National Academies Press: OpenBook

Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment (1989)

Chapter: 1 Biologic Significance of DNA Adducts and Protein Adducts

« Previous: Executive Summary
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 6
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 7
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 8
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 9
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 10
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 11
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 12
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 13
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 14
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 15
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 16
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 17
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 18
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 19
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 20
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 21
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 22
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 23
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 24
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 25
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 26
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 27
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 28
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 29
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 30
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 31
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 32
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 33
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 34
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 35
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 36
Suggested Citation:"1 Biologic Significance of DNA Adducts and Protein Adducts." National Research Council. 1989. Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/773.
×
Page 37

Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

1 Biologic Significance of DNA Abducts and Protein AdJucts Current evidence suggests associations between the occurrence of adducts formed by specific compounds and various types of toxicity, such as mu- tation, cancer, and developmental effects. Clinical expression of the toxic effect is usually tissue-specific and can be delayed. DNA adducts form in many tissues, but some of them might be early markers of disease that could be reversed (NRC, 19871. This chapter 'describes what is known about mech- anisms and rates of DNA-adduct formation and removal, the significance of the adduct's position on the DNA, And the correlation of of adducts of certain specific compounds with toxic effects. In addition, protein adducts are dis- cussed as possible markers of exposure. Studies of laboratory animals and human chemotherapy patients have sug- gested that DNA adducts can serve as biologic dosimeters in providing es- timates of exposure, dose to the target tissue, and sometimes mutagenicity and carcinogenicity (Anderson, 1987; Wogan, 19881. For example, corre- lations between DNA-adduct formation and exposure, hepatocyte initiation, and hepatocellular carcinoma have been observed in experiments with di- ethylnitrosamine (Figures 1 1-1-3) (Dyroff et al., 1986), 2-acetylaminoflu- orene (Beland et al., 1988), aflatoxin Be (Croy and Wogan, 1981; Kensler et al., 1986), and N-methyl-4-aminoazobenzene (Tullis et al., 1987~. De- tection of unique DNA abducts in a population at risk would yield qualitative evidence of exposure. And the use of DNA adducts could perhaps reduce the uncertainty in quantitative risk assessment by providing better dose in- formation for dose-response evaluation. The use of DNA adducts to measure biologically effective dose is scientifically appealing. DNA adducts can in- dicate a measurable dose at a target site and thus make it possible to bypass 6

Biologic Significance of DNA Adducts and Protein Adducts 7 ~ 10-5 ~ . ~ z _ ~ ~ _{ ~_ o 1 o-6 _ , , , , , , , , 0 10 20 30 40 50 60 70 DURATION OF DEN EXPOSURE (days) FIGURE 1-1 Relationship of diethylnitrosamine (DEN) exposure to DEN alkylation in 4-week- old Fischer-344 rats. Data points represent mean concentrations of moles of 04-ethyldeoxythymidine (04-EtdT) to moles of deoxythymidine (dT) + the standard error of the mean (SEM) for 2-4 animals. Adapted from Dyroff et al., 1986, with permission. or to confirm considerations of absorption, distribution, metabolic acti- vation, and detoxification (Hoer et al., 19831. The estimation of carcinogenic risk usually involves two basic pieces of information (NRC, 1983~. A chronic animal bioassay measures the admin- istered doses of a chemical and correlates dose with tumor incidence to provide a quantitative evaluation of carcinogenic hazard at the doses and in 500 cod ~ 400 a) - o 300 200 100 o t -; ~ I I I I I I I 0 10 20 30 40 50 60 70 DURATION OF DEN EXPOSURE (days) FIGURE 1-2 Relationship of DEN exposure to hepatocyte initiation in 4-week-old Fischer-344 rats. Data points represent mean ~y-glutamyl transferase-positive (GOT + ) foci per cubic centimeter + SEM for 10-12 animals. The plateau in initiation represents a steady state, where the number of newly initiated hepatocytes equals the number of previously initiated hepatocytes that die. Adapted from Dyroff et al., 1986, with permission.

DRINKING WATER AND HEALTH 100 - o o- - 80 LL A 111 A a: o :D 40 20 60 o f 0 10 20 30 40 50 ED 70 DURATION OF DEN EXPOSURE (days) FIGURE 1-3 Relationship of DEN exposure to hepatocellular carcinoma in 4-week-old Fischer- 344 rats. Adapted from Dyroff et al., 1986, with permission. the species tested. Carcinogenic hazard is then combined with information about human exposure to estimate the human risk associated with the chem- ical. Unfortunately, animal bioassays are limited both practically and eco- nomically to measuring tumor incidences at exposures that are much higher than would be acceptable in human populations. Because these models are based on high experimental doses, the resulting data must be extrapolated to permit estimation of the dose-response relationship at doses far below those used in the bioassay. The selection of models that best represent true dose-response relationships in humans at low exposures is controversial. All the mathematical models now used yield similar estimates at high doses, but estimates for low doses deviate widely. The rates and routes of metabolic activation and detoxification of chemicals differ between sexes, species, and tissues and between high and low doses. Measuring DNA adducts provides one way to understand and even measure those differences. The following are examples: · Male mice produce different types of DNA adducts from, and more hepatocarcinomas than, female mice after exposure to the same doses of the hepatocarcinogen N-hydroxy-2-acetylaminofluorene (B Bland et al., 19821. · At equimolar doses, rat tissues have higher aflatoxin B~-adduct con- centrations than mouse tissues, possibly because mice have a higher rate of detoxification (Degan and Neumann, 1981; Monroe and Eaton, 1987~. · Rat hepatocytes have a much greater metabolic ability than hepatic sinusoidal cells to activate diethylnitrosamine and thus form DNA adducts (Lewis and Swenberg, 19831. Dose-dependent changes in rates of metabolic activation and detoxification themselves can affect the relation between administered dose and formation

Biologic Significance of DNA Adducts and Protein Adducts 9 of DNA abducts. For example, the tobacco carcinogen 4-(N-methyI-N-ni- ~osamino)-1-~3-pyr~dyl)-1-butanone (NNK) is more efficient per unit dose in producing 06-methylguanine at low doses than it is at high doses, perhaps because enzymes reach their capacity for activation of a xenobiotic com- pound. Thus, higher concentrations of the compound do not necessarily result in greater numbers of adducts (Belinsky et al., 19871. In contrast, the effi- ciency of benzo~aipyrene (BaP) (Adriaenssens et al., 1983) and formalde- hyde (Casanova-Schmitz et al., 1984) in forming DNA adducts and in binding covalently to DNA is greater per unit dose at high exposures, but in a nonlinear fashion. The effect of DNA repair on DNA-adduct accumulation might also be different at high and low doses. The 06-alkylguanine DNA alkyltransferases efficiently remove small amounts of the promutagenic adduct 06-alkyldeoxy- guanosine from DNi\, but become saturated as the concentration of o6- alkyIdeoxyguanosine in DNA increases (Peg", 1983~..As noted above, spe- cies, tissues, and cell types can differ in their concentrations of and abilities to induce these enzymes. For example, human livers have intrinsic concen- trations of 06-alky~guanine DNA alkyltransferase nearly lo times greater than Hose in rat livers (Peg", 19831. New unsensitive methods of detection make it possible to monitor DNA adducts in animals at exposures below those feasible in chronic bioassays and closer to those expected in the human population. Mathematical models that use such biologic dosimeters might yield more accurate extrapolations aIld thus improve quantitative risk assessment. Some problems in using DNA adducts to estimate human risks are related to differences between rodents and humans. We can calculate the risk as- sociated with DNA adducts in experimental animals, but interspecies ex- trapolations remain difficult to validate. Many experiments cannot ethically be performed in humans, and DNA adducts in human target cells or tissues would be expected to vary widely because of individual variations in DNA metabolism and repair. DYNAMICS OF DNA-ADDUCT FORMATION AND REMOVAL The chain of causation from toxic chemicals in drinking water or air to alterations of DNA in mammalian cells involves many pharmacokinetic steps. The rate constants of those steps depend on the chemical, species, sex, tissue, and, within a given tissue, cell type. Figure 1-4 shows how metabolic ac- tivation and detoxification affect the relationship between external concen- tration and DNA-adJuct concentration in three hypothetical cases of chronic exposure. The overall estimation of DNA adducts might not be useful, unless one can determine the ratio of biologically important to unimportant adducts.

|0 DRINKING WATER AND H"LTH 1. - 4, . C' ~ :~$ Z o ~ F C, Z ~ In as Use Z O Z ~ - INCREASING EXTERNAL EXPOSURE CONCENTRATION ~ A . c 1 if: C,9 X al oo O ~ ~0 lo: F Z ~ _ ~ ~ Z =8 B c - INCREASING EXTERNAL EXPOSURE CONCENTRATION ~ FIGURE 1-4 Relations between chronic external exposures and DNA-adduct concentration for steady state of adduct formation and repair in thme hypothetical cases: (a) neither fonnation nor repair reaches capacity at high concentration; (b) metabolic activation (adduct formation) reaches capacity at high external concentration; (c) DNA repair or detoxification reaches capacity at high concentration. Both scales are linear scales. The best example of such a classification is demonstrated by the adducts produced by methylating agents; the major DNA abduct formed is N7-meth- y~guanine (N7-MG), but this abduct is not involved in base-pairing and thus is relatively innocuous biologically. A minor adJuct, 06-methylguanine (o6- MG), which is involved in base-pairing, more closely reflects the mutagen- icity and carcinogenicity of methylating agents. The ratio of the two adducts depends critically on the chemical nature of the methylating agent. Hence, the concentration of N7-MG is not particularly useful as a measure of ex- posure without information on the proportion of N7-MG to 06-MG and on the nature of the methylating agent itself. DNA-AdJuct Formation Rates In chronic exposures, the rate of formation of DNA abducts depends on the concentration of compound in the tissue and the rate constant of formation (kf3. The rate of formation varies over time, because of changes in the tissue concentration of reactants that reflect their absorption, transport, and elim- ination. Low chronic exposures generally do not produce concentrations of xenobiotic compounds at which metabolic activation or detoxification sys- tems reach capacity, so the rate of formation of DNA adducts, ciAIdt, can be considered roughly proportional to the concentration of a toxicant that ultimately reacts with DNA, which in turn is proportional to the extracellular

Biologic Significance of DNA Adducts and Protein Adducts 11 concentration of the parent compound. If C is the time-weighted average concentration of the toxicant that ultimately reacts with DNA, the average rate of formation of adducts at low chronic exposures is given by: dAldt= kfC, (1) where A is the average DNA adduct concentration (e.g., adducts per 10~° nucleotides). At high doses, when capacity limitation might be reached, a more elaborate analysis is needed (Travis et al., 19891. The concentration C in the target cell might vary with time and tissue. In addition, it will probably vary with the person whose DNA is investigated, because concen- trations of activating and detoxifying enzymes vary widely among people. DNA Repair DNA adducts are not necessarily stable; some decompose spontaneously at body temperature. For example, alkylation of the nitrogen in purines tends to labilize the glycosidic bond and gives rise to apurinic sites. In addition, enzymatic DNA repair systems can directly remove the adduct itself, remove the DNA base that contains the adduct (base excision repair), or remove nucleotides that contain the adducted base (nucleotide excision repair) (Fried- berg, 1985~. The DNA repair systems probably arose as evolutionary consequences of damage to DNA that resulted from ultraviolet radiation (repaired by nucleo- tide excision), other naturally occurring alkylating agents and mutagens in food (NRC, 1973), and endogenous chemical or enzymatic reactions. The latter reactions are so numerous that, if DNA repair did not occur, 10% of all human DNA bases would be altered in an average lifetime (Tice and Setlow, l9SS). The enzymatic DNA repair mechanisms all seem to have capacities far in excess of what is needed to handle the low rate of damage from endogenous reactions and low chronic exposures to most exogenous agents (Table 1-11. At chronic low doses, rates of DNA repair (he) are generally limited not by the capacities of repair systems, but by the time for repair enzymes or repair complexes to "find" an adduct. The rate of removal of adducts by repair may be expressed as -dAl~t = krA . (2) For chronic exposures, a steady state is reached when the rate of removal of adducts (Equation 2) equals the rate of production (Equation 1~: krA = kfC and A = (kflFr)C. (3) Under conditions of chronic low exposure, the maximal rate of repair is much

