods, such as 32p_ postlabeling, have been developed for use with high-molecular-weight (bulky) adducts, especially polycyclic aromatic hydrocarbons and aromatic amines. Use of this assay with low-molecularweight alkylating agents is not now feasible. The published literature has been searched for specific information re- garding the reactivity of 16 compounds that have been identified in drinking water and are known to be carcinogenic, mutagenic, teratogenic, or genet- ically toxic in experimental animals. Thirteen were recently reviewed by the NRC (1986) for EPA. Chemical structure, use, occurrence or source of exposure, association with DNA adduct formation, tissue distribution, car- cinogenicity, mutagenicity, and other health effects were the data elements sought. The results are summarized in Appendix A, which can be referred to for some evidence for the theoretical assessments. Table 2-5 summarizes speculations about the ability of the 16 compounds to form DNA adducts. With the exception of benzoLalpyrene, for which considerable data exists concerning adduct analysis, the chemicals have not been the subject of ex- tensive DNA-binding studies. Epidemiology and Human Monitoring Proper investigation of relationships between disease in humans and ex- posure to drinking water contaminants has been hampered by the difficulty of assessing exposure to contaminants appropriately and by the limitations inherent in the use of traditional end points, such as the development of cancer, which are both rare and characterized by long latency. Interest in the incorporation of biologic markers into studies of human exposure to xenobiotic substances has increased, with the hope that use of such markers will enable scientists to characterize the empirical associations between ex- posures and outcomes, improve the accuracy of exposure assessment, en- hance understanding of toxic mechanisms, increase the ability to detect early subclinical effects of exposure, and make better use of data from laboratory animals in predicting the effects of exposures of humans (NRC, 19871. Protein and DNA adducts in humans have been proposed as markers both
50 DRINKING WATER AND HEALTH TABLE 2-4 Evaluation of New Molecular Methods in DNA- or Protein- Adduct Technology - Method of Biologic Monitoringa for DNA Adducts Physical Immunochemical 32P-Post- For Protein Characteristic Methods Methods Labeling Adducts . . Appropriateness for measuring exposure Qualitative ( + ) + + + Recent (1-week) internal dose ? + + + Long-term body burden ? + + (+) Dose at target site ? + + Appropriateness for assessing health effects Reversible ? ( - ) ( - ) ( - ) Irreversible ( - ) ( - ) ( - ) ( - ) Interpretation of results On individual basis + + + + On group basis + + + + Precision of method Technical reproducibility ? ( + ) ( + ) + Stability (+) (+) + (+) Interlaboratory reproducibility ? ( + ) ( + ) + Sensitivity For some environmental exposures For occupational exposures For acute exposures Chemical specificity Absence of confounding factors Absence of background adducts Simplicity Ease of sample storage Current applicability In research In routine use ? (+) b + + + + ? ? + + (+) (-) b (+) ( - ) b (+) (-) b + (+) ( - ) + + + (+) (+) aSymbols: +, applicable or true; (+), probably applicable or true; - , not applicable or not true; ( - ), not now applicable or not now true; ?, unknown. bCannot be generalized.
