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OCR for page 6
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
OCR for page 7
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.
OCR for page 8
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
OCR for page 9
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.
OCR for page 10
|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
OCR for page 11
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
OCR for page 12
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
OCR for page 13
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,
OCR for page 14
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
OCR for page 15
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
OCR for page 16
|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
OCR for page 27
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
OCR for page 28
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-
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Representative terms from entire chapter:
protein adducts