12 DRINKING WATER AND HEALTH TABLE 1-1 Approximate Rates of DNA Damage and Repair in Human Cells at Body Temperature. Estimated Estimated Maximal Occurrences Repair Rate, of Damage Base Pairs per Hour per Hour Type of Damage per Celia . per Cella References Endogenous Depur~nation 1,000 b Setlow, 1987; Tice and Setlow, 1985 Depyr~midination 55 b Tice and Setlow, 198S Cytosine deamination 15 b Setlow, 1987; Tice and Setlow, 1985 Single-strand breaks 5,000 2x 105 Setlow, 1987; Tice and Setlow, 1985 N7-methylguanine 3,500 Not reported Saul and Ames, 1986 O6-methylguanine 130 104 Setlow, 1987; Tice and Setlow, 1985 Oxidation products 120 105 Saul and Ames, 1986; Setlow, 1987 Exogenous Background ionizing radiation Single-strand breaks Oxidation damage Ultraviolet irradiation of skin (noon Texas sunlight) Primidine dimers 10-4 2 x 105 10-4-10-3 105 5x104 5X104 Setlow, 1987 Saul and Ames, 1986 Setlow, 1987; Tiee and Setlow, 1985 aMight be higher or lower by a factor of 2 (Setlow, 1983). bNot reported, but the rates are at least 104, to judge from the concentration of repair activities in cell extracts. greater than the rate of introduction of damage (Table 1-1), so the steady- state value of A is low. Sensitive techniques are needed to detect these low values. At low exposure rates, DNA-adduct concentrations are proportional to C and hence to exposure concentrations or dose rates. The ratio of A to exposure concentration is constant (a curves in Figure 1-41. For exposures at high dose rates, the capacities for adduct formation, detoxification, and repair might be reached. If adduct formation reaches capacity, but repair does not, the rate of formation approaches a constant KfmaX; at the steady state, KfmaX = krA and A = KfmaxIkr

Biologic Significance of DNA Adducts and Protein Adducts 13 A is independent of exposure concentration, and the ratio of A to exposure concentration approaches zero as the latter continues to increase (b curves in Figure 1-4~. However, if detoxification or the repair rate reaches capacity at lower concentrations than the activation rate, adducts continue to increase with time, adduct concentration rises without limit, and the biologic system deteriorates (c curves in Figure 1-4~. DNA that contains adducts has altered template properties, so the rate of introduction of mutations depends on the rate of DNA synthesis. The rate of introduction of altered RNA (possibly leading to changes in gene expres- sion) depends on the rate of transcription. The rates of introduction of errors in replication or transcription depend on both A and the rates of replication and transcription. Increased rates of cell replication are frequently associated with high-dose toxicity. Furthermore, DNA synthesis, transcription, and repair vary from one tissue to another and from one subject to another. The magnitudes of the variations depend on the particular repair system involved, genetic and environmental factors, and the pharmacokinetic and toxic prop- erties of the chemical agent producing the adducts (Wogan, 19881. In bacterial systems, exposure to mutagens at low concentrations often induces synthesis of new repair enzymes and an increase in repair rate. Such an adaptation is well documented for ultraviolet irradiation, whose effects are repaired by nucleotide excision (Friedberg, 1985, pp. 431, 432~. An increase in the rate of repair of DNA damage can also be produced by aLkylating agents and such other agents as benzoLa~pyrene that yield high- molecular-weight (bulky) DNA adducts. Adaptation increases the value of k2 in Equation (2) and results in a decrease in the steady-state value of A. Adaptation reactions in human cells have not been well documented. Insofar as some DNA adducts have been shown to be important in mu- tagenesis and carcinogenesis, estimates of long-term risk would be expected to be proportional to the steady-state concentration of such adducts. The constant of proportionality depends not only on rates of transcription of RNA and replication of DNA, but on biologic factors, such as the location of abducts in the genome and the presence of endogenous promoters or inhib- itors. SITE RELEVANCE Many carcinogens and mutagens react at more than one site on DNA, producing several types of DNA adducts (Figure 1-54. As stated above, adducts at different sites can differ greatly in the rates at which they are formed and repaired and in their efficiency in causing mutations. Thus, data on overall covalent binding or a covalent binding index (Lutz, 1979) could be misleading. It is important to consider all available relevant biologic data,

14 DRINKING WATER AND HEALTH DNA Components Binding Sites _ NH2 INS ~| Simple AJkyla20rs Aromatic Amines PAHe Epoxides Drugs Adenine: ~ ,1_ ~ N / N ~ 0H Guanine: J, 3 N NH2 N \ Cytosine: ~ ~ O ~ POOH I` N/~ CH3 Thymine: ~13 ll ~ OH'\ N / o 11 11 Phosphate: -0-P-O- ~ OH Cytostatic N1/N3/N6/N7 N1/N6/C8 N1/N6 N1/N3/N6 N3 N1_N6 N1 / N2 / N3 / o6 N1 / N2 / o6 N2 / N7 N7 / N1-N7 / N7 / N7-N7 N7 C8 o2 / N3 _ o2 / o4 / N3 _ + - + + N1-N2 N3 / N4 N3 / N3 N4 N3 FIGURE 1-5 Potential sites of binding in DNA. Specific nitrogen (N), oxygen (O), and carbon (C) atoms on the DNA components have different susceptibilities to binding. Adapted from Singer (1985) with additional information from Beland and Kadlubar (1985), Delclos et al. (1987), and Hemminki (1983). including mutagenic efficiency, when choosing DNA adducts to be used as molecular dosimeters or for risk assessment. Alkylation In DNA, the N7 position of guanine is the most nucleophilic site, and it is by far the site most often alkylated by electrophiles. All: the ring nitrogens

Biologic Significance of DNA Adducts and Protein Adducts 15 of the DNA bases, except the nitrogen attached to the deoxyribose sugar, have been shown to be alkylated to some extent by a variety of agents (Singer, 1975~. Figure 1-5 shows all the potential sites for alkylation in the four bases found in DNA, as well as on its phosphate backbone. These sites include the N1, N3, N7, and CS of guanine; the N1, N3, N7, and CS of adenine; the N3 of thymine; and the N3 of cytosine. In addition, all the exocyclic nitrogens and oxygens can be alkylated; these sites include the N2 and o6 of guanine, the N6 of adenine, the o2 and 04 of thymine, and the o2 and N4 of cytosine. Some chemicals, such as ethyl nitrosourea (ENU), have also been shown to alkylate the phosphate oxygens on the DNA backbone, forming phosphotriesters. With ENU, about 60% of total DNA ethylation occurs on the phosphate group (Singer, 19821. All the nucleophilic sites in DNA mentioned above are potential sites of aLkylation, as determined by in vitro experiments, but not all are significantly affected in vivo. Configuration and secondary structure of DNA can play a major role in chemical reactivity (Brown, 1974; Singer and Fraenkel-Conrat, 19691. Other factors, such as the size of the binding electrophile and the association of proteins with chromosomal DNA, also appear to affect the sites or magnitude of DNA alkylation in vivo (Singer, 1982; Swenson and Lawley, 19781. Although many chemicals can alkylate DNA directly, others, such as aromatic amines and polycyclic aromatic hydrocarbons, often undergo com- plex enzymatic modifications before they can alkylate DNA (Brookes, 1977; Kriek and Westra, 1979; Miller, 1978; Sims and Grover, 1974~. There are some striking differences between the DNA adducts produced by enzymat- ically modified chemicals and the adducts formed by simple alkylating agents (Hemminki, 1983~. Not only are many of the adducts formed by enzymat- ically modified chemicals large and aromatic, but for polycyclic aromatic hydrocarbons, the preferred site of reaction in DNA is different. They gen- erally alkylate exocyclic amino groups, particularly the N2 of guanine, whereas the preferred site of aromatic amines is the C8 of guanine. Base Mispairing During DNA replication and in newly synthesized DNA, hydrogen bonds become less stable, and mispairing can occur; thus, alkylation of the DNA bases at sites involved in hydrogen binding is potentially mutagenic (Kroger and Singer, 1979; Singer et al., 1978a, 1979, 1983a,b). Those sites include the N1, N2, and o6 of guanine; the o2, N3, and N4 of cytosine; the N1 and N6 of adenine; and the N3 and O4 of thymine. For example, alkylation of the o6 of guanine can cause miscoding by DNA and RNA polymerases (Abbott and Saffhill, 1979; Gerchman and Ludlum, 19739. O6-Alkylguanine has been shown to direct the misincorporation of substantial amounts of

|6 DRINKING WATER AND HEALTH thymine, instead of the expected cytosine, into newly synthesized DNA (Abbott and Saffhill, 1979; Green et al., 1984; Lawley, 1974; Loechler et al., 1984~. There is also evidence that 06-alkylguanine can direct some misincorporation of adenine (Snow et al., 1983~. Bulky adducts distort the DNA, again increasing the likelihood of misincorporation. Hydrolysis The N3 and N7 alkylpurines can be hydrolyzed from DNA as a conse- quence of the instability of their glycosyl bonds, even at neutral pH. The half-lives of those adducts in DNA can range from a few hours to several days (Singer and Grunberger, 1983~. Their rates of spontaneous hydrolysis are about 106 times greater than the rates for the unmodified purines. The glycosyl bonds of pyrimidines are 100 times more stable than those of the purines. As a consequence, depyrimidination of even the most labile alkyl- pyrimidine, 02-alkylcytosine, has a half-life about 35 times that of N7- alkylguanine (Singer et al., 197Sb). Nevertheless, depyrimidination of o2- alkylcytosine can contribute significantly to the formation of apyrimidinic sites. If apurinic or apyrimidinic sites are present in DNA at the time of replication, any base can be misincorporated into the newly synthesized DNA opposite the gap in the parental strand (Langley and Brookes, 1963~. Phosphate AdJucts The formation of alkyl phosphotriesters, first measured by Bannon and Verly (1972) and later by Sun and Singer (1975), on the phosphate backbone of DNA does not make the chain unstable. Alkyl phosphotriesters have been reported to repair with a half-life of several days in rat liver (O'Connor et al., 1973, 1975) and rat brain (Gosh and Rajewsky, 1974), perhaps as a result of enzymic excision of these products. Miller et al. (1971, 1974) and Kan et al. (1973) reported that triesters exhibit changes in a number of properties that are likely to affect normal replication. However, Rajewsky et al. ~ 1977) found no correlation between the persistence of phosphotriesters in DNA of brain and liver and the sensitivity of these organs to carcinogenesis by ENU. Cross-Links DNA-DNA cross-links can be created by bifunctional or polyfunctional alkylating agents. Brookes and Lawley (1961) demonstrated that di~guanin- 7-yl) derivatives could be formed in DNA exposed to bifunctional alkylating agents. The cross-linking is normally expected to occur between guanines on opposite strands of DNA. Formation of such an adduct is generally be