DNA-Adduct Technology 51 TABLE 2-5 Classification of 16 Dnnking Water Contaminants According to Their Presumed Ability to Form DNA Adducts Definite Probable Possible Ability Ability Ability Insufficient Data Acrylamide Tnchlorfon Diallate Arsensic Chromium Sulfallate Nitrofen Benzo[a]pyrene 1,2-Dichloropropane Pentachlorophenol Dibromochloro- ~ (1,2-DCP) propane (DBCP) 1 ,2,3-Trichloro Ethylene di- propane (1 ,2,3-TCP) bromide (EDB) 1,3-Dichloropropene (1,3-DCP) Di(2-ethylhexyl) phthalate (DEHP) Mono(2-ethylhexyl) phthalate (MEHP) for use in epidemiologic studies to assess the risks associated with exposure to potential genetic toxicants and for use in monitoring exposed populations. However, all the studies that have measured protein or DNA adducts have focused on humans exposed to carcinogens occupationally, environmen- tally, or otherwise. In principle, incorporation of measurements of carcinogen-DNA adduct formation into epidemiologic studies could offer at least two kinds of benef~ts: · The use of sensitive methods, such as immunoassays and 32P-postIa- beling, might afford an opportunity to detect early, subtle effects of small exposures. · Human studies incorporating DNA-adduct assays might provide infor- mation on target-molecule dose that reflects exposure, absorption, metabo- lism, and DNA-adduct formation and repair rates. Although the measurement of DNA-adduct formation in humans holds substantial promise for epidemiologic and monitoring studies, interpretation of data derived from DNA-adduct measurements is extremely complex, par- ticularly in humans. In general, further experimental work is required before measurements of DNA adducts can be successfully incorporated into studies that assess toxicity from drinking water contaminants in humans. The fol- lowing issues are of particular concern in considering the potential appli- cations of DNA-adduct technology in human studies to evaluate potential toxicity of drinking water contaminants: · The paucity of information about the kinetics, dose-response relation- ships, and interindividual and intraindividual variability of DNA-adduct for- mation in humans renders the proper design and interpretation of human
52 DRINKING WATER AND HEALTH studies using DNA-adduct technology very difficult. Studies that characterize the variability of adduct levels in humans due to such factors as age, sex, ethnicity, diet, tobacco use, and such medical conditions as liver disease are needed. Additionally, studies are needed for proper characterization of base- line adduct levels in the general population. · Because numbers of DNA adducts reflect not only exposure, but also rates of metabolism (in the case of indirect carcinogens) and DNA-adduct formation and repair, DNA-adduct concentrations are likely to involve com- plex dynamics. When technically feasible, the use of protein adducts might prove more appropriate for exposure assessment; such adducts in the he- moglobin of red blood cells have demonstrated chemical stability and linear dose-response relationships for a variety of compounds and thus can provide integrated exposure information. To date, the use of protamine adducts in germ cells has been limited to studies of small alkylating agents in mice. Further studies are needed to evaluate the use of human protamines in do- simetry. Protamine dosimetry might help to identify the exposures that pose germinal risks. · In animal studies, it is possible to study DNA-adduct concentrations in target tissue, but the target tissue of interest in humans is often inaccessible. Circulating white blood cells or lymphocytes are used as surrogates for determination of DNA-adduct concentrations. However, the validity of using surrogate tissue, particularly for human risk assessment, has not been ade- quately evaluated. · Some of the better-character~zed chemicals that produce DNA adducts, such as BaP, are ubiquitous in the environment. That presents difficulties in epidemiologic studies, because, even with proper selection of controls, back- ground DNA-adduct concentrations might mask slight differences in con- centrations between "exposed" and "unexposed" populations. REFERENCES Adamkiewicz, J., G. Eberle, N. Huh, P. Nehls, and M. F. Rajewsky. 1985. Quantitation and visualization of alkyl deoxynucleosides in the DNA of mammalian cells by monoclonal antibodies. Environ. Health Perspect. 62:49-55. Adams, J., M. David, and R. W. Giese. 1986. Pentafluorobenzylation of 04-ethylthymidine and analogs by phase-transfer catalysis for determination by gas chromatography with elec- tron capture detection. Anal. Chem. 58:345-348. Adriaenssens, P. I., C. M. White, and M. W. Anderson. 1983. Dose-response relationships for the binding of benzo(a)pyrene metabolites to DNA and protein in lung, liver, and forestomach of control and butylated hydroxyanisole-treated mice. Cancer Res. 43:3712- 3719. Baan, R. A., P. H. M. Lohman, A. M. J. Fichtinger-Schepman, M. A. Muysken-Schoen, and J. S. Ploem. 1986. Immunochemical approach to detection and quantitation of DNA adducts resulting from exposure to genotoxic agents. Prog. Clin. Biol. Res. 207:135-146.
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