Biologic Significance of DNA Adducts and Protein Adducts 17 lieved to be lethal, since it would prevent DNA strand separation at repli- cation. However, no evidence has been presented to relate the occurrence of mutations or cancers to the formation of dialkyl-base adducts. DNA ADDUCTS AND TOXIC EFFECTS Relating information concerning DNA-adduct site and molecular biologic consequences of adduct formation to multistep processes like mutagenesis and carcinogenesis is difficult at best. Specific toxic effects of specific DNA adducts must be correlated with the induction of gene mutation, germ cell mutation, or tumor formation in animal models before the impact of DNA- adduct formation can be assessed in humans. DNA-adduct dosimetry studies are available for different chemical classes of mutagens and carcinogens in various bioassays, including in vitro short-term and single- and multiple- dose whole animal exposures. Some specific carcinogen-DNA-adduct re- lationships have been demonstrated in humans exposed to carcinogens oc- cupationally, environmentally, or otherwise. In Vitro Short-Term Bioassay The correlations between cytotoxicity, mutation frequency, and binding of BPDE I, which is the anti-isomer of benzoLa~pyrene (BaP) diol epoxide (BPDE), to DNA have been examined in normal diploid human fibroblasts in culture and xeroderma pigmentosum cells (Yang et al., 1980; McCormick and Maher, 1985), the latter of which are deficient in excision repair. Those studies seem to show that BPDE I-deoxyguanosine (BPDE I-dG) caused the observed cytotoxicity and mutations and that the relationship between BPDE I-DNA binding and frequency of induced mutations (in tests for resistance to the toxic effects of 6-thioguanine) is linear in normal cells. Similar linear relationships between mutation frequency and binding of BPDE I or BPDE II (the isomer of BPDE) to DNA have been reported in Salmonella typhi- murium strains TA98 and TA100 (Fatal et al., 1981~. Newbold et al. (1979) used an in vitro short-term bioassay to construct curves of mutation frequency versus DNA binding for 7-bromomethylbenzEalanthracene and BPDE I. They demonstrated linear to curvilinear relationships between total DNA binding in Chinese hamster V79 cells and mutation frequency (in tests for resistance to the toxic effects of 8-azaguanine) in the same cell type. A study by Arce et al. (1987) related overall BaP-DNA-adduct and BPDE I-dG-DNA-adduct concentrations to a variety of end points in four different cellculture systems: gene mutation and sister-chromatic exchange in Chinese hamster V79 cells, gene mutation and chromosomal aberrations in mouse lymphoma L5178Y cells (thymidine kinase [TK + / - ]), virus transformation in Syrian hamster embryo cells, and structural transformation in mouse em

18 DRINKING WATER AND HEALTH bryo C3H1OT,/2 fibroblasts. The relationship between the genetically toxic effect and BaP-DNA binding or BPDE I-dG was linear in each assay. Each genetic end point was induced with a different efficiency on a per-adduct basis, whether expressed as total BaP-DNA binding or as specific BPDE I-dG-DNA adducts. Even when standardized by expression of the number of BPDE I-dG adducts per unit of DNA required to double the frequency of induced biologic response, results were the same; the BPDE I-dG-DNA adduct had different potencies in different cell cultures and for different biologic end points. Similar results have been obtained with aromatic amines and amides (Arce et al., 1987; Heflich et al., 19861. Studies of the relationships between carcinogen-DNA adducts and muta- genesis have not been limited to high-molecular-weight (bulky) aromatics like BaP. Van Zeeland et al. (1985) reported on the relationships between 06-ethylguanine (06-EG) formation induced by ethylating agents in Chinese hamster V79 cells (in tests for resistance to the toxic effects of 6-thioguanine), in Escherichia cold (in tests for nalidixic acid resistance), and in mice (spe- cific-locus mutations). For each compound, very similar mutation frequencies per locus were observed in all three assays; this suggests that 06-EG might be a good marker for monitoring exposure to chemical mutagens and for predicting mutation induction by a methylating agent. In Vivo Germ Cell Mutation Little is known about the effect of DNA adducts on germ cell mutation. In two studies of male germ cells, the frequency of sex-linked recessive lethal mutations in Drosophila melanogaster (Aaron and Lee, 1978) and of specific- locus mutations in mice (Van Zeeland et al., 1985) increased linearly with increasing DNA ethylation. Studies comparing the effects of single and re- peated exposures were not as clear-cut. Russell (1984) and Russell et al. (1982) found that ENU was a potent inducer of specific-locus mutations in mouse spermatogonial stem celIs and that the mutation frequency was 5.8 times greater after a single 100-mg/kg exposure than after 10 week]y 10-mg/ kg exposures. In contrast, molecular-dosimetry studies with ENU (Sega et al., 1986) found that the amount of 06-EG fo~ed in testicular DNA by ENU at 100 mg/kg was only 1.4 times that expected in response to 10 weekly 10-mg/kg exposures. That result did not support the idea that 06-EG was an important mutagenic lesion in the germ cells. In fact, the molecular-dosimetry data fit the genetic data well, if two hits on the DNA, not involving the o6 of guanine, are assumed necessary to produce an effect. Thus, in correlating DNA-adduct formation with in vivo germ cell mutation, one must consider both acute and chronic exposure. This has been clearly demonstrated with regard to tumo- rigenesis as the end point.

Biologic Significance of DNA Adducts and Protein Adducts 19 Considerable evidence is accumulating that, for many agents, the induction of mutations does not occur randomly over the chromosome, but that a variety of mutations are formed at preferential "hot spots" in the chromosome (e.g., Benzer, 1961; Drobetsky et al., 1987; Richardson et al., 1987; Skopek et al., 1985; Thilly, 1985; Vrieling et al., 19881. Whether they differ between high and low doses and between species is not yet known, but the effect may be due to site-specific variations in DNA-adduct formation and repair. Tumorigenesis in Animal Models Models of tumor initiation, promotion, and progression have been devel- oped from studies in experimental carcinogenesis. In those models, DNA adducts appear to be of prime importance in the initiation stage of tumor formation, although DNA lesions might also facilitate conversion of initiated cells into tumors or of benign cells into malignant cells. Tumor initiation is often considered to be a single first step in tumor formation. However, initiation is a complex, multistep process. The elec- trophilic reactivity of chemicals or their metabolites with DNA does not lead stoichiometrically to mutation or cancer. Cell replication must occur prior to repair of DNA adducts for mutations to be induced. Many types of DNA lesions are induced by genetically toxic agents, and all lesions are not repaired equally. Information about these relationships is generally scanty, except for the case of some monofunctional adulating agents, where the induction of DNA damage and the ultimate induction of mutation in target cells are directly related quantitatively. Persistent DNA lesions should be considered good markers for measuring exposure; whether or not they are also markers of tumor initiation depends on the properties of the specific genetic toxicant of concern. Many carcinogenic compounds form DNA adducts that can be measured in rodent tissues after single and multiple doses or after chronic exposure (Bedell et al., 1982; Boucheron et al., 1987; Poirier and Beland, 1987; Swenberg et al., 1984; Wogan and Gorelick, 19851. Detecting all DNA adducts derived from a single carcinogen is complex, however, because they can form at many sites on all four DNA bases and on the phosphate backbone of DNA, and they are repaired at different rates (Wogan, 1988~. Few studies have shown a correlation between adduct formation early in carcinogen ex- posure and tumor formation in the same biologic system (Beland et al., 1988; Croy and Wogan, 1981; Dyroff et al., 1986; Kensler et al., 1986; Tullis et al., 1987), but maximal total adduct levels in target tissues of laboratory animals usually reflect carcinogen potency and may or may not be linearly related to dose over a wide range (Swenberg et al., 1987; Wogan, 1988~. This suggests that DNA adducts are generally necessary but not sufficient for tumor formation(Belandet al., 1988;Branstetter, 1987;Neumann, 1983;

20 DRINKING WATER AND HEALTH Wogan and Gorelick, 19851. Thus, some adducts might be found in organs that are not targets for tumorigenesis, and the same or smaller numbers of adducts might be found in organs that are targets (Beland et al., 19884. For example, Goth and Rajewsky (1974) and Kleihues and Margison (1974) demonstrated that 06-alkylguanine persisted in rat brain, but not in liver or kidney, after exposure to ENU or methylnitrosourea (MNU), which primarily produce brain tumors. Kleihues et al. (1974) used methyl methanesulfonate, a chemical much weaker than MNU in inducing brain tumors, to demonstrate that tumor induction was proportional to the amount of 06-methylguanine (06-MG) in the brain. Other experiments implicating 06-alkylguanine as a potentially mutagenic and carcinogenic lesion have been reported (Bedell et al., 1982; Beranek et al., 1983; Cairns et al., 1981; Dodson et al., 1982; Frei et al., 1978; Lawley and Martin, 1975; Lewis and Swenberg, 1980; Newbold et al., 1980; Swenberg et al., 19821. However, Kleihues and Ra- jewsky (1984) showed that persistence of 06-MG does not always result in the production of brain tumors. Mouse, gerbil, and hamster brains can show concentrations of 06-MG similar to those in rats, but have low susceptibility to brain-tumor induction by MNU. Even with today's supersensitive analytical methods, DNA adducts are sometimes unmeasurable at doses that are tumorigenic (Boucheron et al., 19871. Furthermore, tumors can occur spontaneously without chemical ex- posure and known DNA-adduct formation. In addition, the persistence of DNA adducts may or may not be related to susceptibility and target tissue specificity (Wogan, 1988~. Thus, the relationship between DNA-adduct for- mation and tumorigenesis is by no means clearly established. Differences in DNA-adduct formation and persistence appear to provide an explanation for some target site specificities in carcinogenesis (Beland and Kadlubar, 1985; Swenberg and Fennell, 1987~. For example, rats are more susceptible than mice to the carcinogenic effect of aflatoxin B~, and the difference is correlated with the relative concentrations of aflatoxin-DNA adducts in the liver (Croy and Wogan, 19814. Cell specificity of tumor induction in both mice (liver) and rats (liver and lung) has been attributed to differences in DNA-adduct accumulation and persistence among various cell types (Belinsky et al., 1987; Lewis and Swenberg, 1980; Lindamood et al., 19821. Tumors induced by chronic exposures to methylating hepatocarcinogens are predominantly hemangiosarcomas (involving nonparenchymal cells), whereas exposures to ethylating agents cause hepatocellular carcinomas (in- volving hepatocytes) (Richardson et al., 1985; Swenberg et al., 1984~. In the case of methylating agents, the nonparenchymal cells accumulate o6- MG, and the hepatocytes do not. Exposure to ethylating agents, however, leads to accumulation of 04-ethylthymine (04-ET), but not 06-ethylguanine (06-EG), in hepatocytes. Thus, it appears that 04-ET, but not 06-EG, is

Biologic Significance of DNA Adducts and Protein Adducts 21 correlated with occurrence of cancer in hepatocytes of rats. This primarily reflects differences in the abilities of these cell types to repair 06-alkyl- guanine. In vivo studies of BaP binding and tumorigenesis performed on mouse skin (Ashurst et al., 1983; Cohen et al., 1979) and other organs (Adriaenssens et al., 1983; Anderson et al., 1981; Stowers and Anderson, 1985; Wogan and Gorelick, 1985) yielded mixed results. BaP-DNA binding was observed in tissues both susceptible and nonsusceptible to the tumorigenic effects of BaP. Such results indicate that the presence of adducts alone might not necessarily lead to tumor formation. Potential germ cell mutagenesis must be regarded as a major burden for the human population (NRC, 1986, p. 69), even though heritable mutations caused by chemical exposures have not been detected in humans. In somatic cells, cancer is a heritable process, and DNA carries heritable information. DNA adducts might be sensitive indicators of early genetic effects that could be correlated with chromosomal damage in the target organ. Data from in viva experiments on mouse liver with benzidine-DNA adducts (Talaska et al., 1987) support the hypothesis that carcinogen-DNA adducts induce chro- mosomal aberrations and perhaps other toxic effects, including neoplasia. Single-Dose Exposures of Animals In contrast with the uncertainty of the relationship between DNA-adduct formation and tumorigenesis, the presence of unique, measurable DNA ad- ducts always indicates that exposure has occurred. In animals, single doses of carcinogens that do not saturate metabolic activation, detoxification, or DNA repair are linearly correlated with the numbers of DNA adducts mea- sured in different organs (Figure 1-4A, curve a). That is true of a wide range of doses of BaP given either topically (Pereira et al., 1979; Shugart, 1985) or orally (Dunn, 19831. Similar studies with aflatoxins administered orally (Wild et al., 1986) or intraperitoneally (Appleton et al., 1982) have shown liver DNA adducts to increase stoichiometrically with dose. When metabolic activation reaches capacity, a less than linear dose response is evident (Ap- pleton et al., 19829; and when detoxification or DNA repair reaches capacity, a greater than linear dose response occurs (Adriaenssens et al., 1983; Ca- sanova-Schmitz et al., 19841. Chronic Exposures of Animals Dose-response curves for DNA adducts formed in animals during chronic carcinogen administration have characteristic profiles that reflect the com- bined processes of DNA-adduct formation and their removal by DNA repair systems. Several studies have demonstrated that exposure by continuous

22 DRINKING WATER AND H"LTH feeding or frequent injection generally results in a DNA-adduct dose-response curve that is initially linear but levels off at higher doses, where the net rate of change in the concentration of adducts reaches a steady state (Figure 1- 4A, curve b). That is true for the liver carcinogens aflatoxin (Croy and Wogan, 1981; Wild et al., 1986), 2-acetylaminofluorene (Poirier et al., 1984), and diethylnitrosamine (Boucheron et al., 1987) and for the lung, nasal mucosa, and liver carcinogen 4-(N-methyl-N-nitrosamino)-1-~3-pyri- dyl)-l-butanone (NNK) (Belinsky et al., 1986, 1987~. In contrast, when Neumann (1984) injected trans-4-acetylaminostilbene into rats at 3- to 4-day intervals, he found that DNA-adduct formation in four organs had not reached a plateau within 6 weeks. It has been suggested that the failure to reach a plateau might be due to the time between exposures, as well as to the liver's relative efficiency in removing adducts formed from aromatic amines (Poirier et al., 19841. Chronic feeding studies show that the plateau might vary with the con- centration of carcinogen in the diet. For example, when diethylnitrosamine was given continuously to male Fischer-344 rats in drinking water, an increase in concentration from 0.4 ppm to 40 ppm caused a 100-fold increase in the steady-state adduct concentration in the livers of male Fischer-344 rats; how- ever, further exposures up to 100 ppm did not cause any additional increase in adduct formation (Boucheron et al., 1987~. This nonlinearity in dose- response primarily reflects killing of cells that contain adducts. Exposure to 100 ppm of diethylnitrosamine causes a 20-fold increase in liver cell prolif- eration. Again, the presence of DNA adducts indicates carcinogen exposure; but the magnitude of exposure cannot be determined from a single adduct measurement, if the sample is taken after either formation or removal of adducts has exceeded the straight linear portion of the dose-response rela- tionship in either dose or time. Studies in Humans in many studies in which DNA adducts have been measured in humans, the exposure was to a ubiquitous compound producing delayed clinical ef- fects, thus making cumulative exposure unknown and unexposed control populations unidentifiable. However, cancer patients receiving chemotherapy are one human cohort in which drug dosage is known and unexposed controls are easy to identify. Cisplatin is a known carcinogen in rodents (Leopold et al., 1979), but has not been reported to produce secondary tumors in cancer patients receiving platinum-based chemotherapeutic agents. Nucleated blood cell adducts in DNA of testicular- and ovarian-cancer patients receiving cisplatin have been measured, and a dose-response relationship for adduct formation has been observed (Poirier et al., 1985, 19871. Although only about 60% of the persons studied had measurable adducts, the adducts were

\ Biologic Significance of DNA Adducts and Protein Adducts 23 correlated with dose; but the absence of adducts in other patients was not related to absence of exposure. In fact, the presence of adducts was shown to be correlated with tumor remission (Reed et al., 1987) a finding of clinical importance. A limited number of carcinogen-DNA-adduct relationships have been successfully demonstrated in humans with occupational or environmental carcinogens; however, in many of these studies, information about dose was scanty or nonexistent, and the results obtained had to be interpreted carefully because of the nature of the assay system used. For example, an antiserum against BaP-DNA adducts has substantial cross-reactivity with other PAH- DNA adducts. Therefore, whenever this antiserum is used with an enzyme- linked immunosorbent assay (ELISA) or an ultrasensitive enzymatic radioim- munoassay (USERIA) to detect BaP-DNA adducts, it is actually detecting a variety of PAM-DNA adducts. This antiserum was used in a pilot study (Perera et al., 1982) in which lung tissues of patients with and without lung cancer were assayed by ELISA. The results showed that lung DNA from 4 of 14 patients with lung cancer was positive in this test, indicating the presence of adducts, probably from a variety of hydrocarbons. Shamsuddin et al. (1985) used the same antiserum in a USERIA to measure BaP-DNA antigenicity in the white blood cells of roofers and foundry workers, two groups known to have substantial occupational exposure to BaP. Samples from 7 of 28 roofers and from 7 of 20 foundry workers were positive for BaP-DNA antigenicity. Samples from 2 of 9 volunteer controls were also positive, and both of those were smokers. In a cross-sectional analysis that lacked controls, Harris et al. (1985) detected BaP-DNA antigenicity in the peripheral lymphocytes of coke-oven workers using a USERIA and syn- chronous fluorescence spectrophotometry (SFS) with the same antiserum. Of 27 coke-oven workers, 18 showed BaP-DNA antigenicity with a USERIA and 9 did not; of 41, 31 showed detectable adduct formation with SFS and 10 did not. Antibodies to BaP-DNA adducts were found in the serum of 28% of the workers (Harris et al., 19851. Haugen et al. (1986) studied BaP-DNA antigenicity in peripheral lym- phocytes of 38 top-side coke-oven workers and conducted personal air sam- pling for PAHs inside and outside the respirators of 4 of those workers. By SFS, samples from 4 of 38 workers had putative adducts; and by USERIA, 13 of 38 samples had detectable antigenicity. Inside the respirators, the concentration of total PAH ranged from 51 to 162 ~g/m3, and the concen- tration of BaP ranged from 1 to 4 ,ug/m3. Monoclonal antibodies have been used to detect O6-methyldeoxyguanosine adducts (presumably produced from nitrosamines formed in food) in the DNA of esophageal and stomach tissue of subjects from China (Umbenhauer et al., 1985~. A total of 37 malignant and nonmalignant specimens were ob- tained from esophageal and stomach tissue of patients undergoing surgery

24 DRINKING WATER AND H"LTH for cancer of the esophagus in Linxian Province, China (an area of high incidence of esophageal and gastric cancer), for study with a radioimmu- noassay to detect the presence of O6-methyldeoxyguanosine adducts. Ad- ditionally, 12 human tissue samples from Europeans were studied. Of the 37 samples from China, 27 had detectable adducts, and 5 of the 12 European samples had detectable adducts. Perera et al. (1987a) used ELISA to measure BaP-DNA antigenicity in multiple samples of white blood cells of 22 smokers and 24 nonsmokers. They also assayed 4-aminobiphenyl-hemoglobin adducts with negative chem- ical-ionization mass spectrometry. In sample 1, 5 of 22 smokers (23%) and 7 of 24 nonsmokers (29%) showed detectable BaP-DNA antigenicity; in sample 2, 4 of 20 smokers (20%) exhibited detectable antigenicity; in sample 3, 4 of 21 smokers (19%) and 4 of 21 nonsmokers showed detectable anti- genicity. Of the subjects with PAM-DNA adducts, the quantity of adducts was higher in smokers than in nonsmokers and higher in women than in men. Data on exposure of nonsmokers to environmental tobacco smoke were not available. 4-Aminobiphenylhemoglobin adduct formation correlated best with indexes of active smoking. More recently, Perera et al. (1987b) have utilized an ELISA to study BaP- DNA antigenicity among 22 foundry workers and 10 non-occupationally exposed controls. Foundry workers were characterized as having high, me- dium, and low exposures. Mean levels of PAM-DNA adducts (femtomoles/ microgram) increased with exposure (low = 0.32, medium = 0.53, high = 1.2), and there was a significant difference between control (0.063 and pooled exposure (0.60) group means. 32P-postlabeling has been used to investigate the presence of DNA adducts in placentas from smokers and nonsmokers (Everson et al., 1986~. Several modified nucleotides were detected with 32P-postlabeling; one was strongly related to maternal smoking, but only weakly related to either historical or biochemical measures of intensity of smoking. 32P-postlabeling has also been used to evaluate formation of PAM-DNA adducts among nonsmoking pregnant women with exposure to residential wood combustion smoke (RWC), and among unexposed, nonsmoking preg- nant women (Ready et al, 1987~. DNA was isolated from specimens from 12 exposed women (8 white blood cells, 4 placentas) and specimens from 13 unexposed women (8 white blood cells, 5 placentas). Comparison of exposed subjects with controls did not reveal exposure-related adducts; how- ever, all placentas contained unidentified adducts which were not present in maternal white blood cells. Phillips et al. (1988) studied the same group of foundry workers examined by Perera et al. (1987b), utilizing 32P-postlabeling. Foundry workers were classified as having high (>0.2 fig BP/m3), medium (0.05-0.2 1lgBP/m3), or low (<0.05 fig BP/m3) BaP exposure based on historical industrial hygiene

Biologic Significance of DNA Adducts and Protein Adducts 25 measurements and job title. Aromatic adducts were found in DNA from 3 of 4 samples from the high-exposure group, ~ of 10 samples of the medium- exposure group, 4 of 18 samples from the low-exposure group, and 1 of 9 samples from unexposed controls. No differences due to smoking habits were observed. Genetic heterogeneity of metabolic activation and exposures to exogenous compounds that may alter metabolism and adduct formation will contribute to interindividual variability in adduct formation. For example, heterogeneity of human polycyclic aromatic hydrocarbon activation is well established, and the extent of DNA-adduct formation varies over a 1,000-fold range (Harris et al., 19821. In addition, some compounds, such as antioxidants and other dietary ingredients, modulate metabolism and thereby alter adduct for- mation. Ethoxyquin, an antioxidant in cabbage and other plants, decreased adduct formation in rats by 95% when ingested with aflatoxin BY in a rat- liver tumorigenesis study (Kensler et al., 19851. A concomitant decrease in preneoplastic foci was observed. It was concluded that the cabbage content of the diet may be important in the relationship between aflatoxin-DNA adducts and liver cancer in the Chinese. PROTEIN ADDUCTS In protein, the amino acids most likely to be alkylated are cysteine, his- tidine, lysine, and the N-terminal amino acid. Hemoglobin adducts were suggested as suitable for monitoring dose by Osterman-Golkar et al. in 1976; unlike DNA adducts, which can be removed by repair mechanisms, protein adducts had been observed to persist over the 40-day lifespan of the eryth- rocyte in the mouse (Osterman-GoLkar et al., 19761. Ehrenberg and Oster- man-Golkar ( 1980) reviewed the use of protein alkylation to detect mutagenic agents. Such use requires that the exposure result in stable, covalent deriv- atives of amino acids, that the target protein be found in easily accessible fluids, such as blood, and that the derivatives be present in adequate con- centrations. Pereira and Chang (1981), studied the ability of 15 carcinogens and mu- tagens in a wide range of chemical classes to bind covalently to hemoglobin in rats. They used radiolabeled test compounds to demonstrate covalent binding of all the mutagens and carcinogens to hemoglobin. The extent of binding with the different compounds had a range of a factor of 100, but the fact that protein adducts were formed from all the compounds studied indicates the potential usefulness of this molecular target for measuring ex- posure. Segerback et al. (1978) showed that alkylation of hemoglobin in mice by methyl methanesulfonate (MMS) was a linear function of the injected dose. Similarly, the production of N-3-~2-hydroxyethyl~histidine in hemoglobin

26 DRINKING WATER AND H"LTH was found to be a linear function of ethylene oxide (EtO) inhalation exposure (Osterman-Golkar et al., 19831. Dose-response relationships have also been established for 4-aminobiphenyl (Tannenbaum et al., 1983), which showed a linear rate of binding to hemoglobin over a 10,000-fold exposure range; for trans-4-dimethylaminostilbene (Neumann et al., 1980), which had a linear rate of binding to rat hemoglobin over a 100,000-fold exposure range; and for chloroform (Pereira and Chang, 1982), which showed a linear rate of binding to rat and mouse hemoglobin over a 1,000-fold exposure range. Although the amount of hemoglobin alkylation can be related to chemical exposure, it can be used as an indication of risk of genetic toxicity only if hemoglobin alkylation is correlated with alkylations at mutationally important targets, such as DNA. For example, Neumann et al. (1980) showed that the binding of trans-4-dimethylaminostilbene to plasma proteins and hemoglobin was proportional to its binding to liver DNA, and Pereira et al. (1981) found that dose-response curves for the binding of 2-acetylaminofluorene to rat hemoglobin and to liver DNA were closely related over a large dose range. Thus, it might be possible to estimate ONA binding through measurement of protein binding. A more direct use of protein adducts to estimate the risk of genetic toxicity is to select a protein that can be a genetically significant target. Sega and Owens (197S, 1983) found that exposure of male mice to ethyl methane- sulfonate (EMS) and MMS produced significant increases in alkylation in late spermatids and early spermatozoa (the most genetically sensitive germ cell stages) that could not be attributed to increased DNA alkylation but were correlated with sperm protamine alkylation and dominant lethal mutations. Sega and Owens (1987) found that the temporal pattern of alkylation produced by ethylene oxide in protamine, but not DNA, of the maturing sperm stages of mice is correlated with the pattern of dominant lethal mu- tations produced by ethylene oxide in the same stages. Thus, measurement of chemical adducts in human sperm protamine might be a useful means of assessing human germinal exposure to genetic toxicants. Sega (Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn., personal communication, 1987) also studied the binding of ~4C-ac- rylamide to developing spermiogenic stages in mice. The temporal pattern of acrylamide binding in the different spermiogenic stages paralleled the temporal pattern of induced dominant lethal mutations and heritable trans- locations noted by Shelby et al. (1986, 1987), with the greatest binding in late spermatid and early spermatozoa! stages. Binding of acrylamide to DNA was not statistically measurable in the different stages, and binding to pro- tamine could account for essentially all the germ cell alkylation. The above-described studies of methyl methanesulfonate, ethyl methane- sulfonate, ethylene oxide, and acrylamide provide compelling evidence that protamine alkylation is temporally associated with dominant lethal mutations.

Biologic Significance of DNA Adducts and Protein Adducts 27 Sega noted that the proportion of DNA adducts formed in the sensitive stages of sperrniogenesis is small (e.g., MMS, EMS, and EtO) or not measurable (e.g., acrylamide). However, late spermatogenic cells are known to be repair- deficient, and it is possible that dominant lethal mutations occur because a small number of DNA lesions remain unrepaired. Further research is needed to investigate the mechanism by which these low-molecular-weight muta- genic compounds cause dominant lethal mutations and elucidate the relative roles of protamine alkylation and DNA alkylation. SUMMARY To use DNA adducts in risk estimation, one must relate them to other biologic events, such as germ cell mutation, tumorigenesis, or developmental effects. Experimental data correlating tumorigenesis with profiles of DNA- adduct dosimetry in the same animal tissues are sparse (they include studies on diethylnitrosamine and the liver carcinogens 4-(N-methyl-N-nitrosamino)- 1-~3-pyndyl)-1-butanone, 2-acetylaminofluorene, and aflatoxin). Some cor- relations have been observed between persistence of DNA adducts in target tissues and the induction of tumors, but with some compounds no correlations have been noted. This probably reflects the need to incorporate more biologic processes than DNA-adduct formation into risk assessment. No proof exists that developmental effects occur in humans; however, they are presumed to represent a percentage of the genetic damage known to occur. One immediate problem is the lack of appropriate data~sets from which models can be constructed and validated. Both acute and chronic testing should be performed over a wide dose range to acquire knowledge of the points at which detoxification and DNA repair reach their capacities and thus cause nonlinearities in dose-response relationship curves. Dose-response re- lationships for single exposures over a dose range of 103 have been established for tumor induction on only three carcinogens: dimethylnitrosamine, dieth- ylnitrosamine, and benzoLa~pyrene. Several compounds have been studied in bioassays in which the dose ranged over a factor of 100, but bioassays on most carcinogens use doses that range over a factor of 10 or less- including the largest study ever performed, the effective-dose (EDo~) bioassay of 2-acetylaminofluorene (Staffa and Mehlman, 19791. Few DNA-adduct studies have covered dose ranges and used exposure protocols that could be compared. Although a broader dose range is not always possible because of the occurrence of toxic effects, present adduct-detection methods are probably now capable of measuring the results of testing with very low doses. Correlations between DNA-adduct dose-response relationships and biol- ogic effects seem to be compound-specific and independent of chemical cIass or biologic end point. Even for a single compound, quantitative comparisons of chemical-DNA binding and hazard assessment are complicated. One re

28 DRINKING WATER AND HEALTH lationship will not accurately describe all situations; it will vary with the compound, the specific target tissue, the organism's exposure history, the duration and time of exposure, etc. Individual rates of metabolic activation of carcinogens (particularly PAHs) and repair capacities are variable and moderated by personal exposure histones. Thus, because the same chemical exposure can produce widely varying numbers of adducts, prediction of the extent of exposure or the resultant cancer risk is much more difficult in humans on the basis of DNA adducts than in homogeneous laboratory ani- mals. In addition, for many toxic chemicals, the mutagenic or tumongenic adduct has not been identified and can occur among many others that may not produce deleterious effects; thus, measuring overall DNA binding at- tributable to a specific chemical could lead to errors in the estimation of hazard. Despite current gaps in knowledge, DNA-adduct research represents a very promising means to improve risk assessment. When more extensive data become available, they might be used in individual risk assessment to confirm suspected exposures, improve estimates of target tissue dose, and reveal metabolic activation and detoxification parameters that moderate the formation of DNA adducts by a specific carcinogen. In general risk assess- ment, they could be valuable in estimating dosimetry and systemic distri- bution and in establishing possible target tissues or organs and the potential for irreversible toxicity, such as cancer, mutation, or developmental effects. They might improve estimates of the rates of tumor and adduct formation in animals in response to low doses on the basis of high-dose effects and provide better models for predicting mechanisms in humans. Large-scale DNA-adduct dosimetry studies in humans are now becoming possible, but they must be validated and their limitations defined. In addition, protein adducts, such as those found in sperm protamine and hemoglobin, are ap- parently stable for the lifetime of the cell, accurately indicate recent exposure, and should be considered in the estimation of genetic or carcinogenic risk whenever they can be correlated with DNA binding. Monitoring protein adducts has generally been considered to be a good surrogate procedure for measuring DNA-adduct formation in the target organ, but this should be validated in laboratory animals for each compound of interest. REFERENCES Aaron, C. S., and W. R. Lee. 1978. Molecular dosimetry of the mutagen ethyl methanesul- fonate in Drosophila melanogaster spermatozoa: Linear relation of DNA alkylation per sperm cell (dose) to sex-linked recessive lethals. Mutat. Res. 49:27-44. Abbott, P. J., and R. Saffhill. 1979. DNA synthesis with methylated poly (dC-dG) templates: Evidence for a competitive nature to miscoding by 06-methylguanine. Biochim. Biophys. Acta. 562:51-61.

Biologic Significance of DNA AdJucts and Protein Adducts 29 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. Anderson, M. W. 1987. Carcinogen-DNA adducts as a measure of biological dose for risk analysis of carcinogenic data. Pp. 221-228 in National Research Council, Pharmacokinetics in Risk Assessment. Dnnking Water and Health, Vol. 8. Washington, D.C.: National Academy Press. Anderson, M. W., M. Boroujerdi, and A. G. E. Wilson. 1981. Inhibition in viva of the formation of adducts between metabolites of benzo(a)pyrene and DNA by butylated hy- droxyanisole. Cancer Res. 41:4309-4315. Appleton, B. S., M. P. Goetchius, and T. C. Campbell. 1982. Linear dose-response curve for the hepatic macromolecular binding of aflatoxin Be in rats at very low exposures. Cancer Res. 42:3659-3662. Arce, G. T., J. W. Allen, C. L. Doerr, E. Elmore, G. G. Hatch, M. M. Moore, Y. Sanef, D. Grunberger, and S. Nesnow. 1987. Relationships between benzo(a)pyrene-DNA adduct levels and genotoxic effects in mammalian cells. Cancer Res. 47:3388-3395. Ashurst, S. W., G. M. Cohen, S. Nesnow, J. DiGiovanni, and T. J. Slaga. 1983. Formation of benzo(a)pyrene/DNA adducts and their relationship to tumor initiation in mouse epidermis. Cancer Res. 43: 1024-1029. Bannon, P., and W. Verly. 1972.:ALkylation of phosphates and stability of phosphate triesters in DNA. Eur. J. Biochem. 31:103-111. Bedell, M. A., J. G. Lewis, K. C. Billings, and J. A. Swenberg. 1982. Cell specificity in hepatocarcinogenesis: Preferential accumulation of O6-methylguanine in target cell DNA during continuous exposure of rats to 1,2-dimethylhydrazine. Cancer Res. 42:3079-3083. Beland, F. A., and F. F. Kadlubar. 1985. Formation and persistence of arylamine DNA adducts in vivo. Environ. Health Perspect. 62:19-30. Beland, F. A., K. L. Dooley, and C. D. Jackson. 1982. Persistence of DNA adducts in rat liver and kidney after multiple doses of the carcinogen N-hydroxy-2-acetylaminofluorene. Cancer Res. 42: 1348-1354. Beland, F. A., N. F. Fullenon, T. Kinouchi, and M. C. Poirier. 1988. DNA adduct formation dunng continuous feeding of 2-acetylaminofluorene at multiple concentrations. Pp. 175- 180 in Methods for Detecting DNA Damaging Agents in Humans: Applications in Cancer Epidemiology and Prevention, H. Bartsch, K. Hemminki, and I. K. O'Neill, eds. IARC Scientific Publications No. 89. Lyon: International Agency for Research on Cancer. Belinsky, S. A., C. M. White, J. A. Boucheron, F. C. Richardson, J. A. Swenberg, and M. Anderson. 1986. Accumulation and persistence of DNA adducts in respiratory tissue of rats following multiple administrations of the tobacco specific carcinogen 4-(N-methyl-N-nitros- amino)-1-(3-pyridyl)-1-butanone. Cancer Res. 46: 1280-1284. Belinsky, S. A., C. M. White, T. R. Devereux, J. A. Swenberg, and M. W. Anderson. 1987. Cell selective alkylation of DNA in rat lung following low dose exposure to the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res. 47:1143-1148. Benzer, S. 1961. On the topography of the genetic fine structure. Proc. Natl. Acad. Sci. USA 47:403-415. Beranek, D. T., R. H. Heflich, R. L. Kodell, S. M. Morris, and D. A. Casciano. 1983. Correlation between specific DNA-methylation products and mutation induction at the HGPRT locus in Chinese hamster ovary cells. Mutat. Res. 110: 171 - 180. Boucheron, J. A., F. C. Richardson, P. H. Morgan, and J. A. Swenberg. 1987. Molecular

30 DRINKING WATER AND H"LTH dosimetry of O4-ethyldeoxythymidine in rats continuously exposed to diethylnitrosamine. Cancer Res. 47-1577-1581. Branstetter, D. G., G. D. Stoner, H. A. J. Schut, D. Senitzer, P. B. Conran, and P. J. Gold- blatt. 1987. Ethylnitrosourea-induced transplacental Carcinogenesis in the mouse: Tumor response, DNA binding, and adduct formation. Cancer Res. 47:348-352. Brookes, P. 1977. Mutagenicity ofpolycyclic aromatic hydrocarbons. Mutat. Res. 39:257- 284. Brookes, P., and P. D. Lawley. 1961. The reaction of mono- and all-functional alkylating agents with nucleic acids. Biochim. J. 80:496-503. Brown, D. M. 1974. Chemical reactions of polynucleotides and nucleic acids. Pp. 1-90 in Basic Principles in Nucleic Acid Chemistry, Vol. II, P. O. P. Ts'O, ed. New York: Academic Press. Cairns, J., P. Robins, B. Sedgwick, and P. Talmud. 1981. The inducible repair of alkylated DNA. Progr. Nucl. Acid Res. Mol. Biol. 26:237-244. Casanova-Schmitz, M., T. B. Starr, and H. d'A. Heck. 1984. Differentiation between met- abolic incorporation and covalent binding in the labeling of macromolecules in the rat nasal mucosa and bone marrow by inhaled [TIC]- and [3H]formaldehyde. Toxicol. Appl. Phar- macol. 76:26-44. Cohen, G. M., W. M. Bracken? R. P. Iyer, D. L. Berry, J. K. Selkirk and T. J. Slaga. 1979. Anticarcinogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on benzo(a)pyrene and 7,12- dimethylbenz(a)-anthracene tumor initiation and its relationship to DNA binding. Cancer Res. 39:4027-4033. Croy, R. G., and G. N. Wogan. 1981. Temporal patterns of covalent DNA adducts in rat liver after single and multiple doses of aflatoxin Be. Cancer Res. 41:197-203. Degen, G. H., and H. G. Neumann. 1981. Differences in aflatoxin B~-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatox B~-epoxide. Car- cinogenesis 2:299-306. Delclos, K. B., D. W. Miller, J. O. Lay, Jr., D. A. Casciano, R. P. WaLker, P. P. Fu, and F. F. Kadlubar. 1987. Identification of C8-modified deoxyinosine and N2-and C8-modified deoxyguanosine as major products of the in vitro reaction of N-hydroxy-~aminochrysene with DNA and the formation of these adducts in isolated rat hepatocytes treated with 6- nitrochrysene and 6-aminochrysene. Carcinogenesis 8:1703-1709. Dodson, L. A.. R. S. Foote, S. Mitra, and W. E. Masker. 1982. Mutagenesis of bacteriophage T7 in vitro by incorporation of 06-methylguanine during DNA synthesis. Proc. Natl. Acad. Sci. USA 79:7440-7444. Drobetsky,E. A.,A. J.Grosovsky,andB. W.Glickman. 1987.ThespecificityofUV-induced mutations at an endogenous locus in mammalian cells. Proc. Natl. Acad. Sci. USA 84:9103- 9107. Dunn, B. P. 1983. Wide-range linear dose-response curve for DNA binding of orally~admin- istered benzo(a)pyrene in mice. Cancer Res. 43:265~2658. Dyroff, M. C., F. C. Richardson, J. A. Popp, M. A. Bedell, and J. A. Swenberg. 1986. Correlation of O4-ethyldeoxythymidine accumulation, hepatic initiation and hepatocellular carcinoma induction in rats continuously administered diethylnitrosamine. Carcinogenesis 7:241-246. Ehrenberg, L., and S. Osterman-Golkar. 1980. Alkylation of macromolecules for detecting mutagenic agents. Teratogenesis Carcinog. Mutagen. 1: 105- 127. Ehrenberg, L., S. Osterman-Golkar, D. Segerback, K. Svensson, and C. J. Calleman. 1977. Evaluation of genetic risks of alkylating agents. III. Alkylation of haemoglobin after met- abolic conversion of ethene to ethene oxide in vivo. Mutat. Res. 45:175-184.

Biologic Significance of DNA Adducts and Protein Adducts 31 Everson, R. B., E. Randerath, R. M. Santella, R. C. Cefalo, T. A. Avitts, and K. Randerath. 1986. Detection of smoking-related covalent DNA-adducts in human placenta. Science 231:5~57. Fahl, W. E., D. G. Scarpelli, and K. Gill. 1981. Relationship between benzo(a)pyreneinduced DNA base modification and frequency of reverse mutations in mutant strains of Salmonella typhimurium. Cancer Res. 41:3400-3406. Frei, J. V., D. H. Swenson, W. Warren, and P. D. Lawley. 1978. Alkylation of deoxyn- bonucleic acid in viva in various organs of C57B1 mice by the carcinogens N-methyl-N- nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulphonate in relation to induction of thymic lymphoma. Biochim. J. 174:1031-1044. Frieberg, E. C. 1985. DNA Repair. New York: W. H. Freeman. Gerchman, L. L., and D. B. Ludlum. 1973. The properties of O6-methylguanine in templates for RNA polymerase. Biochim. Biophys. Acta. 308:310-316. Goth, R., and M. F. Rajewsky. 1974. Persistence of O6-ethylguanine in rat brain DNA: Correlation with nervous system-specif~c carcinogenesis by ethylnitrosourea. Proc. Natl. Acad. Sci. USA 71:639-643. Green, C. L., E. L. Loechler, K. W. Fowler, and J. M. Essigmann. 1984. Construction and characterization of extrachromosomal probes for mutagenesis by carcinogens: Site-specific incorporation of 06-methylguanine in viral and plasmid genomes. Proc. Natl. Acad. Sci. USA 81:13-17. Harris, C. C., B. F. Trump, R. Grafstrom, and H. Autrup. 1982. Differences in metabolism of chemical carcinogens in cultured human epithelial tissues and cells. J. Cell. Biochem. 18:285-294. Harris, C. C., K. Vahakangas, M. J. Newman, G. E. Trivers, A. Shamsuddin, N. Sinopoli, D. L. Mann, and W. E. Wright. 1985. Detection of benzo(a)pyrene diol epoxide-DNA adducts in peripheral blood lymphocytes and antibodies to the adducts in serum from coke oven workers. Proc. Natl. Acad. Sci. USA 82:6672-6676. Haugen, A., G. Becher, C. Benestad, K. Vahakangas, G. E. Trivers, M. J. Newman, and C. C. Hams. 1986. Determination of polycyclic aromatic hydrocarbons in the unne, benzo(a)pyrene diol epoxide-DNA adducts in lymphocyte DNA, and antibodies to the ad- ducts in sera from coke oven workers exposed to measured amounts of polycyclic aromatic hydrocarbons in the work atmosphere. Cancer Res. 46:4178-4183. Heflich, R. H., S. M. Moms, D. T. Beranek, L. J. McGamty, J. J. Chen, and F. A. Beland. 1986. Relationships between the DNA adducts and the mutations and sister-chromatic ex- changes produced in Chinese hamster ovary cells by N-hydroxy-2-aminofluorene, N-hy- droxy-N~-acetyl benzidine 1-nitrosopyrine. Mutagenesis 1:201-206. Hemminki, K. 1983. Nucleic acid adducts of chemical carcinogens and mutagens. Arch. Toxicol. 52:249-285. Hoel, D. G., N. L. Kaplan, and M. W. Anderson. 1983. Implication of nonlinear kinetics on risk estimation in carcinogenesis. Science 219:1032-1037. Kan, L. S., J. C. Barrett, P. S. Miller, and P. O. P. Ts'O. 1973. Proton magnetic resonance studies of the conformational changes of dideoxynucleoside ethyl phosphotriesters. Biopo- lymers 12:2225-2240. Kensler, T. W., P. A. Egner, M. A. Trush, E. Bueding, and J. D. Groopman. 1985. Mod- if~cation of aflatoxin B~ binding to DNA in vivo in rats fed phenolic antioxidants, ethoxyquin and a dithiothione. Carcinogenesis 6:759-763. Kensler, T. W., P. A. Egner, N. E. Davidson, B. D. Roebuck, A. Pikul, and J. D. Groopman. 1986. Modulation of aflatoxin metabolism, aflatoxin-N7-guanine formation, and hepatic tumorigenesis in rats fed ethoxyquin: Role of induction of glutathione-S-transferases. Cancer Res. 46:3924-3931.

32 DRINKING WATER AND H"LTH Kleihues, P., and G. P. Margison. 1974. Careinogenieity of N-methyl-N-nitrosourea: Possible r4le-of excision repair of O6-methylguanine from DNA. J. Natl. Caneer Inst. 53: 1839- 1841. Kleihues, P., and M. F. Rajewsky. 1984. Chemical neurooneogenesis: Role of structural DNA modifications, DNA repair and neural target cell population. Prog. Exp. Tumor Res. 27:1- - 16. Kleihues, P., K. Patzsehke, G. P. Margison, L. W. Wegner, and C. Mende. 1974. Reaction of methyl methanesulphonate with nucleic acids of fetal and newborn rats in viva. Z. Krebsforseh. 81 :273-283. Kriek, E., and J. G. Westra. 1979. Metabolic activation of aromatic amines and amides and interaction with nucleic acids. Pp. 1-28 in Chemical Carcinogens and DNA, Vol II, P. L. Grover, ed. Boea Raton, Fla.: CRC Press. Kroger, M., and B. Singer. 1979. Ambiguity and transcriptional errors as a result of methylation of the N-1 of purines and N-3 of pyrimidines. Bioehemistry 18:3493-3500. Lawley, P. D. 1974. Alkylation of nucleic acids and mutagenesis. Pp. 17-33 in Molecular and Environmental Aspects of Mutagenesis, L. Prakash, F. Sherman, M. W. Miller, C. W. Lawrence, and H. W. Taber, eds. 6th Rochester International Conference on Environmental Toxicity. Springfield, Ill.: Charles C Thomas. Lawley, P. D., and P. Brookes. 1963. Further studies on the alkylation of nucleic acids and their constituent nueleotides. Bioehim. J. 89:127-138. Lawley, P. D., and C. N. Martin. 1975. Molecular mechanisms in alkylation mutagenesis: Induced reversion of bacteriophage T4rII AP72 by ethyl methanesulphonate in relation to extent and mode of ethylation of purines in bacteriophage deoxyribonucleic acid. Bioehim. J. 145:85-91. Leopold, W. R., E. C. Miller, and J. A. Miller. 1979. Careinogenieity of antitumor cis- platinum(II) coordination complexes in the mouse and rat. Caneer Res. 39:913-918. Lewis, J. G., and J. A. Swenberg. 1980. Differential repair of O6-methylguanine in DNA of rat hepatoeytes and non-parenehymal cells. Nature 288: 185- 187. Lewis, J. G., and J. A. Swenberg. 1983. The kinetics of DNA alkylation, repair and replication in hepatoeytes, Kupffer cells, and sinusoidal endothelial cells in rat liver during continuous exposure to 1,2-dimethylhydrazine. Carcinogenesis 4:529-536. Lindamood, C.. III, M. A. Bedell, K. C. Billings, and J. A. Swenberg. 1982. Alkylation and de mono synthesis of liver cell DNA from C3H mice during continuous dimethylnitrosamine exposure. Caneer Res. 42:4153-4157. Loeehler, E. L., C. L. Green, and J. M. Essigmann. 1984. In vivo mutagenesis by O6-methyl- guanine built into a unique site in a viral genome. Proc. Natl. Aead. Sei. USA 81:6271- 6275. Lutz, W. K. 1979. In vivo covalent binding of organic chemicals to DNA as a quantitative indicator in the process of chemical carcinogenesis. Mutat. Res. 65:289-356. McCormick, J. J., and V. M. Maher. 1985. Cytotxie and mutagenic effects of specific ear- einogen-DNA adduets in diploid human f~broblasts. Environ. Health Perspeet. 62: 145- 155. Miller, E. C. 1978.. Some current perspectives on chemical eareinogenesis in humans and experimental animals: Presidential address. Caneer Res. 38:1479-1496. Miller, P. S., K. N. Fang, N. S. Kondo, and P. O. P. Ts'O. 1971. Synthesis and properties of adenine and thymine nueleoside alkyl phosphotriesters, the neutral analogs of dinucleoside monophosphates. J. Am. Chem. Soc. 93:6657-6665. Miller, P. S., J. C. Barrett, and P. O. P. Ts'O. 1974. Synthesis of oligodeoxyribonueleotide ethyl phosphotriesters and their specific complex formation with transfer ribonueleic acid. Bioehemistry 13:4887-4896. Monroe, D. H., and D. L. Eaton. 1987. Comparative effects of butylated hydroxyanisole on

Biologic Significance of DNA Adducts and Protein Adducts 33 hepatic in vivo DNA binding in vitro biotransformation of aflatoxin B. in the rat and mouse. Toxicol. Appl. Pharmacol. 90:401-409. Neumann, H.-G. 1983. Role of extent and persistence of DNA modifications in chemical carcinogenesis by aromatic amines. Recent Results Cancer Res. 84:77-89. Neumann, H.-G. 1984. Analysis of hemoglobin as a dose monitor for alkylating and arylating agents. Arch. Toxicol. 56:1-6. Neumann, H.-G., H. Baur, and R. Wirsing. 1980. Dose relationship in the primary lesion of strong electrophilic carcinogens. Arch. Toxicol. 3(Suppl.):69-77. Newbold, R. F., P. Brookes, and R. G. Harvey. 1979. A quantitative comparison of the mutagenicity of carcinogenic polycyclic hydrocarbons derivates in cultured mammalian cells. Int. J. Cancer 24:203-209. Newbold, R. F., TV. Warren, A. S. C. Metcalf, and J. Amos. 1980. Mutagenicity of carcin- ogenic methylating agents is associated with a specific DNA modification. Nature 283:596- 599. NRC (National Research Council). 1973. Toxicants Occurring Naturally in Foods, 2nd ed. Washington, D.C.: National Academy of Sciences. 624 pp. NRC (National Research Council). 1983. Risk Assessment in the Federal Government: Man- aging the Process. Washington, D.C.: National Academy Press. 191 pp. NRC (National Research Council). 1986. Drinking Water and Health, Vol. 6. Washington, D.C.: National Academy Press. 457 pp. NRC (National Research Council). Committee on Biological Markers. 1987. Biological mark- ers in environmental health research. Environ. Health Perspect. 74:3-9. O'Connor, P. J., M. J. Capps, and A. W. Craig. 1973. Comparative studies of the hepato- carcinogen N,N-dimethylnitrosamine in vivo: Reaction sites in rat liver DNA and the sig- nificance of their relative stabilities. Br. J. Cancer 27:153-166. O'Connor, P. J., G. P. Margison, and A. W. Craig. 1975. Phosphotriesters in rat liver de- oxyribonucleic acid after the administration of the carcinogen N,N-dimethylnitrosamine in viva. Biochem. J. 145:475-482. Osterman-Golkar, S., P. B. Farmer, D. Segerbck, E. Bailey, C. J. Calleman, K. Svensson, and L. Ehrenberg. 1983. Dosimetry of ethylene oxide in the rat by quantitation of alkylated histidine in hemoglobin. Teratogenesis Carcinog. Mutagen. 3:395-405. Osterman-Golkar, S. L., L. Ehrenberg, D. Segerback, and I. Hallstrom. 1976. Evaluation of genetic risks of alkylating agents. II. Haemoglobin as a dose monitor. Mutat. Res. 34:1- 10. Pegg, A. E. 1983. Alkylation and subsequent repair of DNA after exposure to dimethylnitro- samine and related carcinogens. Rev. Biochem. Toxicol. 5:83-133. Pereira, M. A., and L. W. Chang. 1981. Binding of chemical carcinogens and mutagens to rat hemoglobin. Chem.-Biol. Interact. 33:301-305. Pereira, M. A., and L. W. Chang. 1982. Binding of chloroform to mouse and rat hemoglobin. Chem.-Biol. Interact. 39:89-99. Pereira, M. A., F. J. Burns, and R. E. Albert. 1979. Dose response for benzo(a)pyrene in mouse epidermal DNA. Cancer Res. 39:2556-2559. Pereira, M. A., L.-H. C. Lin, and L. W. Chang. 1981. Dose dependency of 2-acetylami- nofluorene binding to liver DNA and hemoglobin in mice and rats. Toxicol. Appl. Pharmacol. 60:472-478. Perera, F. P., M. C. Poirier, S. H. Yuspa, J. Nakayama, A. Jaretzki, M. M. Curnen, D. M. Knowles, and I. B. Weinstein. 1982. A pilot project in molecular cancer epidemiology: Determination of benzo[a]pyrene-DNA adducts in animal and human tissues by immu- noassays . Carcinogenesis 3: 1405 - 1410.

34 DRINKING WATER AND HEALTH Perera, F. P., R. M. Santella, D. Brenner, M. C. Poirier, A. A. Munshi, H. K. Fischman, and J. Van Ryzin. 1987a. DNA adducts, protein adducts, and sister chromatic exchange in cigarette smokers and nonsmokers. J. Natl. Cancer Inst. 79:449-456. Perera, F. P., K. Hemminki, R. M. Santella, D. Brenner, and G. Kelly. 1987b. DNA adducts in white blood cells of foundry workers (Meeting abstract). Proc. Annul Meet. Am. Assoc. Cancer Res. 28:94. Phillips, D. H., K. Hemminki, A. Alhonen, A. Hewer, and P. L. Grover. 1988. Monitoring occupational exposure to carcinogens: Detection by 32P-postlabeling of aromatic DNA ad- ducts in white blood cells from iron foundry workers. Mutat. Res. 204:531-541. Poirier, M. C., and F. A. Beland. 1987. Determination of carcinogen-induced macromolecular adducts in animals and humans. Prog. Exp. Tumor Res. 31:1-10. Poirier, M. C., J. M. Hunt, B. A. True, B. A. Laishes, J. F. Young, and F. A. Beland. 1984. DNA adduct formation, removal and persistence in rat liver during one month of feeding 2-acetylaminofluorene. Carcinogenesis 5:1591-1596. Poirier, M., E. Reed, L. Zwelling, R. Ozols, C. Litterest, and S. Yuspa. 1985. Polyclonal antibodies to quantitate cis-diamminedichloroplatinum (II)-DNA-adducts in cancer patients and animal models. Environ. Health Perspect. 62:89-94. Poirier, M. C., E. Reed, R. F. Ozols, T. Fasy, and S. H. Yuspa. 1987. DNA adducts of cisplatin in nucleated peripheral blood cells and tissues of cancer patients. Prog. Exp. Tumor Res.31:104-113. Rajewsky, M. F., L. H. Augenlicht, H. Biessmann,-R. Goth, D. F. Huelser, O. D. Laerum, and L. Y. Lomakina. 1977. Nervous system-specific carcinogenesis by ethyl nitrosourea in the rat: Molecular and cellular aspects. Pp. 709-726 in Origins of Human Cancer, H. H. Hiatt, J. D. Watson, and J. A. Winsten, eds. New York: Cold Spring Harbor Laboratory. Reddy, M. V., P. C. Kenny, and K. Randerath. 1987. 32P-assay of DNA adducts in white blood cells (WBC) and placentas of pregnant women exposed to residential wood combustion (RWC) smoke (Meeting abstract). Proc. Annul Meet. Am. Assoc. Cancer Res. 28:97. Reed, E., R. F. Ozols, R. Tarone, S. H. Yuspa, and M. C. Poirier. 1987. Platinum-DNA adducts in leukocyte DNA correlate with disease response in ovarian cancer patients receiving platinum-based chemotherapy. Proc. Natl. Acad. Sci. USA 84:5024-5028. Richardson, F. C., M. C. Dyroff, J. A. Boucheron, and J. A. Swenberg. 1985. Differential repair of 04-alkylthymidine following exposure to methylating and ethylating hepatocarcin- ogens. Carcinogenesis 6:625-629. Richardson, K. K., F. C. Richardson, R. M. Crosby, J. A. Swenberg, and T. R. Skopek. 1987. DNA base changes and alkylation following ~n vivo exposure of Escherichia cold to N-methyl-N-nitrosourea orN-ethyl-N-nitrosourea. Proc. Natl. Acad. Sci. USA 84:344-348. Russell, W. L. 1984. Dose-response, repair and no-effect dose levels in mouse germ-cell mutagenesis. Pp. 153-160 in Problems of Threshold in Chemical Mutagenesis, Y. Tazima, S. Kondo, and Y. Kuroda, eds. Tokyo: Environmental Mutagen Society of Japan. Russell, W. L., P. R. Hunsicker, D. A. Carpenter, C. V. Cornett, and G. M. Guinn. 1982. Effect of dose fractionation on the ethylnitrosourea induction of specific locus mutations in mouse spermatogonia. Proc. Natl. Acad. Sci. USA 79:3592-3593. Saul, R. L., and B. N. Ames. 1986. Background levels of DNA damage in the population. Pp. 529-535 in Mechanisms of DNA Damage and Repair, M. G. Simic, L. Grossman, and A. C. Upton, eds. Basic Life Sciences, Vol. 38. New York: Plenum. Sega, G. A., and J. G. Owens. 1978. Ethylation of DNA and protamine by ethyl methane- sulfonate in the germ cells of male mice and the relevancy of these molecular targets to the induction of dominant lethals. Mutat. Res. 52:87-106. Sega, G. A., and J. G. Owens. 1983. Methylation of DNA and protamine by methyl meth- anesulfonate in the germ cells of male mice. Mutat. Res. 111 :227-244.

Biologic Significance of DNA Adducts and Protein Adducts 35 Sega, G. A., and J. G. Owens. 1987. Binding of ethylene oxide in spermiogenie germ cell stages of the mouse after low-level inhalation exposure. Environ. Moleeul. Mutag. 10:119- 127. Sega, G. A., C. R. Rohrer, H. R. Harvey, and A. E. JeKon. 1986. Chemical dosimetry of ethyl nitrosourea in the mouse testis. Mutat. Res. 159:65-74. Segerbaek, D., C. J. Calleman, L. Ehrenberg, G. Lofroth, and S. Osterman-Golkar. 1978. Evaluation of genetic risks of alkylating agents. IV. Quantitative determination of alkylated amino acids in haemoglobin as a measure of the dose after treatment of mice with methyl methanesulfonate. Mutat. Res. 49:71-82. Setlow, R. B. 1983. Variations in DNA repair among humans. Pp. 231-254 in Human Car- einogenesis, C. C. Harris and H. N. Autrup, eds. New York: Academic Press. Setlow, R. B. 1987. Theory presentation and background summary. Pp. 177-182 in Modern Biological Theories of Aging, H. R. Warner, R. N. Butler, R. L. Sprott, and E. L. Sehnei- der, eds. New York: Raven. Shamsuddin, A. K., N. T. Sinopoli, K. Hemminki, R. R. Boeseh, and C. C. Harris. 1985. Detection of benzo(a)pyrene: DNA-adduets in human white blood cells. Cancer Res. 45:66- 68. Shelby, M. D., K. T. Cain, L. A. Hughes, P. W. Braden, and W. M. Generoso. 1986. Dominant lethal effects of aerylamide in male mice. Mutat. Res. 173:35-40. Shelby, M. D., K. T. Cain, C. V. Cornett, and W. M. Generoso. 1987. Aerylamide: Induction of heritable translocations in male mice. Environ. Mutag. 9:363-368. Shugart, L. 1985. Quantitating exposure to chemical carcinogens: In viva alkylation of he- moglobin by benzo[a]pyrene. Toxicology 34:211-220. Sims, P., and P. L. Grover. 1974. Epoxides in polycyelie aromatic hydrocarbon metabolism and careinogenesis. Adv. Cancer Res. 20: 165-274. Singer, B. 1975. The chemical effects of nucleic acid alkylation and their relationship to mutagenesis end careinogenesis. Prog. Nucleic Acid Res. Mol. Biol. 15:219-284. Singer, B. 1982. Mutagenesis from a chemical perspective: Nucleic acid reactions, repair, translation, and transcription. Basic Life Sci. 20:1-42. Singer, B. 1985. In viva formation and persistence of modified nucleosides resulting from alkylating agents. Environ. Health Perspect. 62:41-48. Singer, B., and H. Fraenkel-Conrat. 1969. The role of conformation in chemical mutagenesis. Prog. Nucleic Acid Res. Mol. Biol. 9:1-29. Singer, B., and D. Grunberger. 1983. Molecular Biology of Mutagens and Carcinogens. New York: Plenum. 347 pp. Singer, B., H. Fraenkel-Conrat, and J. T. Kusmierek, 1978a. Preparation and template activ- ities of polynucleotides containing o2- and 04-alkyluridine. Proe. Natl. Aead. Sei. USA 75: 1722-1726. Singer, B., M. Kroger, and M. Carrano. 1978b. o2- and 04-alkyl-pyrimidine nueleosides: Stability of the gyleosyl bond and of the alkyl group as a function of pH. Biochemistry 17: 1246-1250. Singer, B., R. G. Pergolizzi, and D. Grunberger. 1979. Synthesis and coding properties of dinueleoside diphosphates containing alkyl pyrimidines which are formed by the action of carcinogens on nucleic acids. Nucleic Acids Res. 6:1709-1719. Singer, B., J. Sagi, and J. T. Kusmierek. 1983a. Escherichia cold polymerase I can use o2- methyldeoxythymidine or 04-methyl-deoxythymidine in place of deoxy~ymidine in primed poly (dA-dT).poly(dA-dT) synthesis. Proe. Natl. Aead. Sei. USA 80:4884-4888. Singer, B., J. T. Kusmierek, and H. Fraenkel-Conrat. 1983b. In vitro discrimination of rep

36 DRINKING WATER AND HEALTH lieases acting on eareinogen-modified polynueleotide templates. Proe. Natl. Aead. Sei. USA 80:969-972. Skopek, T. R., R. D. Wood, and F. Hutehinson. 1985. Sequence specificity of mutagenesis in the Cat gene of bacteriophage lambda. Environ. Health Perspeet. 62:157-161. Snow, E. T., R. S. Foote, and S. Mitral 1983. Replication and demethylation of O6-meth- ylguanine in DNA. Prog. Nucleic Acid Res. Mol. Biol. 29:99-103. Staffa, J. A., and M. A. Mehlman, eds. 1979. Innovations in Cancer Risk Assessment (EDGE Study). Park Forest South, Ill.: Pathotox. 246 pp. Stowers, S. J., and M. W. Anderson. 1985. Formation and persistence of benzo(a)pyrene metabolite-DNA adduets. Environ. Health Perspeet. 62:31-39. Sun, L., and B. Singer. 1975. The specificity of different classes of ethylating agents toward various sites of HeLa cell DNA in vitro and in vivo. Biochemistry 14:1795-1802. Swenberg, J. A., and T. R. Fennell. 1987. DNA damage and repair in mouse liver. Arch. Toxieol. (Suppl.) 10:162-171. Swenberg, J. A., M. A. Bedell, K. C. Billings, D. R. Umbenhauer, and A. E. Pegg. 1982. Cell speeif~e differences in 06-alkylguanine DNA repair activity during continuous exposure to carcinogen. Proe. Natl. Aead. Sei. USA 79:5499-5502. Swenberg, J. A., M. C. Dyroff, M. A. Bedell, J. A. Popp, N. Huh, A. Kirstein, and M. F. Rajewsky. 1984. 04-ethyldeoxythymidine, but not O6-ethyldeoxyguanosine, accumulates in hepatoeytes of DNA of rats exposed continuously to diethylnitrosamine. Proe. Natl. Aead. Sei. USA 81: 1692-1695. Swenberg, J. A., F. C. Richardson, J. A. Boueheron, and M. C. Dyroff. 1985. Relationships between DNA abduct formation and eareinogenesis. Environ. Health Perspeet. 62: 177- 183. Swenberg, J. A., F. C. Richardson, J. A. Boueheron, F. H. Deal, S. A. Belinsky, M. Char- bonneau, and B. G. Short. 1987. High- to low-dose extrapolation: Critical determinants involved in the dose-response of carcinogenic substances. Environ. Health Perspeet. 76:57- 63. Swenson, D. H., and P. D. Lawley. 1978. Alkylation of deoxyribonucleic acid by carcinogens dimethyl sulphate, ethyl methanesulphonate, N-ethyl-N-nitrosourea and N-methyl-N-nitro- sourea. Bioehim. J. 171:575-587. Talaska, G., W. W. Au, J. B. Ward, Jr., K. Randerath, and M. S. Legator. 1987. The correlation between DNA abducts and chromosomal aberrations in the target organ of ben- zidine exposed, partially-hepateetomized mice. Carcinogenesis 8: 1899- 1905. Tannenbaum, S. R., P. L. Skipper, L. C. Green, M. W. Obiedzinski, and F. Kadlubar. 1983. Blood protein abducts as monitors of exposure to 4-aminobiphenyl. Abstract 271. Proe. Am. Assoc. Cancer Res. 24:69. Thilly, W. G. 1985. The potential use of gradient denaturing gel electrophoresis to obtain mutation spectra in human cells. Pp. 511-528 in The Role of Chemicals and Radiation in the Etiology of Cancer, E. Huberman, ed. Carcinogenesis, Vol. 10. New York: Raven Press. Tice, R. B., and R. B. Setlow. 1985. DNA repair and replication in aging organisms and cells. Pp. 173-224 in Handbook of the Biology of Aging, 2nd ed, C. E. Finch and E. L. Schneider, eds. New York: Van Nostrand Reinhold. Travis, C. C., R. K. White, A. D. Arms. 1989. A physiologically-based pharmacokinetic approach to assessing the cancer risk of tetrachloroethylene. Pp. 769-795 in The Risk Assessment of Environmental and Human Health Hazards: A Textbook of Case Studies, D. Paustenbach, ed. New York: John Wiley & Sons. Tullis, D. L., K. L. Dooley, D. W. Miller, K. P. Baetcke, and F. F. Kadlubar. 1987. Char- acterization and properties of the DNA abducts formed from N-methyl-4-aminoazobenzene in rats during a carcinogenic treatment regimen. Carcinogenesis 8:577-583.

Biologic Significance of DNA Adducts and Protein Adducts 37 Umbenhauer, D., C. P. Wild, R. Montesano, R. Saffhill, J. M. Boyle, N. Huh, U. Kirstein, J. Thomale, M. F. Rajewsky, and S. H. Lu. 1985. O6-methyldeoxyguanosine in oesophageal DNA among individuals at high risk of oesophageal cancer. Int. J. Cancer 36:661-665. van Zeeland, A. A., G. R. Mohn, A. Neuhauser-Klaus, and U. H. Ehling. 1985. Quantitative comparison of genetic effects of ethylating agents on the basis of DNA adduct formation: Use of O6-ethylguanine as molecular dosimeter for extrapolation from cells in culture to the mouse. Environ. Health Perspect. 62:163-169. Vrieling, H., J. W. I. M. Simons, and A. A. van Zeeland. 1988. Nucleotide sequence de- termination of point mutations at the mouse HPRT locus using in vitro amplification of HPRT mRNA sequences. Mut. Res. 198: 107-113. Wild, C. P., R. C. Garner, R. Montesano, and F. Tursi. 1986. Aflatoxin B~ binding to plasma albumin and liver DNA upon chronic administration to rats. Carcinogenesis 7:853-858. Wogan, G. N. 1988. Detection of DNA damage in studies on cancer etiology and prevention. Pp. 32-54 in Methods for Detecting DNA Damaging Agents in Humans: Applications in Cancer Epidemiology and Prevention, H. Bartsch, K. Hemminki, and I. K. O'Neill, eds. IARC Scientific Publications No. 89. Lyon: International Agency for Research on Cancer. Wogan, G. N., and N. J. Gorelick. 1985. Chemical and biochemical dosimetry of exposure to genotoxic chemicals. Environ. Health Perspect. 62:5-18. Yang, L. L., V. M. Maher, and J. J. McCormick. 1980. Error-free excision of the cytotoxic, mutagenic N2-deoxyguanosine DNA adduct formed in human fibroblasts by ( +)-7B38a2- dihydroxy-9a2,10a2-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene. Proc. Natl. Acad. Sci. USA 10:5933-5937.

Next: 2 DNA-Adduct Technology »
Drinking Water and Health, Volume 9: Selected Issues in Risk Assessment Get This Book
×
Buy Paperback | $60.00
MyNAP members save 10% online.
Login or Register to save!
Download Free PDF

The National Research Council closes the landmark series Drinking Water and Health with Volume 9, published in two parts:

Part I: DNA Adducts provides an overview of DNA adducts and their effects on human health, explores the techniques currently in use for detecting them, offers an outlook on future toxicity testing, and lists specific recommendations for action.

Part II: Mixtures explores the issues surrounding multiple-chemical exposure from drinking water and reviews options for grouping compounds so their toxicity in mixtures can be reliably assessed. The book describes alternative approaches and considers the option of developing a modified "hazard index" for chemical compounds.

  1. ×

    Welcome to OpenBook!

    You're looking at OpenBook, NAP.edu's online reading room since 1999. Based on feedback from you, our users, we've made some improvements that make it easier than ever to read thousands of publications on our website.

    Do you want to take a quick tour of the OpenBook's features?

    No Thanks Take a Tour »
  2. ×

    Show this book's table of contents, where you can jump to any chapter by name.

    « Back Next »
  3. ×

    ...or use these buttons to go back to the previous chapter or skip to the next one.

    « Back Next »
  4. ×

    Jump up to the previous page or down to the next one. Also, you can type in a page number and press Enter to go directly to that page in the book.

    « Back Next »
  5. ×

    To search the entire text of this book, type in your search term here and press Enter.

    « Back Next »
  6. ×

    Share a link to this book page on your preferred social network or via email.

    « Back Next »
  7. ×

    View our suggested citation for this chapter.

    « Back Next »
  8. ×

    Ready to take your reading offline? Click here to buy this book in print or download it as a free PDF, if available.

    « Back Next »
Stay Connected!